Micron 59 (2014) 52–59

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Green synthesis of silver nanoparticles by Phoma glomerata Aniket Gade a,1 , Swapnil Gaikwad a , Nelson Duran b,c , Mahendra Rai a,∗ a

Department of Biotechnology, SGB Amravati University, Amravati 444 602, Maharashtra, India Center of Natural and Human Sciences, Universidade Federal do ABC, Santo André, SP, Brazil c Institute of Chemistry, Biological Chemistry Laboratory, Universidade Estadual de Campinas, Campinas, SP, Brazil b

a r t i c l e

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Article history: Received 15 August 2013 Received in revised form 23 November 2013 Accepted 4 December 2013 Keywords: Mycofabrication Silver nanoparticles Phoma glomerata FTIR TEM

a b s t r a c t We report an extracellular synthesis of silver nanoparticles (SNPs) by Phoma glomerata (MTCC-2210). The fungal filtrate showed rapid synthesis in bright sunlight. The Fourier transform infrared spectroscopy (FTIR) revealed the presence of a protein cap on the silver nanoparticle, which leads to increase stability of SNP in the silver colloid. X-ray diffraction (XRD) analysis showed the number of Bragg’s reflection, which are due to the face centered cubic structure of the crystalline SNPs. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), nanoparticle tracking and analysis (NTA) demonstrated the synthesis of polydispersive and spherical SNPs. Energy dispersive X-ray spectroscopy (EDX) was used to confirm the elemental composition of the sample and Zeta potential measurement was carried out to determine the stability of mycofabricated SNPs. The alkaline pH, room temperature, sunlight demonstrated optimum synthesis. Apart from the physical conditions, concentration of silver nitrate and amount of fungal filtrate affects the mycofabrication process. The study of cultural and physical parameters during the mycofabrication of SNPs by P. glomerata will be helpful in order to increase the yield of mycofabricated SNPs of desired shape and size. The process of mycofabrication of SNPs by P. glomerata was found to be eco-friendly, safe and cost-effective nature. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The Nanobiotechnology has emerged as an important branch of nanotechnology. One of the important aspects in the field of nanotechnology is the development of more reliable processes for the synthesis of nanomaterials over a range of sizes (with good monodispersity) and chemical composition (Rai et al., 2011). The biosynthesis of nanoparticles is in the limelight in modern nanotechnology as it is the greener approach (Gade et al., 2011). Various strategies are employed to synthesize semiconductor and transition metal nanoparticles. Foremost among these are chemical methods because of their inherent advantage in producing large quantities of nanoparticles in relatively short periods of time with a fairly good control on the size distribution.

∗ Corresponding author at: Department of Biotechnology SGB Amravati University, Amravati 444 602, Maharashtra. INDIA Present address: Institute of Chemistry, Biological Chemistry Laboratory, Universidade Estadual de Campinas, Campinas, SP, Brazil. /. E-mail addresses: [email protected] (A. Gade), [email protected], [email protected] (S. Gaikwad), [email protected] (N. Duran), [email protected], [email protected] (M. Rai). 1 Present address: Department of Biology, Utah State University, Logan, UT 84322, USA. 0968-4328/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2013.12.005

Moreover, in chemical synthesis an array of shapes of particles can be synthesized by adjusting the concentration of reacting chemicals and controlling the reaction environment (Matijevic´ and Cimaˇs, 1987) .Current chemical methods for synthesis of nanoparticles are energy intensive, use toxic chemicals and frequently yield particles with nonpolar organic solution thereby precluding biomedical applications. On the other hand, in physical methods of nanoparticles synthesis (Ayyub et al., 2001) such as sputter deposition, laser ablation or cluster beam deposition, thin films are directly grown. However, narrow size distribution of the particles or clusters is often difficult to achieve. Moreover, these methods are time consuming and still under development. Microorganisms typically live under comfortable conditions of temperature, pressure and acidity. These are candidates for the development of manufacturing techniques that are more eco-friendly than today’s often hot, high-pressure and caustic processes. Knowing the importance of emerging eco-friendly nanoparticle synthesis methods, more researchers have turned to synthesis by using microorganisms (Gade et al., 2010; Gaikwad et al., 2013a). Metal ions reduce into metallic nanoparticles when both bacteria and fungi interact with it. This has fueled the researchers to look at the biological systems. Initially, the utilization of prokaryotic cell has emerged as novel methods for the synthesis of nanomaterials. Although, several reports of the use of microbes have been addressed for nanoparticle synthesis, the exposure of different inorganic salts to these

