Process Biochemistry 43 (2008) 713–721

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Process Biochemistry
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Hexavalent chromate reductase activity in cytosolic fractions of Pseudomonas

sp. G1DM21 isolated from Cr(VI) contaminated industrial landfill
Chirayu Desai, Kunal Jain, Datta Madamwar *
BRD School of Biosciences, Sardar Patel Maidan, Vadtal Road, P. Box No. 39, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India

A R T I C L E I N F O A B S T R A C T

Article history: Hexavalent chromate reductase activity was localized and characterized in vitro in cytosolic fraction of a
Received 30 March 2007 newly isolated Pseudomonas sp. G1DM21. The suspended culture of the bacterium reduced 99.7% of
Received in revised form 21 December 2007 500 mM Cr(VI) and 93.06% of 1000 mM Cr(VI) in 48 h. The suspended culture repeatedly reduced 100 mM
Accepted 21 February 2008
Cr(VI) within 6 h up to four consecutive inputs. The permeabilized cells of the bacterium reduced 92% of
100 mM Cr(VI) within 6 h. The cell-free extracts (CFE) reduced 90% of 100 mM Cr(VI) in 120 min. The Km
Keywords: and Vmax determined for chromate reductase activity in the CFE were 175 mM Cr(VI) and 1.6 mmoles/min/
Pseudomonas sp. G1DM21
mg of protein, respectively, the Km and Vmax determined in the presence of 0.5 mM NADH were 150 mM
Cr(VI) reduction
Cr(VI) and 2.0 mmoles/min/mg of protein, respectively. Hexavalent chromate reductase activity was
Cell-free extracts
Chromate reductase maximum at 30 8C and pH 7.0. Relative molecular mass (Mr) of the native Cr(VI) reductase in the cytosolic
fraction was estimated as 61.7 kDa. The Cr(VI) reductase activity increased in the presence of metal ions
like Cu2+, Mg2+, Na+ and electron donors like citrate, succinate, acetate and was significantly inhibited in
the presence of metal ions like Hg2+, Ag+, Cd2+, disulfide reducers like 2-mercaptoethanol, while the
respiratory inhibitors had a minute effect on the activity. Scanning probe atomic force microscopy (AFM)
analysis indicates that exposure of Pseudomonas sp. G1DM21 to 1 mM Cr(VI) for 24 h, leads to an increase
in cell length and height.
ß 2008 Elsevier Ltd. All rights reserved.

1. Introduction consequences of the chromate induced toxicity. The intracellular

Hexavalent chromium Cr(VI) ionic forms, such as: HCrO4,
CrO42, and Cr2O72 are ubiquitously employed in many industrial
processes like chrome leather tanning, chrome plating, ceramics,
dyes, paints and pigments manufacturing, textile processing, metal
finishing, wood processing, photographic sensitizer manufacturing
and others. Disposal of these industrial wastes containing high
levels of toxic chromates results in anthropogenic contamination
of pristine environments [1–4]. Most persistent forms of chro-
mium in the environment are the soluble, mobile and most toxic
hexavalent species Cr(VI), usually found as oxyanions, whereas,
trivalent chromium Cr(III) is hundred times less toxic, less soluble
and less mobile, mostly found as oxides, hydroxides or sulfates,
generally bound to organic matter in soils [1,5].
Hexavalent chromates are strong oxidizing agents, mutagens,
teratogens and listed as class A human carcinogens by the US-EPA
[3,6,7]. Chromate oxyanions, analogous in structures with sulfate
and phosphate ions, can readily permeate through biological
membranes and their intracellular reduction results in the dire
* Corresponding author. Tel.: +91 2692 229380; fax: +91 2692 231042.
E-mail addresses: chirayurdesai@gmail.com (C. Desai),
datta_madamwar@yahoo.com (D. Madamwar).
reduction of Cr(VI) generates Cr(V), Cr(III) valence states and
reactive oxygen species (ROS), the molecular mechanisms of
mutagenesis involve the formation of ternary adducts of intracel-
lular Cr(III) with DNA, proteins and oxidative damage of DNA by
Cr(V) and ROS [8–10].
Environmental clean-up strategies for Cr(VI) removal involve
physicochemical or biological detoxification. Major limitations of
physicochemical processes are the high energy inputs, different
chemical treatments and generation of unnecessary sludge, reactive
chemical species as secondary wastes. These problems can be
overcome by biological Cr(VI) detoxification which is more
ecofriendly and an economically feasible technology [5,11,12].
Bioreduction and biosorption of Cr(VI) using bacterial, fungal, yeast
or plant biomass are amongst the most lucrative strategies currently
employed for removal of chromium by biological means [12–16].
Bioreduction of Cr(VI) has been demonstrated in several
bacterial species including Pseudomonas sp. [17–23], Escherichia
coli [24], Bacillus sp. [11,25–27], Desulfovibrio sp. [28], Micro-
bacterium sp. [4], Shewanella sp. [29,30], Achromobacter sp. [31] and
Arthrobacter sp. [32,33]. Direct bacterial reduction of Cr(VI) to
Cr(III) is the most promising practice with proved expediency in
bioremediation. Elucidation of the enzymatic mechanisms
involved in direct chromate reduction is crucial in designing an

