Materials Sciences and Applications, 2010, 1, 1-7
doi:10.4236/msa.2010.11001 Published Online April 2010 (
Copyright © 2010 SciRes. MSA
Synthesis of Crystalline Ag Nanoparticles (AgNPs)
from Microorganisms
Seema Sharma1, Naheed Ahmad2, Anuradha Prakash3, Vidya Nand Singh4, Ashok Kumar Ghosh3,
Bodh Raj Mehta4
1Department of Physics, A.N College, Patna, India; 2University Department of Botany, Patna University, Patna, India; 3Department
of Water Management, A.N College, Patna, India; 4Thin Film Laboratory, Department of Physics, Indian Institute of Technology,
New Delhi, India.
Received January 26th, 2010; revised February 5th, 2010; accepted February 9th, 2010.
Bacteria obtained from the isolates of the biodiversity of riverine coast of Ganga identified as Bacillus Koriensis, when
challenged with silver nitrate solution accumulated silver nanoparticles on the surface of its cell wall. These nanoparti-
cles showed an absorption peak at 438 nm in UV-visible spectrum corresponding to the plasmon resonance of AgNPs.
The transmission electron micrographs of nanoparticles in aqueous solution showed the production of reasonably
monodisperse AgNPs (average particle size: 9.92 ± 1.311 nm) by the bacteria. X-ray diffraction spectrum of the
nanoparticles confirmed the formation of metallic silver.
Keywords: Crystal Structure, Nanostructures, Metal, AgNPs
1. Introduction
Nanostructured materials are being viewed as the future
material and for various diverse applications in areas
such as biomedical science, optics, mechanics, magnetics
catalysts, biosensors and energy science [1-3]. However
most synthesis techniques employed involve huge inputs
in terms of capital and energy. In the era of growing
awareness about increasing pollution and global warming,
developing new methods of synthesis of nanomaterials
with green technologies is a challenge. Biodiversity is the
best resource for biotechnological innovations as organic
materials of nanometer dimensions form the basis of life.
This diversity of life especially microorganisms is being
used as ecofriendly nanofactories for bioproduction and
synthesis of different compounds of nanometer size [4-6].
The concept of exploiting locally available species with
potential to synthesize extracellular nanoparticles is a
novel concept for bioprospecting. It provides valuable
leads for new product development and new applications
of biological species that have not been studied earlier.
The metal microbe interaction has made the material
scientist aware of the immense potential of the microorg-
anisms as ecofriendly nanofactories [6-8]. Biological sy-
nthesis of such nanomaterials has gained significant in-
terest due to the use of mild experimental conditions of
temperature, pH and pressure. If harnessed to their full
potential, biological synthesis could present extra advan-
tages over chemical methods such as higher productivity
and lower cost. One new dimension in the metal-microbial
interaction has emerged in the last few years, is that the syn-
thesis of metallic nanoparticles have been reported from bac-
teria, yeast, fungi, and other biological sources [9-16].
Bacteria are ubiquitous and constantly exposed to str-
essful situations; hence they develop diverse ways to res-
ist and survive. When exposed to metals or other toxic
substances beyond a certain level they develop many me-
chanisms such as efflux, alteration in the solubility and
toxicity by change in redox state of the metalion, extrac-
ellular complexation or precipitation of metals and the
lack of specific metal transport systems [17-19]. The
exact mechanism leading to the formation of reduction to
silver ions and formation of nanoparticles is not fully
understood. However, it is known that the tolerance of
bacterial cells and formation of mineral particles is very
much dependant on the composition of the growth envi-
Various workers have exploited various microorgan-
isms for nanosynthesis. Bacteria as for instance Bacillus
subtilis 168 was reported to reduce Au+ to nanoscale di-
mensions [20,21].Other bacterial strains like Pseudomo-
nas stutzeri Ag 259 were found to be silver resistant and
were able to produce nanosize silver [22].Other workers
have used the fungi like Fusarium oxysporum and Veri-
Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms
cillium to produce Magnetite, Silica and Titania [23-25].
Klebsiella aerogens was manipulated to produce Cds
nanoparticles extracellularly. In addition to gold and sil-
ver nanoparticles synthesis of semiconductors like CdS,
ZnS and PbS have been obtained from bioorganisms.
Clostridium thermoaceticum precipitates was observed
to precipitate CdS at the cell surface as well as the me-
dium from CdCl2 in presence of cystenine hydrochloride
in the growth media [26]. It was also reported that the
monodispersity of silver/gold nanoparticles produced eit-
her intra or extracellularly by these bioorganisms is not
very high or inferior to those obtained by the conven-
tional chemical methods [27].
