We have investigated two approaches to enhance and extend H 2 photoproduction yields in heterocystous, N 2 -fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO 2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H 2 production over time. Our results demonstrated that alginate-entrapped ⌬hupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ⌬hupL mutant, and Calothrix strain 336/3 films were supplemented with N 2 by periodic treatments of air, or air plus CO 2 . These treatments restored the photosynthetic activity of the cells and led to a high level of H 2 production in Calothrix 336/3 and ⌬hupL cells (except for the treatment air plus CO 2 ) but not in the Anabaena PCC 7120 strain (for which H 2 yields did not change after air treatments). The highest H 2 yield was obtained by the air treatment of ⌬hupL cells. Notably, the supplementation of CO 2 under an air atmosphere led to prominent symptoms of N deficiency in the ⌬hupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O 2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.