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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A minimal SufB 2 C 2 complex functions as a [4Fe-4S] cluster scaffold in methanogenic archaea

Iron-sulfur clusters are essential cofactors in all domains of life, yet their biogenesis in obligately anaerobic archaea remains poorly understood. Here, we characterized the minimal two-protein SUF system in methanogenic archaea, composed solely of SufB and SufC. Using Methanococcus maripaludis as a model, we demonstrate that the SUF proteins from its native host form a stable SufB 2 C 2 heterotetramer that binds a [4Fe-4S] cluster via three conserved cysteines in SufC. Mutations of conserved cysteine and histidine residues of SufB do not impair cluster binding. The complex interacts with the SAM-containing methanogenesis marker protein 10 (MmpX), suggesting direct Fe-S cluster transfer from SufB 2 C 2 to target proteins. Mutational analysis of Methanothermococcus thermolithotrophicus proteins confirmed that SufC is the primary cluster-binding component, while SufB enhances ATPase and cluster transfer activities. Evolutionary comparisons suggest that this two-protein SUF system represents an ancestral form of Fe-S cluster biogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identification of key steps in the evolution of anaerobic methanotrophy in Candidatus Methanovorans (ANME-3) archaea

Despite their large environmental impact and multiple independent emergences, the processes leading to the evolution of anaerobic methanotrophic archaea (ANME) remain unclear. This work uses comparative metagenomics of a recently evolved but understudied ANME group, “Candidatus Methanovorans” (ANME-3), to identify evolutionary processes and innovations at work in ANME, which may be obscured in earlier evolved lineages. We identified horizontal transfer of hdrA homologs and convergent evolution in carbon and energy metabolic genes as potential early steps in Methanovorans evolution. We also identified the erosion of genes required for methylotrophic methanogenesis along with horizontal acquisition of multiheme cytochromes and other loci uniquely associated with ANME. The assembly and comparative analysis of multiple Methanovorans genomes offers important functional context for understanding the niche-defining metabolic differences between methane-oxidizing ANME and their methanogen relatives. Furthermore, this work illustrates the multiple evolutionary modes at play in the transition to a globally important metabolic niche.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

CABO-16S—a Combined Archaea, Bacteria, Organelle 16S rRNA database framework for amplicon analysis of prokaryotes and eukaryotes in environmental samples

Abstract Identification of both prokaryotic and eukaryotic microorganisms in environmental samples is currently challenged by the need for additional sequencing to obtain separate 16S and 18S ribosomal RNA (rRNA) amplicons or the constraints imposed by “universal” primers. Organellar 16S rRNA sequences are amplified and sequenced along with prokaryote 16S rRNA and provide an alternative method to identify eukaryotic microorganisms. CABO-16S combines bacterial and archaeal sequences from the SILVA database with 16S rRNA sequences of plastids and other organelles from the PR2 database to enable identification of all 16S rRNA sequences. Comparison of CABO-16S with SILVA 138.2 results in equivalent taxonomic classification of mock communities and increased classification of diverse environmental samples. In particular, identification of phototrophic eukaryotes in shallow seagrass environments, marine waters, and lake waters was increased. The CABO-16S framework allows users to add custom sequences for further classification of underrepresented clades and can be easily updated with future releases of reference databases. Addition of sequences obtained from Sanger sequencing of methane seep sediments and curated sequences of the polyphyletic SEEP-SRB1 clade resulted in differentiation of syntrophic and non-syntrophic SEEP-SRB1 in hydrothermal vent sediments. CABO-16S highlights the benefit of combining and amending existing training sets when studying microorganisms in diverse environments.

Eitel, Eryn M. (ORCID:0009000723919297)↗
DOE OSTI2025

Global Archaeal Diversity Revealed Through Massive Data Integration: Uncovering Just Tip of Iceberg