Ag Nanoparticles (AgNPs) have received considerable
attention due to their attractive physicochemical proper-
ties.The surface plasmon resonance and large effective
scattering cross section of individual AgNPs make them
ideal candidates for molecular labelling [28]. The exist-
ing physical methods for AgNPs synthesis, such as gas
condensation and irradiation by ultraviolet or gradiation,
are usually associated with a low production rate and
high expense. Further, the largescale synthesis of silver
nanomaterial suffers from issues such as polydispersity
and stability, especially if the reduction is carried out in
aqueous media. Therefore the extracellular biological sy-
nthesis of AgNPs could be an attractive and ecologically
friendly alternative method for the preparation of large
quantities because it offers the advantage of easy downst-
ream processing. Moreover, bacteria are easy to handle
and can be manipulated genetically without much diffic-
ulty. Considering these advantages, a bacterial system
could prove to be an excellent alternative for the extrace-
llular synthesis of AgNPs. Ionic silver is highly toxic to
most bacterial cells and has long been used as a potent
bactericidal agent [29]. However, several silver-resistant
bacterial strains have been reported and even shown to
accumulate AgNPs in their periplasmic space [4,14,15].
In this paper, we explore the local biodiversity for co-
mmercially valuable biological and genetic resources, as
they offer great opportunity for searching more environ-
ment friendly biomolecules as an answer to cleaner tech-
nologies for sustainable development. Biosynthesis of
nanomaterials is carried out from the microbial diversity
obtained from the state of India, Bihar (rich fertile allu-
vial Gangetic plain). Bacillus species has been used to
synthesize silver nanoparticles which was isolated from
the Gangetic riverine belt was seen to synthesize silver
nanoparticles extracellularly. This is the first report when
the microbial diversity of the Gangetic plain has been
tapped for nanosynthesis. The extracellular synthesis off-
ers a great advantage over an intracellular process of
synthesis from the application point of view. Since the
nanoparticles formed inside the biomass would have re-
quired additional step of processing for release of the
nanoparticles from the biomass by ultrasound treatment
or by reaction with suitable detergents. The extracellular
synthesis of nanoparticle makes it possible to harness and
immobilize/deposit onto desired solid support for the use
of different practical purposes.
2. Experimental Details
2.1 Culture
Soil samples were collected from the banks of Ganges at
various places in Patna, capital of Bihar during an ex-
haustive screening programme undertaken in our labora-
tory to isolate microorganisms capable of synthesizing
metal-based nanoparticles.The intracellular silver accu-
mulation was negligible (< 1%). However, extracellular
precipitation of black silver was observed with no loss in
the viability of cells. The bacteria selected for this study
was gram positive and rod shaped, forming chain like co-
lonies and was identified as Bacillus koriensis (Figure 1).
The selection was based on the criteria that this bacte-
rium was tolerating and reducing heavy metals.
2.2 Production of Silver Nanoparticles
Serial dilutions were made and plated on nutrient agar pl-
ates for culture of the bacteria. The plates were incubated,
after the requisite period the colonies were picked up and
pure cultures were isolated. The cultures were then che-
cked for their silver resistant properties by spot inocula-
tion with increasing concentrations of AgNO3.The se-
lected culture Bacillus Koriensis sp. was then inoculated
in at 0.5% level in 2 l Erlenmeyer flasks containing MG-
YP (Malt extract 1%, glucose 1%, yeast extract 0.3%,
Peptone 0.5%) media and incubated at room temperature
till the absorbance of the culture was between 0.8-0.9 and
the media was observed to be turbid. Colour changes
were observed both before and after exposure to sunlight.
Silver in form of AgNO3 from 1 mM to 8 mM was then
Figure 1. Photograph of Bacillus koriensis sp
Copyright © 2010 SciRes. MSA
Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms3
added and left for further incubation.The culture super-
natant that was obtained at different time intervals after
the addition of aqueous AgNO3 solution was centrifuged
at 3000 rpm for 5 min prior to harvesting bacterial cells;
the supernatant was filtered through a 0.22-mm filter,
and centrifuged again at 8000 rpm for 15 min to precipi-
tate AgNPs. While the Bacillus Koriensis sp. incubated
with deionized water (positive control) retained its origi-
nal colour, the silver nitrate treated organism turned dark
red after 24 h due to the deposition of silver nanoparti-
cles.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.
2.3 Characterization of AgNPs
2.3.1 UV–Visible Spectral Analysis
The optical absorbance of silver nano particles suspended
in distilled water was recorded on UV VIS spectrophoto-
meter (Systronics 2202 double beam model) from wave-
lengths 200-800 nm. The formation and quality of com-
pounds were checked by XRD technique.
