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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Unraveling the depth-dependent causal dynamics of methanogenesis and methanotrophy in a high-latitude fen peatland

The dynamics of methane (CH 4 ) cycling in high-latitude peatlands through different pathways of methanogenesis and methanotrophy are still poorly understood due to the spatiotemporal complexity of microbial activities and biogeochemical processes. Additionally, long-term in situ measurements within soil columns are limited and associated with large uncertainties in microbial substrates (e.g. dissolved organic carbon, acetate, hydrogen). To better understand CH 4 cycling dynamics, we first applied an advanced biogeochemical model, ecosys , to explicitly simulate methanogenesis, methanotrophy, and CH 4 transport in a high-latitude fen (within the Stordalen Mire, northern Sweden). Next, to explore the vertical heterogeneity in CH 4 cycling, we applied the PCMCI/PCMCI+ causal detection framework with a bootstrap aggregation method to the modeling results, characterizing causal relationships among regulating factors (e.g. temperature, microbial biomass, soil substrate concentrations) through acetoclastic methanogenesis, hydrogenotrophic methanogenesis, and methanotrophy, across three depth intervals (0–10 cm, 10–20 cm, 20–30 cm). Our results indicate that temperature, microbial biomass, and methanogenesis and methanotrophy substrates exhibit significant vertical variations within the soil column. Soil temperature demonstrates strong causal relationships with both biomass and substrate concentrations at the shallower depth (0–10 cm), while these causal relationships decrease significantly at the deeper depth within the two methanogenesis pathways. In contrast, soil substrate concentrations show significantly greater causal relationships with depth, suggesting the substantial influence of substrates on CH 4 cycling. CH 4 production is found to peak in August, while CH 4 oxidation peaks predominantly in October, showing a lag response between production and oxidation. Overall, this research provides important insights into the causal mechanisms modulating CH 4 cycling across different depths, which will improve carbon cycling predictions, and guide the future field measurement strategies.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Hypophosphite Is a Naturally Occurring Selective Inhibitor of Syntrophic Methanogenesis

Microbial methanogenesis is a major contributor to global warming, and methane fluxes represent a loss of energy and electrons from industrial ecosystems. The chemical space of methane control strategies is still underexplored. Most known methanogenesis inhibitors target methanogenic archaeal enzymes. However, interference with the exchange of syntrophic electron carriers (H 2 or formate) in methanogenic systems presents an additional target for methane control. Here, we show that hypophosphite (H 2 PO 2 − ), an inorganic formate analogue, is a potent and selective inhibitor of syntrophic methanogenesis versus primary fermentation in rice field sediments and cattle rumens. Hypophosphite is also generally recognized as safe and relatively nontoxic to plants and animals. Genetic screens and physiological assays in the model methanogen Methanococcus maripaludis S2 implicate formate metabolism as the target of hypophosphite inhibition. Currently, there is no known biological pathway for anaerobic hypophosphite oxidation, and hypophosphite is stable in anoxic sediments for weeks to months. Given its widespread natural occurrence, we propose that hypophosphite may modulate the carbon cycle in natural environments. Taken together, our results suggest that hypophosphite could be used as a safe, inexpensive strategy for methane control in syntrophic methanogenic ecosystems.

chemical inhibitor↗
DOE OSTI2025

Methanogenesis and Acetogenesis in Hydrogenotrophy with Carbonate Minerals: Dependence on Mineral Surface Area, Biofilm Growth, and Microbial Community

The production, storage, and use of hydrogen are anticipated to grow substantially to achieve energy and climate goals. Consequently, microbial communities in many terrestrial and subsurface Earth environments could be exposed to elevated hydrogen concentrations. Hydrogen stimulates metabolic processes that reduce aqueous chemical species, such as bicarbonate or sulfate, that can exchange with solid mineral phases, but the controls on microbial hydrogenotrophy with mineral sources of electron acceptors are not fully understood. Herein, we applied laboratory experiments and biogeochemical modeling to study the response of a natural microbial community to an elevated partial pressure of hydrogen in the presence of carbonate minerals of varying composition, solubility, and size. Experimental incubations and simulation results showed that hydrogen consumption by microbial communities was initially dominated by sulfate reduction and, subsequently, transitioned to acetogenesis and methanogenesis. The rates of acetogenesis and methanogenesis were not correlated with the solubility of carbonate minerals. Instead, we observed strong linear correlations between the rates and surface area of carbonate minerals. Methane and acetate production slowed down in all incubations after about 2 weeks of incubation, although biogeochemical modeling predicted that the metabolic processes were not thermodynamically limited. Electron microscopy and infrared spectroscopy showed that biofilms with diverse microorganisms grew on the carbonates during this period. The methane δ13C value significantly increased, consistent with slower growth at elevated pH. This work highlights that microbial communities form biofilm on carbonate mineral surfaces as a response to hydrogen and that biofilm formation could pose a strong kinetic limitation to hydrogenotrophic metabolism utilizing carbonate minerals.

