Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin–Benson–Bassham cycle

Photobiological H 2 production has the potential of becoming a carbon-free renewable energy source, because upon the combustion of H 2 , only water is produced. The [Fe–Fe]-type hydrogenases of green algae are highly active, although extremely O 2 -sensitive. Sulphur deprivation is a common way to induce H 2 production, which, however, relies substantially on organic substrates and imposes a severe stress effect resulting in the degradation of the photosynthetic apparatus. We report on the establishment of an alternative H 2 production method by green algae that is based on a short anaerobic induction, keeping the Calvin–Benson–Bassham cycle inactive by substrate limitation and preserving hydrogenase activity by applying a simple catalyst to remove the evolved O 2 . Cultures remain photosynthetically active for several days, with the electrons feeding the hydrogenases mostly derived from water. The amount of H 2 produced is higher as compared to the sulphur-deprivation procedure and the process is photoautotrophic. Our protocol demonstrates that it is possible to sustainably use algal cells as whole-cell catalysts for H 2 production, which enables industrial application of algal biohydrogen production.