BiocatalysisBacillus sp. IntroductionHexavalent chromium [Cr(VI)] compounds are used in a wide variety of commercial processes and unregulated disposal of chromium containing effluents has led to the contamination of soil, aquatic sediments, and surface and groundwater environments. Chromium, a steel-grey, lustrous, hard and brittle metal, occurs in nature in the bound form that constitutes 0.1-0.3 mg/kg of the Earth's crust. It has several oxidation states ranging from (ÀII) to (þVI), the trivalent and hexavalent states being the most stable. A maximum acceptable concentration of 50 mg/L for Cr(VI) in drinking water has been established on the basis of health considerations (Kiilunen, 1994). In some American states, the exposure limit for Cr(VI) is as low as 15 mg/L for humans and 10 mg/L for aquatic organisms (Levitskaia et al., 2008) which is below the detection limit for most low cost colorimetric methods. Cr(VI) concentrations above the allowable limit cause cancer in humans and aquatic fauna, and is acutely toxic at much higher concentrations (U.S. EPA, 1978; Federal Register, 2004 A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / w a t r e s ARTICLE IN PRESS0043-1354/$ -see front matter ª

A mixed-culture of bacteria collected from a wastewater treatment plant in Brits, North-West Province (South Africa) biocatalytically reduced Cr(VI) at much higher concentrations than previously observed in cultures isolated in North America. Complete Cr(VI) reduction in aerobic cultures was achieved at a high concentration of 200 mg/L after incubation for only 65 hours. Under anaerobic conditions up to 150 mg, Cr(VI)/L was completely removed after incubating for 130 to 155 hours, still higher than the Cr(VI) reduction achieved with previous cultures where complete removal was only observed in cultures with the added Cr(VI) concentration not greater than 30 mg/L. Cr(VI) reduction capability of the cultures was verified in purified cultures. Consortium cultures were characterised using 16S rRNA partial sequence analysis. Results showed that the gram-positive Bacillus genera predominated under aerobic conditions with a small composition of the gram-negative Microbacterium sp. There was more biodiversity observed in the anaerobic cultures with the marked appearance of Enterococcus, Arthrobacter, Paenibacillus and Oceanobacillus species. The results showed that Cr(VI) reduction rate in the new culture was up to eight times higher than that previously observed in other Cr(VI) reducing cultures isolated from Cr(VI) contaminated soil environments in Newark (New Jersey) and other sites in North America.

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Microbial Cr(VI) reduction in groundwater aquifer media was investigated in microcosm reactors extracted from Cr(VI) contaminated sites in South Africa. The reactors were operated under an influent Cr(VI) concentration of 40 mg/L to simulate the current Cr(VI) level at the contaminated site. Near complete Cr(VI) removal was observed in microcosm reactors inoculated with Cr(VI) reducing bacteria from dried activated sludge collected from a treatment plant receiving periodic loadings of Cr(VI). The best performance was observed under low hydraulic loading (flow rate, Q=0.310 cm(3)/hr). Microbial culture characterisation results showed a change in culture composition after 17 days of reactor operation, indicating Bacillus and Lysinibacillus species as the most dominant organisms in reactors that reduced Cr(VI). The predominance of Bacillus and Lysinibacillus species was either due to resilience against toxicity or adaptation to the changing conditions in the reactor. This research was the initial step towards the development of an in situ bioremediation process to contain the spread of a Cr(VI) plume in a groundwater aquifer at contaminated site in Brits, South Africa. South Africa holds about 72% percent of the world's chromium resources, the majority of which is mined in the North Eastern region of the country formally known as Transvaal.

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