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RESEARCH ARTICLE
Evaluation of Acinetobacter sp. B9 for Cr (VI) resistanceand
detoxification with potential application in bioremediationof
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) detoxificationand
resistance mechanism of a newly isolated strain (B9)
ofAcinetobacter sp. Bioremediation potential of the strain B9
isshown by simultaneous removal of major heavy metals in-cluding
chromium from heavy-metals-rich metal finishingindustrial
wastewater. Strain B9 tolerate up to 350 mg L1 ofCr (VI) and also
shows level of tolerance to Ni (II), Zn (II), Pb(II), and Cd (II).
The strain was capable of reducing 67 % ofinitial 7.0 mg L1 of Cr
(VI) within 24 h of incubation, whilein presence of Cu ions 100 %
removal of initial 7.0 and10 mg L1 of Cr (VI) was observed with in
24 h. pH in therange of 6.08.0 and inoculum size of 2 % (v/v) were
deter-mined to be optimum for dichromate reduction. Fourier
trans-form infrared spectroscopy and transmission
electronmicroscopy studies suggested absorption or intracellular
ac-cumulation and that might be one of the major mechanismsbehind
the chromium resistance by strain B9. Scanning elec-tron microscopy
showed morphological changes in the straindue to chromium stress.
Relevance of the strain for treatmentof 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, after144 h of treatment in a batch mode.
Keywords Chromium (VI) . Heavymetals . Resistance .
Bioaugmentation . Acinetobacter sp. B9 .
Industrialwastewater
Introduction
Heavy metals are the major toxic constituent of various
in-dustrial wastewaters and pose greater risk for the environmentif
not treated properly prior to their disposal. In the midst
ofvarious heavy metals, chromium (VI) is considered as
highlyhazardous metal due to its oxidizing, mutagenic, and
carcino-genic properties and almost every statutory body in the
worldhas listed Cr (VI) as priority toxic chemical for
control(Cheung and Gu 2007). Moreover, the persistent stabilitydue
to non-biodegradable nature and high solubility in aque-ous
environment increases the toxicity and contaminationability of this
heavy metal (Cheung and Gu 2007; Desai etal. 2008; Colin et al.
2012; Liu et al. 2012). Electroplating,leather tanning processes,
chromate ore processing, dyes andpigments, wood preservation, alloy
making, and metalfinishing industries are major source of Cr (VI)
and its com-pounds into the environment (Suksabye et al. 2008;
Quintelaset al. 2009; Ye et al. 2010). As per US-EPA, the
permissiblelimit of less than 0.05 mg L1 of Cr (VI) has to be
attainedbefore disposal of chromate containing wastewater into
natu-ral environment (Srivastava and Thakur 2007; Dhal et al.2010;
Sharma and Adholeya 2011).
Various conventional physico-chemical processes such aschemical
reduction followed by precipitation, ion exchange,adsorption (coal,
activated carbon, fly ash, alum, and agri-cultural waste), reverse
osmosis, membrane separation, andsolvent extraction are available
for treatment of chromiumand 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
totheir tendency to cause secondary pollution along with highcost
and high energy requirement, the focus has been shiftedto better
alternative ways of treatment, i.e., biological
Responsible editor: Robert Duran
A. Bhattacharya :A. Gupta (*)University School of Environment
Management, Guru GobindSingh Indraprastha University, Sector
16-C,Dwarka, New Delhi 110078, Indiae-mail: [email protected]
Environ Sci Pollut Res (2013) 20:66286637DOI
10.1007/s11356-013-1728-4
-
method of treatment using chromium-resistant microbes(Pei et al.
2009; Liu et al. 2012). These methods are notonly economic and
efficient but also address the problemsassociated with these types
of pollutants containing waste-water in a natural way. Various
reports on use of metal-tolerant viable microbes for treatment of
Cr (VI) containingwastewater have been reported by different
authors in thelast 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
as-sociated with Cr (VI) by adopting a number of
detoxificationstrategies like biotransforming Cr (VI) to less toxic
form of Cr(III) using enzymes/metabolites, adsorption (absorption),
orintracellular accumulation inside the cell (Sharma andAdholeya
2011; Sagar et al. 2012). The selection of efficientmicrobial
strain, capable of tolerating and reducing high con-centration of
toxicants and study of microbetoxicant interac-tion, is
prerequisite for development of efficient bioremediationstrategy
(Pei et al. 2009; Das and Mishra 2010). The presentwork was
attempted to study the Cr (VI) tolerance and detox-ification
potential of a newly isolated strain (B9) ofAcinetobacter sp., and
elucidation of its chromium resistancemechanism using various
analytical techniques like scanningelectron 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 chromiumis also shown by treatment of real metal
finishing industrialwastewater using the isolated bacteria.
