1. ORIGINAL PAPER
Novel polyhedral gold nanoparticles: green synthesis, optimization
and characterization by environmental isolate of Acinetobacter sp.
SW30
Sweety A. Wadhwani • Utkarsha U. Shedbalkar •
Richa Singh • Meena S. Karve • Balu A. Chopade
Received: 31 January 2014 / Accepted: 23 June 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract Gold nanoparticles have enormous applications
in cancer treatment, drug delivery and nanobiosensor due
to their biocompatibility. Biological route of synthesis of
metal nanoparticles are cost effective and eco-friendly.
Acinetobacter sp. SW 30 isolated from activated sewage
sludge produced cell bound as well as intracellular gold
nanoparticles when challenged with HAuCl4 salt solution.
We first time report the optimization of various physio-
logical parameters such as age of culture, cell density and
physicochemical parameters viz HAuCl4 concentration,
temperature and pH which influence the synthesis of gold
nanoparticles. Gold nanoparticles thus produced were
characterized by various analytical techniques viz. UV–
Visible spectroscopy, X-ray diffraction, cyclic voltamme-
try, transmission electron microscopy, selected area elec-
tron diffraction, high resolution transmission electron
microscopy, environmental scanning electron microscopy,
energy dispersive X-ray spectroscopy, atomic force
microscopy and dynamic light scattering. Polyhedral gold
nanoparticles of size 20 ± 10 nm were synthesized by
24 h grown culture of cell density 2.4 9 109
cfu/ml at
50 °C and pH 9 in 0.5 mM HAuCl4. It was found that most
of the gold nanoparticles were released into solution from
bacterial cell surface of Acinetobacter sp. at pH 9 and
50 °C.
Keywords Acinetobacter sp. Á Optimization Á Polyhedral
gold nanoparticles Á Atomic force microscopy
Introduction
Nanotechnology is currently a frontier of research due to
wide applications of nanomaterials in biomedical, agri-
cultural, catalysis, optical and electronic fields (Kannan
and Subbalaxmi 2011; Ghosh et al. 2012; Kitture et al.
2012). Recently inorganic nanoparticles (NP) have invoked
a lot of interest owing to their distinct physical, chemical
and biological properties as compared to the respective
bulk materials (Bhattacharya and Mukherjee 2008). Metal
nanoparticles are mostly studied because of their physico-
chemical and optoelectronic properties (Krolikowska et al.
2003).There are various physical and chemical methods
available for synthesis of metal NP (Shankar et al. 2004;
Panacek et al. 2006). However, they are costly and gen-
erate toxic byproducts (Shedbalkar et al. 2014; Gade et al.
2010). Therefore biological synthesis mediated by plants,
bacteria, fungi and algae is gaining more acceptance in
research because of its cost effectiveness and eco-friendly
nature (Gaidhani et al. 2013; Nagajyothi and Lee 2011;
Mukherjee et al. 2001). It has been hypothesized that
synthesis of NP can be one of the defense mechanism
adapted by microorganisms when subjected to higher metal
salt concentrations (Venkataraman et al. 2011).
S. A. Wadhwani Á U. U. Shedbalkar Á R. Singh Á
B. A. Chopade (&)
Department of Microbiology, University of Pune,
Pune 411007, Maharashtra, India
e-mail: bachopade@gmail.com; chopade@unipune.ac.in
S. A. Wadhwani
e-mail: sweety.wadhwani123@gmail.com
U. U. Shedbalkar
e-mail: utkarsha.shedbalkar@gmail.com
R. Singh
e-mail: richasngh316@gmail.com
M. S. Karve
Institute of Bioinformatics and Biotechnology, University of
Pune, Pune 411007, Maharashtra, India
e-mail: meenaskarve@gmail.com
123
World J Microbiol Biotechnol
DOI 10.1007/s11274-014-1696-y

2. Microorganisms present in activated sewage sludge are
of more interest to microbiologists due to their diversity
and efficient enzymatic activity (Li and Chro´st 2006).
Sewage sludge has complex composition including differ-
ent metals and toxic substances from the community dis-
charge. Hence the microorganisms present in activated
sludge may differ in their properties from the normal
microflora of environment. Acinetobacter sp. is normal
inhabitant of sewage, water, soil, food and humans (Carr
et al. 2003; Saha and Chopade 2001; Patil et al. 2001).