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prokaryotic cells resulted in synthesis of nanoparticles intracellularly. Fungi are easy to culture in bulk due to which they have much importance. Bacterial system comprises use of sophisticated instruments to obtain clear filtrate from colloidal broth where as, in fungal system it secrets enzyme extracellularly which is having additional advantage in the downstream processing and handling of biomass (Gade et al., 2008). Moreover, fungi are excellent secretors of protein compared to bacteria and actinomycetes, resulting into higher yield of nanoparticles (Sastry et al., 2003). Due to these dissimilatory properties of fungi, it could be widely used for the rapid and eco-friendly biosynthesis of metal nanoparticles. Rai et al. (2009) proposed the term “Myconanotechnology” to include research carried out on nanoparticles synthesized by fungi. After an extensive literature survey, it is clear that the fungal system is the better alternative for the biological synthesis of metal nanoparticles. If the nanoparticles could be synthesized extracellularly directly in the aqueous medium, such biotransformation-based nanoparticle synthesis approach would have better commercial viability. In the present study, we have synthesized SNPs by Phoma glomerata. Metallic silver in the form of SNPs has made a remarkable comeback as a potential antimicrobial as well as antiviral agent (Gaikwad et al., 2013b). As the several pathogenic bacteria have developing resistance against various antibiotics, the SNPs could be used against such bacteria. SNPs have been studied and applied in many areas including biomedical, agricultural, electronic fields, and textile, etc. Researchers have been working on new, eco-friendly and cheaper methods to make products of colloidal silver. Due to the ease of biological method, metal nanoparticles have been studied by many researchers that produce small particles stabilized by protein. The mechanism involved in this production has not yet been revealed, however hypothetical mechanisms have been proposed in the literature. The present study has been focused on synthesis of SNPs by P. glomerata. The effect of physical factors, salt concentration and filtrate amount was also evaluated.

2. Materials and methods 2.1. Fungal culture Phoma glomerata (MTCC 2210) was procured from IMTECH (Chandigarh), India and grown on potato dextrose agar at 25 ◦ C for 48–72 h and then the pure culture of Phoma glomerata was maintained on potato dextrose agar slants at 4 ◦ C.

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2.3. Characterization of SNPs 2.3.1. UV–vis absorption spectroscopy UV–vis spectroscopy is one of the most important and simplest ways to confirm the formation of nanoparticles. The absorption peak shifts toward higher energy with a decrease in the size of mycofabricated nanoparticles. Absorbance spectrum of colloidal samples was taken from 200 to 800 nm, with the help of UV–vis spectrometer (Perkin Elmer, Lambda-950). 2.3.2. Fourier transform infrared spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) measurements (Thermo Scientific, NICOLET-6700) were made to identify the possible biomolecules responsible for the reduction of the Ag+ ions and capping of the mycofabricated SNPs. The colloidal solution of mycofabricated SNPs was dried overnight in an oven at 60 ◦ C so that a film could be prepared on a glass slide. The excessive drying of the sample could lead to the formation of powder; film prepared on the glass shows less noise as compared to the powdered sample. FTIR spectra were recorded using a Diamond smart orbit accessory (30,000–200 cm−1 ). The scans recorded were an average of 150 scans, and the contribution of the background accounted for each sample was measured in a transmission mode at a resolution of 4 cm−1 . 2.3.3. X-ray diffractometer (XRD) The X-ray diffractometer also provides information regarding the structures or phase of crystals. The colloidal silver nanoparticles were treated with acetone leading to precipitation of SNPs. The precipitate collected after centrifugation was air-dried to obtain a powder. Improper drying can lead to the formation of aggregates. The dried SNPs were used for powder XRD analysis. The diffraction patterns were recorded in the range of 20◦ –80◦ using a Bruker D8 Advance diffractometer. 2.3.4. Transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX) TEM was performed to determine the shape and size of the mycofabricated SNPs and EDX provided the information regarding the elemental composition of the sample. A drop of solution containing mycofabricated SNPs was placed on the carbon coated copper grids and kept in infrared light until sample get dried before loading them onto a specimen holder. TEM micrographs were taken by Philips CM 200 super twin’s TEM operating at 200 kV (0.23 nm resolution) instrument.

