J Nanopart Res (2009) 11:1811–1815
DOI 10.1007/s11051-009-9621-2
BRIEF COMMUNICATION
Microbial synthesis of silver nanoparticles by Bacillus sp.
Nalenthiran Pugazhenthiran Æ Sambandam Anandan Æ
Govindarajan Kathiravan Æ Nyayiru Kannaian Udaya Prakash Æ
Simon Crawford Æ Muthupandian Ashokkumar
Received: 31 October 2008 / Accepted: 2 March 2009 / Published online: 13 March 2009
Ó Springer Science+Business Media B.V. 2009
Abstract A silver resistant Bacillus sp. was isolated
through exposure of an aqueous AgNO3 solution to
the atmosphere. Silver nanoparticles were synthe-
sized using these airborne bacteria (Bacillus sp.).
Transmission electron microscopy (TEM) and energy
dispersive X-ray (EDX) analyses confirmed that
silver nanoparticles of 5–15 nm in size were depos-
ited in the periplasmic space of the bacterial cells; a
preferable cell surface location for the easy recovery
of biogenic nanoparticles.
Keywords Bacteria
Nanoparticles
Bacillus sp.

Silver
Nanobiotechnology
N. Pugazhenthiran
S. Anandan (&)
Nanomaterials & Solar Energy Conversion Lab,
Department of Chemistry, National Institute of
Technology, Trichy 620015, India
e-mail: sanand@nitt.edu
G. Kathiravan
N. K. Udaya Prakash
Marina Labs, Choolaimedu, Chennai 600094, India
S. Crawford
School of Botany, University of Melbourne, Melbourne,
Victoria 3010, Australia
M. Ashokkumar (&)
School of Chemistry, University of Melbourne,
Melbourne, Victoria 3010, Australia
e-mail: masho@unimelb.edu.au
Introduction
Nanomaterials are at the leading edge of the rapidly
developing field of nanotechnology. The synthesis of
nanomaterials of specific composition and size is a
burgeoning area of materials science research. The
properties of these materials in applications as diverse
as catalysis, sensors and medicine depend critically on
the size and composition of the nanomaterials. New
routes to the preparation of these materials extend the
choice of properties that can be obtained. Several
synthetic techniques that usually employ atomic,
molecular, and particulate processing in a vacuum
or in a liquid medium are in use (Daniel and Astruc
2004; Yonezawa and Toshima 1995; Link et al. 1999;
Silvert et al. 1996; Mizukoshi et al. 1997; Vinodgopal
et al. 2006; Anandan et al. 2008; Treguer et al. 1998;
Hodak et al. 2000; Chen and Yeh 2001; Mandal et al.
2006). Most of the techniques are capital intensive, as
well as inefficient in materials and energy use. Hence,
there is an ever-growing need to develop clean, non-
toxic, and environmentally benign synthetic proce-
dures. Consequently, researchers have used biological
synthesis, since this technique provides particles with
good control over the size distribution. The main
reason for this may be that the processes devised by
nature for the synthesis of inorganic materials on
nano- and micro- scales have contributed to the
development of a relatively new and largely unex-
plored area of research based on the use of microbes in
the biosynthesis of nanomaterials (Mandal et al. 2006;
123
1812
Sastry et al. 2004). Among the microorganisms,
prokaryotic bacteria have received the most attention
in the area of bio-synthesis of nanoparticles. Micro-
bial resistance against heavy metal ions such as Fe,
Co, Ni, Cu, Zn, As, Cd, Hg, Pb or U has been explored
for bioleaching processes of ores (Dopson and Lind-
strom 1999; Bacelar-Nicolau and Johnson 1999;
Lundgren and Silver 1980) and for biological metal
recovery systems (White et al. 1997, 1998; Misra
1992; Nies 1992; Jeong et al. 1997). Early studies
reveal that Bacillus subtilis 168 is able to reduce Au3?
ions to produce octahedral gold particles of nanoscale
dimensions (5–25 nm) within the bacterial cells by
incubation of the cells with gold chloride solution
(Beveridge and Murray 1980; Southam and Beveridge
1994; Fortin and Beveridge 2000) under ambient
conditions. Silver is highly toxic to most microbial
cells and can be used as biocide or antimicrobial agent
(Slawson et al. 1992a, b). Nevertheless, it has been
reported that several bacterial strains are silver
resistant (Pooley 1982; Slawson et al. 1992a, b) and
may even accumulate silver at the cell wall to as much
as 25% of the dry weight biomass, thus indicating
their use for industrial recovery of silver from ore
materials (Pooley 1982). The silver resistant bacterial
strain Pseudomonas stutzeri AG259 accumulates sil-
ver nanoparticles in the size range, 35–46 nm, along
with some silver sulphide, in the cell (Haefeli et al.
