Molecular characterization of chromium (VI) reducing potential in Gram positive bacteria isolated from contaminated sites

Abstract

Hexavalent chromium [Cr(VI)] is highly toxic, teratogenic and carcinogenic to man and other animals. Some bacterial species have the ability to reduce Cr(VI) to a stable speciation state of trivalent chromium [Cr(III)], which is insoluble and comparatively less toxic. Therefore, the reduction of Cr(VI) thus provides potential as a means for environmental bioremediation of Cr(VI) pollution. In the present study bacteria isolated from chromium and diesel contaminated sites were found to have the ability to rapidly reduce highly toxic concentrations of Cr(VI) to Cr(III) when grown in minimal medium supplemented with glucose as the sole carbon source. Partial chromate reductase gene sequences were retrieved after PCR amplification of genomic DNA extracted from three Gram positive isolates which were highly similar (>99% sequence similarity) to chromate reductase genes found in Gram negative bacteria, more specifically those identified from Escherichia coli and Shigella spp. whole-genome studies. The isolated bacteria were putatively identified by 16S rRNA gene sequencing as Arthrobacter aurescens strain MM10, Bacillus atrophaeus strain MM20, and Rhodococcus erythropolis strain MM30.

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.