2.2. Synthesis of nanoparticles To prepare biomass for biosynthesis studies, P. glomerata was grown aerobically in a potato dextrose broth (PDB). The flasks were inoculated with P. glomerata, incubated on orbital shaker at 25 ◦ C and agitated at 120 rpm. The biomass was harvested after 96 h of growth by filtering through Whatman filter paper No. 1. The harvested biomass was rinsed three to four times with distilled water to remove any medium component from the biomass. The biomass thus obtained (approx. 20 g, fresh weight) was mixed with 100 mL of distilled water for 24 h at 25 ◦ C in Erlenmeyer flasks and agitated at 120 rpm. After the incubation, the cell filtrate was separated from the biomass by passing it through Whatman filter paper No. 1. For synthesis of nanoparticles, AgNO3 , 1 mM final concentration (50 mL) was mixed with 50 mL of cell filtrate in a 250-mL Erlenmeyer flask and the biomass was also mixed with 50 mL of 1 mM AgNO3 solution, incubated and agitated at 25 ◦ C and 120 rpm, respectively, in the sunlight. Control (without the silver ion, only filtrate) was also kept along with the experimental flasks. All the experiments were performed in triplicate.

2.3.5. Nanoparticle tracking and analysis (NTA) The preliminary study about particle size and distribution was executed by Nanoparticles Tracking and Analysis (NTA) using LM20 (NanoSight Ltd. UK). The size distribution of nanoparticles, which can be obtained on a particle-by-particle basis by LM-20, was studied. NTA enables separation of the particle population by size and intensity, microscopically visualizing individual nanoparticles in suspension and simultaneously determining their Brownian motion. The NTA calculates the particle size by the distance traveled by it. Size calculation was based on Stokes-Einstein equation, applied to particles with its size. For each distribution, data are given in mean (the average particle size measured) and the mode (most frequent particle size found) terms. 2.3.6. Zeta potential measurement Zeta potential was measured with the Malvern instrument Zetasizer (Malvern Zetasizer 3000 HS) using zeta dip cell. The sample preparation for zeta analysis involves the mixing of mycofabricated SNPs in 1 mM KCL in 1:10 proportion, total volume of the

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sample was 1000 ␮l taken in the clear disposable zeta cell for the measurement of zeta potential. 2.4. Influence of different conditions For large scale production and stable mycofabrication of SNPs, it is necessary to study the influence of different cultural and physical conditions on synthesis. Several experiments were carried out concerning the rate of synthesis and stability of SNPs. The parameters such as pH, temperature, and light intensity, concentration of silver nitrate, the volume of filtrate and time of reaction was studied. For each condition, respective controls were maintained. All experiments were performed in triplicate. 2.4.1. Effect of hydrogen-ion-concentrations Better synthesis and stability of SNPs was studied by suspending biomass in different pH including pH 3, pH 5, pH 7, pH 9 and pH 11 and pH of the filtrate was maintained by 1 N HCl and 1 M NaOH. 2.4.2. Effect of temperatures Effect of different temperatures on the rate of synthesis of SNPs was studied in which the fungal filtrate after treating with 1 mM silver nitrate kept at different temperature ranging from 0 ◦ C, 20 ◦ C, 40 ◦ C, 60 ◦ C, 80 ◦ C and 90 ◦ C. 2.4.3. Effect of light intensity Effect of different light intensities like 15.5, 93.1, 141.3, 190.7 lux on the rate of nanoparticle synthesis was studied. The SNPs were also synthesized in sunlight (beyond range) and in the dark. Light intensity was measured using Lux meter (range 200 lux).

