Molecular characterization of chromium (VI) reducing potential in Gram positive bacteria isolated from contaminated sites
Introduction
Contamination of the soil, surface water and groundwater with hexavalent chromium [Cr(VI)] is an issue of potential concern due to its toxicity (DEFRA, 2002). It is well known for its toxic, mutagenic, carcinogenic and teratogenic effects on human beings and other living organisms and is classified under priority pollutants in many countries (Ye and Shi, 2001, Avudainayagam et al., 2003). The toxicity of chromium to non-tolerant soil microorganisms inhibits the bioremediation of organic pollutants in contaminated soils (Kourtev et al., 2009). Chromate is generated as a by-product of a large number of industries including those engaged in welding, paper and pigment production, leather-tanning, chrome plating and thermonuclear weapons manufacturing. Cr(VI) bears structural similarity to sulphate (SO42−), and is readily taken up by bacterial and mammalian cells through the sulphate transport system (Singh et al., 1998, Cervantes et al., 2001). It is more toxic than its reduced trivalent form [Cr(III)] which is rather considered essential for some biological functions (Krishna and Philip, 2005).
Metal pollutants, unlike organic contaminants, cannot be degraded. So, their detoxification can be achieved either by adsorption/accumulation or by conventional physicochemical treatments but they are quite expensive and cumbersome (Malik, 2004). Thus, biological detoxification of Cr(VI), transforming it to a less toxic oxidation state Cr(III), is considered as an ecofriendly and cost-effective technique for the environmental clean-up of this heavy metal contaminant (Camargo et al., 2003, Megharaj et al., 2003). A number of microorganisms have been reported to resist/tolerate Cr(VI) by periplasmic biosorption, intracellular bioaccumulation, and/or biotransformation to a less toxic speciation state through direct enzymatic reaction or indirectly with metabolites, and include members within the genera Pseudomonas, Aeromonas, Streptomyces, Microbacterium, Desulfovibrio, Enterobacter, Escherichia, Shewanella, and Bacillus (Cervantes and Silver, 1992, Camargo et al., 2003, Thacker et al., 2007) and have attracted considerable interest for their potential use in the bioremediation of chromate-containing industrial waste waters. Thus, biotransformation of Cr(VI) to the non-toxic trivalent form by chromium-reducing bacteria (CRB) therefore offers an option for Cr(VI) detoxification to achieve bioremediation of contaminated environment (Pal et al., 2005). Most of the studies carried out so far on Cr(VI) reduction by environmental isolates or microbial cultures describe the enzyme kinetics in different media (Bo et al., 2009, Thacker et al., 2007), location of the enzyme (Dhakephalkar et al., 1996), influence of physicochemical or cultural factors (Parameswari et al., 2009), or nutrient supplementation (Bhide et al., 1996) etc. but lack details of possible genetic mechanisms responsible for the Cr(VI) reduction.
Several mechanisms of Cr(VI) reduction have been identified in bacteria and these include reduction by DT-diaphorase, aldehyde oxidase in the cell cytoplasm, Cr(VI) reductase and cytochrome P450 on cell membrane as well as nitroreductase (Kwak et al., 2003, Cheung and Gu, 2007). Although Cr(VI) reductase from Pseudomonas ambigua has been purified and characterized (Suzuki et al., 1992), the identity of the genes involved in Cr(VI) reductase, and its expression, has not been published with respect to an individual organism or environmental consortia. This paper describes Cr(VI) reducing potential of bacteria isolated from contaminated environments, the method developed for identification of specific genes responsible for Cr(VI) reduction and their characterization. The standardized protocol could be applied to screen the bacteria collected from contaminated environments to verify if the organism or consortium has the gene of interest for remediation of Cr(VI) contaminated environments.
