potential of novel bacterial consortium for the remediation of chromium contamination

Potential of Novel Bacterial Consortium for the Remediationof Chromium Contamination

Periyasamy Panneerselvam & Girish Choppala &

Anitha Kunhikrishnan & Nanthi Bolan

Received: 19 September 2012 /Accepted: 6 June 2013 /Published online: 20 November 2013# Springer Science+Business Media Dordrecht 2013

Abstract This study was aimed to examine the efficien-cy of a novel bacterial consortium on the reduction of toxichexavalent chromium [Cr(VI)] to non-toxic trivalent Cr[Cr(III)]. Six Cr(VI)-resistant bacteria (IS1-IS6) were iso-lated from a tannery waste disposal site at Mount Barker,South Australia, of which three viz., IS1, IS2 and IS3wereselected based on Cr(VI) reduction ability in minimalsalt medium. The isolates were identified as Bacillusendophyticus (IS1), Microbacterium paraoxydans (IS2)andBacillus simplex (IS3) by 16S rRNAgene sequencing.

All three isolates were able to tolerate chromium (Cr(VI),300–400 mg L−1), arsenic (As(V), 1,000 mg L−1), copper(Cu(II), 300–400mg L−1) and lead (Pb(II), 1,000mg L−1).The isolates were evaluated both as an individual and as aconsortia for Cr(VI) reduction inminimal salt medium andstorm water, both spiked with 100 mg Cr(VI)L−1. In bothcases, the rate of Cr(VI) reduction was found to be signif-icantly higher in the bacterial consortium inoculation (t½=8.45 for minimal salt medium; 6.02 h for storm water),compared to inoculation with individual isolates (t½=53.3–115.5 h for minimal salt medium; 8.77–9.76 h forstormwater). The rate of Cr(VI) reduction in bothminimalsalt medium and storm water was found to be higher inbacterial consortium inoculation (IS1+IS2+IS3) than inindividual isolate inoculation. This experiment demon-strated that bacterial consortium prepared by using B.endophyticus, M. paraoxydans and B. simplex was moreeffective in Cr(VI) detoxification than application of indi-vidual bacterium. This experiment also proved that abacterial consortium was more effective in Cr(VI)detoxification than the application of individual bacteri-al strain.

Keywords Cr(VI) reduction .Bacillus sp .

Microbacterium sp. . Bacterial consortium .

Tannery waste . Bioremediation

1 Introduction

Chromium is released to the environment through theactivities of various industries including steel, timbertreatment, electroplating, refractory inorganic chemicals

Water Air Soil Pollut (2013) 224:1716DOI 10.1007/s11270-013-1716-9

Guest Editors: R Naidu, Euan Smith, MH Wong, MegharajMallavarapu, Nanthi Bolan, Albert Juhasz, and Enzo Lombi

This article is part of the Topical Collection on Remediation ofSite Contamination

P. Panneerselvam (*)Indian Institute of Horticultural Research,Hessaraghatta Lake Post, Bangalore 560089, Indiae-mail: [email protected]

P. Panneerselvam :G. Choppala :N. BolanCentre for Environmental Risk Assessment andRemediation, University of South Australia,Mawson Lakes, SA 5095, Australia

G. Choppala :N. BolanCooperative Research Centre for Contaminants Assessmentand Remediation of the Environment, University of SouthAustralia,Mawson Lakes, SA 5095, Australia

A. KunhikrishnanChemical Safety Division, Department of Agro–FoodSafety, National Academy of Agricultural Science,Suwon-si, Gyeonggi-do 441-707, Republic of Korea

production and textile manufacturing (Adriano 2001).Chromium exists in several oxidation states; however,only two predominant oxidation states, trivalent Cr[Cr(III)] and hexavalent Cr [Cr(VI)], are dominant inthe environment. Chromium(VI) is a strong oxidizingagent; it is reduced to Cr(III) in biological systems andreacts with nucleic acids to produce mutagenic andcarcinogenic effects (McLean and Beveridge 2001).Furthermore, Cr(VI) is highly mobile and water solublecompared to Cr(III), which is relatively inert, chemicallymore stable and less bioavailable due to its lower cellpermeability (Pal et al. 2005). Since Cr(VI) poses a greatthreat to humans, cleaning up this contaminant from soiland water is essential (Bolan et al. 2003; Choppala et al.2013).

A number of conventional methods are available forremoving Cr(VI) from industrial effluents includingchemical precipitation, chemical reduction, ion exchange,filtration, electrochemical treatment, reverse osmosis,membrane technologies and evaporation recovery(Ahluwalia and Goyal 2007). These processes may beineffective or expensive especially when the concentra-tion of Cr(VI) is low (Nourbakhsh et al. 1994). Therefore,it is important to develop innovative, low-cost, and eco-friendly technologies for the removal of toxic Cr(VI)from the waste water sources and soils.

Recently, microbial remediation of heavy metal(loid)shas gained importance, as it results in processes withclean technologies producing maximum benefits, due toits adaptability and environment-friendly nature. Hence,the search for identifying suitable microbial strains con-tinues; however, only a limited number of microbialspecies in the environment have been examined. Bacte-rial strains have been reported to reduce Cr(VI) to Cr(III)under aerobic, anaerobic or both conditions (Ishibashiet al. 1990; Pattanapipitpaisal et al. 2001; Srinath et al.2001). Reduction of Cr(VI) has been demonstrated invarious bacterial species including Bacillus sp. (Masoodand Malik 2011; Liu et al. 2004) Pseudomonas sp.(Ganguli and Tripathi 2002), Microbacterium sp.(Pattanapipitpaisal et al. 2001) and Arthrobacter sp.(Asatiani et al. 2004).

Avast number of researchers have demonstrated theefficiency of individual microorganism on Cr(VI) re-duction, but only very few studies are available on theeffects of bacterial consortia. The advantage of selectingindigenous bacteria from contaminated environments isthat, they will be better adapted to the relevant contam-inants than alien microbes. Several researchers have

suggested that bacterial Cr(VI) reduction and resistanceare independent processes (Ohtake et al. 1987; Boppand Ehrlich 1988; Megharaj et al. 2003). Chromiumcontamination exerts a selective pressure on microor-ganisms of tannery waste-contaminated soils (Viti et al.2003). Therefore, the present study has been set up withthe objectives of isolating and identifying Cr(VI)-resis-tant bacteria from tannery effluent-contaminated soilsand to examine the ability of a bacterial consortium toreduce Cr(VI) in minimal salt medium and storm water.