Material and methods
Material
The media components were obtained from Hi-MediaLaboratories
(Mumbai, India). Potassium dichromate(K2Cr2O7) was used as source
of Cr (VI) and procured fromSISCO research laboratories (Mumbai,
India). Diphenylcarbazide 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 fromthe
wastewater of local common effluent treatment plantsituated in New
Delhi, India. The isolate was identifiedusing 16S r-RNA sequencing
from Microbial Type CultureCollection and Gene Bank (MTCC),
Institute of MicrobialTechnology (IMTECH) Chandigarh, India. The
strain B9was maintained on nutrient agar slants at 4 C and
sub-cultured every 20 days interval.
Preparation of mother culture
B9 inoculum was prepared by transferring a loopful of
stockculture into sterile nutrient broth (pH 7.5) containing(g L1):
peptone, 5.0; NaCl, 5.0; yeast extract, 1.5; beefextract, 1.5
followed by overnight incubation at 30 C and200 rpm in an orbital
shaker (Innova, Brunswick).
Chromium tolerance studies
The minimum inhibitory concentration (MIC) of Cr (VI) forstrain
B9 was studied at various initial Cr (VI) concentrations,ranging
from 35 to 425 mg L1. Sterile nutrient broth (50 mL)taken in
250-mLErlenmeyer flasks were amendedwith varyingCr (VI)
concentrations, separately. All the flasks were asepti-cally
inoculated with 2 % (v/v) B9 mother cultures followed byincubation
at 30 C and 200 rpm in an orbital shaker. The MICwasmeasured on the
basis of growth (A600 nm) observedwith in48 h. The minimal
concentration of Cr (VI) inhibiting completegrowth 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 250-mL
Erlenmeyer flasks as batch reactors under aerobic condi-tions. The
potential of strain B9 for Cr (VI) reduction wasassessed 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
withconstant shaking at 200 rpm in an orbital shaker for 96 h.
Inorder 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)
mediainoculated with autoclaved B9 cells. During incubation,
ali-quots of samples (1.0 mL) were withdrawn periodically (12,24,
48, 72, and 96 h) from all the flasks for estimation ofresidual Cr
(VI). The samples were centrifuged at 2,376g,4 C for 10 min and
supernatants thus obtained were used forthe analysis of residual Cr
(VI). The reduction rate was calcu-lated using formula: reduction
rate=C0Ct/t (Pang et al. 2011),where C0initial Cr (VI)
concentration (mg L
1), CtCr (VI)concentration (mg L1) at time t, and tincubation
time (h).
The estimation of viable cells was done by using spreadplate
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 theisolate,
the effects of initial pH (5.0, 6.0, 7.0, 8.0, 9.0, and10.0) and
inoculum size (1, 2, 4, and 6 %; v/v) were
Environ Sci Pollut Res (2013) 20:66286637 6629
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monitored on Cr (VI) reduction. In both the cases, initial7.0 mg
L1 of Cr (VI) was used. Samples were withdrawnafter 24 h of
incubation for residual Cr (VI) determination.
Effect of heavy metals and metabolic inhibitors on
chromatereduction
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 supplementedwith
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 ionswere found to stimulate Cr (VI)
reduction, its effect wasfurther studied with higher initial Cr
(VI) concentration of10, 14, and 21 mg L1. Flasks were then
inoculated with 2 %(v/v) of B9 inoculum followed by incubation as
describedabove and analyzed for residual chromium at 24 h time
period.
As Cr (VI) reduction is reported to be associated
withrespiratory chain of the bacteria in some cases (Wani et
al.2007; Dey and Paul 2012), the effect of
variousmetabolite/respiratory inhibitors was tested to evaluate the
roleof respiratory chain on Cr (VI) reduction in Acinetobacter
sp.B9 strain also. NaN3 and NaF (inhibitor of electron
transportchain) at 1 mM concentration were supplemented to
culturemedia (pH 7.0) having initial 7.0 mg L1 of Cr (VI)
individu-ally followed by inoculation with 2 % (v/v) of B9 cells.