The domain of Archaea has gathered significant interest for its ecological and biotechnological potential and its role in helping us to understand the evolutionary history of Eukaryotes. In comparison to the bacterial domain, the number of adequately described members in Archaea is relatively low, with less than 1000 species described. It is not clear whether this is solely due to the cultivation difficulty of its members or, indeed, the domain is characterized by evolutionary constraints that keep the number of species relatively low. Based on molecular evidence that bypasses the difficulties of formal cultivation and characterization, several novel clades have been proposed, enabling insights into their metabolism and physiology. Given the extent of global sampling and sequencing efforts, it is now possible and meaningful to question the magnitude of global archaeal diversity based on molecular evidence. To do so, we extracted all sequences classified as Archaea from 500 thousand amplicon samples available in public repositories. After processing through our highly conservative pipeline, we named this comprehensive resource the ‘Global Archaea Diversity’ (GAD), which encompassed nearly 3 million molecular species clusters at 97% similarity, and organized it into over 500 thousand genera and nearly 100 thousand families. Saline environments have contributed the most to the novel taxa of this previously unseen diversity. The majority of those 16S rRNA gene sequence fragments were verified by matches in metagenomic datasets from IMG/M. These findings reveal a vast and previously overlooked diversity within the Archaea, offering insights into their ecological roles and evolutionary importance while establishing a foundation for the future study and characterization of this intriguing domain of life.

59 BASIC BIOLOGICAL SCIENCES↗
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 OSTI2026

Circularization of 23S rRNA but not 16S rRNA within archaeal ribosomes

Background Processing of archaeal 16S and 23S rRNAs is believed to involve excision of individual rRNAs from polycistronic precursors, circularization of excised rRNAs, and re-linearization before the incorporation into ribosomes. However, all the knowledge is derived from several isolated species, leaving open the possibility that different processes may occur in other archaeal groups. Results Here, we investigate rRNAs from diverse and mostly uncultivated archaea. Sequencing of total cellular RNA from eight phylum-level lineages indicates that archaeal circular 23S rRNA transcript abundances vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggest that ribosomes contain circular 23S rRNAs. Thus, we directly sequence RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans, and confirm that the 23S rRNAs in the ribosomes are circular. Structural modeling places the 5′ and 3′ ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their existence as circular molecules. We also confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups, suggesting that certain archaea do not circularize 16S rRNA during processing. Conclusions Our findings uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.

Archaea↗
DOE OSTI2025

Cyclization of archaeal membrane lipids impacts membrane protein activity and archaellum formation

Enhancement of the cyclization of membrane lipids GDGTs (glycerol dialkyl glycerol tetraethers) is a critical strategy for archaea to adapt to various environmental stresses. However, the physiological function of membrane lipid cyclization remains unclear. Here, we reported that the GDGT ring synthases mutant, deficient in GDGT cyclization, inhibited archaellum formation and reduced cell motility in thermoacidophilic crenarchaeon Sulfolobus acidocaldarius . This inhibition was caused by decreased transcription of the archaellum operon, likely due to cleavage of the C-terminal domains in transmembrane proteins ArnRs, the transcription factors that regulate archaellum operon expression. The transcriptomic and proteomic analysis showed deficiency of GDGT cyclization broadly impacted the expression of membrane associate proteins, including respiratory chain proteins, and decreased cellular ATP concentration. Moreover, phylogenetic analysis demonstrated that the correlation between GDGT cyclization and archaellum formation is widespread among (hyper)thermophilic archaea, and this was further verified in the euryarchaeon Thermococcus kodakarensis. Our findings suggested that archaea modify their membrane lipids to profoundly alter cellular appendages and cell physiology to adapt to environmental fluctuations.

Yang, Wei (ORCID:0000000262755981)↗
DOE OSTI2025

An archaeal genetic code with all TAG codons as pyrrolysine

Multiple genetic codes developed during the evolution of eukaryotes and bacteria, yet no alternative genetic code is known for archaea. We used proteomics to confirm our prediction that certain archaea consistently incorporate pyrrolysine (Pyl) at TAG codons, supporting an alternative archaeal genetic code that we designate the Pyl code. This genetic code has 62 sense codons encoding 21 amino acids. In contrast to monophyletic genetic code distributions in bacteria, the archaeal Pyl code occurs sporadically, indicating that it arose independently in multiple lineages. We discovered that more than 1800 archaeal proteins contain Pyl, increasing the number of such proteins by two orders of magnitude. Additionally, five Pyl transfer RNA (tRNA) pyrrolysyl–tRNA synthetase pairs from Pyl-code archaea were used to introduce Pyl analogs into proteins in Escherichia coli.

