Springer 2006
Biotechnology Letters (2006) 28: 247–252
DOI 10.1007/s10529-005-5526-z
Reduction of Cr(VI) by a Bacillus sp.
R. Elangovan1, S. Abhipsa2, B. Rohit3, P. Ligy1 & K. Chandraraj2,*
1
Department of Civil Engineering, Indian Institute of Technology Madras, Chennai 600 036, India
Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
3
Department of Chemical Engineering, Indian Institute of Technology Madras, Chennai 600 036, India
*Author for correspondence (E-mail: kcraj@iitm.ac.in)
2
Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005
Key words: Bacillus sp., chromate reductase, chromium tolerance, Cr(VI) bioremediation
Abstract
A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside
the cell. When Cr(VI) was 10 and 40 lg ml)1, >95% of the total Cr(VI) was reduced in 24 and 72 h of
growth, respectively, whereas at 80 lg Cr(VI) ml)1 only 50% of Cr(VI) was reduced. However growth was
not affected; the cell mass was 0.7–0.8 mg ml)1 in all cases. The cell-free extract showed Cr(VI) reducing
enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also
reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was
optimal at pH 6.0 and 30 C. The enzyme was stable up to 30 C and from pH 5.5 to 8, but from pH 4 to 5
the enzyme was severely destabilized. Its Km and Vmax were 14 lM and 3.8 nmol min)1 mg)1 respectively.
The enzyme activity was enhanced by Cu2+ and Ni2+ and inhibited by Hg2+.
Introduction
Hexavalent chromium, a widespread industrial
waste, is a strong oxidant and is toxic. It is
highly soluble and therefore easily spreads in
the environment, whereas Cr(III) is less toxic
and less soluble (Cervantes et al. 2001). Thus,
one way of detoxifying Cr(VI) is reduction of
Cr(VI)–Cr(III), which is possible by chemical or
microbial means. Chemical reduction requires
high energy input and leads to a large quantity
of chemical sludge formation, while the microbial method does not require high energy input
and involves reduction or bio-accumulation or
absorption and an efflux pump (Philip et al.
1998, Alvarez et al. 1999). Bacteria facilitate
reduction while fungi and algae are good for
biosorption during chromium detoxification.
Bacteria reduce chromium using NADH or glutathione as enzyme co-factors. The enzymatic
reduction of Cr(VI) involves a soluble cytosolic
chromate reductase under aerobic conditions or
a membrane-bound chromate reductase during
anaerobic respiration where chromate acts as
the terminal electron acceptor (Wang et al.
1990, Camargo et al. 2004). Though microbes of
genera Pseudomonas, Arthrobacter, Escherichia
and Bacillus have been reported to reduce
Cr(VI) through soluble chromate reductase, only
a few of these enzymes have been characterized
(Park et al. 2000, Camargo et al. 2003, Megharaj et al. 2004, Bae et al. 2005). The chromate
reductase from Bacillus sp. has not been fully
characterized to date and properties of a crude
chromate reductase enzyme from Bacillus sp.
ES29 has been reported (Camargo et al. 2003).
The present article describes characterization of
a crude chromate reductase enzyme from a new
Bacillus sp. isolated from chromium contaminated soil.
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Materials and methods
Chromate reductase assay
Microorganism and growth conditions
Chromate reductase activity was assayed using
NADH as an electron donor. The reaction mixture contained 0.1 ml crude enzyme solution,
0.1 mM NADH and 3.4 lM Cr(VI) in 50 mM
potassium phosphate buffer (pH 6) in a total
volume of 1 ml. Assay mixtures comprising similar composition given above except enzyme or
NADH were used as respective controls. The
assay mixtures were incubated at 30 C for
30 min. The amount of residual Cr(VI) in the
reaction mixture was quantified as described.
The organism was isolated from chromate contaminated soil which contained 12 mg g)1 of total chromium and 5.6 mg g)1 of Cr(VI). The
isolate was deposited at MTCC, India, with an
accession number MTCC 7048. The 16S rRNA
gene fragment of the isolate was amplified by
PCR using genomic DNA as the template and
sequenced according to Pattanapipitpaisal et al.