2 Materials and Methods

2.1 Soil Samples and Isolation of Bacteria

Composite soil samples (around 10 kg) were collectedfrom a long-term tannery waste-contaminated site atMount Barker, South Australia. The collected soilsamples were characterised for pH, CEC and total or-ganic matter following the procedures described byRayment and Higginson (1992). Soil samples wereserially diluted with sterile water and plated ontoLuria–Bertani (LB) agar (Rehman et al. 2008) amendedwith filter sterilized 200 mg L−1 Cr(VI) as K2Cr2O7 andincubated at 30 °C for 48–72 h to isolate Cr(VI)-tolerantbacteria. Colonies of different morphologies were thenselected from LB agar plates containing Cr(VI). Thestorm water used in this experiment was collected usinga 1-L glass beaker, approximately 20 min after the onsetof rainfall at Mawson Lakes, South Australia and char-acterized for pH, EC and total metal(loid) concentrations.

2.2 Chromium(VI) Reduction Ability of BacterialIsolates in Minimal Salt Medium

A total of six bacteria were isolated from the soilsamples of tannery waste-contaminated site. All sixbacterial isolates (IS1–IS6) were purified and evaluatedfor Cr(VI) reduction in minimal salt medium. Twenty-four hours old bacterial isolates (2.1×109 cells mL−1) wereseparately inoculated (3 % inoculum v/v) into 100 mLliquid minimal salt medium (Na2HPO4—6 g; KH2PO4—3 g; NH4Cl—1 g; NaCl—0.5 g; MgSO4.7H2O—0.25 g;CaCl2—0.01 g; MQ water—1,000 mL) amended with35 mg L−1 Cr(VI) and incubated at 30 °C for 72 h in anorbital shaker. A set of uninoculated controls were pre-pared following the same conditions and procedures.Samples were taken at regular intervals (6, 12, 18, 24,

1716, of 11 Water Air Soil Pollut (2013) 224:1716

48, 66 and 72 h of incubation) and divided equally intotwo sets. One portion was centrifuged at 6,500×g for5 min, and Cr(VI) in the supernatant was determinedcalorimetrically using 1, 5-diphenylcarbazide as the col-our developing reagent (Fulladosa et al. 2006). Theabsorbance was measured at 540 nm by UV–visiblespectrophotometer (Broadway et al. 2010). The solidmaterial remaining in the centrifuge tube was extractedwith 1 M KH2PO4 and analysed for Cr(VI) adsorbed bythe bacterial cells. The amount of Cr(VI) reduction wascalculated from the difference between the amount addedand the amount remaining in solution. In order tomonitorany abiotic Cr(VI) reduction, bacterial cell-free controlswere used. The second portion was used to evaluate cellgrowth by measuring optical density at 600 nm.

The amount of Cr(VI) reduction was estimated fromthe decrease in the concentration of Cr(VI) in mediumsolution using Eq. (1).

Cr ¼ Ci−C f � V

W

� �� ð1Þ

where Cr is the amount of reduced Cr(VI) (mg L−1), Ci

is the initial concentration Cr(VI) (mg L−1) and Cf is theconcentration of KH2PO4 extractable Cr(VI) (mg L−1),V is the solution volume (mL) and W is the weight ofsolid material remaining after extraction (mg).

Based on Cr(VI) reduction ability at 35 mg L−1 anda compatibility test, three isolates viz., Bacillusendophyticus (IS1), Microbacterium paroxytones (IS2)and Bacillus simplex (IS3) were selected and assessedfor their individual and combined performance in Cr(VI)reduction in 100 mL minimal salt medium amended with100 mg L−1 Cr(VI). In this experiment, the three bacterialisolates (IS1, IS2 and IS3) and four bacterial consortiums(IS1+IS2, IS1+IS3, IS2+IS3 and IS1+IS2+IS3) wereevaluated. Three percent (v/v) inoculums of 24-h-oldbacterial cultures (optical density of 0.658 at 600 nm)were inoculated separately into 250-mL flasks containing100 mL medium. Following the inoculation, all flaskswere incubated in amechanical shaker (150 rpm) at 30 °Cfor 120 h. Samples were taken at regular time intervals (1,24, 42, 72 and 120 h) and analysed for residual Cr(VI)concentration by the method as described above.

2.3 Identification of Chromium-Resistant Bacteria

Among six Cr(VI)-resistant bacterial strains (IS1–IS6),three isolates (IS1, IS2 and IS3) were selected based on

Cr(VI) reduction efficiency in minimal salt medium formolecular identification. Twenty-four-hour-old bacte-rial isolates grown in LB agar (IS1, IS2 and IS3) wereused for DNA extraction using a Qiagen BacterialDNA Kit. Bacterial 16S rRNA gene primers 27R5CGGCTACCTTGTTACGACT3 and 1502F 5GAGTTTGATCCTGGCTCAG3 were used for polymerasechain reaction (PCR) amplification of the 16S rRNAgene. The PCR products were purified using the TIAquick midi purification kit and sequenced at the SouthPath and Flinders Sequencing Facility, Flinders MedicalCentre, South Australia. The sequences were initiallyanalysed at the NCBI server (http://www.ncbi.nlm.nih.gov/) using the BLAST tool and corresponding se-quences were downloaded. The bacteria nucleotide se-quences were aligned using the CLUSTALX program.Phylogenetic trees (Figs. 1 and 2) were constructed bythe neighbour-joiningmethod using theMEGA packageversion (Tamura et al. 2007). The sequences were de-posited at GenBank.

