Fermentations of lactose, glucose and galactose using Escherichia coil WDHL, a hydrogen over producer strain, were performed. With glucose as substrate pyruvate was mainly routed to the lactate pathway, resulting in hydrogen production and yield of 1037 mL and 0.30 mol H-2/mol of glucose, respectively. When galactose was the substrate, the pyruvate formate lyase pathway was the main route for pyruvate and a fermentation yield of 1.12 mol H-2/mol of galactose and a hydrogen production of 2080 mL were obtained. The fermentation of lactose or glucose plus galactose showed a similar yield of 1.02 mol H-2/mol of hexose consumed. This work clearly demonstrated that the kinetics of hydrogen and metabolites production as well as the hydrogen yield were affected by the type of sugar used as substrate as reflected by the deviations from the metabolic hydrogen-production pathway. (C) 2012 Elsevier Ltd. All rights reserved.