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Background: Current understanding of the carbon cycle in methanogenic environments involves trophic interactions such as interspecies H 2 transfer between organotrophs and methanogens. However, many metabolic processes are thermodynamically sensitive to H 2 accumulation and can be inhibited by H 2 produced from co-occurring metabolisms. Strategies for driving thermodynamically competing metabolisms in methanogenic environments remain unexplored., Results: To uncover how anaerobes combat this H 2 conflict in situ, we employ metagenomics and metatranscriptomics to revisit a model ecosystem that has inspired many foundational discoveries in anaerobic ecology-methanogenic bioreactors. Through analysis of 17 anaerobic digesters, we recovered 1343 high-quality metagenome-assembled genomes and corresponding gene expression profiles for uncultured lineages spanning 66 phyla and reconstructed their metabolic capacities. We discovered that diverse uncultured populations can drive H 2 -sensitive metabolisms through (i) metabolic coupling with concurrent H 2 -tolerant catabolism, (ii) forgoing H 2 generation in favor of interspecies transfer of formate and electrons (cytochrome- and pili-mediated) to avoid thermodynamic conflict, and (iii) integration of low-concentration O 2 metabolism as an ancillary thermodynamics-enhancing electron sink. Archaeal populations support these processes through unique methanogenic metabolisms-highly favorable H 2 oxidation driven by methyl-reducing methanogenesis and tripartite uptake of formate, electrons, and acetate., Conclusion: Integration of omics and eco-thermodynamics revealed overlooked behavior and interactions of uncultured organisms, including coupling favorable and unfavorable metabolisms, shifting from H 2 to formate transfer, respiring low-concentration O 2 , performing direct interspecies electron transfer, and interacting with high H 2 -affinity methanogenesis. These findings shed light on how microorganisms overcome a critical obstacle in methanogenic carbon cycles we had hitherto disregarded and provide foundational insight into anaerobic microbial ecology. Video Abstract.

Methanospirillum hungatei strain JF1 (DSM 864) is a methane-producing archaeon and is the type species of the genus Methanospirillum, which belongs to the family Methanospirillaceae within the order Methanomicrobiales. Its genome was selected for sequencing due to its ability to utilize hydrogen and carbon dioxide and/or formate as a sole source of energy. Ecologically, M. hungatei functions as the hydrogen- and/or formate-using partner with many species of syntrophic bacteria. Its morphology is distinct from other methanogens with the ability to form long chains of cells (up to 100 μm in length), which are enclosed within a sheath-like structure, and terminal cells with polar flagella. The genome of M. hungatei strain JF1 is the first completely sequenced genome of the family Methanospirillaceae, and it has a circular genome of 3,544,738 bp containing 3,239 protein coding and 68 RNA genes. The large genome of M. hungatei JF1 suggests the presence of unrecognized biochemical/physiological properties that likely extend to the other Methanospirillaceae and include the ability to form the unusual sheath-like structure and to successfully interact with syntrophic bacteria.

Syntrophobacter fumaroxidans strain MPOB(T) is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project.