Cytosolic Reduction of Toxic Cr (VI) by Indigenous Microorganism
In the present study chromium resistant bacterial strain Bacillus cereus S-6 isolated from effluents of tannery was used for the reduction of toxic Cr (VI) into less toxic Cr (III). It could resist very high concentration of K2CrO4 i.e., up to 40 mg mL-1 on nutrient agar, 25 mg mL-1 in nutrient broth and up to 1.5 mg mL-1 of K2CrO4 in acetate-minimal medium. At an initial Cr (VI) concentration of 100 μg mL-1, the cytosol and membrane preparation of the strain were able to reduced almost 67 and 43% of Cr (VI) within 24 h incubation period while the heat killed cytosol and membrane preparation reduced 24 and 18% within the same time period. At high initial K2CrO4 concentration (500 μg mL-1), the reduction percentage decreased and cytosol reduce 36% and membrane preparation 18% of the total chromium supplied after 24 h. After heat shock these reduction values were 13 and 9%, respectively.
Increased industrialization posed harsh effects on the environment through the rough elimination of heavy metals containing wastes. Metals like nickel, cadmium, lead, chromium, copper, mercury, uranium etc. found in waste waters are harmful to the environment and their effects on biological system are too severe (Panchal et al., 2006). Chromium occurs in oxidation states Cr (0) to Cr (VI) but two forms Cr (III) and Cr (VI) are biologically important. Trivalent chromium, Cr (III) is an essential element required for normal carbohydrate and lipid metabolism (Mihaela et al., 2005). Several factors make Cr (VI) contamination as a matter of intense concern, particularly its toxic, mutagenic (James et al., 2005), carcinogenic (Max and Klean, 2006) and terartogenic effects above critical level (Asmatullah et al., 1998). Removal of such toxic metal by its reduction to the much less harmful and physically immobile state is now our concern. Several bacterial strains (Pseudomonas ambigua, Desulfovibrio vulgaris, Enterobacter cloacae HO-1, Alcaligenes eutrophus, Dinococcus radiodurans R1) has been described for their ability to reduce hexavalent chromium into insoluble low valence form Cr (III) both aerobically and aerobically (Branco et al., 2004; Dmitrenko et al., 2006; Elangovan et al., 2006). Hence the chromate reducing bacteria are most crucial for the immobility of soluble hexavalent chromium in the environment. The goal of present study was to develop a system to describe chromate reduction alone and in the presence of other toxic contaminants.
MATERIALS AND METHODS
Bacterial Strains and Culture Conditions
Bacterial strain Bacillus cereus, used in this study was isolated
and identified by Faisal and Hasnain (2004). The chromate resistance level in
this strain was checked both in nutrient as well as in acetate-minimal media
(Pattanapipitpaisal et al., 2002) supplemented with different concentrations
Cr (VI) Reduction Experiments
In this study two initial K2CrO4 concentrations (100
and 500 μg mL-1) were used. Overnight culture of Bacillus
cereus was taken and washed thrice with saline water and the resultant pellet
was sonicated. The mixture was centrifuged at 10,000 rpm for 10 min. Supernatant
(cytosol) and pellet (membrane preparation) were taken to check chromium reduction
ability before and after heat shock at 121°C for 15 min. For reduction experiments,
DeLeo and Ehrlich (1994) medium (grams per litre: tryptone 10, yeast extract
5, NaCl 5, citric acid 1, Na2HPO4 6.9) was used. After
24 h incubation period samples were taken aseptically and were analyzed for
Cr (VI) reduction. Chromium (VI) reduction was monitored with the help of a
spectrophotometer at 540 nm using diphenylcarbazide as complexing agent following
method of Clesceri et al. (1998).
Standard errors of the means and LSD were calculated following Steel and
Present study was conducted in 2005-2006 at Microbiology and Molecular Genetics Research Lab, Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan where a chromium resistant/reducing bacterial Bacillus cereus was examined for its capacity to reduced hexavalent chromium.
Isolation, Characterization and Identification of Strain
The present work deals with bacterial strain Bacillus cereus. Bacillus
cereus was isolated from chromium contaminated soil sample from Sheikhupura,
Pakistan (Faisal and Hasnain, 2004). It could resist very high concentration
of K2CrO4 in nutrient broth (up to 25 mg mL-1)
as well as on nutrient agar (up to 50 mg mL-1). It is a spore former
motile strictly aerobic rod.
Cr (VI) Reduction
In this experiment the reduction of Cr (VI) by the cytosol (supernatant)
and pellet (membrane preparation) after the sonication of the bacterial cell
pellet before heat shock and after heat shock were observed after 24 h incubation.