2.4 Determination of Cross Resistance to VariousHeavy Metal(loid)s

The cross heavy metal(loid) resistance of the three bac-terial isolates (IS1, IS2 and IS3) was determined usingstandard methods (Rehman et al. 2008; Rajkumar et al.2008) against the different metal(loid)s Cr(VI), arsenate(As(V)), lead (Pb(II)) and copper (Cu(II)) at differentconcentrations (0–1,000 mg L−1). In this experiment,48-well polystyrene microplates (Iwaki polystyrene,sterile, non-treated, flat bottom with lid) were used.Sterile minimal medium was amended with each heavymetal(loid) and inoculated with 24-h-old bacterial cul-tures (v/v). The test microplates were incubated at 30 °Con an orbital shaker at 150 rpm. Bacterial growth wasmeasured in terms of optical density at 600 nm for 5 daysat 24-h intervals using the Bio-Tek®SynergyTM HTMulti-Detection Microplate Reader equipped withKC4 software.

2.5 Chromium(VI) Reduction by Bacterial Isolatesin Storm Water

Storm water spiked with 100 mg L−1 Cr(VI) asK2Cr2O7 was used as the test material. In this experi-ment, the individual isolates and a consortium of thebacterial isolates (IS1+IS2+IS3) were evaluated fortheir performance in Cr(VI) reduction in 100 mL storm

Water Air Soil Pollut (2013) 224:1716 of 11, 1716

http://www.ncbi.nlm.nih.gov/

http://www.ncbi.nlm.nih.gov/

water. Around 3 % inoculum (24-h-old bacterialculture) was introduced into the storm water andincubated in a mechanical shaker (150 rpm) at30 °C for 96 h. Samples were collected at regularintervals (0, 12, 42, 60, 72 and 96 h) and analysedfor residual Cr(VI) concentration by the method asdescribed above in Section 2.2. The uninoculatedstorm water and storm water inoculated with aheat-killed bacterial consortium were maintained ascontrols.

2.6 Statistical Analysis

Estimation of Cr(VI) half-life was calculated basedon the first-order decay rate equation as describedby Choppala et al. (2012). In this equation, the netrate of change in residual Cr(VI) at any instanttime will be equal to the decay rate (Vdec), whichis expressed by the following first-order differentialequation:

Vdec ¼ −dC

dt

� �¼ kC ð2Þ

where k is the first-order decay rate constant (per time),and C is the concentration of residual Cr(VI) in themedium at that instant time. The equation has thefollowing solution:

C ¼ C0e kt ð3Þwhere C0 is the initial amount of Cr(VI) recoveredin the medium at day 0 of the experiment. The rate

constant k was estimated by the nonlinear leastsquares method. The half-life (time taken by theCr(VI) to reduce its concentration to half of the initialvalue) was calculated as:

t1=2 ¼ 0:693

Kð4Þ

All observations, including pH, CEC, metal(loid)contents and Cr(VI) reduction were calculated fromtriplicates of each treatment. Calculations and stan-dard deviations of the replicates were determinedusing Microsoft Excel. Data were subjected to one-wayanalysis of variance. Treatment differences were evalu-ated using least significant difference at p<0.05. Therelationships between bacterial growth and the opticaldensity of different bacterial culture mediums werederived using Grapher software (version 7; GoldenSoftware, Golden, CO).

3 Results and Discussion

3.1 Soil and Storm Water Physical and ChemicalProperties

The pH of the contaminated soil was slightly alkaline(7.64) with a CEC of 48.7 cmol kg−1, a total Cr of 8.1 %and an organic matter content of 51 g kg−1. The pH ofstorm water used in this experiment was neutral (6.96),and its EC and dissolved organic carbon (DOC) were11.4 dS m−1 and 34.21 mg L−1, respectively.

Fig. 1 Phylogenetic tree showing the evolutionary relationshipbetween M. paraoxydans PAN 1974 and other representativenumbers of the genus Microbacterium. The tree has beenconstructed by the neighbor-joining method using the software

MEGA 4.0. Bootstrap values showing significant alignment aredisplayed at the nodes (n=1,000). GenBank accession numberis Pan 1974-HM235673. The bar represents 0.01 nucleotidesper substitution


3.2 Isolation and Identification of Chromium-ResistantBacterial Isolates

In the isolation step, six different Cr(VI)-resistant bac-teria (IS1 to IS6) were isolated from soil samples froma tannery effluent-contaminated site. Based on theCr(VI) reduction ability of these isolates in minimalsalt medium, three isolates (IS1, IS2 and IS3), whichshowed high Cr(VI) reduction, were selected and usedfor molecular identification. The partially amplifiedand sequenced 16S rRNA genes from the isolates ofIS1, IS2 and IS3 were used to determine phylogeneticaffiliations by employing the BLAST program (Figs. 1and 2). The results indicated that the selected isolates(IS1, IS2 and IS3) and their 16S rRNA genes showed100, 99 and 100 % sequence homology to those in thegenus of B. endophyticus, M. paraoxydans and B.simplex, respectively. The nucleotide sequence codingfor the 16S rRNA gene of bacteria, IS1, IS2 and IS3 hasbeen deposited in the GenBank database under accessionnumbers of Pan 1975 HM235674, Pan 1973 HM235672and Pan 1974 HM235673, respectively.

Chromium-resistant bacteria are reported to be verycapable at Cr(VI) reduction when isolated from Cr-contaminated environments (Srinath et al. 2001).Kaushik et al. (2012) reported that M. paraoxydanscould tolerate 100-mg L−1 concentrations of cobalt,nickel, zinc, Cr, selenium and tin; and it might playan important role in the bioremediation of soils. There

was not much information about B. simplex and B.endophyticus on metal(loid) resistance; however, B.simplex was found to be a potential isolate for removalof various metals from wastes (Valentine et al. 1996).