Thesamples 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 andabsence of
35 mg L1 of Cr (VI) at 30 C and 200 rpm.Cells were harvested at 4 C
after 48 h of growth usingcentrifugation at 9,503g for 10 min. The
cell pellets werewashed by suspending in 2 mL of sterile saline
solutionfollowed by vortexing for 2 min. The resultant cell
suspensionwas again centrifuged at 9,503g and 4 C for 10 min to
pelletdown the cell mass. This washing procedure was repeatedthrice
followed by overnight fixation at 4 C in modifiedKarnovskys
fixative (David et al. 1973) containing 1 %glutraldehyde and 4 %
paraformaldehyde in 0.1 M phosphatebuffer (pH 7.4). After removal
of fixative, the cells werefinally suspended in 0.1 M phosphate
buffer and subsequentlyprocessed at All India Institute of Medical
Sciences (AIIMS),New Delhi, India for SEM analysis. Scanning
electron micro-graphs were recorded by using Leo 435VP SEM at
AIIMS.
Transmission electron microscopy
B9 cells were grown in basal media with and without10.6mg L1 of
Cr (VI). After 48 h of growth at 30 C incubation
temperature and 200 rpm shaking speed, the cells wereharvested
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 wereafterward processed at
advanced instrumentation research facil-ity, Jawaharlal Nehru
University (JNU), New Delhi, India, forTEM analysis. Transmission
electron micrographs wererecorded 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
cellsgrown in the presence of chromium (VI), the FT-IR spec-trum of
the normal and Cr (VI)-treated B9 cells were studiedusing FT-IR
spectroscopy (Varian 7000 FTIR).
To carry out the analysis, bacterial culture samples grownfor 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 pelletdown the cell mass. The cells
pellets were washed withsaline prior to drying overnight at 60 C
(Pei et al. 2009;Dhal et al. 2010). The dried pellet was crushed to
finepowder using mortar and pestle. The resultant powderedbiomass
was mixed with KBr in the ratio of about 1:100(one part biomass and
100 part KBr) and pressed to formKBr disks. The FT-IR spectra of
dried biomass in KBr phasewas recorded by using FT-IR spectrometer
in the range of5004,000 cm1.
Application of strain B9 in treatment of chromium-richindustrial
wastewater
Physico-chemical characterization of wastewater
Raw industrial wastewater was collected from localelectroplating
and metal-based industry located in nationalcapital region of
Delhi, India. The wastewater was charac-terized for following
parameters: pH, chemical oxygen de-mand (COD), total suspended
solids (TSS), total dissolvedsolids (TDS), and heavy metal content
viz, Cr (VI), total Cr,Ni, Cu, Fe, Pb, and Cd. The physico-chemical
parameterslike COD, TSS, and TDS were estimated according
tostandard APHA method (APHA 1998). Heavy metals anal-ysis 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
highconcentration of Cr (VI), total Cr, and Ni, the sample
wasdiluted (1:1) with mineral salt medium (Dong et al. 2008) tomake
the final Cr (VI), total Cr, and Ni concentrations ofabout 30, 246,
and 51 mg L1, respectively.
6630 Environ Sci Pollut Res (2013) 20:66286637
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For inoculum preparation, 4.0 mL of overnight grown B9mother
culture was centrifuged to pellet down the cell mass.The pellet was
washed twice with sterile saline solution andfinally suspended in 2
mL of saline solution. The resultantbacterial suspension was used
to inoculate 100 mL of dilutedindustrial wastewater supplemented
with 0.5 % (w/v) of glu-cose as carbon source in 250-mL Erlenmeyer
flask. Thetreatment was carried out at 30 C under constant shaking
of200 rpm for 144 h. Control flask containing 100 mL of
dilutedwastewater with 0.5 % (w/v) glucose but without B9 cells
wasalso incubated under similar conditions to monitor the role
ofindigenous microorganisms in heavy metals reduction.During
incubation, aliquots of samples were withdrawn peri-odically (24,
48, 66, 96, 120, and 144 h) for estimation ofresidual Cr (VI) in
the control and experimental setups.
Analytical techniques
The Cr (VI) concentration was estimated colorimetrically at540
nm using 1, 5 diphenyl carbazide method (APHA1998). Quantification
of total chromium and other heavymetals were made using atomic
absorption spectrophotom-eter (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 differ-ences
in their individual results in each set of experiments wereless
than 5 %. The errors bars shown in figures representstandard
deviations, calculated by using Microsoft Excel.
