Synthesis and Characterization of Silver Nanoparticles Using Bio Waste and its Antibacterial Activity

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1 JOURNAL OF MODERN BIOTECHNOLOGY, VOL.4, NO. 1, pp 8 12, January 2015 Copyright 2015, by Apex Biotechnology Training and Research Institute. All Right Reserved. Research Article Synthesis and Characterization of Silver Nanoparticles Using Bio Waste and its Antibacterial Activity Arul doss Vijayakumar and PuthupalayamThangavelu Kalaichelvan Centre for Advanced Studies in Botany, Guindy Campus, University of Madras, Chennai , India. *Corresponding Author arulvijai@gmail.com Received 15 November 2014; Revised 14 December 2014; Accepted 24 December 2014 Abstract In the present study, synthesis of silver nanoparticles and its activity on pathogenic bacteria were investigated. Silver nanoparticles were rapidly synthesized in 60 minutes using various bio wastes such as lemon fruits peel, banana fruits peel, peduncle of mint leaves, jambul fruits seed, peduncle of curry leaves and coir pith. The results recorded from UV Vis spectrum, Transmission electron microscope (TEM) and X-ray diffraction (XRD) support the biosynthesis. Further, the antibacterial activity of silver nanoparticles showed effective inhibitory activity against human pathogenic bacteria. Keywords: Silver nanoparticles, Bio wastes, Pathogenic bacteria, Transmission electron microscope (TEM) and X-ray diffraction (XRD), Antimicrobial activity INTRODUCTION Nanoparticle synthesis and the study of their size and properties are of fundamental importance in the advancement of recent research (Nath et al., 2008; Cao, 2004; Chang, 2005). It is found that the optical, electronic, magnetic, and catalytic properties of metal nanoparticles depend on their size, shape and chemical surroundings Cao, 2004; Chang, 2005]. The development of reliable green process for the synthesis of silver nanoparticles is an important aspect of current nanotechnology research. Nanomaterials such as Ag, Au, Pt and Pd have been synthesized by different methods, including hard template (Zhou et al., 1999), using bacteria (Husseiny et al., 2007), fungi (Sastry et al., 2003) and plants (Sharma et al., 2007). The development of green processes for the synthesis of nanoparticles is evolving into an important branch of nanotechnology (Raveendran et al., 2006; Armendariz et al., 2002). Biogenic synthesis, which involves the action of biological material produce metal nanoparticles that remains pertinent in present context. This method of synthesis is clean, cost effective, employs ambient conditions and less energy intensive (Saxena et al., 2010). The diverse application of nanocrystalline silver ranges from biosensing and diagnostics (Songping and Shuyuan, 2005; Schultz et al., 2000), antimicrobial (Pal et al., 2007). In one study, antimicrobial activity of silver nanoparticles was shown to promote wound healing (Ghosh et al., 2010). An anti-proliferative activity of silver nanoparticles against human cancer cells have been reported (Rani et al., 2009). 8 VOLUME 4 NUMBER 1 JANUARY 2015 JOURNAL OF MODERN BIOTECHNOLOGY

