[FeFe]-hydrogenases are the enzymes responsible for high yield H-2 production during dark fermentation in bio-hydrogen production plants. The culturable bacterial population present in a pilot-scale plant efficiently producing H-2 from waste materials was isolated, classified and identified by means of 16S rDNA gene analysis. The culturable part of the mixed population consists of nine bacterial species that include non-hydrogen producers (Lactobacillus, Enterococcus and Staphylococcus) and several Clostridium that are directly responsible for H-2 production. An extensive analysis of the expression of [FeFe]-hydrogenases in the three best producer strains was achieved by RT-PCR, covering the complete set of known genes for each species. This revealed that during H-2 production there are several different [FeFe]-hydrogenases simultaneously expressed, with genes belonging to the same phylogenetic and structural classification sharing similar transcriptional profiles. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.