The experimental investigation aimed at the study of the microbial population during the continuous operation of a complete mixed reactor in large lab-scale (30 L) by variation of the Organic Loading Rate (OLR) and Sludge Retention Time (SRT) ranging from 10 g sucrose/(L.d) to 30 g/(L.d) and from 12 h to 48 h respectively. H(2) yield reached to 1.72 mol H(2)/mol hexose for HRT = 1.6 d and OLR = 20 g sucrose/(L.d). In each phase the dominant microbial genera were identified by sequencing after a Polymerase Chain Reaction (PCR) with universal primers for the domains of Archaea and Eubacteria and specific for Clostridium species and genetic material isolation by Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The phylogenetic analyses showed that hydrogen producing Clostridium species could be affiliated in all experimental phases. Other dominant genera were affiliated mainly to Ethanoligenes harbinense and uncultured Prevotella and Selonomonas species. Bio-hydrogen production was associated to a mixed butyric/ethanol type fermentation facilitated mainly by Clostridium tyrobutyricum and E. harbinense in the presence of lactate as intermediate metabolic product. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.