The ecophysiology of one candidate methanogen class WSA2 (or Arc I) remains largely uncharacterized, despite the long history of research on Euryarchaeota methanogenesis. To expand our understanding of methanogen diversity and evolution, we metagenomically recover eight draft genomes for four WSA2 populations. Taxonomic analyses indicate that WSA2 is a distinct class from other Euryarchaeota. None of genomes harbor pathways for CO2-reducing and aceticlastic methanogenesis, but all possess H2 and CO oxidation and energy conservation through H2-oxidizing electron confurcation and internal H2 cycling. As the only discernible methanogenic outlet, they consistently encode a methylated thiol coenzyme M methyltransferase. Although incomplete, all draft genomes point to the proposition that WSA2 is the first discovered methanogen restricted to methanogenesis through methylated thiol reduction. In addition, the genomes lack pathways for carbon fixation, nitrogen fixation and biosynthesis of many amino acids. Acetate, malonate and propionate may serve as carbon sources. Using methylated thiol reduction, WSA2 may not only bridge the carbon and sulfur cycles in eutrophic methanogenic environments, but also potentially compete with CO2-reducing methanogens and even sulfate reducers. These findings reveal a remarkably unique methanogen ‘Candidatus Methanofastidiosum methylthiophilus’ as the first insight into the sixth class of methanogens ‘Candidatus Methanofastidiosa’.

Anaerobic digestion is a common technology to biologically stabilize wasted solids produced in municipal wastewater treatment. Its efficiency is usually evaluated by calculating the reduction in volatile solids, which assumes no biomass growth associated with digestion. To determine whether this assumption is valid and further evaluate digestion efficiency, this study sampled 35 digester sludge from different reactors at multiple time points together with the feed biomass in a full-scale water reclamation plant at Chicago, Illinois. The microbial communities were characterized using Illumina sequencing technology based on 16S rRNA and 16S rRNA gene (rDNA). 74 core microbial populations were identified and represented 58.7% of the entire digester community. Among them, active populations were first identified using the ratio of 16S rRNA and 16S rDNA (rRNA/rDNA) for individual populations, but this approach failed to generate consistent results. Subsequently, a recently proposed mass balance model was applied to calculate the specific growth rate (μ) and this approach successfully identified active microbial populations in digester (positive μ) that could play important roles than those with negative μ. It was further estimated that 82% of microbial populations in the feed sludge were digested in comparison with less than 50% calculated using current equations.

Background: Current understanding of the carbon cycle in methanogenic environments involves trophic interactions such as interspecies H₂ transfer between organotrophs and methanogens. However, many metabolic processes are thermodynamically sensitive to H₂ accumulation and can be inhibited by H₂ produced from co-occurring metabolisms. Strategies for driving thermodynamically competing metabolisms in methanogenic environments remain unexplored. Results: To uncover how anaerobes combat this H₂ 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 H₂-sensitive metabolisms through (i) metabolic coupling with concurrent H₂-tolerant catabolism, (ii) forgoing H₂ generation in favor of interspecies transfer of formate and electrons (cytochrome- and pili-mediated) to avoid thermodynamic conflict, and (iii) integration of low-concentration O₂ metabolism as an ancillary thermodynamics-enhancing electron sink. Archaeal populations support these processes through unique methanogenic metabolisms—highly favorable H₂ 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 H₂ to formate transfer, respiring low-concentration O₂, performing direct interspecies electron transfer, and interacting with high H₂-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.

Microbial community assembly in engineered biological systems is often simultaneously influenced by stochastic and deterministic processes, and the nexus of these two mechanisms remains to be further investigated. Here, three lab-scale activated sludge reactors were seeded with identical inoculum and operated in parallel under eight different sludge retention time (SRT) by sequentially reducing the SRT from 15 days to 1 day. Using 16S rRNA gene amplicon sequencing data, the microbial populations at the start-up (15-day SRT) and SRT-driven (≤10-day SRT) phases were observed to be noticeably different. Clustering results demonstrated ecological succession at the start-up phase with no consistent successional steps among the three reactors, suggesting that stochastic processes played an important role in the community assembly during primary succession. At the SRT-driven phase, the three reactors shared 31 core operational taxonomic units (OTUs). Putative primary acetate utilizers and secondary metabolizers were proposed based on K-means clustering, network and synchrony analysis. The shared core populations accounted for 65% of the total abundance, indicating that the microbial communities at the SRT-driven phase were shaped predominantly by deterministic processes. Sloan’s Neutral model and a null model analysis were performed to disentangle and quantify the relative influence of stochastic and deterministic processes on community assembly. The increased estimated migration rate in the neutral community model and the higher percentage of stochasticity in the null model implied that stochastic community assembly was intensified by strong deterministic factors. This was confirmed by the significantly different α- and β-diversity indices at SRTs shorter than 2 days and the observation that over half of the core OTUs were unshared or unsynchronized. Overall, this study provided quantitative insights into the nexus of stochastic and deterministic processes on microbial community assembly in a biological process.