Environ Sci Pollut Res (2013) 20:66286637

DOI 10.1007/s11356-013-1728-4

RESEARCH ARTICLE

Evaluation of Acinetobacter sp. B9 for Cr (VI) resistance

and detoxification with potential application in bioremediation
of heavy-metals-rich industrial wastewater
Amrik Bhattacharya & Anshu Gupta

Received: 5 February 2013 / Accepted: 8 April 2013 / Published online: 26 April 2013
# Springer-Verlag Berlin Heidelberg 2013

Abstract Present work demonstrates Cr (VI) detoxification

and resistance mechanism of a newly isolated strain (B9) of
Acinetobacter sp. Bioremediation potential of the strain B9 is
shown by simultaneous removal of major heavy metals including chromium from heavy-metals-rich metal finishing
industrial wastewater. Strain B9 tolerate up to 350 mg L1 of
Cr (VI) and also shows level of tolerance to Ni (II), Zn (II), Pb
(II), and Cd (II). The strain was capable of reducing 67 % of
initial 7.0 mg L1 of Cr (VI) within 24 h of incubation, while
in presence of Cu ions 100 % removal of initial 7.0 and
10 mg L1 of Cr (VI) was observed with in 24 h. pH in the
range of 6.08.0 and inoculum size of 2 % (v/v) were determined to be optimum for dichromate reduction. Fourier transform infrared spectroscopy and transmission electron
microscopy studies suggested absorption or intracellular accumulation and that might be one of the major mechanisms
behind the chromium resistance by strain B9. Scanning electron microscopy showed morphological changes in the strain
due to chromium stress. Relevance of the strain for treatment
of heavy-metals-rich industrial wastewater resulted in 93.7,
55.4, and 68.94 % removal of initial 30 mg L1 Cr (VI),
246 mg L1 total Cr, and 51 mg L1 Ni, respectively, after
144 h of treatment in a batch mode.
Keywords Chromium (VI) . Heavy metals . Resistance .
Bioaugmentation . Acinetobacter sp. B9 . Industrial
wastewater
Responsible editor: Robert Duran
A. Bhattacharya : A. Gupta (*)
University School of Environment Management, Guru Gobind
Singh Indraprastha University, Sector 16-C,
Dwarka, New Delhi 110078, India
e-mail: anshurcy@yahoo.com

Introduction
Heavy metals are the major toxic constituent of various industrial wastewaters and pose greater risk for the environment
if not treated properly prior to their disposal. In the midst of
various heavy metals, chromium (VI) is considered as highly
hazardous metal due to its oxidizing, mutagenic, and carcinogenic properties and almost every statutory body in the world
has listed Cr (VI) as priority toxic chemical for control
(Cheung and Gu 2007). Moreover, the persistent stability
due to non-biodegradable nature and high solubility in aqueous environment increases the toxicity and contamination
ability of this heavy metal (Cheung and Gu 2007; Desai et
al. 2008; Colin et al. 2012; Liu et al. 2012). Electroplating,
leather tanning processes, chromate ore processing, dyes and
pigments, wood preservation, alloy making, and metal
finishing industries are major source of Cr (VI) and its compounds into the environment (Suksabye et al. 2008; Quintelas
et al. 2009; Ye et al. 2010). As per US-EPA, the permissible
limit of less than 0.05 mg L1 of Cr (VI) has to be attained
before disposal of chromate containing wastewater into natural environment (Srivastava and Thakur 2007; Dhal et al.
2010; Sharma and Adholeya 2011).
Various conventional physico-chemical processes such as
chemical reduction followed by precipitation, ion exchange,
adsorption (coal, activated carbon, fly ash, alum, and agricultural waste), reverse osmosis, membrane separation, and
solvent extraction are available for treatment of chromium
and other heavy-metals-containing wastewater (Gupta et al.
2009; Owlad et al. 2009; Dhal et al. 2010; Ye et al. 2010;
Sharma and Adholeya 2011; Liu et al. 2012). But due to
their tendency to cause secondary pollution along with high
cost and high energy requirement, the focus has been shifted
to better alternative ways of treatment, i.e., biological

