Photosystem ratio imbalance promotes direct sustainable H2 production in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii can photoproduce H-2 gas for only a few minutes under anaerobic conditions due to the inhibition of hydrogenase by O-2 produced by Photosystem II (PSII). A few days of sustained H-2 production can only be achieved when O-2 and H-2 production are temporally separated under two-stage processes such as sulfur deprivation. Under sulfur deprivation, H-2 production is initiated after the over-reduction of the plastoquinone pool and decreased PSII activity in the thylakoid membrane. As a result, activated hydrogenase consumes the excess of electrons produced by PSII [Volgusheva et al., Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 7223]. Here, we report that similar conditions can be achieved by simply altering the ratio between photosystem I (PSI) and PSII. In the C3 mutant of C. reinhardtii, we found a lower PSI/PSII ratio than in the wild type, 0.33 vs. 0.85, respectively. This imbalance of photosystems resulted in the over-reduced state of the plastoquinone pool and activation of hydrogenase in the C3 mutant that allowed the photoproduction of H-2 continuously for 42 days. This is an unprecedented duration of H-2 production in green algae under standard growth conditions without any nutrient limitation. Photosynthetic electron flow from PSII to hydrogenase was closely regulated during this long-term H-2 production. The amount of PSII was decreased and the amount of PSI was increased reaching a PSI/PSII ratio of more than 5 as shown by EPR and fluorescence spectroscopy. This fine-tuning of photosystems allows to sustain the long-term production of H-2 in C. reinhardtii by a direct photosynthetic pathway.