Two initial K2CrO4 concentrations were used i.e., 100
and 500 μg mL-1. The reduction of hexavalent chromium by the
Bacillus cereus in medium resulted in the production of offwhite residues
that was sign of chromate reduction. The strain exhibits variation in reduction
potential of hexavalent chromium. At an initial Cr (VI) concentration of 100
μg mL-1, the supernatant and pellet of the strain was able to
reduced almost 67 and 43% of Cr (VI) within 24 h incubation period. With an
increase in K2CrO4 concentration (500 μg mL-1)
a decrease in Cr (VI) reduction was observed both in supernatant and pellet
(36 and 18%, respectively) of the total chromium supplied after 24 h (Fig.
1). Figure 2 showed that after giving heat shock, the
heat killed supernatant and pellet was able to reduced 24 and 18% at an initial
K2CrO4 concentration 100 μg mL-1 after
24 h incubation.
Percentage Cr (VI) reduction in the supernatant (cytosol)
and pellet (membrane preparation) of Bacillus cereus at an initial
Cr (VI) concentrations of 100 and 500 μg mL-1
Percentage Cr (VI) reduction in the supernatant (cytosol)
and pellet (membrane preparation) of Bacillus cereus after giving
heat shock at 121°C for 15 min. The initial Cr (VI) concentrations
used were 100 and 500 μg mL-1
At high K2CrO4 concentration (500 μg mL-1),
supernatant reduced 13% and pellet 9% of the total chromium supplied after 24
h (Fig. 2).
Wide growing of industrial activities has lead to widespread chromium contamination
within soils and water. Chromate is a widespread contaminant that has deleterious
impacts on human health, the mobility and toxicity of which are diminished by
reduction to Cr (III) (Elangovan et al., 2006). Bacillus cereus
showed resistance to very high level of K2CrO4 both in
nutrient broth (Up to 25 mg mL-1) and on nutrient agar (50 mg mL-1).
Such a high level resistance was not reported previously in the literature.
The chromate resistance level of this strain is also very high in acetate-minimal
medium relative to the strains reported by other researchers. Chromium resistant
bacteria isolated by Basu et al. (1997) from effluent of tanneries were
resistant up to 250 μg mL-1 of Cr (VI). Megharaj et al.
(2003) also isolated bacterial strains from chromium-contaminated soil which
were able to grow at a concentration of up to 100 μg mL-1 of
Cr (VI) in minimal medium. Most Cr (VI)-resistant microorganisms
are tolerant up to 10 to 1500 mg L-1 of Cr (VI) (McLean
and Beveridge, 2001). In the present study, besides Cr (VI), strains also have
a broad ranges of heavy metals (Mn, Ni, Zn, Pb, Cu, Co and Hg) and antibiotics
(streptomycin, ampicillin, kanamycin, tetracycline and chloramphenicol) resistances
(data not shown), which shows a positive sign for the successfulness of this
strain in the treatment of industrial effluents. However, if this strain exhibit
sensitive behavior (toward different metals and antibiotics), then first of
all there must be a primary system to remove other metals contaminants before
the treatment of target metal with these strains.
The Cr (VI) concentrations also affect the Cr (VI) reduction. In this case at two different K2CrO4 concentrations 100 and 500 μg mL-1 were used. Chromium (VI) reduction in supernatants and pellets before and after heat shock was observed. Cr (VI) reducing activity was found both in the supernatant and pellet of the bacterial cells. The cell free extract of Bacillus sp. reduced 50% Cr (VI) under anaerobic conditions, using NADH as an electron donor and produced a soluble a chromate reducing enzyme stimulated by copper Cu+2 (Camargo et al., 2003). In the present study highest Cr (VI) reduction potential was observed in supernatant (cytosol) at an initial Cr (VI) concentration 100 μg mL-1. In anaerobic systems, membrane preparations reduce Cr (VI) and Cr (VI) has been shown to serve as a terminal electron acceptor (Lovley and Phillips, 1994). Assays with permeabilized (treated with toluene or triton x 100) cells and crude extracts verified that the Cr (VI) reduction was mostly associated with the soluble protein fraction of the cell (Megharaj et al., 2003). An enrichment consortium almost completely (98.5%) reduced 0.6 mM Cr (VI) in 168 h and rate of reduction was 0.5 g Cr (VI) g protein-1 h-1 (Cheung et al., 2003). Microbacterium sp. at a concentration of 2.4x109 cells mL-1, 100 μM sodium chromate was reduced within 30 h however; the maximum specific reduction rate was obtained at lower initial cell concentrations (Pattanapipitpaisal et al., 2001). After giving heat shock supernatant and pellet also retain their capability of Cr VI) reduction. In this case supernatant (cytosol) reduced maximum chromium as compared to pellet (membrane preparation). It was observed that Cr (VI) activity was lost when the Cr (VI)-reducing cell extracts were heated at 100°C (Ohtake and Hardoyo, 1992). When the cytosol and membrane was heat shocked at 121°C for 15 min, the Cr (VI) reduction property was drastically decreased. It means that the chromium reduction ability of this strain is related with some chromate reductase enzyme, which lost its activity after giving heat shock.
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