Process Biochemistry 42 (2007) 919–923

www.elsevier.com/locate/procbio

Short communication
Rapid synthesis of silver nanoparticles using culture supernatants
of Enterobacteria: A novel biological approach
Ahmad R. Shahverdi a,*, Sara Minaeian a,b, Hamid Reza Shahverdi c,
Hossein Jamalifar a, Ashraf-Asadat Nohi b
a
Department of Pharmaceutical Biotechnology and Medical Nanotechnology Research Center, Faculty of Pharmacy,
Medical Sciences/University of Tehran, Tehran, Iran
b
Department of Material Science, Faculty of Engineering, Tarbiat Modares University, Tehran, Iran
c
Division of Microbiology, Azad University of Science and Research Units, Tehran, Iran
Received 4 November 2006; received in revised form 14 February 2007; accepted 15 February 2007

Abstract
The development of reliable processes for the synthesis of silver nanomaterials is an important aspect of current nanotechnology research.
Reports on the cell-associated biosynthesis of silver nanoparticles using microorganisms have been published, but these methods of synthesis are
rather slow. In this paper, we report on the rapid synthesis of metallic nanoparticles of silver using the reduction of aqueous Ag+ ion using the
culture supernatants of Klebsiella pneumonia, Escherichia coli, and Enterobacter cloacae (Enterobacteriacae). The synthetic process was quite
fast and silver nanoparticles were formed within 5 min of silver ion coming in contact with the cell filtrate. Through a limited screening process
involving a number of common microorganisms, we observed that the culture supernatants of different bacteria from Enterobacteriacae were
potential candidates for the rapid synthesis of silver nanoparticles; further, we revealed that this method of synthesis requires far less time than
previously published biological methods. Our investigation also showed that piperitone can partially inhibit the reduction of Ag+ to metallic silver
nanoparticles by Enterobacteriacae.
# 2007 Elsevier Ltd. All rights reserved.

Keywords: Silver nanoparticles; Escherichia coli; Enterobacter cloacae; Klebsiella pneumonia; Nitroreductase; Synthesis

