Central Instrumentation Laboratory, Directorate of Research office, Sardarkrushinagar Dantiwada Agricultural

Transcription

1 Journal of Cell and Tissue Research Vol. 16(1) (2016) (Available online at ISSN: ; E-ISSN: Original Article IN VITRO ANALYSIS OF ANTIMICROBIAL PROPERTY OF SILVER AND SILVER COATED SILICA NONO PARTICLES FROM MICROORGANISM? YUVRAJSINH, K. V., FALDU, H. G., DEDHROTIYA, A. T. AND TALEKARN, S Central Instrumentation Laboratory, Directorate of Research office, Sardarkrushinagar Dantiwada Agricultural 2 University, Sardarkrushinagar Gujarat, ; Biotechnology laboratory, BRD School of Biosciences, 3 Sardar Patel University, Vallabh Vidyanagar ; Department of Genetics and Plant Breeding,C.P. College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Gujarat, E. mail: yuvrajsinh.bt@gmail.com, Cell: Received: February 24, 2016; Revised: February 29, 2016; Accepted: March 10, 2016 Abstract: Nanotechnology can be defined as the design, synthesis, and application of materials and devices whose size and shape have been engineered at the nanoscale. It exploits unique chemical, physical, electrical, and mechanical properties that emerge when matter is structured at the nanoscale. Certain nano powders possess antimicrobial properties. When these powders contact cells of E. coli, or other bacteria species and viruses, over 90% are killed within a few minutes. Due to their antimicrobial effect, nanoparticle of silver and titanium dioxide (<100nm) are assessed as coatings for surgical masks. In present work isolation and screening of microorganisms capable of synthesizing nanoparticles and after purification apply as antimicrobial property on Microorganisms. Key words: Antimicrobial, Silver nanoparticles. INTRODUCTION Nano-particle is defined as a small object that behaves as a whole unit in terms of its transport and properties, there is no strict dividing line between nanoparticles and non-nanoparticles (bulk material). The size at which materials display different properties to the bulk material is material dependent and can certainly be claimed for many materials much larger in size than 100 nm. Two primary factors cause nanomaterials to behave significantly different than bulk materials: surface effects (causing smooth properties scaling due to the fraction of atoms at the surface) and quantum effects (showing discontinuous behavior due to quantum confinement effects in materials with delocalized electrons) [1]. These factors affect the chemical reactivity of materials, as well as their mechanical, optical, electric, and magnetic properties. Morphological characteristics taken into account are: flatness, sphericity, and aspect ratio. A general classification exists between highand low-aspect ratio particles. High aspect ratio nanoparticles include nanotubes and nanowires, with various shapes, such as helices, zigzags, belts, or perhaps nanowires with diameter that varies with length. Small-aspect ratio morphologies include spherical, oval, cubic, prism, helical, or pillar. Collections of many particles exist as powders, suspension, or colloids. The study of biosynthesis of nanomaterials offers valuable contribution into materials chemistry. The ability of some microorganisms such as bacteria 5393