They harbor multiple plasmids and can produce biosur-
factants and bioemulsifiers (Patil et al. 2001; Deshpande
and Chopade 1994). Besides tolerance to extreme condi-
tions they are resistant to multiple antibiotics and metal
salts (Deshpande and Chopade 1994). In view of this, we
proposed that Acinetobacter sp. isolated from sludge may
have potential to synthesize metal NP.
Gold nanoparticles (AuNP) have been synthesized by
many bacteria (Kalimuthu et al. 2009; Nangia et al. 2009;
Suresh et al. 2011; Shedbalkar et al. 2014). However, there
are no reports using member of genus Acinetobacter. Few
researchers have tried to optimize AuNP synthesis using
fungi and algae (Mittal et al. 2013; Gericke and Pinches
2006; Pimprikar et al. 2009). Surprisingly, there are no
reports about optimization of bacteria mediated AuNP
synthesis. This is the first extensive study for optimization
of polyhedral AuNP employing Acinetobacter sp. The aim
of present study is to optimize the process for synthesis of
monodispersed AuNP by studying the physiological
parameters such as culture age, cell density and physico-
chemical parameters, viz HAuCl4 concentration, tempera-
ture and pH.
Materials and methods
Isolation and identification of Acinetobacter sp. SW30
from activated sewage sludge
Fresh activated sewage sludge was collected in sterile
bottles (Schott duran, Germany) from Pune Municipal
Corporation, sewage treatment plant, Erandwane, Pune,
Maharashtra, India. Enrichment of culture was carried out
in Baumann’s enrichment medium (Baumann 1968). Five
milliliter of freshly collected sludge was inoculated in
100 ml of media and incubated at 30 °C at 200 rpm for
48 h. After every 24 h aliquots of enriched broth were
serially diluted and 100 ll from 10-6
, 10-8
and 10-10
dilutions were spread plated on cysteine lactose electrolyte
deficient agar (CLED) (HiMedia, India). The plates were
incubated at 30 °C for 48 h.
Gram’s staining, motility, oxidase test and capsule
staining were performed for preliminary identification of
isolates. Cultures resembling microbiologically to Acine-
tobacter were further identified by 16 s rRNA sequencing.
The identified culture was routinely subcultured and
maintained on Luria–Bertani (LB) (HiMedia, India) agar at
4 °C and in glycerol stocks stored at -80 °C.
Screening for synthesis of metal nanoparticles
A loopful of culture was inoculated in 100 ml LB broth and
incubated at 30 °C, 200 rpm for 24 h. Cells were harvested
by centrifugation (5,000 rpm for 6 min at 10 °C) and
washed three times with sterile distilled water (D/W). Cell
pellet was suspended in sterile D/W and challenged inde-
pendently with metal salt solutions viz. AgNO3, CuSO4
(HiMidia, Mumbai, India), HAuCl4, H2PtCl6 and Na2PdCl4
(S.D. Fine Chemicals, Mumbai, India) so as to get the final
concentration of 1 mM and incubated at 30 °C, 180 rpm.
After every 24 h, 200 ll aliquots were withdrawn and UV–
Visible (UV–Vis) spectrum (Jasco V-530, USA) was
recorded from 200 to 800 nm. All the experiments were
performed in triplicates using 24 h grown culture of
2.4 9 109
cfu/ml with 1 mM HAuCl4 at 30 °C and
180 rpm in dark, unless otherwise specified.
Characterization of gold nanoparticles
AuNP were characterized by various analytical techniques.
The nature of NP was analyzed by X-ray diffraction
(XRD). Thoroughly dried thin film of AuNP solution was
made on glass slide and observed under D8 Advance
Brucker X-ray diffractometer with Cu Ka (1.54 A˚ ) source.
