Extracellular biosynthesis of silver nanoparticles

Extracellular biosynthesis of silver nanoparticles using a novel and non-pathogenic fungus, Neurospora intermedia: controlled synthesis and antibacterial activity Sepideh Hamedi, Seyed Abbas Shojaosadati, Soheila Shokrollahzadeh & Sameereh Hashemi-Najafabadi World Journal of Microbiology and Biotechnology ISSN 0959-3993 Volume 30 Number 2 World J Microbiol Biotechnol (2014) 30:693-704 DOI 10.1007/s11274-013-1417-y

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Author's personal copy World J Microbiol Biotechnol (2014) 30:693–704 DOI 10.1007/s11274-013-1417-y

ORIGINAL PAPER

Extracellular biosynthesis of silver nanoparticles using a novel and non-pathogenic fungus, Neurospora intermedia: controlled synthesis and antibacterial activity Sepideh Hamedi • Seyed Abbas Shojaosadati • Soheila Shokrollahzadeh • Sameereh Hashemi-Najafabadi

Received: 8 January 2013 / Accepted: 23 June 2013 / Published online: 26 September 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract In the present study, the biosynthesis of silver nanoparticles (AgNPs) using Neurospora intermedia, as a new non-pathogenic fungus was investigated. For determination of biomass harvesting time, the effect of fungal incubation period on nanoparticle formation was investigated using UV–visible spectroscopy. Then, AgNPs were synthesized using both culture supernatant and cell-free filtrate of the fungus. Two different volume ratios (1:100 and 1:1) of the culture supernatant to the silver nitrate were employed for AgNP synthesis. In addition, cell-free filtrate and silver nitrate were mixed in presence and absence of light. Smallest average size and highest productivity were obtained when using equal volumes of the culture supernatant and silver nitrate solution as confirmed by UV– visible spectra of colloidal AgNPs. Comparing the UV– visible spectra revealed that using cell-free filtrate for AgNP synthesis resulted in the formation of particles with higher stability and monodispersity than using culture supernatant. The absence of light in cell-free filtrate mediated synthesis led to the formation of nanoparticles with the lowest rate and the highest monodispersity. The presence of elemental silver in all prepared samples was confirmed using EDX, while the crystalline nature of synthesized particles was verified by XRD. FTIR results showed the presence of functional groups which reduce

S. Hamedi S. A. Shojaosadati (&) S. Hashemi-Najafabadi Biotechnology Group, Chemical Engineering Faculty, Tarbiat Modares University, P.O. Box: 14115-114, Tehran, Iran e-mail: [email protected] S. Shokrollahzadeh (&) Department of Chemical Technologies, Iranian Research Organization for Science and Technology, P.O. Box: 15815-3538, Tehran, Iran e-mail: [email protected]

Ag? and stabilize AgNPs. The presence of nitrate reductase was confirmed in the cell-free filtrate of the fungus suggesting the potential role of this enzyme in AgNP synthesis. Synthesized particles showed significant antibacterial activity against E. coli as confirmed by examining the growth curve of bacterial cells exposed to AgNPs. Keywords Antibacterial assay Extracellular biosynthesis Silver nanoparticles Neurospora intermedia Nitrate reductase enzyme

Introduction Nanoparticles are regarded as fundamental molecular building blocks for preparing many nanostructures and devices in nanotechnology (Saravanan et al. 2011). Noble metal nanoparticles such as gold and silver show unique optical, electrical, mechanical, magnetic and chemical properties that are significantly different from those of bulk materials (Aswathy Aromal and Philip 2012; Kouvaris et al. 2012). These materials have applications in various areas, including biology (biolabelling and DNA sequencing), medicine (antimicrobial and antibacterial actions), non-linear optics, catalysis and surface-enhanced raman scattering (SERS). (Edison and Sethuraman 2012; Gurunathan et al. 2009; Otari et al. 2012; Philip 2011; Vijayakumar et al. 2012). Various physical and chemical procedures exist for the synthesis of silver nanoparticles. Some of these methods include thermal decomposition in organic solvents, chemical reduction and photoreduction in reverse micelles (Hussain et al. 2011; Wang et al. 2005). However, the above-mentioned methods are extremely expensive and also involve using or generation of highly toxic and hazardous materials. These drawbacks may