Results
Identification of isolate B9 and its metal tolerance
Isolate B9 was isolated from the wastewater of commoneffluent
treatment plant (CETP), located in New Delhi,India. Since this CETP
receives wastewater from nearbyindustries majority of which are
metal-based, it was likelythat such environment will provide
natural adaptation toheavy-metals-resistant microbes. A number of
bacterialstrains could successfully be isolated from this
heavy-metal-contaminated site and one potential strain B9
wasselected for detailed studies. The isolate B9 was identifiedas
Acinetobacter baumannii using 16S rRNA sequencing(1,432 bp and
99.86 similarity) from Microbial TypeCulture Collection and Gene
Bank (MTCC), Institute ofMicrobial Technology (IMTECH), Chandigarh,
India, anddeposited at its collection bank with accession No.
MTCC10506.
The bacterial strain B9 was checked for its ability to growin
the presence of various heavy metals. It was found toshow very good
growth when incubated on culture mediawith varying concentration of
Cr (VI). The minimum
inhibitory concentration of strain B9 towards Cr (VI)
wasdetermined to be 350 mg L1. The isolate also had a degreeof
tolerance to Zn (II), Pb (II), Ni (II), and Cd (II) asdetermined by
its ability to grow in presence of these heavymetals (data not
shown). Unless otherwise mentioned, stud-ies on MIC determination
for Cr (VI) and degree of toler-ance to other heavy metals were
done only in case of B9among the various isolated bacterial
strains.
Cr (VI) removal potential of Acinetobacter sp. B9
Cr (VI) removal potential of the isolated bacterium wasstudied
by using nutrient broth as basal media with initial7.0 mg L1 of Cr
(VI). The isolate B9 was able to signifi-cantly remove Cr (VI) with
overall rate of 0.194 mg L1 h1
at 7.0 mg L1 concentration. However, complete reductionof Cr
(VI) could not be obtained even after 96 h of incuba-tion. At 24 h,
67 % reduction of Cr (VI) was observed,which remained almost
constant till 96 h (Fig. 1 and 2). Tosee the effects of media
components and other abiotic fac-tors on Cr (VI) removal, two types
of control set ups wererun in parallel. Cr (VI) reductions in case
of both thecontrols were observed to be less then 10 %. This
showedthat the Cr (VI) reduction observed in case of
experimentalsetup was due to the live cells of Acinetobacter sp. B9
andmedia components and other abiotic factors did not
playsignificant role in the Cr (VI) reduction.
Effect of varying initial Cr (VI) concentrations
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
0
1
2
3
4
5
6
7
8
0 10 24 48 72Time (h)
[ Cr (
VI) ]
, mg
L-1
Uninoculated mediaInoculated with autoclaved cellsInoculated
with live cells
Fig. 1 Time course reduction of Cr (VI) by Acinetobacter sp. B9.
2 %(v/v) inoculum size of B9 cells were inoculated in nutrient
mediumcontaining initial 7.0 mg L1 of Cr (VI) and incubated at 30 C
and200 rpm. Controls in the form of un-inoculated media and
mediainoculated with autoclaved B9 cells (2 %,v/v) were also
incubated withexperimental setup
Environ Sci Pollut Res (2013) 20:66286637 6631
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of Cr (VI), 67 % of chromium reduction was observed atfirst 24
h. With further increase in concentration of Cr (VI)to 14, 21, 28,
and 35 mg L1, the reduction percentages ofrespective initial
chromium content was found to decline to35, 21, 21, and 15 %,
respectively, at 24 h of incubation.The Cr reduction was found to
remain stationary at furthertime period of 48, 72, and 96 h at all
Cr concentrations. Thisbehavior might be attributed to decrease in
number of viablecells after 24 h of bacterial growth. At higher
concentrationof 35 mg L1 of Cr (VI), the viable cells were found
todecrease from 2.0108 CFU mL1 at 12 h and 1.7108 CFU mL1 at 24 h
to 11.0105 CFU mL1 at 48 h.The value was further decreased to
4.5105 CFU mL1 at72 h. Similarly, at low concentration of 7.0 mg L1
Cr (VI)tested, the viable cell counts decreased from 7.5107 CFU mL1
at 12 h and 3.5107 CFU mL1 at 24 h to3.0105 CFU mL1 at 48 h.
Since no major Cr (VI) reduction was observed after 24 hof
incubation at all Cr concentrations, a 24-h time periodwas used for
subsequent studies viz. effect of pH, inoculumsizes, heavy metals,
and metabolic inhibitors on chromiumreduction.