2 Synthesis of silver nanoparticles using bio waste A plant species Gliricidia sepium used for the synthesis of silver nanoparticles, showed absorption maximum at 440 nm (Raut et al., 2009). Green synthesis of silver nanoparticles using Argimone maxicana leaves broth generated particles of 20 nm and found to be effective against many bacterial and fungal pathogens (Khandelwal et al., 2010). A plant species Solanum torvum produced silver nanoprticles of 14 nm dimension and showed the absorbance peak at 434 nm. The antimicrobial activity of synthesized nanoparticles was tested against Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus flavus and Aspergillus niger, showing a zone of inhibition (Govindaraju et al., 2010). Weeds such Ipomoea aquatica, Enhydra fluctuans and Ludwigia adscendens were used as a precursor for the synthesis of silver nanoparticles showed absorbance peak between 400 to 480 nm (Roy and Barik, 2010). Silver nanoparticles synthesized from Boswellia ovalifoliolata stem bark showed UV-Vis analysis absorption maxima at 430 nm and their size varied from 30 to 40 nm (Ankanna et al., 2010). Dried leaves of Cinnamomum camphora have been implicated in synthesis 55 to 80 nm of silver nanoparticles (Huang et al., 2007). Bio-reduction of silver using various plant extracts such as Helianthus annus, Basella alba, Oryza sativa, Saccharum officinarum, Sorghum bicolor and Zea mays have been studied (Leela and Vivekanandan, 2008). Leaf extract of Parthenium hysterophorus synthesized silver nanoparticles of average size of 50 nm (Parashar et al., 2009). An aqueous extract of Azadirachta indica (Neem) leaves too was studied for the biogenic synthesis of silver nanoparticles, showed maximum absorbance between 440 to 500 nm (Mukherjee et al., 2009). The present study was aimed to rapid synthesis of silver nanoparticles using aqueous extracts from biowastes and evaluates its antibacterial activity against human pathogens such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, and Salmonella typhi. MATERIALS AND METHODS Materials Various bio wastes such as lemon fruits peel, banana fruits peel, peduncle of mint leaves, jambul fruits seed, peduncle of curry leaves and coir pith were used. AgNO3, D-sorbitol was purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. The bacterial cultures obtained from American Type Culture Collection and Microbial Type Culture Collection culture (Manassas) VA/USA and (Chandigarh) India, respectively. Preparation of bio waste and extract The collected bio wastes were washed with running tap water three times and air dried to remove moisture. Four gram (4g) of each of them was weighed and added to 40 ml distilled water and boiled with 2 minutes. It was then filtered through Whatman No. 1 filter paper. This was centrifuged at 5000 rpm for 5 minutes and the supernatant was collected and stored at 4 C in airtight bottle for further use. Synthesis of silver nanoparticle and its characterization 3ml of bio waste extract and 1ml of D- sorbitol 0.01 M (10-2 ) was added to 40 ml of 10-3 M AgNO 3 solution. All the reaction was carried out in the dark at 30 C. Periodically, aliquots of the reaction solution were removed and the absorptions were measured using a UV-Vis spectrophotometer. Further, the reaction mixture was subjected to centrifugation at g for 20 min; resulting pellet was dissolved in sterilized glass distilled water and filtered through Millipore filter (0.45µm). An aliquot of this filtrate containing silver nanoparticles was used for TEM and XRD studies. For electron microscopic studies, 25µg of samples was sputter coated on copper stub and the image of nanoparticles was studied using TEM (JEOL, Model JFC-1600). For XRD studies, dried nanoparticles were coated on XRD grid and the spectra was recorded by using Philips PW 1830 X-ray generator operated at a voltage of kv and a current of 30 ma with Cu K 1 radiation. Antibacterial activity 9 JOURNAL OF MODERN BIOTECHNOLOGY VOLUME 4 NUMBER 1 JANUARY 2015

3 Synthesis of silver nanoparticles using bio waste The antimicrobial activity of silver nanoparticles synthesized from mint leaves peduncle was screened against Micrococcus lettuce (lab culture), E.coli MTCC1687, Salmonella typhi and S.aureus MTCC96, K.pneumoniae MTCC109 and B.subtilis (lab culture) were used. In vitro antimicrobial activity was screened by using Muller Hinton agar plates and well of 7mm in diameter. To which 25, µl and 50µl of the synthesized silver nanoparticles loaded on the pathogen swabbed plates. The plates were then incubated at 37 C for 24 hrs and the zone of incubation was recorded. RESULTS AND DISCUSSION Different bio wastes like lemon fruits peel, banana fruits peel, peduncle of mint leaves, jambul fruits seed, peduncle of curry leaves and coir pith were used in the current study for the synthesis of silver nanoparticles. All the bio waste can synthesis silver nanoparticles and when added to aqueous silver nitrate at room temperature resulted in the formation of silver nanoparticles (within 5 minutes). The reaction due to the reduction of silver ions is evidenced by the change in colour from greenish to yellowish brown (Sastry et al., 1998). Reaction was stopped at 60 minutes of incubation (Figure 1). nm after 60 min of reaction, which is a characteristic surface plasmon resonance (SPR) band of silver nanoparticles possibly due to the excitation of longitudinal plasmon vibrations in silver nanoparticles in the solution (Mulvaney, 1996; Mock et al., 2002). The intensity of the SPR band increased as the reaction proceeded for 60 min and there was no increase in the intensity after 60 min, suggesting the completion of the reaction. The size and shape of silver nanoparticles was reported to influence their optical properties (Jensen et al., 2000). The UV visible spectroscopy results indicated that the obtained silver nanoparticles were spherical and nano-sized. The silver nanoparticle solution synthesized from mint leaf peduncle was extremely stable, with evidence of flocculation of the particles even several weeks after reaction. (Relative maximum absorbance at 440 nm of several bio waste extractions). Destabilization of the nanoparticles is evident in the case of all other extracts after1 hr. Figure 1: Silver nanoparticle synthesis using different bio wastes Figure 2: UV-visible spectroscopy synthesis of silver nanoparticle using bio wastes UV-Vis Spectroscopy analysis The reduction of Ag +ions was monitored by UV visible spectroscopy and the spectra were recorded at room temperature (28 C) as a function of time of the reaction of culture supernatant with silver nitrate recorded at every 3 minutes interval and is shown in Figure 2. The nanoparticles exhibited an absorption peak around 440 TEM analysis A TEM image analysis (Figure 3) revealed that silver nanoparticles were are spherical in shape with a smooth surface morphology. The diameter of the nanoparticle is found to be approximately in the size range 20 to 40 nm. TEM image also shows that the synthesized nanoparticles are more or less uniform in size and shape. 10 JOURNAL OF MODERN BIOTECHNOLOGY VOLUME 4 NUMBER 1 JANUARY 2015