Environ Sci Pollut Res (2013) 20:66286637

method of treatment using chromium-resistant microbes

(Pei et al. 2009; Liu et al. 2012). These methods are not
only economic and efficient but also address the problems
associated with these types of pollutants containing wastewater in a natural way. Various reports on use of metaltolerant viable microbes for treatment of Cr (VI) containing
wastewater have been reported by different authors in the
last decades (Ganguli and Tripathi 2002; Srinath et al. 2002;
Pazouki et al. 2007; Ahmad et al. 2010; Machado et al.
2010; Sharma and Adholeya 2011; Naik et al. 2012).
These chromium-resistant microbes combat the toxicity associated with Cr (VI) by adopting a number of detoxification
strategies like biotransforming Cr (VI) to less toxic form of Cr
(III) using enzymes/metabolites, adsorption (absorption), or
intracellular accumulation inside the cell (Sharma and
Adholeya 2011; Sagar et al. 2012). The selection of efficient
microbial strain, capable of tolerating and reducing high concentration of toxicants and study of microbetoxicant interaction, is prerequisite for development of efficient bioremediation
strategy (Pei et al. 2009; Das and Mishra 2010). The present
work was attempted to study the Cr (VI) tolerance and detoxification potential of a newly isolated strain (B9) of
Acinetobacter sp., and elucidation of its chromium resistance
mechanism using various analytical techniques like scanning
electron microscopy (SEM), transmission electron microscopy
(TEM), and Fourier transform infrared spectroscopy (FT-IR),
in order to develop a potential Cr (VI) remediation tool.
Potential application of strain for bioremediation of chromium
is also shown by treatment of real metal finishing industrial
wastewater using the isolated bacteria.

Material and methods

Material
The media components were obtained from Hi-Media
Laboratories (Mumbai, India). Potassium dichromate
(K2Cr2O7) was used as source of Cr (VI) and procured from
SISCO research laboratories (Mumbai, India). Diphenyl
carbazide was a product of Molychem (Mumbai, India).
All chemicals used were of analytical grade.
Microorganism
The strain B9 used in the present study was isolated from
the wastewater of local common effluent treatment plant
situated in New Delhi, India. The isolate was identified
using 16S r-RNA sequencing from Microbial Type Culture
Collection and Gene Bank (MTCC), Institute of Microbial
Technology (IMTECH) Chandigarh, India. The strain B9
was maintained on nutrient agar slants at 4 C and subcultured every 20 days interval.

6629

Preparation of mother culture

B9 inoculum was prepared by transferring a loopful of stock
culture into sterile nutrient broth (pH 7.5) containing
(g L1): peptone, 5.0; NaCl, 5.0; yeast extract, 1.5; beef
extract, 1.5 followed by overnight incubation at 30 C and
200 rpm in an orbital shaker (Innova, Brunswick).
Chromium tolerance studies
The minimum inhibitory concentration (MIC) of Cr (VI) for
strain B9 was studied at various initial Cr (VI) concentrations,
ranging from 35 to 425 mg L1. Sterile nutrient broth (50 mL)
taken in 250-mL Erlenmeyer flasks were amended with varying
Cr (VI) concentrations, separately. All the flasks were aseptically inoculated with 2 % (v/v) B9 mother cultures followed by
incubation at 30 C and 200 rpm in an orbital shaker. The MIC
was measured on the basis of growth (A600 nm) observed with in
48 h. The minimal concentration of Cr (VI) inhibiting complete
growth of the bacterial isolate was taken as MIC.
Media and culture conditions for Cr (VI) removal
All experiments for Cr (VI) removal were performed in 250mL Erlenmeyer flasks as batch reactors under aerobic conditions. The potential of strain B9 for Cr (VI) reduction was
assessed by using nutrient broth as basal media. Two percent
(v/v) of the overnight grown B9 mother culture (A600 nm 2.0)
was used to inoculate 50 mL of sterile media (pH 7.0)
containing various initial concentrations of Cr (VI) [3.5, 7.0,
14, 21, 28, and 35 mg L1] and incubated at 30 C with
constant shaking at 200 rpm in an orbital shaker for 96 h. In
order to see the role of any abiotic factors on Cr (VI) reduction,
two types of control sets were run in parallel to the test solution:
(A) control flask with un-inoculated media, and (B) media
inoculated with autoclaved B9 cells. During incubation, aliquots of samples (1.0 mL) were withdrawn periodically (12,
24, 48, 72, and 96 h) from all the flasks for estimation of
residual Cr (VI). The samples were centrifuged at 2,376g,
4 C for 10 min and supernatants thus obtained were used for
the analysis of residual Cr (VI). The reduction rate was calculated using formula: reduction rate=C0 Ct/t (Pang et al. 2011),
where C0initial Cr (VI) concentration (mg L1), CtCr (VI)
concentration (mg L1) at time t, and tincubation time (h).
The estimation of viable cells was done by using spread
plate method using heterotrophic plate count technique
(APHA 1998).
Effect of initial pH and inoculum size on Cr (VI) removal
To characterize the Cr (VI) reduction efficiency of the
isolate, the effects of initial pH (5.0, 6.0, 7.0, 8.0, 9.0, and
10.0) and inoculum size (1, 2, 4, and 6 %; v/v) were