(2001). The gene sequence was submitted to
GenBank with the accession number DQ268872.
The organism was grown in nutrient medium composed of (in g l)1) peptone-5; yeast extract-1; beef extract-2 and NaCl-1. About 2 ml
of seed culture (16 to 18 h old) was diluted to
100 ml with fresh nutrient medium containing
potassium dichromate (10–160 mg l)1) in 500 ml
culture flasks and incubated (200 rpm) at
35 C. Growth was monitored by measuring
optical density at 600 nm (OD600 of 1=340 mg
cell dry wt l)1).
Estimation of chromium
Hexavalent chromium in the culture supernatant
was measured using 1,5-diphenylcarbazide (DPC)
reagent (APHA 1995). Total chromium was measured using atomic absorbance spectrophotometry
after acid digestion of the sample (APHA 1995).
Enzyme preparation
Cells from culture grown for 18 h in nutrient
medium plus potassium dichromate (5 lg ml)1)
as described above were harvested and re-suspended in 50 mM potassium phosphate buffer of
pH 6 (1 g cells wet wt in 6 ml buffer). The cells
were lysed by sonication (5 cycles of 40 s on and
40 s off at 175 W), centrifuged (10,000 g and
4 C) and the cell-free extract (CFE) was collected. The CFE was fractionated with ammonium
sulfate and the precipitate corresponding to 30 to
40% (w/v) salt concentration was re-dissolved in
the same phosphate buffer (1/10 vol of CFE). The
fractionated sample was dialyzed against the
same buffer overnight and was used as crude
chromate reductase. Total protein was estimated
by Lowry’s method.
Characterization of chromate reductase
Chromate reductase activity was measured at
30 C and at different pH using various buffers
(50 mM sodium acetate, pH 4–5.5; 50 mM sodium phosphate, pH 5.5–8; 50 mM sodium carbonate, pH 8–10). The enzyme sample was
incubated in different buffers (pH 4.5–9.0) at
4 C for 24 h and the residual activity was
measured to check the pH stability. Effect of
temperature was studied by measuring enzyme
activity between 10 and 70 C and at pH 6.
For thermal stability, enzyme was incubated at
10–70 C and at pH 6 for 30 and 60 min,
cooled in ice bath and the residual enzyme
activity was measured. The enzyme sample was
treated with metal ions (1 mM) at pH 6 and
30 C for 10 min and the residual activity was
assayed as described. Activity of the chromate
reductase without pretreatment was considered
as 100%. The kinetic parameters Km and Vmax
were estimated from Lineweaver–Burk plot.
The rate of reaction was expressed as the number of nmoles of Cr(VI) reduced in 1 min by
1 mg of total protein in the enzyme sample.
Results
Organism
A mixed culture of bacteria resistant to chromate
was obtained from chromium contaminated soil
located near tanneries. A pure bacterial strain
resistant to chromate with Cr(VI) reducing ability
was isolated from the mixed culture. The isolate
was Gram-positive and rod shaped. 16S rRNA
249
gene sequence of the isolate was compared with
known sequences in the nucleotide databases
using BLASTN which showed closest match
(>99%) to that of Bacillus sp. and hence this
strain was identified as Bacillus sp.
Cr(VI) reduction
Time course of Cr(VI) reduction during the
growth of Bacillus sp. is shown in Figure 1.
More than 95% of Cr(VI) reduction was obtained at 24 h with 10 lg Cr(VI) ml)1 and the
growth time extended to 42 h with 20 lg
Cr(VI) ml)1. After 64 h, Cr(VI) reduction was
80%
with
40 lg Cr(VI) ml)1,
but
with
80 lg Cr(VI) ml)1, maximum Cr(VI) reduction
was 50%. There was no Cr(VI) reduction or
growth at 160 lg Cr(VI) ml)1. The cell mass
reached maximum at 18 and 24 h of growth with
Cr(VI) of 10–20 and 40–80 lg ml)1 respectively.