4 Synthesis of silver nanoparticles using bio waste E.coli MTCC1687 (3.5 mm). Salmonella typhi and S.aureus MTCC96 (3.0mm) and B.subtilis (lab culture) (1.9mm) were observed. Minimum inhibition K.pneumoniae MTCC109 (1.0mm) was observed after 38 hrs of incubation. Mint leaves peduncle synthesised silver nanoparticle suggesting the broad spectrum nature of their antimicrobial activity. The antimicrobial activity of silver nanoparticles was reported to be due to the Figure 3: TEM showing particle size distribution of the silver nanoparticles XRD analysis The X- ray diffraction (XRD) spectrum of silver nanoparticle (Figure 4) exhibited the characteristic Bragg peaks of the silver nanocrystallites observed at 2ɵ values of 32.1, 37.9, 44.0 and 45.9 corresponding to (111), (200), (220) and (311) facets of the face centred cubic (fcc) silver nanoparticle which conforms the SAED results. Figure 4: X- ray diffraction (XRD) spectrum of silver nanoparticle Antimicrobial activity Antimicrobial activity of silver nanoparticles (Graph 2) was evaluated against pathogenic Gram-positive, Gramnegative bacteria like maximum inhibition was observed Micrococcus lettuce (lab culture) (6.1mm), followed by 11 JOURNAL OF MODERN BIOTECHNOLOGY penetration into the bacteria and damage of cell membrane and release of cell contents (Panacek et al., 2006). Another possibility suggested (Kim et al., 2009; Li et al., 2010) was the release of silver ions from the nanoparticles, which may contribute to the bactericidal properties of silver nanoparticles. CONCLUSION Even though gold/silver nanoparticles have been synthesized using prokaryotes and eukaryotes such as fungi, bacteria (Mukherjee et al., 2001a and b), the nanoparticles grow intracellularly, except in the case of a recent report like Enterobacteriaceae, in which the nanoparticles grew extracellularly (Minaeian et al., 2008). In the current work, all the bio wastes including peduncle of mint leaves showed the capability of synthesizing silver nanoparticle with silver nitrate at room temperature. This is one of the simplest and cheapest processes for obtaining novel way of synthesis of silver nanoparticles. The UV visible spectrum showed a surface plasmon resonance peak at around 440 nm, which is characteristic of silver nanoparticles. The silver nanoparticles formed were spherical in shape and smooth surface with an average particle size of nm, crystalline in nature, and the particle; these properties were confirmed by TEM, XRD and FTIR analysis. The silver nanoparticle solution synthesized from mint leaf peduncle was extremely stable and has a broad spectrum antimicrobial activity against Micrococcus luteus, E.coli, Klebsiella pneumonia, Staphylococcus aureus, Salmonella typhi and Bacillus subtilis. ACKNOWLEDGEMENTS The authors acknowledge the financial assistance provided by Council of Scientific and Industrial Research (CSIR), New Delhi. (Meritorious Fellowship). VOLUME 4 NUMBER 1 JANUARY 2015

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