Effect of inactivation of genes involved in ammonium regulation on the biohydrogen production of Rhodobacter capsulatus

Hydrogen production by nitrogenase is an energetically expensive process for the cell, hence strictly controlled at different levels. Ammonium is one of the substances regulating nitrogenase activity. The key proteins in the regulation of nitrogenase by ammonium are two regulatory proteins; GlnB and GlnK. In order to increase hydrogen production of Rhodobacter capsulatus DSM1710 (wild type strain) grown on agricultural materials/wastes, ammonium inhibition of nitrogenase enzyme has to be eliminated. In this study, GlnB and GlnK were targeted to be inactivated by in frame site-directed mutagenesis. The glnB mutant R. capsulatus (GP1 strain) was obtained at the end of mutagenesis studies. In the case of glnK, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained.The effect of ammonium on the growth and hydrogen production of R. capsulatus GP1 was investigated and compared with DSM1710. Both DSM1710 and GP1 strains were effectively utilized acetate. The mutation did not affect cell growth significantly at different ammonium levels. Ammonium negatively affected hydrogen production of GP1 strain as well as the DMS1710. However, hydrogen production was significantly low in GP1 strain. The ammonium inhibition of hydrogen production could not be removed in glnB mutant probably due to the presence of an active GlnK protein in the cell. Therefore, GlnK has much more important role in the ammonium dependent control of nitrogenase than GlnB does. The growth and hydrogen production kinetics of R. capsulatus DSM1710 and GP1 were modelled. They were shown to fit to Logistic Model and Modified Gompertz Model, respectively.

► NH4+ inhibits photofermentative hydrogen production due to repression of nitrogenase. ► Removal of GlnB in R. capsulatus cannot remove ammonium inhibition on nitrogenase. ► GlnK might be an essential protein for the cell, since glnK− strain could not be achieved. ► Logistic Model well represents the growth data of R. capsulatus strains. ► Modified Gompertz Model well represents hydrogen production data of R. capsulatus strains.