As seen in the Figure 1, the biomass level was
not affected drastically by different concentrations of Cr(VI) in the medium.
fraction showed no Cr(VI) reduction. Addition
of NADH to the reaction mixture improved the
chromate reductase to 1.1 U mg)1 under similar
conditions. Chromate reductase activity was also
observed when NADPH was used as an electron
donor and there was no substantial difference in
the level of chromate reductase activity between
NADH and NADPH. The protein from ammonium sulfate fractionation showed 2.2-fold
increase in specific activity (2.4 U mg)1) of chromate reductase and this sample was used for further studies using NADH as an electron donor.
Characteristics of chromate reductase
After cell lysis by ultrasonication the CFE had
0.24 U chromate reductase activity mg)1 for
5 lg Cr(VI) ml)1, while the cell membrane
Chromate reductase activity was observed in a
narrow pH range with an optimum at 6.0 (Figure 2). More than 85% of the maximum activity
was lost when the pH was 5.0 and 7.0. As seen
in Figure 2, acidic pH drastically decreased the
enzyme stability while alkaline pH slightly reduced the stability with retention of around 80%
residual activity. At the optimum pH of 6.0,
maximum chromate reductase activity was found
at 30 C (Figure 3). Activity at 20–25 C was
85% of the optimal activity and at 10 C activity
was only 35% of the optimum. Rapid decrease
in chromate reductase activity was observed
above 30 C. At optimum pH 6, chromate reductase was most stable below 30 C; as seen in
Fig. 1. Kinetics of growth and Cr(VI) reduction. Bacillus sp.
was cultured with Cr(VI) at 10 (re), 20 (jh), 40 (ds) and
80 (Dm) lg ml)1 in nutrient media as described. Solid and
broken lines refer to cell density and Cr(VI) respectively.
Data are mean of two independent growth experiments and
the error bar denotes standard deviation.
Fig. 2. Effect of pH on the activity (j) and stability (d) of
chromate reductase (sp. act. 2 Æ 4 U mg)1).
Chromate reductase activity of cell free extract
(CFE)
250
Fig. 3. Effect of temperature on the activity (d) and stability
(j) of chromate reductase (sp. act. 2 Æ 4 U mg)1).
Figure 3, after 60 min of incubation at 30 C,
which is its optimum temperature for activity,
80% of the residual activity was retained. Further increase of temperature resulted in rapid loss
of stability. Among the metal ions tested, Cu2+
and Ni2+ enhanced the activity of chromate
reductase by 51 and 14%, respectively. Mg2+
and Ca2+ had no effect and other metal ions
Hg2+, Ag+, Fe2+, Ba2+ and Zn2+ inhibited
chromate reductase activity at various levels (40–
75%) (Figure 4).
Fig. 4. Effect of metal ions on the activity of chromate reductase (sp. act. 2 Æ 4 U mg)1). Data are means of triplicate assays and the error bars indicate standard deviation. Analysis
of the difference between groups was performed according to
Tukey’s test with a confidence range of 95% using XLSTAT.
Bars with the same letter on top do not have significant difference in means.
Cr(VI) reduction by chromate reductase
showed strong dependence on substrate concentration. For Cr(VI) concentration from 2.5 to
10 lg ml)1 atleast 50% of Cr(VI) reduction was
observed within 3 h of incubation. At
2.5 lg ml)1, Cr(VI) reduction was highest
(>90%) and at 10 lg Cr(VI) ml)1, 50% of the
Cr(VI) reduction was observed after 7 h of
reaction. The initial velocity of the chromate
reductase linearly increased with chromate concentration and it followed saturation enzyme
kinetics up to 76 lM Cr(VI). When the concentration of Cr(VI) was increased to 100 lM and
above, the rate of Cr(VI) reduction decreased
suggesting possible substrate inhibition. The kinetic parameters Km and Vmax were 14 lM and
3.8 nmol
min)1 (mg protein))1 respectively
(Figure 5).
