Heterocyst-forming cells of the cyanobacterium Anabaena sp. strain PCC 7120 Delta Hup, lacking an uptake hydrogenase, photobiologically produce H-2 by nitrogenase. Under N-2-rich atmosphere, the nitrogenase activity declines in a rather short time due to the sufficiency of combined nitrogen. From the parental Delta Hup strain, site-directed double-crossover variants, dc-Q193S and dc-R284H, were created with amino acid substitutions presumed to be located in the vicinity of the FeMo-cofactor of nitrogenase. Unlike the case for the Delta Hup strain, H-2 production activities of the variants were not decreased by the presence of high concentrations of N-2 and they continuously produced H-2 over 21 days with occasional headspace gas replacement. This property of N-2 insensitivity is a potentially useful strategy for reducing the cost of the culture gas in future practical applications of sustainable biofuel production. This Anabaena strain has only the Mo-containing nitrogenase which reduces acetylene to ethylene, but the dc-Q193S variant also produced ethane at low but measurable rates along with greater rates of ethylene production. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.