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Synthesis and characterization of silver nanoparticles from Penicillium sps.
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 4 (2017) 11923–11932
www.materialstoday.com/proceedings
ICNANO 2016
Synthesis and characterization of silver nanoparticles from Penicillium sps. Shareef J. Ua, Navya Rani Mb, Anand Sc, Dinesh Rangappad* a
Department of Nano Technology, Srinivas Institute of Technology, Mangalore-574143, India b Department of Biotechnology, RGIT, Bangalore-560032, Inida c Department of Biotechnology, Saptagiri College of Engineering, Bangalore-560057, Inida d Department of Nano Technology, PG Centre Bangalore Region, Visvesvaraya Technological University, Visvesvaraya Institute of Advanced Technology (VIAT), Muddenhalli post, Chickballapur District 562101 India.
Abstract Among the different methods employed for synthesis of nanoparticles, the biological method is most favourable and well established. Fungi provide many advantages in this context. In this study, extracellular synthesis of silver nanoparticles from Penicillium sps. was carried out. Nanoparticles were produced due to reduction of silver ions from silver nitrate, the formation of which was monitored by UV-visible spectrophotometry. The optimization of the biosynthesis procedure with respect to substrate concentration, pH, and salinity were carried out. The size distribution was determined using zetasizer Nano S90 and the dimensions were observed to be around 75nm by AFM and morphology was characterized by SEM. Then the synthesized silver nanoparticles are subjected to XRD analysis. The efficiency of activity of antibiotics is increased by several folds by conjugating the AgNPs with the different types of antibiotics by using zone of inhibition method. © 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON NANOTECHNOLOGY (ICNANO2016). Keywords: Silver nanoparticles, Penicillum sp, Spectrophotometry, zetasizer Nano S90, AFM, SEM, XRD.
* Corresponding author. Tel.: +91 9632764659; E-mail address: [email protected] 2214-7853© 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON NANOTECHNOLOGY (ICNANO-2016).
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1. Introduction Silver is medically considered as one of the most powerful elements due to its activity against mammalian tissues where it acts as an antiseptic agent [1]. Silver, in its metallic as well as ionic forms, exhibits cytotoxicity against several microorganisms and hence used as an anti-microbial agent [2]. Some microorganisms are also able to accumulate silver by adsorption. Silver nanoparticles have been exploited for their unique properties and their vast applications in biomedicine. Environmentally benign methods have been developed for the synthesis of these nanoparticles which eliminate the use of toxic chemicals during their synthesis process [3]. Ag nanoparticles are usually synthesized from bacteria and fungi. The latter posses more advantages which include filamentous fungal tolerance towards metals, their high binding capacity and intracellular uptake of metals [4]. Further, the mass production of fungi is easy, for synthesis of nanoparticles [5]. Ag nanoparticles have been widely employed due to their physicochemical properties [6] and are currently used as anti-bacterial agents in food storage, textile and health industries, for biolabeling and as biosensors. The anti-microbial activity of silver nanoparticles has now been well established and they are confirmed to posses anti-inflammatory, anti-viral, anti-platelet and anti-fungal activity [7]. One of the major issues to be considered during synthesis of nanoparticles is their size, depending on which, the applications may vary [8]. Synthesis may be routed through either intracellular or extracellular pathway. The latter is disadvantageous for harvesting the product and recovery may be cumbersome and expensive. For extracellular synthesis, it is reported that a reductase enzyme is released into solution which brings about reduction of Ag+ ions [9]. Therefore the extracellular synthesis of Ag nanoparticles from fungi is most widely adapted. The present study deals with the extracellular synthesis of Ag nanoparticles from the fungus Penicillium sp, the formation of which was monitored by spectroscopy. This was followed by microscopic characterization. Thus, silver nanoparticles are the metal of choice as they have the capability to kill microbes effectively. The strong toxicity of silver against wide range of microorganisms is well known and silver nanoparticles have shown to be an effective antimicrobial agent. 2. Experimental Procedure 2.1 Collecting Sample Three soil specimen are gathered Soil of wet environment is preferred, one soil sample from balehannu kere situated west part of Bangalore was collected and the other two from sapthagiri college garden. Aseptic method was employed to avoid contamination throughout taking the specimen. Four distinctive specimens were gathered from the depth of 6 inches of the ground by utilizing Random Sampling Method. 2.2 Isolation of the specimen The sample was mixed with Distilled tap water to make solution sample. This solution sample was diluted up to 10-9 serial dilution. Serial dilution is a process of diluting a sample several times. 2.3 Serial dilution method The sterile Petri dish is labelled. Then 9ml of phosphate buffer is transferred into each of 5 tubes using sterile pipettes (10ml) with aseptic technique. By using sterile pipette (1ml), 1ml of water sample is transferred into 10-1 tube. Mix the test tube properly. Continue dilution with aseptic technique for test 10-2 until 10-5. After that each of serial dilution is transferred into Potato Dextrose Agar (PDA) plate by using spread plate method.
