Abstract
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.