Metabolically engineered microbial strains can be usefully employed to give higher yields, but this also requires development of a suitable bioprocess. Maximization of product yield during fermentation requires that a number of process parameters, some of which may interact, be optimized. Here we report the effects of different fermentative process conditions; pH, temperature and glucose concentration, on the molar hydrogen yield by a genetically optimized Escherichia coli strain, DJT135. In order to simultaneously reduce the number of the experiments, and to obtain the interactions between the variables important for achieving maximum hydrogen production, a 3(K) full factorial Box-Behnken design and response surface methodology (RSM) were employed for experimental design and analysis. A maximum molar hydrogen yield of 1.69 mol H(2) mol(-1) glucose was obtained under the optimal conditions of 75 mM glucose, 35 degrees C and pH 6.5. Thus. RSM with Box-Behnken design is a useful method for achieving higher molar hydrogen yields by metabolically engineered organisms. (C) 2009 Elsevier Ltd. All rights reserved.