Bacterial community composition during steady-state, fermentative H(2) production was compared across a range of organic loading rates (OLRs) of 0.5-19 g COD l(-1) h(-1) in a 2-l continuous flow reactor at degrees 0'C. The varied OLRs were achieved with glucose concentrations of 2.5-10 g l(-1) and hydraulic retention times of 1-10 h. The synthetic wastewater feed was amended with L-cysteine and maintained at a pH of 5.5. For each run at a given glucose concentration, the reactor was inoculated with an aliquot of well-mixed agricultural topsoil that had been heat-treated to reduce numbers of vegetative cells. At OLRs less than 2 g COD l(-1) h(-1), DNA sequences from ribosomal RNA intergenic spacer analysis profiles revealed more diverse and variable populations (Selenomonas, Enterobacter, and Clostridium spp.) than were observed above 2 g COD l(-1) h(-1) (Clostridium spp. only). An isolate, LYH1, was cultured from a reactor sample (10 g glucose l(-1) at a 10-h HRT) on medium containing L-cysteine. in confirming H(2) production by LYH1 in liquid batch culture, lag periods for H(2) production in the presence and absence Of L-cysteine were 5 and 50 h, respectively. The 16S rRNA gene sequence of LYH1 indicated that the isolate was a Clostridium sp. affiliated with RNA sulocluster Ic, with >99% similarity to Clostridium sp. FRB1. In fluorescent in situ hybridization tests, an oligonucleotide probe complementary to the 16S rRNA of LYH1 hybridized with 90% of cells observed at an OLR of 2 g COD h(-1), compared to 26% of cells at an OLR of 0.5 g COD l(-1) h(-1). An OLR of 2 g COD l(-1) h(-1) appeared to be a critical threshold above which clostridia were better able to outcompete Enterobacteriaceae and other organisms in the mixed soil inoculum. Our results are discussed in light of other biohydrogen studies employing pure cultures and mixed inocula. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.