ABSTRACT
Biogas, a mix of CO
2
, CH
4
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.