AimThe aim of this research was to create a work scheme for the isolation of the different micro-organisms commonly found in hydrogen-producing reactors and to test its effectiveness. Methods and ResultsMethods were selected to isolate anaerobic spore-forming fermenters, anaerobic fermenters that do not form spores, facultative aerobic fermenters and lactic acid bacteria. The methods were tested in two samples taken from a hydrogen-producing reactor fed with cheese whey. 16S rRNA gene sequences from isolates were compared with pyrosequencing analysis from the same samples. The isolates represented more than 88% of the abundance detected by pyrosequencing. Organisms from the genera Clostridium, Rahnella, Megasphaera, Lactobacillus, Propionibacterium, Bifidobacterium, Chryseobacterium and Acetobacter were isolated. Hydrogen-producing capacity was confirmed for the Clostridium, Rahnella and Megasphaera isolates. Coculture experiments indicate that Megasphaera prevented the total inhibition of Clostridium by Lactobacillus. ConclusionThe work scheme proposed was effective to isolate most of the micro-organisms detected by pyrosequencing analysis. Physiological studies suggested a key role of Megasphaera. Significance and Impact of the StudyWe showed the high culturability of the microbial communities from hydrogen-producing bioreactors. The isolates can be used to perform physiological studies to understand the H-2-producing process.