Bioremediation of hexavalent chromium using gram-negative bacteria.

Hexavalent chromium (Cr (VI)), the most toxic form of chromium, is widely used in industrial processes. As a result substantial amounts of Cr (VI) contaminated wastes are produced. The use of microbial cells as bioremediation of heavy metals is a potential alternative to conventional chemical methods.In this work, laboratory- scale experiments were performed to investigate Cr (VI) removal using five environmental Gram-negative bacterial strains, three of which were nosocomial strains. The potential of live and autoclaved bacterial strains was investigated to mitigate Cr (VI) from its initial concentration of 2.54 mg/1. The autoclaved bacteria were used to determine whether Cr (VI) removal was dependent upon metabolism of the cells or a simple chemical reaction. The results showed notable reduction in Cr (VI) concentration (up to 87% and 23% using live and autoclaved bacteria, respectively). Proteus mirabilis and Methylococcus capsulatus (Bath) bacterial strains were selected for further detailed analyses to investigate the enzyme system that is responsible for Cr (VI) reduction. To locate the cell compartment in which Cr (VI) removal took place in P. mirabilis, a standard bacterial cell fractionation method was used. The highest Cr (VI) removal activity resided in the cytoplasm, and there was also some activity in the cell membrane. No chromium VI removal was observed in the cell wall fraction.The removal by M. capsulatus of Cr (VI) in high copper sulfate media was more rapid than in low copper sulfate media. Phenylacetylene, an inhibitor of soluble methane monooxygenase, completely inhibited Cr (VI) removal. The results reveals that pMMO, sMMO or other enzymes that induced by copper were involved in reducing or otherwise removing Cr (VI). The di-heme cytochrome c peroxidase is also a possible candidate enzyme of reducing chromium (VI), since it is known to be present in the periplasm and to play a role in reducing peroxides generated by oxidative metabolism.Inductively coupled plasma mass spectrometry coupled with ion chromatography, for the determination of chromium species in P. mirabilis and M. capsulatus, showed that Cr (VI) was reduced and detoxified to less toxic and less soluble Cr (III). Furthermore, prominent changes in the polysaccharide, fatty acids, phosphate and proteins wereobserved in FTIR spectra of P. mirabilis and M. capsulatus (Bath) with potassium dichromate. These changes were consistent with the adsorption of chromium.BLAST searches using known chromate (VI) reducing enzymes from other bacteria showed a presence of four significant potential chromate reductase genes in the genome sequence of P. mirabilis.During the growth of M. capsulatus (Bath), it was noticed that a contaminant bacterium appeared in some cell cultures. The contaminant bacterium was identified as Bacillus licheniformis (100%) using PCR and 16S rRNA sequencing. The mixed culture that contains M. capsulatus (Bath) plus Bacillus licheniformis was also tested for Cr (VI) mitigation.The results of this work are a step forward in understanding the potential of environmental microorganisms for remediation of hexavalent chromium contamination. The future work may reveal more about the mechanism of Cr (VI) removal by the bacteria studied here as well as how they can be exploited for practical bioremediation.