Materials Letters 61 (2007) 1413 – 1418

www.elsevier.com/locate/matlet

Biological synthesis of silver nanoparticles

using the fungus Aspergillus flavus
N. Vigneshwaran ⁎, N.M. Ashtaputre, P.V. Varadarajan, R.P. Nachane,
K.M. Paralikar, R.H. Balasubramanya
Nanotechnology Research Group, Central Institute for Research on Cotton Technology, Adenwala Road, Matunga, Mumbai, 400 019, India
Received 29 May 2006; accepted 14 July 2006
Available online 2 August 2006

Abstract

The fungus, Aspergillus flavus when challenged with silver nitrate solution accumulated silver nanoparticles on the surface of its cell wall
in 72 h. These nanoparticles dislodged by ultrasonication showed an absorption peak at 420 nm in UV–visible spectrum corresponding to
the plasmon resonance of silver nanoparticles. The transmission electron micrographs of dislodged nanoparticles in aqueous solution showed the
production of reasonably monodisperse silver nanoparticles (average particle size: 8.92 ± 1.61 nm) by the fungus. X-ray diffraction spectrum of the
nanoparticles confirmed the formation of metallic silver. The Fourier transform infrared spectroscopy confirmed the presence of protein as
the stabilizing agent surrounding the silver nanoparticles. These protein-stabilized silver nanoparticles produced a characteristic emission peak at
553 nm when excited at 420 nm in photoluminescence spectrum. The use of fungus for silver nanoparticles synthesis offers the benefits of eco-
friendliness and amenability for large-scale production.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Aspergillus flavus; Biological synthesis; Biosorption; Electron microscopy; Luminescence; Silver nanoparticles

1. Introduction very much effective in this process. Various microbes are

Research in nanotechnology mostly deals with the synthesis
and stabilization of various nanoparticles by physical and
chemical processes. Currently, there is a growing need to
develop an eco-friendly process for nanoparticle synthesis and
hence the focus turned towards ‘green’ chemistry and
bioprocesses. Inspiration from nature comes through magneto-
tactic bacteria synthesizing magnetite nanoparticles [1], diatoms
synthesizing siliceous materials [2] and S-layer bacteria
producing gypsum and calcium carbonate layers [3]. The
nucleation and growth of these inorganic structures are mostly
controlled by the proteins and other biomacromolecules [4–6].
Silver nanoparticles, like its bulk counterpart, is an effective
antimicrobial agent against various pathogenic microorganisms.
Though various chemical and biochemical methods are being
explored for silver nanoparticles production [7], microbes are
⁎ Corresponding author. Tel.: +91 22 24127273; fax: +91 22 24130835.
E-mail address: nvw75@yahoo.com (N. Vigneshwaran).
0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.matlet.2006.07.042
known to reduce the metals, most of them are found to be
spherical particles as reported earlier [8–10]. The resistance
conferred by bacteria to silver is determined by the ‘sil’ gene in
plasmids [11] while a nitrate-dependent reductase and a shuttle
quinone extracellular process were reported for the reduction of
silver ions by several Fusarium oxysporum strains [12].
Bio-recovery of metals from solution, a process referred to as
“biosorption”, occurs by either active or passive mechanisms.
Active metal transformation processes require viable microbes,
enzymatically catalyzing the alteration of the metal, leading to
sequestration or concentration. One possible (passive) role of
the microorganisms is in providing a multitude of nucleation
centers; establishing conditions for obtaining highly disperse
nanoparticle systems. In addition, they slow down aggregation,
or entirely prevent it by immobilizing the particles, and
providing viscous medium [13]. Thus produced nanoparticles
have highly intricate architectures and are ordered during
assembly. In some cases, the particles have a well-defined shape
formed within a narrow size range and have orientational and
1414 N. Vigneshwaran et al. / Materials Letters 61 (2007) 1413–1418

geometrical symmetry [14]. Even though many biotechnolog-

ical applications such as the remediation of toxic metals employ
microorganisms such as bacteria and fungi, such microorgan-
isms are recently found as possible eco-friendly nanofactories
[15]. This study demonstrated the extracellular synthesis of
stable silver nanoparticles using the fungus, A. flavus.

