Rokubacteria is a phylogenetic clade of as-yet-uncultivated prokaryotes, which are detected in diverse terrestrial habitats and are commonly addressed as members of the rare biosphere. This clade was originally described as a candidate phylum; however, based on the results of comparative genome analysis, was later defined as the order-level lineage, Rokubacteriales, within the phylum Methylomirabilota. The physiology and lifestyles of these bacteria are poorly understood. A dataset of 16S rRNA gene reads retrieved from four boreal raised bogs and six eutrophic fens was examined for the presence of the Rokubacteriales; the latter were detected exclusively in fens. Their relative abundance varied between 0.2 and 4% of all bacteria and was positively correlated with pH, total nitrogen content, and availability of Ca and Mg. To test an earlier published hypothesis regarding the presence of methanotrophic capabilities in Rokubacteria, peat samples were incubated with 10% methane for four weeks. No response to methane availability was detected for the Rokubacteriales, while clear a increase in relative abundance was observed for the conventional Methylococcales methanotrophs. The search for methane monooxygenase encoding genes in 60 currently available Rokubacteriales metagenomes yielded negative results, although copper-containing monooxygenases were encoded by some members of this order. This study suggests that peat-inhabiting Rokubacteriales are neutrophilic non-methanotrophic bacteria that colonize nitrogen-rich wetlands.

Methanotrophic verrucomicrobia of the order Methylacidiphilales are known as extremely acidophilic, thermophilic or mesophilic bacteria that inhabit acidic geothermal ecosystems. The occurrence of verrucomicrobial methanotrophs in other types of acidic environments remains an open question. Notably, Methylacidiphilales-affiliated 16S rRNA gene sequences are commonly retrieved from acidic (pH 3.5–5.5) peatlands. In this study, we compared the patterns of verrucomicrobial diversity in four acidic raised bogs and six neutral fens located in European North Russia. Methylacidiphilales-like 16S rRNA gene reads displaying 83–86% similarity to 16S rRNA gene sequences of currently described verrucomicrobial methanotrophs were recovered exclusively from raised bogs. Laboratory incubation of peat samples with 10% methane for 3 weeks resulted in the pronounced increase of a relative abundance of alphaproteobacterial methanotrophs, while no response was detected for Methylacidiphilales-affiliated bacteria. Three metagenome-assembled genomes (MAGs) of peat-inhabiting Methylacidiphilales bacteria were reconstructed and examined for the presence of genes encoding methane monooxygenase enzymes and autotrophic carbon fixation pathways. None of these genomic determinants were detected in assembled MAGs. Metabolic reconstructions predicted a heterotrophic metabolism, with a potential to hydrolyze several plant-derived polysaccharides. As suggested by our analysis, peat-inhabiting representatives of the Methylacidiphilales are acidophilic aerobic heterotrophs, which comprise a sister family of the methanotrophic Methylacidiphilaceae.

Upland forest ecosystems are recognized as net sinks for atmospheric methane (CH₄), one of the most impactful greenhouse gases. Biological methane uptake in these ecosystems occurs due to the activity of aerobic methanotrophic bacteria. Russia hosts one-fifth of the global forest area, with the most extensive forest landscapes located in West Siberia. Here, we report seasonal CH₄ flux measurements conducted in 2018 in three types of stands in West Siberian middle taiga–Siberian pine, Aspen, and mixed forests. High rates of methane uptake of up to −0.184 mg CH₄ m⁻² h⁻¹ were measured by a static chamber method, with an estimated total growing season consumption of 4.5 ± 0.5 kg CH₄ ha⁻¹. Forest type had little to no effect on methane fluxes within each season. Soil methane oxidation rate ranged from 0 to 8.1 ng CH₄ g_DW⁻¹ h⁻¹ and was negatively related to water-filled pore space. The microbial soil communities were dominated by the Alpha- and Gammaproteobacteria, Acidobacteriota and Actinobacteriota. The major group of 16S rRNA gene reads from methanotrophs belonged to uncultivated Beijerinckiaceae bacteria. Molecular identification of methanotrophs based on retrieval of the pmoA gene confirmed that Upland Soil Cluster Alpha was the major bacterial group responsible for CH₄ oxidation.

Recently discovered fields of seeps in western Siberia are an important, previously unknown source of methane. Seeps are located in the floodplains of small rivers and vary in shape, size and localization. Methane fluxes from seeps may be high, making them the sources of high regional importance. However, the data on the seep bacterial community composition are scarce, concentrating mainly on methanotrophic bacteria. In the present work, the overall bacterial diversity in the sediments of shallow-water seeps at the floodplain of the Bolshaya Rechka River was studied using high-throughput 16S rRNA gene sequencing. Molecular analysis revealed that Gammaproteobacteria and Actinobacteria were the most abundant bacterial groups (28.5–33.8 and 11–13.2% of total 16S rRNA genes, respectively). A significant part of the sequences belonged to Chloroflexi, Desulfobacterota, and Bacteroidota. Acidobacteriota and Verrucomicrobiota were responsible for 1.5–2.7 and 1.0–1.9%, respectively. The methanotrophic community was dominated by bacteria of the genus Methylobacter. The most numerous species-level OTU (8% of all 16S rRNA gene sequences) belonged to Methylobacter tundripaludum (97% identity). Methanotrophic Alphaproteobacteria were not detected in the seeps. Our results confirm the presence of an active bacterial community in the sediments of shallow-water seeps, with predominance of methanotrophic Gammaproteobacteria.