1984; Klaus et al. 1999; Silver 2003). The exact
reaction mechanisms leading to the formation of
silver nanoparticles by the silver resistant bacteria is
yet to be elucidated. In this study, we have made an
attempt to corroborate the microbial reduction of
water soluble Ag? to Ag0 using an airborne bacteria
(Bacillus sp.) present in the atmosphere.
Experimental details
Bacterial strain and growth conditions
Atmospheric bacteria were grown on nutrient agar
substrate containing 3.5 mM AgNO3 (Aldrich) under
aerobic conditions at 30 °C. A single colony was
isolated from a dozen petridishes exposed. The isolated
bacteria were identified as Bacillus sp. through their
morphology and biochemical studies (Holt et al. 1994).
The isolated colony was sub-cultured into 50 ml of
nutrient broth containing 3.5 mM AgNO3. The broth
123
J Nanopart Res (2009) 11:1811–1815
was inoculated with a loopful of bacteria and incubated
for a period of 7 days in darkness at room temperature.
The bacteria were then harvested by centrifugation
(10,000 rpm) at room temperature. The harvested cells
were analyzed by TEM. For comparison, petridishes
containing only the culture supernatant without silver
nitrate solution and only silver nitrate (without culture
supernatant) were incubated under similar experimen-
tal conditions. Upon visual observation, the culture
supernatant incubated in the presence of silver nitrate
showed a colour change from yellow to brown whereas
no colour change could be observed in culture
supernatant without silver nitrate and silver nitrate
solution without the culture. These control experi-
ments indicate that the Ag? ion reduction is not just a
thermal process.
TEM, EDX and electron diffraction analyses
Pellets of freshly harvested Ag-loaded bacteria were
fixed in 2.5% (w/v) aqueous glutaraldehyde, centri-
fuged, re-suspended in 1.5 ml of 0.1 M phosphate
buffer (pH-7.2) at 4 °C and post fixed in 1% Osmium
tetraoxide at 4 °C in 0.1 M phosphate buffer (60 min)
for TEM. Samples were dehydrated using a graded
series of acetone. After two 15 min washes in acetone,
cells were embedded in fresh araldite followed by
polymerization at 60 °C for 24 h. Ultrathin sections
(90 nm) were cut on a Leica Ultracut R microtome,
and mounted on pioloform-coated Cu grids. The
sections were stained with 2% aqueous uranyl acetate
for 10 min and triple lead stain for 5 min. Micro-
graphs were taken with a Philips CM120 transmission
electron microscope at 120 kV using a Gatan Multi-
scan 600CW digital camera. Energy-dispersive X-ray
(EDX) spectra were acquired using TECNAI G2
model transmission electron microscopy.
Results and discussion
To confirm silver precipitation, Bacillus sp. were
observed using TEM after exposure to a 3.5 mM
aqueous AgNO3 solution. Figure 1 (a–e) shows the
TEM images of thin sections of Bacillus sp. after
exposure to a 3.5 mM aqueous AgNO3 solution. As
shown in low magnification TEM images (Fig. 1a–c)
silver nanoparticles were formed in most bacterial
cells.
