9
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 HAuCl 4 salt solution. We first time report the optimization of various physio- logical parameters such as age of culture, cell density and physicochemical parameters viz HAuCl 4 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 10 9 cfu/ml at 50 °C and pH 9 in 0.5 mM HAuCl 4 . 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: [email protected]; [email protected] S. A. Wadhwani e-mail: [email protected] U. U. Shedbalkar e-mail: [email protected] R. Singh e-mail: [email protected] M. S. Karve Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, Maharashtra, India e-mail: [email protected] 123 World J Microbiol Biotechnol DOI 10.1007/s11274-014-1696-y

Wadhwani et al 2014

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ORIGINAL PAPER

Novel polyhedral gold nanoparticles: green synthesis, optimizationand 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

� 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. � Optimization � Polyhedral

gold nanoparticles � 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 � U. U. Shedbalkar � R. Singh �B. A. Chopade (&)

Department of Microbiology, University of Pune,

Pune 411007, Maharashtra, India

e-mail: [email protected]; [email protected]

S. A. Wadhwani

e-mail: [email protected]

U. U. Shedbalkar

e-mail: [email protected]

R. Singh

e-mail: [email protected]

M. S. Karve

Institute of Bioinformatics and Biotechnology, University of

Pune, Pune 411007, Maharashtra, India

e-mail: [email protected]

123

World J Microbiol Biotechnol

DOI 10.1007/s11274-014-1696-y

Page 2: Wadhwani et al 2014

Microorganisms present in activated sewage sludge are

of more interest to microbiologists due to their diversity

and efficient enzymatic activity (Li and Chrost 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 HAuCl4solution 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

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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

World J Microbiol Biotechnol

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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

Abs

orba

nce

0

50

100

150

200

250

300

20 30 40 50 60 70 80

Inte

nsity

(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

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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 1.21.5 1.8 2.1 2.4 2.7

Time (h)

A54

0

109cfu/ml(b)(a)

0

0.5

1

1.5

12 24 36 48 60 72 84 96400 500 600 700 800

12h18h24h6h

wavelength (nm)

Abs

orba

nce

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 vi i

0

1

2

400

500

600

700

0.1mM 0.3mM 0.5mM 0.7mM0.9mM 1.0mM 1.5mM 2.0mM2.5mM 3.0mM 3.5mM 4.0mM

Wavelength (nm)

Abs

orba

nce

(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 HAuCl4concentration

World J Microbiol Biotechnol

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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 C37 C 50 C60 C

Wavelength (nm)

Abs

orba

nce

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

Abs

orba

nce

0

1

2

350 450 550 650

pH2 pH4

pH6 pH7

pH8 pH9

pH10

Wavelength (nm)

(c) (d)

Size (nm)

% In

tens

ity

Fig. 6 Effect of pH on AuNP

synthesis with 0.5 mM HAuCl4concentration 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)

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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; Balcazar

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

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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.

References

Aldous L, Silvester DS, Villagra C, Pitner WR, Compton RG,

Lagunas MC et al (2006) Electrochemical studies of gold and

chloride in ionic liquids. New J Chem 30:1576–1583

Balcazar GM, Gonzalez TJ, Villalobos TI, Morales-Hernandez MJ,

Castaneda GF (2012) Carbon electrode-supported au nanoparti-

cles for the glucose electrooxidation: on the role of crystallo-

graphic orientation. J Nanomater doi:10.1155/2012/387581

Baumann P (1968) Isolation of Acinetobacter from soil and water.

J Bacteriol 96:39–42

Bhambure R, Bule NS, Madhusudan KR (2009) Extracellular

biosynthesis of gold nanoparticles using Aspergillus niger its

characterization and stability. J Chem Eng Technol 32:

1036–1041

Bhattacharya R, Mukherjee P (2008) Biological properties of

‘‘naked’’ metal nanoparticles. J Adv Drug Deliv Rev 60:

1289–1306

Carr E, Kampfer P, Patel B, Gurtler V, Seviour R (2003) Seven novel

species of Acinetobacter isolated from activated sludge. Int J

Syst Evol Microbiol 53:953–963

Das SK, Marsili E (2011) Bioinspired metal nanoparticle: synthesis,

properties and application. In: Mohammed R (ed) Nanomateri-

als. doi:10.5772/25305

Deshpande LM, Chopade BA (1994) Plasmid mediated silver

resistance in Acinetobacter baumanii. Biometals 7:49–56

Du L, Jiang H, Liu X, Wang E (2007) Biosynthesis of gold

nanoparticles assisted by Escherichia coli DH5a and its

application on direct electrochemistry of hemoglobin. Electro-

chem Commun 9:1165–1170

Gade A, Ingle A, Whiteley C, Rai M (2010) Mycogenic metal

nanoparticles: progress and applications. Biotechnol Lett 32:593–600

Gaidhani S, Singh R, Singh D, Patel U, Shevade K, Yeshvekar R et al

(2013) Biofilm disruption activity of silver nanoparticles

synthesized by Acinetobacter calcoaceticus. Mater Lett 108:

