Sediment samples from hot spring were enriched for hydrogen-producing bacteria with xylose, glucose and mixed of both sugars at high temperature (60 degrees C). Enriched cultures encoded as PGR and YLT showed high cumulative hydrogen production in xylose glucose mixed and xylose with hydrogen evolution of 1506 and 1487 mL H(2)/L-medium and hydrogen yields of 301.3 and 297.4 mL H(2)/g sugar(consumed), respectively. The enriched cultures coded PGR and YLT were produced acetate and butyrate as main soluble metabolites with high hydrogen production. PCR-DGGE profiling showed that the enriched cultures gave best hydrogen production from xylose glucose mixed comprised of three groups of hydrogen-producing species, (A) relating to genus Bacillus sp. and Anoxybacillus sp., (B) relating to genus Caloramator sp. and Clostridium sp. and (C) relating to efficient hydrogen-producing species (Thermoanaerobacterium sp. and Caldicellulosiruptor sp.). These microbial consortia developed show promising to apply for biohydrogen production from lignocellulosic hydrolysate containing xylose-glucose mixed. Crown Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.