Colloids and Surfaces B: Biointerfaces 47 (2006) 160–164

Extracellular biosynthesis of silver nanoparticles

using the fungus Aspergillus fumigatus
Kuber C. Bhainsa, S.F. D’Souza ∗
Enzyme and Microbial Technology Section, Nuclear Agriculture and Biotechnology Division,
Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India
Received 13 September 2005; received in revised form 25 November 2005; accepted 29 November 2005
Available online 18 January 2006

Abstract
Development of reliable and eco-friendly process for synthesis of metallic nanoparticles is an important step in the filed of application of
nanotechnology. One of the options to achieve this objective is to use natural processes such as use of biological systems. In this work we
have investigated extracellular biosynthesis of silver nanoparticles using Aspergillus fumigatus. The synthesis process was quite fast and silver
nanoparticles were formed within minutes of silver ion coming in contact with the cell filtrate. UV–visible spectrum of the aqueous medium
containing silver ion showed a peak at 420 nm corresponding to the plasmon absorbance of silver nanoparticles. Transmission electron microscopy
(TEM) micrograph showed formation of well-dispersed silver nanoparticles in the range of 5–25 nm. X-ray diffraction (XRD)-spectrum of the
silver nanoparticles exhibited 2θ values corresponding to the silver nanocrystal. The process of reduction being extracellular and fast may lead to
the development of an easy bioprocess for synthesis of silver nanoparticles.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Extracellular biosynthesis; Silver; Nanoparticles; Aspergillus fumigatus; Nanocrystal

1. Introduction
Increased industrialization and urbanization has damaged the
environment by introducing a number of harmful and unwanted
substances. Microorganisms have been exposed to a variety of
such pollutants in the environment (including water and soil).
Of which, metal ion, being non-biodegradable and persistent
in nature often causes toxicity and inhibits microbial growth.
However, even at high metal ion concentration microorgan-
isms can survive and grow due to their ability to fight the
metal stress. The mechanisms include: efflux systems; alteration
of solubility and toxicity via reduction or oxidation; biosorp-
tion; bioaccumulation; extracellular complexation or precipi-
tation of metals and lack of specific metal transport systems
[1,2]. These metal–microbe interactions have important role
in several biotechnological applications including the fields of
bioremediation, biomineralization, bioleaching and microbial
corrosion. However, it is only recently that microorganisms have
been explored as potential biofactory for synthesis of metal-
∗ Corresponding author. Tel.: +91 22 25593632; fax: +91 22 25505151.
E-mail address: sfdsouza@apsara.barc.ernet.in (S.F. D’Souza).
lic nanoparticles such as cadmium sulfide, gold and silver [3].
Though there are several physical and chemical methods for
synthesis of metallic nanoparticles, to achieve the objective of
developing simple and eco-friendly technology researchers in
this field have turned to biological systems. Holmes et al. have
shown that the bacteria, Klebsiella aerogenes when exposed to
cadmium ions resulted in intracellular formation of CdS parti-
cles in the range of 20–200 nm [4]. Sastry et al. [3] have reported
that fungus Verticillium sp. and Fusarium oxysporum, when
exposed to gold and silver ions, reduced the metal ion fairly
rapidly and formed respective metallic nanoparticles. Klaus et
al. have observed that the Pseudomonas stutzeri AG259, iso-
lated from a silver mine, when placed in a concentrated solution
of silver nitrate produced silver nanoparticles of well-defined
size and distinct morphology within the periplasmic space of the
bacteria [5]. Synthesis of nanoparticles was found to be intracel-
lular in all the examples given above except F. oxysporum. This
makes the job of downstream processing difficult and beats the
purpose of developing a simple and cheap process. Therefore,
in this study focus has been given to development of an extra-
cellular process. Filamentous fungi are very good candidate for
such processes and furthermore, these biomasses are easy to
handle. Silver nanoparticles have several important applications

0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2005.11.026
 K.C. Bhainsa, S.F. D’Souza / Colloids and Surfaces B: Biointerfaces 47 (2006) 160–164 161

in the field of biolabelling, sensors, antimicrobial agents and

filters [6]. Considering all these factors, an extensive screen-
ing study was carried out involving several filamentous, fungal
biomasses to identify a biological system for the extracellular
biosynthesis of silver nanoparticles. Preliminary results (data not
shown) indicated Aspergillus fumigatus as a potential candidate
for this purpose. Hence, in the present work we have inves-
tigated extracellular biosynthesis of silver nanoparticles using
the filamentous fungus, A. fumigatus. The study includes kinet-
ics of synthesis, spectroscopic and microscopic characterization
of the silver nanoparticles. It was the same fungal biomass that
had exhibited very good binding capacity for uranium reported
earlier by our group [7].