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impose considerable environmental and biological risks which can limit the use of AgNPs for clinical applications (Dwivedi and Gopal 2010; Gopinath et al. 2012; Yilmaz et al. 2011). Consequently, green synthesis of nanoparticles has been explored as an alternative method for the synthesis of such particles (Kaviya et al. 2011; Zaki et al. 2011). One of the most promising techniques for the green synthesis of silver nanoparticles is extracellular biosynthesis using microorganisms such as bacteria, yeasts, and fungi (Alani et al. 2012; Das et al. 2012). Extracellular biosynthesis of AgNPs is more economical than the intracellular approach due to the ease of downstream processing (Bai et al. 2011; Fayaz et al. 2011). Filamentous fungi have advantages over other microorganisms for biosynthesis of nanoparticles due to their high secretion of proteins, enzymes and metabolites, high growth rates, easy handling in large-scale production and low-cost requirements for production procedures (Kashyap et al. 2013; Musarrat et al. 2010). In addition, nanoparticles synthesized using fungi present fair monodispersity and stability compared to other microorganisms (Sanghi and Verma 2009a). As a result, different fungal species have been utilized in the synthesis of silver nanoparticles, some of which include Verticillium sp. (Mukherjee et al. 2001), Trichoderma asperellum (Mukherjee et al. 2008), and some species of Aspergillus (Saravanan and Nanda 2010; Vigneshwaran et al. 2007), Penicillium (Nayak et al. 2011), and Fusarium oxysporum (Ahmad et al. 2003; Khosravi and Shojaosadati 2009; Mohammadian et al. 2007). Despite the presence of several published studies on biosynthesis of AgNPs, the exact mechanism of AgNP formation is still not well understood; however, it has been postulated that NADH–dependent nitrate reductase is an important factor in the biosynthesis of metal nanoparticles (Duran et al. 2011; Shaligram et al. 2009). The main objective of the present study was to evaluate the potency of a new non-pathogenic fungus, Neurospora intermedia, for the production of AgNPs. To this aim, the efficiency of AgNP production using the culture supernatant and cell-free filtrate of this fungus was investigated. Colloidal AgNPs synthesized by culture supernatant and cell-free filtrate of N. intermedia were characterized by UV–visible spectroscopy, scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) patterns, dynamics light scattering (DLS) and X-ray diffraction (XRD) analyses. FTIR spectra of reaction solutions before and after nanoparticle synthesis were recorded to provide information on the mechanism of AgNP biosynthesis. Moreover, the level of nitrate reductase enzyme in the cell-free filtrate of the fungus was examined to understand the role of this enzyme in AgNP synthesis. Finally, the antibacterial activity of AgNPs against gramnegative bacterium Escherichia coli was evaluated.

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Materials and methods Microorganism and chemicals Neurospora intermedia (PTCC 5291) was obtained from Persian Type Culture Collection, Iranian Research Organization for Science and Technology. The pure culture was maintained on a potato dextrose agar (PDA) supplemented with 0.5 % (w/v) yeast extract at 24 °C, and the resulting medium was stored at 4 °C. This strain was sub-cultured periodically to sustain its viability during the period of this study. All chemicals, including silver nitrate, potassium nitrate, N-(1-naphthyl) ethylene diamine dihydrochloride (NEED), sulphanilamide (SA) were of analytical grade and purchased from Sigma-Aldrich. Fungal cultivation conditions Neurospora intermedia inoculums (107 spore (cfu)/ml) were transferred into 250 ml shake flasks containing 50 ml potato dextrose broth (PDB) medium supplemented with 0.5 % (w/v) yeast extract as nitrogen source. The pH was set to 5.8 ± 0.2, its optimal value for N. intermedia growth, using 1 N HCl. Twenty flasks containing the inoculated medium were incubated at 24 °C with agitation on an orbital shaker operating at 200 rpm for 100 h. Every 10 h, two flasks were removed simultaneously and their biomass content was harvested by vacuum filtration. In order to measure the dry weight, the resultant wet mycelia were dried at 60 °C to a constant weight. The growth curve was obtained by plotting the fungal dry weights versus incubation periods. To determine the dependence of AgNP biosynthesis on growth stages of fungus, the biomass free medium obtained at various incubation periods were mixed with silver nitrate solution and the nanoparticle formation was quantified by using UV–Vis spectroscopy. The UV– visible spectra with the highest absorbance and the lowest wavelength were selected and the related incubation periods were considered as the optimal condition for AgNP synthesis. Biosynthesis of AgNPs using culture supernatant N. intermedia fungus was grown aerobically in liquid media containing PDB ?0.5 % (w/v) yeast extract. The final pH of the medium was adjusted to 5.8 using 1 N HCl. After the cultivation period, the fungal culture was centrifuged at 6,000 rpm for 20 min and the supernatant was collected. The supernatant was added to 1 mM and 2 mM silver nitrate solutions with volume ratios of 1:100 and 1:1, respectively (these ratios were selected based on some preliminary experiments). Finally, the mixtures were incubated at 28 °C (ambient temperature) and agitated at

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200 rpm to complete the biosynthesis of the nanoparticles. All reactions were carried out in the presence of light.