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2.4 Spread plate method 2.4.1 Preparation of media Media used here is PDA (potato dextrose agar), it is used to grow fungus, 200ml of PDA is prepared by using 40gms of Potato, 4gms of Dextrose and 4gms of Agar. The solution is then subjected to autoclave at 1210 C for 15mins. Then the sterile media is used for the inoculation. 2.4.2 Procedure One millilitre of an appropriately diluted culture is spread over the surface of agar using sterile glass spreader. The plate is then incubated until the colonies appear. It is important that the surface of the plate be fairly dry so that the spread liquid soaks in. 2.4.3 Pure culture Different types of colonies are observed by spread plate method among those single colony is selected and then it is inoculated by streaking the plate. Then incubated at 290C for 3days and the colony is selected for the inoculation to liquid broth.A culture of Penicillium sps. Was used for the extra cellular production of silver nanoparticles 2.5 Production of bio-mass The preparation of biomass for biosynthesis of silver nanoparticles from Penicillium sp involved the aerobic culturing of fungus in a liquid broth composed of KH2PO4,(7.0g/L), K2HPO4(2.0g/L), MgSO4. 7H2O (0.1g/L), (NH4)2SO4 (1.0g/L) yeast extract, (0.6g/L) and glucose (10g/L). 2.5.1 Culturing of fungus in liquid broth Liquid broth is prepared for 250ml and divided into two sample. One colony is selected from pink pure culture and it is inoculated in 1st sample and other colony from green pure culture is inoculated in 2nd sample. The flask containing this culture was incubated at 290C for about 8 days. The biomass thus obtained was harvested, followed by extensive washing with Milli Q water. 2.6 Synthesis of silver nanoparticles utilizing silver nitrate Biomass of 15gms was re-suspended in 150ml milli Q water and incubated in shaker for 72 hours at 290C. Then the biomass was filtered utilizing a Whattman channel paper No1. The pH of filtrate was discovered to be 5.84. To the 150ml of filtrate silver nitrate was added to give an overall concentration of 1mM (0.017g/100ml). The reaction was carried out in dark. The control with just the cell filtrate and without silver particle was run with the test sample. Time dependent formation of silver nanoparticles was measured by using UV-visible spectrophotometer at 24, 48 and 72 hours and the absorbance was measured at wave lengths extending from 200 to 600nm. 2.7 Optimization of silver nanoparticle synthesis The filtrate containing silver nanoparticles were subjected to further studies. The impact of substrate concentration, pH and salinity, on the production of silver nanoparticles was optimized d by fluctuating the parameters, such as substrate concentration (0.5, 1.0, 1.5, 2.0, 2.5mM of AgNO3), pH (5,6,7,8) and salinity 0.1, 0.2, 0.3, 0.4, 0.5% Nacl) at different time intervals. 300µl of the sample was withdrawn to measure the optical thickness at a wavelength of 450nm with an UV-visible spectrophotometer.
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2.8 Characterization and Examination of silver nanoparticles After incubation of 72 hours, the filtrate which has silver nanoparticles were characterized using spectrophotometry and zetasizer Nano S90. The latter is used to predict the size of nanoparticles. The sample was prepared by centrifuging the cell filtrate and making a smear with the pellet obtained. This was then subjected to AFM and SEM analysis. Then the sample is analysed by XRD pattern for structural confirmation. 2.9 Studies on the Antimicrobial activity of silver nanoparticles An examination was done to focus the synergistic impact of antimicrobials against the gram positive and gram negative microorganisms with the silver nanoparticles synthesized. Standard antibiotic discs were used and plated onto Muller-Hinton Agar along with the silver nanoparticles. Silver nanoparticles solution of 20µl were used along with the standard antibiotic disc. Overnight the test sample was inoculated and incubated at 370C overnight. The zone of restraint was measured. 3 Results and Discussion Several physical and chemical methods exist for this nanoparticle synthesis [10]. However recently there has been an increasing interest in developing and promoting an eco-friendly route for their synthesis. This involves using biological systems like bacteria, fungi and yeast, which show the ability to reduce metal ions to metallic nanoparticles. Fungi are widely used as tools for this purpose and are more advantageous in both processing as well as handling the biomass [11].
Figure 1: Cell filtrate with AgNO3 (right) and control (left)
Figure 3.1 show flasks containing cell filtrate with silver nitrate solution and the positive control flask (only filtrate without silver nitrate) after an incubation of 24 hours. The change in colour of test filtrate from pale pink to yellowish brown was observed visually. The colour remained yellowish brown in case of the positive one. A negative control with only silver nitrate was incubated simultaneously and this remained colourless after 24 hours. This shows the probable formation of silver nanoparticles in the test sample. 3.1 UV spectroscopy The presence of nanoparticles was confirmed using UV-visible Biospectrophotometry. The absorbance measurements of the test filtrate for 24, 48, and 72hours are given in Figure 3.2, for a complete wavelength scan of 200 to 600 nm. Results shows that the absorbance values peak between 420-430 nm at all three time intervals with maximum values being observed after a period of 48 hours. The fluctuation in absorbance values after 48 hours is not very significant, showing that the attainment of reaction equilibrium after 72 hours. The enzyme activity probably reaches its maximum by this time.
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Figure 2: UV- spectral recordings at 24, 48 and 72 hours
The observations in this study indicate that enzyme nitrate reductase is probably released into cell filtrate. Upon substrate addition, silver ions are reduced to silver nanoparticles by the enzyme action thus bringing about extracellular biosynthesis. 3.2 Zetasizer NanoS90 3.2.1 Size distribution of Silver nanoparticles with supernatant
Figure 3. Average particle size distribution in the supernatant sample
3.3 Optimization of silver nanoparticles 3.3.1 Effect of substrate concentration silver nanoparticles The impact of substrate focus was concentrated on by using different concentration of silver nitrate to synthesize the nanoparticles. Cell filtrate was examined with 0.5, 1.0, 1.5, 2.0, 2.5mM of AgNO3 and incubated. The greatest generation was seen at 1.5mm fixation as demonstrated in Figure 3.4. The present study accomplished the additional cell bio-synthesis of silver nanoparticles in 2 hours of incubation with silver particles. Production of nanoparticles was checked at different concentrations of the substrate, viz., silver nitrate, but maximum production, as again confirm by absorbance estimations, was seen at a concentration of 1.5mM. At lower AgNO3 concentrations,
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maximum amount of enzyme may have been available within the system, however higher production might not have happened because of absence of substrate molecules.
