Work scheme to isolate the different micro-organisms found in hydrogen-producing reactors: a study of effectiveness by pyrosequencing analysis.

AIM: The 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 RESULTS: Methods 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. CONCLUSION: The 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 STUDY: We showed the high culturability of the microbial communities from hydrogen-producing bioreactors. The isolates can be used to perform physiological studies to understand the H2 -producing process.