1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.02.015
714 C. Desai et al. / Process Biochemistry 43 (2008) 713–721
efficient Cr(VI) bioreduction process and such mechanisms have
been reported in Pseudomonas ambigua G-1 [20], Pseudomonas
putida [21,22], E. coli ATCC 33456 [24], Bacillus sp. AND 303 [34],
Bacillus sp. ES 29 [11], Bacillus sp. QC-2 [27], Bacillus sp. [25].
Several studies implicate the role of hydrogenases [35], nitror-
eductases [36], quinone reductase [37] to function as chromate
reductases. Molecular and proteomic analysis of response to toxic
Cr(VI) stress in E. coli K-12 [38], Shewanella oneidensis MR-1 [39]
indicate several cellular and metabolic changes exerted by Cr(VI).
Minimizing these stress factors affecting the Cr(VI) reducing
bacterium will help in increasing the Cr(VI) bioreduction efficiency
of the bacterium in question.
The present study elucidates the sub-cellar localization and in
vitro characterization of the enzymatic chromate reductase
activity in cytosolic fractions of a newly isolated Pseudomonas
sp. G1DM21. In addition, the morphological changes exerted by
Cr(VI) stress on Pseudomonas sp. G1DM21 have been exemplified
at a nanoscale using atomic force scanning probe microscopy.
2. Materials and methods
2.1. Bacterial isolation and culture conditions
Hexavalent chromium resistant and Cr(VI) reducing bacterial strain was isolated
from long-term chromium polluted landfill site of Gorwa Industrial Estate,
Vadodara, Gujarat, India, receiving chromated wastes for more than 40 years.
Sediment samples of these landfills were contaminated with a chromium level of
10,703 mg/kg as estimated by inductively coupled plasma atomic emission
spectroscopy (ICP-AES) using PerkinElmer ICP Optima-3300RL (PerkinElmer,
Norwalk, Conn). The bacterial strain was screened from the serial dilutions
(101 to 107) of the sediment suspensions plated onto plate count agar media
(Difco, Detroit, MI). The isolate was repeatedly streaked and maintained on 0.5 mM
Cr(VI) containing Luria agar plates. Bacterial cultures grown in Luria-Bertani broth
[tryptone (1 g), NaCl (1 g), yeast extract (0.5 g) dissolved in 100 ml distilled water,
pH 7.0] at 37 8C up to A660 of 1.0 were used as primary inoculums. All the reduction
experiments and enzymatic assays were performed in triplicates with abiotic
controls.
2.2. Phylogenetic identification by 16S rRNA gene sequencing and biochemical
characterization
Bacterial genomic DNA was extracted following a previously described method
[40]. The genomic DNA was diluted appropriately to (20–50 ng) and used as
template in (30 ml) PCR reactions consisting of 1X GeneAmp PCR Gold Buffer
(150 mM Tris–HCl, pH 8.0, 500 mM KCl, 25 mM MgCl2), 50 mM each of GeneAmp1
dNTPs, 20 pmoles each of universal eubacteria primers 16F27N (50 -CCA GAG TTT
GAT CMT GGC TCA G-30 ) and 16R1525 (50 -TTC TGC AGT CTA GAA GGA GGT GWT
CCA GCC-30 ) custom synthesized (MWG Biotech, Ebersberg, Germany) and 1.5 U of
AmpliTaq1 Gold DNA polymerase (Applied Biosystems, Foster City, CA).
Amplification cycles (25 cycles in total) consisted of initial denaturation step at
94 8C for 5 min, followed by a 2 min denaturation step at 94 8C, a 1 min annealing
step at 55 8C, and a 1.5 min elongation step at 72 8C and a final extension step at
72 8C for 15 min were performed using Eppendorf Mastercycler1 gradient PCR
cycler. The purified PCR product was subjected to sequencing by automated DNA
Analyzer 3730 using ABI PRISM1 BigDyeTM Cycle Sequencing kit (Applied
Biosystems, Foster City, CA). The nearly complete sequence (>95%) of bacterial
16S rRNA gene (1504 bp) was submitted to GenBank at NCBI with an accession no.
of DQ416798. BLAST(n) program at NCBI server [41] was used to identify and
download the nearest neighbor sequences from the NCBI database. All the
sequences were aligned using Clustal W 1.6 program at (http://www.ebi.ac.uk/
clustalw), the alignment file thus obtained were analyzed and edited using DAMBE
(Data Analysis in Molecular Biology and Evolution) software package [42]. The
phylogenetic tree was constructed using aligned sequences by the neighbor joining
algorithm using Kimura 2 parameter distance and more than 1000 replicates in
Molecular Evolutionary Genetics Analysis 3.1 software [43]. Biochemical char-
acteristics of the bacterial strain were studied using Rapid IDE strips for biochemical
tests in an automated API system for differential substrate utilization [44].
2.3. Time-course Cr(VI) reduction and growth of Pseudomonas sp. G1DM21
Time-course Cr(VI) reduction was monitored in 50 ml sterile Luria-Bertani (LB)
broth [45–47]. Bacterial cultures were grown for overnight to an A660 of 1.0 in
sterile LB broth. Five milliliters of these suspended cultures were inoculated into
50 ml of sterile LB broth (pH 7.0) in 250 ml flasks amended with 250, 500, 750 and
1000 mM of Cr(VI) concentrations as potassium chromate (K2CrO4). Potassium