Figure 4: Effect of substrate on Ag concentration
Alternatively, the presence of substrate in the medium may also serve to induce the release of enzyme from penicillium, which may in turn covert silver in the substrate to nanoparticles. As more of the substrate molecules are supplied to the medium, the enzyme secretion by the fungi may proportionately increase till a threshold concentration which in this case was observed to be 1.5mM. 3.3.2 Effect of pH on silver nanoparticles Figure 3.5 indicates effect of pH in nanoparticle synthesis and the optimum value for maximum synthesis was found to be 6 compared to other levels (pH 5, 7, 8). It was also proved that maximum production happened at pH 6, as the maximum absorbance was seen at this pH. At higher and lower pH, the absorbance was proportionately lower. The enzyme reductase catalyzing the synthesis is probably deactivated gradually as the conditions become alkaline, and this may be the reason for reduced synthesis and lower absorbance which is observed at higher pH values.
Figure 5: Cell filtrate with varying pH Figure 6: Effect of pH on AgNP
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3.3.3 Effect of salinity on silver nanoparticles Synthesis was studied at different salinity levels (0.1, 0.2, 0.3, 0.4, 0.5% NaCl) (Figure 4.8). Maximum production was observed at a salt concentration of 0.1%. It was also observed that concentrations of NaCl above 0.1% were unfavorable for the nanoparticle synthesis. Deactivation of enzyme by excess salt may have reflected in lower absorbance values. The concentration of 0.1% NaCl favored the production and stability of metal nanoparticles.
Figure 7: Cell filtrate with varying salt
Figure 8: Effect of salinity on Ag concentration
3.4 AFM Characterization of silver nanoparticles was carried out using AFM. It was observed that, nanoparticles of size 75 nm were formed (Figure 3.9).
Figure 9: AFM image of silver nanoparticle having a size of 75nm
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3.5 SEM Characterization of silver nanoparticles was carried out using SEM.
Figure 10: SEM image of silver nanoparticle
By observing the figure 3.10 we can conclude that the most of the silver nanoparticles are found to be triangle, some pyramid shaped and some are spherical in nature. 3.6 XRD The dry powders of silver nanoparticles are used for XRD analysis. The intensities were recorded from 200 to 900 at 2 theta angles. The diffraction pattern in fig 3.10 corresponds to pure silver metal powder.Analysis of silver nanoparticles precipitate by powder-XRD revealed four peaks at 380(111), 420(200) and 620(220) 780(311) and 820(222). The peaks were examined with X-ray diffraction database. The particle size histogram resulted silver nanoparticles shows wide appropriation of particle size. The size range went from 50-100 nm and the normal particle size comes out to be 75nm. The X-ray diffraction pattern got for silver nanoparticles produced by fungi demonstrates that the synthesized nanoparticles are crystalline in nature.
Figure 11 shows XRD analysis of synthesized nanoparticles
Shareef J. U et al./ Materials Today: Proceedings 4 (2017) 11923–11932
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3.7 Zone of inhibition Table 1 and 2: Zone of inhibition for different antibiotics against the test organisms
Methicillin(10µg/disc) Met Met+AgNP Fold increase (zone in mm) (zone in mm) (in %) S.aureas P.mirabilis
0 0
0 0
0 0
Erythromycin (10µg/disc) Ery Ery+AgNP Fold increase (zone in mm) (zone in mm) (in %) 0 2
Vanomycin(10µg/disc)
0 4
0 100
Bacitracin (10µg/disc)
Van
Van+AgNP
Fold increase
Bac
Bac+AgNP
Fold increase
(zone in mm)
(zone in mm)
(in %)
(zone in mm)
(zone in mm)
(in %)
S.aureas
13
16
63.07
18
20
11.11
P.mirabilis
0
0
100
0
0
0
The antibacterial action of the Ag-NPs was done to assess the adequacy of these nanoparticles against a gram positive and gram negative microbes. The measurement of the inhibitory zone and the rate of increment in fold region were measured. The anti-toxins were utilized as a positive control within correlation with that of Ag-NPs. The antibacterial action of Ag-NPs in mixture with erythromycin, vancomycin and bacitracin was expanded, while no zone of restraint was seen if there should be an occurrence of methicillin against both the test life forms (Table 1). This was likewise evaluated by demonstrating the expand in rate fold zone. It was watched that erythromycin demonstrated the greatest expand in fold region (Figure 3.11). Comparable study was completed to focus the antifungal action of silver nanoparticle in mix with fluconazole [6]. The impact of silver nanoparticles on gram negative microscopic organisms was indicated to be improved over that of the gram positive. A comparative case was explored by Singh et al [14].
Figure 12: Fold increase in antibacterial activity of antibiotics with AgNP against test orgs
Literatures show that the exact mechanism of action of Ag-NPs is understood only to some extent. There are different theories proposed to evaluate the same. Matsumura et al proposed that silver ions interacts with the thiol groups of some of the major enzymes and inactivates them [13]. The findings of Feng et al revels that the replication ability of DNA is lost as a result of interaction of silver ions with the bacterium. Their other studies showed that there were some structural changes in the cell membrane due to silver ions which was responsible for the antibacterial activity of the Ag-NPs [15].