(Cary 100, Varian) with a resolution of 1 nm in the range of 200–800 nm.

Biosynthesis of AgNPs using cell-free filtrate

Dynamic light scattering (DLS)

The biomass from N. intermedia cultures was harvested by filtration and then washed extensively with sterilized distilled water to remove any remaining media components. Then, 10 g of wet fungal mycelia were suspended in 100 ml sterilized distilled water and incubated at 24 °C with agitation on an orbital shaker operating at 200 rpm for 72 h. Then, the cell-free filtrate was collected by filtration of this suspension through Whatman filter paper No. 1. Finally, 50 ml of the cell-free filtrate was added to 50 ml of a 2 mM silver nitrate solution. The mixtures were incubated at 28 °C under shaking at 200 rpm until complete bioreduction of Ag? was achieved. These experiments were done both in the presence and absence of light and in triplicates.

The size distribution and average size of the synthesized AgNPs were determined by dynamic light scattering (DLS) (Malvern, UK). DLS measurements were carried out for size ranges from 0.3 nm to 104 nm. Polydispersity indexes determined from DLS experiments were used as a measure of particle aggregation. Polydispersity index (PDI) is a dimensionless criterion to define the size distribution of nanoparticles obtained from a photon correlation spectroscopic analysis. It ranges from a value of 0.01 for monodispersed particles and up to values of 0.5–0.7 (Nidhin et al. 2008).

Nitrate reductase assay Nitrate reductase in fungal filtrate was assayed according to the procedure described by Harley (Jaidev and Narasimha 2010). After determination of the incubation period, mycelia were separated from media by filtration and washed extensively with sterile distilled water to prevent any contamination from media components. For enzyme assay, a mixture of wet mycelia and distilled water (10 % w/v) was incubated at 24 °C for 120 h (5 days). The 5-day filtrate was obtained by passing the mycelial mixture through Whatman filter paper No. 1. Then, 10 ml aliquot of the 5-day fungal filtrate was mixed with 10 ml of assay medium (30 mM KNO3 and 5 % propanol in 0.1 M phosphate buffer of pH 7.5) and incubated at 30 °C in the absence of light for 1 h. Then, 5 ml of SA solution and 5 ml of NEED solution were added to determine the formation of nitrites in the solution. Finally, the absorbance of resultant pink solutions at 540 nm was measured using UV–visible spectrophotometer. The enzyme activity was determined based on the increase in nitrite content of the solution over 1 h for the amount of the initial sample (10 ml) and expressed as nmol nitrite h-1ml-1.

X-ray diffraction (XRD) X-ray diffraction was carried out using a Philips PW-1730 system (Philips, Netherlands) equipped with Co Ka radi˚ ) operating at 30 mA and 40 kV. The ation (k = 1.7889 A diffractograms were recorded over the range 20°–80° (2h) angles. Samples were prepared by casting a drop of colloidal silver solutions on a silicon substrate, allowing time for liquid evaporation and then exposing it to X-ray. Scanning electron microscopy (SEM) and energydispersive X-ray spectroscopy (EDX) The size and morphological characterization of the synthesized silver nanoparticles were accomplished by SEM (KYKY-EM3200, China). The SEM images were recorded at 25.00 kV. Thin film was prepared by drop coating of purified biologically synthesized silver nanoparticles from all prepared solutions onto carbon coated copper SEM grids. Five minutes were allowed for evaporation of solvent and then the extra sample solution was removed using a blotting paper. SEM images were acquired at 30,0009 magnification. The presence of the elemental silver was confirmed through EDX (Hitachi S 4160, USA) analysis. Fourier-transform infrared spectroscopy (FTIR)

Characterizations of synthesized AgNPs UV–visible spectral analysis The color changes of reaction mixtures were used as evidence for AgNP formation. Therefore, 2 ml samples were withdrawn from flasks containing colloidal AgNPs at predetermined time intervals, and the absorbance was measured by a double beam UV–visible spectrophotometer

The characterization of functional groups on the surface of AgNPs was performed by fourier-transform infrared spectroscopy (FTIR) (Perkin-Elmer, Germany). The spectra were scanned in the 500–4,000 cm-1 range with a resolution of 4 cm-1. For FTIR analysis, the solutions containing AgNPs were centrifuged at 10,000 rpm for 30 min and the pellets were dispersed in deionized water. The centrifugation and redispersion were repeated three times.

