APPLIED PHYSICS LETTERS 94, 233901 共2009兲

Facile biosynthesis of phosphate capped gold nanoparticles by a bacterial

isolate Stenotrophomonas maltophilia
Yogesh Nangia,1 Nishima Wangoo,1 Saurabh Sharma,2 Jin-Song Wu,2 Vinayak Dravid,2,a兲
G. S. Shekhawat,2,a兲 and C. Raman Suri1,a兲
1
Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160036, India
2
Department of Material Science and Engineering and International Institute for Nanotechnology,
Northwestern University, Evanston, Illinois 60208, USA
共Received 11 March 2009; accepted 21 April 2009; published online 8 June 2009兲
We report intracellular biosynthesis of gold nanoparticles 共GNPs兲 by a strain Stenotrophomonas
maltophilia 共AuRed02兲 isolated from the soil samples of Singhbhum gold mines, India. An aqueous
solution of gold chloride was reduced to metallic gold in a suspension of disrupted cell mass of
AuRed02, which progressively turns into cherry red within 8 h of incubation at 25 ° C. The optical
spectrum showed the plasmon resonance at 530 nm and analysis by transmission electron
microscopy and dynamic light scattering confirmed the formation of around 40 nm GNPs. Zeta
potential and Fourier transform infrared measurements confirmed GNPs are capped by negatively
charged phosphate groups of NADP. © 2009 American Institute of Physics.
关DOI: 10.1063/1.3141519兴

Gold nanoparticles 共GNPs兲 have attracted considerable
attention in recent years, owing to their various applications
in catalysis, chemical sensing, photonics, biolabeling, and
the most imminent cancer imaging, diagnostics, and
therapeutics.1–4 Many chemical synthetic routes to GNPs re-
quire highly toxic solvents, harmful precursors, high tem-
peratures, and anoxic conditions.5–8 Conventional reactions
to synthesize GNPs employs sodium borohydride or citrate
reduction of tetrachloroauric acid 共HAuCl4兲 in the presence
of a capping ligand 共e.g., small organic molecules, polymers,
and biomacromolecules兲 containing at least one –NH2 or one
–SH group in a nonpolar medium. Such reaction conditions
introduce toxic impurities and limit the use of synthesized
GNPs for clinical applications.9
Biological methods using microorganisms, such as bac-
teria and cyanobacteria, for the synthesis of nanoparticles are
relatively new areas that are currently being explored.10–12
These microorganisms can survive and grow at elevated
metal ion concentrations and have developed specific de-
fense mechanisms to quell metal induced stresses. However,
the actual mechanism for the biosynthesis of GNPs by dif-
ferent microorganisms is still not well understood.13,14 It has
been suggested that the ability to reduce Au3+ remains to be
species specific, and closely related organisms with analo-
gous response against a range of metals have differing activi-
ties against Au3+. For example, P. islandicum effectively re-
duces Au3+ as compared to its close relative, P. aerophilum,
which was not able to reduce Au3+. Here the mechanism for
Au3+ reduction remains unknown and clearly necessitate for
a specific microorganism strain to effectively synthesize
GNPs.15 Recently, it has been hypothesized that hydrogenase
are directly involved in noble metal reduction through the
generation of NADPH. Earlier studies reported that a pos-
sible mechanism involved in the reduction of silver nitrate to
silver metal is the electron shuttle enzymatic metal reduction
a兲
Authors to whom correspondence should be addressed. Electronic ad-
dresses: g-shekhawat@northwestern.edu, v-dravid@northwestern.edu, and
raman@imtech.res.in.
process involving nitrate reductase present in
micro-organism.16,17
In this work, we report isolation and characterization of
a strain Stenotrophomonas maltophilia 共AuRed02兲, which
was isolated from the actual gold mine site 共Singhbhum,
Jharkhand state of India兲. The soil samples obtained from the
gold enriched sites were used as the inoculums and plated
onto Nutrient Agar media 共Himedia, India兲 by serial dilution
and pour-plate method on agar. The pH of the medium was
adjusted to 7.4. The morphological and physiological char-
acterizations of the selected isolate were carried out by bio-
chemical tests using the Bergeys Manual of Systematic
Bacteriology.18 Further characterization of isolate was done
by means of 16S rRNA gene analyses.19 The 16S rDNA
sequencing was done by M/s Bangalore Genei, India.
The characterized isolate was inoculated into sterile nu-
trient broth and 1 g of wet micro-organisms was harvested
after 16 h of inoculation. The biomass obtained was washed
thrice with de-ionized water and transferred to a 100 ml Er-
lenmeyer flask containing 1 mM gold chloride 共Sigma, India兲
solution prepared in de-ionized water spiked with 400 mg
yeast extract. The mixture was incubated at 25 ° C for 8 h
and then centrifuged to collect the cell mass. Cells were then
disrupted using 0.2% 共v / v兲 Triton X-100 followed by cen-
trifugation at 3500 rpm for 15 min at 4 ° C to isolate the
synthesized GNPs from the supernatant.