2 J. Cell Tissue Research and fungi to control the synthesis of metallic nanoparticles should be employed in the synthesis of new materials [2-3]The biosynthetic methods are investigated as an alternative to chemical and physical ones. These methods can be divided on two categories depending on the place where the nanoparticles or nanostructures are created. It is known that many microorganisms can transform inorganic materials either intracellularly or extracellularly [3]. Many fungi that exhibit these characteristic properties, in general, are capable of reducing Au (III) or Ag (I) [4]. Besides these extracellular enzymes, several naphthoquinones [5-7] and anthraquinones [8] with excellent redox properties, were reported in F. oxysporum that could act as electron shuttle in metal reductions [9]. The metabolic activity of microorganisms can lead to the precipitation in the external environment of the cell; the fungi being extremely good candidates for these processes. The extracellular synthesis of silver and gold nanoparticles by the fungus Colletotrichum sp. [9] or Aspergillus fumigatus has been reported [10]. A novel biological method for synthesis of silver nanoparticles using Vericillum was proposed by Mukherjee and coworkers [11] and the two-step mechanism was suggested. The 2+ first step involved trapping of the Ag ions at the surface of the fungal cells. In the second step, the enzymes present in the cell, reduced silver ions. In present investigation eighteen different fungal cultures were used for primary screening amongst which seven cultures viz., A. flavus,f. oxysporium, T. versicolor, P. ostreatus, Alt. brunsii 1, isolate 1072 and isolate 6 were found to synthesize silver nanoparticles. MATERIALS AND METHODS All the chemicals and media used in the present study were of pure analytical grade and were prepared in glass distilled water. Silver nitrite was purchased from Sigma-Aldrich, U.K. Organisms used in this study: Organisms used in this study was isolated from different sources viz., (1) Lab No. 208, BRD School of Bioscience, Sardar Patel University, Vallabh Vidyanagar, Anand ( ), Gujarat (2) Lab No. 103, BRD School of Bioscience, Sardar Patel University, Vallabh Vidyanagar, Anand ( ), Gujarat, India and (3) Garden Soil and Contaminated Soil area around, BRD School of Bioscience, Sardar Patel University, Vallabh Vidyanagar, Anand ( ) ( see Table 1) Isolation of fungal cultures from soil: Fungus was isolated from highly contaminated soil. The soil sample were collected and suspended in sterile distilled water & allowed to settled down for min. and supernatant from this suspension was serially diluted like 10,10-,10-,10 to10 in sterile distilled water and 0.1 ml aliquots of these diluted sample were spreaded on potato dextrose agar (PDA) plates using sterile glass spreader. The plates were incubated at 28C-30C for 24-48h. From these plate spores from isolated fungal colonies were transferred on fresh PDA plates repeatedly to get pure cultures. Inoculum preparation: 50 ml of sterile potato dextrose broth in 250 ml Erlenmeyer flasks were inoculated with 1 agar block (10 mm) of fungal growth from a PDA plate in each flask. The flasks were then incubated on orbital shaker (150 rpm) at 30C for 48 hours. Screening for silver reducing fungus: The 48 hours old fungal culture in potato dextrose broth was filtered using cheese cloth and washed repeatedly with sterile distilled water to remove salts from the surface of the biomass. Approximately 10 g of weight biomass was taken in a conical flask containing 100 ml of sterile distilled water and spiked with AgNO 3 to a final concentration of 1mM and incubated in dark on orbital shaker (150 rpm) at 30C. At regular intervals of 24 h, 5mL aliquots were withdrawn, centrifuged and UV-Visible absorbance spectra were recorded using a Diode Array UV-Vis spectrophotometer (Hewlett-Packard 8453). The silver nanoparticles were characterized by scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) at a voltage of 20 kv. Chemical synthesis of silver coated silica nanoparticle: Silver coated silica nanoparticles were synthesized by hydrolysis of tetra ethoxy silane in presence of AgNO in sonicator bath. The 3 hydrolysis was performed using 25% NH OH. The 4 reaction was stopped after the reaction medium became turbid. The silica nanoparticles were harvested by centrifugation at 10,000 rpm for 30 minutes. The nanoparticles were resuspended in acetone and washed twice with acetone. The nanoparticles were then dried at 60 C. Antimicrobial activity: Study on microbial effect Dried powder of Silver nano practical apply on 5394

