Hydrogen production from glucose via single-stage photofermentation was examined with the photosynthetic bacterium Rhodobacter capsulatus JP91 (hup-). Response surface methodology with Box-Behnken design was used to optimize the independent experimental variables of glucose concentration, glutamate concentration and light intensity, as well as examining their interactive effects for maximization of molar hydrogen yield. Under optimal condition with a light intensity of 175 W/m(2), 35 mM glucose, and 4.5 mM glutamate, a maximum hydrogen yield of 5.5 (+/- 0.15) mol H-2/mol glucose, and a maximum nitrogenase activity 01 246 (+/- 3.5) nmol C2H4/ml/min were obtained. Densitometric analysis of nitrogenase Fe-protein expression under different conditions showed significant variation in Fe-protein expression with a maximum at the optimized central point. Even under optimum conditions for hydrogen production, a significant fraction of the Fe-protein was found in the ADP-ribosylated state, suggesting that further improvement in yields might be possible. (C) 2012 Elsevier Ltd. All rights reserved.