6630

monitored on Cr (VI) reduction. In both the cases, initial

7.0 mg L1 of Cr (VI) was used. Samples were withdrawn
after 24 h of incubation for residual Cr (VI) determination.
Effect of heavy metals and metabolic inhibitors on chromate
reduction
In order to determine the effect of heavy metals on Cr (VI)
reduction efficacy of strain B9, the culture media (pH 7.0)
containing initial 7.0 mg L1 of Cr (VI) was supplemented
with 7.0 mg L1 each of Cu (II) [CuSO45H2O], Ni (II)
[NiSO47H2O], Zn (II) [ZnSO47H2O], and Pb (II) [Pb
(NO3)2] individually. Since in the present study, the Cu ions
were found to stimulate Cr (VI) reduction, its effect was
further studied with higher initial Cr (VI) concentration of
10, 14, and 21 mg L1. Flasks were then inoculated with 2 %
(v/v) of B9 inoculum followed by incubation as described
above and analyzed for residual chromium at 24 h time period.
As Cr (VI) reduction is reported to be associated with
respiratory chain of the bacteria in some cases (Wani et al.
2007; Dey and Paul 2012), the effect of various
metabolite/respiratory inhibitors was tested to evaluate the role
of respiratory chain on Cr (VI) reduction in Acinetobacter sp.
B9 strain also. NaN3 and NaF (inhibitor of electron transport
chain) at 1 mM concentration were supplemented to culture
media (pH 7.0) having initial 7.0 mg L1 of Cr (VI) individually followed by inoculation with 2 % (v/v) of B9 cells. The
samples were taken after 24 h of incubation for residual Cr (VI)
analysis.
Scanning electron microscopy
B9 cells were grown in basal media in the presence and
absence of 35 mg L1 of Cr (VI) at 30 C and 200 rpm.
Cells were harvested at 4 C after 48 h of growth using
centrifugation at 9,503g for 10 min. The cell pellets were
washed by suspending in 2 mL of sterile saline solution
followed by vortexing for 2 min. The resultant cell suspension
was again centrifuged at 9,503g and 4 C for 10 min to pellet
down the cell mass. This washing procedure was repeated
thrice followed by overnight fixation at 4 C in modified
Karnovskys fixative (David et al. 1973) containing 1 %
glutraldehyde and 4 % paraformaldehyde in 0.1 M phosphate
buffer (pH 7.4). After removal of fixative, the cells were
finally suspended in 0.1 M phosphate buffer and subsequently
processed at All India Institute of Medical Sciences (AIIMS),
New Delhi, India for SEM analysis. Scanning electron micrographs were recorded by using Leo 435VP SEM at AIIMS.
Transmission electron microscopy
B9 cells were grown in basal media with and without
10.6 mg L1 of Cr (VI). After 48 h of growth at 30 C incubation

Environ Sci Pollut Res (2013) 20:66286637

temperature and 200 rpm shaking speed, the cells were

harvested using centrifugation at 9,503g, 4 C for 10 min.
The cells were washed with saline and fixed as described above.
The fixed samples suspended in 0.1 M phosphate buffer were
afterward processed at advanced instrumentation research facility, Jawaharlal Nehru University (JNU), New Delhi, India, for
TEM analysis. Transmission electron micrographs were
recorded by using JEOL 2100F, TEM at JNU.
FT-IR analysis of B9 cells
In order to determine the changes in surface characteristics
(conformational changes in functional groups) of the cells
grown in the presence of chromium (VI), the FT-IR spectrum of the normal and Cr (VI)-treated B9 cells were studied
using FT-IR spectroscopy (Varian 7000 FTIR).
To carry out the analysis, bacterial culture samples grown
for 48 h in presence and absence of 10.6 mg L1 of Cr (VI)
were centrifuged at 9,503g and 4 C for 10 min to pellet
down the cell mass. The cells pellets were washed with
saline prior to drying overnight at 60 C (Pei et al. 2009;
Dhal et al. 2010). The dried pellet was crushed to fine
powder using mortar and pestle. The resultant powdered
biomass was mixed with KBr in the ratio of about 1:100
(one part biomass and 100 part KBr) and pressed to form
KBr disks. The FT-IR spectra of dried biomass in KBr phase
was recorded by using FT-IR spectrometer in the range of
5004,000 cm1.
Application of strain B9 in treatment of chromium-rich
industrial wastewater
Physico-chemical characterization of wastewater
Raw industrial wastewater was collected from local
electroplating and metal-based industry located in national
capital region of Delhi, India. The wastewater was characterized for following parameters: pH, chemical oxygen demand (COD), total suspended solids (TSS), total dissolved
solids (TDS), and heavy metal content viz, Cr (VI), total Cr,
Ni, Cu, Fe, Pb, and Cd. The physico-chemical parameters
like COD, TSS, and TDS were estimated according to
standard APHA method (APHA 1998). Heavy metals analysis was carried out using atomic absorption spectrometry
(AAS) after acid digestion of sample.
Treatment of wastewater with Acinetobacter sp. B9 cells
Since the industrial wastewater was found to contain high
concentration of Cr (VI), total Cr, and Ni, the sample was
diluted (1:1) with mineral salt medium (Dong et al. 2008) to
make the final Cr (VI), total Cr, and Ni concentrations of
about 30, 246, and 51 mg L1, respectively.