3 Yuvrajsinh et al. E.coli and study on zone of inhibition by cup borer method. Table 1: Name and Source of organisms used for synthesis of silver and silver coated silica for identification of antimicrobial activity Sr. Name of organism Source No. 1 Aspergillus parasiticus Lab No. 208, BRD School of 2 Aspergillus flavus Bioscience, Sardar Patel University, 3 Alterneria brunsii 1 Vallabh Vidyanagar, Anand ( ), Gujarat, India 4 Alterneria brunisi 2 5 Trametes versicolor 6 Pleurotus ostreatus 7 Fungus Candida albicans Lab No. 103, BRD School of Bioscience, Sardar Patel University, 9 Fusarium oxsporium Vallabh Vidyanagar, Anand ( ), Gujarat, India 10 Fungus 1 Garden Soil and Contaminated 11 Fungus 2 Soil area around,brd School 12 Fungus 3 of Bioscience, Sardar Patel University, Vallabh Vidyanagar, 13 Fungus 4 Anand ( ), Gujarat, India 14 Fungus 5 15 Fungus 6 16 Fungus 7 17 Fungus 8 18 Yeast RESULTS In order to isolated microorganisms capable of synthesizing silver nanoparticles, primary screening was performed using eighteen different fungal cultures. As listed in table 1, some of the Table 2: Screening of fungal cultures for synthesis of silver nanoparticles at different time interval viz., 24hrs, 48 hrs, 72hrs and 96 hrs Name of organism 24 hrs 48hrs 72hrs 96hrs Aspergillus parasiticus Aspergillus flavus Alterneria brunsii Alterneria brunsii Trametes versicolor Pleurotus ostreatus Fungus Candida albicans Fusarium oxysporium Fungus Fungus Fungus Fungus Fungus Fungus Fungus Fungus Yeast fungal cultures were isolated from garden soil and others were obtained from the collection of our department. All the eighteen fungal cultures were initially cultivated in potato dextrose broth upto mid-logarithmic phase and then harvested by filtration through cheese cloth. The fungal biomass was then washed with sterile distilled water, twiceto remove salts from the biomass and then 10g of biomass was suspended in 100 ml of sterile distilled water, spiked with 1mM AgNO 3 and incubated in dark under on orbital shaker (150 rpm) at 30 C. 5 ml aliquots were removed at regular intervals of 24 hours and were analyzed for presence of silver nanoparticles by UV-visible Fig. 1: UV-visible spectra of fungal filtrate containing silver nanoparticles: cell filtrate + silver nitrate; biomass + silver nitrate; control (cell filtrate); Silver nitrate (1 mm). 5395

4 J. Cell Tissue Research a b Fig. 2: Scanning Electron Microscopy (SEM): (a) Ag-NPs synthesized fromfungus Fusarium oxysporium(biologically synthesized)and (b) silver coated silica nanoparticle by challenging silver nitrate (chemically synthesized) (1 mm). 5396

5 Yuvrajsinh et al. Fig. 3: Spectrum of silver nano-particles obtained by EDX spectroscopy.sil2,3, silicon (Si); Oka, oxygen (O).Energydispersive x-ray (EDX) spectroscopy analysis for the confirmation of elemental silver was carried out for the detection of elemental silver. ions extracellularly. Fig. 4: Potential Antimicrobial Effect of Silver Nanoparticles on E. Coli. (Zone of inhibition is 15 mm) spectrophotometry. Optical absorption spectroscopy has proved to be a very useful technique for analysis of nanoparticles. It has been reported that silver nanoparticles exhibit a surface plasmon peak at around 420 nm. Amongst all cultures tested, Aspergillus flavus, Alternaria brunsii1, Fusarium oxysporium, Trametes versicolor, Pleurotus ostreatus, Isolate 1072 and isolate 6 were found to synthesize silver nanoparticles extracellularly Table 2. Amongst all the isolates, Fusarium oxysporium, isolate 1072 and Alternaria brunsii 2+ exhibited higher efficiency of reduction of Ag Fig. 1 shows the UV-Visible spectra of extracellular filtrate of Fusarium oxysporium. A strong surface plasmon peak at 414 nm could be seen which may be due to silver nanoparticles. Fig. 2 shows the SEM image of silver nanoparticles produced by Fusarium oxysporiumand strain isolate 6 wherein 500 nm silver nanoparticles are clearly visible. In the present work antimicrobial activity of biosynthesized silver nanoparticles studied against the E. coli using standard zone of inhibition. MDR isolate E. coli showed maximum zone of inhibition of 15 mm for 20μg/ml of silver nanoparticle concentration (Fig. 4). Fig. 3 shows the plot of the spectra intensity versus X-ray energy on a semi-log scale for the EDX analysis carried out for fungal filtrate. From the spectra, it can be seen that silicon at K = 1.739KeV is clearly the dominant element in the fungal filtrate. DISCUSSION It is known that microorganisms such as bacteria, yeast and fungi play an important role in remediation of toxic metals through reduction of the metal ions and thus are explored for synthesis of nanoparticles [10]. Using these dissimilar proper- ties of fungi, the biosynthesis of inorganic nanomaterials may be used to grow nanoparticle of 5397

