The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 +/- 0.62 mmol H-2/g CMCadded and 73.4 +/- 3.8 mL H-2/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 degrees C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 +/- 3.22 mmol H-2/g cellulose(added). The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.