Unveiling the ecology, taxonomy and metabolic capabilities of MBA03, a potential key player in anaerobic digestion

Biogas, a mix of CO2, CH4 and small proportions of other gases, is a biofuel obtained by anaerobic digestion (AD). Biogas production is often considered a black box process, as the role and dynamics of some of the microorganisms involved remain undisclosed. Previous metataxonomic studies in the frame of the MICRO4BIOGAS project (www.micro4biogas.eu) revealed that MBA03, an uncharacterised and uncultured bacterial taxon, was very prevalent and abundant in industrial full-scale AD plants. Surprisingly, no culturable specimen or genome of this taxon has ever been reported, so its role in AD has remained unclear. In the present work, thirty samples derived from anaerobic digesters were sequenced, allowing the reconstruction of 108 metagenome-assembled genomes (MAGs) potentially belonging to MBA03. According to phylogenetic analyses and genomic similarity indices, MBA03 constitutes a new bacterial order, proposed as Darwinibacteriales ord. nov., which includes Darwinibacter acetoxidans gen. nov., sp. nov. of the family Darwinibacteriaceae fam. nov., along with Wallacebacter cryptica gen. nov., sp. nov. of the Wallacebacteriaceae fam. nov. Ecotaxonomic studies determined that AD processes are the main ecological niche of Darwinibacteriales. Moreover, metabolic predictions identified Darwinibacteraceae members as putative syntrophic acetate oxidising bacteria (SAOB), as they encode for the reversed Wood-Ljungdahl (W-L) pathway coupled to the glycine cleavage system. This suggests that Darwinibacteraceae members work in collaboration with hydrogenotrophic archaea to produce methane in industrial biogas plants. Overall, our findings present Darwinibacteriales as a potential key player in anaerobic digestion and pave the way towards the complete characterisation of this newly described bacterial taxa.