UV-vis spectroscopic measurements of the GNPs were
carried out using a Hitachi U2800 spectrophotometer. The
cell morphology was investigated using JEOL scanning elec-
tron microscopy 共SEM兲. Transmission electron microscopic
共TEM兲 measurements of intact bacteria were carried out us-
ing a JEOL 2100F transmission electron microscope operat-
ing at 120 kV accelerating voltage. Samples were prepared
by placing a drop of GNP solutions on carbon-coated TEM
grids. Particle size and charge distribution was analyzed us-
ing dynamic light scattering system 共Beckman Coulter,
USA兲 by illuminating the colloidal gold solution with He–Ne
laser 共633 nm兲 in a sample cell. Fourier transform infrared
共FTIR兲 spectra of biosynthesized GNPs were measured using
Perkin–Elmer FTIR spectrophotometer in AgCl plates to

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 233901-2 Nangia et al.
1µm
Strain AuRed02
FIG. 1. 共Color online兲 Phylogenetic tree made in MEGA 3.1 software using
Neighbor joining method. Inset shows the morphology of Stenotrophomonas
maltophilia as imaged by SEM.
confirm the capping of GNPs by NADPH. For cryo-TEM
measurements, bacteria samples suspended in de-ionized wa-
ter were spun down at 1000 g for 10 min in an Eppendorf
tube. The resulting pellet was fixed using Karnovsky’s for-
mula 关2.5% glutaraldehyde, 2% paraformeldehyde in a
0.01M phosphate buffer saline 共PBS兲兴. Microwave process-
ing was carried out in a Pelco BioWave microwave, set at
82 W for 4 min. 共1 off, 1 on兲. The fixed micro-organisms
were rinsed twice with 0.01M PBS in the microwave 共82 W,
two changes, 4 min each兲 followed by two water rinses
共same parameters兲 and then “enrobed” in 2% agar. The plugs
were dehydrated in a graded acetone series 共30%, 50%, 70%,
90%, and 100%, respectively兲 using microwave for 40 s for
each change. Specimens were infiltrated with a graded series
of EMBed 812 resin in acetone 共2:1 and 1:1 acetone:resin
steps were processed at 82 W in the microwave for 3 min
each. The 1:2 mixture was processed room temperature for
1 h兲. Samples were held in 100% resin overnight at room
temperature. Afterward, the samples were transferred to fresh
resin in BEEM capsules, then polymerized in a 60 ° C oven
for 24 h. Ultrathin sections 共⬃90 nm兲 were cut using a Leica
Ultracut and collected on copper mesh grids. Specimens
were observed and imaged using a JEOL 1230 TEM at 80
kV.
The bacterial characterization confirms that the isolate
was a gram negative, oval shaped, with discrete lipopolysac-
charides layers, as evident from the scanning electron photo-
micrograph 共inset of Fig. 1兲. Optimum biomass growth was
observed at 37 ° C and pH 7.0. The strain was found to be
catalase positive, oxidase negative, citrate positive, and ure-
ase negative. Most of the chemotaxonomic properties indi-
cate that AuRed02 were typical members of the family
␥-proteobacteria. Based on alignment of 16S rDNA sequenc-
ing 共1.460 kb兲 and phylogenetic analysis of the strain Au-
Red02, it was revealed that the strain AuRed02 clustered
homolog being Pseudomonas Sp. 共Acc. No. FJ211222兲. At
species level, strain AuRed02 shows the highest level of se-
quence similarity with Stenotrophomonas maltophilia BL-15
共99%兲 as shown in phylogenetic tree 共Fig. 1兲.
The GNP synthesis by AuRed02 was very rapid as at
25 ° C as yellowish gold chloride solution progressively
transformed to cherry red with 8 h contact time. Control
experiments, without the addition of biomass as well as with
heat-killed cells, showed no change in color of suspension
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Appl. Phys. Lett. 94, 233901 共2009兲
(i) (ii) (iii
)
(a) (b)
Intensity
Zeta Potential (mV)
(c) (d)
FIG. 2. 共Color online兲 共a兲 GNPs synthesized by incubating the solution of
HAuCl4 in presence of cell mass suspension 共a兲. Control experiments 关共b兲
and 共c兲兴: HAuCl4 incubated without the addition of biomass and with heat-
killed cells respectively. 共b兲 UV-vis spectra of GNPs solutions prepared by
resuspending the biomass for different time intervals in the presence of
HAuCl4. 共c兲 and 共d兲 depict the zeta potential measurement and IR spectrum
of biosynthesized GNPs, respectively.
关Fig. 2共a兲兴 confirming that the cherry red color formation of
GNPs was due to the addition of active AuRed02. The UV-
vis spectra revealed a strong absorption at nearly 530 nm at 8
h of incubation at 25 ° C, gradually showing a redshift with
time when kept at 25 ° C 共curves bottom to top兲. The char-
acteristic intense plasmon resonance band at 530 nm indi-
cates the formation of GNPs of approximately 40 nm size.