J Nanopart Res (2009) 11:1811–1815 1813

Fig. 1 TEM images of

Bacillus sp. reduced Ag
nanoparticles [low
resolution images (a–c) and
high resolution images (d
and e)]. Lattice fringes of
silver nanoparticles were
observed from HRTEM
image (e). FFT pattern (f)
confirms (110) plane of
silver nanoparticles. SAED
spectrum (g) confirms
Bacillus sp. reduce Ag? to
Ag0

123
1814
In a higher magnification TEM image (Fig. 1d–e),
biogenic nanoparticles 10–15 nm in size were
observed in the periplasmic space of the bacterial
cells, which is between the outer and inner cell
(plasma) membranes. However, in contrast to many
other reports of metal-resistant bacteria, for which
efflux of toxic ions is the main detoxification mech-
anism (Silver and Phung 1996; Gupta et al. 1999), the
majority of the accumulated silver here is deposited as
seed like particles between the outer membrane and
the plasma membrane. The sizes of the deposits in our
results is so small (5–15 nm) compared to previous
studies using P. stutzeri Ag259 (35–46 nm), which
may be caused by different cell growth and metal
incubation conditions (Haefeli et al. 1984). The FFT
pattern (Fig. 1f) confirms the plane (110) of silver
nanoparticle and thus from the electron diffraction
pattern it is evidenced that the silver nanoparticles are
crystalline in nature. Also, the lattice fringes observed
in micrograph (Fig 1f) supports the crystalline nature
of the silver nanoparticles. The selected area electron
diffraction (SAED) (Fig 1g) results related to the
metallic silver nanoparticles indicates the reduction of
Ag? to Ag0 by Bacillus sp. Therefore, it can be
concluded that the resting cells of Bacillus sp. can
reduce Ag? ions to elemental silver (Ag0) in their
periplasmic space.
In summary, a simple microbial reduction method
has been developed for synthesizing Ag nanoparticles.
This methodology could be used for synthesizing a
number of metallic nanoparticles involving other
metals with good size and shape morphology.
Acknowledgement The research described herein was
supported by the Department of Innovation, Industry, Science
and Research, Australia and Department of Science and
Technology, India.
References
Anandan S, Grieser F, Ashokkumar M (2008) Sonochemical
synthesis of Au-Ag core-shell bimetallic nanoparticles. J
Phys Chem C 112:15102–15105. doi:10.1021/jp806960r
Bacelar-Nicolau P, Johnson DB (1999) Leaching of pyrite by
acidophilic heterotrophic iron-oxidizing bacteria in pure
and mixed cultures. Appl Environ Microbiol 65:585–590
Beveridge TJ, Murray RGE (1980) Sites of metal deposition in
the cell wall of Bacillus subtilis. J Bacteriol 141:876–877
Chen YH, Yeh CS (2001) A new approach for the formation of
alloy nanoparticles: laser synthesis of gold–silver alloy
123
J Nanopart Res (2009) 11:1811–1815
from gold–silver colloidal mixtures. Chem Commun
(Camb) 371–372. doi:10.1039/b009854j
Daniel MC, Astruc D (2004) Gold nanoparticles: assembly,
supramolecular chemistry, quantum-size-related proper-
ties, and applications toward biology, catalysis, and
nanotechnology. Chem Rev 104:293–346. doi:10.1021/
cr030698?
Dopson M, Lindstrom EB (1999) Potential role of thiobacillus
caldus in arsenopyrite bioleaching. Appl Environ Microbiol
65:36–40
Fortin D, Beveridge TJ (2000) From biology to biotechnology
and medical applications. In: Baeuerien E (ed) Biomin-
eralization. Wiley-VCH, Weinheim, pp 7–22
Gupta A, Matsui K, Lo JF, Silver S (1999) Molecular basis for
resistance to silver cations in Salmonella. Nat Med 5:183–
188. doi:10.1038/5545
Haefeli C, Franklin C, Hardy K (1984) Plasmid-determined
silver resistance in Pseudomonas stutzeri isolated from a
silver mine. J Bacteriol 158:389–392
Hodak JH, Henglein A, Giersig M, Hartland GV (2000) Laser-
induced inter-diffusion in AuAg core-shell nanoparticles.
J Phys Chem B 104:11708–11718. doi:10.1021/jp002438r
Holt et al (1994) Bergey’s manual of determination bacteriol-
ogy. William & Wilkins, Baltimore
Jeong BC, Hawes C, Bonthrone KM, Macaskie LE (1997)
Localization of enzymically enhanced heavy metal accu-
mulation by Citrobacter sp. and metal accumulation
in vitro by liposomes containing entrapped enzyme.
Microbiology 143:2497–2501
Klaus T, Joerger R, Olsson E, Granqvist CG (1999) Silver-
based crystalline nanoparticles, microbially fabricated.
