To identify the key bacterial populations in hydrogen fermentation of galactose, a fermentor seeded with a heat-treated sludge was operated. After 27 h of fermentation, the proportion of butyric acid increased to 69.4 wt.% and the gas production yield reached 1.0 mol H-2/mol galactose. In the pyrosequencing of 16S rDNA, an increase of the proportion of the phylum Firmicutes from 4.2% to 92% (mostly cluster I Clostridium) was observed. To verify the predominance and the ubiquity of the cluster, five fermentors seeded with different heat-treated anaerobic sludges having different feedstock compositions and digestion temperatures were investigated using qPCR analyses. The abundance of the cluster increased >100-fold during the fermentation, regardless of the inocula. Moreover, the abundance was negatively correlated with the lag time of hydrogen production and positively correlated with the hydrogen production rate, demonstrating the relevance of the cluster to hydrogen production. Taken together, the results clearly revealed the importance of cluster I Clostridium in the hydrogen fermentation of galactose. (C) 2014 Elsevier Ltd. All rights reserved.