Environ Sci Pollut Res (2013) 20:66286637

For inoculum preparation, 4.0 mL of overnight grown B9

mother culture was centrifuged to pellet down the cell mass.
The pellet was washed twice with sterile saline solution and
finally suspended in 2 mL of saline solution. The resultant
bacterial suspension was used to inoculate 100 mL of diluted
industrial wastewater supplemented with 0.5 % (w/v) of glucose as carbon source in 250-mL Erlenmeyer flask. The
treatment was carried out at 30 C under constant shaking of
200 rpm for 144 h. Control flask containing 100 mL of diluted
wastewater with 0.5 % (w/v) glucose but without B9 cells was
also incubated under similar conditions to monitor the role of
indigenous microorganisms in heavy metals reduction.
During incubation, aliquots of samples were withdrawn periodically (24, 48, 66, 96, 120, and 144 h) for estimation of
residual Cr (VI) in the control and experimental setups.
Analytical techniques
The Cr (VI) concentration was estimated colorimetrically at
540 nm using 1, 5 diphenyl carbazide method (APHA
1998). Quantification of total chromium and other heavy
metals were made using atomic absorption spectrophotometer (PerkinElmer AAS-700) after centrifugation (9,503g)
and acid digestion of samples (Srivastava and Thakur 2007).
Each experiment was done at least two times and the differences in their individual results in each set of experiments were
less than 5 %. The errors bars shown in figures represent
standard deviations, calculated by using Microsoft Excel.

6631

inhibitory concentration of strain B9 towards Cr (VI) was

determined to be 350 mg L1. The isolate also had a degree
of tolerance to Zn (II), Pb (II), Ni (II), and Cd (II) as
determined by its ability to grow in presence of these heavy
metals (data not shown). Unless otherwise mentioned, studies on MIC determination for Cr (VI) and degree of tolerance to other heavy metals were done only in case of B9
among the various isolated bacterial strains.
Cr (VI) removal potential of Acinetobacter sp. B9
Cr (VI) removal potential of the isolated bacterium was
studied by using nutrient broth as basal media with initial
7.0 mg L1 of Cr (VI). The isolate B9 was able to significantly remove Cr (VI) with overall rate of 0.194 mg L1 h1
at 7.0 mg L1 concentration. However, complete reduction
of Cr (VI) could not be obtained even after 96 h of incubation. At 24 h, 67 % reduction of Cr (VI) was observed,
which remained almost constant till 96 h (Fig. 1 and 2). To
see the effects of media components and other abiotic factors on Cr (VI) removal, two types of control set ups were
run in parallel. Cr (VI) reductions in case of both the
controls were observed to be less then 10 %. This showed
that the Cr (VI) reduction observed in case of experimental
setup was due to the live cells of Acinetobacter sp. B9 and
media components and other abiotic factors did not play
significant role in the Cr (VI) reduction.
Effect of varying initial Cr (VI) concentrations

Identification of isolate B9 and its metal tolerance

Isolate B9 was isolated from the wastewater of common
effluent treatment plant (CETP), located in New Delhi,
India. Since this CETP receives wastewater from nearby
industries majority of which are metal-based, it was likely
that such environment will provide natural adaptation to
heavy-metals-resistant microbes. A number of bacterial
strains could successfully be isolated from this heavymetal-contaminated site and one potential strain B9 was
selected for detailed studies. The isolate B9 was identified
as Acinetobacter baumannii using 16S rRNA sequencing
(1,432 bp and 99.86 similarity) from Microbial Type
Culture Collection and Gene Bank (MTCC), Institute of
Microbial Technology (IMTECH), Chandigarh, India, and
deposited at its collection bank with accession No. MTCC
10506.
The bacterial strain B9 was checked for its ability to grow
in the presence of various heavy metals. It was found to
show very good growth when incubated on culture media
with varying concentration of Cr (VI). The minimum

Figure 2 shows the effect of varying Cr (VI) concentrations

(3.535 mg L1) on Cr (VI) reduction by Acinetobacter sp.
B9. At lower initial concentrations, i.e., 3.5 and 7.0 mg L1
8
Uninoculated media
Inoculated with autoclaved cells
Inoculated with live cells

7
6

[ Cr (VI) ], mg L-1

Results

5
4
3
2
1
0
0

10

24
Time (h)

48

72

Fig. 1 Time course reduction of Cr (VI) by Acinetobacter sp. B9. 2 %

(v/v) inoculum size of B9 cells were inoculated in nutrient medium
containing initial 7.0 mg L1 of Cr (VI) and incubated at 30 C and
200 rpm. Controls in the form of un-inoculated media and media
inoculated with autoclaved B9 cells (2 %,v/v) were also incubated with
experimental setup

6632

Environ Sci Pollut Res (2013) 20:66286637

40

30
[Cr (VI) ], mg L-1

and high inoculum concentrations, lower level of metal

removal was observed. The chromate reduction potential
of the strain decreased to 40 and 38 %, respectively at
inoculum sizes of 4 and 6 % (v/v). Whereas, at lower
inoculum size of 1 % (v/v), only 34 % reduction of chromate
was found.