2. Experimental

2.1. Preparation

The fungus culture, Aspergillus flavus (NCIM 650) was

obtained from National Chemical Laboratory culture collection,
Pune, India. All chemicals used were of analytical grade. A.
flavus was grown in yeast malt broth containing dextrose, 10 g/
L; peptic digest of animal tissue, 5 g/L; yeast extract, 3 g/L; and
malt extract, 3 g/L. The final pH was adjusted to 6.2 ± 0.2. The
flasks were incubated in the environmental shaker at 200 rpm at
37 °C. After 5 days of incubation, the mycelium was separated
by filtration and washed thrice with Milli-Q deionized water.
The washed mycelium (5 g fresh weight) was challenged with
100 mL of 1 mM silver nitrate solution (prepared in deionized
water) and incubated in shaker at 200 rpm in dark condition at
Fig. 2. X-ray diffraction pattern of the dried fungal mycelium with and without
37 °C. Simultaneously, a positive control of incubating the
the impregnated silver nanoparticles. In spectrum ‘a’ (recorded for pure metallic
fungus mycelium with deionized water and a negative control silver powder), the diffractions at 38.5°, 44° and 64.5° 2θ can be indexed to the
containing only silver nitrate solution were maintained under (111), (200) and (220) planes of the face-centered cubic (fcc) silver, respectively.
same conditions. Spectrum ‘b’ corresponds to the fungal mycelial mat without silver nitrate
treatment. Spectrum ‘c’ corresponds to the fungal mycelial mat after being
treated with silver nitrate. Here, the filled circles (●) indicate the peaks
2.2. UV–visible spectral analysis
corresponding to that of fungal mycelial mat (semi-crystalline chitin
micro fibrils), stars (⁎) indicate the peaks produced by metallic silver, while
Change in colour was observed in the silver nitrate solu- two new peaks (?) are formed due to the interaction of silver with the fungal
tion incubated with the A. flavus. The UV–visible spectra of cell wall.
this solution was recorded in Specord 50 ANALYTIKJENA®
Spectrophotometer, from 200 to 800 nm, at an interval of 24 h.
The silver nanoparticles dispersed in water by ultrasonication absorption at 420 nm was measured continuously, to determine
was kept at room temperature (37 °C) for 3 months and the their stability.

2.3. X-ray diffraction analysis

The fungal mycelium embedded with the silver nanoparticles

was freeze-dried, powdered and used for XRD analysis. The
spectra were recorded in Philips® automatic X-ray Diffractom-
eter with Philips® PW 1830 X-ray generator. The diffracted
intensities were recorded from 30° to 80° 2θ angles.
Simultaneously, the control fungal sample was also analyzed
as per the above-mentioned procedure. For standard, the silver
metal powder was subjected to XRD analysis.

2.4. SEM, TEM and electron diffraction analysis

The freeze-dried mycelial mats (positive control and silver

Fig. 1. UV–visible spectra of fungal filtrate as a function of time. Curve A
corresponds to positive control, while B, C, D and E correspond to that of
incubations with silver nitrate (1 mM) after 24 h, 48 h, 72 h and 96 h,
respectively. The peak at 420 nm corresponds to the surface plasmon resonance
of silver nanoparticles. The inset is a plot of the absorbance at 420 nm as a
function of time, corresponding to the spectra shown in the main figure.
nitrate treated sample) were mounted on specimen stubs with
double-sided adhesive tape and coated with gold/palladium in a
sputter coater and examined under Philips® XL 30 SEM at 12–
15 kV with a tilt angle of 45°. For TEM, a drop of aqueous
solution containing the silver nanomaterials was placed on the
carbon coated copper grids and dried under Infrared lamp.
 N. Vigneshwaran et al. / Materials Letters 61 (2007) 1413–1418 1415