chromate (K2CrO4) stock solutions were used as source of Cr(VI) and filter sterilized
using 0.22 mm filters (Millipore, Bedford, MA). Reduction experiments were
followed from 0 to 48 h under gyratory shaking of 150 rpm at 37 8C. Samples (2 ml)
were removed after every 6 h up to 48 h and subjected to centrifugation (6000  g
for 10 min) and supernatants were used to measure remaining Cr(VI) following 1,5-
diphenyl carbazide method by reading absorbance at 540 nm using Spectronic
20D+ spectrophotometer (Milton Roy, USA) [48]. The growth in presence of Cr(VI)
was monitored after every 6 h by measuring absorbance at 660 nm (A660 of
1 = 48 mg/ml cell-mass).
2.4. Repeated detoxification of Cr(VI) by Pseudomonas sp. G1DM21
Bacterial culture grown for overnight to an A660 of 1.0 in 100 ml sterile LB broth
was amended with 100 mM Cr(VI) as final concentration and incubated at 37 8C
under gyratory shaking of 150 rpm. After every 6 h of the incubation time (2 ml)
culture suspensions were withdrawn to measure Cr(VI) remaining as described
above and the culture flasks were repeatedly added with increments of 100 mM
Cr(VI) until saturation in Cr(VI) reduction was observed.
2.5. Cr(VI) reduction by resting cells of Pseudomonas sp. G1DM21
Culture suspensions of Pseudomonas sp. G1DM21 were grown for overnight in
100 ml LB broth (pH 7.0) and harvested by centrifugation at 4000  g at 4 8C, cell
pellets (2 ml) obtained on centrifugation were washed twice with 100 mM
potassium phosphate buffer (pH 7.0) and resuspended in same buffer. Triplicates of
these suspended cell pellets were spiked with Cr(VI) concentrations of 25–200 mM,
vortexed for 5 min and incubated at 30 8C for 6 h. Heat killed (2 ml) culture pellets
were used as control. After incubation of 6 h, the tubes were centrifuged and 200 ml
aliquots were withdrawn from each sample to estimate the remaining Cr(VI) by 1,5-
diphenyl carbazide (DPC) method [48].
2.6. Cr(VI) reduction by permeabilized cells of Pseudomonas sp. G1DM21
Bacterial culture of Pseudomonas sp. G1DM21 was grown for overnight,
harvested and washed with potassium phosphate buffer (pH 7.0) as described
above. The suspended culture pellets were treated with 0.2% (v/v) Tween 80 and
0.2% (v/v) Triton X-100 by vortexing for 20 min to achieve cell permeabilization.
Permeabilized cell suspensions (1 ml) were then added with 25, 50, 100, 150, 200,
250 mM Cr(VI) as final concentrations and incubated for 6 h at 30 8C. Experiments
with each set of permeabilization treatment and Cr(VI) concentrations were
performed in triplicates.
2.7. Preparation of cell-free extracts
Cell-free extracts of Pseudomonas sp. G1DM21 were prepared by modifying the
previously published protocols [34,47]. Bacterial suspensions grown for overnight
in 100 ml LB broth were harvested at 6000  g at 4 8C for 10 min, washed and
resuspended in 100 mM potassium phosphate buffer (pH 7.0). The culture pellets
thus obtained were resuspended in the 5% (v/v) of the original culture volume in
100 mM potassium phosphate buffer (pH 7.0). These cell suspensions were placed
in ice bath and disrupted using an Ultrasonic Probe (Sonics Vibra Cell 500, USA) with
amplitude of 35% at 50 W with 9 s pulses and 1 s off mode for 35 min. The sonicate,
thus obtained was then centrifuged at 32,000  g for 40 min at 4 8C. The
supernatant or the cytosolic fraction thus obtained was then filtered through
0.22 mm filters (Millipore, Bedford, USA) to give the cell-free extracts devoid of
membrane fractions. The sonicated cell pellets were accordingly resuspended in
same volume of phosphate buffer (pH 7.0) and used for subsequent assays.
2.8. Localization and characterization of chromate reductase activity
Cell-free extracts or cytosolic fractions and sonicated pellet or membrane
fractions were used as aliquots (200 ml) for chromate reductase assay in order to
localize the chromate reductase activity.
2.9. Chromate reductase assay
Enzymatic chromate reduction was estimated as described previously using a
standard curve of Cr(VI) 0–20 mM [22,34]. Assay mixtures were modified from those
described in previous studies [34,47]. The reaction system (1.0 ml) was made up of
varying Cr(VI) final concentrations (50–300 mM) in 0.8 ml of 100 mM potassium
phosphate buffer (pH 7.0) added with 0.2 ml aliquots of CFE for chromate reduction.
The system volume of 1.0 ml was kept constant for all experiments. Assay
conditions were kept constant with a reaction time of 30 min and 30 8C unless
stated for assays at differential time intervals and temperature conditions. Unit
enzyme activity for chromate reductase was derived as amount of enzyme that
reduces 1 mmole of Cr(VI) per min at 30 8C. Specific activity was defined as unit
chromate reductase activity per mg protein concentration in the CFE. Protein
concentrations were estimated using Bio-Rad protein assay kit by reading
absorbance at 595 nm, following the principle of Bradford [49]. The enzyme
 C. Desai et al. / Process Biochemistry 43 (2008) 713–721 715

