This work investigated how heat and acid pretreatment affected thermophilic biohydrogen production by a mixed culture present in the sludge of a sugarcane vinasse treatment plant. Heat- (80 degrees C for 15 min) or acid- (pH 3 for 24h) treated sludge samples were employed to seed batch bioreactors along 36 days. The heat- and acid-treated sludge samples afforded H-2 production rates (HPRs) of 8.4 and 9.5 mmo/L.d, respectively. The lactic acid concentration increased in the bioreactor seeded with heat-treated sludge, whilst the lactic acid concentration diminished in the bioreactor inoculated with the acid-treated sludge. Identification of the microbial community revealed that Clostridiaceae predominated in all the sludge samples. The most abundant Clostridiaceae in the heat- and acid-treated sludge samples were Clostridium and Thermoanaerobacterium, respectively. Acid pretreatment modified the microbial community and enhanced consumption of lactic acid, a metabolite that is normally associated with decreased H2 production. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.