Acetobacterium↗
DOE OSTI2025

Effects of soluble electron shuttles on microbial iron reduction and methanogenesis

In many aquatic and terrestrial ecosystems, iron (Fe) reduction by microorganisms is a key part of biogeochemical cycling and energy flux. The presence of redox-active electron shuttles in the environment potentially enables a phylogenetically diverse group of microbes to use insoluble iron as a terminal electron acceptor. We investigated the impact that different electron shuttles had on respiration, microbial physiology, and microbial ecology. We tested eight different electron shuttles, seven quinones and riboflavin, with redox potentials between 0.217 and −0.340 V. Fe(III) reduction coupled with acetate oxidation was observed with all shuttles. Once Fe(III) reduction began to plateau, a rapid increase in acetate consumption was observed and coincided with the onset of methane production, except in the incubations with the shuttle 9,10-anthraquinone-2-carboxylic acid (AQC). The rates of iron reduction, acetate consumption, methanogenesis, and the microbial communities varied significantly across the different shuttles independent of redox potential. In general, shuttles appeared to reduce the overall diversity of the community compared to no shuttle controls, but certain shuttles were exceptions to this trend. Geobacteraceae were the predominant taxonomic family in all enrichments except in the presence of AQC or 1,2-dihydroxyanthraquinone (AQZ), but each shuttle enriched a unique community significantly different from the no shuttle control conditions. This suggests that the presence of different redox-active electron shuttles can have a large influence on the microbial ecology and total carbon flux in the environment.

Anaerobic Redox Reactions↗
DOE OSTI2025

The stable carbon isotope fractionation of methanogenesis products at complete carbon consumption

The stable carbon isotope signature (δ 13 C) of methane (CH 4 ) is used to discriminate between biological, thermogenic, and abiotic sources. Methanogens, or methane producing archaea, inhabit a broad range of chemical conditions. Many of these environments are replete in dissolved inorganic carbon (DIC), causing isotopically depleted δ 13 C biogenic CH 4 . However, some extreme environments inhabited by methanogens, such as serpentinising systems, exhibit low carbon dioxide (CO 2 ) availability, replete H 2 , and isotopically enriched δ 13 C CH 4 that is outside the known biogenic range. We measured the δ 13 C of CO 2 , biomass, lipids, and CH 4 during hydrogenotrophic methanogenesis under hydrogen replete conditions with a limited carbon pool to investigate carbon isotope dynamics at complete DIC consumption. As theory predicts, we found that the final, accumulated methane δ 13 C values closely reflect the δ 13 C of the initial DIC supply, and that methane is more 13 C enriched than biomass and lipids. This provides the first experimental evidence that methanogens can achieve complete carbon consumption and thus can produce accumulated CH 4 products that isotopically reflect the initial CO 2 . These data show that the range of possible δ 13 C values from biogenic methane needs to be expanded for natural environments impacted by extreme carbon limitation.

biomass↗
DOE OSTI2025

Spatial evidence of cryptic methane cycling and methylotrophic metabolisms along a land–ocean transect in salt marsh sediment

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 35 S-SO 4 , 14 C-mono-methylamine, and 14 C-CH 4 . 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 CH 4 production rates in the sub-nanomolar to nanomolar range per cm 3 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 14 C-CH 4 , 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 14 C-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.