Cyclic voltammetry (CV) (PGSTAT 302) was used to
confirm the complete reduction of HAuCl4 salt to AuNP,
where electrochemical response of AuNP and HAuCl4
solution was recorded. In CV, HAuCl4 and AuNP solutions
were immersed in three electrode system consisting of
glassy carbon electrode as working electrode, Ag/AgCl as
reference electrode and platinum wire as counter electrode
with scan rate 100 mV/s. The exact morphology, size and
fringes pattern of AuNP was determined by transmission
electron microscopy (TEM, Technai G2, 20 ultra win FEI,
Netherland) and high resolution transmission electron
microscopy (HRTEM, JEM-2100 (JEOL)) respectively
using carbon coated copper grid. Selected area electron
diffraction (SAED) pattern of AuNP was also studied.
Surface morphology of AuNP with cells was observed by
environmental scanning electron microscopy (ESEM, Joel
JSM-6360A, USA) and elemental composition was detec-
ted by energy dispersive X-ray spectroscopy (EDXS).
Surface morphology was also confirmed by atomic force
microscope (AFM, NTEGRA, NT-MDT, Russia.) equip-
ped with 10 9 10 mm scanner and operated in semi con-
tact mode in air was used for AFM experiments.
World J Microbiol Biotechnol
123

3. Commercial golden silicon NSG 11 cantilevers (NT-MDT)
had a nominal radius 10 nm. The NOVA software (NT-
MDT, Russia) was used for image processing the scan
angle was 0° and scan rate was typically 1.5 Hz with 256
lines.
Optimization of parameters for obtaining
monodispersed gold nanoparticles
The effect of various physicochemical parameters such as
culture age, cell density, HAuCl4 concentration, tempera-
ture and pH was checked on the rate of synthesis and
morphology of AuNP. The effect of culture age was
studied by incubating it for 6, 12, 18 and 24 h in LB broth.
The culture was harvested and challenged with HAuCl4.
Synthesis of AuNP was monitored up to 96 h using UV–
Vis spectral analysis with an interval of 24 h. The effect of
cell density was studied by adjusting the density corre-
sponding to 0.3, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.4 and
2.7 9 109
cfu/ml as per McFarland’s standards (Scott
2011). HAuCl4 concentration was optimized using various
concentrations viz. 0.1, 0.3, 0.5, 0.7, 0.9, 1.0, 1.5, 2.0, 2.5,
3.0, 3.5 and 4.0 mM.
To study the effect of temperature, cell suspension was
challenged with 0.5 mM HAuCl4 concentration and incu-
bated at different temperatures such as 20, 30, 37, 50 and
60 °C. Optimized temperature (50 °C) and 0.5 mM
HAuCl4 concentration was used to study the effect of pH.
The pH of cell suspension was adjusted at 2, 4, 6, 7, 8, 9
and 10 using 0.1 N HCl and NaOH (Himedia, India). At
each stage, AuNP obtained were characterized by UV–Vis
spectroscopy and TEM. The particle size distribution of
optimized AuNP solution was studied by dynamic light
scattering technique (Particle sizing systems, Inc. Santa
Barbara, CA, USA). The concentration of AuNP synthe-
sized by Acinetobacter sp. was calculated according to the
formula of Liu et al. 2007.
Determination of toxicity of HAuCl4 and AuNP
on Acinetobacter sp. SW30
The minimum inhibitory concentration (MIC) was deter-
mined by broth micro dilution method given by the Clinical
Laboratory Standards Institute (CLSI). Two-fold serial
dilutions of HAuCl4 and AuNP (1- 1024 ug/ml) were
prepared using Mueller–Hinton (MH) broth in 96-well
microtiter plate. To each well, 50 ll of culture
(5 9 105
cfu/ml) was added. The microtiter plates were
incubated at 37 °C for 20 h, and results were recorded. The
lowest concentration completely inhibiting the growth was
reported as the MIC. From the above assay, a 5 ll aliquot
was taken from the wells showing no visual growth after
incubation and spotted onto MH agar plates. The lowest
concentration showing no growth on the MH agar after
20 h of incubation at 37 °C was recorded as the minimum
bactericidal concentration (MBC) (Singh et al. 2013). Total
viability count (TVC) was determined after synthesis of
AuNP.