3.3 Effect of Cr(VI) on the Growth of DifferentBacteria

The six Cr(VI)-resistant bacterial isolates were exposedto two different concentrations of Cr(VI) (35 and100 mg Cr(VI) L−1), and their bacterial optical density(OD600) was measured and compared with control inwhich no Cr(VI) was added. The growth pattern ofbacteria was studied in 35mgCr(VI) L−1, and the resultsindicated that among six isolates (IS1–IS6), IS1 (B.endophyticus), IS2 (M. paraoxydans) and IS3 (B. sim-plex) showed higher growth after 96 h of incubationcompared to other isolates (IS4, IS5 and IS6; Fig. 3).However, growth was inhibited by 7.68±0.12 to 9.73±0.15 % compared to the growth of bacteria in Cr(VI)-free medium. Based on the growth performance at35 mg Cr(VI) L−1 concentration, the three isolates,IS1, IS2 and IS3, were further evaluated at 100 mg L−1

Cr(VI) concentrations. It was observed that the growth ofbacteria at higher concentration of Cr(VI) was furtherinhibited by 24±0.38, 22±0.34 and 26±0.46 % in IS1,IS2 and IS3, respectively, compared to the control (Fig. 4).As the reduction of Cr(VI) proceed further, the bacterial

Fig. 2 The neighbor-joiningtree showing the evolution-ary relationship between B.simplex PAN 1973 and B.endophyticus PAN 1975with other related gram-positive Bacillus sp. The treehas been constructed by theneighbor-joining methodusing the software MEGA4.0. Bootstrap values show-ing significant alignment aredisplayed at the nodes(n=1,000). GenBank acces-sion numbers are PAN 1973-HM235672 and PAN 1975-HM235674. The bar repre-sents 0.01 nucleotides persubstitution


growth of individual species as measured by opticaldensity was increased (Fig. 5).

Chaturvedi (2011) reported that B. circulans wasfound to tolerate Cr(VI) concentration as high as4,500 mg L−1, but growth was majorly reduced wheninitial Cr(VI) concentration was increased from 1,110to 4,500 mg L−1; nevertheless, microbial cell growthwas not inhibited in the Cr(VI) concentration rangebetween 500 and 1,110 mg L−1. This observation hasindicated that these microbes can tolerate Cr(VI) up tothis extent, but higher concentrations of Cr(VI) de-crease microbial growth due to extreme toxicity.Garbisu et al. (1998) noticed that Cr(VI) at 52 mg L−1

significantly affected the growth of Bacillus subtiliscells, but growth rate was not measured. High initialconcentrations (1,110 and 4,500 mg L−1) of Cr(VI) were

toxic to the bacterial cells (Chaturvedi, 2011). In thepresent study, a decrease in microbial growth in100 mg L−1 Cr(VI) when compared to 35 mg L−1 maybe due to Cr stress. The inhibitory effect of Cr (VI) onthe growth of microorganisms was dependent upon theCr concentration. It was clearly proved that higherCr(VI) concentration prevented multiplication of bacte-ria (Bopp and Ehrlich 1988; Ezaka and Anyanwu 2011).The inhibition of microbes varies and depends on themetal(loid) and its concentrations in the medium(Hassen et al. 1998). The heavy metals may be toxic tomicroorganisms due to their strong affinity to formcomplexes with the cell membrane constituents, causingloss of integrity and impairment of their functions.However, the microbial resistance to heavy metals isattributable to a variety of detoxifying mechanisms de-veloped by resistant microorganisms (Ezaka andAnyanwu 2011).

3.4 Chromium Reduction of Different BacterialIsolates in the Minimal Salt Medium

Initially, the Cr(VI) reduction ability of the bacterialisolates (IS1–IS6) was studied in minimal salt mediumcontaining 35 μg mL−1 Cr(VI) concentration. Thehalf-life for Cr(VI) reduction (Table 1) revealed thatamong six isolates, IS2 recorded the lowest t1/2(4.22±0.14 h) followed by IS3 (4.50±0.23 h) and IS1(5.72±0.26 h), whereas the half-life of Cr(VI) reductionwas 16.50±0.74, 8.34±0.25 and 13.58±0.63 h in IS4,IS5 and IS6 inoculated treatments, respectively.Based on the Cr(VI) reduction ability at 35 mg L−1

0 40 80 120Time (hours)

0

0.4

0.8

1.2

1.6

Opt

ical

den

sity

at

600

nm

IS-1IS-2IS-3IS-4IS-5IS-6

Fig. 3 Growth pattern of different chromium-tolerant bacterialisolates in minimal salt medium (35 mg L−1 of Cr(VI))

0 40 80 120Time (hours)

0

0.4

0.8

1.2

1.6

Opt

ical

den

sity

at

600

nm

IS-1IS-2IS-3

IS-1 + IS-2IS-1 + IS-3IS-2 + IS-3

IS-1 + IS-2 + IS-3

Fig. 4 Growth of individual and consortium of bacterial isolatesin minimal salt medium (100 mg L−1 of Cr(VI))

0 20 40 60Time (hours)

0

10

20

30

40

Cr(

VI)

red

ucti

on (

mg

L-1

)

0

0.4

0.8

1.2

1.6

Opt

ical

den

sity

at

600

nm

IS-1IS-2IS-3IS-4IS-5IS-6ControlIS-1IS-2IS-3IS-4IS-5IS-6

Fig. 5 The relationship between reduction of Cr(VI) and thebacterial growth as measured by optical density


Cr(VI) concentration, the isolates IS1, IS2 and IS3were further evaluated for their Cr(VI) reductionability at 100 mg L−1 Cr(VI) concentration in minimalsalt medium.

The results demonstrated that the individual isolatesIS1, IS2 and IS3 required 115.5±5.38, 53.3±1.87 and57.7±3.08 h, respectively, for 50 % reduction of Cr(VI)from the initial concentration of 100 mg L−1, whereas itwas in the range of 17.32–21.0 h in inoculated samplescontaining two bacterial cultures (Table 2). Interesting-ly, the half-life of Cr(VI) reduction was found to besignificantly lower (8.45 h) in the bacterial consortium-inoculated treatment comprising three isolates (IS1+IS2+IS3). Molokwane et al. (2008a, b) reported thatmixed cultures of microorganisms (Bacillus sp. andMicrobacterium sp.) achieved higher reduction rates(three to eight times) compared to individual microor-ganisms. Similarly, Muneer et al. (2009) concluded thatthree types of microorganisms (Bacillus thuringiensis,Candida etchellsii and Stylonychia mytilus) grown to-gether in a culture medium could collectively uptake90% of Cr(VI) from the culture medium as against 82%by the combination of two cultures. A wide variety ofmicrobial species have been reported to reduce thehighly soluble and toxic Cr(VI) to the sparingly solubleand non-toxic Cr(III) under aerobic and/or anaerobicconditions (Cheung and Gu 2003). Some of the micro-organisms were not effective in Cr(VI)-reducing capac-ity at elevated levels of Cr(VI) (Pattanapipitpaisal et al.2001). Bader et al. (1999) reported that even a lowconcentration of Cr(VI) was also toxic to microorganismsand inhibited the cell growth.

