Summary We report on the genomic characterization of three novel classes in the phylum Desulfobacterota. One class (proposed name Candidatus ‘Anaeroferrophillalia’) was characterized by heterotrophic growth capacity, either fermentatively or utilizing polysulfide, tetrathionate or thiosulfate as electron acceptors. In the absence of organic carbon sources, autotrophic growth via the Wood–Ljungdahl (WL) pathway and using hydrogen or Fe(II) as an electron donor is also inferred for members of the ‘Anaeroferrophillalia’. The second class (proposed name Candidatus ‘Anaeropigmentia’) was characterized by its capacity for growth at low oxygen concentration, and the capacity to synthesize the methyl/alkyl carrier CoM, an ability that is prevalent in the archaeal but rare in the bacterial domain. Pigmentation is inferred from the capacity for carotenoid (lycopene) production. The third class (proposed name Candidatus ‘Zymogenia’) was characterized by fermentative heterotrophic growth capacity, broad substrate range and the adaptation of some of its members to hypersaline habitats. Analysis of the distribution pattern of all three classes showed their occurrence as rare community members in multiple habitats, with preferences for anaerobic terrestrial, freshwater and marine environments over oxygenated (e.g. pelagic ocean and agricultural land) settings. Special preference for some members of the class Candidatus ‘Zymogenia’ for hypersaline environments such as hypersaline microbial mats and lagoons was observed.

An overwhelming majority of bacterial life remains uncharacterized. Recent efforts to assemble genomes from metagenomes have provided invaluable insights into these yet-uncultured bacterial lineages. We report on the characterization of 30 genomes belonging to three novel classes within the phylum Desulfobacterota. One class (proposed name Candidatus 'Anaeroferrophillalia') was characterized by the capacity for heterotrophic growth, either fermentatively or utilizing polysulfide, tetrathionate and thiosulfate as electron acceptors. Autotrophic growth using the Wood Ljungdahl pathway and hydrogen or Fe(II) as an electron donor could also occur in absence of organic carbon sources. The second class (proposed name Candidatus “Anaeropigmentia”) was characterized by its capacity for fermentative or aerobic growth at low oxygen thresholds using a broad range of sugars and amino acids, and the capacity to synthesize the methyl/alkyl carrier CoM, an ability that is prevalent in the archaeal but rare in the bacterial domain. Pigmentation is inferred from the capacity for carotenoids (lycopene) production. The third class (proposed name Candidatus 'Zymogenia') was characterized by the capacity for heterotrophic growth fermentatively using broad sugars and amino acids as carbon sources, and the adaptation of some of its members to hypersaline habitats. Analysis of the distribution pattern of all three classes showed their occurrence as rare community members in multiple habitats, with preferences for anaerobic terrestrial, freshwater, and marine environments, over oxygenated (e.g. pelagic ocean and agricultural land) settings. Special preference for some members of the class Candidatus 'Zymogenia' to hypersaline environments, e.g. hypersaline microbial mats and lagoons was observed.