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Bacterial strains and cultivation conditions
Bacterial isolates were grown from single colonies using the methods of Eisenstadt et al. (1994), from surface soil samples (0–15 cm depth) collected from a long term tannery waste contaminated site at Mount Barker, South Australia and sediment samples (0–5 cm depth) from a diesel contaminated site from Perth, Western Australia. Isolation and subsequent growth experiments were carried out with a minimal salt medium (Megharaj et al., 2003) supplemented with Cr(VI) (100 mg/l chromium as K2Cr2O7)
Cr(VI) reducing ability of bacterial strain
Two Gram positive Cr(VI) resistant bacterial strains (MM10 and MM20) were isolated from long term tannery waste contaminated soil and another Gram positive strain (MM30) from diesel contaminated sediment in Western Australia by selection on minimal medium agar plates amended with 50 mg/l Cr(VI) with 0.5% glucose as the sole carbon source. Fig. 1 shows the graphical representation of the density of the bacterial isolates and % Cr(VI) reduction at different time intervals. The increase in
Discussion
The ever increasing concern about the toxicity of Cr(VI) and the potential biotransformation technology for detoxification of Cr(VI) through in situ microbial reduction of Cr(VI) to less toxic and insoluble Cr(III) that circumvents the limitations posed by physical and chemical treatment methods (Viamajala et al., 2002) coerce the isolation of Cr(VI) reducing bacteria from contaminated sites and characterization of chromate reductase genes in Cr(VI) reducing bacteria (CRB).
In the present
Acknowledgement
The first author is thankful to Department of Biotechnology, Ministry of Science and Technology, India for financial support in the form of DBT Overseas Associateship (2006–07) Award. MB and MM thank Information Technology, Engineering and Environment (ITEE) Division, University of South Australia for funding under Divisional small grants scheme.
References (43)
- et al.
Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale
Chemosphere
(2007) - et al.
Essential residues in the chromate transporter ChrA of Pseudomonas aeruginosa
FEMS Microbiology Letters
(2004) - et al.
Interactions of chromium with microorganisms and plants
FEMS Microbiology Reviews
(2001) - et al.
Plasmid chromate resistance and chromate reduction
Plasmid
(1992) - et al.
Bioreduction of chromium (VI) by Bacillus sp. isolated from soils of iron mineral area
European Journal of Soil Biology
(2009) - et al.
Mechanism of hexavalent chromium detoxification by microorganisms and bioremediation application potential: a review
International Biodeterioration & Biodegradation
(2007) - et al.
Evaluation of in vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill
Bioresource Technology
(2008) - et al.
Bioremediation of Cr(VI) in contaminated soils
Journal of Hazardous Materials B
(2005) Metal bioremediation by growing cells
Environment International
(2004)- et al.
Nucleotide sequence and expression of a plasmid-encoded chromate resistance determinant from Alcaligenes eutrophus
Journal of Biological Chemistry
(1990)
Efflux-mediated heavy metal resistance in prokaryotes
FEMS Microbiology Reviews
A bacterial flavin reductase system reduces chromate to a soluble chromium(III)–NAD+ complex
Biochemistry and Biophysics Research Communications
Reduction of chromate by cell-free extract of Brucella sp. isolated from Cr(VI) contaminated sites
Bioresource Technology
Chromate efflux by means of the ChrA chromate resistance protein from Pseudomonas aeruginosa
Journal of Bacteriology
Chemistry of chromium in soils with emphasis on tannery waste sites
Reviews of Environmental Contamination and Toxicology
Behaviour of chromium in solids. III. Oxidation
Journal of Environmental Quality
Microbiological process for the removal of Cr(VI) from Cr(VI)-bearing cooling tower effluent
Biotechnology Letters
Kinetic study and mathematical modeling of chromium(VI) reduction and microorganism growth under mixed culture
Current Microbiology
Chromate reduction by chromium resistant bacteria isolated from soils contaminated with dichromate
Journal of Environmental Quality
Hydrogenases in sulphate-reducing bacteria function as chromium reductase
Applied Microbiology and Biotechnology
Prominent role of DT-diaphorase as a cellular mechanism reducing chromium(VI) and reverting its mutagenicity
Cancer Research
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