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Finally, the purified suspension was freeze-dried to obtain dried powder and analyzed after mixing with KBr. Antibacterial assay of synthesized AgNPs Escherichia coli BL21 was obtained from Tarbiat Modares University Culture Collection, Tehran, Iran. Fresh colonies of E. coli from a Luria–Bertani (LB) agar plate were inoculated into 10 ml of LB broth medium. The flask was incubated overnight at 37 °C and agitation rate of 200 rpm. The fresh inoculum was transferred to six shake flasks each containing 20 ml of LB media. The culture media of four flasks was supplemented with 50 lg/ml AgNPs synthesized by either culture supernatant (two replicates) or cell-free filtrate (two replicates). Two flasks were considered as control without addition of nanoparticles. Finally, all flasks were incubated at 37 °C with continuous shaking at 200 rpm. The optical densities of all media at 600 nm (OD600) were measured every 1 h. The antibacterial activity of silver nanoparticles was assessed by determining the growth curve of E. coli in the presence and absence of AgNPs.

Results Effect of fungal incubation period on the synthesis of AgNPs The non-pathogenic fungus, Neurospora intermedia, was used for the extracellular biosynthesis of highly stable silver nanoparticles as a new fungus in this application. The evaluation of the growth profile showed that there was no considerable change in the dry cell weight of N. intermedia after 72 h (data not shown). Simultaneously, the influence of fungal incubation period on the synthesis of AgNPs was investigated by mixing the silver nitrate

Fig. 1 a UV–visible spectra of colloidal AgNPs originated from culture medium of fungus at mid logarithmic (I), late logarithmic (II) and stationary (III) phases, b Color changes of the culture supernatant

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solution with the biomass free medium obtained at various growth stages. UV–visible spectra of colloidal AgNPs obtained at different growth phases (stationary, late logarithmic and mid logarithmic) are presented in Fig. 1a. UV– visible spectra of these solutions showed that the peak area and the height of the spectra originated from the biomass free medium after 72 h of incubation (i.e. the early stationary phase), were considerably higher. Therefore, the early stationary phase of growth was chosen for biomass harvesting and colloidal AgNPs were synthesized by using the culture supernatant and the cell-free filtrate of N. intermedia obtained at this phase. The properties of the synthesized AgNPs, such as size and size distribution, were compared as well as the productivity of the preparatory methods. UV–visible analysis As shown in Fig. 1b, the preliminary detection of AgNP formation was carried out by visual observation of the color changes of reaction solutions. UV–visible spectroscopy is an important technique to investigate surface plasmon resonance (SPR) and thus, the morphology, size and stability of synthesized nanoparticles. The UV–visible spectra of AgNPs synthesized at different time intervals are presented in Fig. 2. As shown in this figure, there is no noticeable change in the intensities of the spectra after 120 h under the condition of cell-free filtrate mediated synthesis in the absence of light, and after 72 h in other conditions, which indicates the reaction completion. Figure 2a and b show the culture supernatant mediated synthesis of AgNPs with 1:1 and 1:100 volume ratios of supernatant: AgNO3. As shown in these figures, a strong SPR is observed near 403 and 460 nm for 1:1 and 1:100 volume ratios, respectively. Also, the highest and the lowest plasmon resonance peaks are acquired from the mixtures of culture supernatant and silver nitrate with 1:1

(I) and cell-free filtrate (II) of the fungus N. intermedia after addition of silver nitrate


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Fig. 2 UV-visible spectra of colloidal AgNPs in reaction solutions containing a 1:1 volume ratio of N. intermedia culture supernatant and AgNO3; b 1:100 volume ratio of culture supernatant and AgNO3; c cell-free filtrate and AgNO3 in light; and d cell-free filtrate and

AgNO3 in absence of light; at 12 h (I), 24 h (II), 48 h (III), 72 h (IV), 120 h (V), after 2 months storage at room temperature (VI). Insets show the profiles of AgNP biosynthesis at different times

and 1:100 volume ratios, respectively. UV–visible spectra of AgNPs synthesized by cell-free filtrate of N. intermedia in the presence and absence of light are shown in Fig. 2c and d, respectively. As shown in these figures, the SPR bands are centered at 420 and 406 nm. The plasmon bands observed for AgNPs formed using the cell-free filtrate are more symmetrical than using the culture supernatant. Comparison of Fig. 2c and d reveals that the AgNPs synthesized in the absence of light have a more symmetrical band. The insets in Fig. 2 show the kinetic profiles of AgNP formation under various conditions. The highest rate of AgNP formation is observed in the cell-free filtrate mediated method in the presence of light as verified by early color change of the reaction solution. The stability of AgNPs synthesized by this fungus was assessed by measuring the absorbance intensities and kmax of the spectra of the reaction solutions over a period of 2 months at room temperature. No significant changes in the absorbance intensity and kmax of the colloidal AgNPs were observed during this period as demosntrated in Fig. 2a, c and d.

