Elimination of the flavodiiron electron sink facilitates long-term-H2 photoproduction in green algae

Background: The development of renewable and sustainable biofuels to cover the future energy demand is one of the most challenging issues of our time. Biohydrogen, produced by photosynthetic microorganisms, has the potential to become a green biofuel and energy carrier for the future sustainable world, since it provides energy without CO2 emission. The recent development of two alternative protocols to induce hydrogen photoproduction in green algae enables the function of the O-2-sensitive [FeFe]-hydrogenases, located at the acceptor side of photosystem I, to produce H-2 for several days. These protocols prevent carbon fixation and redirect electrons toward H-2 production. In the present work, we employed these protocols to a knockout Chlamydomonas reinhardtii mutant lacking flavodiiron proteins (FDPs), thus removing another possible electron competitor with H-2 production. Results: The deletion of the FDP electron sink resulted in the enhancement of H-2 photoproduction relative to wildtype C. reinhardtii. Additionally, the lack of FDPs leads to a more effective obstruction of carbon fixation even under elongated light pulses. Conclusions: We demonstrated that the rather simple adjustment of cultivation conditions together with genetic manipulation of alternative electron pathways of photosynthesis results in efficient re-routing of electrons toward H-2 photoproduction. Furthermore, the introduction of a short recovery phase by regular switching from H-2 photoproduction to biomass accumulation phase allows to maintain cell fitness and use photosynthetic cells as long-term H-2-producing biocatalysts.