The time required for complete reduction of Cr(VI)increased with increasing concentrations in the medi-um. Similar findings were previously reported withEnterobacter cloacae (Wang et al. 1989), Escherichiacoli (Shen and Wang 1994), Pseudomonas fluorescensand Bacillus sp. (Wang and Xiao 1995). In the presentstudy, the evaluation of individual bacterium (IS1, IS2and IS3) on Cr(VI) reduction indicated that the half-lifefor Cr(VI) reduction was 4.22±0.14–5.72±0.26 at thelower concentration (35 mg L−1 ) of Cr(VI), whereas atthe higher concentration, the half-life was 53.3±1.87–115.5±5.38 h. This observation clearly indicated thatthe higher concentration of Cr(VI) might have inhibitedthe bacterial cells which in turn decreased the rate ofCr(VI) reduction. However, the rate of Cr(VI) reductionwas observed to be 6.8–13.6 times higher in bacterialconsortium-inoculated treatment (IS1+IS2+IS3) com-pared to individual isolate in minimal salt mediumamended with 100 mg L−1 of Cr(VI).

Different genera of bacteria such as Pseudomonassp. (Rajkumar et al. 2005), Bacillus sp. (Dhal et al.2010), Arthrobacter sp. (Gutiérrez et al. 2010) andMicrobacterium sp. (Pattanapipitpaisal et al. 2001;Gutiérrez et al. 2010) have been reported to be in-volved in Cr(VI) reduction. Reduction of Cr(VI) mayalso be carried out by the microbial-associated com-pounds such as enzymes, amino acids, nucleotides,sugars, vitamins, organic acids or glutathione (Cervanteset al. 2001). The rate of Cr(VI) reduction under aerobicand anaerobic conditions has been shown to increase

Table 1 Half-life of Cr(VI) reduction by different bacterialisolates in minimal salt medium at 35 mg L−1 concentration

Bacteria Equation R2 Half-life of Cr(VI)reduction (h)

IS1 y=40.387e−0.121x 0.9719 5.72±0.26

IS2 Y=25.795e−0.164x 0.9730 4.22±0.14

IS3 Y=49.227e−0.154x 0.9695 4.50±0.23

IS4 y=20.97e−0.042x 0.9156 16.50±0.74

IS5 Y=33.105e−0.083x 0.9439 8.34±0.25

IS6 Y=20.494e−0.051x 0.9569 13.58±0.63

CV (%) 9.12

CD (0.05) 1.43

Values are mean of three replicates±standard deviation

CV coefficient variation, CD critical difference at 5 %

Table 2 Half-life of Cr(VI) reduction by individual and consor-tium of bacterial isolates in minimal salt medium at 100 mg L−1

concentration

Bacteria Equation R2 Half-life of Cr(VI)reduction (h)

IS1 Y=100.77e−0.006x 0.9884 115.5±5.38

IS2 Y=99.09e−0.013x 0.9781 53.30±1.87

IS3 Y=102.26e−0.012x 0.987 57.75±3.08

IS1+IS2 Y=104.16e−0.037x 0.992 18.72±0.84

IS1+IS3 Y=120.28e−0.033x 0.9567 21.00±0.64

IS2+IS3 Y=88.633e−0.04x 0.9601 17.32±0.80

IS1+IS2+IS3 Y=52.031e−0.082x 0.851 8.45±0.29

CV (%) 10.20

CD (0.05) 7.67

Values are mean of three replicates±standard deviation

CV coefficient variation, CD critical difference at 5 %


with an increase in the microbial population (McLeanand Beveridge 2001; Wang and Xiao 1995). In thepresent study, Cr(VI) reduction was increased by 2.0–2.08-fold in the bacterial consortium inoculation (IS1+IS2+IS3) compared to individual isolate, which indicatesthat some synergistic process might have increased theCr(VI) reduction in the mixed bacterial culture. Thisobservation also suggested that inoculation with a bacte-rial consortium was more effective in Cr(VI) reductionthan inoculation with an individual organism.

3.5 Multi-metal(loid) Resistance of Bacterial Isolates

The bacterial isolates IS1, IS2 and IS3 were evaluatedfor their multi-metal(loid) resistance to various heavymetal(loid)s such as As(V), Cr(VI), Cu(II) and Pb(II).The bacterial isolates were found to be resistant toAs(V) (1,000 mg L−1) Cr(VI) (300–400 mg L−1),Pb(II) (1,000 mg L−1) and Cu(II) (300–400 mg L−1;Table 3), and the order of resistance of bacterial isolatesregarding the metal(loid) concentration was As(V)>Pb(II)>Cr(VI)>Cu(II).

All three bacterial isolates (IS1, IS2 and IS3) couldgrow in a medium containing both Pb(II) and As(V) at

1,000 mg L−1; this observation indicates that theseisolates could tolerate even more than 1,000 mg L−1

of both Pb(II) and As(V). Natural habitats are generallycharacterized by the coexistence of a large number oftoxic and nontoxic cations, and therefore, it is necessaryto study the effects of multiple metal(loid)s on microor-ganisms (Verma and Singh 1995). Tolerances of mi-crobes to other metal(loid)s are an added advantage asthis allows them to tolerate the presence of these metal-lic ions while performing their metabolic activities(Masood and Malik 2011). The present findings werecorroborated with the earlier findings of Rehman et al.(2008), who reported that Bacillus sp.ev3 could tolerateCr(VI) (4,800 mg L−1), Pb(II) (800 mg L−1),Cu(II) (200 mg L−1), Cd(II) (50 mg L−1), Zn(II)(400 mg L−1), Ni(II) (4,000 mg L−1) and Hg(II)(50 mg L−1). M. paraoxydans was found to be resistantto many metal(loid)s like arsenite, cobalt, nickel, zinc,Cr, selenium and tin (Kaushik et al. 2012).