6 J. Cell Tissue Research gold and silver [11-12] intracellularly or extracellularly. Recently, it was found that aqueous chloroaurate ions could be reduced extracellularly using the fungus F. oxysporum, to generate extremely stable gold or silver nanoparticles in water [11-12]. R.: Angew. Chem. Int. Ed. 40: (2001). [12] Mukherjee, P., Ahmad, A., Mandal, D., Senapati, S., Sainkar, S.R., Khan, M.I., Parischa, R., Ajayakumar, P.V., Alam, M., Kumar, R. and Sastry, M.: Nano. Lett. 1: (2001). In order to determine the time of incubation for maximum reduction of silver ions, and optimum amount of biomass required for maximum synthesis of nanopartilces, experiment was performed where in silver nanoparticles synthesis was monitored at different time intervals using different amount of biomass for three different fungi (Table 2). CONCLUSION Isolated fungus was capable of synthesizing silver nanoparticles. Eighteen different fungal cultures were used for primary screening amongst which seven cultures viz., A. flavus,f. oxysporium, T. versicolor, P. ostreatus, Alt. brunsii 1, isolate 1072 and isolate 6 were found to synthesize silver nanoparticles. Amongst all cultures, Fusarium oxysporiumwas found to be most potent and exhibited extracellular synthesis of silver nanoparticles. The production of silver nanoparticles was investigated using UV-Visible spectrophotometry and SEM. REFERENCES [1] oduner, E.: Chem. Soc. Rev (2006). [2] Mandal, D., Bolander, M.E., Mukhopadhyay, D., Sarkar, G. and Mukherjee, P.: Appl. Microbiol. Biotechnol. 69: (2006). [3] Pantidos, N. and Horsfall, L.E.: J. Nanomed. Nanotechnol. 5: 1-10 (2014). [4] Iravani, S.: Intern. Scholarly Res. Notic : 1-18 (2014). [5] M e d e n t s e v, A. G. a n d A l i m e n k o, V. K. : Phytochemistry, 47: (1998). [6] Duran, N., Teixeira, M.F.S., De Conti, R. and Esposito, E.: Crit. Rev. Food. Sci. Nutr., 42: (2002). [7] Bell, A.A., Wheeler, M.H., Liu, J., Stipanovic, R.D., Puckhaber, L.S. and Orta, H.: Pest Manag. Sci., 59: (2003). [8] Huang, Q., Lu, G., Shen, H.M., Chung, M.C. and Ong, C.N.: Med. Res. Rev. 27(5): (2007). [9] Thakker, J.N., Pranay, D. and Pinakin, C.D.: ISRN Biotechnol. (515091): 1-55 (2013). [10] Bala, M. and Arya, V.: Intern. J. Nanomat. Biostruc. 3(2): (2013). [11] Mukherjee, P., Ahmad, A., Mandal, D., Senapati, S., Sainkar, S.R., Khan, M.I., Ramani, R., Parischa, R., Ajaykumar, P.V., Alam, M., Sastry, M. and Kumar, 5398