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Methylotrophic methanogenesis in the sulfate-rich zone of coastal and marine sediments couples with anaerobic oxidation of methane (AOM), forming the cryptic methane cycle. This study provides evidence of cryptic methane cycling in the sulfate-rich zone across a land–ocean transect of four stations–two brackish, one marine, and one hypersaline–within the Carpinteria Salt Marsh Reserve (CSMR), southern California, USA. Samples from the top 20 cm of sediment from the transect were analyzed through geochemical and molecular (16S rRNA) techniques, in-vitro methanogenesis incubations, and radiotracer incubations utilizing 35S-SO4, 14C-mono-methylamine, and 14C-CH4. Sediment methane concentrations were consistently low (3 to 28 µM) at all stations, except for the marine station, where methane increased with depth reaching 665 µM. Methanogenesis from mono-methylamine was detected throughout the sediment at all stations with estimated CH4 production rates in the sub-nanomolar to nanomolar range per cm3 sediment and day. 16S rRNA analysis identified methanogenic archaea (Methanosarcinaceae, Methanomassiliicoccales, and Methanonatronarchaeacea) capable of producing methane from methylamines in sediment where methylotrophic methanogenesis was found to be active. Metabolomic analysis of porewater showed mono-methylamine was mostly undetectable (<3 µM) or present in trace amounts (<10 µM) suggesting rapid metabolic turnover. In-vitro methanogenesis incubations of natural sediment showed no linear methane buildup, suggesting a process limiting methane emissions. AOM activity, measured with 14C-CH4, overlapped with methanogenesis from mono-methylamine activity at all stations, with rates ranging from 0.03 to 19.4 nmol cm− 3 d− 1. Geochemical porewater analysis showed the CSMR sediments are rich in sulfate and iron. Porewater sulfate concentrations (9–91 mM) were non-limiting across the transect, supporting sulfate reduction activity (1.5–2,506 nmol cm− 3 d− 1). Porewater sulfide and iron (II) profiles indicated that the sediment transitioned from a predominantly iron-reducing environment at the two brackish stations to a predominantly sulfate-reducing environment at the marine and hypersaline stations, which coincided with the presence of phyla (Desulfobacterota) involved in these processes. AOM activity overlapped with sulfate reduction and porewater iron (II) concentrations suggesting that AOM is likely coupled to sulfate and possibly iron reduction at all stations. However, 16S rRNA analysis identified anaerobic methanotrophs (ANME-2) only at the marine and hypersaline stations while putative methanogens were found in sediment across all stations. In one sediment horizon at the marine station, methanogen families (Methanosarcinaceae, Methanosaetaceae, Methanomassiliicoccales, and Methanoregulaceae) and ANME 2a,2b, and 2c groups were found together. Collectively, our data suggest that at the brackish stations methanogens alone may be involved in cryptic methane cycling, while at the marine and hypersaline stations both groups may be involved in the process. Differences in rate constants from incubations with 14C-labeled methane and mono-methylamine suggest a non-methanogenic process oxidizing mono-methylamine to inorganic carbon, likely mediated by sulfate-reducing bacteria. Understanding the potential competition of sulfate reducers with methanogens for mono-methylamine needs further investigation as it might be another important process responsible for low methane emissions in salt marshes. 