Results
Twenty well isolated bacterial colonies were selected from
CLED agar plates. One of them was found to be nonmotile,
Gram negative, encapsulated, coccobacilli and oxidase
negative; which was further confirmed to be Acinetobacter
sp. by 16S rRNA gene sequencing and sequence is sub-
mitted to NCBI as Acinetobacter sp. SW30 (GenBank
KF421246). Cell suspension of Acinetobacter sp. SW30
could efficiently reduce Au?3
ions in HAuCl4 to AuNP
which was evident due to change in color from colorless to
purple exhibiting surface plasmon resonance (SPR) peak at
540 nm (Fig. 1a) after 24 h of incubation, while it could
not reduce AgNO3, CuSO4, H2PtCl6 and Na2PdCl4 salts up
to 120 h.
The XRD pattern showed four distinct peaks at 2h val-
ues of 38.10°, 44.1°, 64.5° and 77.6° (Fig. 1b) corre-
sponding to [111], [200], [220], [311] planes of Au,
indicating face centered cubic crystal structure of AuNP
(JCPDS 04-0783). Similar results were obtained with
SAED analysis (Fig. 2c).
In CV HAuCl4 showed two (1,2) reduction peak indi-
cating reduction of Au?3
to Au?
and Au?
to Au0
and two
(3,4) oxidation peaks indicating oxidation of HAuCl4 from
Au0
to Au?
and Au?
to Au?3
whereas there was no peak
found in AuNp solution indicating complete reduction of
HAuCl4 to AuNP (Fig. 1c).
TEM analysis revealed triangles, rods, spherical and
polyhedral shaped AuNP on bacterial cell surface (Fig. 2a,
b). Lattice fringes were studied by HRTEM, which showed
the distance between two lattice was 0.23 nm which con-
firms crystalline nature of AuNP (Fig. 2c, inset). ESEM
images (Fig. 2d) showed cell bound AuNP which was also
confirmed by AFM (Fig. 2f). The presence of gold was
indicated by peak at 2 keV in EDXS (Fig. 2e).
AuNP synthesis was observed in cultures grown for 6,
12, 18 and 24 h with maximum in 24 h grown culture
(Fig. 3a). Increase in culture age resulted in increased rate
of NP synthesis. Synthesis of AuNP was started in
0.6 9 109
cfu/ml and reached to its maxima in
2.4 9 109
cfu/ml and decreased thereafter. Hence, for
further studies cell density was adjusted to 2.4 9 109
cfu/ml (Fig. 3b).
Synthesis of AuNP was observed at 0.5–1.0 mM
HAuCl4 concentration with intense purple color. Above
1 mM and below 0.5 mM HAuCl4 concentration AuNP
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4. synthesis was not observed (Fig. 4b) and was confirmed by
UV–Vis spectroscopy (Fig. 4a). In TEM analysis, AuNP
synthesized at 0.5 mM HAuCl4 concentration (Fig. 4c)
were found to be small sized compared to AuNP synthe-
sized at higher concentrations of HAuCl4. Further, at
higher HAuCl4 concentrations AuNP were more irregular
0
0.5
1
400 500 600 700
Absorbance
0
50
100
150
200
250
300
20 30 40 50 60 70 80
Intensity
(111)
(220)
(311)
(200)
2 (Theta)Wavelength (nm)
(b)(a) (c)
Fig. 1 Characterization of AuNP synthesized by Acinetobacter sp.
SW30. a UV–Vis spectrum of AuNP synthesized by cell suspension
of Acinetobacter sp. with 1 mM HAuCl4 at 30 °C; Inset T: Color
change in cell suspension after addition of HAuCl4. C: Control.
b XRD pattern of AuNP. c Cyclic voltammetry of HAuCl4 and AuNP
with scan rate of 100 mV/s
200nm
2.0um
2.001/nm
(a) (b) (c)
(e)(d) (f)
200nm 100nm
Fig. 2 Characterization of AuNP. a TEM image of single bacterium
with triangle, rod and spherical shaped AuNP. b Triangle and
polyhedral shaped AuNP released from cells. c SAED pattern of
AuNP; Inset HRTEM image of AuNP. d ESEM image of cell bound
AuNP. e EDX spectrum. f AFM image
World J Microbiol Biotechnol
123

5. and polydispersed (Fig. 4d–f). Hence, the optimum
HAuCl4 concentration was 0.5 mM for synthesis of AuNP.
Synthesis of AuNP was observed at 30, 37 and 50 °C
temperatures as per UV–Vis spectroscopy (Fig. 5a). The
synthesis of polyhedral, pointed edged AuNP was initiated
in 12 h at 50 °C (Fig. 5a, b), which was the optimum
temperature.