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4. Conclusion Penicillium sps. was exploited for the biological synthesis of nanoparticles extracellularly. This green chemistry approach towards the synthesis of silver nanoparticles has many advantages. Green synthesis approach for synthesis is advantageous over chemical methods as it is economical and also eco-friendly. Applications of such eco-friendly nanoparticles are bactericidal, medical and electronic applications make this method potentially exciting for the large scale synthesis of other inorganic materials (nanomaterials). Penicillium sps. was observed to have maximum production of nanoparticles at a pH 6, at a salt concentration of 0.1% with a substrate concentration of 1.5mM silver nitrate. The size of the silver nanoparticles was observed to be 75 nm by AFM. Then the morphological studies are carried out by SEM. Also the sample is characterized by XRD for structural conformation. Antimicrobial activity of silver nanoparticles produced by penicillium sps were studied against four antibiotics where in Erythromycin showed highest fold increase with silver nanoparticles produced by Penicillium sps. Acknowledgement We acknowledge the support from the department of Biotechnology Saptagiri College of Engineering and VTUCPGS, Muddenahalli, for providing the facilities for the project work. I also thank to VIT Vellore and NIT Calicut for providing opportunity to use XRD, SEM and AFM. References [1] C. Sundaramoorth, M. Kalaivani, D.M. Mathews, S. Palanisamy, A. Kalaiselvan and Rajasekaran A, “Biosynthesis of silver nanoparticles from Aspergillis niger and evaluation of its wound healing activity in experimental rat model,” International Journal of PharmTech Research, vol. 1, pp. 1523- 1529, 2009. [2] M. Valodkar, A. Bhadorai, J. Pohnerkar, M. Mohan and S. Thakore, “Morphology and antibacterial activity of carbohydrate stabilized silver nanoparticles,” Carbohydrate Research, vol. 345, pp. 1767-1773, 2010. [3] A.M. Fayaz, K. Balaji, M. Girilal, R. Yadav, P.T. Kalaichelvan and R. Venketesan, “Biogenic synthesis of silver nanoparticles and their synergestic effect with antibiotics: a study against gram-positive and gram-negative bacteria,” Nanomedicine: Nanotechnology, Biology, and Medicine, vol. 6, pp. 103-109, 2010. [4] M.A. Dias, I.C. Lacerda, P.F. Pimentel, H.F. De Castro and C.A. Rosa, “Removal of heavy metal by an Aspergillus terreus strain immobilized in a polyurethane matrix,” Letters in Applied Microbiology, vol. 34, pp. 46-50, 2002. [5] J.C. Chen, Z.H. Lin and X.X. Ma, “Evidence of the production of silver nanoparticles via pretreatment of Phoma sp.3.2883 with silver nitrate,” Letters in Applied Microbiology, vol. 37, pp. 105-108, 2003. [6] D.S. Balaji, S. Basavaraja, R. Deshpande, D.B. Mahesh, B.K. Prabhakar and A. Venkataraman, “Extracellular biosynthesis of functionalized silver nanoparticles by strains of Clostridium cladosporiodes fungus,” Colloids and Surfaces B: Biointerfaces, vol. 68, pp. 88-92, 2009. [7] K. Kalishwaralal, V. Deepak, S.R.K. Pandian, M. Kottaisamy, S.B. Manikanth, B. Karthikeyan and S. Gurunathan, “Biosynthesis of silver and gold nanoparticles using Brevibacterium casei,” Colloids and Surfaces B: Biointerfaces, vol. 77, pp. 257-262, 2010. [8] S. Gurunathan, K. Kalishwarlal, R. Vaidhyanathan, V. Deepak, S.R.K. Pandian, J. Muniyandi, N. Hariharan and S.H. Eom, “Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli,” Colloids and Surfaces B: Biointerfaces, vol. 74, pp, 328335, 2009. [9] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar and M. Sastry, “Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum,” Colloids and Surfaces B: Biointerfaces, vol. 28, pp. 313-318, 2003. [10] K.C. Bhainsa and S.F. D’Souza, “Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigates,” Colloids and Surfaces B: Biointerfaces, vol. 47, pp. 160-164, 2006. [11] N. Duran, P.D. Marcato, O.I. Alves, G.I. DeSouza and E. Esposito, “Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporium strains,” Journal of Nanobiotechnology, vol. 3, pp. 8, 2005. [12] K. Vahabi, G.A. Mansoori and S. Karimi, “Biosynthesis of silver nanoparticles by fungus Trichoderma reesei,” Insciences Journal, vol. 1, pp. 65-79, 2011. [13] Y. Matsumura, K. Yoshikata, S. Kunisaki and T. Tsuchido, “Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate,” Applied and Environmental Microbiology, vol. 69, pp. 4278-4281, 2003. [14] M. Singh, S. Singh, S. Prasad and I.S. Gambhir, “Nanotechnology In Medicine And Antibacterial Effect Of Silver Nanoparticles,” Digest Journal of Nanomaterials and Biostructures, vol. 3, pp. 115 – 122, 2008. [15] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim and J.O. Kim, “A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus,” Journal of Biomedical Materials Research, vol. 52, pp. 662-668, 2000.