3. Results and discussion

Biological systems, masters of ambient condition chemistry,

synthesize inorganic materials that are hierarchically organized from
the nano- to the macroscale [17]. In this work, the fungus A. flavus is
used for the synthesis of stable silver nanoparticles. While the fungal
mat incubated with deionized water (positive control) retained its
original colour, the silver nitrate treated fungus turned dark red after 72 h
due to the deposition of silver nanoparticles. This colour is primarily
due to the surface plasmon resonance of deposited silver nanoparticles.
In case of negative control (silver nitrate solution alone), no change in
colour was observed even after 10 days.
Fig. 1 shows the UV–visible spectra of the silver nitrate solutions
challenged with the fungus. While no absorption band was observed in
both controls (positive and negative), a characteristic surface plasmon
absorption band at 420 nm was observed at 24 h that attained the
maximum intensity after 72 h. After 72 h of incubation, no change in
intensity at 420 nm was observed indicating complete reduction of
silver ions (Fig. 1 — inset). Apart from this, two absorption peaks were
observed in the UV region corresponding to 220 and 280 nm. While the
peak at 220 nm may be due to the amide band, the other peak at 280 nm
may be attributed to the tryptophan and tyrosine residues present in the
protein that might have stabilized the nanoparticles [18]. The plasmon
bands are broad with an absorption tail in the longer wavelengths,

Fig. 3. Scanning electron micrographs of the fungal mycelium incubated with

deionized water (a) and fungal mycelium incubated with 1.0 mM silver nitrate
solution (b) for 72 h. Arrows in (b) shows the mold-like (1) and yeast-like (2)
morphologies of silver nitrate treated fungal mycelium. Scale bar corresponds to
20 μm. Inset shows the magnified view (scale bar in insets correspond to 1 μm).
The white arrows in inset show the silver nanoparticles deposited on the surface
of fungal mycelium incubated with silver nitrate solution.

Micrographs were obtained using a Philips® EM208 operating

at 200 kV.

2.5. FTIR analysis

The fungal filtrate containing the silver nanoparticles was

freeze-dried and the dried powder was diluted with potassium
bromide in the ratio of 1:100 and recorded the spectrum in
IRPrestige-21® Fourier Transform Infrared Spectrophotometer
using the diffuse reflectance accessory. The spectrum was sub-
jected to Kubelka–Munk correction to get back the transmission
spectrum.

2.6. Photoluminescence analysis

Photoluminescence spectra were recorded in Perkin Elmer

LS55® Spectrofluorimeter using 90° illumination. Initially,
prescan was performed [16] to find out the excitation and emis-
sion maxima for the silver nanoparticles. Based on the excitation
maximum (420 nm), emission scan was carried out in the range
of 500 to 750 nm. The excitation and emission slit widths were
kept at 2.5 and 5.0 nm, respectively. The excitation spectrum was
Fig. 4. TEM image (a) of silver nanoparticles produced by Aspergillus flavus.
recorded from 280 to 450 nm with emission scan wavelength at Bar represents 50 nm. Inset shows the electron diffraction pattern of the selected
553 nm. The entire scanning was done at the speed of 100 nm / area. Histogram (b) represents the particle size distribution determined from the
min. The data were analyzed using the FL Winlab® software. TEM micrographs, for about 1000 particles.
1416 N. Vigneshwaran et al. / Materials Letters 61 (2007) 1413–1418

Fig. 5. FTIR spectrum of silver nanoparticles synthesized by A. flavus, after 72 h. The bands seen at 3280 cm− 1 and 2924 cm− 1 correspond to the stretching vibrations
of primary and secondary amines, respectively; while their corresponding bending vibrations were seen at 1651 cm− 1 and 1548 cm− 1. The two bands observed at
1379 cm− 1 and 1033 cm− 1 can be assigned to the C–N stretching vibrations of aromatic and aliphatic amines, respectively.