Kivenson, Veronika [University of California, Berk↗
DOE OSTI2026

Rerouting reductant flux via protein tethering enhances biohydrogen production in Thermococcus kodakarensis

Microbes that generate copious amounts of hydrogen (H 2 ) via dark fermentation are a promising means to evolve and improve renewable biofuels. Many anaerobic hyperthermophilic archaea, such as the fast-growing, genetically tractable, heterotroph Thermococcus kodakarensis, produce generous quantities of H2 and provide an idealized platform to further optimize naturally high levels of biohydrogen reduction. Precise genetic manipulations and modifications to growth conditions have already resulted in substantial increases to H2 output but additional improvements are desired. An unexamined and potentially valuable route towards increased H 2 production is to tether select electron donor and acceptor proteins together to reroute and maximize the flow of electrons towards H 2 production. Such strategies have shown promise in Bacteria and Eukarya but have not yet been investigated in thermophilic Archaea. Here, we generate and evaluate twelve novel T. kodakarensis strains wherein a proteinaceous electron carrier (a ferredoxin, Fd) is physically tethered to the membrane-bound-hydrogenase (MBH), the sole H 2 producing enzyme, to direct electron flux towards biohydrogen generation. Growth assessments and H 2 output measurements demonstrate that strains encoding protein-fusions evolve up to ~ 40% more H2 per cell than the host strain. Eliminating H 2 consumption and alternative routes of electron sinks in concert with protein tethering further increased H2 output per cell for a maximum increase of ~ 66% over the host strain. Our results demonstrate that rerouting electron flux via protein tethering coupled with the elimination of reductant sinks is a promising means towards improved biohydrogen production in T. kodakarensis. KEY POINTS: Protein tethering between redox proteins can reroute electron flux in vivo. Enforced protein proximity results in ~ 40% increases in H2 production per cell. Protein-tethering provides a generalizable framework to redirect redox metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Archaeal protein containing domain of unknown function 2193 undergoes oligomeric reconfiguration upon iron–sulfur cluster binding

Methanogenic archaea are particularly rich in iron–sulfur proteins, yet their roles remain largely enigmatic. Here, we characterized aMethanococcus voltae(Mvo) protein from the domain of unknown function (DUF) 2193 family, a group of proteins present primarily in archaea and characterized by a conserved cysteine‐rich C‐terminal motif.MvoDUF2193 was heterologously expressed and characterized by a range of spectroscopic and analytical methods. The results demonstrate thatMvoDUF2193 binds a single [4Fe–4S] cluster per subunit and that cluster occupancy regulates the transition from an apo tetramer to a [4Fe–4S] monomeric form. We hypothesize thatMvoDUF2193 serves a regulatory role in the cell, mediated by [Fe–S] cluster binding and changes in oligomeric state.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Photosynthetic Biohybrid System for Enhanced Abiotic N 2 -to-NH 3 Conversion under Ambient Conditions

Photosynthetic biohybrid systems (PBSs) offer an eco-friendly approach to transforming solar energy into value-added products by integrating biological entities with inorganic semiconductors. However, the chemical conversion capacity of most PBSs has inherent limitations, as whole-cell bacteria and isolated enzymes require fine-tuning of environmental conditions. Here, in this study, we report a new PBS developed by introducing free-standing ceria nanoparticles into the purple membrane (PM) of Halobacterium salinarum archaea, which can unidirectionally transfer charge carriers in response to incident photons, even after separation from living archaea at various conditions. Our microscopy, spectroscopy, and synchrotron X-ray scattering analyses confirm that the electrostatic assembly between ceria and PM creates seamless interfacial contact, thereby enhancing the photocatalytic capacity of ceria. Although the conversion of dinitrogen (N 2 ) to ammonia (NH 3 ) is thermodynamically challenging due to the triple bond in N 2 and a series of charge-transfer reactions, our PM–ceria (PMC) hybrid nanoparticle efficiently produces NH 3 by reducing N 2 using solar energy even under atmospheric pressure and room temperature while simultaneously converting glycerol into value-added derivatives. Additionally, our PMC nanoparticle involves neither toxic/precious metals nor bioengineering processes to achieve enhanced photocatalytic N 2 -to-NH 3 conversion. This study sheds light on the new aspect of PBSs by employing PM to potentially resolve the global energy and environmental challenges posed by the conventional Haber–Bosch process.