2.4.4. Effect of different concentrations of silver nitrate and filtrate volume In order to increase the synthesis of the SNPs, effect of different concentrations of silver nitrate 0.1 mM, 0.2 mM, 0.3 mM up to 1 mM and effect of filtrate volume 0.1 mL, 0.3 mL, 0.5 mL upto 1.9 mL (at fixed concentration of 1 mM AgNO3 ) were studied. The time course of SNPs synthesis was studied by measuring UV–vis spectra after regular interval of 1 min up to 10 min.

3. Results Phoma glomerata maintained on potato dextrose agar slants at 4 ◦ C was used for the synthesis of SNPs. The reduction of Ag+ ions occurs rapidly, almost completing within few minutes of reaction in the presence of sunlight, whereas the incubation in the dark could take almost 24 h for the complete reduction. Mycofabricated SNPs were detected by measuring UV–vis spectrum, which revealed the absorbance around 420 nm, a characteristic absorbance for the SNPs. The FTIR analysis revealed bands at 1623, 1525, 1228 and 952 cm−1 , whereas the fungal filtrate (control) spectrum revealed bands at 1558, 1417 and 1018 cm−1 (Fig. 1A). XRD analysis exhibited peaks at 38◦ , 44◦ , 64◦ and 77◦ , 2 values that can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) facets of silver (Fig. 1B). NTA analysis of mycofabricated SNPs by P. glomerata showed the mean size of 66 nm with standard deviation (SD) of 30 and mode of 16.6 nm, the concentration of mycofabricated SNPs was found to be 1.6 × 1010 particles/mL (Fig. 1C and D). The size determined by SEM analysis was found to be 65 nm (Fig. 2A) and by TEM analysis was found to be 19 nm (Fig. 2C). EDX analysis confirms the presence of elemental silver in the SEM sample (Fig. 2B). Also zeta

Fig. 1. Characterization of mycosynthesized SNPs from Phoma glomerata (MTCC-2210), where (A) FTIR spectrum of SNPs, (B) XRD graph of SNPs, (C) NTA Particle size distribution and (D) NTA 3D plot of particle size distribution.

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Fig. 2. Characterization of mycosynthesized SNPs from Phoma glomerata (MTCC-2210), where (A) SEM micrograph (scale bar 100 nm), (B) EDX, (C) TEM micrograph (scale bar 100 nm) and (D) Zeta potential graph.

potential revealed the biosynthesis of stable SNPs having zeta value −30.7 mV at pH 7 (Fig. 2D). Effect of light on rate of SNPs synthesis by P. glomerata (MTCC2210) clearly demonstrated better synthesis of SNPs in sunlight as compared with synthesis in the absence of light and light intensity with 15.5 (moonlight), 141.39 (bulb), 190.7 (tubelight) lux unit (Fig. 3A). The time course analysis of the synthesis process of SNPs by P. glomerata clearly demonstrates the rapid synthesis of SNPs (Fig. 3B). The UV–vis spectrum analysis showed the most effective synthesis of SNPs by P. glomerata with the bottle wrapped with light blue color cellophane paper followed by dark-blue, green and yellow color (Fig. 3C). UV–visible spectra of mycofabricated SNPs at different pH values were obtained and shown in Fig. 4A, which clearly indicate the suitability of alkaline pH for the synthesis of SNPs. The characteristic absorbance for SNPs was noticed for the fungal filtrate and silver nitrate incubated at different temperature like 4, 25, 37, 65, and 90 ◦ C by P. glomerata. On the basis of UV–vis studies, P. glomerata demonstrated better synthesis at 25 ◦ C (Fig. 4B). The different silver nitrate concentration used for the mycofabrication of SNPs by P. glomerata includes 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 mM. Synthesis of SNPs was monitored by UV–visible spectroscopy. A broad absorption peak was observed around 420 nm. It could have been due to the excitation of surface plasmon which is typical of the SNPs. The synthesis of silver nanoparticles in P. glomerata increases with increase in substrate concentration up to 0.8 mM, with the optimum synthesis of SNPs at 0.8 mM silver nitrate concentration (Fig. 4C). The different amounts of fungal filtrate were used for the synthesis of SNPs by P. glomerata, the quantities of fungal filtrate studied were 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7 and 1.9 mL of the