Anaerobic oxidation of methane↗
DOE OSTI2025

Sulfide stress tolerance as a controller of methane production in temperate wetlands

Abstract Wetlands are a major source of methane emissions and contribute to the observed increase in atmospheric methane over the last 20 years. Methane production in wetlands is the final step of carbon decomposition performed by anaerobic archaea. Although hydrogen/carbon dioxide and acetate are the substrates most often attributed to methanogenesis, other substrates—such as methylated compounds—may additionally play important roles in driving methane production in wetland systems. Here we conducted mesocosm experiments combined with genome-resolved metatranscriptomics to investigate the impact of diverse methanogenic substrate amendment on methanogenesis in two high methane-emitting wetlands with distinct geochemistry, termed P7 and P8. Methanol amendment resulted in high methane production at both sites, whereas acetate and formate amendment only stimulated methanogenesis in P7 mesocosms, where aqueous sulfide concentrations were lower. In P7 sediments, formate amendment fueled acetogenic microbes that produced acetate, which was subsequently utilized by acetoclastic methanogens. In contrast to expression profiles in P7 mesocosms, active methylotrophic methanogen genomes from P8 showed increased expression of genes related to membrane remodeling and DNA damage repair, indicative of stress tolerance mechanisms to counter sulfide toxicity. Methylotrophic methanogenesis generates higher free energy yields than acetoclastic methanogenesis, which likely enables allocation of more energy toward stress responses. These findings contribute to the growing body of literature highlighting methylotrophic methanogenesis as an important methane production pathway in wetlands. By using less competitive substrates like methanol that provide greater energy yields, methylotrophic methanogens may invest in physiological strategies that provide competitive advantages across a range of environmental stresses.

Environmental Sciences & Ecology↗
DOE OSTI2024

Transcriptional response of Methanosarcina acetivorans to repression of the energy-conserving methanophenazine: CoM-CoB heterodisulfide reductase enzyme HdrED

ABSTRACT Methane-producing archaea are key organisms in the anaerobic carbon cycle. These organisms, also called methanogens, grow by converting substrate to methane gas in a process called methanogenesis. Previous research showed that the reduction of the terminal electron acceptor is the rate-limiting step in methanogenesis by Methanosarcina acetivorans . In order to gain insight into how the cells sense and respond to the availability of the terminal electron acceptor, we designed an experiment to deplete cells of the essential terminal oxidase enzyme, HdrED. We found that the depletion of HdrED in vivo results in a higher abundance of transcripts for methyltransferases ( mtaC2, mtaB3, mtaC3 ), coenzyme B biosynthesis, C1 metabolism, and pyrimidine compounds. In most cases, these changes were distinct from transcript abundance changes observed during the transition from exponential growth to stationary phase cultures. These data implicate the methylotrophic methanogenesis regulator MsrC (MA4383) in CoM-S-S-CoB heterodisulfide sensing and indicate cells have a specific mechanism to sense intracellular ratio of CoM-S-S-CoB, coenzyme M, and coenzyme B thiols and further suggest transcripts encoding translation and methanogenesis functions are controlled by feed-forward regulation depending on substrate availability. IMPORTANCE Methanosarcina is an emerging model archaeon and synthetic biology platform for the production of renewable energy and sustainable chemicals to reduce dependence on petroleum. Research into metabolic networks and gene regulation in this organism and other methanogens will inform genome-scale metabolic modeling and microbial function prediction in uncultured or non-model anaerobes and archaea. This study suggests methanogens use unknown mechanisms to efficiently couple methanogenesis to gene regulation via CoM-S-S-CoB and ATP availability.

Buan, Nicole R. (ORCID:000000027560973X)↗
DOE OSTI2026

Hydrogen production from full-strength corn stover fermentation effluent in single-chamber replaceable-cathode microbial electrolysis cells