Proc Natl Acad Sci USA 968:13611–13614. doi:10.1073/
pnas.96.24.13611
Link S, Wang ZL, El-Sayed MA (1999) Alloy formation of
gold-silver nanoparticles and the dependence of the
plasmon absorption on their composition. J Phys Chem B
103:3529–3533. doi:10.1021/jp990387w
Lundgren DG, Silver M (1980) Ore leaching by bacteria. Annu
Rev Microbiol 34:263–283. doi:10.1146/annurev.mi.34.
100180.001403
Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Muk-
herjee P (2006) The use of microorganisms for the
formation of metal nanoparticles and their application.
Appl Microbiol Biotechnol 69:485–492. doi:10.1007/
s00253-005-0179-3
Misra TK (1992) Bacterial resistances to inorganic mercury
salts and organomercurials. Plasmid 27:4–16. doi:10.1016/
0147-619X(92)90002-R
Mizukoshi Y, Okitsu K, Maeda Y, Yamamoto TA, Oshima R,
Nagata Y (1997) Sonochemical preparation of bimetallic
nanoparticles of gold/palladium in aqueous solution. J
Phys Chem B 101:7033–7037. doi:10.1021/jp9638090
Nies DH (1992) Resistance to cadmium, cobalt, zinc, and
nickel in microbes. Plasmid 27:17–28. doi:10.1016/0147-
619X(92)90003-S
Pooley FD (1982) Bacteria accumulate silver during leaching
of sulphide ore minerals. Nature 296:642–643. doi:10.
1038/296642a0
Sastry M, Ahmad A, Khan MI, Kumar R (2004) Microbial
nanoparticle production. In: Niemeyer CM, Mirkin CA (eds)
J Nanopart Res (2009) 11:1811–1815
Nanobiotechnology. Wiley-VCH, Weinheim, Germany,
pp 126–135
Silver S (2003) Bacterial silver resistance: molecular biology
and uses and misuses of silver compounds. FEMS
Microbiol Rev 27:341–353. doi:10.1016/S0168-6445(03)
00047-0
Silver S, Phung LT (1996) Bacterial heavy metal resistance: new
surprises. Annu Rev Microbiol 50:753–789. doi:10.1146/
annurev.micro.50.1.753
Silvert PY, Vijayakrishnan V, Vibert P, Herrara-Urbina R,
Elhsissen KT (1996) Synthesis and characterization of
nanoscale Ag-Pd alloy particles. Nanostruct Mater 7:611–
618. doi:10.1016/0965-9773(96)00039-6
Slawson RM, Trevors JT, Lee H (1992a) Silver accumulation
and resistance in Pseudomonas stutzeri. Arch Microbiol
158:398–404. doi:10.1007/BF00276299
Slawson RM, Van Dyke MI, Lee H, Trevors JT (1992b)
Germanium and silver resistance, accumulation, and tox-
icity in microorganisms. Plasmid 27:72–79. doi:10.1016/
0147-619X(92)90008-X
Southam G, Beveridge TJ (1994) The in vitro formation of
placer gold by bacteria. Geochim Cosmochim Acta
58:4527–4530. doi:10.1016/0016-7037(94)90355-7
1815
Treguer M, de Cointet C, Remita H, Khatouri J, Mostafavi M,
Amblard J, Mostafavi M, Amblard J, Belloni J, de Keyzer
R (1998) Dose rate effects on radiolytic synthesis of gold-
silver bimetallic clusters in solution. J Phys Chem B
102:4310–4321. doi:10.1021/jp981467n
Vinodgopal K, He Y, Ashokkumar M, Grieser F (2006)
Sonochemically prepared platinum-ruthenium bimetallic
nanoparticles. J Phys Chem B 110:3849–3852. doi:
10.1021/jp060203v
White C, Sayer JA, Gadd GM (1997) Microbial solubilization
and immobilization of toxic metals: key biogeochemical
processes for treatment of contamination. FEMS Micro-
biol Rev 20:503–516. doi:10.1111/j.1574-6976.1997.
tb00333.x
White C, Sharman AK, Gadd GM (1998) An integrated
microbial process for the bioremediation of soil contam-
inated with toxic metals. Nat Biotechnol 16:572–575. doi:
10.1038/nbt0698-572
Yonezawa T, Toshima N (1995) Mechanistic consideration
of formation of polymer-protected nanoscopic bimetallic
clusters. J Chem Soc Faraday Trans 91:4111–4119. doi:
10.1039/ft9959104111
123