fungal filtrate in a total reaction mixture of 10 mL with the incubation of 20 min in bright sunlight. The effect of fungal filtrate on the synthesis of SNPs was tracked by monitoring changes in color with UV–vis spectroscopy; absorbance around 420 nm was used to quantify SNPs concentrations (Fig. 4D). The rate of synthesis of SNPs by P. glomerata increased with an increase in fungal filtrate concentration.

4. Discussion The complete reduction of silver ions into the SNPs was confirmed by performing qualitative analysis for the presence of free Ag+ ions with NaCl in the supernatant formed after the purification of SNPs. It is well known that the metal nanoparticles of size ranging from 2 to 100 nm exhibit strong but broad surface plasmon peak. As the particles size increases, the optical absorption spectra of metal nanoparticles that are dominated by surface plasmon resonances (SPR), shifts toward longer wavelengths. The position of absorption band also mainly depends upon dielectric constant of the medium and surface-adsorbed species (Xia and Halas, 2005). According to Mie’s theory, single SPR band will result in the absorption spectra of spherical nanoparticles, while particles could give rise to two or more SPR bands depending on the shape of the particles (Mie, 1908). In the present investigation, reaction mixtures of P. glomerata showed a single SPR band and with spherical shape of SNPs on confirmation with electron microscopy (SEM and TEM) images. The number of SPR peaks increases as the symmetry of the nanoparticle decreases. Therefore, triangular nanoparticles, disks, and spherical nanoparticles of silver will demonstrate three, two, and one peak, respectively.

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Fig. 3. Effect of different parameters on mycofabrication of SNP by P. glomerata (MTCC-2210), where (A) effect of different light intensity (sunlight, tubelight, in dark, moonlight, bulb), (B) effect of different time interval (1, 2, 3, 4, 5, 6, 7, 8, 9, 20 min), (C) effect of different light color in sunlight (white, green, light blue, yellow, blue, red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

In comparison with the FTIR spectrum of control, the shift in the band at 1623 cm−1 in the experimental was due to the stretch vibration of C C (Huang et al., 2007), 1525 was due to Amide II (Bansal et al., 2005), 1228 was due to amide III (Sanghi and Verma, 2009) and 952 was due to stretch vibration of C O C bond (Huang et al., 2007). Similarly, Bawaskar et al. (2010), Gajbhiye et al. (2009), Ingle et al. (2009), Sanghi and Verma (2009) and Sastry et al. (2003) have reported that bonds or functional groups such as C O C , amide and C C are derived from heterocyclic compounds like amino acids, the building blocks of protein, which are present in the fungal extract and acts as the capping ligands to the mycofabricated SNPs. The corresponding electron diffraction pattern obtained by XRD analysis confirmed the “fcc” crystalline structure of metallic silver of mycofabricated SNPs. The variation in the size determined by different methods was due to the fact that these methods rely on different physical principles and/or detection methods. In addition, electron microscopy probes dry particles, i.e. the metallic core only, whereas the NTA probe the hydrodynamic radius, which is always larger. The possibilities and limitations of different analytical methods for the size

determination of metallic nanoparticles is available (Mahl et al., 2011). The synthesis of SNPs does occur at light intensity with 190.7 and 141.3 lux unit but took more time (few hours) for the synthesis of SNPs. Therefore, in the presence of bright sunlight, synthesis of SNPs by P. glomerata was very fast. The mycofabrication was evident within a few minutes. Variety of photo-induced synthetic method for metal nanoparticle synthesis and nanostructure has been developed. Rapid mycofabrication of SNPs in sunlight may be resulted due to the photosensitization of aromatic molecules present in the fungal filtrate, the free electrons generated would be utilized for the mycofabrication SNPs. The characteristic absorbance for the SNPs due to surface plasmon resonance phenomenon in case of P. glomerata appears after the incubation of few minutes. There are several reports of synthesis of SNPs using fungal system; the different fungal system required different time for the synthesis. Birla et al. (2009) reported 2 h of incubation time for the synthesis of SNPs by P. glomerata. Nayak et al. (2011) have reported SNPs synthesis by

