Characterisation of the Bioremediation of Chromate by Methylococcus capsulatus

Chromate (VI) is an oxidising pollutant that is harmful to humans and the environment. Reduction of chromate (VI) produces chromium (III), which is less toxic, less soluble and less bioavailable. Methylococcus capsulatus Bath is an example of a diverse group of methane oxidising bacteria that are widespread in the environment and have potential for bioremediation of a wide range organic and inorganic pollutants, including reduction of chromium (VI) to chromium (III). Cells of Mc. capsulatus were broken and centrifugally fractionated during the bioremediation reaction. HPLC-ICP-MS analysis showed that the concentration of chromium (VI) in the culture supernatant progressively declined and there was a corresponding increase in the concentration of chromium (III) in the cytoplasm + membranes fraction. Further fractionation showed that the distribution of chromium (III) was approximately two thirds in the cell membrane fraction and one third in the cytoplasm fraction. The cellular distribution and speciation of chromium were further investigated using transmission electron microscopy- energy dispersive X-ray (TEM-EDX), high-angle annular dark-field-scanning transmission electron microscopy (HAADF-STEM), Electron energy loss spectroscopy (EELS) and X-ray photoelectron spectroscopy (XPS) imaging of whole and sectioned cells. Mc. capsulatus Bath took up Cr (III) that has been added to the culture medium via a process that was promoted by the growth substrate methane and inhibited by the metabolic inhibitor sodium azide. Accumulation of chromium (III) within ii the Mc. capsulatus cells may reduce its bioavailability and help to prevent its re-oxidation to the toxic chromium (VI). The cell fractions of Mc. capsulatus Bath were studied for their chromium (VI) removal activity at various temperatures and the optimum temperature of the fractions was found at 30 °C. All cellular fractions reduced chromium (VI) to chromium (III); reduction activity was greatest in the cytoplasm fraction. The chromium (VI)-removing activity, which was enhanced by the presence of NADH, was purified from the cytoplasmic fraction by means of DEAE Anion exchange and Capto Blue Dye Affinity chromatography to yield fractions that contained a single 17-kDa protein. Three new methanotroph strains were isolated from the Leeds and Liverpool canal and a railway location near Doncaster. One of these (isolated from canal sediment via enrichment at 45 °C) is another strain of Mc. capsulatus that is capable of Cr (VI) removal.