which could be in principle due to the size distribution of the particles both mold-like (Fig. 3b — arrow 1) and yeast-like (Fig. 3b — arrow 2)
[19]. Since the varying intensity of the plasmon resonance depends on morphologies.
the cluster size [20], the number of particles cannot be related linearly Transmission electron microscopy has provided further insight
to the absorbance intensities. The stability of the synthesized silver into the morphology and size details of the silver nanoparticles. A
nanoparticles was studied by measuring its intensity at 420 nm over a representative TEM image recorded from the silver nanoparticles so-
period of 3 months in room temperature. No significant change in the lution is shown in Fig. 4a. In general, the particles are isotropic (i.e.,
intensity was observed which proved its stability over a period of low aspect ratio) in shape and reasonably monodisperse. All the
3 months (data not shown). nanoparticles are well separated and no agglomeration was noticed.
The diffraction pattern in Fig. 2a corresponds to that of pure silver From the TEM images we obtained the average size of silver
metal powder while Fig. 2b and c correspond to that of control fungal nanoparticles of 8.92 ± 1.61 nm. The selected area diffraction pattern
mycelial mat and silver nanoparticles embedded fungal mycelial mat, (Fig. 4a — inset) confirms the ‘fcc’ crystalline structure of metallic
respectively. In Fig. 2a, the diffractions at 38.5°, 44° and 64.5° 2θ can silver. The histogram of the nanoparticles size distribution (Fig. 4b)
be indexed to the (111), (200) and (220) planes of the face-centered was obtained by measuring the size of about 1000 nanoparticles.
cubic (fcc) silver, respectively. The diffraction peaks in Fig. 2b are Fig. 5 shows the FTIR spectrum recorded from the freeze-dried
primarily due to the semi-crystalline chitin micro fibrils present in the powder of silver nanoparticles, formed after 72 h of incubation with the
fungal cell wall that are embedded in an amorphous matrix of β-glucan
[21]. In Fig. 2c, the diffraction peaks of both silver metal and fungal
cell wall were seen; in addition, two new peaks were formed due to the
interaction of silver nitrate with fungal cell wall matrix. Also, the
broadening of the X-ray peaks is primarily due to the small particle size
of silver. The lattice constant calculated from this pattern (Fig. 2c) was
4.087 Å, a value in agreement with the literature report (a = 4.086 Å,
Joint Committee on Powder Diffraction Standards file no. 04-0783).
Fig. 3a shows the scanning electron micrograph of the fungal
mycelium treated as positive control (incubated with deionized water
for 72 h) while Fig. 3b shows fungal mycelium treated with 1.0 mM
silver nitrate solution for 72 h. The insets show the magnified view of
corresponding micrographs. The surface deposited silver nanoparticles
(indicated by white arrows in the inset of Fig. 3b) are seen clearly at a
higher magnification in the silver nitrate treated fungal mycelium. Fig. 6. Photoluminescence spectra of silver nanoparticles synthesized by A.
Dimorphism is reported to be dependent on temperature and carbon flavus. Excitation spectrum (a) shows the maximum at 420 nm similar to its
dioxide concentration [22]. Here, the dimorphism was observed when absorption peak. Emission maximum (b) was obtained at 553 nm while excited
the fungus was challenged with the silver nitrate solution, exhibiting at 420 nm.
 N. Vigneshwaran et al. / Materials Letters 61 (2007) 1413–1418
fungus. The amide linkages between amino acid residues in proteins
give rise to the well-known signatures in the infrared region of the
electromagnetic spectrum. The bands seen at 3280 cm− 1 and 2924 cm− 1
were assigned to the stretching vibrations of primary and secondary
amines, respectively; while their corresponding bending vibrations
were seen at 1651 cm− 1 and 1548 cm− 1, respectively. The two bands
observed at 1379 cm− 1 and 1033 cm− 1 can be assigned to the C–N
stretching vibrations of aromatic and aliphatic amines, respectively. The