2.3.2 X-Ray Diffraction Analysis
The X-ray diffraction (XRD) pattern measurements of
drop-coated films AgNPs on glass substrate were recor-
ded in a wide range of Bragg angles 2 at a scanning rate
of 20min-1, carried out on a Philips PW 1830 instrument
that was operated at a voltage of 40 kV and a current of
30 mA with Cu Kα radiation (1.5405Å).
2.3.3 TEM and Electron Diffraction Analysis
High Resolution Transmission Electron Microscopy (HR-
TEM) was performed by TECHNAI G20-STWIN (200 KV)
machine with a line resolution 2.32 (in angstrom). These
images were taken by drop coating AgNPs on a carbon-
coated copper grid. Energy Dispersive Absorption Spec-
troscopy photograph of AgNPs were carried out by the
HRTEM equipment as mentioned above.
3. Results and Discussions
Figure 2(a) shows the characteristic Surface Plasmon
Resonance (SPR) peak for AgNPs [30] obtained from
Baccilus koriensis, which was absent in all appropriate
simultaneously run controls. The AgNP production was
not detected even in a closely related genus of the same
family of E. coli, which indicates that the phenomenon
could be specific to silver-resistant Baccilus koriensis.
Production optimization of AgNPs was performed with
respect to concentrations of Ag+ ions, which ranged from
1 to 8 mM. It clearly showed an increase in the intensity
of the SPR up to 4 mM concentration, and an intensity
decrease at higher concentrations. The results clearly
indicated that 44 mM concentration of Ag+ ions was
most appropriate for the synthesis of AgNPs from Bac-
cilus koriensis. While no absorption band was observed
250 450 650 850
Wav e le ng th(n m)
250 450 650 850
Wavelength (nm)
200 300 400 500600 700 800
Wavelength (nm)
15 mins
Figure 2. UV/Vis spectra recorded from the culture super-
natant that shows the production of AgNPs (a) after 24 h of
reaction; (b) stability on storage of AgNPs
in both controls (positive and negative), a characteristic
surface Plasmon absorption band at 438 nm was ob-
served at 6 h that attained the maximum intensity after 24
h. After 24 h of incubation, no change in intensity at 438
nm was observed indicating complete reduction of silver
ions. The Plasmon bands are broad with an absorption
tail in the longer wavelengths, which could be in princi-
ple due to the size distribution of the particles [31]. Since
the varying intensity of the plasmon resonance depends
on the cluster size, the number of particles cannot be re-
lated linearly to the absorbance intensities [31].
Nanoparticles find various applications in electronics,
optoelectronics etc. in some of these applications it is a
requirement that the particles are stable in certain organic
solvents. Hence, the stability of the culture in organic
solvents such as ethanol, acetone and diethyl ether was
determined. It was observed that the Ag solution was
soluble in ethanol and no shift in absorbance maxima
was observed when the absorbance measurements of the
silver nanoparticles suspended in ethanol were taken.
However, the particles did not disperse uniformly in
acetone and diethyl ether and precipitated. The stability
of the synthesized silver nanoparticles was studied by
Copyright © 2010 SciRes. MSA
Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms
measuring its intensity over a period of 1 month in room
temperature. A narrow peak at 280 nm can be attributed
to the presence of contaminating proteins in the sample.
No significant change in the intensity was observed
which proved its stability over a period of 1 month indi-
cating that the coating polymer is not allowing the ag-
glomeration of the particles (Figure 2(b)). This may be
described by the fact that bacterial cells to protect itself
from the toxic environment of the silver takes advantage
of the detoxification mechanism by precipitation of silver
to elemental silver, following the interaction of metal
with chemical reactive group located at the bacterial sur-
face or its translocation into the cell. These sites nulcle-
ate deposition of metal chemical precipitate, thus leading
to formation of more metals as chemical precipitate and
lead to the formation of mineralized crystalline particles.
Such a response results in detoxification of Ag+ because
AgO is less toxic. The silver cation (Ag+) is a highly re-
active chemical structure which binds strongly to elec-
tron donor groups containing sulfur, oxygen, or nitrogen
and these bindings with biomolecules like protein could
restrict the size of the particle [32]. Whatever the mecha-
nism, the production of silver based crystalline particles
seems to be connected with the ability of bacterial cell to
survive in an environment that would be highly toxic to
other bacteria.
The bacteria were found to be reducing the silver extr-
acellularly. The biosynthesis and reduction of metal was
indicated by change in colour. The colour changed from
golden yellow which turned to tea brown on addition of
AgNO3 after 24 hours (Figure 3).There was perceptible
reduction in time of biosynthesis when the bacteria in log
phase with AgNO3 was exposed to sunlight.
