Uncoupling CR(VI) Reduction in Mixed-Culture Biofilm Systems: Electron-Flow Pathway Analysis

The electron transport pathway for microbial Cr(VI) reduction was investigated using specific inhibitors of the trans-membrane enzymes in a culture of Escherichia coli ATCC 33456 grown in a coculture with the phenol degrader, Pseudomonas putida DMP-1. The analysis involved blocking the electron flow pathway through the inner cell membrane at specific enzyme locations using known metabolic inhibitors such as azide (N3-) and 21-oligomycin A. Cr (VI) reduction resulted from the utilization of NADH as an intermediate electron donor. Therefore, the electron pathway through NADH-dehydrogenase and cytochrome system was targeted. Results showed that 2,4-DNP restored Cr(VI) reduction activity in a culture previously inactivated by 21 oligomycin A. However, Cr(VI) reduction under uncoupler could not be sustained for a long period probably due to cell death as a result of lack of maintenance. This research contributes a new understanding of the biochemistry of Cr(VI) reduction in living cells and a more accurate representation of Cr(VI) reduction for future kinetic modeling.

Microorganisms have over billion of years evolved biochemical processes for harnessing energy from organic and inorganic compounds, sunlight, geothermal heat, radioactive decay, and other less likely sources. The archaic biochemical mechanism of respiration, which utilizes the differential in the redox potential between solutions and interiors of organisms, and between dissolved compounds and the medium, has been used by almost all living organisms and has been conserved faithfully during evolution of all earthbound species over the eons (Dickerson, 1980; De Duve, 1991; Li and Graur, 1991). Scientists later realized that microorganisms are capable of utilizing parts of this complex apparatus to transfer electrons to and from other potentially toxic compounds in order to neutralize the detrimental effects of these compounds. The oldest known cases include the removal of the toxic reactive oxygen species during aerobic oxygen reduction in cells and the deposition of iron on the sea-bed in the aftermath of the evolution of photosynthesis 2.5 to 3 billion years ago (Schidlowski, 1980).

In recent studies, it was suggested that the reduction of toxic forms of Fe(III), Cr(VI), Mn(IV), Tc(VII), and U(VI) may be catalyzed by enzymes associated with the membrane respiratory chain pathway (Ishibashi et al., 1990; Horitsu et al., 1987; Lloyd, 2003).

In this study, two mechanisms have so far been studied for Cr(VI) reduction in anaerobic and aerobic cultures of bacteria. The first mechanism involves Cr(VI) reduction mediated by a soluble reductase with nicotinamide adenine dinucleotide-hydrogen (NADH) serving as the electron donor either by necessity (Horitsu et al., 1987) or for maximum activity (Ishibashi et al., 1990). The NADH-dehydrogenase pathway was shown to predominate under aerobic conditions (Ishibashi et al., 1990). In the second mechanism, Cr(VI) acts as an electron acceptor in a process mediated by a membrane-bound Cr(VI) reductase activity (Lovley and Philip, 1994). Figure 1 illustrates the relationship between the two mechanisms described above.

A spin-off of the study evaluated the sustainability of a coculture based on utilization of toxic organic copollutants of heavy metals as electron donors in the metal detoxification process. In this study, two toxic compounds, Cr(VI) and phenol, which are commonly discharged together from several industrial processes, were simultaneously removed from wastewater using a coculture of bacteria supplemented with the Cr(VI) reducing bacteria, Escherichia coli ATCC 33456, as terminal organisms. E. coli utilized byproducts of phenol degradation by the phenol degraders in the anaerobic consortium for growth and Cr(VI) reduction.

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