Lignocellulosic residual biomass generated by the agricultural sector is an abundant feedstock for biohydrogen production via dark fermentation. However, this process is intrinsically inefficient, converting only ~30% of the reductant energy into H2 and leaving substantial amounts of reduced byproducts. These byproducts, mostly found in the fermentation effluents, can be further valorized in microbial electrolysis cells (MECs) to enhance the overall H2 recovery. However, current MEC configurations are typically dual- or single-chamber systems, yet both suffer from key inefficiencies. Dual-chamber systems rely on proton exchange membranes that are costly and prone to rapid biofouling, whereas single-chamber, membraneless systems are limited by reduced productivity due to H2 recycling and methanogenic consumption In this study, three single-chamber, 50-mL replaceable-cathode microbial electrolysis cells (RC-MECs) were 3D-printed and equipped with a physical separator to isolate anode and cathode compartments and limit H2 migration. Full-strength milled corn stover (MCS) fermentation effluent (COD of ~23.8 g-COD/L) was treated in fed-batch mode over two operational periods spanning 21 (Run 1) and 80 (Run 2) days. The RC-MECs exhibited comparable performance in both runs: after biofilm maturation, current densities exceeded 100 A/m²_cathode, COD removal reached up to 43%. Notably, extended RC-MECs operation led to a substantial methanogenic activity with the CH4 fraction in the cathode gas increasing to as high as 80% of the total biogas. Additions of a methanogenesis inhibitor 2-bromoethanesulfonate (2-BES) produced transient increases in hydrogen yields (11.51 and 5.12 L-H2/L_reactor/day in Runs 1 and 2, respectively); however, sustained 2-BES addition in subsequent cycles reduced total biogas production, decreased COD removal, and led to volatile fatty acid accumulation. Overall, single-chamber MECs can treat high-strength dark fermentation effluents while improving H2 recovery, but methanogenesis remains a key bottleneck, and complete long-term inhibition may be operationally unsustainable.

Hydrogen Production↗
DOE OSTI2025

Northern peatland microbial communities exhibit resistance to warming and acquire electron acceptors from soil organic matter

The response of microbial communities that regulate belowground carbon turnover to climate change drivers in peatlands is poorly understood. Here, we leverage a whole ecosystem warming experiment to elucidate the key processes of terminal carbon decomposition and community responses to temperature rise. Our dataset of 697 metagenome-assembled genomes (MAGs) represents the microbial community from the surface (10 cm) to 2 m deep into the peat column, with only 3.7% of genomes overlapping with other well-studied peatlands. Community composition has yet to show a significant response to warming after 3 years, suggesting that metabolically diverse soil microbial communities are resistant to climate change. Surprisingly, abundant and active methanogens in the genus Candidatus Methanoflorens, Methanobacterium , and Methanoregula show the potential for both acetoclastic and hydrogenotrophic methanogenesis. Nonetheless, the predominant pathways for anaerobic carbon decomposition include sulfate/sulfite reduction, denitrification, and acetogenesis, rather than methanogenesis based on gene abundances. Multi-omics data suggest that organic matter cleavage provides terminal electron acceptors, which together with methanogen metabolic flexibility, may explain peat microbiome composition resistance to warming.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Metabolic Redox Coupling Controls Methane Production in Permafrost‐Affected Peatlands Through Organic Matter Quality‐Dependent Energy Allocation

ABSTRACT Permafrost thaw represents one of Earth's largest climate feedback risks, potentially releasing vast carbon (C) stores as greenhouse gases (GHG). However, our ability to predict emissions remains limited by poor understanding of how changing organic matter (OM) composition affects microbial carbon processing. We test a metabolism‐centered redox framework, which views microbial processes as coupled oxidative‐reductive reactions, to mechanistically explain how organic matter metabolite quality controls greenhouse gas production in permafrost‐affected peatland ecosystems. Rather than relying solely on geochemical redox measurements, our approach examines how microbes balance electron flow through metabolic pathways. Using active layer peat (9–19 cm) from contrasting environments (bog and fen), we employed multi‐omics approaches, including metabolomics, metagenomics, and metatranscriptomics, to link OM chemistry to microbial function. Our results reveal distinct dissolved organic matter metabolite composition, with fen systems enriched in compounds with higher substrate quality (low molecular weight (MW) sugars with high H:C ratios and low aromaticity) and bog systems dominated by compounds with lower substrate quality (high MW phenols with lower H:C ratios and higher aromaticity). In fen samples, these sugar‐like compounds correlated with higher oxidative metabolism and methanogenesis, supported by increased glycolysis gene expression. Initially, electrons from increased oxidative metabolism were balanced through nitrate and sulfate reduction, but as these electron acceptors were depleted, methanogenesis increased to maintain redox balance. Fen samples showed rapid degradation of both high‐ and low‐substrate‐quality compounds, suggesting sufficient energy for efficient C cycling. Conversely, bog samples exhibited more polyphenolic compounds, lower glycolysis activity, and higher stress‐related gene expression, suggesting energy was diverted towards cell maintenance under acidic conditions rather than C processing. This approach suggests that predicting greenhouse gas emissions requires an understanding of how organic matter quality shapes microbial energy allocation strategies, providing a mechanistic framework for improving emission predictions from permafrost‐affected peatlands and similar ecosystems.

