Dark fermentation using mixed cultures is a promising biotechnology for producing hydrogen (H(2)) from renewable organic waste at a low cost. The impact of the characteristics of carbohydrates was evaluated on H(2) production and the associated changes in clostridial populations. A series of H(2)-producing batch experiments was performed from mono-, di- to tri-saccharides (i.e. fructose, glucose, sucrose, maltose, cellobiose, maltotriose). Both chain length and alpha- or beta-linkage of carbohydrates impacted H(2) production performance as well as the patterns of hydrogenases. The H(2) yield, ranging from 1.38 to 1.84 mol-H(2)/mol-hexose, decreased with the increasing chain length of the carbohydrates, showing a negative effect of the hydrolysis step on H(2) production efficiency. Changes in H(2) yield were associated with a specialization of clostridial species, which used different metabolic routes. The rise in H(2) production was associated with butyrate and acetate increases while H(2) consumption was related to caproate formation. Both clostridia] [FeFe]- and [NiFe]-hydrogenases were identified in cellobiose cultures by a proteomic approach. This is the first study that combines genetic and proteomic analyses focused on H(2)-producing bacteria under various conditions and it opens very interesting perspectives to better understand and optimize H(2) production using mixed cultures. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.