Jang, Jinhyeong [Argonne National Laboratory (ANL)↗
DOE OSTI2026

Carbon monoxide oxidation expands the known metabolic capacity in anaerobic methanotrophic consortia

Consortia of anaerobic methane-oxidizing archaea (ANME-2) and sulphate-reducing bacteria (SRB) represent globally relevant syntrophic associations capable of growing with minimal amounts of free energy and can persist when methane becomes limiting. Carbon monoxide (CO) has been reported in seep environments and represents a thermodynamically favourable alternative electron donor due to its low reduction potential. Here, we show that environmental ANME-SRB consortia can oxidize CO in the absence of methane, in anoxic microcosm experiments using a combination of stable isotope geochemical tracers, metatranscriptomics, and single cell activity measurements (FISH–nanoSIMS). The oxidation of CO was coupled with sulphate-reduction by syntrophic consortia, and, in the absence of sulphate, through CO 2 reduction to methane by ANME-2. Under these conditions, the production of methane was one ninth the rate of methanotrophy coupled to sulphate-reduction. Paired single cell FISH-nanoSIMS analysis of anabolic activity indicates that CO respiration appears to support cell maintenance rather than active growth, consistent with the observed down-regulation of energy generating complexes in ANME (e.g., mtr, rnf, etc.). The versatile capability of CO oxidation by anaerobic methanotrophic consortia broadens our understanding of carbon cycling in methane seeps and highlights potential mechanisms of resilience by methanotrophic archaea under changing geochemical regimes.

03 NATURAL GAS↗
DOE OSTI2025

A dynamic protein interactome drives energy conservation and electron flux in Thermococcus kodakarensis

ABSTRACT Life is supported by energy gains fueled by catabolism of a wide range of substrates, each reliant on the selective partitioning of electrons through redox ( red uction and ox idation) reactions. Electron flux through tunable and regulated protein interactions provides dynamic routes for energy conservation, but how electron flux is regulated in vivo , particularly for archaeal metabolisms that support rapid growth at the thermodynamic limits of life, is poorly understood. Identification of bona fide in vivo protein assemblies and how such assemblies dictate the totality of electron flux is critical to our understanding of the regulation imposed on metabolism, energy production, and energy conservation. Here, 25 key proteins in central metabolic redox pathways in the model, genetically accessible, hyperthermophilic archaeon Thermococcus kodakarensis , were purified to reveal an extensive, dynamic, and tightly interconnected network of protein interactions that responds to environmental cues (such as the availability of various reductive sinks) to direct electron flux to maximize energetic gains. Interactions connecting disparate functions suggest many catabolic and anabolic activities occur in spatial proximity in vivo , and while protein complexes have been historically defined under optimal conditions, many of these complexes appear to maintain alternative partnerships in changing conditions. The totality of the results obtained redefines our understanding of in vivo assemblies driving ancient metabolic strategies supporting the growth of modern Archaea. IMPORTANCE Given the potential for rational genetic manipulations of biofuel- and biotech-promising archaea to yield transformative results for major markets, it is a priority to define how the metabolisms of such species are controlled, at least in part, by in vivo protein assemblies, and from such, define routes of energy flux that can be most efficiently altered toward biofuel or biotechnological gains. Proteinaceous electron carriers (PECs, such as ferredoxins) offer the potential for specific protein–protein interactions to coordinate selective reductive flow. Employing the model, genetically accessible, hyperthermophilic archaeon, Thermococcus kodakarensis , we establish the metabolic protein interactome of 25 key redox proteins, revealing that each redox active protein has a dynamic partnership profile, suggesting catabolic and anabolic activities may occur in concert and in temporal and spatial proximity in vivo . These results reveal critical importance in evaluating the newly identified partnerships and their role and utility in providing regulated redox flux in T. kodakarensis .

Williams, Sere A. (ORCID:0000000235509590)↗
DOE OSTI2025

Microbial ecology of acidic, biogenic gypsum: community structure and distribution of extremophiles on freshly formed and relict sulfate deposits in a hydrogen sulfide-rich cave

Sulfate minerals are abundant on the Martian surface, and many of these evaporite deposits are thought to have precipitated from acidic fluids. On Earth, gypsum (CaSO 4 •2H 2 O) and other sulfates sometimes form under acidic conditions, so exploring the extremophilic life that occurs in these mineral environments can help evaluate the astrobiological potential of acid sulfate depositional settings. Here, we characterized the microbial communities associated with acidic gypsum deposits in a sulfuric acid cave, where sulfate precipitation is driven by sulfide-oxidizing bacteria and archaea. We used 16S rRNA gene sequencing and cell counts to characterize gypsum-associated microorganisms in freshly formed and relict deposits throughout the cave, to test how microbial community composition and abundance would vary with distance from the sulfidic water table and with the concentration of H 2 S(g) and other gases in the cave atmosphere. We found that actively forming gypsum in the lower cave levels was colonized by low-diversity communities that have few cells compared to other environments in the cave. The most abundant taxa were Acidithiobacillus, Metallibacterium, Mycobacteria, and three different Thermoplasmatales-group archaea, which occupied distinct niches based on proximity to sulfidic streams and the concentration of gases in the cave air. By contrast, deposits in older cave levels had more diverse communities that were distinct from those associated with freshly formed gypsum and likely represent a community reliant on different energy resources. These findings show that acidic sulfate deposits serve as habitats for extremophilic microorganisms and broaden our knowledge of the life associated with terrestrial sulfates.