ScienceDirect Materials Today: Proceedings 4 (2017) 11923–11932
www.materialstoday.com/proceedings
ICNANO 2016
Synthesis and characterization of silver nanoparticles from Penicillium sps. Shareef J. Ua, Navya Rani Mb, Anand Sc, Dinesh Rangappad* a
Department of Nano Technology, Srinivas Institute of Technology, Mangalore-574143, India b Department of Biotechnology, RGIT, Bangalore-560032, Inida c Department of Biotechnology, Saptagiri College of Engineering, Bangalore-560057, Inida d Department of Nano Technology, PG Centre Bangalore Region, Visvesvaraya Technological University, Visvesvaraya Institute of Advanced Technology (VIAT), Muddenhalli post, Chickballapur District 562101 India.
Abstract Among the different methods employed for synthesis of nanoparticles, the biological method is most favourable and well established. Fungi provide many advantages in this context. In this study, extracellular synthesis of silver nanoparticles from Penicillium sps. was carried out. Nanoparticles were produced due to reduction of silver ions from silver nitrate, the formation of which was monitored by UV-visible spectrophotometry. The optimization of the biosynthesis procedure with respect to substrate concentration, pH, and salinity were carried out. The size distribution was determined using zetasizer Nano S90 and the dimensions were observed to be around 75nm by AFM and morphology was characterized by SEM. Then the synthesized silver nanoparticles are subjected to XRD analysis. The efficiency of activity of antibiotics is increased by several folds by conjugating the AgNPs with the different types of antibiotics by using zone of inhibition method. © 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON NANOTECHNOLOGY (ICNANO2016). Keywords: Silver nanoparticles, Penicillum sp, Spectrophotometry, zetasizer Nano S90, AFM, SEM, XRD.
* Corresponding author. Tel.: +91 9632764659; E-mail address: [email protected] 2214-7853© 2017 Elsevier Ltd. All rights reserved. Selection and Peer-review under responsibility of INTERNATIONAL CONFERENCE ON NANOTECHNOLOGY (ICNANO-2016).
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1. Introduction Silver is medically considered as one of the most powerful elements due to its activity against mammalian tissues where it acts as an antiseptic agent [1]. Silver, in its metallic as well as ionic forms, exhibits cytotoxicity against several microorganisms and hence used as an anti-microbial agent [2]. Some microorganisms are also able to accumulate silver by adsorption. Silver nanoparticles have been exploited for their unique properties and their vast applications in biomedicine. Environmentally benign methods have been developed for the synthesis of these nanoparticles which eliminate the use of toxic chemicals during their synthesis process [3]. Ag nanoparticles are usually synthesized from bacteria and fungi. The latter posses more advantages which include filamentous fungal tolerance towards metals, their high binding capacity and intracellular uptake of metals [4]. Further, the mass production of fungi is easy, for synthesis of nanoparticles [5]. Ag nanoparticles have been widely employed due to their physicochemical properties [6] and are currently used as anti-bacterial agents in food storage, textile and health industries, for biolabeling and as biosensors. The anti-microbial activity of silver nanoparticles has now been well established and they are confirmed to posses anti-inflammatory, anti-viral, anti-platelet and anti-fungal activity [7]. One of the major issues to be considered during synthesis of nanoparticles is their size, depending on which, the applications may vary [8]. Synthesis may be routed through either intracellular or extracellular pathway. The latter is disadvantageous for harvesting the product and recovery may be cumbersome and expensive. For extracellular synthesis, it is reported that a reductase enzyme is released into solution which brings about reduction of Ag+ ions [9]. Therefore the extracellular synthesis of Ag nanoparticles from fungi is most widely adapted. The present study deals with the extracellular synthesis of Ag nanoparticles from the fungus Penicillium sp, the formation of which was monitored by spectroscopy. This was followed by microscopic characterization. Thus, silver nanoparticles are the metal of choice as they have the capability to kill microbes effectively. The strong toxicity of silver against wide range of microorganisms is well known and silver nanoparticles have shown to be an effective antimicrobial agent. 2. Experimental Procedure 2.1 Collecting Sample Three soil specimen are gathered Soil of wet environment is preferred, one soil sample from balehannu kere situated west part of Bangalore was collected and the other two from sapthagiri college garden. Aseptic method was employed to avoid contamination throughout taking the specimen. Four distinctive specimens were gathered from the depth of 6 inches of the ground by utilizing Random Sampling Method. 2.2 Isolation of the specimen The sample was mixed with Distilled tap water to make solution sample. This solution sample was diluted up to 10-9 serial dilution. Serial dilution is a process of diluting a sample several times. 2.3 Serial dilution method The sterile Petri dish is labelled. Then 9ml of phosphate buffer is transferred into each of 5 tubes using sterile pipettes (10ml) with aseptic technique. By using sterile pipette (1ml), 1ml of water sample is transferred into 10-1 tube. Mix the test tube properly. Continue dilution with aseptic technique for test 10-2 until 10-5. After that each of serial dilution is transferred into Potato Dextrose Agar (PDA) plate by using spread plate method.
