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: anshurcy@yahoo.com

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

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

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

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

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

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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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