An anaerobic syntrophic substrate-degrading coculture system was used to isolate uncultivated methanogens that can provide hydrogen at a very low partial pressure. Primary enrichments for methanogens were prepared from ten environmental samples with ethanol, butyrate, and propionate, which are degraded by syntrophic association with anaerobic syntrophic bacteria and hydrogenotrophic methanogens. For comparison, enrichments with hydrogen at a high partial pressure were also prepared with the same inoculum. To identify methanogens in the enrichments, we applied 16S rRNA gene-based clone analysis to all the enrichments. The clone analysis revealed that almost all the clonal sequences obtained from the hydrogen enrichments were closely related to those of previously isolated methanogens, such as species belonging to the genera Methanobacterium and Methanospirillum. However, several clones retrieved from the syntrophic substrate-degrading enrichments were affiliated with uncultured lineages within the order Methanomicrobiales and with the candidate order Rice Cluster I. After several attempts at isolating the methanogens, we successfully isolated a methanogen affiliated with Rice Cluster I, and obtained a highly purified culture mainly consisting of a methanogen belonging to the family ‘Methanolineaceae’. These results indicate that anaerobic syntrophic substrate-degrading coculture system could be used as an effective method of isolating uncultivated methanogens.
