Clean and sustainable H2 production is crucial to a carbon–neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not large-scalable because of lacking desirable strains with both optimal H2 productivity and sufficient knowledge of underlying molecular mechanism. We hereby carried out extensive and in-depth investigations of H2 photoproduction of hpm91 mutant lacking PGR5 (Proton Gradient Regulation 5) toward its up-scaling and fundamental mechanism issues. We show that hpm91 is at least 100-fold scalable (up to 10 L) with continuous H2 collection of 7287 ml H2/10L-HPBR in averagely 26 days under sulfur deprivation. Also, we show that hpm91 is robust and active during sustained H2 photoproduction, most likely due to decreased intracellular ROS relative to wild type. Moreover, we obtained quantitative proteomic profiles of wild type and hpm91 at four representing time points of H2 evolution, leading to 2229 and 1350 differentially expressed proteins, respectively. Compared to wild type, major proteome alterations of hpm91 include not only core subunits of photosystems and those related to anti-oxidative responses but also essential proteins in photosynthetic antenna, C/N metabolic balance, and sulfur assimilation toward both cysteine biosynthesis and sulfation of metabolites during sulfur-deprived H2 production. These results reveal not only new insights of cellular and molecular basis of enhanced H2 production in hpm91 but also provide additional candidate gene targets and modules for further genetic modifications and/or in artificial photosynthesis mimics toward basic and applied research aiming at advancing solar-H2 technology.

Clean and sustainable H2 production is essential toward a carbon-neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not scalable because of lacking ideal strains. Here, we explore hpm91, a previously reported PGR5-deletion mutant with remarkable H2 production, that possesses numerous valuable attributes towards large-scale application and in-depth study issues. We show that hpm91 is at least 100-fold scalable (upto 10 liter) with H2 collection sustained for averagely 26 days and 7287 ml H2/10L-HPBR. Also, hpm91 is robust and active over the period of sulfur-deprived H2 production, most likely due to decreased intracellular ROS relative to wild type. Moreover, quantitative proteomic analysis revealed its features in photosynthetic antenna, primary metabolic pathways and anti-ROS responses. Together with success of new high-H2-production strains derived from hpm91, we highlight that hpm91 is a potent strain toward basic and applied research of algal-H2 photoproduction.