Effect of pH and inoculum size
The effect of different initial pH of media on Cr (VI)reduction
showed maximum Cr (VI) reduction by the strainin the pH range of
6.0 to 8.0 with 70, 67, and 66 % reductionat pH 6.0, 7.0, and 8.0,
respectively. With further increase inpH to 9.0 and 10, the Cr (VI)
reductions percentages werefound to be decreased to 50 and 47 %,
respectively. While atacidic pH of 5.0, only 40 % of Cr (VI)
reduction wasobserved.
Inoculum size of 2 % (v/v) was determined to be opti-mum for
chromate reduction using strain B9. While at low
and high inoculum concentrations, lower level of metalremoval
was observed. The chromate reduction potentialof the strain
decreased to 40 and 38 %, respectively atinoculum sizes of 4 and 6
% (v/v). Whereas, at lowerinoculum size of 1 % (v/v), only 34 %
reduction of chromatewas found.
Effect of heavy metals
From range of heavy metals selected to monitor the influ-ence of
heavy metals on Cr (VI) removal ability of strainB9, the Cu was
found to stimulate the process. In presenceof Cu, the initial Cr
(VI) content of 7.0 mg L1 was reducedto non-detectable level after
24 h of incubation, i.e., 100 %chromate reduction was observed
(Fig. 3a). Whereas, pres-ence of Ni, Zn, and Pb showed inhibitory
effect on chromatereduction as only 34.6, 10.25, and 9 % Cr (VI)
reductionwas observed in presence of Ni, Zn, and Pb,
respectively.
0
5
10
15
20
25
30
35
40
0 24 48 72 96Time (h)
[Cr (
VI) ]
, mg
L-1
3.5 mg/l7.0 mg/l14 mg/l21 mg/l28 mg/l35 mg/l
Fig. 2 Effect of varying Cr (VI) concentrations on Cr (VI)
reductionpotential of Acinetobacter sp. B9. B9 cells (2 %, v/v)
were inoculatedin nutrient media (pH 7.0) containing varying Cr
(VI) concentrationsfollowed by incubation at 30 C and 200 rpm
0
20
40
60
80
100
120
Control Cu Ni Zn PbHeavy metals
Cr (V
I) re
duct
ion
(%)
0
20
40
60
80
100
120
7 10 14 21[Cr (VI)], mg L-1
Cr (V
I) red
uct
ion (%
)a
b
Fig. 3 a Effect of different heavy metals on Cr (VI) reduction
byAcinetobacter sp. B9. Different heavy metals (7.0 mg L1)
weresupplemented to nutrient media containing initial 7.0 mg L1 of
Cr(VI) followed by inoculation of 2 % (v/v) of B9 cells and
incubation at30 C and 200 rpm. b Effect of varying Cr (VI)
concentrations on Cr(VI) reduction by Acinetobacter sp. B9 in
presence of Cu ions. Twopercent B9 cells were inoculated in media
containing 7.0 mg L1 of Cu(II) while concentration of Cr (VI) was
varied
6632 Environ Sci Pollut Res (2013) 20:66286637
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Since Cu was found to stimulate the reduction process, itwas
worthwhile to study the effect of Cu at higher concen-trations of
Cr (VI). Figure 3b shows that complete, i.e.,100 % Cr (VI)
reduction was observed with initial10 mg L1 of Cr (VI), while 95
and 78 % of chromatereduction 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 pres-ence of
NaN3 and NF, respectively, as compared to 67 % ofthat in their
absence. Hence, presence of sodium azide(NaN3) and sodium fluoride
(NF) in culture media remark-ably inhibited Cr (VI) reduction
potential of strain B9.
Scanning electron microscopy
Figure 4 shows the scanning electron micrographs of B9cells
grown in the presence and absence of Cr (VI). In
absence of Cr (VI), the cells appeared to be distinctly clearand
discrete, with an average length and width of 0.95 and0.69 m,
respectively. On the other hand, appearance ofcells adherence or
clump formation was observed in thepresence of chromium. The
average length and width ofcells, grown in the presence of chromium
was estimated tobe 1.25 and 0.69 m, respectively. Thus, the cells
turn out tobe elongated lengthwise with no change in width in
pres-ence of chromium stress.
