Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H-2/mol xylose. In CSTR the highest H-2 yield and production rate at 45 degrees C were 1.97 mol H-2/mol xylose and 7.3 mmol H-2/h/L, respectively, and at 37 degrees C, 1.18 mol H-2/mol xylose and 1.7 mmol H-2/h/L, respectively. At 45 degrees C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 degrees C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H-2 production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 degrees C for both mesophiles and thermophiles. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.