AuNP were synthesized at all the tested pH with max-
imum at pH 9 (Fig. 6a). Polyhedral AuNP with smooth
edges were produced at pH 9 having average size of
20 ± 10 nm, measured using image J software (Fig. 6b)
from TEM images. The size of AuNP was well correlated
with particle size distribution obtained from DLS technique
(Fig. 6c) which gave 20.7 nm as mean diameter. It was
found that at pH 9 and 50 °C most of the AuNP were
released into solution from bacterial cell surface. At other
pH large irregular shaped AuNP were observed. Acineto-
bacter sp. could produce 0.084 mol of AuNP per cfu.
MIC value for HAuCl4 salt was 8 ug/ml (0.024 mM)
and MBC value was 256 ug/ml (0.768 mM). Cells were
0
0.5
1
1.5
2
2.5
<0.3 0.3 0.6 0.9 1.2
1.5 1.8 2.1 2.4 2.7
Time (h)
A540
109
cfu/ml
(b)(a)
0
0.5
1
1.5
12 24 36 48 60 72 84 96400 500 600 700 800
12h
18h
24h
6h
wavelength (nm)
Absorbance
Fig. 3 Optimization of
biosynthesis of AuNP. a UV–
Vis spectrum of AuNP
synthesized using cells of
different age. b Time course of
biosynthesis of AuNP at 30 °C
using culture of different cell
density
iii iii iv v vii
0
1
2
400
500
600
700
0.1mM 0.3mM 0.5mM 0.7mM
0.9mM 1.0mM 1.5mM 2.0mM
2.5mM 3.0mM 3.5mM 4.0mM
Wavelength (nm)
Absorbance
(a)
(b)
vii viii ix x xi xii
(c)
100nm
(f)
100nm
(e)
100nm
(d)
100nm
Fig. 4 Effect of HAuCl4 concentration on AuNP synthesis. a UV–
Vis spectrum of AuNP using different HAuCl4 concentration at
30 °C. b Color change in AuNP solution with different salt
concentration. (i) 0.1 mM (ii) 0.3 mM (iii) 0.5 mM (iv) 0.7 mM
(v) 0.9 mM (vi) 1.0 mM (vii) 1.5 mM (viii) 2.0 mM (ix) 2.5 mM
(x) 3.0 mM (xi) 3.5 mM (xii) 4.0 mM. Lower panel showing TEM
image of AuNP synthesized at c 0.5, d 0.7, e 0.9, f 1 mM HAuCl4
concentration
World J Microbiol Biotechnol
123

6. not viable after 48 h of synthesis of AuNP. Hence, as
the gold salt is toxic to cells and cells may have pro-
duced NP as defence mechanism but produced AuNP
were non toxic to Acinetobacter sp, according to MIC
and MBC.
Discussion
There are several reports on bacteria isolated from acti-
vated sewage sludge (Carr et al. 2003); however, their
potential of metal NP synthesis has not reported. We
50nm
0
1
2
3
4
300 500 700
20 C 30 C
37 C 50 C
60 C
Wavelength (nm)
Absorbance
50nm
i ii iii iv v (b)(a) (c)°
°
°
°
°
Fig. 5 Effect of temperature on AuNP synthesis. a UV–Vis spectrum
of AuNP with different temperatures. Inset color change in AuNP
solution with respect to temperature. (i) 20 °C, (ii) 30 °C, (iii) 37 °C,
(iv) 50 °C, (v) 60 °C. b TEM image of AuNP synthesized with
0.5 mM HAuCl4 concentration at 50 °C. c Enlarged view of (b)
50nm
(b)
(a)
50nm
I ii iii iv v vi vii
Absorbance
0
1
2
350 450 550 650
pH2 pH4
pH6 pH7
pH8 pH9
pH10
Wavelength (nm)
(c) (d)
Size (nm)
%Intensity
Fig. 6 Effect of pH on AuNP
synthesis with 0.5 mM HAuCl4
concentration at 50 °C. a UV–
Vis spectrum of AuNP with
different pH. Inset color change
of AuNP solution with respect
to pH. (i) pH2, (ii) pH4, (iii)
pH6, (iv) pH 7, (v) pH8, (vi)
pH9, (vii) pH10. b TEM image
of AuNP synthesized with
0.5 mM HAuCl4 concentration
at 50 °C and pH 9. c Particle
size distribution by DLS,
d Enlarged view of (b)
World J Microbiol Biotechnol
123

7. hereby, first time report the isolation of novel Acineto-
bacter sp. from activated sewage sludge having potential to
synthesize AuNP. Also it is the first report on synthesis and
optimization of AuNP using Acinetobacter sp. for obtain-
ing monodispersed AuNP. So far, few members of genus
Acinetobacter isolated from soil and water have been used
for extracellular synthesis of silver nanoparticles (AgNP)
and Mn2O3 NP (Gaidhani et al. 2013; Singh et al. 2013;
Hosseinkhani and Emtiazi 2011).
