The microbial electrolysis cell (MEC) biocathode has shown great potential as alternative for expensive metals as catalyst for H-2 synthesis. Here, the bacterial communities at the biocathode of five hydrogen producing MECs using molecular techniques were characterized. The setups differed in design (large versus small) including electrode material and flow path and in carbon source provided at the cathode (bicarbonate or acetate). A hydrogenase gene-based DNA microarray (Hydrogenase Chip) was used to analyze hydrogenase genes present in the three large setups. The small setups showed dominant groups of Firmicutes and two of the large setups showed dominant groups of Proteobacteria and Bacteroidetes. The third large setup received acetate but no sulfate (no sulfur source). In this setup an almost pure culture of a Promicromonospora sp. developed. Most of the hydrogenase genes detected were coding for bidirectional Hox-type hydrogenases, which have shown to be involved in cytoplasmatic H-2 production. (C) 2014 Elsevier Inc. All rights reserved.