ABSTRACT While wetlands are major sources of biogenic methane (CH₄), our understanding of resident microbial metabolism is incomplete, which compromises the prediction of CH₄emissions under ongoing climate change. Here, we employed genome-resolved multi-omics to expand our understanding of methanogenesis in the thawing permafrost peatland of Stordalen Mire in Arctic Sweden. In quadrupling the genomic representation of the site’s methanogens and examining their encoded metabolism, we revealed that nearly 20% of the metagenome-assembled genomes (MAGs) encoded the potential for methylotrophic methanogenesis. Further, 27% of the transcriptionally active methanogens expressed methylotrophic genes; forMethanosarcinalesandMethanobacterialesMAGs, these data indicated the use of methylated oxygen compounds (e.g., methanol), while forMethanomassiliicoccales, they primarily implicated methyl sulfides and methylamines. In addition to methanogenic methylotrophy, >1,700 bacterial MAGs across 19 phyla encoded anaerobic methylotrophic potential, with expression across 12 phyla. Metabolomic analyses revealed the presence of diverse methylated compounds in the Mire, including some known methylotrophic substrates. Active methylotrophy was observed across all stages of a permafrost thaw gradient in Stordalen, with the most frozen non-methanogenic palsa found to host bacterial methylotrophy and the partially thawed bog and fully thawed fen seen to house both methanogenic and bacterial methylotrophic activities. Methanogenesis across increasing permafrost thaw is thus revised from the sole dominance of hydrogenotrophic production and the appearance of acetoclastic at full thaw to consider the co-occurrence of methylotrophy throughout. Collectively, these findings indicate that methanogenic and bacterial methylotrophy may be an important and previously underappreciated component of carbon cycling and emissions in these rapidly changing wetland habitats. IMPORTANCEWetlands are the biggest natural source of atmospheric methane (CH₄) emissions, yet we have an incomplete understanding of the suite of microbial metabolism that results in CH₄formation. Specifically, methanogenesis from methylated compounds is excluded from all ecosystem models used to predict wetland contributions to the global CH₄budget. Though recent studies have shown methylotrophic methanogenesis to be active across wetlands, the broad climatic importance of the metabolism remains critically understudied. Further, some methylotrophic bacteria are known to produce methanogenic by-products like acetate, increasing the complexity of the microbial methylotrophic metabolic network. Prior studies of Stordalen Mire have suggested that methylotrophic methanogenesis is irrelevantin situand have not emphasized the bacterial capacity for metabolism, both of which we countered in this study. The importance of our findings lies in the significant advancement toward unraveling the broader impact of methylotrophs in wetland methanogenesis and, consequently, their contribution to the terrestrial global carbon cycle. 

Abstract. The recently discovered cryptic methane cycle in the sulfate-reducing zone of marine and wetland sediment couples methylotrophic methanogenesis to anaerobic oxidation of methane (AOM). Here we present evidence of cryptic methane cycling activity within the upper regions of the sulfate-reducing zone, along a depth transect within the Santa Barbara Basin, off the coast of California, USA. The top 0–20 cm of sediment from each station was subjected to geochemical analyses and radiotracer incubations using 35S–SO42-, 14C–mono-methylamine, and 14C–CH4 to find evidence of cryptic methane cycling. Methane concentrations were consistently low (3 to 16 µM) across the depth transect, despite AOM rates increasing with decreasing water depth (from max 0.05 nmol cm−3 d−1 at the deepest station to max 1.8 nmol cm−3 d−1 at the shallowest station). Porewater sulfate concentrations remained high (23 to 29 mM), despite the detection of sulfate reduction activity from 35S–SO42- incubations with rates up to 134 nmol cm−3 d−1. Metabolomic analysis showed that substrates for methanogenesis (i.e., acetate, methanol and methylamines) were mostly below the detection limit in the porewater, but some samples from the 1–2 cm depth section showed non-quantifiable evidence of these substrates, indicating their rapid turnover. Estimated methanogenesis from mono-methylamine ranged from 0.2 to 0.5 nmol cm−3 d−1. Discrepancies between the rate constants (k) of methanogenesis (from 14C–mono-methylamine) and AOM (from either 14C–mono-methylamine-derived 14C–CH4 or from directly injected 14C–CH4) suggest the activity of a separate, concurrent metabolic process directly metabolizing mono-methylamine to inorganic carbon. We conclude that the results presented in this work show strong evidence of cryptic methane cycling occurring within the top 20 cm of sediment in the Santa Barbara Basin. The rapid cycling of carbon between methanogenesis and methanotropy likely prevents major build-up of methane in the sulfate-reducing zone. Furthermore, our data suggest that methylamine is utilized by both methanogenic archaea capable of methylotrophic methanogenesis and non-methanogenic microbial groups. We hypothesize that sulfate reduction is responsible for the additional methylamine turnover, but further investigation is needed to elucidate this metabolic activity. 