Microorganisms isolated from natural environmentscontaminated with heavy metal(loid)s often exhibittolerance to multiple pollutants as they have adaptedto such environments (Abou-Shanab et al. 2007). Thebacterial isolates may have developed metal(loid) re-sistance to protect sensitive cellular components. Themechanisms by which microorganisms exhibit resis-tance include exclusion of metal(loid) ions, extracellularprecipitation, binding of metal(loid) ions to the outersurface of bacteria, enzymatic transformation, precipita-tion by oxidation/reduction reaction and biosynthesis ofmetal(loid) binding proteins or extracellular polymers(Patel et al. 2007; Kaushik et al. 2012). In the presentstudy, the variation of multi metal(loid)-resistant char-acteristics of bacterial isolates (IS1, IS2 and IS3) mightbe characteristic of the organisms and will be very useful

Table 3 Cross heavy metal(loid)s resistance of selected bacterialisolates

Heavy metal(loid)s Resistance of bacterial isolates tometal(loid)s

IS1 IS2 IS3

Chromium (mg L−1) 300 400 400

Lead (mg L−1) 1,000 1,000 1,000

Arsenate (mg L−1) 1,000 1,000 1,000

Copper (mg L−1) 400 400 300

Table 4 Half-life of Cr(VI)reduction by individual andconsortium of bacterial isolatesin storm water at 100 mg L−1

concentration

Values are mean of threereplicates±standard deviation

CV coefficient variation, CDcritical difference at 5 %

Treatments Equation R2 Half-life of Cr(VI)reduction (h)

Storm water+IS1 Y=105.9e−0.072x 0.974 9.62±0.44



Storm water+(IS1+IS2+IS3) Y=86.365e−0.115x 0.995 6.02±0.27

Storm water+(heat-killed bacterialcultures of IS1+IS2+IS3)

Y=98.399e−0.027x 0.890 25.66±0.78

Storm water alone Y=96.754e−0.028x 0.906 24.75±1.15

CV (%) 9.25

CD (0.05) 2.32


for the remediation of sites with mixed contamination ofheavy metal(loid)s. In general, the efflux mechanism ofmicrobes could minimize the damage to cellular com-ponents due to various species of Cr (Shi et al. 1991;Suzuki et al. 1992). In some cases, bacteria harbouringplasmid will give resistance to different metal(loid)s, forexample P. aeruginosa RA65 harboured a single plas-mid (9.5 kb) which mediated metal(loid) resistance(Mohamed and Abo-Amer 2012).

3.6 Bacterial Consortium Inoculation on Cr(VI)Reduction in Storm Water

The effect of individual strains and a bacterial consor-tium (IS1+IS2+IS3) on Cr(VI) reduction in stormwater revealed that the half-life of Cr(VI) reductionwas 9.62±0.44, 8.77±0.30 and 9.76±0.52 h in IS1,IS2 and IS3 bacterial isolate-inoculated treatments,respectively (Table 4), while the half-life of Cr(VI)reduction was decreased to 6.02±0.27 h in the bacterialconsortium-inoculated treatment (IS1+IS2+IS3). Inthe control, the half-life of Cr(VI) reduction was in therange of 24.75±1.15 to 25.66±0.78 h. Among the threeisolates, there was no significant variation in Cr(VI)reduction rate in storm water; however, reduction wassubstantially higher than the uninoculated control. Thisobservation clearly indicated that the rate of Cr(VI)reduction was significantly higher in the bacterialconsortium-inoculated treatment than with the individ-ual isolates. These findings were in accordance withGutiérrez et al. (2010)), where the application of abacterial consortium to the industrial effluent resultedin a higher rate of Cr(VI) reduction than with theindividual isolates.

The reduction of Cr(VI) in the control could beattributed to the presence of DOC in the storm water.These findings indicated that the faster rate of Cr(VI)reduction in storm water inoculated with bacterial con-sortium (IS1+IS2+IS3) than with individual isolatesmight be due to the synergistic effect of the microbes.Inoculation of three types of microbes together in aculture medium effectively remediated Cr(VI)-contam-inated water, better than individual microbial organ-isms (Muneer et al. 2009). Similar observations havebeen made in the case of co-cultures of E. coli ATCC33456 and Pseudomonas putida DMP-1 as reported byShen and Wang (1995). Mauricio et al. (2010) reportedthat a bacterial consortium was capable of 93.8 %Cr(VI) reduction of 81 μg L−1 Cr(VI) of the industrial

effluent under aerobic condition, which reveals theirpossible use in environmental cleanup. Similarly, amixed culture of bacteria isolated from a wastewatertreatment plant in Brits, North-West Province in SouthAfrica could bio-catalytically reduce Cr(VI) at muchhigher concentrations (Molokwane et al. 2008a, b).

4 Conclusions

Six novel Cr(VI)-resistant bacteria were isolated fromsoil samples from a tannery waste disposal site, ofwhich three isolates viz., IS1, IS2 and IS3 wereselected based on Cr(VI) reduction ability in minimalsalt medium. The isolates were identified as B.endophyticus (IS1), M. paraoxydans (IS2) and B. sim-plex (IS3). All three isolates had the ability to toleratedifferent metal(loid)s [Cr(VI), 300–400 mg L−1,As(V), 1,000 mg L–1, Cu(II), 300–400 mg L−1 andPb(II), 1,000 mg L−1] with large degree of variousconcentrations. Among the three bacterial isolates, M.paraoxydans and B. simplex were found to be moreeffective in Cr(VI) reduction when compared to B.endophyticus in minimal salt medium, but there wasno significant (p<0.05) difference in the rate of Cr(VI)reduction in storm water. In the bacterial consortiuminoculation (IS1+IS2+IS3), the rate of Cr(VI) reduc-tion was found to be 84.1–92.6 and 31.3–38.3 %higher in minimal salt medium and storm water,respectively, compared to individual isolate inocula-tion. The bacterial consortium prepared by using B.endophyticus, M. paraoxydans and B. simplex strainsmay have potential applications in the bioremediationof Cr(VI)-contaminated waters. Overall, the resultsof this study clearly showed the potential of nat-urally occurring microorganisms operating togetheras a consortium, in the detoxification of Cr(VI)-contaminated sites.

