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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 isolatedthrough exposure of an aqueous AgNO
3
solution tothe atmosphere. Silver nanoparticles were synthe-sized using these airborne bacteria (
 Bacillus
sp.).Transmission electron microscopy (TEM) and energydispersive X-ray (EDX) analyses confirmed thatsilver nanoparticles of 5–15 nm in size were depos-ited in the periplasmic space of the bacterial cells; apreferable cell surface location for the easy recoveryof biogenic nanoparticles.
Keywords
Bacteria
Á
Nanoparticles
Á
Bacillus
sp.
Á
Silver
Á
Nanobiotechnology
Introduction
Nanomaterials are at the leading edge of the rapidlydeveloping field of nanotechnology. The synthesis of nanomaterials of specific composition and size is aburgeoning area of materials science research. Theproperties of these materials in applications as diverseas catalysis, sensors and medicine depend critically onthe size and composition of the nanomaterials. Newroutes to the preparation of these materials extend thechoice of properties that can be obtained. Severalsynthetic techniques that usually employ atomic,molecular, and particulate processing in a vacuumor in a liquid medium are in use (Daniel and Astruc2004; Yonezawa and Toshima1995; 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 Yeh2001; Mandal et al. 2006). Most of the techniques are capital intensive, aswell 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 biologicalsynthesis, since this technique provides particles withgood control over the size distribution. The mainreason for this may be that the processes devised bynature for the synthesis of inorganic materials onnano- and micro- scales have contributed to thedevelopment of a relatively new and largely unex-plored area of research based on the use of microbes inthe biosynthesis of nanomaterials (Mandal et al.2006;
N. Pugazhenthiran
Á
S. Anandan (
&
)Nanomaterials & Solar Energy Conversion Lab,Department of Chemistry, National Institute of Technology, Trichy 620015, Indiae-mail: sanand@nitt.eduG. Kathiravan
Á
N. K. Udaya PrakashMarina Labs, Choolaimedu, Chennai 600094, IndiaS. CrawfordSchool of Botany, University of Melbourne, Melbourne,Victoria 3010, AustraliaM. Ashokkumar (
&
)School of Chemistry, University of Melbourne,Melbourne, Victoria 3010, Australiae-mail: masho@unimelb.edu.au
 123
J Nanopart Res (2009) 11:1811–1815DOI 10.1007/s11051-009-9621-2
 
Sastry et al.2004). Among the microorganisms,prokaryotic bacteria have received the most attentionin 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 exploredfor bioleaching processes of ores (Dopson and Lind-strom1999; Bacelar-Nicolau and Johnson1999; Lundgren and Silver1980) and for biological metalrecovery systems (White et al.1997,1998; Misra 1992; Nies1992; Jeong et al.1997). Early studies reveal that
Bacillus subtilis
168 is able to reduce Au
3
?
ions to produce octahedral gold particles of nanoscaledimensions (5–25 nm) within the bacterial cells byincubation of the cells with gold chloride solution(Beveridge and Murray1980; Southam and Beveridge1994; Fortin and Beveridge2000) under ambient conditions. Silver is highly toxic to most microbialcells and can be used as biocide or antimicrobial agent(Slawson et al.1992a,b). Nevertheless, it has been reported that several bacterial strains are silverresistant (Pooley1982; Slawson et al.1992a,b) and may even accumulate silver at the cell wall to as muchas 25% of the dry weight biomass, thus indicatingtheir use for industrial recovery of silver from orematerials (Pooley1982). The silver resistant bacterialstrain
Pseudomonas stutzeri
AG259 accumulates sil-ver nanoparticles in the size range, 35–46 nm, alongwith some silver sulphide, in the cell (Haefeli et al.1984; Klaus et al.1999; Silver2003). The exact reaction mechanisms leading to the formation of silver nanoparticles by the silver resistant bacteria isyet to be elucidated. In this study, we have made anattempt to corroborate the microbial reduction of water soluble Ag
?
to Ag
0
using an airborne bacteria(
 Bacillus
sp.) present in the atmosphere.
Experimental details
Bacterial strain and growth conditionsAtmospheric bacteria were grown on nutrient agarsubstrate containing 3.5 mM AgNO
3
(Aldrich) underaerobic conditions at 30
°
C. A single colony wasisolatedfromadozenpetridishesexposed.Theisolatedbacteria were identified as
Bacillus
sp. through theirmorphologyandbiochemicalstudies(Holtetal.1994).The isolated colony was sub-cultured into 50 ml of nutrient broth containing 3.5 mM AgNO
3
. The brothwasinoculatedwithaloopfulofbacteriaandincubatedforaperiodof7 daysindarknessatroom temperature.The bacteria were then harvested by centrifugation(10,000 rpm)atroomtemperature.Theharvestedcellswere analyzed by TEM. For comparison, petridishescontaining only the culture supernatant without silvernitrate solution and only silver nitrate (without culturesupernatant) were incubated under similar experimen-tal conditions. Upon visual observation, the culturesupernatant incubated in the presence of silver nitrateshowedacolourchangefromyellowtobrownwhereasno colour change could be observed in culturesupernatant without silver nitrate and silver nitratesolution without the culture. These control experi-ments indicate that the Ag
?
ion reduction is not just athermal process.TEM, EDX and electron diffraction analysesPellets of freshly harvested Ag-loaded bacteria werefixed in 2.5% (w/v) aqueous glutaraldehyde, centri-fuged, re-suspended in 1.5 ml of 0.1 M phosphatebuffer (pH-7.2) at 4
°
C and post fixed in 1% Osmiumtetraoxide at 4
°
C in 0.1 M phosphate buffer (60 min)for TEM. Samples were dehydrated using a gradedseries of acetone. After two 15 min washes in acetone,cells were embedded in fresh araldite followed bypolymerization 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. Thesections were stained with 2% aqueous uranyl acetatefor 10 min and triple lead stain for 5 min. Micro-graphs were taken with a Philips CM120 transmissionelectron microscope at 120 kV using a Gatan Multi-scan 600CW digital camera. Energy-dispersive X-ray(EDX) spectra were acquired using TECNAI G
2
model transmission electron microscopy.
Results and discussion
To confirm silver precipitation,
Bacillus
sp. wereobserved using TEM after exposure to a 3.5 mMaqueous AgNO
3
solution. Figure1(a–e) shows theTEM images of thin sections of 
Bacillus
sp. afterexposure to a 3.5 mM aqueous AgNO
3
solution. Asshown in low magnification TEM images (Fig.1a–c)silver nanoparticles were formed in most bacterialcells.
1812 J Nanopart Res (2009) 11:18111815
 123
 
Fig. 1
TEM images of 
 Bacillus
sp. reduced Agnanoparticles [lowresolution images (
a
c
) andhigh resolution images (
d
and
e
)]. Lattice fringes of silver nanoparticles wereobserved from HRTEMimage (
e
). FFT pattern (
)confirms (110) plane of silver nanoparticles. SAEDspectrum (
g
) confirms
 Bacillus
sp. reduce Ag
?
toAg
0
J Nanopart Res (2009) 11:18111815 1813
 123

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