kinetics was studied using the enzymatic progress curve and Lineweaver Burk plot characterization showed the isolate to be an encapsulated, non-
derived using specific activity of chromate reduction by the CFE. Abiotic control
motile, gram-negative short rod exhibiting UV-fluorescence.
reaction mixtures consisted of 100 mM potassium phosphate buffer and respective
Cr(VI) concentrations without the addition of CFE, and subjected to same assay Biochemical characterization performed by automated API system
conditions as those followed for experimental reactions. for differential substrate utilization by the strain G1DM21, showed
a substrate utilization profile similar to related Pseudomonas sp. in
2.10. Cr(VI) reduction by CFE as a function of time the API system database, the list of substrates utilized by the
bacterium is shown in Table 1. The DNA sequencing and BLAST
Cr(VI) reductase activity in the CFE was measured at initial Cr(VI) concentrations of
25, 50, 100, 150 and 200 mM with increasing incubation time intervals (0–120 min). analysis of complete 16S rRNA gene sequence (1.5 kb) of the
strain G1DM21 showed maximum sequence homology (99%) with
2.11. Effect of temperature on Cr(VI) reduction by CFE the complete sequence of Pseudomonas fragi 16S rRNA gene
(D84014). Phylogenetic analysis indicates that the newly isolated
Cr(VI) reductase activity in the CFE was measured at initial Cr(VI) concentrations
of 100 and 200 mM Cr(VI) with 30 min incubation time at different temperatures
strain G1DM21 is affiliated to Pseudomonas species. The phylogeny
(20–70 8C). Non-enzymatic Cr(VI) reduction was checked following inactivation of cluster of G1DM21 along with related Pseudomonas and Gamma-
CFE at 100 8C for 5 min. proteobacteria is depicted in Fig. 1. The genus Pseudomonas has
been reported to exhibit enzymatic machinery for chromate
2.12. Effect of pH on Cr(VI) reduction by CFE bioreduction in previous studies [20–22].
The cell-free extracts were prepared in respective buffers of differential pH range
and the same buffers were used for Cr(VI) reductase assays with 100 mM Cr(VI) as 3.2. Cr(VI) reduction and growth in culture flasks
initial concentration. The buffers used for Cr(VI) reductase assays were potassium
phosphate buffers (pH 5.8–8.0), Tris–HCl buffers (pH 7.2–8.8) and citrate phosphate
The suspended cultures of the bacterium were efficient in Cr(VI)
buffers (pH 4.0–7.0).
reduction even at higher initial chromium concentrations. The
2.13. Effect of metal cations, electron donors and inhibitors on Cr(VI) reduction by CFE Pseudomonas sp. G1DM21 upon incubation for 48 h at 37 8C with
gyratory shaking of 150 rpm, could achieve exponential growth
Cell-free extracts of Pseudomonas sp. G1DM21 were characterized for differential
and efficiently reduce more than 99% of Cr(VI) at the initial
specific activity following Cr(VI) reductase assays in the presence of (1 mM each)
metal ions (Cu2+,Cd2+, Co2+, Pb2+, Zn2+, Mg2+, Na+, Hg2+, Ag+, Ni2+, Ca2+, Ba2+) concentrations of 250 and 500 mM, and more than 93% of 750 and
supplemented as CuCl2, CdCl2, CoCl2, Pb(NO3)2, ZnCl2, MgCl2, Na2SO4, HgCl2, AgCl2, 1000 mM of Cr(VI) as seen in Table 2. An exponential growth was
NiSO4, CaCl2, BaCl2, electron donors (NADH, citrate, succinate, acetate, lactose, observed even at higher initial Cr(VI) concentrations of 750 and
fructose, carbonate, glucose, formate), protein denaturants (2-mercaptoethanol, 1000 mM, but after 24 h of incubation a little increase in the cell
EDTA) and respiratory inhibitors (KCN, NaN3).
density was observed, which was even followed by a decrease in
cell density after 42 h of incubation as reported in previous studies
2.14. Estimation of molecular mass of native Cr(VI) reductase in cell-free extracts
[26,50,32]. The reason for such a growth pattern can be attributed
The relative molecular mass (Mr) of native enzyme responsible for Cr(VI) to the toxicity and stress exerted by chromium on the Cr(VI)
reductase activity in the crude cell-free extract was estimated using gel-filtration reducing bacterium [1,38,39]. Repeated detoxification of 100 mM
chromatography [24]. The crude cell-free extract was loaded onto Sephadex G-150
column (Pharmacia Fine Chemicals, Uppsala, Sweden) with a fractionation range of
Table 1
5–300 kDa and a bed volume of 10 ml. The gel-filtration column was pre-
Biochemical characterization based on differential substrate utilization tests
equilibrated with 100 mM potassium phosphate buffer. (pH 7.0) and calibrated
performed by automated API system for bacterial identification
using non-denatured molecular weight protein standards; alcohol dehydrogenase
(150 kDa), bovine albumin fraction V (66 kDa), carbonic anhydrase (29 kDa), List of substrates Results
myoglobin (17 kDa), RNase A (13.7 kDa) and blue dextran (2000 kDa) (Sigma, USA).
The column elution was carried out using 0.1 M potassium phosphate buffer at a Mannitol Negative
constant flow-rate of 0.2 ml/min at 25 8C. A total of 35 fractions (0.5 ml) were D-Glucose Positive
collected for each of the protein standards and crude cell-free extract. Absorbance of Salicin Negative
the eluted fractions was measured at 280 nm and the chromate reductase activity in D-Melibiose Negative
the CFE fractions supplemented with 0.5 mM NADH was measured as described L-Fucose Negative
above. The elution volume of the CFE fraction giving the highest Cr(VI) reductase D-Sorbitol Negative
activity was used for the relative molecular mass estimation of the native chromate L-Arabinose Negative
reductase enzyme within the cytoplasm of Pseudomonas sp. G1DM21 using the Propionate Negative
standard calibration curve of logarithm of Mr versus elution volumes. Caparate Positive
Valerate Positive
Citrate Positive
2.15. Atomic force microscopy to exemplify morphological changes in Pseudomonas sp.
Histidine Negative
G1DM21 induced by Cr(VI) stress
2-Ketogluconate Negative
Suspended culture of Pseudomonas sp. G1DM21 was grown in 100 ml LB broth for 3-Hydroxy butyrate Positive
24 h in the presence of 1000 mM Cr(VI) which served as experimental sample and in 4-Hydroxy benzoate Negative
L-Proline Positive
the absence of Cr(VI) in LB broth served as control sample. The slide preparations were
made from the experimental and control samples from same stage of growth after Rhamnose Negative
fixation with 4% paraformaldehyde for overnight [39]. Cell suspensions were dried on N-Acetyl glucosamine Negative
D-Ribose Negative
polylysine-coated microscopic slides and then mounted on the stage and adjusted for
imaging with the scanning probe AFMtips. One-dimensional flattened AFM images Inositol Positive
D-Sucrose Negative
were recorded in contact mode at room temperature using siliconnitrideAFMtips
(Veeco Digital Instruments, Santa Barbara, CA). Height distributions were recorded for Maltose Positive
individual cell surfaces, deviations in height values represent areas of the surface Itaconate Positive
extended above the surface baseline (0 nm). Suberate Negative
Malonate Positive
Acetate Positive
3. Results and discussion
DL-Lactate Negative
L-Alanine Positive
3.1. Chromate reducing bacterium 5-Ketogluconate Negative
Glycogen Positive
The Cr(VI) reducing bacterial strain screened from the above- 3-Hydroxy benzoate Negative
L-Serine Negative
mentioned site could tolerate up to 7 mM Cr(VI). Microscopic
716 C. Desai et al. / Process Biochemistry 43 (2008) 713–721

Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequence of Pseudomonas sp. G1DM21 (accession no. DQ416798) and sequences of closest phylogenetic neighbors
obtained by NCBI BLAST(n) analysis, numbers in the parenthesis indicate accession numbers of corresponding sequences. The tree was constructed using neighbor joining
algorithm with Kimura 2 parameter distances in MEGA 3.1 software. E. coli K12 has been taken as an out-group. Numbers at nodes indicate percent bootstrap values above 50
supported by more than 1000 replicates. The bar indicates the Jukes-Cantor evolutionary distance.

Table 2
Hexavalent chromium reduction and growth of Pseudomonas sp. G1DM21 in the presence of different chromate concentrations at temperature of 37 8C and pH 7.0

Time (h) Cr(VI) concentration (mM)

250 500 750 1000

Cr(VI) remaining Cell mass Cr(VI) remaining Cell mass Cr(VI) remaining Cell mass Cr(VI) remaining Cell mass
(mM) (mg/ml) (mM) (mg/ml) (mM) (mg/ml) (mM) (mg/ml)

0 250  0.7 1.5  1 500  0.9 1.5  0.6 750  0.8 1.5  0.5 1000  0.8 1.5  0.9
6 16  0.5 8  0.5 69  1.5 5  1.6 453  1.5 4  0.5 654  0.5 4  0.9
12 15  0.5 10  0.6 57  1.4 8  1.5 47  1 8  0.5 105  1.3 5  0.7
18 10  0.7 45  1 54  1.3 9  1.2 45  0.8 9  0.6 98  0.8 7  0.9
24 5  0.6 50  0.5 45  1.4 43  1.3 43  1.2 38  0.7 88  0.7 37  0.6
30 3  0.5 56  1.5 27  1.2 45  1.1 42  0.7 39  0.5 82  1.2 38  1
36 0.6  0.5 66  1.1 9  0.9 55  0.8 41  0.5 58  1 78  0.5 43  1.3
42 0.5  0.5 84  1.2 3  0.5 78  0.9 40  0.8 45  1 74  0.7 40  1
48 0.2  0.1 97  0.5 1  0.8 83  1 37  0.6 39  1.1 69  0.9 28  0.8

Cr(VI) could be achieved up to four consecutive inputs as shown in
Fig. 2, suggesting the utility of the bacterium for a continuous
Cr(VI) bioreduction process.
3.3. Cr(VI) reductase activity in Pseudomonas sp. G1DM21
reductase activity of the CFE prepared from Cr(VI) induced cells did
not show any significant increase, suggesting the constitutive nature
of the soluble chromate reductase as reported for Pseudomonas CRB5
[23] and Bacillus sp. AND 303 [34]. Bacterial chromate reductases
have been localized either in the membrane fraction [51,54] or in the