Acknowledgments The senior author thanks the IndianCouncil of Agricultural Research (ICAR), India for deput-ing overseas training in the field of Bioremediation underNAIP project. The authors are grateful to the CooperativeResearch Centre for Contamination Assessment and Reme-diation of the Environment (CRC CARE), Australia forfunding this research work in collaboration with Universityof South Australia, Mawson Lakes Campus, Adelaide, SA,Australia. The Postdoctoral fellowship program (PJ008650042012)at the National Academy of Agricultural Science, RuralDevelopment Administration, Republic of Korea, supportedDr Kunhikrishnan’s contribution.


References

Abou-Shanab, R. A. I., Van Berkum, P., & Angle, J. S. (2007).Heavy metal resistance and genotypic analysis of metalresistance genes in gram-positive and gram-negative bacte-ria present in Ni-rich serpentine soil and in the rhizosphereof Alyssum murale. Chemosphere, 68, 360–367.

Adriano, D. C. (2001). Trace elements in terrestrial environments:biogeochemistry, bioavailability, and risks of metals. NewYork: Springer.

Ahluwalia, S. S., & Goyal, D. (2007). Microbial and plantderived biomass for removal of heavy metals from waste-water. Bioresour Technol, 98, 2243–2257.

Asatiani, N. V., Abuladze, M. K., & Kartvelishvili, T. M. (2004).Effect of chromium(VI) action on Arthrobacter oxydans.Curr Microbiol, 49, 321–326.

Bader, J. L., Gonzalez, G. L., Goodell, P. C., Ali, A. M., & Pillai,S. D. (1999). Aerobic reduction of hexavalent chromium insoil by indigenous microorganisms. Bioremediation Journal,3, 201–211.

Bolan, N. S., Adriano, D. C., Natesan, R. & Koo, B. J. (2003).Effects of organic amendments on the reduction andphytoavailability of chromate in mineral soil. J EnvironQual, 32, 120–128.

Bopp, L. H., & Ehrlich, H. L. (1988). Chromate resistance andreduction in Pseudomonas fluorescens strain LB300. ArchMicrobiol, 150, 426–431.

Broadway, A., Cave, M. R., Wragg, J., Fordyce, F. M., Bewley, R.J., Graham, M. C., Ngwenya, B. T., & Farmer, J. G. (2010).Determination of the bioaccessibility of chromium in Glasgowsoil and the implications for human health risk assessment.Science of the Total Environment, 409, 267–277.

Cervantes, C., Campos-Garcia, J., Debars, S., Gutierrez-Corona, F., Loza-Tavera, H., Carlos-Tarres-Guzman, M.,& MorenoSanchez, R. (2001). Interaction of chromiumwith microorganisms and plants. FEMS Microbiol Rev, 25,335–347.

Chaturvedi, M. K. (2011). Studies on chromate removal bychromium-resistant Bacillus sp. isolated from tannery efflu-ent. Journal of Environmental Protection, 2, 76–82.

Cheung, K.H., &Gu, J. D. (2003). Reduction of chromate (CrO42−)

by an enrichment consortium and an isolate of marine sulfate-reducing bacteria. Chemosphere, 52, 1523–1529.

Choppala, G. K., Bolan, N. S., Megharaj, M., Chen, Z., & Naidu,R. (2012). The influence of biochar and black carbon onreduction and bioavailability of chromate in soils. J EnvironQual, 41, 1175–1184.

Choppala, G., Bolan, N. S., & Park, J. H. (2013). Chromiumcontamination and its risk assessment in complex environ-mental settings. Adv Agron, 120, 129–172.

Dhal, B., Thatoi, H., Das, N., & Pandey, B. D. (2010). Reductionof hexavalent chromium by Bacillus sp. isolated from chro-mite mine soils and characterization of reduced product. JChem Technol Biotechnol, 85, 1471–1479.

Ezaka, E., & Anyanwu, C. U. (2011). Chromium (VI) toleranceof bacterial strains isolated from sewage oxidationditch. International Journal of Environmental Sciences,1, 1725–1734.

Fulladosa, E., Desjardin, V., Murat, J. C., Gourdon, R., &Villaescusa, I. (2006). Cr (VI) reduction into Cr(III) as a

mechanism to explain the low sensitivity of Vibrio fischeribioassay to detect chromium pollution. Chemosphere, 65,644–650.

Ganguli, A., & Tripathi, A. K. (2002). Bioremediation of toxicchromium from electroplating effluent by chromate reduc-ing Pseudomonas aeruginosa A2Chr in two bioreactors.Appl Microbiol Biotechnol, 58, 416–420.

Garbisu, C., Alkorta, I., Llama, M. J., & Serra, J. L. (1998).Aerobic chromate reduction by Bacillus subtilis. Biodegra-dation, 9, 133–141.

Gutiérrez, A. M., Cabriales, Peña, J. J., & Maldonado, V. M.(2010). Isolation and characterization of hexavalent chro-mium reducing rhizospheric bacteria from a wetland. Int JPhytoremediation, 12, 317–334.

Hassen, A., Saidi, N., Cherif, M., & Boudabous, A. (1998).Effects of heavy metals on Pseudomonas aeruginosa andBacillus thuringiensis. Bioresour Technol, 65, 73–82.

Ishibashi, Y., Cervantes, C., & Silver, S. (1990). Chromiumreduction in Pseudomonas putida. Appl Environ Microbiol,56, 2268–2270.

Kaushik, P., Rawat, N., Mathur, M., Raghuvanshi, P., Bhatnagar,P., Swarnkar, H., & Flora, S. (2012). Arsenic hyper-tolerance in fourMicrobacterium species isolated from soilcontaminated with textile effluent. Toxicology International,19, 188–194.

Liu, Y. G., Xu, W. H., Zeng, G. M., Tang, C. F., & Li, C. F.(2004). Experimental study on Cr(V) reduction by Pseudo-monas aeruginosa. J Environ Sci, 16(5), 797–801.

Masood, F., & Malik, A. (2011). Hexavalent chromium re-duction by Bacillus sp. strain FM1 isolated from heavy-metal contaminated soil. Bull Environ Contam Toxicol,86, 114–119.

Mauricio, G. A., Peña, C. J. J., & Maldonado, V. M. (2010).Isolation and characterization of hexavalent chromium-reducing rhizospheric bacteria from a wetland. Int JPhytoremediation, 12(4), 317–334.