Biodiversity & Conservation↗
DOE OSTI2025

Cultivation of Methanonezhaarchaeia, the third class of methanogens within the phylum Thermoproteota

Methane is a potent greenhouse gas, largely produced by methanogenic archaea, contributing to Earth’s dynamic climate and biogeochemical cycles. In the past decade, metagenomics revealed that lineages outside of the Euryarchaeota superphylum encode genes for methanogenesis. This was recently confirmed through the cultivation of two classes of methanogenic Thermoproteota. Thus far, all methanogens within the Thermoproteota are predicted or were shown to be methylotrophic. The only exception to this are the Nezhaarchaea, for which metagenomic predictions suggest they are CO 2 -reducing methanogens. Here, we demonstrate methanogenic activity in a third class of Thermoproteota, the Methanonezhaarchaeia. Contrary to genomic predictions for this class, we cultivated a methylotrophic species, Candidatus Methanonezhaarchaeum fastidiosum YNP3N, highlighting the importance of testing metagenomic hypotheses through experimentation. We investigate the metabolic diversity of Methanonezhaarchaeia, including metabolic modifications accompanying frequent loss of methanogenesis in this class. This highlights gaps in our understanding of the biochemistry, diversity, and evolution of thermoproteotal methanogens and their contributions to carbon cycling.

Kohtz, Anthony J. [Montana State Univ., Bozeman, M↗
DOE OSTI2025

From the top: surface-derived carbon fuels greenhouse gas production at depth in a peatland

Tropical peatlands play an important role in global carbon (C) cycling, but little is known about factors driving carbon dioxide (CO 2 ) and methane (CH 4 ) emissions from these ecosystems, especially production in deeper soils. This study aimed to identify source material and processes regulating C emissions originating deep in three sites in a peatland on the Caribbean coast of Panama. We hypothesized that (1) surface-derived organic matter transported down the soil profile is the primary C source for respiration products at depth and that (2) high lignin content results in hydrogenotrophic methanogenesis as the dominant CH 4 production pathway throughout the profile. We used radiocarbon isotopic values to determine whether CO 2 and CH 4 at depth are produced from modern substrates or ancient deep peat, and we used stable C isotopes to identify the dominant CH 4 production pathway. Peat organic chemistry was characterized using 13 C solid-state nuclear magnetic resonance spectroscopy ( 13 C-NMR). We found that deep peat respiration products had radiocarbon signatures that were more similar to surface dissolved organic C (DOC) than deep solid peat. These results indicate that surface-derived organic matter was the dominant source for gas production at depth in this peatland, likely because of vertical transport of DOC from the surface to depth. Lignin, which was the most abundant compound (55 %–70 % of C), increased with depth across these sites, whereas other C compounds like carbohydrates did not vary with depth. These results suggest that there is no preferential decomposition of carbohydrates but instead preferential retention of lignin. Stable isotope signatures of respiration products indicated that hydrogenotrophic rather than acetoclastic methanogenesis was the dominant production pathway of CH 4 throughout the peat profile. These results show that deep C in tropical peatlands does not contribute greatly to surface fluxes of carbon dioxide, with compounds like lignin preferentially retained. This protection of deep C helps explain how peatland C is retained over thousands of years and points to the vulnerability of this C should anaerobic conditions in these wet ecosystems change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

A minor respiratory process with major global implications: is atmospheric methane oxidation in tree stems driven by stem respiration rather than microbial methanotrophy?

