Three mutants were constructed in this study employing R. sphaeroides HY01 as wildtype strain: a cbbR gene partially deleted mutant assigned HR02 (cbbR(.)), a transposon mutant based on HR02 with insertional inactivation on pycA gene named HRT16 (cbbR(.),Delta ApycA) and a double-deletion mutant HDR02 (cbbR(.), pycA(.)). The effects of double-gene disruption in R. sphaeroides HY01 on the growth and H-2 generation were studied. The growth of R. sphaeroides HR02 under light anaerobic condition with NH4Cl as nitrogen source was repressed obviously. The H-2 yields of transposon insertion mutant HRT16 with better growth characteristic were enhanced by 2.0%, 10.5%, and 26.8% comparing with HY01 under different NH4+-N concentration conditions (0.6, 1.2 and 1.8 mM NH4Cl supplemented, respectively), while the corresponding maximum H-2 production rates were improved by 15.9%, 6.2% and 46.1%. With 1.2 mM NH4Cl, the H-2 production of HDR02 was increased by 3.6% comparing with its parental strain HY01, and its maximum H-2 production rate approached to that of HRT16. Double-gene mutants revealed enhanced H-2 production performance by improving the ammonium tolerance, contrarying to the single mutant strain. These results implied the superposition effect of genes and the validity of transposon mutagenesis for screening mutants with enhanced H-2 production. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.