3.5 mg/l
7.0 mg/l
14 mg/l
21 mg/l
28 mg/l
35 mg/l

35

25
20
15

Effect of heavy metals

10

24

48
Time (h)

72

96

Fig. 2 Effect of varying Cr (VI) concentrations on Cr (VI) reduction

potential of Acinetobacter sp. B9. B9 cells (2 %, v/v) were inoculated
in nutrient media (pH 7.0) containing varying Cr (VI) concentrations
followed by incubation at 30 C and 200 rpm

of Cr (VI), 67 % of chromium reduction was observed at

first 24 h. With further increase in concentration of Cr (VI)
to 14, 21, 28, and 35 mg L1, the reduction percentages of
respective initial chromium content was found to decline to
35, 21, 21, and 15 %, respectively, at 24 h of incubation.
The Cr reduction was found to remain stationary at further
time period of 48, 72, and 96 h at all Cr concentrations. This
behavior might be attributed to decrease in number of viable
cells after 24 h of bacterial growth. At higher concentration
of 35 mg L1 of Cr (VI), the viable cells were found to
decrease from 2.0 108 CFU mL 1 at 12 h and 1.7
108 CFU mL1 at 24 h to 11.0105 CFU mL1 at 48 h.
The value was further decreased to 4.5105 CFU mL1 at
72 h. Similarly, at low concentration of 7.0 mg L1 Cr (VI)
tested, the viable cell counts decreased from 7.5
107 CFU mL1 at 12 h and 3.5107 CFU mL1 at 24 h to
3.0105 CFU mL1 at 48 h.
Since no major Cr (VI) reduction was observed after 24 h
of incubation at all Cr concentrations, a 24-h time period
was used for subsequent studies viz. effect of pH, inoculum
sizes, heavy metals, and metabolic inhibitors on chromium
reduction.

a 120
100
Cr (VI) reduction (%)

From range of heavy metals selected to monitor the influence of heavy metals on Cr (VI) removal ability of strain
B9, the Cu was found to stimulate the process. In presence
of Cu, the initial Cr (VI) content of 7.0 mg L1 was reduced
to non-detectable level after 24 h of incubation, i.e., 100 %
chromate reduction was observed (Fig. 3a). Whereas, presence of Ni, Zn, and Pb showed inhibitory effect on chromate
reduction as only 34.6, 10.25, and 9 % Cr (VI) reduction
was observed in presence of Ni, Zn, and Pb, respectively.

80
60
40
20
0
Control

Cu

Ni
Heavy metals

Zn

Pb

b120
100
Cr (VI) reduction (%)

80

60

40

Effect of pH and inoculum size

20

The effect of different initial pH of media on Cr (VI)

reduction showed maximum Cr (VI) reduction by the strain
in the pH range of 6.0 to 8.0 with 70, 67, and 66 % reduction
at pH 6.0, 7.0, and 8.0, respectively. With further increase in
pH to 9.0 and 10, the Cr (VI) reductions percentages were
found to be decreased to 50 and 47 %, respectively. While at
acidic pH of 5.0, only 40 % of Cr (VI) reduction was
observed.
Inoculum size of 2 % (v/v) was determined to be optimum for chromate reduction using strain B9. While at low

0
7

10

[Cr (VI)], mg L

14

21

-1

Fig. 3 a Effect of different heavy metals on Cr (VI) reduction by

Acinetobacter sp. B9. Different heavy metals (7.0 mg L1) were
supplemented to nutrient media containing initial 7.0 mg L1 of Cr
(VI) followed by inoculation of 2 % (v/v) of B9 cells and incubation at
30 C and 200 rpm. b Effect of varying Cr (VI) concentrations on Cr
(VI) reduction by Acinetobacter sp. B9 in presence of Cu ions. Two
percent B9 cells were inoculated in media containing 7.0 mg L1 of Cu
(II) while concentration of Cr (VI) was varied

