The effects of pH on hydrogen fermentation of glucose by newly isolated H-2-producing bacterium Clostridium tyrobutyricum JM1 were investigated in batch cultivations. The changes of carbon material and energy balances by pH conditions provided useful information for understanding and interpreting the regulatory system of the microorganism, and for optimization of a desired product, in this case, molecular hydrogen. The most probable metabolic pathways of C. tyrobutyricum JM1 were determined through an accurate analysis of stoichiometry and the consistency of the experimental data, checked by high carbon recovery. The carbon material and energy balances were adequately applied to estimate the carbon-flow distribution. They suggested that pH 6.3 was appropriate to maximize hydrogen production with a high concentration of butyrate and balanced activities of NADH. (c) 2008 Elsevier Ltd. All rights reserved.