X-ray diffraction (XRD) pattern (Figure 4) shows in-
tense Bragg’s reflections that can be indexed on the basis
of the fcc structure of silver [33]. The XRD pattern thus
obtained clearly shows [111], [200], [220], and [311]
planes, and exhibit that the synthesized AgNPs by the
Bacillus koriensis were crystalline in nature. The diffrac-
tion peaks were found to be broad around their bases ind-
icating that the silver particles are in nanosizes. The peak
broadening at half maximum intensity of the X-ray dif-
fraction lines is due to a reduction in crystallite size, flat-
tening and micro-strains within the diffracting domains.
Scherrer’s equation for broadening resulting from a
small crystalline size, the mean, effective or apparent di-
mension of the crystalline composing the powder is
Phkl = k / 1/2 cos
where and have their usual meaning, is the breadth
of the pure diffraction profile in radians on 2 scale and
k is a constant approximately equal to unity and related
both to the crystalline shape and to the way in which is
defined. The best possible value of k has been estimated
as 0.89. The particle sizes of all the samples in our study
Figure 3. Photographs showing change in colour after add-
ing AgNO3. (a) after 6hrs; (b) after 24hrs
Figure 4. X-ray diffractogram of silver nanoparticles: (a)
angular and background corrected raw data with structure
factors of Ag represented by vertical bars
have been estimated by using the above Scherrer’s equa-
tion and was found to be ~10nm for the strongest peak.
Transmission electron microscopy (TEM) images of
AgNPs that were synthesized by Bacillus koriensis, indi-
cated that the nanoparticles were in the size range of ~10
Copyright © 2010 SciRes. MSA
Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms
Copyright © 2010 SciRes. MSA
nm (Figures 5(a) and (b)). Selected area electron dif-
fraction (SAED) spots that corresponded to the (from
inside to outside of the central ring) [111], [200], [220],
[311] and [222] planes of the face-centered cubic (fcc)
structure of elemental silver [21] are clearly seen in Figure
5(c). HRT-EM image (Figure 5(d)) shows the d spacing
of 2.02Å, which matches with the [200] plane of AgNPs.
to understand the biochemical and molecular mechanism
of the synthesis of the nanoparticles by the cell filtrate in
order to achieve better control over size and polydisper-
sity of the nanoparticles.
4. Conclusions
Biosorption mechanism of metal ions by microorganisms
includes ion exchange, precipitation and complexation.
Reduction 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 Bacillus koriensis. These nanoparticles that can be
attributed to surface binding of stabilizing materials secr-
eted by the Bacteria.
Figure 6 shows the Energy Dispersive Absorption Sp-
ectroscopy photograph of AgNPs. All the peaks of Ag
are observed and are assigned. Peaks for Cu and C are fr-
om the grid used and the peaks for S, P and N correspond
to the protein capping over the AgNPs. It is reported ear-
lier that proteins can bind to nanoparticles either through
free amine groups or cysteine residues in the proteins [34,
35] and via the electrostatic attraction of negatively cha-
rged carboxylate groups in enzymes present in the cell
wall of mycelia [36] and therefore, stabilization of the
silver nanoparticles by protein is a possibility. The amide
linkages between amino acid residues in proteins give ri-
se to the well-known signatures in the infrared region of
the electromagnetic spectrum and have been shown by
the FTIR spectrum [37]. In future, it would be important
In this study, extracellular synthesis of AgNPs has be-
en shown from silver-resistant Bacillus k sp. XRD analy-
sis showed that nanoparticles were crystalline and metal-
lic in nature, respectively. HRTEM analysis showed that
most of the particles were spherical in shape with size
~10 nm. The extracellular bacterial synthesis of AgNPs
(a) (b)
(c) (d)
Figure 5. (a) and (b) Transmission electron microscopy images of AgNPs from Bacillus koriensis at different magnifications;
(c) Selected area electron diffraction showing the characteristic crystal planes of elemental silver; (d) HRTEM image showing
characteristic d spacing for the [200] plane
Synthesis of Crystalline Ag Nanoparticles (AgNPs) from Microorganisms
Figure 6. Energy dispersive absorption spectroscopy photograph of AgNPs
has many advantages and might be an excellent means of
developing an ecologically friendly protocol. The bacte-
rium was highly resistant to silver cations. Thus it can be
safely said that metal microbe interactions have an im-
portant role in many biotechnological applications inclu-
ding biomineralization. So, today it seems to be a cheap
and viable method for the production of ecofriendly nan-
5. Acknowledgements
The authors wish to acknowledge University Grants Co-
mmission, New Delhi, India for the financial support
under the major research project scheme.
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