The resting and permeabilized cells of the bacterium were

expedient in reducing 25–250 mM Cr(VI) concentrations in 6 h as
shown in Fig. 3. The cell permeabilization significantly increased
the Cr(VI) reduction by the resting cells, as the Tween 80
permeabilized cells could reduce greater than 95% of 25 and
50 mM Cr(VI), greater than 90% of 100 mM Cr(VI) and greater than
70% of 150, 200 and 250 mM of Cr(VI) within 6 h, suggesting an
efficient intracellular mechanism of chromate reduction. The
localization of Cr(VI) reductase activity was made by performing
the assays using the ultrasonicated sub-cellular fractions. The
Cr(VI) reductase activity in the cytosolic fractions (CFE) of cells
grown in absence and presence of 1 mM Cr(VI) was 0.52  0.01 and
0.46  0.01 mmoles/min/mg protein, respectively. Whereas, a negli-
gible Cr(VI) reductase activity of 0.01  0.03 and 0.02  0.01 mmoles/
min/mg protein, respectively, was detected in the membrane
fractions (sonicated cell pellet) in the absence and presence of
1 mM Cr(VI), respectively. These results indicate that the Cr(VI)
reductase activity was solely associated with the CFE (cytosolic Fig. 2. Repeated detoxification of 100 mM Cr(VI) by Pseudomonas sp. G1DM21 at
fractions) and not with the membrane fractions. Also, the Cr(VI) 37 8C under 150 rpm of shaking.
 C. Desai et al. / Process Biochemistry 43 (2008) 713–721
Fig. 3. Resting and permeabilized cell assays for Cr(VI) reduction by Pseudomonas
sp. G1DM21 performed at initial concentrations of 25–250 mM Cr(VI), pH 7.0 and
30 8C.
cytosolic fractions [20,22,24,25,34,41] of the Cr(VI) reducing bacteria.
The Cr(VI) reduction by the cell-free extracts as a function of time is
shown in Fig. 4; wherein, the cell-free extracts could reduce 90% of
100 mM Cr(VI) and 76.5% of 200 mM Cr(VI) in 120 min, suggesting an
effective enzymatic mechanism of Cr(VI) reduction in the cytosolic
fractions of the bacterium. The specific activity of Cr(VI) reduction by
the cell-free extracts increased with an increase in the initial Cr(VI)
concentrations as observed in Bacillus sphaericus AND 303 [34] and
Ochrobacterium intermedium SDCr-5 [50]. The chromate reductase
activity was enhanced by 37% at initial Cr(VI) concentration of
200 mM in the presence of 0.5 mM NADH. The enzymatic progress
curves of the specific Cr(VI) reductase activity in the CFE exhibit a
marked increase in the presence of 0.5 mM NADH with increasing
initial Cr(VI) concentrations as seen in Fig. 5. The dependence of the
Cr(VI) reductases on NADH has been discovered in several previous
reports as in the case of Pseudomonas sp. [20,22], E. coli ATCC 33456
[24] and Bacillus sp. ES 29 [47]. As, studied in E. coli it has been
suggested that the intracellular Cr(VI) reduction using NADH as an
electron donor results in the formation of soluble Cr(III)–NAD+
complexes [52], whereas, it has been deduced that soluble chromate
reductase of P. ambigua G-1 catalyzes three-electron reduction of
Cr(VI) to Cr(III) consuming three molecules of NADH [20]. Following
the enzyme kinetic analysis for the Cr(VI) reductase activity in the CFE
the Km and Vmax were determined to be 175 mM Cr(VI) and
1.6 mmoles/min/mg of protein, respectively, to study the NADH
dependence the Km and Vmax were also determined in the presence of
0.5 mM NADH. The apparent Km and Vmax in the presence of NADH
were 150 mM Cr(VI) and 2.0 mmoles/min/mg of protein. The Vmax for
the chromate reductase activity in the CFE of the Pseudomonas sp.
Fig. 4. Cr(VI) reduction by cell-free extracts as a function of time determined with
100 and 200 mM Cr(VI) as initial concentrations at pH 7.0 and 30 8C.
717
Fig. 5. Cr(VI) reductase activity in cell-free extracts of Pseudomonas sp. G1DM21 in
absence and presence of 0.5 mM NADH at initial concentrations of 50–600 mM
Cr(VI), at pH 7.0 and 30 8C.
G1DM21 is 27 times higher than that reported for P. putida [21], 5
times higher than that of Pseudomonad CRB5 [23] and 59 times higher
than that reported for the purified chromate reductase of P. ambigua
G-1 [20] suggesting the presence of an efficient chromate reductase in
Pseudomonas sp. G1DM21. The relative molecular mass (Mr) of the
native protein responsible for Cr(VI) reductase activity was estimated
to be 61.7 kDa as shown in Table 3. The cytoplasmic Cr(VI) reductases
of P. ambigua G-1 [20], P. putida [22], and E. coli ATCC 33456 [24], have
been reported to exhibit a native molecular mass of 65, 50 and 84 kDa,
respectively.
3.4. Effect of different in vitro conditions on the chromate
reductase activity
The functioning of the chromate reductase of Pseudomonas sp.
G1DM21 was characterized in different in vitro conditions. To
define the optimal pH the Cr(VI) reductase assays were carried in
potassium phosphate, citrate phosphate, Tris–HCl buffers of
differential pH ranges, of the different buffers used the potassium
phosphate buffer showed a characteristic pH curve for the
enzymatic activity with an optimum pH range of pH 7–7.4, as
depicted from Fig. 6. The optimal temperature for the Cr(VI)
reductase activity was 30 8C but, the reductase activity was not
altered significantly up to 50 8C but, when the assays were
performed at 70 8C temperature the reductase activity was
Fig. 6. Effect of pH on Cr(VI) reductase activity in cell-free extracts of Pseudomonas
sp. G1DM21 determined in potassium phosphate buffers (pH 5.8–8.0) with initial
concentration of 100 mM Cr(VI), at 30 8C.
718 C. Desai et al. / Process Biochemistry 43 (2008) 713–721