324–327

Gericke M, Pinches A (2006) Biological synthesis of metal nanopar-

ticles. Hydrometallurgy 83:132–140

Ghosh S, Patil S, Ahire M, Kitture R, Gurav DD, Jabgunde AM et al

(2012) Gnidia glauca flower extract mediated synthesis of gold

nanoparticles and evaluation of its chemocatalytic potential.

J Nanobiotechnol 10:17. doi:10.1186/1477-3155-10-17

He S, Zhang Y, Guo Z, Gu N (2008) Biological synthesis of gold

nanowires using extract of Rhodopseudomonas capsulate. Bio-

technol Prog 24:476–480

Hezard T, Fajerwerg K, Evrard D, Vincent Colliere V, Philippe Behra

P, Gros P (2012) Gold nanoparticles electrodeposited on glassy

carbon using cyclic voltammetry: application to Hg(II) trace

analysis. J Electroanal Chem 664:46–52

Hosseinkhani B, Emtiazi G (2011) Synthesis and characterization of a

novel extracellular biogenic manganese oxide (Bixbyite-like

Mn2O3) nanoparticle by isolated Acinetobacter sp. Curr Micro-

biol 63:300–305

Huang D, Liao F, Molesa S, Redinger D, Subramanian V (2003)

Plastic-compatible low resistance printable gold nanoparticle

conductors for flexible electronics. J Electrochem Soc 150:G412

Husseiny M, El-Aziz M, Badr Y, Mahmoud M (2007) Biosynthesis of

gold nanoparticles using Pseudomonas aeruginosa. Spectrochim

Acta A 67:1003–1006

Kalimuthu K, Venkataraman D, Sureshbabu R, Pandian K, Guruna-

than S (2009) Biological synthesis of gold nanocubes from

Bacillus licheniformis. Bioresour Technol 100:5356–5358

Kannan N, Subbalaxmi S (2011) Biogenesis of nanoparticles: a

current perspective. Rev Adv Mater Sci 27:99–114

Kitture R, Ghosh S, Kulkarni P, Liu XL, Maity D, Patil DJ et al

(2012) Fe3O4-citrate-curcumin: promising conjugates for super-

oxide scavenging, tumor suppression and cancer hyperthermia.

J Appl Phys 111. doi:10.1063/1.3696001

Krolikowska A, Kudelski A, Michota A, Bukowska J (2003) SERS

studies on the structure of thioglycolic acid monolayers on silver

and gold. Surf Sci Rep 532:227–232

Lengke M, Southam G (2006) Bioaccumulation of gold by sulfate

reducing bacteria cultured in the presence of gold (I)–thiosulfate

complex. J Geochim Cosmochim Acta 70:3646–3661

Li Y, Chrost RJ (2006) Microbial enzymatic activities in aerobic

activated sludge model reactors. Enz Micro Technol 39:568–572

Liu X, Atwater M, Wang J, Huo Q (2007) Extinction coefficient of

gold nanoparticles with different sizes and different capping

ligands. Colloids Surf B Biointerfaces 58:3–7

World J Microbiol Biotechnol

123

Page 9: Wadhwani et al 2014

Mishra A, Tripathy S, Wahab R, Jeong S, Hwang I, Yang Y et al

(2011) Microbial synthesis of gold nanoparticles using the

fungus Penicillium brevicompactum and their cytotoxic effects

against mouse blast cancer C2C12 cells. J Appl Microbiol

Biotechnol 92:617–630

Mittal AK, Kaler A, Mulay AV, Banerjee UC (2013) Synthesis of

gold nanoparticles using whole cells of Geotrichum candidum.

J Nanopart. doi:10.1155/2013/150414

Mukherjee P, Ahmad A, Mandal D, Senapati S, Sainkar SR, Khan MI

et al (2001) Bioreduction of AuCl by the fungus, Verticillium sp.

and surface trapping of the gold nanoparticles formed. Angew

Chem Int Ed Engl 40:3585–3588

Murphy CJ, Gole AM, Stone JN, Sisco PN, Alkilany AM, Goldsmith

EC et al (2008) Gold nanoparticles in biology: beyond toxicity to

cellular imaging. Acc Chem Res 41:1721–1730

Nagajyothi PC, Lee KD (2011) Synthesis of plant-mediated silver

nanoparticles using Dioscorea batatas rhizome extract and

evaluation of their antimicrobial activities. J Nanopart. doi:10.

