Enhanced hydrogen production performance of cbbR & pycA inactived R.sphaeroides mutant by improving the ammonium tolerance

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^-,ΔpycA) and a double-deletion mutant HDR02 (cbbR^-, pycA^-). The effects of double-gene disruption in R. sphaeroides HY01 on the growth and H₂ generation were studied. The growth of R. sphaeroides HR02 under light anaerobic condition with NH₄Cl as nitrogen source was repressed obviously. The H₂ 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 H₂ production rates were improved by 15.9%, 6.2% and 46.1%. With 1.2 mM NH₄Cl, the H₂ production of HDR02 was increased by 3.6% comparing with its parental strain HY01, and its maximum H₂ production rate approached to that of HRT16. Double-gene mutants revealed enhanced H₂ 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 H₂ production.