Purple non-sulfur (PNS) bacteria produce hydrogen by photofermentation of organic acids in waste-water. However, NH4+ in wastewater may inhibit hydrogen synthesis by repressing the expression and activity of nitrogenase, the enzyme catalyzing hydrogen production in PNS bacteria In this study, the Rhodobacter sphaeroides 6016 glnA gene encoding glutamine synthetase (GS) was knocked out by homologous recombination, and the effects on hydrogen production and nitrogenase activity were examined. Using 3 mM glutamine as the nitrogen source, hydrogen production (1,245-1,588 mL hydrogen/L culture) and nitrogenase activity were detected in the mutant in the presence of relatively high NH4+ concentrations (15-40 mM), whereas neither was detected in the wild-type strain under the same conditions. Further analysis indicated that high NH4+ concentrations greatly inhibited the expression of mfA and nitrogenase gene in the wild-type strain but not in the glnA1(-) mutant. These observations suggest that GS is essential to NH4+ repression of nitrogenase and that deletion of glnA1 results in the complete depression of nitrogenase by preventing NH4+ assimilation in vivo, thus relieving the inhibition of mfA and nitrogenase gene expression. Knocking out glnA1 therefore provides an efficient approach to removing the inhibitory effects of ammonium ions in R. sphacroides and possibly in other hydrogen producing PNS bacteria. Biotechnol. Bioeng. 2010, 106: 564-572 (C) 2010 Wiley Periodicals, Inc.