UV–Vis spectroscopy results were similar to previous
reports (Kalimuthu et al. 2009; Mukherjee et al. 2001).
Plasmon frequency is sensitive to dielectric nature of its
interface with the local medium. Any change in the sur-
roundings of these particles viz. surface modification,
aggregation, medium refractive index, etc. leads to color-
imetric change in the dispersion (Murphy et al. 2008).
Hence, there are few reports on deviation of SPR peak from
540 nm (He et al. 2008).
AuNP produce different shades of colors from yellow
(large particles) to red (small particles) and even mauve
(purple) based on their size, shape and monodispersity.
These vibrant colors are due to interaction of AuNP with
visible light; which are strongly dictated by the environ-
ment, size and physical dimensions of AuNP (Huang et al.
2003; Thompson 2007; Murphy et al. 2008; Peng et al.
2009; Shedbalkar et al. 2014). Moreover, there are reports
on microbially synthesized AuNP exhibiting purple color
(Gericke and Pinches 2006; Kalimuthu et al. 2009; Oza
et al. 2012a, b). In our case, Acinetobacter sp. SW30
synthesized AuNP with purple color and presence of AuNP
was also confirmed by TEM analysis.
Same XRD pattern was observed in AuNP synthesized
by Escherichia coli (Du et al. 2007), cell filtrate of Peni-
cillium brevicompactum (Mishra et al. 2011) and Rhodo-
pseudomonas capsulate (He et al. 2008). Four peaks for
electrochemical response of HAuCl4 were not observed in
AuNP solution indicating complete reduction of HAuCl4 to
AuNP (Aldous et al. 2006). CV has been used so far in
designing nano biosensors (Hezard et al. 2012; Balca´zar
et al. 2012); however, till day no one has used CV as a
technique for AuNP characterization. Location of AuNP
can be clearly seen in TEM and ESEM images. Bacteria
can synthesize AuNP extracellularly (Bhambure et al.
2009; Husseiny et al. 2007), intracellularly (Gericke and
Pinches 2006) as well as cell bound (Du et al. 2007) while,
Acinetobacter sp. SW30 was found to synthesize cell
bound as well as intracellular AuNP as seen in TEM and
ESEM images. It is important to note that intracellular
formation of AuNP has not been understood clearly up till
now. However, it has been proposed that metal ions bind to
cell surface through electrostatic interactions; these
adsorbed ions get reduced due to membrane bound proteins
(Das and Marsili 2011). Biologically synthesized AuNP
can be of various shapes such as triangles, spherical, cubes,
nanoplates and nanowires (Du et al. 2007; He et al. 2008;
Kalimuthu et al. 2009; Lengke and Southam 2006).
AuNP obtained from Acinetobacter sp. SW30 were
polydispersed. However, for nanomedicine applications
monodispersed NP are required (Singh et al. 2013), which
can be obtained after optimization of various physico-
chemical parameters (Mittal et al. 2013; Gericke and Pin-
ches 2006; Pimprikar et al. 2009). The culture age and cell
density has significant effect on synthesis of AuNP as per
previous reports where these studies have been performed
using fungi like Geotrichum candidum, Verticillium lute-
oalbum and Yarrowia lipolytica (Mittal et al. 2013; Gericke
and Pinches 2006; Pimprikar et al. 2009). However; we
first time report the optimization of culture age and cell
density of Acinetobacter sp. SW30 for AuNP synthesis.
