Photofermentation by the photosynthetic bacterium Rhodopseudomonas palustris has been used to convert the crude glycerol fraction from biodiesel production to hydrogen as a means of converting this large resource to useful energy. In the present study response surface methodology was applied to investigate the interactive effects among several important process parameters; light intensity, and the concentrations of crude glycerol and glutamate, on the stoichiometric conversion of crude glycerol to hydrogen. Under optimal conditions, a light intensity of 175 W/m(2), 30 mM glycerol, and 4.5 mM glutamate, 6.69 mol hydrogen/mole of crude glycerol were obtained, a yield 96% of theoretical. Determination of nitrogenase activity and expression levels showed that there was relatively little variation in levels of nitrogenase protein with changes in process variables whereas nitrogenase activity varied considerably, with maximal nitrogenase activity (228 nmol of C2H4/ml/min) at the optimal central point. (C) 2011 Elsevier Ltd. All rights reserved.