Abstract Background Microbial colonization of subsurface shales following hydraulic fracturing offers the opportunity to study coupled biotic and abiotic factors that impact microbial persistence in engineered deep subsurface ecosystems. Shale formations underly much of the continental USA and display geographically distinct gradients in temperature and salinity. Complementing studies performed in eastern USA shales that contain brine-like fluids, here we coupled metagenomic and metabolomic approaches to develop the first genome-level insights into ecosystem colonization and microbial community interactions in a lower-salinity, but high-temperature western USA shale formation. Results We collected materials used during the hydraulic fracturing process (i.e., chemicals, drill muds) paired with temporal sampling of water produced from three different hydraulically fractured wells in the STACK ( S ooner T rend A nadarko Basin, C anadian and K ingfisher) shale play in OK, USA. Relative to other shale formations, our metagenomic and metabolomic analyses revealed an expanded taxonomic and metabolic diversity of microorganisms that colonize and persist in fractured shales. Importantly, temporal sampling across all three hydraulic fracturing wells traced the degradation of complex polymers from the hydraulic fracturing process to the production and consumption of organic acids that support sulfate- and thiosulfate-reducing bacteria. Furthermore, we identified 5587 viral genomes and linked many of these to the dominant, colonizing microorganisms, demonstrating the key role that viral predation plays in community dynamics within this closed, engineered system. Lastly, top-side audit sampling of different source materials enabled genome-resolved source tracking, revealing the likely sources of many key colonizing and persisting taxa in these ecosystems. Conclusions These findings highlight the importance of resource utilization and resistance to viral predation as key traits that enable specific microbial taxa to persist across fractured shale ecosystems. We also demonstrate the importance of materials used in the hydraulic fracturing process as both a source of persisting shale microorganisms and organic substrates that likely aid in sustaining the microbial community. Moreover, we showed that different physicochemical conditions (i.e., salinity, temperature) can influence the composition and functional potential of persisting microbial communities in shale ecosystems. Together, these results expand our knowledge of microbial life in deep subsurface shales and have important ramifications for management and treatment of microbial biomass in hydraulically fractured wells. 

Abstract Drought impacts on microbial activity can alter soil carbon fate and lead to the loss of stored carbon to the atmosphere as CO₂and volatile organic compounds (VOCs). Here we examined drought impacts on carbon allocation by soil microbes in the Biosphere 2 artificial tropical rainforest by tracking¹³C from position-specific¹³C-pyruvate into CO₂and VOCs in parallel with multi-omics. During drought, efflux of¹³C-enriched acetate, acetone and C₄H₆O₂(diacetyl) increased. These changes represent increased production and buildup of intermediate metabolites driven by decreased carbon cycling efficiency. Simultaneously,¹³C-CO₂efflux decreased, driven by a decrease in microbial activity. However, the microbial carbon allocation to energy gain relative to biosynthesis was unchanged, signifying maintained energy demand for biosynthesis of VOCs and other drought-stress-induced pathways. Overall, while carbon loss to the atmosphere via CO₂decreased during drought, carbon loss via efflux of VOCs increased, indicating microbially induced shifts in soil carbon fate. 

Abstract Microorganisms play vital roles in modulating organic matter decomposition and nutrient cycling in soil ecosystems. The enzyme latch paradigm posits microbial degradation of polyphenols is hindered in anoxic peat leading to polyphenol accumulation, and consequently diminished microbial activity. This model assumes that polyphenols are microbially unavailable under anoxia, a supposition that has not been thoroughly investigated in any soil type. Here, we use anoxic soil reactors amended with and without a chemically defined polyphenol to test this hypothesis, employing metabolomics and genome-resolved metaproteomics to interrogate soil microbial polyphenol metabolism. Challenging the idea that polyphenols are not bioavailable under anoxia, we provide metabolite evidence that polyphenols are depolymerized, resulting in monomer accumulation, followed by the generation of small phenolic degradation products. Further, we show that soil microbiome function is maintained, and possibly enhanced, with polyphenol addition. In summary, this study provides chemical and enzymatic evidence that some soil microbiota can degrade polyphenols under anoxia and subvert the assumed polyphenol lock on soil microbial metabolism.