Novel properties of photofermentative biohydrogen production by purple bacteria Rhodobacter sphaeroides: effects of protonophores and inhibitors of responsible enzymes

BACKGROUND: Biohydrogen (H(2)) production by purple bacteria during photofermentation is a very promising way among biological H(2) production methods. The effects of protonophores, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,4-dinitrophenol (DNP), and inhibitors of enzymes, involved in H(2) metabolism, metronidazole (Met), diphenyleneiodonium (DPI), and dimethylsulphoxide (DMSO) on H(2) production by Rhodobacter sphaeroides MDC6522 isolated from Jermuk mineral springs in Armenia have been investigated in both nitrogen-limited and nitrogen-excess conditions. RESULTS: With the increase of inhibitors concentrations H(2) yield gradually decreased. The complete inhibition of H(2) production was observed in the presence of DPI and CCCP. DPI’s solvent—DMSO in low concentration did not significantly affect H(2) yield. N,N′-dicyclohexylcarbodiimide (DCCD)-inhibited the F(O)F(1)-ATPase activity of bacterial membrane vesicles was analyzed in the presence of inhibitors. Low concentrations of DPI and DMSO did not affect ATPase activity, whereas Met and CCCP stimulated enzyme activity. The effect of DNP was similar to CCCP. CONCLUSIONS AND SIGNIFICANCE: The results have shown the low concentration or concentration dependent effects of protonophores and nitrogenase and hydrogenase inhibitors on photofermentative H(2) production by Rh. sphaeroides in nitrogen-limited and nitrogen-excess conditions. They would be significant to understand novel properties in relationship between nitrogenase, hydrogenase and the F(O)F(1)-ATPase in Rh. sphaeroides, and regulatory pathways of photofermentation. The inhibitors of nitrogenase and hydrogenase can be used in biotechnology for regulation of H(2) production in different technology conditions and development of scale-up applications, for biomass and energy production using purple bacterial cells.