Table 3
Estimation of relative molecular mass (Mr) of native Cr(VI) reductase fraction from
crude cell-free extracts of Pseudomonas sp. G1DM21 on basis of elution volumes
against molecular mass of gel-filtration protein standards

Analytes Molecular Elution

mass (kDa) volume (ml)

Alcohol dehydrogenase (Yeast) 150 3.4

Serum albumin fraction V (Bovine) 66 6
Carbonic anhydrase (Bovine) 29 10
Myoglobin (Horse) 17 11
Ribonuclease A (Bovine) 13.7 12.5
Cr(VI) reductase fraction of cell-free extract 61.7 6.3

markedly affected as exhibited in Fig. 7. The heat-killed CFE treated

for 5 min at 100 8C did not exhibit any Cr(VI) reductase activity.
The effect of different metal cations, electron donors, respira-
tory inhibitors and protein denaturants on the chromate reductase
Fig. 8. Effect of electron donors on Cr(VI) reductase activity in cell-free extracts of
activity of Pseudomonas sp. G1DM21 was determined as exhibited
Pseudomonas sp. G1DM21 at pH 7.0 and 30 8C.
in Figs. 8–10. Amongst the metal ions tested Mg2+, Na+ and Cu2+
stimulated the Cr(VI) reductase activity of the CFE by 15, 16 and
33%, respectively. Stimulation of chromate reductase activity by Hg2+ and Ag+ strongly inhibited the Cr(VI) reductase activity of
Cu2+ has been observed in case of Bacillus sp. ES 29 [47] and the CFE by 90 and 80%, respectively. Mercurial salts have been
Pseudomonad CRB5 [23], whereas, Cu2+ has also been reported to reported to decrease the chromate reductase activity in P. putida
inhibit the membrane associated chromate reductase activity of [21], Bacillus sp. [25,47] and E. coli ATCC 33456 [24] owing to their
Enterobacter cloacae [53]. The role of Cu2+ in stimulation of Cr(VI) action as disulfide reducers causing denaturation of the reductase
reductases has been attributed to its action as a redox centre protein, these results were further confirmed by using 2-
shuttling electrons between protein subunits or as a protective mercaptoethanol (2-ME) which again decreased the Cr(VI)
agent against oxygen [47]. However, the divalent cations of Zn2+, reductase specific activity by 80%. Inhibition of bacterial Cr(VI)
Ni2+, Ca2+, Ba2+ and Co2+ did not exhibit any significant effect on the reductases by Ag+ has been found in other gram-negative bacteria
reductase activity. Whereas, in the case of Bacillus sphaericus AND like E. coli ATCC 33456 [24] and P. putida [21]. The Cr(VI) reductase
303 metal ions like Ni2+, Cu2+ have been reported to exhibit strong
inhibition on the Cr(VI) reductase activity in the cell-free extracts
and Zn2+ has been reported to inhibit Cr(VI) reduction in
Ochrobacterium sp. [50], Ochrobacterium intermedium SDCr-5
[50], Bacillus sp. [25] and E. coli ATCC 33456 [24]. Similarly, the
reductase activity increased on supplementation in the reaction
mixtures with electron donors like citrate, succinate and acetate by
25, 21 and 14%, respectively. Whereas, other electron donors like
formate, fructose, carbonate, lactose and glucose showed negli-
gible effect on the reductase activity, although in previous reports
on Bacillus sp. glucose has been reported to act as an electron donor
and has been demonstrated to increase Cr(VI) reduction [26,34],
also formate-dependent Cr(VI) reductases have been reported in
Shewanella putrefaciens MR-1 [29].

Fig. 9. Effect of different metal ions on Cr(VI) reductase activity in cell-free extracts
of Pseudomonas sp. G1DM21 at pH 7.0 and 30 8C.