Culture age of 24 h was optimum for AuNP synthesis, may
be due to highest expression of reductants. In Geotrichum,
candidum, 48 h grown culture was found to give maximum
synthesis of AuNP (Mittal et al. 2013). AuNP synthesis
was decreased with increasing age of culture in case of
Verticillium luteoalbum, in early exponential phase of
culture more AuNP was observed under TEM (Gericke and
Pinches 2006).
HAuCl4 concentration has shown to have an effect on
morphology and rate of AuNP synthesis. Small AuNP were
obtained at lower concentration (0.5 mM) in Acinetobacter
sp. SW30. Similar results were reported in Verticillium sp.
(Gericke and Pinches 2006), where small uniform AuNP
were obtained at 250 and 500 mg/l HAuCl4 and at higher
concentration (2,500 mg/l) very large and irregular AuNP
were synthesized (Gericke and Pinches 2006). In Coriolus
versicolor, increased salt concentration, led to increase in
synthesis rate without affecting the morphology (Sanghi
and Verma 2010). In Geotrichum candidum 1 mM con-
centration of HAuCl4 was found to be optimum (Mittal
et al. 2013).
Temperature played an important role by increasing
AuNP synthesis rate in Acinetobacter sp. SW30. It may be
due to maximum activity of proteins involved in reduction
of HAuCl4. Synthesis decreased at 60 °C due to inactiva-
tion of the cells leading to their death. Comparable results
were reported in Verticillium luteoalbum (Gericke and
Pinches 2006) where increase in temperature has shown to
accelerate the AuNP synthesis. The time required for
synthesis of AuNP has been reduced from 12 h at 37 °C to
2 h at 50 °C. In Chlorella pyrenoidusa it was shown that
100 °C was effective temperature (Oza et al. 2012a). On
the contrary in Sargassum wightii synthesis was not
observed at 100 °C (Oza et al. 2012b). Similar studies were
performed with AgNP where synthesis increases with
increase in temperature (Soni and Prakash 2011; Singh
et al. 2013). pH has shown to have an effect on NP
World J Microbiol Biotechnol
123

8. morphology. Acinetobacter sp. synthesized polyhedral
AuNP of uniform size (20 ± 10 nm) at pH 9. In Verticil-
lium luteoalbum produced uniform sized AuNP at acidic
pH and increased pH led to polydispersity (Gericke and
Pinches 2006). Effect of pH on localization of NP has been
reported in which acidic pH promoted intracellular syn-
thesis while extracellular synthesis was observed in alka-
line conditions (Sanghi and Verma 2010).
Synthesis of AuNP can be one of the mechanisms to
resist HAuCl4 toxicity in Acinetobacter sp. SW30 (Prakash
et al. 2010) hence, HAuCl4 salt was found to be toxic to
Acinetobacter sp. SW30 while synthesized AuNP were
nontoxic.
Conclusions
Acinetobacter sp. SW30 was found to synthesize cell
bound as well as intracellular AuNP which were charac-
terized by different physicochemical techniques. It was
found that physiological parameters such as culture age and
cell density as well as physicochemical conditions viz.
HAuCl4 concentration, temperature and pH had great
influence on morphology of AuNP and its rate of synthesis.
Monodispersed polyhedral AuNP of size 20 ± 10 nm were
synthesized by 24 h grown culture of cell density
2.4 9 109
cfu/ml at 50 °C and pH 9 in 0.5 mM HAuCl4.
The Acinetobacter strain can be used for synthesis of
AuNP in large scale and scale up study is in progress.
Acknowledgments S.W. and R.S. acknowledge University Grants
Commission (UGC), New Delhi, India for awarding research fel-
lowship. U.S. is thankful to UGC awarding UGC-DSK-PDF. Authors
are thankful to DST-FIST for providing transmission electron
microscopy (TEM) at Department of Physics, University of Pune,
India. Authors are also thankful to Professor Avinash Kumbhar,
Department of Chemistry, University of Pune, Pune for explaining
cyclic voltammetry of HAuCl4 salt. Part of this work was funded by
UPE Phase II: Focus area : Biotechnology (2012–2017) awarded to
University of Pune, India.
Conflict of interest The authors declare no competing financial
interest.
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