overall observation confirms the presence of protein in the samples of
silver nanoparticles. It is reported earlier that proteins can bind to
nanoparticles either through free amine groups or cysteine residues in
the proteins [23,24] and via the electrostatic attraction of negatively
charged carboxylate groups in enzymes present in the cell wall of
mycelia [10] and therefore, stabilization of the silver nanoparticles by
protein is a possibility.
Study of photoluminescence (PL) spectra is an effective method to
evaluate the optical property of silver nanoparticles as photonic
materials. Fig. 6 shows the typical PL spectra of silver nanoparticles
suspended in water, measured at room temperature. The excitation peak
(Fig. 6a) was observed at 420 nm while the emission peak (Fig. 6b) was
observed at 553 nm with very broad base. We have earlier reported
similar PL spectra of silver nanoparticles stabilized by soluble starch
[25]. Since the silver nanoparticles are known to enhance the fluores-
cence emission intensity and photostability of nearby fluorophores
[26], the emission peak intensity (at 553 nm) of the organic matrix
(analyzed to be protein by FTIR) bound to silver nanoparticles might
have gotten enhanced.
Klaus et al. [6] reported the formation of triangular, hexagonal and
spheroidal Ag-containing nanoparticles at different cellular binding
sites of the bacterium, Pseudomonas stutzeri AG259. Though the
mechanism of silver resistance offered by bacteria using silver binding
protein [11] is well studied, extraction and purification posed a problem
in large-scale production. Hence, it is advantageous to work with fungus
for the large-scale production and purification of silver nanomaterials.
Enzymes like silver reductase [27] and the amino acids (present in
proteins/enzymes) such as aspartic acid and tryptophan [24] have been
reported in various microbial systems responsible for the reduction of
metal ions. Though the earlier reports demonstrated the biosorption of
heavy metal ions by A. niger [28], Phaenerochaete chrysosporium ME-
446 [29] and other fungal biomass [30–32], the extraction and
characterization of the biosorbed metal ions were not studied. Present
study indicates the avenues towards the preparation of nanostructured
materials with uniform spherical structures using the fungus, A. flavus.
A model Gram-negative bacterium E. coli, when treated with silver
nanoparticles, showed the formation of “pits” in its cell wall with the
accumulation of nanoparticles in the cell membrane, leading to
increased permeability and cell death [33]. Hence, the synthesized
nanoparticles could be of immense use as antimicrobial agent.
4. Conclusions
Biosorption mechanism of metal ions by microorganisms
includes ion exchange, precipitation and complexation. Reduc-
tion and surface accumulation of metals may be a process by
which microorganisms protect themselves from the toxic effects
of metallic ions. This study shows the biosorption of silver in
the form of nanoparticles by the fungus A. flavus. These nano-
particles are found to be stable in water for more than 3 months
that can be attributed to surface binding of stabilizing materials
secreted by the fungus. The possibility of protein as a stabilizing
material in silver nanoparticles is revealed by FTIR analy-
1417
sis. The average size of the nanoparticles was estimated to be
8.92 ± 1.61 nm. These silver nanoparticles are found to have
characteristic absorption peak at 420 nm and emission peak at
553 nm. This process of nanoparticle production is eco-friendly
as it is free from any solvent or toxic chemicals; also easily
amenable for large-scale production.
Acknowledgements
We are grateful to Dr. S. Sreenivasan, Director, CIRCOT for
helpful discussions and Mr. Virendra Prasad, Mr. Hadge, Mr.
Sekar and Mr. Vivekanandan for their help rendered in carrying
out the experiments. We acknowledge the support extended by
Sophisticated Analytical Instrumentation Facility, IIT Mumbai
in analyzing the samples by TEM.
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