McLean, J., & Beveridge, T. J. (2001). Chromate reduction byPseudomonads isolated from a site contaminated withchromated copper arsenate. Appl Environ Microbiol, 67,1076–1084.

Megharaj, M., Avudainayagam, S., & Naidu, R. (2003). Toxicityof hexavalent chromium and its reduction by bacteria iso-lated from soil contaminated with tannery waste. CurrMicrobiol, 47, 51–54.

Molokwane, P. E., Meli, K. C., & Nkhalambayausi-Chirwa, E.M. (2008a). Chromium(VI) reduction in activated sludgebacteria exposed to high chromium loading: Brits culture(South Africa). Water Res, 42, 4538–4548.

Molokwane, P. E., Meli, C. K., & Chirwa, E. M. (2008b).Chromium (VI) reduction in activated sludge bacteria ex-posed to high chromium loading. Water Sci Technol, 58(2),399–405.

Mohamed, R. M., & Abo-Amer, A. E. (2012). Isolation andcharacterization of heavy-metal resistant microbes from road-side soil and phylloplane. Journal of Basic Microbiolology,52(1), 53–65.

Muneer, B., Rehman, A., Shakoori, F. R., & Shakoori, A.R. (2009). Evaluation of consortia of microorganismsfor efficient removal of hexavalent chromium fromindustrial wastewater. Bull Environ Contam Toxicol,82, 597–600.


Nourbakhsh, M., Sag, Y., Ozer, D., Aksu, Z., Kutsal, T., &Calgar, A. (1994). A comparative study of variousbiosorbents for removal of chromium (e) ions from industrialwastewater. Process Biochem, 29, 1–5.

Ohtake, H., Cervantes, C., & Silver, S. (1987). Decreased chro-mate uptake in Pseudomonas fluorescens carrying a chromateresistance plasmid. J Bacteriol, 169, 3853–3856.

Pal, A., Dutta, S., & Paul, A. K. (2005). Reduction of hexavalentchromium by cell free extract of Bacillus sphaericusAND 303 isolated from serpentine soil. Curr Microbiol,51, 327–330.

Patel, P. C., Goulhen, F., Boothman, C., Gault, A. G., Charnock,J. M., Kalia, K., & Lloyd, J. R. (2007). Arsenate detoxifi-cation in a Pseudomonad hypertolerant to arsenic. ArchMicrobiol, 187, 171–183.

Pattanapipitpaisal, P., Brown, N. L., & Macaskie, L. E. (2001).Chromate reduction and 16SrRNA identification of bacteriaisolated from a Cr (VI) contaminated site. Appl MicrobiolBiotechnol, 57, 257–261.

Rajkumar, M., Ma, Y., & Freitas, H. (2008). Characteriza-tion of metal-resistant plant-growth promoting Bacillusweihenstephanensis isolated from serpentine soil inPortugal. J Basic Microbiol, 48, 500–508.

Rajkumar, M., Nagendran, R., Lee, K. J., & Lee, W. H. (2005).Characterization of a novel Cr6+ reducing Pseudomonassp. with plant growth promoting potential. Curr Microbiol,50, 266–271.

Rayment, G. E., & Higginson, F. R. (1992). Australian laboratoryhandbook of soil and water chemical methods. Melbourne:Reed International Books Australia P/L, Inkata Press.

Rehman, A., Zahoor, A., Muneer, B., & Hasnain, S. (2008).Chromium tolerance and reduction potential of a Bacillussp.ev3 isolated from metal contaminated wastewater. BullEnviron Contam Toxicol, 81, 25–29.

Shen, H., &Wang, Y. T. (1994). Biological reduction of chromium byE. coli. Journal of Environmental Engineering, 120, 560–572.

Shen, H., & Wang, Y. T. (1995). Simultaneous chromium reduc-tion and phenol degradation in a coculture of Escherichia

coli ATCC 33456 and Pseudomonas putida DMP-1. ApplEnviron Microbiol, 61, 2754–2758.

Shi, X. L., Dalal, N. S., & Vallyathan, V. (1991). One-electronreduction of carcinogen chromate by microsomes, mito-chondria, and Escherichia coli: identification of Cr(V) andOH radical. Arch Biochem Biophys, 290, 381–386.

Srinath, T., Khare, S., & Ramteke, P. W. (2001). Isolation ofhexavalent chromium-reducing facultative anaerobes from tan-nery effluent. Journal of General and Applied Microbiology,47, 307–312.

Suzuki, T., Miyata, N., Horitsu, H., Kawai, K., Takamizawa, K.,Tai, Y., & Okazaki, M. (1992). NAD(P)H-dependent chro-mium (VI) reductase of Pseudomonas ambigua G-1: aCr(V)intermediate is formed during the reduction of Cr(VI)to Cr(III). J Bacteriol, 174, 5340–5345.

Tamura, K., Dudley, J., & Nei, M. (2007). MEGA 4: molecularevolutionary genetics analysis (MEGA) software version4.0. Mol Biol Evol, 24, 1596–1599.

Valentine, N. B., Bolton, H. J., Kingsley, M. T., Drake, G. R.,Balkwill, D. L., & Plymale, A. E. (1996). Biosorption ofcadmium, cobalt, nickel, and strontium by a Bacillus sim-plex strain isolated from the vadose zone. J Ind Microbiol,16(3), 189–196.

Verma, S. K., & Singh, S. P. (1995). Multiple metal resistance inthe cynobacterium, Nostoc muscorum. Bull EnvironContam Toxicol, 54, 614–619.

Viti, C., Pace, A., & Giovanetti, L. (2003). Characterizationof Cr (VI)-resistant bacteria isolated from chromium-contaminated soil by tannery activity. Curr Microbiol,46, 1–5.

Wang, P. C., Mori, T., Komori, K., Sasatsu, M., Toda, K., &Ohtake, H. (1989). Isolation and characterization of anEnterobacter cloacae strain that reduces hexavalent chro-mium under anaerobic conditions. Appl Environ Microbiol,55, 1665–1669.

Wang, Y. T., & Xiao, C. (1995). Factors affecting hexavalentchromium reduction in pure cultures of bacteria.Water Res,29, 2467–2474.


potential of novel bacterial consortium for the remediation of chromium contamination

Documents