In this work, a semi-continuous biological system was established to produce hydrogen and generate electricity by coupling the bioreactor to a fuel cell. Heat and acid pretreatments (at 35 and 55 degrees C) of a seed sludge used as inoculum were performed in order to increase hydrogen producers. Different initial glucose concentrations (IGC) were tested for heat pretreated inoculum at 35 degrees C to determine the optimum concentration of glucose that supported the highest hydrogen production. Results showed that the heat pretreated inoculums (35 degrees C) reached the highest hydrogen molar yield of 2.85 mol H(2)/mol glucose (0.014 L/h), which corresponds to the acetic acid pathway. At the optimum IGC (10 g/L, 35 degrees C) the hydrogen molar yield was 3.6 mol H(2)/mol glucose (0.023 L/h). The coupled bioreactor-fuel cell system yielded an output voltage of 1.06 V, power of 0.1 W (25 degrees C) and a current of 68 mA. The overall results suggest that high hydrogen molar yields can be obtained through the acetic acid pathway and that is feasible to generate electricity using hydrogen from the semi- continuous bioreactor. (c) 2009 Elsevier Ltd. All rights reserved.