Particle size analysis The average particle size, size distribution, and polydispersity index (PDI) of the synthesized AgNPs were determined by DLS (Fig. 3). As illustrated in Fig. 3a, the average sizes of AgNPs produced by volume ratios of 1:1 and 1:100 (culture supernatant: AgNO3) are 19 and 84 nm, and their PDI are 0.3 and 0.4, respectively. The average size and PDI of AgNPs produced by the cell-free filtrate (under light) are 30 nm and 0.2, respectively (Fig. 3b). Synthesizing AgNPs by the cell-free filtrate in the absence of light, lead to the formation of nanoparticles with average size of 24 nm and PDI of 0.15. X-ray diffraction (XRD) The phase of the synthesized nanoparticles was investigated by the XRD technique, and the corresponding patterns are shown in Fig. 4. AgNPs produced by the culture supernatant of fungus with 1:1 volume ratio (Fig. 4a) shows distinguished XRD peaks with 2h values of 64.17,

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38.7, 46.33 and 77, whereas AgNPs formed by the cell-free filtrate at light environment shows peaks with 2h values of 64.71, 37.59, 44.52 and 76.6 (Fig. 4b). These peaks are assigned to the (220), (111), (200), and (311) reflection planes of face-centered-cubic (fcc) silver, respectively. Scanning electron microscopy (SEM) and energydispersive X-ray spectroscopy (EDX) Scanning electron microscopy images of AgNPs formed by the culture supernatant (Fig. 5a, b) and the cell-free filtrate (Fig. 5c, d) of N. intermedia are presented in Fig. 5. Insets in Fig. 5a and d show the SEM images of the synthesized nanoparticle after 24 h of the reaction. The biosynthesis of AgNPs by N. intermedia fungus was further characterized by EDX analysis, which provided further evidence on the formation of elemental silver (Fig. 5e). A similar EDX spectrum was obtained for each sample analyzed. Fourier-transform infrared spectroscopy (FTIR) FTIR spectra of samples were recorded in order to obtain insight about the interactions between biomolecules and silver nanoparticles. Figure 6a shows the IR spectra of the pure culture supernatant, and AgNPs prepared using the culture supernatant. IR spectra of the cell-free filtrate alone and synthesized AgNPs using the cell-free filtrate are presented in Fig. 6b.


Antibacterial assay The antibacterial activity of synthesized AgNPs using the culture supernatant and the cell-free filtrate of N. intermedia are presented in Fig. 7. The presence of AgNPs in both culture media reduces the growth rate of E. coli. Also, the growth of bacterial cells treated with AgNPs produced using the cell-free filtrate is slower than that of cells treated with AgNPs synthesized by culture supernatant.

Discussion Biosynthesis of silver nanoparticles This study presents the extracellular synthesis of proteinstabilized silver nanoparticles using the culture supernatant and the cell-free filtrate of a non-pathogenic and novel fungus, N. intermedia, under ambient conditions. As indicates in Fig. 1a, nanoparticle formation depends on the growth phase of the fungal cells and increases at the stationary phase as compared with that grown in other phases. Increased formation of AgNPs at the early stationary phase

Nitrate reductase assay The presence of extracellular enzyme, nitrate reductase in Neurospora intermedia was confirmed by using the Harley method, which suggested the involvement of this enzyme in the formation of nanoparticles. The enzyme activity was measured in the filtrate of biomass which was harvested at the early stationary phase where the maximum nanoparticle formation occurred. The nitrate reductase activity of the fungal cell-free filtrate was measured as 340 nmol h-1 ml-1. Fig. 3 DLS analysis of colloidal AgNPs generated by the mixtures of a culture supernatant of N. intermedia and AgNO3 with volume ratios of 1:1 and 1:100; b cell-free filtrate of N. intermediaand AgNO3 in the presence and absence of light

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Fig. 4 XRD patterns of AgNPs synthesized by a culture supernatant and AgNO3 with an equal volume ratio; b cell-free filtrate of N. intermedia. The numbers in the parenthesis show the face center cubic planes of AgNPs