Tree stem surfaces are widely recognized as sites of carbon dioxide (CO₂) efflux and oxygen (O₂) influx, reflecting the dynamics of aerobic respiration of photosynthate substrates, such as sugars, delivered via the phloem. Stems are also largely considered passive conduits for methane (CH₄) produced in anoxic soils via microbial methanogenesis, where CH₄ is thought to be transported upward through the transpiration stream and/or diffusion and emitted through stem surfaces and the canopy. However, recent observations from dynamic stem chambers suggest that stems may also act as active sinks for atmospheric CH₄. Despite these findings, the extent and drivers of stem CH₄ consumption remain poorly characterized across biomes, species, and environmental gradients, and its quantitative relationship to stem respiration has not been established. Moreover, previous studies captured only snapshot fluxes, leaving diurnal patterns of CH₄ exchange uncharacterized. Here, we address these limitations by combining real-time measurements of stem CH₄ and O₂ uptake under ambient conditions in a California cherry tree, using a dynamic stem gas exchange system with three chambers receiving a continuous flow of ambient air and automated chamber and reference air sampling every 10 min. Our results confirm that stems of upland trees can actively consume both atmospheric CH₄ and O₂, but with decreasing temperature sensitivity as daily temperatures increase. Early mornings were marked by rapid influxes of both gases, followed by declining uptake as temperatures rose further. Methane uptake was tightly coupled with O₂ influx and represented a minor (0.012% ± 0.002%) fraction of stem respiratory activity, as determined by concurrent O₂ uptake. These findings suggest that while atmospheric CH₄ oxidation is a minor respiratory process in stems, it is strongly linked with stem physiological activity. This challenges the current assumption that terrestrial CH₄ uptake is driven solely by microbial methanotrophy and raises the possibility that living stem tissues may contribute to CH₄ oxidation through an as-yet-unidentified plant-based mechanism.

Atmospheric greenhouse gases↗
DOE OSTI2026

Data driven investigation to understand the influence of total solids on biological biogas upgrading

In situ biogas upgrading achieves CO 2 conversion to CH 4 via hydrogenotrophic methanogenesis; however, gas-liquid mass transfer constraints limit the upgrading performance. Recognizing that optimization studies often underrepresent the effects of total solids (TS) and organic loading rate (OLR), this study undertook a holistic, statistics driven assessment of operating conditions for in situ H 2 assisted biogas upgrading, centering the analysis on TS and OLR. A dataset of 31 studies was compiled and comprised 99 observations. A rigorous analytical framework was employed, combining data standardization, fixed- and random-effects (REML) weighted regressions with cluster-robust errors, stratified analyses, and machine learning. Mixed-effects meta regression indicated that TS was the main factor explaining differences of methane fraction (CH 4 %) when considering the between studies heterogeneity. Focusing on a near-stoichiometric subset (H 2 /CO 2 ≈ 4:1), TS remained significant. Stratified results showed a stronger negative relationship between TS and CH 4 % in UASB reactors than in CSTRs, with a negative effect under mesophilic conditions and no significant effect under thermophilic conditions. A Random Forest model corroborated the statistical findings, consistently ranking H 2 /CO 2 ratio, OLR, TS, and hydrogen injection rate (HIR) as the most influential predictors. These findings delineate trends across increasing TS levels, particularly between 1% and 10%, and provide preliminary insights for TS above 15% in in situ biogas upgrading. They further provide insights for the influence of TS by reactor type and temperature, thereby advancing the evidence base for implementing biological CO 2 conversion to CH 4 in practice.

In situ biogas upgrading↗
DOE OSTI2025

Ecology of methyl-coenzyme M reductase encoding Thermoproteota

The recent demonstration that members of at least three classes of archaea affiliated with the Thermoproteota superphylum are involved in the production of the climate-active gas methane has sparked discussions about how well we understand the diversity of methanogens. Here, in this work, we show that members of all three of these lineages, as well as several other, yet uncultured and physiologically uncharacterized groups within the Thermoproteota that encode the key enzyme of anaerobic methane cycling, methyl-coenzyme M reductase (MCR), are widely distributed in anoxic ecosystems. We postulate that the taxonomic, metabolic, and ecological diversity of methanogenic and MCR-encoding Thermoproteota are poorly understood, and that the contribution of methylotrophic and thermoproteotal methanogenesis to methane production is largely unknown. We hypothesize that thermoproteotal methanogens could contribute, potentially substantially, to methane emissions in many anoxic environments that harbor methylated precursors, including wetlands, sediments, peat, rice paddies, wastewater sludge, and geothermal systems. We highlight the necessity to experimentally test the (eco)physiology of these widely distributed archaea using both culture-dependent ( in vitro ) and culture-independent ( in situ ) approaches to assess their potential contribution to methane emissions. Last, we stress the importance of remaining agnostic about the physiology of MCR-encoding Thermoproteota in the absence of experimental data because most of these archaea also carry the genetic potential to grow non-methanogenically.