1155/2011/573429

Nangia Y, Wangoo N, Sharma S, Wu J, Dravid V, Shekhawat GS et al

(2009) Facile biosynthesis of phosphate capped gold nanopar-

ticles by a bacterial isolate Stenotrophomonas maltophilia. Appl

Phys Lett 94:233901

Oza G, Pandey S, Mewada A, Kalita G, Sharon M, Phata J,

Ambernath W, Sharon M (2012a) Facile biosynthesis of gold

nanoparticles exploiting optimum pH and temperature of fresh

water algae Chlorella pyrenoidusa. Adv Appl Sci Res

3:1405–1412

Oza G, Pandey S, Shah R, Sharon M, Phata J, Ambernath W, Sharon

M (2012b) A mechanistic approach for biological fabrication of

crystalline gold nanoparticles using marine algae, Sargassum

wightii. Eur J Exp Biol 2:505–512

Panacek A, Kvitek L, Prucek R, Kolar M, Vecerova R, Pizurova N

et al (2006) Silver colloid nanoparticles: synthesis, character-

ization and their antibacterial activity. J Phy Chem B

110:16248–16253

Patil JR, Jog NR, Chopade BA (2001) Isolation and characterization

of Acinetobacter spp. from upper respiratory tract of healthy

humans and demonstration of lectin activity. Indian J Med

Microbiol 19:30–35

Peng G, Tisch U, Adams O, Hakim M, Shehada N, Broza YY et al

(2009) Diagnosing lung cancer in exhaled breath using gold

nanoparticles. Nat Nanotechnol 4:669

Pimprikar PS, Joshi SS, Kumar AR, Zinjarde SS, Kulkarni SK (2009)

Influence of biomass and gold salt concentration on nanoparticle

synthesis by the tropical marine yeast Yarrowia lipolytica NCIM

3589. Colloids Surf B Biointerfaces 74:309–316

Prakash A, Sharma S, Ahmad N, Ghosh A, Sinha P (2010) Bacteria

mediated extracellular synthesis of metallic nanoparticles. Int

Res J Biotechnol 1(5):071–079

Saha SC, Chopade BA (2001) Studies on occurrence and distribution

of Acinetobacter sp. and other Gram-negative bacteria from

meat. J Food Sci Technol 38:17–22

Sanghi R, Verma P (2010) pH dependant fungal proteins in the

‘green’ synthesis of gold nanoparticles. Adv Mater Lett

1:193–199

Scott S (2011) Measurement of microbial cells by optical density

(Winter). J Valid Technol 17:46–49

Shankar SS, Ahmad A, Rai A, Sastry M (2004) Rapid synthesis of

Au, Ag and bimetallic Au core–Ag shell nanoparticles by using

neem (Azadirachta indica) leaf broth. J Colloid Interface Sci

275:496–502

Shedbalkar U, Singh R, Wadhwani S, Gaidhani S, Chopade BA

(2014) Microbial synthesis of gold nanoparticles: current status

and future prospects. Adv Colloid Interface Sci 209:40–48

Singh R, Wagh P, Wadhwani S, Gaidhani S, Kumbhar A, Bellare J

et al (2013) Synthesis, optimization and characterization of silver

nanoparticles from Acinetobacter calcoaceticus and their

enhanced antibacterial activity with antibiotics. Int J Nanomed

8:4277–4290

Soni N, Prakash S (2011) Factors affecting the geometry of silver

nanoparticles synthesis in Chrysosporium tropicum and Fusar-

ium oxysporum. Am J Nanotechnol 2:112–121

Suresh AK, Pelletier DA, Wang W, Broich ML, Moon JW, Gu B et al

(2011) Biofabrication of discrete spherical gold nanoparticles

using the metal-reducing bacterium Shewanella oneidensis. Acta

Biomater 7:2148–2152

Thompson DT (2007) Using gold nanoparticles for catalysis. Nano

Today 2:40

Venkataraman D, Kalishwaralal K, Pandian SR, Gurunathan S (2011)

An insight into the bacterial biogenesis of silver nanoparticles,

industrial production and scale-up. In: Rai M, Duran N (eds)

Metal nanoparticles in microbiology. Springer, Berlin, pp 17–35

World J Microbiol Biotechnol

123