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a

200 nm m

25 kV V, 60 k kX

b

X 15 kV V, 1100 kX

3300

c

2000 n nm

25 k kV V, 600 kX X

d

25 kV V, 660 k kX

nm 2000 n m

e

Fig. 5 SEM images of colloidal AgNPs in reaction solutions containing a equal volume ratio of culture supernatant of N. intermediaand AgNO3; b 1:100 volume ratio of supernatant and AgNO3; c cell-free filtrate and AgNO3 in light; and d cell-free filtrate

and AgNO3 in absence of light, e EDX spectrum of AgNPs synthesized by fungus N. intermedia.Insets show the SEM images of AgNPs 24 h after the beginning of the reaction

is likely due to the higher secretion of extracellular enzymes in this phase. These results revealed that the efficiency of AgNP synthesis depends upon the growth phases of the fungus which is in agreement with previous reports (Bai et al. 2009; Natarajan and Selvaraj 2010). The

color changes of all samples (Fig. 1b) are evidence for the formation of silver nanoparticles and are attributed to the surface plasmon resonance (SPR) arising due to the collective oscillation of free conduction electrons induced by an interacting electromagnetic field (Shukla et al. 2012).

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Fig. 6 Comparison of FTIR spectra for a culture supernatant of N. intermedia before (I) and after mixing by AgNO3 (II); and b cell-free filtrate of N. intermedia before (I) and after mixing by AgNO3 (II)

1.8 1.6 1.4

OD600

1.2 1 0.8 0.6 0.4 0.2 0 0

1

2

3

4

5

6

7

8

9

10

11

12

13

Time (h)

Fig. 7 Growth curves of E. coli in LB culture media supplemented (filled triangle) with 50 lg/ml AgNPs synthesized using cell-free filtrate; (filled square) with 50 lg/ml AgNPs synthesized using culture supernatant; (filled diamond) without AgNPs

These bands have been previously used to confirm the presence of AgNP in colloidal solutions (Pandey et al. 2012). The presence of a single SPR band in all UV– visible spectra (Fig. 2) corresponds to the production of isotropic nanoparticles (Sadhasivam et al. 2010). The intensities of these spectra steadily increased as a function of time, indicating the formation of increased number of AgNPs in solutions (Sathishkumar et al. 2010). Also, the absorption band near 380 nm in Fig. 2a and b corresponds to the transverse plasmon vibration in the AgNPs (Basavaraja et al. 2008). The exhibition of absorption band at 265 nm, in UV–visible spectra of the solutions attribute to the aromatic amino acids of proteins (data not shown) that arises due to electronic excitations in tryptophan and tyrosine residues of the proteins (Bhainsa and D’Souza 2006). This observation can be due to the secretion of proteins into the solution by N. intermedia and suggests a possible mechanism for the reduction of silver ions present in the solution. It is reported that the shape and size of the synthesized AgNPs have strong effect on the SPR band. On the other hand, the shorter-wavelength region implies the formation of smaller nanoparticles (Prathna et al. 2011). In this study, using culture supernatant with a 1:1

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volume ratio (Fig. 2a) and cell-free filtrate in absence of light (Fig. 2d) resulted in the formation of the smallest AgNPs. The shift of the SPR band to higher wavelengths (nearly 460 nm) in Fig. 2b indicates that AgNP synthesis with the 1:100 volume ratio of the culture supernatant to the AgNO3 solution led to the formation of nanoparticles with different shapes and sizes. The absorbance intensity of UV–visible spectra provides insight into the bioreduction of Ag? and consequently, the productivity of each method (Ghaseminezhad et al. 2012). The mixture of the culture supernatant and the silver nitrate with a 1:1 volume ratio (Fig. 2a) showed the highest AgNP productivity. This may be due to the higher amounts of proteins, enzymes and also the presence of metabolites in the culture supernatant compared to the cell-free filtrate. These biomolecules may act as reducing and/or stabilizing agents in the AgNP synthesis. Figure 2b also reveals that the mixture of culture supernatant and silver nitrate solution with the volume ratio of 1:100 lead to the formation of AgNPs with the lowest productivity and the largest size. This can be attributed to the dilution of the culture supernatant in reaction mixture which results in a decrease in the amount of reducing and stabilizing agents. Similar findings about the effect of the volume ratio on AgNP characteristics have been reported in the literature (Philip 2010; Philip and Unni 2011). The presence of a symmetrical plasmon band in UV–visible spectra can be attributed to the narrower size distribution of the particles produced in this method (Hamedi et al. 2012) which reveals that the size distribution of AgNPs formed using the cell-free filtrate both in presence and absence of light was narrower than those synthesized using the culture supernatant. Moreover, the synthesized AgNPs using the cell-free filtrate in the absence of light have a narrower size distribution. This can be related to lower nucleation rate of nanoparticles in the absence of light. The different rates of AgNP formation may also be due to different mechanisms involved in the silver nanoparticle formation. Molecular studies are needed and are currently being performed in our laboratory to shed light on these mechanisms.