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2.4 Spread plate method 2.4.1 Preparation of media Media used here is PDA (potato dextrose agar), it is used to grow fungus, 200ml of PDA is prepared by using 40gms of Potato, 4gms of Dextrose and 4gms of Agar. The solution is then subjected to autoclave at 1210 C for 15mins. Then the sterile media is used for the inoculation. 2.4.2 Procedure One millilitre of an appropriately diluted culture is spread over the surface of agar using sterile glass spreader. The plate is then incubated until the colonies appear. It is important that the surface of the plate be fairly dry so that the spread liquid soaks in. 2.4.3 Pure culture Different types of colonies are observed by spread plate method among those single colony is selected and then it is inoculated by streaking the plate. Then incubated at 290C for 3days and the colony is selected for the inoculation to liquid broth.A culture of Penicillium sps. Was used for the extra cellular production of silver nanoparticles 2.5 Production of bio-mass The preparation of biomass for biosynthesis of silver nanoparticles from Penicillium sp involved the aerobic culturing of fungus in a liquid broth composed of KH2PO4,(7.0g/L), K2HPO4(2.0g/L), MgSO4. 7H2O (0.1g/L), (NH4)2SO4 (1.0g/L) yeast extract, (0.6g/L) and glucose (10g/L). 2.5.1 Culturing of fungus in liquid broth Liquid broth is prepared for 250ml and divided into two sample. One colony is selected from pink pure culture and it is inoculated in 1st sample and other colony from green pure culture is inoculated in 2nd sample. The flask containing this culture was incubated at 290C for about 8 days. The biomass thus obtained was harvested, followed by extensive washing with Milli Q water. 2.6 Synthesis of silver nanoparticles utilizing silver nitrate Biomass of 15gms was re-suspended in 150ml milli Q water and incubated in shaker for 72 hours at 290C. Then the biomass was filtered utilizing a Whattman channel paper No1. The pH of filtrate was discovered to be 5.84. To the 150ml of filtrate silver nitrate was added to give an overall concentration of 1mM (0.017g/100ml). The reaction was carried out in dark. The control with just the cell filtrate and without silver particle was run with the test sample. Time dependent formation of silver nanoparticles was measured by using UV-visible spectrophotometer at 24, 48 and 72 hours and the absorbance was measured at wave lengths extending from 200 to 600nm. 2.7 Optimization of silver nanoparticle synthesis The filtrate containing silver nanoparticles were subjected to further studies. The impact of substrate concentration, pH and salinity, on the production of silver nanoparticles was optimized d by fluctuating the parameters, such as substrate concentration (0.5, 1.0, 1.5, 2.0, 2.5mM of AgNO3), pH (5,6,7,8) and salinity 0.1, 0.2, 0.3, 0.4, 0.5% Nacl) at different time intervals. 300µl of the sample was withdrawn to measure the optical thickness at a wavelength of 450nm with an UV-visible spectrophotometer.
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2.8 Characterization and Examination of silver nanoparticles After incubation of 72 hours, the filtrate which has silver nanoparticles were characterized using spectrophotometry and zetasizer Nano S90. The latter is used to predict the size of nanoparticles. The sample was prepared by centrifuging the cell filtrate and making a smear with the pellet obtained. This was then subjected to AFM and SEM analysis. Then the sample is analysed by XRD pattern for structural confirmation. 2.9 Studies on the Antimicrobial activity of silver nanoparticles An examination was done to focus the synergistic impact of antimicrobials against the gram positive and gram negative microorganisms with the silver nanoparticles synthesized. Standard antibiotic discs were used and plated onto Muller-Hinton Agar along with the silver nanoparticles. Silver nanoparticles solution of 20µl were used along with the standard antibiotic disc. Overnight the test sample was inoculated and incubated at 370C overnight. The zone of restraint was measured. 3 Results and Discussion Several physical and chemical methods exist for this nanoparticle synthesis [10]. However recently there has been an increasing interest in developing and promoting an eco-friendly route for their synthesis. This involves using biological systems like bacteria, fungi and yeast, which show the ability to reduce metal ions to metallic nanoparticles. Fungi are widely used as tools for this purpose and are more advantageous in both processing as well as handling the biomass [11].
Figure 1: Cell filtrate with AgNO3 (right) and control (left)
Figure 3.1 show flasks containing cell filtrate with silver nitrate solution and the positive control flask (only filtrate without silver nitrate) after an incubation of 24 hours. The change in colour of test filtrate from pale pink to yellowish brown was observed visually. The colour remained yellowish brown in case of the positive one. A negative control with only silver nitrate was incubated simultaneously and this remained colourless after 24 hours. This shows the probable formation of silver nanoparticles in the test sample. 3.1 UV spectroscopy The presence of nanoparticles was confirmed using UV-visible Biospectrophotometry. The absorbance measurements of the test filtrate for 24, 48, and 72hours are given in Figure 3.2, for a complete wavelength scan of 200 to 600 nm. Results shows that the absorbance values peak between 420-430 nm at all three time intervals with maximum values being observed after a period of 48 hours. The fluctuation in absorbance values after 48 hours is not very significant, showing that the attainment of reaction equilibrium after 72 hours. The enzyme activity probably reaches its maximum by this time.
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Figure 2: UV- spectral recordings at 24, 48 and 72 hours
The observations in this study indicate that enzyme nitrate reductase is probably released into cell filtrate. Upon substrate addition, silver ions are reduced to silver nanoparticles by the enzyme action thus bringing about extracellular biosynthesis. 3.2 Zetasizer NanoS90 3.2.1 Size distribution of Silver nanoparticles with supernatant
Figure 3. Average particle size distribution in the supernatant sample
3.3 Optimization of silver nanoparticles 3.3.1 Effect of substrate concentration silver nanoparticles The impact of substrate focus was concentrated on by using different concentration of silver nitrate to synthesize the nanoparticles. Cell filtrate was examined with 0.5, 1.0, 1.5, 2.0, 2.5mM of AgNO3 and incubated. The greatest generation was seen at 1.5mm fixation as demonstrated in Figure 3.4. The present study accomplished the additional cell bio-synthesis of silver nanoparticles in 2 hours of incubation with silver particles. Production of nanoparticles was checked at different concentrations of the substrate, viz., silver nitrate, but maximum production, as again confirm by absorbance estimations, was seen at a concentration of 1.5mM. At lower AgNO3 concentrations,
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maximum amount of enzyme may have been available within the system, however higher production might not have happened because of absence of substrate molecules.
