Targets of Improvement in Bacterial Chromate Bioremediation

Cr(VI) (chromate) is a widespread, toxic and soluble environmental contaminant. Bacteria can reduce chromate to insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. However, many fundamental parameters that define an organism’s capacity to remediate chromate have not previously been characterized. We have measured the innate ability of a wide range of bacteria to reduce chromate. All bacteria that we tested were able to reduce Cr(VI) through to stable Cr(III) end-products. One of these, Pseudomonas putida MK1, is a promising candidate for chromate bioremediation, and we show that chromate transformation per mass unit increases in these cells as the environmental chromate concentration rises. However, bacterial growth is inhibited by chromate concentrations above 0.8 mM. Furthermore, although cell extracts show increased chromate reductase activity under slow-growth conditions (mimicking nutrient-limited field conditions), the ability of the whole cells to transform chromate is greatly diminished. Chromate reduction by both whole cells and purified ChrR (a P. putida chromate reductase) is also inhibited by co-pollutants of chromate-contaminated sites. Thus, these studies identify several potential areas of improvement for generation of improved chromate-remediating bacteria. INTRODUCTION Hexavalent chromium [Cr(VI); chromate)] is generated as a by-product of a large number of industries, for example, welding, paper and pigment production, and chrome plating. This has resulted in large-scale contamination of the environment by chromate. The manufacture of nuclear weapons also produced vast quantities of chromate, which were either discharged directly into the environment or stored in buried canisters. Rusting and leakage of the latter exacerbated the chromate contamination problem at the U.S. Department of Energy (DOE) waste sites with the result that chromate is second only to lead as the most abundant heavy metal contaminant at these sites. There, chromate concentration is reported to be as high as 173 μM in ground water and 76 mM in sediments (Riley, 1992). Since soil water is stored in small capillary spaces, the latter may amount to much higher concentrations. Estimates for the cost of cleanup of the DOE waste sites alone run up to hundred of billion of dollars (McIlwain, 1996). Chromate is readily taken up by biological cells through the anionic transport systems and is a known toxic and carcinogenic agent. Its stable reduced product Cr(III), on the other hand is not bioavailable, as it is not taken up by the cells. Thus, Cr(III) is much less harmful. Moreover, in contrast to Cr(VI), which is highly soluble, Cr(III) is much less so; thus, while it is very difficult to sequester the former, the latter can readily be confined to discrete regions. For this reason conversion of environmental chromate to Cr(III) offers a solution to the chromate contamination problem.