In the present study, hydrogen-fermentation microflorae were examined in order to develop a microbial mechanism for application of hydrogen fermentation as a new technique for utilisation of beer lees, the main waste product from beer brewing. Stable hydrogen-fermentation microflorae were explored, and 8 influential microflorae, which required no additional treatments, were chosen from 33 environmental microflorae. The maximum hydrogen yield of 29.3 mL H-2/g TS was obtained and was 9-times greater than that of previous studies when pre-treatment was not used. Additionally, the microfloral composition and hydrogen productivity of the main bacterium were analysed by culture-independent and culture-dependent methodologies. Results revealed 41 species belonging to 14 genera. From 151 strains isolated through culture-dependent methods, Clostridium roseum was the most common bacterium emerging as a useful organism for hydrogen production from beer lees. This report represents the first description of a microbiological mechanism for hydrogen fermentation from beer lees. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.