Metagenomic Insights Into the Ecology, Taxonomy and Metabolic Capabilities of ‘ <i>Candidatus</i> Darwinibacteriales’ Ord. Nov. (Formerly <scp>MBA03</scp> ), 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 belonging to phylum Bacillota, was very prevalent and abundant in industrial full-scale AD plants. Despite the efforts, this taxon has not yet been cultivated, which makes the analysis of its taxonomy, ecology and metabolism even more challenging. In the present work, 30 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 was classified as a new bacterial order, proposed as 'Candidatus Darwinibacteriales' ord. nov., which includes 'Candidatus Darwinibacter acetoxidans' gen. nov., sp. nov. of 'Candidatus Darwinibacteriaceae' fam. nov., along with 'Candidatus Wallacebacter cryptica' gen. nov., sp. nov. of the 'Candidatus Wallacebacteriaceae' fam. nov. Ecotaxonomic studies determined that AD processes are the main ecological niche of 'Candidatus 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 could work in collaboration with hydrogenotrophic methanogenic archaea to produce methane in industrial biogas plants. Overall, our findings present 'Candidatus Darwinibacteriales' as a potential key player in anaerobic digestion and pave the way towards the complete characterisation of this newly described bacterial taxon, which has not yet been cultured.