Figure 4: Effect of substrate on Ag concentration
Alternatively, the presence of substrate in the medium may also serve to induce the release of enzyme from penicillium, which may in turn covert silver in the substrate to nanoparticles. As more of the substrate molecules are supplied to the medium, the enzyme secretion by the fungi may proportionately increase till a threshold concentration which in this case was observed to be 1.5mM. 3.3.2 Effect of pH on silver nanoparticles Figure 3.5 indicates effect of pH in nanoparticle synthesis and the optimum value for maximum synthesis was found to be 6 compared to other levels (pH 5, 7, 8). It was also proved that maximum production happened at pH 6, as the maximum absorbance was seen at this pH. At higher and lower pH, the absorbance was proportionately lower. The enzyme reductase catalyzing the synthesis is probably deactivated gradually as the conditions become alkaline, and this may be the reason for reduced synthesis and lower absorbance which is observed at higher pH values.
Figure 5: Cell filtrate with varying pH Figure 6: Effect of pH on AgNP
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3.3.3 Effect of salinity on silver nanoparticles Synthesis was studied at different salinity levels (0.1, 0.2, 0.3, 0.4, 0.5% NaCl) (Figure 4.8). Maximum production was observed at a salt concentration of 0.1%. It was also observed that concentrations of NaCl above 0.1% were unfavorable for the nanoparticle synthesis. Deactivation of enzyme by excess salt may have reflected in lower absorbance values. The concentration of 0.1% NaCl favored the production and stability of metal nanoparticles.
Figure 7: Cell filtrate with varying salt
Figure 8: Effect of salinity on Ag concentration
3.4 AFM Characterization of silver nanoparticles was carried out using AFM. It was observed that, nanoparticles of size 75 nm were formed (Figure 3.9).
Figure 9: AFM image of silver nanoparticle having a size of 75nm
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3.5 SEM Characterization of silver nanoparticles was carried out using SEM.
Figure 10: SEM image of silver nanoparticle
By observing the figure 3.10 we can conclude that the most of the silver nanoparticles are found to be triangle, some pyramid shaped and some are spherical in nature. 3.6 XRD The dry powders of silver nanoparticles are used for XRD analysis. The intensities were recorded from 200 to 900 at 2 theta angles. The diffraction pattern in fig 3.10 corresponds to pure silver metal powder.Analysis of silver nanoparticles precipitate by powder-XRD revealed four peaks at 380(111), 420(200) and 620(220) 780(311) and 820(222). The peaks were examined with X-ray diffraction database. The particle size histogram resulted silver nanoparticles shows wide appropriation of particle size. The size range went from 50-100 nm and the normal particle size comes out to be 75nm. The X-ray diffraction pattern got for silver nanoparticles produced by fungi demonstrates that the synthesized nanoparticles are crystalline in nature.
Figure 11 shows XRD analysis of synthesized nanoparticles
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3.7 Zone of inhibition Table 1 and 2: Zone of inhibition for different antibiotics against the test organisms
Methicillin(10µg/disc) Met Met+AgNP Fold increase (zone in mm) (zone in mm) (in %) S.aureas P.mirabilis
0 0
0 0
0 0
Erythromycin (10µg/disc) Ery Ery+AgNP Fold increase (zone in mm) (zone in mm) (in %) 0 2
Vanomycin(10µg/disc)
0 4
0 100
Bacitracin (10µg/disc)
Van
Van+AgNP
Fold increase
Bac
Bac+AgNP
Fold increase
(zone in mm)
(zone in mm)
(in %)
(zone in mm)
(zone in mm)
(in %)
S.aureas
13
16
63.07
18
20
11.11
P.mirabilis
0
0
100
0
0
0
The antibacterial action of the Ag-NPs was done to assess the adequacy of these nanoparticles against a gram positive and gram negative microbes. The measurement of the inhibitory zone and the rate of increment in fold region were measured. The anti-toxins were utilized as a positive control within correlation with that of Ag-NPs. The antibacterial action of Ag-NPs in mixture with erythromycin, vancomycin and bacitracin was expanded, while no zone of restraint was seen if there should be an occurrence of methicillin against both the test life forms (Table 1). This was likewise evaluated by demonstrating the expand in rate fold zone. It was watched that erythromycin demonstrated the greatest expand in fold region (Figure 3.11). Comparable study was completed to focus the antifungal action of silver nanoparticle in mix with fluconazole [6]. The impact of silver nanoparticles on gram negative microscopic organisms was indicated to be improved over that of the gram positive. A comparative case was explored by Singh et al [14].
Figure 12: Fold increase in antibacterial activity of antibiotics with AgNP against test orgs
Literatures show that the exact mechanism of action of Ag-NPs is understood only to some extent. There are different theories proposed to evaluate the same. Matsumura et al proposed that silver ions interacts with the thiol groups of some of the major enzymes and inactivates them [13]. The findings of Feng et al revels that the replication ability of DNA is lost as a result of interaction of silver ions with the bacterium. Their other studies showed that there were some structural changes in the cell membrane due to silver ions which was responsible for the antibacterial activity of the Ag-NPs [15].