MCR↗
DOE OSTI2025

Structural and Mechanistic Advances in the Chemistry of Methyl-Coenzyme M Reductase (MCR)

Methane represents 34% of U.S. energy consumption and is a major greenhouse gas related to the global carbon cycle and energy production. However, current industrial practices significantly increase atmospheric methane levels, necessitating a deeper understanding of its biosynthesis and oxidation. Methyl-coenzyme M reductase (MCR) is central to biological methane metabolism, catalyzing the final step of methanogenesis and the first step in anaerobic methane oxidation. It is also a key target for strategies to capture and transform methane into value-added chemicals.The active site of MCR is a buried Ni-based cofactor only accessible by the substrates via a 50 Å long tunnel. Although the Ni(I) state is required to initiate catalysis, capturing this state remains a challenge for the current structural techniques. Recent advances in structural biology using X-ray Free-Electron Laser serial crystallography have provided insights into MCR's inactive Ni(II) state at room temperature and show promise for capturing its active Ni(I) form.Our team has established several critical aspects of the MCR mechanism using a combination of experimental and computational studies. MCR uses CH 3 -SCoM and CoBSH as substrates, producing methane and a disulfide product CoMSSCoB. Kinetic analysis showed that productive substrate binding requires CH 3 -SCoM to bind first, inducing conformational changes that optimize the active site for subsequent CoBSH binding. Following substrate binding, four proposed methane production/oxidation mechanisms were examined, establishing whether the reaction proceeds through an organometallic methyl-nickel(III), methyl anion ion, or methyl radical intermediate. Experimental measurements using CoBSH analogs successfully slowed the reaction, allowing for mechanistic insight that demonstrated the methyl radical pathway, where the initial interactions involve homolytic cleavage of the methyl-sulfur bond, generating a methyl radical that quickly abstracts the thiol hydrogen atom of CoBSH to form methane. Computational studies further confirmed that, compared to other mechanisms, the methyl radical mechanism is thermodynamically more favorable and accessible under physiological conditions.Spectroscopic and computational studies challenged the conventional understanding of substrate binding in MCR by proposing an alternative positioning of CH 3 -SCoM and CoMSSCoB in the active site pocket. The research suggested that CH 3 -SCoM (substrate) and CoMSSCoB (product) bind via their sulfonate groups to the Ni(I) center of cofactor F 430 . This binding allows for the reaction without substrate reorganization in the pocket but would require a long-range electron transfer. Altogether, the work summarized in this review reflects our current understanding of the enzyme's catalytic mechanism and structural dynamics. This is essential for developing efficient methane conversion technologies that could mitigate its environmental impact while harnessing energy-storage properties.

03 NATURAL GAS↗
DOE OSTI2026

Technoeconomic and Life Cycle Analysis of an Integrated Fermentation and Microbial Electrochemical Process for Volatile Fatty Acid Production from Food Waste

Techno-economic analysis (TEA) and life cycle assessment (LCA) were conducted for an integrated system designed for the production of volatile fatty acid (VFA) from food waste. The TEA estimated a production cost of $\$$3.12/kg VFA, and the LCA predicted negative greenhouse gas (GHG) emissions of -0.4 kg CO 2 e/kg VFA, driven primarily by diverting organic waste from landfills and avoiding methane emissions while producing valuable chemical products. Hotspot analysis showed arrested methanogenesis (AM) fermentation as the largest contributor to costs (37%) and environmental burden (47%), driven by high sodium hydroxide (NaOH) consumption. Distillation and microbial electrosynthesis (MES) units were the next-largest environmental contributors (28% and 18%). Major cost drivers also included residuals management (biosolids and wastewater) and the equipment and operating costs for AM, MES, and sonication pretreatment units. Although the new integrated system is environmentally benign, its costs and environmental impacts can be further reduced by integrating alternative energy sources, minimizing chemical and energy inputs through process optimization, and improving efficiency. In conclusion, this work highlighted the viability of waste-derived VFA production and provided a clear, data-driven strategy to accelerate the commercialization of waste valorization technology.

Carboxylic Acid Production↗