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 productio n of H-2 gas via the reversible hydrogenase pathway in the green alga Chlam ydomonas reinhardtii. This single-organism, two-stage H-2 production method circumvents the severe O-2 sensitivity of the reversible hydrogenase by te mporally separating photosynthetic O-2 evolution and carbon accumulation(st age 1) from the consumption of cellular metabolites and concomitant H-2 pro duction (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (P SII) and O-2 evolution. Under these conditions, oxidative respiration by th e cells in the light depleted O-2 and caused anaerobiosis in the culture, w hich was necessary and sufficient for the induction of the reversible hydro genase. Subsequently, sustained cellular H-2 gas production was observed in the light but not in the dark. The mechanism of H-2 production entailed pr otein consumption and electron transport from endogenous substrate to the c ytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light abs orption by PSI was required for H-2 evolution, suggesting that photoreducti on of ferredoxin is followed by electron donation to the reversible hydroge nase. The latter catalyzes the reduction of protons to molecular H-2 in the chloroplast stroma.