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In this study we have demonstrated the possibility of phototrophic hydrogen production in C. reinhardtii under N-deprived conditions. When tested under air + CO 2 , and Ar + CO 2 N-deprived C. reinhardtii demonstrated decrease in PSII activity mainly due to over reduction of PQ, in addition no ascorbate accumulation was observed in cells. Under air + CO 2 atmosphere cells accumulated excessive amounts of starch. When incubated under Ar + CO 2 atmosphere cells accumulated starch as nitrogen replete cultures and no hydrogen production was observed. Hydrogen production (86 ml H 2 per one l of culture) occurred under Ar + CO 2 atmosphere when particular two-step illumination protocol was implicated. In oxygen producing and early oxygen consuming stage cells were illuminated under light intensity 169 μE m−2 s−1. When light was switched to 30 μE m−2 s−1, cultures quickly respired all oxygen and transient to anaerobic conditions with subsequent hydrogen production 2 h later. Actual quantum yield of C. reinhardtii cultures was measured in photobioreactor and maximal quantum efficiency of PSII of dark adapted cells together with JIP test were studied. • Photoautotrophic Chlamydomonas reinhardtii under N deprivation were studied. • Under air cultures accumulated starch and moderately decreased PSII activity. • Under Ar cultures decreased PSII activity and transit to anaerobiosis. • Under Ar two step illumination protocol was applied for sustainable H 2 production. • Under this illumination protocol 80 ml of H 2 per one l of culture was produced. [ABSTRACT FROM AUTHOR]