It is well-known that the application of silver nanoparticles, especially in the medical field depends on their timedependent stability. The stability assessment of AgNPs for a period of 2 months (Fig. 2c, d) verified that the synthesized colloidal AgNP were extremely stable with no evidence of particle agglomeration. The decrease in absorbance intensity in Fig. 2b (1:100 volume ratio of culture supernatant to AgNO3) may be assigned to instability and agglomeration of AgNPs. These results indicate that the volume ratio of the culture supernatant and silver nitrate can control the AgNP characteristics such as average size, size distribution, stability and productivity. Moreover, the presence of light in cell-free filtrate mediated synthesis has a significant effect on the size distribution of the prepared AgNPs. Structural and morphological studies of AgNPs The average sizes of the produced AgNPs by N. intermedia in the present study are compared with previous studies with various fungi and are listed in Table 1. A comparison of the average particle size and PDI under various conditions reveals that using the cell-free filtrate produces AgNPs with the highest monodispersity. Also, produced AgNPs in reaction solution containing a 1:1 volume ratio of the culture supernatant to the AgNO3 solution have the smallest average size. These results are in agreement with the UV–visible analysis. The observed 2h values in XRD patterns (Fig. 4) are in agreement with the pure crystalline silver structure’s database of the Joint Committee on Powder Diffraction Standards (JCPDS) file No. 04-0783. XRD results verify the crystalline nature of the synthesized AgNPs by the cell-free filtrate and the culture supernatant of the fungus N. intermedia. Similar results were reported for other fungal or bacterial strains (Narayanan and Sakthivel 2011; Wei et al. 2012). The observed noise and other additional peaks in Fig. 4a and b are attributed to the effect of nanosized particles and the presence of various crystalline biological compounds in two independent extracts (Soni and Prakash 2011; Sukirtha et al. 2012). The broad bottom area of the peaks in Fig. 4a indirectly represents the smaller size of nanoparticles (Binupriya et al. 2010). The peak intensity is higher for the culture supernatant mediated synthesis compared to the cell-free filtrate mediated one. This is an additional proof for the higher productivity of AgNPs by the culture supernatant, which is well-supported by UV–visible spectral data. The shape of AgNPs from both methods is predominantly spherical (Fig. 5). Also, the nanoparticles are not in direct contact even within the aggregates, indicating the stabilization of nanoparticles by a capping agent. Both Fig. 5a and b show the agglomerated nanoparticles in certain places in the case of the culture supernatant mediated biosynthesis. These figures

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also reveal that the particles with the smallest and the largest diameters were produced in the volume ratios of 1:1 and 1:100 of the supernatant to the silver nitrate solution, respectively. As indicated in Fig. 5c and d, the use of the cell-free filtrate resulted in the formation of monodispersed nanoparticles, especially in the absence of light. These observations are corroborated with UV–visible, FTIR and DLS analyses. As indicated above, nanoparticles with the smallest size and the highest monodispersity were synthesized by using culture supernatant with a 1:1 volume ratio and, cell-free filtrate in the absence of light, respectively. Having employed the above mentioned conditions, insets in Fig. 5a and d show the SEM images of colloidal AgNPs recorded 24 h after the beginning of the reaction. These figures confirm the nucleation mechanism of nanoparticle formatin. This mechanism contains three steps of nucleation, growth and stabilization. Firstly, a portion of silver ions (Ag?) in a solution is reduced to silver atoms (Ag0) by the available reducing agents. These atoms act as nucleation centers and catalyze the remaining silver ions which are present in the reaction solution. Clusters are formed via atoms aggregation. Secondly, the surface ions are frequently reduced in order to attain the high values of nuclei leading to the formation of larger particles during the process. Finally, the capping agents in the solution function as stabilizer and prevent the further aggregation of the particles. The EDX spectra (Fig. 5e) show the optical absorption peak at approximately 3 keV, which is typical for the absorption of metallic silver nanocrystals due to SPR. This peak confirms the presence of nanocrystalline elemental silver. Mechanism of AgNP formation The broad bands between 3,300 and 3,500 cm-1 in all FTIR spectra (Fig. 6) are assigned to the –N–H stretching of the amide A band and the O–H stretching of the aromatic amines (Juibari et al. 2011; Sanghi and Verma 2009b). These bands indicate strong hydrogen bonding. Also, the presence of bands between 1,630 and 1,680 cm-1 in all spectra arise from –C=O (carbonyl) stretch vibrations in the amide I linkage of the proteins secreted by the fungus in two extracts (Haris and Severcan 1999; Syed and Ahmad 2012). As shown in Fig. 6a, the FTIR spectrum of AgNPs synthesized by the culture supernatant shows absorption bands associated with the C–N stretch of the amines, the – COO– stretch of the carboxylic acids and the C–H stretch of the methylene groups of the protein at 1,076, 1,404 and 1,455 cm-1, respectively (Bar et al. 2009; Barth 2000; Priya et al. 2011). Also, the band at 1,516 cm-1 has been identified as amide II band, which arose due to C=O (carbonyl) stretch and –N–H– stretch vibrations in the amide linkages of proteins (Gajbhiye et al. 2009). The two

