Microbiota and methanogenic activities in an anaerobic internal circulation reactor: insights into biogas production from brewery wastewater.

In this study, we analyzed the prokaryotic community and methanogenic activities in sludge samples collected from a full-scale internal circulation (IC) reactor used to treat brewery wastewater. The reactor performance was monitored over 15 months, and specific methanogenic activities were periodically measured in fresh sludge samples using CO/H or acetate as substrates. The maximum hydrogenotrophic activities were consistently higher than maximum acetoclastic activities, suggesting the relevance of hydrogenotrophic methanogens in the sludge. Over six months, the prokaryotic community present in four sludge samples was analyzed using amplicon libraries and metagenomics. V4-16S rRNA amplicon libraries revealed the presence of a diverse microbial community dominated by Firmicutes and Bacteroidetes among bacterial phyla, and Halobacterota and Euryarchaeota among archaea. Furthermore, the 16S libraries constructed with cDNA were consistent with the methanogenic activity assays. A genome-centric metagenomics approach was used to assemble 42 high-quality metagenome-assembled genomes (MAGs), among which Methanothrix and Methanobacterium were the dominant archaeal members, and Acidobacteriota, Synergistota, Krumholzibacteriota, and Nitrospirota phyla were among the bacteria. Potential acetogenic members were explored via the fths gene; 15 MAGs contained this marker gene. A combination of methanogenic activity tests, amplicon libraries, and MAG analysis was used to gain insights into the prokaryotic structure and functional potential of the microbial community driving methane production in the reactor.