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Fig. 4. Effect of different conditions on mycofabrication of SNP by P. glomerata (MTCC-2210), where (A) effect of different pH (pH 3, pH 5, pH 7, pH 9, pH 11), (B) effect of different Temperature (4 ◦ C, 25 ◦ C, 37 ◦ C, 65 ◦ C, 90o C), (C) effect of different concentration of silver nitrate (0.1, 0.2, 0.3,. . ., 1.0 mM) and (D) effect of different amount of fungal filtrate (0.1, 0.3, 0.5,. . ., 1.9 mL).

Penicillium purpurogenum after 24 h of incubation, Vahabi et al. (2011) reported from Trichoderma reesei after 12 h of incubation. Minimum time reported for the synthesis of SNPs by fungal system is 2 h, the reports include Pleuorotus sp. (Gade et al., 2007), Fusarium acuminatum (Ingle et al., 2008), Aspergillus niger (Gade et al., 2008), Fusarium solani (Ingle et al., 2009), Alternaria alternata (Gajbhiye et al., 2009) and Fusarium culmorum (Bawaskar et al., 2010). Therefore, the method of mycofabrication of SNPs by P. glomerata was found to be rapid. The least effective synthesis of SNPs was evident by bottle wrapped with red color cellophane paper. The results concluded that the synthesis of SNPs by the P. glomerata is optimum in bluelight and minimum in red light. Fungal system can respond to wavelengths of light from UV–vis to far-red and blue light sensors had been known. The fungal proteins serve as a photoreceptor and comprises a signature motif for flavin binding, and the protein having flavin binding motifs can be photosensitized by blue light. It was observed that the absorption band of mycofabricated SNPs was centered on 420 nm which is the characteristic of spherical SNPs. This feature was noticed for the culture filtrate at pH 5, 7, 9 and 11 for P. glomerata. The main absorption band of SNPs shifted

to higher wavelengths (nearly 460 nm and even higher) along with unresolved absorption band corresponding to 380 nm for the SNPs formed at pH 3 by P. glomerata, indicating that the nanoparticles are not spherical and may be of different size and shape. The presence of the minor absorption band at 380 nm could have been due to polydispersity in the size or shape of the nanoparticles. Overall the alkaline condition (pH 9 and 11) was found to be optimum for the mycofabrication of SNPs by P. glomerata, while in acidic pH aggregates were observed. The study on effect of temperature on mycofabricattion of SNPs by P. glomerata, controverts the findings reported by Deepak and Kalishwaralal (2011), where they reported that the incubation temperature is directly proportional to the dynamics of the ions and nucleation regions due to the rapid dissociation of ions at increased temperature. The increase in the temperature also increases the kinetic energy of the mycofabricated SNPs in the solution, thereby resulting the collision frequency between the particles and this leads to the higher rate of agglomeration (Sarkar et al., 2007). Similarly, the formation of rapid aggregates was reported by Van Hyning et al. (2001) on the basis of surface potential of SNPs being inversely related to temperature. By classical chemical kinetics theory given by Glasstone and Lewis (1960).

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Fig. 5. Three steps mechanism of spherical NPs mycofabrication by P. glomerata.