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4. Conclusion Penicillium sps. was exploited for the biological synthesis of nanoparticles extracellularly. This green chemistry approach towards the synthesis of silver nanoparticles has many advantages. Green synthesis approach for synthesis is advantageous over chemical methods as it is economical and also eco-friendly. Applications of such eco-friendly nanoparticles are bactericidal, medical and electronic applications make this method potentially exciting for the large scale synthesis of other inorganic materials (nanomaterials). Penicillium sps. was observed to have maximum production of nanoparticles at a pH 6, at a salt concentration of 0.1% with a substrate concentration of 1.5mM silver nitrate. The size of the silver nanoparticles was observed to be 75 nm by AFM. Then the morphological studies are carried out by SEM. Also the sample is characterized by XRD for structural conformation. Antimicrobial activity of silver nanoparticles produced by penicillium sps were studied against four antibiotics where in Erythromycin showed highest fold increase with silver nanoparticles produced by Penicillium sps. Acknowledgement We acknowledge the support from the department of Biotechnology Saptagiri College of Engineering and VTUCPGS, Muddenahalli, for providing the facilities for the project work. I also thank to VIT Vellore and NIT Calicut for providing opportunity to use XRD, SEM and AFM. References [1] C. Sundaramoorth, M. Kalaivani, D.M. Mathews, S. Palanisamy, A. Kalaiselvan and Rajasekaran A, “Biosynthesis of silver nanoparticles from Aspergillis niger and evaluation of its wound healing activity in experimental rat model,” International Journal of PharmTech Research, vol. 1, pp. 1523- 1529, 2009. [2] M. Valodkar, A. Bhadorai, J. Pohnerkar, M. Mohan and S. Thakore, “Morphology and antibacterial activity of carbohydrate stabilized silver nanoparticles,” Carbohydrate Research, vol. 345, pp. 1767-1773, 2010. [3] A.M. Fayaz, K. Balaji, M. Girilal, R. Yadav, P.T. Kalaichelvan and R. Venketesan, “Biogenic synthesis of silver nanoparticles and their synergestic effect with antibiotics: a study against gram-positive and gram-negative bacteria,” Nanomedicine: Nanotechnology, Biology, and Medicine, vol. 6, pp. 103-109, 2010. [4] M.A. Dias, I.C. Lacerda, P.F. Pimentel, H.F. De Castro and C.A. Rosa, “Removal of heavy metal by an Aspergillus terreus strain immobilized in a polyurethane matrix,” Letters in Applied Microbiology, vol. 34, pp. 46-50, 2002. [5] J.C. Chen, Z.H. Lin and X.X. Ma, “Evidence of the production of silver nanoparticles via pretreatment of Phoma sp.3.2883 with silver nitrate,” Letters in Applied Microbiology, vol. 37, pp. 105-108, 2003. [6] D.S. Balaji, S. Basavaraja, R. Deshpande, D.B. Mahesh, B.K. Prabhakar and A. Venkataraman, “Extracellular biosynthesis of functionalized silver nanoparticles by strains of Clostridium cladosporiodes fungus,” Colloids and Surfaces B: Biointerfaces, vol. 68, pp. 88-92, 2009. [7] K. Kalishwaralal, V. Deepak, S.R.K. Pandian, M. Kottaisamy, S.B. Manikanth, B. Karthikeyan and S. Gurunathan, “Biosynthesis of silver and gold nanoparticles using Brevibacterium casei,” Colloids and Surfaces B: Biointerfaces, vol. 77, pp. 257-262, 2010. [8] S. Gurunathan, K. Kalishwarlal, R. Vaidhyanathan, V. Deepak, S.R.K. Pandian, J. Muniyandi, N. Hariharan and S.H. Eom, “Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli,” Colloids and Surfaces B: Biointerfaces, vol. 74, pp, 328335, 2009. [9] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar and M. Sastry, “Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum,” Colloids and Surfaces B: Biointerfaces, vol. 28, pp. 313-318, 2003. [10] K.C. Bhainsa and S.F. D’Souza, “Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigates,” Colloids and Surfaces B: Biointerfaces, vol. 47, pp. 160-164, 2006. [11] N. Duran, P.D. Marcato, O.I. Alves, G.I. DeSouza and E. Esposito, “Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporium strains,” Journal of Nanobiotechnology, vol. 3, pp. 8, 2005. [12] K. Vahabi, G.A. Mansoori and S. Karimi, “Biosynthesis of silver nanoparticles by fungus Trichoderma reesei,” Insciences Journal, vol. 1, pp. 65-79, 2011. [13] Y. Matsumura, K. Yoshikata, S. Kunisaki and T. Tsuchido, “Mode of bactericidal action of silver zeolite and its comparison with that of silver nitrate,” Applied and Environmental Microbiology, vol. 69, pp. 4278-4281, 2003. [14] M. Singh, S. Singh, S. Prasad and I.S. Gambhir, “Nanotechnology In Medicine And Antibacterial Effect Of Silver Nanoparticles,” Digest Journal of Nanomaterials and Biostructures, vol. 3, pp. 115 – 122, 2008. [15] Q.L. Feng, J. Wu, G.Q. Chen, F.Z. Cui, T.N. Kim and J.O. Kim, “A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus,” Journal of Biomedical Materials Research, vol. 52, pp. 662-668, 2000.