1. Introduction
The development of biologically inspired experimental
processes for the synthesis of nanoparticles is evolving into an
important branch of nanotechnology [1,2]. Biologically synthe-
sized silver nanoparticles could have many applications; for
example, they might be used as spectrally selective coatings for
solar energy absorption and intercalation material for electrical
batteries, as optical receptors, as catalysts in chemical reactions,
for biolabelling, and as antimicrobials [3–5]. There are several
reports in the literature on the cell-associated biosynthesis of
silver nanoparticles using several microorganisms, particularly
Fusarium oxyporum [1,5–7]. The cell mass of F. oxyporum, and
the leached components from these fungi cells [1,6], has been
reported to reduce silver ion to silver nanoparticles.
* Corresponding author. Tel.: +98 21 66482706; fax: +98 21 66461178.
E-mail address: shahverd@sina.tums.ac.ir (A.R. Shahverdi).
1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2007.02.005
Also, for the past few years, various rapid chemical methods
have been developed for the synthesis of silver nanoparticles
[2,8]. Different natural products such as monosaccharide or
plant extracts have been used as reducing agents during these
studies. However, microbiological methods generate the
nanoparticles at a much slower rate than that observed when
plant extracts or other chemical reducing agents are used. In
studies focussing on the synthesis of silver nanoparticles using
the cell mass of bacteria [9–11] and fungi [6] or their leached
cell components [1], the time required to complete the reaction
(i.e., for a complete reduction of the metal ions) ranged from 24
to 120 h; this lengthy reaction is the one major drawback of
biological synthesis and must be corrected if it is to compete
with chemical procedures for nanoparticle synthesis [2].
Biological nanoparticle synthesis would have greater commer-
cial viability if the nanoparticles could be synthesized more
rapidly in the reaction vessels. To this end, we have screened the
culture supernatants of different microorganisms and have
observed the rapid synthesis of silver nanoparticles by treating
920 A.R. Shahverdi et al. / Process Biochemistry 42 (2007) 919–923
the aqueous silver nitrate with culture supernatants of different
strains of Enterobacteria.
2. Materials and methods
2.1. Bacteria
The test strains were: Bacillus cereus, Bacillus subtilis, Escherichia coli,
Enterobacter cloacae, Klebsiella pneumonia, Lactobacillus acidophilus, Sta-
phylococcus aereus, and Pseudomonas aeroginosa (from our collection);
Candida albicans (ATCC 14053); Aspergillus niger (PIM).
2.2. Preparation of supernatants
Muller-Hinton broth (MHB) was prepared, sterilized, and inoculated with a
fresh batch of test strains. The culture flasks were incubated for 24 h at 35 8C for
bacteria, at 30 8C for 24 h for C. albicans, and at 25 8C for 3 days for A. niger.
After the incubation period, the cultures were centrifuged at 12,000 rpm and
their supernatants were used for further experiments. To investigate its effect on
the synthesis of silver nanoparticles a sub-inhibitory concentration (400 mg/ml)
of piperitone (3-methyl-6-(1-methylethyl)-2-cyclohexen-1-one) was added to
MHB. Piperitone was kindly provided by Charabot, 06332 Grasse, France.
2.3. Synthesis of silver nanoparticles
Aqueous silver nitrate solution (10 3 M) was separately added to reaction
vessels containing different supernatants (1%, v/v) and the resulting mixture
allowed to stand for 5 min at room temperature. The reduction of the Ag+ ions
by biological species in the solutions was monitored by sampling the aqueous
component (2 ml) and measuring the UV–vis spectrum of solutions. The UV–
vis spectra of these samples were measured on a Cecil model 9200 Ultraviolet-
visible spectrophotometer operated at a resolution of 1 nm. Particle-size dis-
tributions of the samples were also obtained using Zetasizer Nano ZS (Malvern
Instruments, Southborough, UK). Furthermore, the silver nanoparticles were
characterized by transmission electron microscopy (model EM 208 Philips) and
energy-dispersive spectroscopy (EDS).
3. Results and discussion
3.1. Silver reduction
The formation of silver nanoparticles by the culture
supernatants of different common microorganisms was
investigated. The appearance of a yellowish-brown color in
the reaction vessels suggested the formation of silver
nanoparticles [1]. Fig. 1 shows two conical flasks with the
supernatant of K. pneumonia (from Enterobacteriaceae) before
(left flask) and after reaction with Ag+ for 5 min (right flask).
Before reaction, the silver containing solution (left flask) is
colorless but changes to a brownish color on completion of the
reaction (right flask). Also the supernatants that were derived
from cultures of other Enterobacteriaceae (E. coli and E.
cloacae) changed the solutions to a brownish color after 5 min
of reaction with Ag+. No brownish color was observed from the
supernatants derived from other test strains under the same
conditions.
Several hydroquinones with excellent redox properties were
reported that could be act as electron shuttle in metal reductions
[5,12]. Thus, it was evident that electron shuttles or other
reducing agents released by Enterobacteria are capable of
reducing silver ions to silver nanoparticles. On the other hand,
Fig. 1. Solutions of silver nitrate (1 mM) before (left) and after (right) exposure
to the culture supernatant of Klebsiella pneumonia (Enterobacteriacae).
the reduction of silver ions also occurs in the absence of
bacterial cells. This clearly indicates that the reducing agents
that are released into the cultures of the aforementioned
Enterobacteria are involved in the reduction process.
3.2. UV–visible spectroscopy
The silver nanoparticles were characterized by UV–visible
spectroscopy. This technique outlined above has proved to be
very useful for the analysis of nanoparticles [13–15]. As
illustrated in Fig. 2(A), a strong, broad peak located between
420 and 430 nm was observed for the silver nanoparticles
prepared using the Enterobacteria group. Observation of this
peak, assigned to a surface plasmon, is well-documented for
various metal nanoparticles with sizes ranging from 2 to
100 nm [13–15]. The strong surface plasmon resonance of K.
pneumonia, E. coli, and E. cloacae was centered at ca. 430, 419,
and 420 nm, respectively. When exposed to the Enterobacteria
supernatants, the aqueous silver ions are rapidly reduced in the
solution. The largest plasmon resonance peak was observed for
K. pneumonia; therefore, this test strain was selected for further
experiments. The surface plasmon resonance was not observed
for culture supernatants of other microorganisms tested during
this investigation. Fig. 2(B) shows the UV–visible spectra
recorded from the aqueous silver nitrate-culture supernatant of
the K. pneumonia reaction medium as a function of time of
reaction. The strong resonance centered at about 430 nm is
clearly seen and increases with time. The sharp drop in reaction
time, from a few days to a few minutes, observed for the culture
supernatant of Enterobacteriacae is a highly significant
advance toward achieving the goal of developing a rapid
method for silver nanoparticle synthesis.
3.3. Particle size and its chemical composition
Fig. 3 shows a representative TEM image recorded from the
drop-coated film of the silver nanoparticles synthesized by
treating the silver nitrate solution with culture supernatants of
 A.R. Shahverdi et al. / Process Biochemistry 42 (2007) 919–923 921

Fig. 2. UV–vis spectra of Ag colloids. (A) Spectra recorded after the addition of culture supernatants of different test strains (1 ml) to 100 ml of silver nitrate solution
(1 mM). The curves are recorded after a period of 5 min. The UV–vis spectra for other microorganisms are not shown. (B) Spectra recorded as a function of time for a
1 mM aqueous solution of silver nitrate that was reacted with culture supernatants of K. pneumonia. The test strain was cultivated in Muller-Hinton broth and was
incubated at 35 8C for 24 h. After the incubation period, the culture was centrifuged at 12,000 rpm and its supernatants used to reduce the silver nitrate solution.