These results suggest that Stenotrophomonas maltophilia
could rapidly synthesize GNPs and size and shape of GNP
can be controlled by varying contact time of HAuCl4 with
the AuRed02 关Fig. 2共b兲兴. The colored solution of GNPs re-
mained stable for more than 2 weeks at 4 ° C indicating the
capping of GNPs with some charged groups. Zeta potential
measurements of synthesized GNPs showed a peak at ⫺16.7
mV confirming the capping of GNPs by some negatively
charged groups 关Fig. 2共c兲兴. FTIR analysis of these GNPs
further confirmed the capping of GNPs by phosphate groups
共900 cm−1兲, as shown in the FTIR spectrum 关Fig. 2共d兲兴.
Cryo-TEM image showed the predominance of GNPs
within the inner cytoplasmic membrane as well as in the
cytosol of the bacteria 关Fig. 3共a兲兴. The micrograph revealed
that gold ions 共Au3+兲 are preferentially taken up by the bac-
(a) (b)
100 nm
n
FIG. 3. 共a兲 TEM images of thin sections of stained Stenotrophomonas mal-
tophilia cells after reaction with gold chloride solution for
8 h. 共b兲 shows the TEM image recorded from a drop-coated film of GNPs
obtained from the disrupted bacteria. Scale bars in both 共a兲 and 共b兲 corre-
spond to 100 nm.
 233901-3 Nangia et al.
FIG. 4. 共Color online兲 共a兲 UV-vis spectra of GNPs prepared by adding
different concentrations of NADPH in the solution of suspended biomass
along with HAuCl4 共tubes S1–S5兲. In controls, either cell mass 共C1兲 or
NADPH 共C2兲 was not added.
teria through ion-transport channel and are reduced by the
enzymes present close to the inner cytoplasmic membrane
and within the cytoplasm.20 Figure 3共b兲 shows the TEM im-
age of the gold particles. The average diameter of the colloi-
dal gold obtained was 40 nm 共⫾ 15%兲.
Evidence of NADP+ binding to the biosynthesized nano-
particles was obtained by incubating biomass with NADPH
and change in color of solution was monitored visually and
by UV-vis spectrophotometry. Control experiments, without
the addition of either cell free extract 共C1兲 or cell free extract
without NADPH 共C2兲, showed no change in color of suspen-
sion. However, addition of NADPH in the cell free extract at
varying concentrations 共0.05 to 0.8 mM NADPH labeled as
S1-S5兲 showed the synthesis of GNPs with gradual increase
in color intensity 共Fig. 4兲. Thus, confirming the formation of
GNPs in presence of both biomass and NADPH together.
These results are very similar to the findings to the cognate
chemistry catalyzed by the specific enzyme auric ion reduc-
tase.
Our study suggested that biosynthesis and stabilization
of GNPs via charge capping involved NADPH-dependent
reductase enzyme that converts Au3+ to Au0 through electron
shuttle enzymatic metal reduction process. The reduction in
the gold chloride solution takes place in two steps. At first
step, AuCl−4 ions are rapidly reduced to Au+ species 关Eq. 共1兲兴.
The latter product is then reduced by NADPH to metallic
gold 关Eq. 共2兲兴 subsequently followed by the encapsulation of
GNPs by charged NADP+ species,
AuCl−4 + NADPH → Au+ + 4Cl− + NADP+ + H+ , 共1兲
2Au+ + NADPH → 2Au0 + NADP+ + H+ . 共2兲
Based on these experimental findings, a schematic represen-
tation of the potential mechanism of GNPs synthesis by
Stenotrophomonas maltophilia through enzymatic reduction
is proposed 共Fig. 5兲.
The approach described in this study offers a novel bio-
synthesis approach to obtain GNPs from the gold chloride
solution. To the best of our knowledge, this is the first study
where Stenotrophomonas maltophilia has been reported to
synthesize stable GNPs. The results suggested a novel
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Appl. Phys. Lett. 94, 233901 共2009兲
FIG. 5. 共Color online兲 Proposed synthesis mechanism of GNPs by Stenotro-
phomonas maltophilia through enzymatic reduction.
mechanism hypothesis involving specific NADPH-
dependent enzyme that reduces Au3+ to Au0 through an elec-
tron shuttling mechanism leading to the synthesis of nearly
monodisperse GNPs.
This work was supported by the National Science Foun-
dation and Department of Science and technology 共DST-
INDIA兲 under NSF Grant No. NSF-INT-0352706 and NSF-
ECS-IREE and NSF-MWN programs. Authors gratefully
acknowledge Mr. Kumar Rajesh for necessary technical sup-
port and NSF-IREE program for hosting Nishima Wangoo
from IMTECH at Northwestern University.
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