Transmission electron microscopy
Transmission electron micrographs of cells grown in pres-ence
and absence of Cr (VI) are presented in Fig. 5. Clearand distinct
electron dense particles can be seen in thecytoplasm and cells wall
of bacterial cells grown in presenceof Cr (VI). Since, the
micrographs were recorded without
Fig. 4 Scanning electron micrographs of Acinetobacter sp. B9
cells. aCells grown in absence of Cr (VI) (control). b Cells grown
in presenceof Cr (VI)
Fig. 5 Transmission electron micrographs of B9 cells. a Cells
grownin 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 showsdeposition of Cr particles
Environ Sci Pollut Res (2013) 20:66286637 6633
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double staining the samples with metals salts of uranylacetate
and lead citrate and no deposition was detected inthe control cells
grown in the absence of Cr (VI), theelectron dense deposition seen
in the Cr (VI) exposed cellsis because of absorption of Cr (VI) by
B9 cells only.
Fourier transform infrared spectroscopy
FT-IR analysis of B9 cells grown in presence and absence
ofchromium was carried out to find the role of functionalgroups
involved in absorption/adsorption of chromium.The FT-IR spectrum of
control and cells exposed to chro-mium are shown in Fig. 6. The
chromium exposed biomassexhibited major changes in the region of
3,4303,270 cm1 and 1,2401,232 cm1. Slight shifting ofpeak/band at
1,079 cm1 and changes in the region of800850 cm1 was observed in
the spectrum of chromium-treated cells as compared to control. A
new peak was alsofound at 786 cm1 in FT-IR spectra of Cr-treated
cells.
Application of Acinetobacter sp. B9 in removal of heavymetal,
chromium from industrial wastewater
The physico-chemical parameters and heavy metals contentof the
wastewater sample are presented in Table 1. Since Cr(VI), total Cr,
and Ni are present in very high concentrationand above the limits
of discharge according to Indian stan-dard
(http://www.cpcb.nic.in/Industry-Specific-Standards/Effluent);
bioremediation of this heavy-metals-rich waste-water sample was
attempted by using Acinetobacter sp. B9cells. During treatment with
B9 cells, the Cr (VI) content ofthe wastewater was found to
decrease rapidly with time ascompared to control (Fig. 7a). Initial
Cr (VI) content wasreduced to 30, 49, 81.3, 91.3, and 93.7 % at 24,
48, 96, 120,and 144 h of bacterial treatment, respectively. While
in caseof control set up (without B9 cells), only 19.6, 32.4,
52.5,
5001000150020002500300035004000Wavenumber (cm-1)
% T
rans
miss
ion
0
5
10
15
20
25
30
35
40
45
50 a
b
Fig. 6 FTIR spectra of biomass grown in (a) presence, and (b)
absenceof Cr (VI)
Table 1 Physico-chem-ical parameters andheavy metals content
ofwastewater
BDL below detectionlimitaExcept pH all valuesare in mg L1
Parameters Value (mg L1)
Color Yellow
pHa 8.3
COD 700
TSS 856
TDS 1,868
Cr (VI) 60
Total Cr 493
Ni 102
Fe 13.7
Cu 1.2
Pb 0.5
Cd BDL
0
10
20
30
40
50
60
70
80
90
100a
b
0 24 48 66 96 120 144Time (h)
Cr (
VI) r
emoval (
%)
0
5
10
15
20
25
30
35
[Cr (VI)],
mg L
-1
Control, % Cr (VI) removal Acinetobacter sp. B9, % Cr (VI)
removalControl, [Cr (VI)], mg/L Acinetobacter sp. B9, [Cr (VI)],
mg/L
0
10
20
30
40
50
60
70
80
90
100
Cr (VI) Total Cr Ni
Rem
ov
al (
%)
ControlAcinetobacter sp. B9
Fig. 7 a Application of Acinetobacter sp. B9 in removal of Cr
(VI)from heavy-metal-based industrial wastewater. B9 cells were
inoculat-ed in wastewater containing initial 30 mg L1 of Cr (VI)
followed byincubation at 30 C and 200 rpm. Control set was not
inoculatedextraneously with B9 cells. b Removal of Cr (VI), total
Cr, and Niafter 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 byincubation at 30 C and 200 rpm.
Control set was without B9 cells
6634 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 foundto
decrease with a reduction percentages of 55.4 and
68.94,respectively, after 144 h of treatment (Fig. 7b).
Controlsetup on the other hand showed merely 13.6 and 20.3
%reduction of total Cr and Ni, respectively at the same timeperiod.
During treatment, the original yellow color of waste-water sample
was observed to fade with time and after 144 hof treatment, the
yellow color was almost diminished in caseof B9 inoculated
experimental set up.