Environ Sci Pollut Res (2013) 20:66286637

Since Cu was found to stimulate the reduction process, it

was worthwhile to study the effect of Cu at higher concentrations of Cr (VI). Figure 3b shows that complete, i.e.,
100 % Cr (VI) reduction was observed with initial
10 mg L1 of Cr (VI), while 95 and 78 % of chromate
reduction was observed at initial 14 and 21 mg L1 of Cr
(VI), respectively in presence of 7.0 mg L1 of Cu.
Effect of metabolic inhibitors
Chromate reduction of 35 and 41 % was observed in presence of NaN3 and NF, respectively, as compared to 67 % of
that in their absence. Hence, presence of sodium azide
(NaN3) and sodium fluoride (NF) in culture media remarkably inhibited Cr (VI) reduction potential of strain B9.
Scanning electron microscopy

6633

absence of Cr (VI), the cells appeared to be distinctly clear

and discrete, with an average length and width of 0.95 and
0.69 m, respectively. On the other hand, appearance of
cells adherence or clump formation was observed in the
presence of chromium. The average length and width of
cells, grown in the presence of chromium was estimated to
be 1.25 and 0.69 m, respectively. Thus, the cells turn out to
be elongated lengthwise with no change in width in presence of chromium stress.
Transmission electron microscopy
Transmission electron micrographs of cells grown in presence and absence of Cr (VI) are presented in Fig. 5. Clear
and distinct electron dense particles can be seen in the
cytoplasm and cells wall of bacterial cells grown in presence
of Cr (VI). Since, the micrographs were recorded without

Figure 4 shows the scanning electron micrographs of B9

cells grown in the presence and absence of Cr (VI). In

Fig. 4 Scanning electron micrographs of Acinetobacter sp. B9 cells. a

Cells grown in absence of Cr (VI) (control). b Cells grown in presence
of Cr (VI)

Fig. 5 Transmission electron micrographs of B9 cells. a Cells grown

in absence of Cr (VI) [scale 20 nm]. b Cells grown in presence of Cr
(VI) [scale 20 nm]. Black spots in case of Cr (VI) exposed cells shows
deposition of Cr particles

6634

Environ Sci Pollut Res (2013) 20:66286637

double staining the samples with metals salts of uranyl

acetate and lead citrate and no deposition was detected in
the control cells grown in the absence of Cr (VI), the
electron dense deposition seen in the Cr (VI) exposed cells
is because of absorption of Cr (VI) by B9 cells only.

Table 1 Physico-chemical parameters and

heavy metals content of
wastewater

Fourier transform infrared spectroscopy

FT-IR analysis of B9 cells grown in presence and absence of
chromium was carried out to find the role of functional
groups involved in absorption/adsorption of chromium.
The FT-IR spectrum of control and cells exposed to chromium are shown in Fig. 6. The chromium exposed biomass
exhibited major changes in the region of 3,430
3,270 cm 1 and 1,2401,232 cm 1. Slight shifting of
peak/band at 1,079 cm1 and changes in the region of
800850 cm1 was observed in the spectrum of chromiumtreated cells as compared to control. A new peak was also
found at 786 cm1 in FT-IR spectra of Cr-treated cells.
Application of Acinetobacter sp. B9 in removal of heavy
metal, chromium from industrial wastewater

BDL below detection

limit
a

Except pH all values

are in mg L1

Parameters

Value (mg L1)

Color
pHa
COD
TSS
TDS
Cr (VI)
Total Cr
Ni
Fe
Cu
Pb
Cd

Yellow
8.3
700
856
1,868
60
493
102
13.7
1.2
0.5
BDL

a 100

35

90

30
25

70
60

20

50

15

40
30

10

[Cr (VI)], mg L-1

The physico-chemical parameters and heavy metals content

of the wastewater sample are presented in Table 1. Since Cr
(VI), total Cr, and Ni are present in very high concentration
and above the limits of discharge according to Indian standard (http://www.cpcb.nic.in/Industry-Specific-Standards/
Effluent); bioremediation of this heavy-metals-rich wastewater sample was attempted by using Acinetobacter sp. B9
cells. During treatment with B9 cells, the Cr (VI) content of
the wastewater was found to decrease rapidly with time as
compared to control (Fig. 7a). Initial Cr (VI) content was
reduced to 30, 49, 81.3, 91.3, and 93.7 % at 24, 48, 96, 120,
and 144 h of bacterial treatment, respectively. While in case
of control set up (without B9 cells), only 19.6, 32.4, 52.5,

Cr (VI) removal (%)

80

20

10

0
0

24

48

66
Time (h)

96

120

144

Control, % Cr (VI) removal

Acinetobacter sp. B9, % Cr (VI) removal

Control, [Cr (VI)], mg/L

Acinetobacter sp. B9, [Cr (VI)], mg/L

b 100

Control
Acinetobacter sp. B9

90
80
70

Removal (%)

50
45
40

% Transmission

35

50
40
30

30
25

60

20

10
0

20

Cr (VI)

Total Cr

Ni

15
10
5
0
4000

3500

3000

2500
2000
Wavenumber (cm-1)

1500

1000

500

Fig. 6 FTIR spectra of biomass grown in (a) presence, and (b) absence
of Cr (VI)

