Pyrosequencing of mcrA and Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA ) and 16S rRNA genes.Methanobacteriales ,Methanomicrobiales , andMethanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of themcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularlyMethanosarcinales , while the other digesters were dominated by hydrogenotrophicMethanomicrobiales . The proposed euryarchaeotal orderMethanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potentialmcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using themcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance ofmcrA transcripts in digesters treating sludge and wastewater samples, supporting themcrA gene as a biomarker for methane yield.