Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii

The work describes a novel approach for sustained photobiological production of H-2 gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H-2 production method circumvents the severe O-2 sensitivity of the reversible hydrogenase by temporally separating photosynthetic O-2 evolution and carbon accumulation(stage 1) from the consumption of cellular metabolites and concomitant H-2 production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O-2 evolution. Under these conditions, oxidative respiration by the cells in the light depleted O-2 and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H-2 gas production was observed in the light but not in the dark. The mechanism of H-2 production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H-2 evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H-2 in the chloroplast stroma.