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

Abstract

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, and Methanosarcinales 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 the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales, while the other digesters were dominated by hydrogenotrophic Methanomicrobiales. The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA 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 the mcrA 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 of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield. Methane from anaerobic digestion is an important source of renewable energy that can circumvent the problems of dwindling fossil fuels and of atmospheric carbon dioxide (CO 2 ) emissions due to fossil fuel combustion (1). Anaerobic digestion is becoming a key part of municipal wastewater treatment, as it allows recovery of energy (biogas) from waste streams to offset onsite energy consumption. The anaerobic digestion of heterogeneous organic substrates for methane production is a complex process involving four major sequential phases: hydrolysis, fermentation, acetogenesis, and methanogenesis (2). Methanogens are strictly anaerobic archaea that produce methane from a limited number of substrates, including hydrogen (H 2 ), acetate, and some C 1 compounds (2). Phylogenetically, methanogens belong to the Euryarchaeota with six established (Methanobacteriales, Methanococcales, Methanomicrobiales, Methanocellales, Methanopyrales, and Methanosarcinales) and one proposed (Methanomassiliicoccales) order(s) (3) and at least 31 genera (4). As well as being major functional components of anaerobic digester communities (5, 6), methanogens are found in other anoxic environments such as peatlands (7), landfills...

Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

“…A high abundance of the acetoclastic Methanosaeta typically indicates lower acetate concentration and/or retention time (47,48) and previously has been observed in GZ and SWH sludge (26). Given the high acetate concentrations observed toward the end of the time course experiment (Fig.…”

Inhibitory Effect of Coumarin on Syntrophic Fatty Acid-Oxidizing and Methanogenic Cultures and Biogas Reactor Microbiomes

“…A customized database was created by downloading all mcrA sequences from the Functional Gene Repository (40) containing taxonomic information to at least the genus level. The mcrA sequences were taxonomically classified against this database using the RDP Classifier 2.2 (41), as described previously (42). Sequence alignment was performed using the MUSCLE algorithm (43).…”

Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen

“…However, one potentially class-level Euryarchaeota clade thought to be capable of methanogenesis, WSA2 (or Arc 1), (Hugenholtz 2002;Chouari et al, 2005) remains uncharacterized, despite the identification through 16S ribosomal RNA (rRNA) sequencing more than 15 years ago (Dojka et al, 1998). Moreover, members of this clade have been observed in a wide range of natural and engineered environments (for example, freshwater and marine sediments, contaminated groundwater and bioreactors; Dhillon et al, 2005;Cheng et al, 2012;Saito et al, 2015;Wilkins et al, 2015). To fill this gap in our understanding of methanogen phylogeny and WSA2's potential roles in anaerobic biogeochemical cycles, we construct the first WSA2 genomes through metagenomics of methanogenic bioreactors treating wastewater.…”