Fig. 3. Transmission electron micrograph (left picture) recorded from a region of a drop-coated film of silver nitrate solution treated with the culture supernatant of K.
pneumonia for 5 min (scale bars correspond to 50 nm). The TEM image shows at least two different areas, one with higher contrast due to the silver nanoparticles and
other with lower contrast probably due to other micro (or even nano) crystals originating from insoluble Ag salts. The particle size distribution histogram obtained
using a Zetasizer Nano ZS (Malvern Instruments, Southborough, UK) is shown on the right.
922 A.R. Shahverdi et al. / Process Biochemistry 42 (2007) 919–923
Fig. 4. EDS spectrum of silver nanoparticles. Different X-ray emission peaks
are labeled. Strong signals from the atoms in the nanoparticles are observed,
while weaker signals from Na, Cl, Ca and Fe atoms are also visible.
K. pneumonia. The particle size histogram of silver particles
(right illustration in Fig. 3) shows that the particles range in size
from 28.2 to 122 nm and possess an average size of 52.5 nm. In
the analysis of the silver nanoparticles by energy dispersive
spectroscopy (EDS), the presence of elemental silver signal was
confirmed (Fig. 4). The Ag nanocrystallites display an optical
absorption band peaking at 3 keV, which is typical of the
absorption of metallic Ag nanocrystallites due to the surface
plasmon resonance [16].
In addition, the TEM image shows at least two different areas,
one with higher contrast due to the silver nanoparticles and other
with lower contrast probably due to other micro (or even nano)
crystals originating from insoluble Ag salts. Several anions such
as Cl , SO42 and MoO42 which are present in the supernatants
can easily form poorly soluble salts with Ag+ cations. This is also
suggested by the EDS spectrum shown in Fig. 4 in which, among
other metals (Na, Ca, and Fe), a peak attributed to Cl in the silver
nanoparticles is present. The stability of silver nanoparticles has
not been analyzed during this study. Nevertheless, the stability of
these particles may be unstable after 5 min and more work is
needed to understand this phenomenon and to increase the
stability for a longer time scale.
3.4. The inhibitory effect of piperitone
NfsA, the major oxygen-insensitive nitroreductase of
Enterobacteriacae, is a flavoprotein that is able to reduce
nitro groups in many different nitroaromatic compounds under
Fig. 5. The structure of piperitone (3-methyl-6-(1-methylethyl)-2-cyclohexen-
1-one).
Fig. 6. UV–vis spectra recorded after the addition of culture supernatants of K.
pneumonia (1 ml) to silver nitrate solution (1 mM; 100 ml) with and without
piperitone (400 mg/ml). The curves were recorded after standing for 5 min.
aerobic conditions [17]. This enzyme is also responsible for
reduction of chromate to less soluble and less toxic Cr(III) [18].
Recently, natural products such as piperitone (Fig. 5) and
menthol have been shown to have inhibitory effects on
nitroreduction activity of Enterobacteriacae [19,20]. Enter-
obacteria as Gram-negative bacteria are usually associated
with intestinal infections, but can be found in almost all natural
habitats [17]. Our preliminary investigation showed that
piperitone can partially inhibit the reduction of Ag+ to metallic
silver nanoparticles by K. pneumoniae (Fig. 6) and other
different strains of Enterobacteriaceae (data not shown).
Certainly, in our case, Ag(0) reduction was mainly due to a
conjugation between the electron shuttles with the reductase
participation. Thus, it appears that nitroreductase enzymes may
be involved in silver ion reduction process.
4. Conclusions
Here, we described a low cost approach for reducing silver
nitrate solution to form nanoparticles. Although several reports
on the biological synthesis of silver nanoparticles have been
published, those reports have focused on the use of bacteria and
fungi cell masses for the synthesis [1,5–7]. This process is slow
because of the time required to complete the silver nitrate
reduction. This is the first report focussing on the use of culture
supernatants of different Enterobacteria strains, and our results
 A.R. Shahverdi et al. / Process Biochemistry 42 (2007) 919–923
prove that this method is much faster than previously published
biological methods. We are currently pursuing further studies in
this area that focus on the proper identification and isolation of
the compounds responsible for the reduction of the metal
nanoparticles.
Acknowledgments
We would like to express our gratitude to the Nanotechnology
Research Center, Faculty of Pharmacy, Medical Sciences/
University of Tehran, Tehran, Iran for its support of this work. We
also wish to thank Dr. S. Sarkar of the Iranian Nanotechnology
Initiative Center for his useful advice and support.
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