Discussion
The isolated strain of Acinetobacter sp. could grow well
inpresence of high concentration of Cr (VI) and other heavymetals
and thus seems to be promising for bioremediation ofwastewaters
contaminated with multiple heavy metals. Thereare few reports
available in literature on Cr (VI) removal byAcinetobacter spp.
(Srivastava and Thakur 2007; Pei et al.2009; Essahale et al. 2012;
Panda and Sarkar 2012; Samuel etal. 2012). For instance, Essahale
et al. (2012) and Panda andSarkar (2012) have reported tannery
isolates belonging toAcinetobacter sp. AB1 and Acinetobacter sp. PD
S2 that cantolerate 400 mg L1 and 4.2 g L1 of Cr (VI),
respectively. Onthe other hand, Pei et al. (2009) reported
Acinetobacterhaemolyticus strain isolated from textile dye effluent
that cantolerate up to 90 mg L1 of Cr (VI).
In the present study, Acinetobacter sp. B9 was also foundto
reduce the toxic concentration of Cr (VI) from syntheticmedia.
Though complete Cr (VI) reduction was not ob-served 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 L1 h1 at 3.5, 7.0, 14,
21, and28 mg L1, respectively). The reduction rate was found
todecline at 35 mg L1 concentration (0.220 mg L1 h1).Similar trend
on chromate reduction have also been reportedby 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 bedue to Cr (VI) toxicity on
Acinetobacter sp. B9 cells as alsoreported by Pang et al. (2011)
and Dey and Paul (2012) incase of Pseudomonas aeruginosa and
Arthrobacter sp. SUK1201, respectively at higher chromium
concentration.
Optimum pH for B9-mediated chromate removal wasfound to be in
the range of 6.08.0. Srivastava and Thakur(2007) and Panda and
Sarkar (2012) have reported pH 7.0 tobe optimum for chromate
removal in case of Acinetobactersp PCP3 and Acinetobacter sp. PD 12
S2, respectively. Incontrast, optimum pH of 10.0 was reported by
Essahale etal. (2012) in case of Acinetobacter sp. AB 1.
During the study on effect of other heavy metals onchromium
reduction, copper ions were found to stimulatechromate reduction
efficiency of strain B9. This stimulatingeffect of Cu might be due
to the fact that Cu works asprosthetic group for many reductases,
and thus helps inprotection of electron transport chain or acts as
an electronredox center and in some cases act as shuttle for
electronstransport between protein subunits (Abe et al. 2001; He
etal. 2009; Dey and Paul 2012). Almost similar effect of Cuions on
the reduction of Cr (VI) is also evident from thestudy of Dey and
Paul (2012) and He et al. (2009) usingArthrobacter sp. SUK 1201 and
Ochrobactrum sp. CsCr-3,respectively. In the present case,
inhibition of chromatereduction by metabolic inhibitors like sodium
azide andsodium fluoride also suggests the possible role of
electrontransport chain in the chromate removal (Wani et al.
2007;Dey and Paul 2012).
Bacterial cells are known to adapt to toxic concentration
ofheavy metals including Cr (VI) by changing their morphology(Naik
et al. 2012). During SEM analysis, Acinetobacter B9cells were also
found to show some morphological changesdue to chromium stress. The
chromium-treated cells appearedto be attached to each other because
of more flexibility andelasticity in the cell wall or
exopolysaccharide layer. Similarobservation in case of
chromium-treated cells is also reportedby Panda and Sarkar (2012).
Increase in cell size due tochromium stress as observed in the
present case is alsoreported by Srivastava and Thakur (2007) and
Samuel et al.(2012) in case of Acinetobacter sp. PCP3 and
Acinetobacterjunii VITSUKMW2, respectively.
TEM of Cr (VI)-treated cells shows chromium absorptionor
accumulation by the cells. Atomic absorption spectros-copy analysis
of the spent media samples also showedreduction in initial total
chromium content, suggesting chro-mium absorption by microbial
cells (data not shown). TheTEM observation is in agreement with the
previous report ofPei et al. (2009) and Srivastava and Thakur
(2007) on A.haemolyticus and Acinetobacter sp. PCP3,
respectively.