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Table 1 The comparison of AgNP characteristics produced by some selected fungi Fungus

Extract

Volume ratio

Condition

Temperature (°C)

Time (h)

Average particle size (nm)

PDIa

Reference

Neurospora intermedia

Culture supernatant

1:1

Light

Ambient

72

19

0.3

Present work

Neurospora intermedia

Culture supernatant

1:100

Light

Ambient

72

84

0.4

Present work

Neurospora intermedia Neurospora intermedia

Cell-free filtarte Cell-free filtrate

1:1 1:1

Light Dark

Ambient Ambient

72 120

30 24

0.2 0.15

Present work Present work

Neurospora crassa

Biomass

–

Light

28

24

11

ND

Castro-Longoria et al. (2011)

Pycnoporus sanguineus

Culture supernatant

1:100

Dark

30

120

63.8

0.091

Chan and Mat Don (2013)

Schizophyllum commune

Culture supernatant

1:100

Dark

30

120

56.5

0.086

Chan and Mat Don (2013)

Coriolus versicolor

Culture supernatant

NDb

Dark

37

72

ND

ND

Sanghi and Verma (2009a)

Aspergillus niger

Cell-free filtrate

ND

Light

28 ± 4

72

10

ND

Jaidev and Narasimha (2010)

Cladosporium cladosporioides

Cell-free filtrate

1:10

Light

27

78

ND

ND

Balaji et al. (2009)

Alternaria alternate

Cell-free filtrate

1:1

Light

Ambient

ND

32.5

ND

Gajbhiye et al. (2009)

a

PDI polydispersity index

b

ND not determined

bands observed at 1,385.26 and 1,074.1 cm-1 in the FTIR spectrum of the cell-free filtrate (Fig. 6b) can be assigned to the C–N stretching vibrations of aromatic amines and primary amines, respectively (Sanghi and Verma 2009a). Comparing the spectra of both functionalized AgNPs and associated solutions in Fig. 6a and b, revealed that peaks obtained by these solutions are repeated in FTIR spectra of functionalized AgNPs. As shown in Fig. 6a and b, the peaks around 3,300–3,500 cm-1 (representing N–H and O–H stretching vibration) in synthesized AgNP spectra became narrower compared to the solutions. This change may be due to the breakage of H–bonds between amide groups present in the media, which are adsorbed onto silver nanoparticle surfaces and tend to form stronger bonds with Ag atoms thus leading to the narrowing of these peaks. These results are similar to those previously reported on the synthesis of AgNP using wool keratin and Nigrospora oryzae (Lu¨ and Cui 2010; Saha et al. 2011). Thus, FTIR studies confirm that the carbonyl group of the amino acid residues and the proteins have a strong ability to bind to the metal and can possibly perform dual functions in the formation and stabilization of AgNPs synthesized by the microbial method. These results reveal that proteins can bind to nanoparticle surfaces either through free amine groups or cysteine residues, which act as a capping agent and stabilize the particles (Lu¨ and Cui 2010). The presence of nitrate reductase in the filtrate is potentially the reason behind the reduction of silver nitrate to silver nanoparticles. The NR enzyme activity was also measured as

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270 nmol h-1 ml-1 for penicillium sp (HemathNaveen et al. 2010). Antibacterial activity of AgNPs In this study, the synthesized AgNPs exhibited a significant antibacterial effect on E. coli. The antibacterial activity of AgNPs may be due to the small size of the nanoparticles, which leads to increased membrane permeability. The mechanism of the bactericidal action of AgNPs is not well understood. Previous reports proposed that the antibacterial activity of AgNPs is related to the formation of ‘‘pits’’ in the cell wall of bacteria and consequently, leading to increased membrane permeability and resulting in cell death (Velmurugan et al. 2013). The increased inhibition action of AgNPs synthesized using the cell-free filtrate may be attributed to the narrower size distribution of these nanoparticles. Acknowledgments The authors wish to acknowledge Iranian Nanotechnology Initiative Council for partial financial support of this research projects.

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