Discussion
The new isolate designated as Bacillus sp.
strain RE was resistant to chromium and
showed Cr(VI) reduction activity. During the
growth in medium containing chromate the
concentration of total chromium remained constant in the medium irrespective of the initial
chromate concentration, which indicated possible existence of efflux pump and the absence of
chromium bio-accumulation. This efflux process
minimizes damage to cellular components due
Fig. 5. Effect of Cr(VI) concentration on chromate reductase
activity (sp. act. 2 Æ 4 U mg)1). The inset denotes the Lineweaver–Burk plot of Cr(VI) reduction. Data are mean of three
independent sets of reactions. The rate (Vo) of Cr(VI) reduction refers to nmol Cr(VI) reduced per min per mg protein.
251
to various species of chromium that could be
formed inside the cell (Shi et al. 1991, Suzuki
et al. 1992). The reduction of Cr(VI) and subsequent efflux of chromium are important steps
in the microbial remediation of Cr(VI) contaminated environments. Though the efficiency of
Cr(VI) reduction by Bacillus sp. RE was affected by the concentration of Cr(VI), the cell
growth was not significantly affected by Cr(VI)
up to 80 lg ml)1 indicating tolerance to this level of Cr(VI). Similar trends of growth has
been reported for A. oxydans, which showed
tolerance up to 50 lg chromate ml)1 (Megharaj
et al. 2004). Chromium tolerance and Cr(VI)
reduction are not related properties of chromate resistant bacteria. Accordingly, this Bacillus sp. showed different level of tolerance
[80 lg Cr(VI) ml)1] and Cr(VI) reducing capacity [40 lg Cr(VI) ml)1].
Cr(VI) reducing activity of the CFE of this
strain was dependent on NADH or NADPH.
Though the activity of chromate reductase decreased significantly at alkaline pH, stability was
not affected. But acidic pH affected both the
activity and stability. However, the optimum pH
and temperature were in the range (pH 5–9 and
30 C) reported for bacterial chromate reductases
(Camargo et al. 2003, 2004, Bae et al. 2005). A
bacterial chromate reductase, active and stable at
high temperature, was also isolated from Pseudomonas putida, which was active and stable between 50 and 80 C and was not active at low
temperatures (Park et al. 2000).
During growth, 100% Cr(VI) reduction was
observed for 10 lg ml)1 of Cr(VI) in the medium,
but enzymatic reduction showed only 50% Cr(VI)
reduction for the corresponding chromate concentration. In both the cases high concentration of
Cr(VI) resulted in decreased efficiency of Cr(VI)
reduction. The decrease in Cr(VI) reduction by
cells was not due to bio-accumulation of chromium since the chromium was pumped out. The
Km (14 lM) of the crude chromate reductase was
close to that of E. coli (17 lM) and 2-fold greater
than that of Bacillus sp. (7 lM), whereas the Vmax
(3.8 nmol min)1 mg)1) was different from the bacterial chromate reductases, which ranges from 18
to 322 nmol min)1 mg)1 (Camargo et al. 2003,
Bae et al. 2005). Enhancement of chromate reductase activity by Cu2+ was similar to that reported
from Bacillus sp. (Camargo et al. 2003). On the
other hand, Cu2+ has been known to inhibit the
chromate reductase from P. putida (Park et al.
2000). The inhibition by Hg2+ suggests possible
involvement of thiol group in the catalysis and
similar observation has been also reported in other
chromate reductases (Park et al. 2000, Megharaj
et al. 2004).
The chromate resistant Bacillus sp. that was
isolated from chromium contaminated soil
showed good chromium tolerance (80 lg ml)1)
and Cr(VI)-reduction through soluble enzyme but
it did not accumulate chromium inside the cell.
This soluble chromate reductase was kinetically
different from other bacterial chromate reductases and was stimulated by copper and nickel ions,
though temperature and pH profile were closer to
others. The Bacillus sp. with both chromate tolerance and chromate reductase activity combined
with subsequent possible chromium efflux mechanism would be useful for bioremediation of chromium contaminated environment.
Acknowledgements
This project was supported by DBT, New Delhi.
Elangovan and Abhipsa are JRF awarded by
DST and IITM respectively.
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