Fig. 7. Effect of temperature on Cr(VI) reductase activity in cell-free extracts of

Pseudomonas sp. G1DM21 with initial concentrations of 100 and 200 mM Cr(VI) at Fig. 10. Effect of different inhibitors on Cr(VI) reductase activity in cell-free extracts
pH 7.0. of Pseudomonas sp. G1DM21 at pH 7.0 and 30 8C.
 C. Desai et al. / Process Biochemistry 43 (2008) 713–721 719

Fig. 11. Scanning probe atomic force microscopy images of control and 1 mM Cr(VI) induced bacterial cells of Pseudomonas sp. G1DM21 (A–D). Height distributions recorded
at a nanoscale for control (A1) and 1 mM Cr(VI) induced experimental samples (B1–D1).
720 C. Desai et al. / Process Biochemistry 43 (2008) 713–721
activity also decreased by 14 and 25% in the presence of Pb2+ and
Cd2+, respectively. Both Pb2+ and Cd2+ have been reported to inhibit
Cr(VI) reduction in Ochrobacterium intermedium SDCr-5 [50],
whereas, Cd2+ has been reported to inhibit Cr(VI) reduction in
Bacillus sphaericus AND 303 [34]. Respiratory inhibitors like azide
(1 mM) and cyanide (1 mM) caused a less than 10% decrease in the
Cr(VI) reductase activity, these results corroborate with those
obtained in previous studies, it has been observed that cyanide and
azide partially inhibited purified chromate reductase of E. coli ATCC
33456 [24] and aerobic chromate reduction by Bacillus subtilis [55]
and inhibited more than 50% of membrane associated chromate
reductase activity of S. putrefaciens MR-1 [29] while no inhibition
was observed in CFE of Bacillus sp. ES29 [47]. Respiratory inhibitors
act on de novo protein synthesis or affect the respiratory chain
intermediates responsible for Cr(VI) reduction, wherein Cr(VI)
serves as a terminal electron acceptor. Hence, it can be deduced
that in Pseudomonas sp. G1DM21 a soluble chromate reductase in
the cell-free system is involved in Cr(VI) reduction which catalyzes
initial one electron transfer to Cr(VI) to form an intermediate Cr(V),
followed by two electron transfer and formation of Cr(III) as
derived for P. putida [22].
3.5. Effect of chromium stress on Cr(VI) reducing Pseudomonas sp.
G1DM21
The effect of Cr(VI) on the cellular morphology and surface
topology has been observed in other gram-negative bacteria like E.
coli K-12 and Shewanella oneidensis MR-1, with formation of snake
like and elongated structures when exposed to toxic Cr(VI) [38,39].
The scanning probe atomic force microscopy analysis of Cr(VI)
induced and uninduced cells of Pseudomonas sp. G1DM21 was
performed in order to determine effects of 24 h exposure to higher
Cr(VI) concentration of 1 mM while growth in aerobic conditions.
The images captured and the height distributions obtained are
representative of the triplicate analysis performed for bacterial
cells at a same stage of growth. As seen from Fig. 11(A–D) a marked
elongation of the bacterial cell is observed in the presence of 1 mM
Cr(VI), also a significant difference in the cell height was observed,
as the uninduced controls showed heights of 170 nm, which was
significantly increased in the presence of 1 mM Cr(VI) to 550, 500
and 650 nm as seen in Fig. 11(A1–D1). The formation of elongated
structures upon a 24 h exposure to 1 mM Cr(VI), also decreased the
Cr(VI) reduction potential of the bacterium, as the initial rate of
reduction of 1 mM Cr(VI) in 6 h of incubation was 34.5% which
increases by 54.9% in 12 h whereas, after 24 h of exposure only
1.7% increase was observed, as shown in Table 2. The decrease in
Cr(VI) reduction potential and formation of elongated structures
upon 24 h exposure to higher Cr(VI) concentration suggest the
toxicity of Cr(VI) exerted on the bacterium as observed in previous
reports [38,39].
4. Conclusions
The present study elucidated the localization and characteriza-
tion of a very efficient Cr(VI) reductase of Pseudomonas sp.
G1DM21. Chromate reductase assays of the cell-free extracts (CFE)
have shown a high Cr(VI) reductase activity, implicating the
localization of enzyme in the cytosolic fraction. The Cr(VI)
reduction potential of the resting cells was increased by cell
permeabilization. The CFE could rapidly reduce more than 90% of
100 mM Cr(VI) in 120 min. Optimum temperature and pH of
chromate reductase activity of the bacterium was found to be 30 8C
and 7.0, respectively, and activity was enhanced in presence of
0.5 mM NADH and 1 mM of metal ions like Cu2+, Mg2+ and Na+. The
Cr(VI) reductase activity was stable in presence of most metal ions
tested except, Hg2+ and Ag2+. The bacterium could resist and
reduce higher Cr(VI) concentrations in suspended cultures and
repeatedly reduce subsequent doses of 100 mM Cr(VI). Interest-
ingly, cells of Pseudomonas sp. G1DM21 formed elongated
structures upon a 24 h exposure to higher Cr(VI) concentration,
which further decreased its Cr(VI) reduction potential. The results
of higher rates of Cr(VI) reduction by the CFE, functionality of the
Cr(VI) reductase at ambient temperatures, pH and in presence of
polymetals indicates a potential application of Pseudomonas sp.
G1DM21 for pragmatic Cr(VI) bioremediation.
Acknowledgements
We gratefully acknowledge Department of Biotechnology,
Ministry of Science and Technology, New Delhi, India and Puri
Foundation, Nottingham, UK for providing financial support.
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