Fig. 7 a Application of Acinetobacter sp. B9 in removal of Cr (VI)

from heavy-metal-based industrial wastewater. B9 cells were inoculated in wastewater containing initial 30 mg L1 of Cr (VI) followed by
incubation at 30 C and 200 rpm. Control set was not inoculated
extraneously with B9 cells. b Removal of Cr (VI), total Cr, and Ni
after 6 days of industrial wastewater treatment with Acinetobacter sp.
B9. B9 cells were inoculated in wastewater containing initial 30, 246,
and 51 mg L1 of Cr (VI), total Cr, and Ni, respectively, followed by
incubation at 30 C and 200 rpm. Control set was without B9 cells

Environ Sci Pollut Res (2013) 20:66286637

60.1, and 72.8 % of Cr (VI) reduction was observed at 24,

48, 96, 120, and 144 h, respectively.
Total Cr and Ni content in the wastewater was also found
to decrease with a reduction percentages of 55.4 and 68.94,
respectively, after 144 h of treatment (Fig. 7b). Control
setup on the other hand showed merely 13.6 and 20.3 %
reduction of total Cr and Ni, respectively at the same time
period. During treatment, the original yellow color of wastewater sample was observed to fade with time and after 144 h
of treatment, the yellow color was almost diminished in case
of B9 inoculated experimental set up.

Discussion
The isolated strain of Acinetobacter sp. could grow well in
presence of high concentration of Cr (VI) and other heavy
metals and thus seems to be promising for bioremediation of
wastewaters contaminated with multiple heavy metals. There
are few reports available in literature on Cr (VI) removal by
Acinetobacter spp. (Srivastava and Thakur 2007; Pei et al.
2009; Essahale et al. 2012; Panda and Sarkar 2012; Samuel et
al. 2012). For instance, Essahale et al. (2012) and Panda and
Sarkar (2012) have reported tannery isolates belonging to
Acinetobacter sp. AB1 and Acinetobacter sp. PD S2 that can
tolerate 400 mg L1 and 4.2 g L1 of Cr (VI), respectively. On
the other hand, Pei et al. (2009) reported Acinetobacter
haemolyticus strain isolated from textile dye effluent that can
tolerate up to 90 mg L1 of Cr (VI).
In the present study, Acinetobacter sp. B9 was also found
to reduce the toxic concentration of Cr (VI) from synthetic
media. Though complete Cr (VI) reduction was not observed even at the lowest concentration used (3.5 mg L1),
but reduction rate was found to increase with increase in Cr
(VI) concentrations of up to 28 mg L1 (0.097, 0.194, 0.202,
0.20, and 0.245 mg L 1 h1 at 3.5, 7.0, 14, 21, and
28 mg L1, respectively). The reduction rate was found to
decline at 35 mg L1 concentration (0.220 mg L1 h1).
Similar trend on chromate reduction have also been reported
by Zakaria et al. (2007) and Dey and Paul (2012) using A.
haemolyticus and Arthrobacter sp. SUK 1201, respectively.
The lower rate of reduction at 35 mg L1 of Cr (VI) could be
due to Cr (VI) toxicity on Acinetobacter sp. B9 cells as also
reported by Pang et al. (2011) and Dey and Paul (2012) in
case of Pseudomonas aeruginosa and Arthrobacter sp. SUK
1201, respectively at higher chromium concentration.
Optimum pH for B9-mediated chromate removal was
found to be in the range of 6.08.0. Srivastava and Thakur
(2007) and Panda and Sarkar (2012) have reported pH 7.0 to
be optimum for chromate removal in case of Acinetobacter
sp PCP3 and Acinetobacter sp. PD 12 S2, respectively. In
contrast, optimum pH of 10.0 was reported by Essahale et
al. (2012) in case of Acinetobacter sp. AB 1.