2. Materials and methods

A. fumigatus (NCIM 902) was obtained from National Chem-

ical Laboratory, Pune, India and maintained on potato dextrose
agar slants. To prepare biomass for biosynthesis studies the fun-
gus was grown aerobically in a liquid media containing (g/l)
KH2 PO4 , 7.0: K2 HPO4 , 2.0; MgSO4 ·7H2 O, 0.1; (NH4 )2 SO4 ,
1.0; yeast extract, 0.6; and glucose, 10.0. The flasks were inoc-
ulated, incubated on orbital shaker at 25 ◦ C and agitated at
150 rpm. The biomass was harvested after 72 h of growth by
sieving through a plastic sieve, followed by extensive washing
with distilled water to remove any medium component from the
biomass. Typically 20 g of biomass (fresh weight) was brought
in contact with 200 ml of Milli-Q deionized water for 72 h at
25 ◦ C in an Erlenmeyer flask and agitated in the same condition
as described earlier. After the incubation, the cell filtrate was
obtained by passing it through Whatman filter paper no. 1. For
synthesis of silver nanoparticles, AgNO3 , 1 mM final concentra-
tion was mixed with 50 ml of cell filtrate in a 250 ml Erlenmeyer
flask and agitated at 25 ◦ C in dark. Control (without the silver
ion, only biomass) was also run along with the experimental
flask. Sample of 1 ml was withdrawn at different time inter-
vals and the absorbance was measured at a resolution of 1 nm
using UV–visible spectrophotometer (UV–vis UV4, UNIVAM
Ltd., UK). After 72 h of incubation the cell filtrates containing
nanoparticles were used for transmission electron microscopy
(TEM). The silver nanoparticles film was formed on carbon-
coated copper TEM grids and analyzed by transmission electron
microscopy (TECNAI 120) at a voltage of 120 kV. Drop-coated
film of cell filtrate containing nanoparticles on silica were sub-
jected to X-ray diffraction (XRD) analysis using Phillips PW
1710 operating in transmission mode, at 30 kV, 20 mA with Cu
K␣ radiation.
3. Results
The detailed study on extracellular biosynthesis of silver
nanoparticles by the Aspergillus biomass was carried out in this
work. The fungal biomass after incubation for 72 h with Milli-Q
water was separated by filtration. The cell filtrate of the fila-
mentous fungus incubated with silver ion at the beginning of
the reaction is represented in Fig. 1a. The flasks being incubated
in the dark in an environmental shaker showed gradual change
Fig. 1. Cell filtrate (72 h) of Aspergillus fumigatus with silver ion (1 mM): (a)
at the beginning of the reaction and (b) after 24 h of reaction. “For interpretation
of the references to color in this figure legend, the reader is referred to the web
version of the article”.
in color of the medium to brown, with intensity increasing dur-
ing the period of incubation. The color of the medium changed
to intense brown after 24 h of incubation, which has been pre-
sented in Fig. 1b. The change in color of the medium was noted
by visual observation. The solution remained as hydrosol and no
precipitation was observed even after 72 h of incubation. Control
(without silver ion) showed no change in color of the cell filtrate
when incubated in the same environmental condition (data not
shown).
The light absorption pattern of the cell filtrate was monitored
in the range of 200–800 nm using a UV–visible spectropho-
tometer. Short term incubation carried out within few minutes
of introducing silver ions into the flask containing the cell fil-
trate resulted in the spectrum of increasing intensity in the range
of 350–600 nm (Fig. 2a). UV–visible spectrum of the medium
was also recorded to study the change in light absorption pro-
file of the medium and change in intensity of the brown color
during long term incubation (72 h) (Fig. 2b). The UV–visible
spectra (Fig. 2a and b) recorded at different time intervals
showed increased absorbance with increasing time of incuba-
tion at around 420 nm. Absorption spectrum (200–800 nm) at
the beginning of the reaction showed three distinct peaks, cen-
tering around 220, 280 and 340 nm. The absorption spectrum
showed increase in intensity over a broad spectrum in the range
of 350–600 nm within 10 min of contact time while the remain-
162 K.C. Bhainsa, S.F. D’Souza / Colloids and Surfaces B: Biointerfaces 47 (2006) 160–164
Fig. 2. UV–visible spectrum of aqueous medium containing cell filtrate and
silver ion (1 mM): (a) short incubation time and (b) long incubation time.