The finding of chromium uptake is further supported byFT-IR, as
FT-IR analysis also showed conformational changesin functional
groups of biomass due to metal absorption.Comparison of control and
Cr-treated biomass spectra showsmajor changes in the region of
3,4303,270 cm1, whichrepresent changes in -OH and -NH groups of
glucose andproteins, respectively (Pei et al. 2009). The
suppression ofband at 1,2401,232 cm1 in chromium-treated cells
might bean effect of chromium association with microbial
phosphatemoieties or SO3 group present in the cell membrane (Pei et
al.2009; Chatterjee et al. 2011). Slight broadening of peak at1,079
cm1 in case of chromium-treated biomass representsthe role of
Pyridine (I)(CH) (Ye et al. 2010). A change in thespectrum of
chromium-treated biomass in the region of 850800 cm1 was also
observed which showed the involvement
Environ Sci Pollut Res (2013) 20:66286637 6635
-
of sulfonate group (Das and Guha 2007; Pei et al. 2009).
Theabove comparative changes in the spectrum of Cr-treatedbiomass
with that of Cr-untreated cells indicate the involve-ment of
chromium with functional groups of bacterial cell(Dhal et al.
2010). The presence of peak at 786 cm1 ischaracteristic of Cr-O
vibration which shows the presence ofCr on the cell wall of
chromium-treated B9 cells. This inter-action is also evident from
the elongation of chromium-treatedB9 cells as shown by scanning
electron microscopy studies.Similar observation of Cr-O vibrations
in the region of 782725 cm1 and its correlation with elongation of
cells due tocells interaction with chromium is also reported by
Samuel etal. (2012) in case of Bacillus subtilis VITSUKMW1, A.
juniiVITSUKMW2, and Escherichia coli VITSUKMW3.
To test the applicability and efficiency of strain B9
inbioremediation of chromium from industrial wastewater, thestrain
B9 was inoculated in chromium and other heavy-metals-laden real
industrial wastewater. The Acinetobacter sp. B9strain along with
the native microorganisms of wastewaterwas observed to rapidly
remove hexavalent chromium, totalchromium, and nickel from the
wastewater as compared tocontrol. During treatment, the original
yellow color of thewastewater (due to the presence of high content
of dichromateions) was found to fade, suggesting the removal of Cr
(VI) fromthe wastewater. The removal of some amount of heavy
metalsin case of control might be due to the presence of
indigenousmicrobes of wastewater. Since same physical and
nutritionalconditions (aeration, temperature, and nutrients) have
beenprovided to both control and experimental setups; that
mighthave arranged favorable conditions for growth of
indigenousmicrobes also. But comparatively lesser or slow Cr and
Niremoval in case of control confirmed that strain B9 augmentedthe
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 ofCr (VI) from
the media. (2) The results of FT-IR and TEMshowed chromium
absorption/accumulation by bacteriumcells grown in presence of Cr.
SEM micrograph also showedvisible morphological changes in the
cells exposed to chro-mium. (3) Simultaneous removal of high
concentrations oftotal Cr, Cr (VI), and Ni was observed when strain
B9 wasapplied for bioremediation of real industrial wastewater.
These studies shows that Acinetobacter sp. B9 could
beeffectively used as bioremediation tool for alleviation of
highconcentration of toxic heavy metals from industrial
wastewater.
Acknowledgments The financial support provided by
UniversityGrants Commission (UGC), Govt. of India in the form of
Senior
Research Fellowship (SRF) to AB is gratefully acknowledged. We
alsoacknowledge electron microscope facility at All India Institute
ofMedical Sciences (AIIMS), New Delhi for SEM and Advanced
Instru-mentation Research Facility at Jawaharlal Nehru University,
NewDelhi for TEM and FTIR facilities.
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Environ Sci Pollut Res (2013) 20:66286637 6637
Evaluation...AbstractIntroductionMaterial and
methodsMaterialMicroorganismPreparation of mother cultureChromium
tolerance studiesMedia and culture conditions for Cr (VI)
removalEffect of initial pH and inoculum size on Cr (VI)
removalEffect of heavy metals and metabolic inhibitors on chromate
reductionScanning electron microscopyTransmission electron
microscopyFT-IR analysis of B9 cellsApplication of strain B9 in
treatment of chromium-rich industrial wastewaterPhysico-chemical
characterization of wastewaterTreatment of wastewater with
Acinetobacter sp. B9 cells
Analytical techniques
ResultsIdentification of isolate B9 and its metal toleranceCr
(VI) removal potential of Acinetobacter sp. B9Effect of varying
initial Cr (VI) concentrationsEffect of pH and inoculum sizeEffect
of heavy metalsEffect of metabolic inhibitorsScanning electron
microscopyTransmission electron microscopyFourier transform
infrared spectroscopyApplication of Acinetobacter sp. B9 in removal
of heavy metal, chromium from industrial wastewater
DiscussionConclusionReferences