The reports in the literature predicts the involvement of an enzyme in the synthesis of SNPs by the fungal system or mycofabrication of SNPs is an enzyme catalyzed reaction (Anil Kumar et al., 2007; Durán et al., 2005; Ingle et al., 2008). Considering the mycofabrication as an enzyme catalyzed reaction, for enzyme catalyzed reaction studies for the effect of substrate (silver nitrate) and enzyme concentration (fungal filtrate) on product (SNPs) formation is required. Moreover, the synthesis of SNPs by using fungal system reported by several researchers (Birla et al., 2009; CastroLongoria et al., 2011; Fayaz et al., 2010; Gade et al., 2008, 2011; Ingle et al., 2008, 2009; Vahabi et al., 2011) have reported synthesis of SNPs by using 1 mM silver nitrate concentration, but they could not give proper justification for using 1 mM silver nitrate concentration for the mycofabrication. To address the issue, synthesis of SNPs by P. glomerata was monitored at different substrate or salt (silver nitrate) concentration by measuring UV–vis spectrum from 200 to 800 nm. The results showed an increase in the mycofabrication of SNPs with an increase in the substrate concentration up to 0.8 mm, with the optimum mycofabrication of SNPs at 0.8 mM silver nitrate concentration. This is the first report of using silver nitrate concentration less than 1 mM for the synthesis of SNPs. The study on the effect of different amounts of fungal filtrate for the mycofabrication demonstrated that amount of fungal filtrate was directly proportional to the nanoparticle synthesis. This might be due to the increase in the secreted protein concentration as the amount of fungal filtrate increases. So we would like to propose three-step mechanism for the synthesis of spherical SNPs by P. glomerata. The first step is activation, which includes the activation by photosensitization of aromatic compounds in the fungal filtrate on exposure to bright sunlight. Second step is nucleation, which involves the role of photosensitized aromatic compounds or proteins to initiate the synthesis of spherical SNPs acting as capping agent, and the third step exhibits actual synthesis by reduction of silver ions to form SNPs. This step includes the donation of electron either by inorganic nitrate or by photosensitized aromatic compounds from fungal filtrate, by donating an electron to silver ions and leading to the reduction of them to form spherical SNPs (Fig. 5). Metal

nitrates can be decomposed due to the heat generated on exposure to bright sunlight, i.e. thermally to the respective oxides. 5. Conclusion From the above study, it can be concluded that rapid synthesis of SNPs was observed in the presence of bright sunlight. From the FTIR analysis of the synthesized SNPs by P. glomerata confirms that the proteins are the biomolecules responsible for the reduction of silver ions and as a capping agent, providing the stability to the silver colloids by avoiding the aggregation of SNPs. NTA analysis confirmed the synthesis of SNPs by P. glomerata, NTA provides the information about the size and concentration but not about the shape of synthesized SNPs. The method was found to be more convenient and easy for the analysis of silver colloids. TEM analysis shows the synthesis of polydispersed spherical SNPs. The mycofabrication of SNPs by P. glomerata is also affected by the parameters like temperature, pH, silver nitrate concentration and fungal filtrate concentration. Synthesis was optimum at alkaline pH and at room temperature incubation. Three-step mechanism for the synthesis of spherical SNPs by P. glomerata has been proposed. The three-step include: (i) activation (ii) nucleation, and (iii) reduction. Finally, the synthesis of SNPs by P. glomerata is rapid, easy and eco-friendly method. Acknowledgement MKR is thankful to FAPESP for a Visiting Scientific Grant at IQUniversidade Estadual de Campinas, SP, Brazil. References Anil Kumar, S., Abyaneh, M.K., Gosavi, S.W., Kulkarni, S.K., Pasricha, R., Ahmad, A., Khan, M.I., 2007. Nitrate reductase-mediated synthesis of silver nanoparticles from AgNO3 . Biotechnol. Lett. 29, 439–445. Ayyub, P., Chandra, R., Taneja, P., Sharma, A.K., Pinto, R., 2001. Synthesis of nanocrystalline material by sputtering and laser ablation at low temperatures. Appl. Phys. A: Mater. Sci. Process. 73, 67–73.

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Green synthesis of silver nanoparticles by Phoma glomerata.

We report an extracellular synthesis of silver nanoparticles (SNPs) by Phoma glomerata (MTCC-2210). The fungal filtrate showed rapid synthesis in brig...
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