ing part of the absorption spectrum in the range of 200–350
and 600–800 nm remained almost unaffected. The intensity in
the range of 350–600 nm continued to increase with increasing
incubation time till 60 min. The absorbance at around 220 and
280 nm maintained the same intensity throughout the incubation
time. The nature of the absorbance spectrum (Fig. 2b) represent-
ing absorbance profile of the medium for longer incubation time
(0–72 h) showed trend similar to Fig. 2a; however, the intensity
of absorbance in the range of 350–600 nm increased significantly
resulting in gradual appearance of a peak at 420 nm. A repre-
sentative TEM micrograph of silver nanoparticles obtained after
72 h of incubation is presented in Fig. 3. The micrograph showed
nanoparticles with variable shape, most of them present in spher-
ical in nature with some others having occasionally triangular
shape. The size of the particle ranged from 5 to 25 nm. Major-
ity of the silver nanoparticles were scattered with only a few
of them showing aggregates of varying sizes as observed under
TEM. Further studies were carried out using X-ray diffraction
to confirm the crystalline nature of the particle and the XRD pat-
tern obtained has been represented in Fig. 4. The XRD pattern
Fig. 3. TEM micrograph of silver particles synthesized by A. fumigatus (scale
bar: 50 nm).
Fig. 4. XRD pattern of nanoparticle film on Si surface obtained from cell filtrate
of A. fumigatus.
showed four intense peaks in the whole spectrum of 2θ value
ranging from 20 to 80.
4. Discussion
Silver nanoparticles have many important applications that
include: spectrally selective coating for solar energy absorption
and intercalation material for electrical batteries [5], as optical
receptors [8], polarizing filters, catalysts in chemical reaction,
biolabelling [9] and as antimicrobial agents [10]. Application of
silver nanoparticles in these fields is dependent on the ability to
synthesize particles with different chemical composition, shape,
size, and monodispersity. Further, the particles should be chem-
ically stable without undergoing degradation such as partial
oxidation or undesired sintering. There are several physical and
chemical methods for synthesis of metallic nanoparticles that
are followed by the material scientists currently [11]. However,
development of simple and eco-friendly synthetic route would
help promoting further interest in the synthesis and application
 K.C. Bhainsa, S.F. D’Souza / Colloids and Surfaces B: Biointerfaces 47 (2006) 160–164
of metallic nanoparticles. In this respect, nature has provided
exciting possibilities of utilizing biological systems for this
purpose. This comes from the fact that microorganisms while
interacting with metal ions have shown to reduce the ions into
metallic particles. Both bacteria and fungi have shown ability
to reduce metal ions to form metallic nanoparticles. However,
it would be advantageous if a fungus is used for the develop-
ment of a process keeping in mind handling of the biomass and
down stream processing of the nanoparticles [3]. In this regard
extracellular biosynthesis of silver nanoparticles achieved in this
study using A. fumigatus may prove to be an important step in
the right direction.
It was observed that upon addition of the silver ion (1 mM)
into the flask containing the cell filtrate, the color of the medium
changed very rapidly to brown (Fig. 1b). The appearance of the
brown color was an indication of formation of colloidal silver
particles in the medium. The brown color of the medium could
be due to the excitation of surface plasmon vibrations, typical
of the silver nanoparticles [12]. As shown in Fig. 1b, the silver
nanoparticles were synthesized in the extracellular cell filtrate
of the filamentous fungus. This offers a great advantage over an
intracellular process of synthesis from the application point of
view. Since the nanoparticles formed inside the biomass would
have required additional step of processing for release of the
nanoparticles from the biomass by ultrasound treatment or by
reaction with suitable detergents.
In the short term incubation studies, the result (Fig. 2a)
showed synthesis of silver nanoparticles within 10 min of sil-
ver ions coming in contact with the filtrate. To the best of our
knowledge, this is the first report showing such rapid synthesis
of nanoparticles within minutes of contact time using a biolog-
