Two continuous anaerobic fluidized bed reactors (AFBRs) were operated under thermophilic (55 degrees C) temperature for 150 days to investigate the effect of dark H-2 fermentation of diluted and raw sugarcane vinasse on H-2 production using mixed seed sludge. Although effective H-2 production (52.8% of H-2; 0.80 L H-2 h(-1) L-1; 0.79 mmol gCOD(added)(-1) was observed using an elevated substrate concentration (30,000 mg COD L-1), the optimal operational conditions were found for the AFBR(1) (10,000 mg COD L-1) fed with diluted sugarcane vinasse (HRT 6 h; OLR 40 kg COD m(-3) d(-1)), achieving a H-2 yield of 2.86 mmol H-2 g CODadded-1. H-2 production was inhibited by elevated volatile fatty acid (VFA) concentrations (butyric and acetic acids = 3.7 and 3.0 g L-1, respectively) in the raw feedstock. Denaturing gradient gel electrophoresis (DGGE) analyses revealed changes in the bacterial population of the expanded clay biofilm as a function of the substrate concentration. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.