6635

During the study on effect of other heavy metals on

chromium reduction, copper ions were found to stimulate
chromate reduction efficiency of strain B9. This stimulating
effect of Cu might be due to the fact that Cu works as
prosthetic group for many reductases, and thus helps in
protection of electron transport chain or acts as an electron
redox center and in some cases act as shuttle for electrons
transport between protein subunits (Abe et al. 2001; He et
al. 2009; Dey and Paul 2012). Almost similar effect of Cu
ions on the reduction of Cr (VI) is also evident from the
study of Dey and Paul (2012) and He et al. (2009) using
Arthrobacter sp. SUK 1201 and Ochrobactrum sp. CsCr-3,
respectively. In the present case, inhibition of chromate
reduction by metabolic inhibitors like sodium azide and
sodium fluoride also suggests the possible role of electron
transport chain in the chromate removal (Wani et al. 2007;
Dey and Paul 2012).
Bacterial cells are known to adapt to toxic concentration of
heavy metals including Cr (VI) by changing their morphology
(Naik et al. 2012). During SEM analysis, Acinetobacter B9
cells were also found to show some morphological changes
due to chromium stress. The chromium-treated cells appeared
to be attached to each other because of more flexibility and
elasticity in the cell wall or exopolysaccharide layer. Similar
observation in case of chromium-treated cells is also reported
by Panda and Sarkar (2012). Increase in cell size due to
chromium stress as observed in the present case is also
reported by Srivastava and Thakur (2007) and Samuel et al.
(2012) in case of Acinetobacter sp. PCP3 and Acinetobacter
junii VITSUKMW2, respectively.
TEM of Cr (VI)-treated cells shows chromium absorption
or accumulation by the cells. Atomic absorption spectroscopy analysis of the spent media samples also showed
reduction in initial total chromium content, suggesting chromium absorption by microbial cells (data not shown). The
TEM observation is in agreement with the previous report of
Pei et al. (2009) and Srivastava and Thakur (2007) on A.
haemolyticus and Acinetobacter sp. PCP3, respectively.
The finding of chromium uptake is further supported by
FT-IR, as FT-IR analysis also showed conformational changes
in functional groups of biomass due to metal absorption.
Comparison of control and Cr-treated biomass spectra shows
major changes in the region of 3,4303,270 cm1, which
represent changes in -OH and -NH groups of glucose and
proteins, respectively (Pei et al. 2009). The suppression of
band at 1,2401,232 cm1 in chromium-treated cells might be
an effect of chromium association with microbial phosphate
moieties or SO3 group present in the cell membrane (Pei et al.
2009; Chatterjee et al. 2011). Slight broadening of peak at
1,079 cm1 in case of chromium-treated biomass represents
the role of Pyridine (I)(CH) (Ye et al. 2010). A change in the
spectrum of chromium-treated biomass in the region of 850
800 cm1 was also observed which showed the involvement

6636

of sulfonate group (Das and Guha 2007; Pei et al. 2009). The
above comparative changes in the spectrum of Cr-treated
biomass with that of Cr-untreated cells indicate the involvement of chromium with functional groups of bacterial cell
(Dhal et al. 2010). The presence of peak at 786 cm1 is
characteristic of Cr-O vibration which shows the presence of
Cr on the cell wall of chromium-treated B9 cells. This interaction is also evident from the elongation of chromium-treated
B9 cells as shown by scanning electron microscopy studies.
Similar observation of Cr-O vibrations in the region of 782
725 cm1 and its correlation with elongation of cells due to
cells interaction with chromium is also reported by Samuel et
al. (2012) in case of Bacillus subtilis VITSUKMW1, A. junii
VITSUKMW2, and Escherichia coli VITSUKMW3.
To test the applicability and efficiency of strain B9 in
bioremediation of chromium from industrial wastewater, the
strain B9 was inoculated in chromium and other heavy-metalsladen real industrial wastewater. The Acinetobacter sp. B9
strain along with the native microorganisms of wastewater
was observed to rapidly remove hexavalent chromium, total
chromium, and nickel from the wastewater as compared to
control. During treatment, the original yellow color of the
wastewater (due to the presence of high content of dichromate
ions) was found to fade, suggesting the removal of Cr (VI) from
the wastewater. The removal of some amount of heavy metals
in case of control might be due to the presence of indigenous
microbes of wastewater. Since same physical and nutritional
conditions (aeration, temperature, and nutrients) have been
provided to both control and experimental setups; that might
have arranged favorable conditions for growth of indigenous
microbes also. But comparatively lesser or slow Cr and Ni
removal in case of control confirmed that strain B9 augmented
the removal of these heavy metals from wastewater.

Conclusion
Overall, the following outcome emerged from this study: (1)
the isolated strain B9 can tolerate high concentration of Cr
(VI) and also able to significantly reduce the concentration of
Cr (VI) from the media. (2) The results of FT-IR and TEM
showed chromium absorption/accumulation by bacterium
cells grown in presence of Cr. SEM micrograph also showed
visible morphological changes in the cells exposed to chromium. (3) Simultaneous removal of high concentrations of
total Cr, Cr (VI), and Ni was observed when strain B9 was
applied for bioremediation of real industrial wastewater.
These studies shows that Acinetobacter sp. B9 could be
effectively used as bioremediation tool for alleviation of high
concentration of toxic heavy metals from industrial wastewater.
Acknowledgments The financial support provided by University
Grants Commission (UGC), Govt. of India in the form of Senior

Environ Sci Pollut Res (2013) 20:66286637

Research Fellowship (SRF) to AB is gratefully acknowledged. We also
acknowledge electron microscope facility at All India Institute of
Medical Sciences (AIIMS), New Delhi for SEM and Advanced Instrumentation Research Facility at Jawaharlal Nehru University, New
Delhi for TEM and FTIR facilities.

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