ical system. In a previous report, Ahmad et al. [12] have shown
extracellular synthesis of silver nanoparticles within hours of
contact time. Generally, other physical and chemical processes
of synthesis of nanoparticles are fast and method of synthesis
being reported in this study could comparable to them.
The absorption spectrum of the medium containing the sil-
ver ions showed increased intensity at 420 nm and after 1 h of
incubation a clear peak was observed (Fig. 2b). The increase
in intensity could be due to increasing number of nanoparticles
formed as a result of reduction of silver ions present in the aque-
ous solution. The fact that silver nanoparticles peak remained
close to 420 nm even after 72 h of incubation indicates that the
particles were well dispersed in the solution and there was not
much aggregation. Monodispersity is an important character-
istic of the nanoparticles. Gold nanoparticles with very good
monodispersity have been reported by Ahmad et al. [13] using
Thermonospora sp. The nanoparticles present in the aqueous
medium were quite stable, even up to 4 months of incubation
at 25 ◦ C. This is an important aspect of synthesis of nanoparti-
cles, since the lack of sufficient stability of many nanoparticles
preparation has to some extent impeded the development of the
real world applications of nanomaterials.
The result obtained from the TEM study (Fig. 3) gives a clear
indication regarding the shape and size of the nanoparticles. As
shown in the picture, majority of the particles were spherical in
shape and appeared to be reasonably monodispersed. The sizes
163
of the silver particles were found to be in the range of 5–25 nm.
Such variation in shape and size of nanoparticles synthesized
by biological systems is common. However, if the process of
silver nanoparticles is to be a viable alternative to the current
chemical method followed, then greater control over particle size
and polydispersity would be required. It is important to know
the exact nature of the silver particles formed and this can be
achieved by measuring the XRD-spectrum of the samples. The
XRD-spectrum measured in this case resulted in four intense
peaks (Fig. 4). The four intense peaks observed in the spectrum
agree to the Braggs’s reflection of silver nanocrystals reported in
literature [14]. This further confirms that the silver nanoparticles
formed in the extracellular filtrate are present in the form silver
nanocrystals.
Application of the biological systems for synthesis of silver
nanoparticles has already been reported earlier [3,5]. However,
the exact reaction mechanism leading to the formation of sil-
ver nanoparticles by all these organisms is yet to be elucidated.
Ahmad et al. [13] have reported that certain NADH dependent
reductase was involved in reduction of silver ions in case of
F. oxysporum. In this study, the UV–vis spectrum study pro-
vides some clues regarding the mechanism of synthesis of silver
nanoparticles. There were two absorbance peaks found in the
UV range corresponding to 220 and 280 nm. While the peak
at 220 nm may be due to absorption by amide bond, the other
peak at 280 nm may be attributed to the tryptophan and tyrosine
residues present in the protein. This indicates secretion of some
proteinic components into the medium by the fungal biomass
which might play important role in the reduction of the metal
ions in the form of nanoparticles. Consequently the proteins also
may bind to the nanoparticles and enhance the stability. Cur-
rently, we are concentrating and separating the proteins released
by the fungus to determine its exact role in the reduction pro-
cesses.
In conclusion, the filamentous fungus, A. fumigatus has
shown potential for extracellular synthesis of fairly monodis-
persed, silver nanoparticles in the range of 5–25 nm. The kinetics
of silver nanoparticles synthesis using the cell filtrate indicates
that the rapid synthesis of nanoparticles would be suitable for
developing a biological process for mass scale production. Fur-
thermore, the extracellular synthesis would make the process
simpler and easier for downstream processing. In future, it would
be important to understand the biochemical and molecular mech-
anism of the synthesis of the nanoparticles by the cell filtrate in
order to achieve better control over size and polydispersity of
the nanoparticles.
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