58 GEOSCIENCES↗
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

Plant-Nitrifier Interactions in Topsoil and Subsoil

Plants can influence soil microbes through resource acquisition and interference competition, with consequences for ecosystem function such as nitrification. However, how plants alter soil conditions to influence nitrifiers and nitrification rates remains poorly understood, especially in the subsoil. Here, coupling the 15N isotopic pool dilution technique, high throughput sequencing and in situ soil O2 monitoring, we investigated how a deep-rooted perennial grass, miscanthus, versus an adjacent shallow-rooted turfgrass reference shapes nitrifier assembly and function along 1 m soil profiles. In topsoil, the suppression of ammonia (NH3) oxidizing archaea (AOA) and gross nitrification rates in miscanthus relative to the reference likely resulted from nitrifiers being outcompeted by plant roots and heterotrophic bacteria for ammonium (NH4+). The stronger tripartite competition under miscanthus may have been caused in part by the lower soil organic matter (SOM) content, which supported lower gross nitrogen (N) mineralization, the major soil process that produces NH4+. In contrast, below 10 cm soil depth, significantly greater gross nitrification rates were observed in miscanthus compared to the reference. This was likely driven by the significantly lower oxygen (O2) in miscanthus than reference subsoil, which selected against aerobic heterotrophic bacteria but in favor of AOA. Overall, we found that plants can regulate AOA community structure and function through different mechanisms in topsoil and subsoil, with suppression of nitrification in topsoil and enhancement of nitrification in subsoil.

Field Data↗
DOE OSTI2025

Whole metagenome sequencing and 16S rRNA gene amplicon analyses reveal the complex microbiome responsible for the success of enhanced in-situ reductive dechlorination (ERD) of a tetrachloroethene-contaminated Superfund site

The North Railroad Avenue Plume (NRAP) Superfund site in New Mexico, USA exemplifies successful chlorinated solvent bioremediation. NRAP was the result of leakage from a dry-cleaning that operated for 37 years. The presence of tetrachloroethene biodegradation byproducts, organohalide respiring genera (OHRG), and reductive dehalogenase (rdh) genes detected in groundwater samples indicated that enhanced reductive dechlorination (ERD) was the remedy of choice. This was achieved through biostimulation by mixing emulsified vegetable oil into the contaminated aquifer. This report combines metagenomic techniques with site monitoring metadata to reveal new details of ERD. DNA extracts from groundwater samples collected prior to and at four, 23 and 39 months after remedy implementation were subjected to whole metagenome sequencing (WMS) and 16S rRNA gene amplicon (16S) analyses. The response of the indigenous NRAP microbiome to ERD protocols is consistent with results obtained from microcosms, dechlorinating consortia, and observations at other contaminated sites. WMS detects three times as many phyla and six times as many genera as 16S. Both techniques reveal abundance changes in Dehalococcoides and Dehalobacter that reflect organohalide form and availability. Methane was not detected before biostimulation but appeared afterwards, corresponding to an increase in methanogenic Archaea. Assembly of WMS reads produced scaffolds containing rdh genes from Dehalococcoides, Dehalobacter, Dehalogenimonas, Desulfocarbo, and Desulfobacula. Anaerobic and aerobic cometabolic organohalide degrading microbes that increase in abundance include methanogenic Archaea, methanotrophs, Dechloromonas, and Xanthobacter, some of which contain hydrolytic dehalogenase genes. Aerobic cometabolism may be supported by oxygen gradients existing in aquifer microenvironments or by microbes that produce O 2 via microbial dismutation. The NRAP model for successful ERD is consistent with the established pathway and identifies new taxa and processes that support this syntrophic process. This project explores the potential of metagenomic tools (MGT) as the next advancement in bioremediation.

59 BASIC BIOLOGICAL SCIENCES↗
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↗