Diversity and function of methyl-coenzyme M reductase-encoding archaea in Yellowstone hot springs revealed by metagenomics and mesocosm experiments

Survey of mcrA genes across physicochemically contrasting geothermal features

The presence and diversity of Mcr-encoding populations was assessed in 100 geothermal features of YNP via mcrA gene amplicon sequencing. Gene amplicons were recovered from 66 sediment and/or microbial mat samples spanning 39 geothermal features located in the Lower Culex Basin (LCB; 61 samples, 35 features), the Mud Volcano Region (MVR; 4 samples, 3 features), and the White Creek Area (WCA; 1 sample, 1 feature). These features were characterized by a wide range of temperature (22–86.3 °C), pH (2.40–9.77), and dissolved methane (40–1784 nM), oxygen (<13–771 µM), and sulfide (<2–27 µM; Figs. 1A, 2, SI Data 1).

Fig. 2: Diversity of mcrA genes detected in 66 samples from 39 geothermal features of YNP.
figure 2

Relative sequence abundance of mcrA gene amplicons affiliated with abundant lineages (relative sequence abundance >1% in at least one sample). Samples selected for metagenomics are underlined in bold. Samples were collected from geothermal features (identified by numbers) in the Lower Culex Basin (LCB, circle), Mud Volcano Region (MVR, diamond), and White Creek Area (WCA, triangle) and consisted of either sediment (black), microbial mat (white), or a mixture of sediment and mat material (grey). Physicochemical parameters of the geothermal water were recorded at the time of sample collection. X: no data available. Clustering based on Bray-Curtis dissimilarity using relative sequence abundance data of the presented lineages. No correlative trends between taxonomic affiliation of mcrA genes and physicochemistry were observed (SI Fig. 2). See SI Data 1 for details.

Generally, the mcrA-containing microbial community in each geothermal feature was composed of a small number [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21] of mcrA ASVs with >1% relative sequence abundance. The alpha diversity of mcrA tended to decrease with increasing temperature (SI Fig. 1, SI Data 1), a trend consistent with previous results based on 16S rRNA gene diversity in geothermal environments [42, 78, 79]. The mcrA-containing populations detected across samples included both confirmed methanogens (Methanomassiliicoccales, Methanosarcinales, Methanomicrobiales, Methanocellales, and Methanobacteriales) and lineages with proposed but untested methane/alkane metabolism (Archaeoglobi, Ca. Methanofastidiosales, Ca. Methanomethylicia, and Ca. Korarchaeia) (Fig. 2, SI Data 1). Confirmed methanogens dominated most samples (46/66) and were frequently identified in geothermal features with moderate temperatures (<60 °C). Other Mcr-encoding lineages prevailed at elevated temperatures (>60 °C) and were exclusively detected in LCB024.1, LCB058.1, and LCB063.2 (SI Fig. 2, SI Data 1). Particularly, Archaeoglobi-affiliated mcrA genes dominated at high temperatures (≥70 °C) and elevated concentrations of dissolved sulfide (≥3 µM; SI Fig. 2), which are conditions similar to those environments in which Archaeoglobi were previously detected [18, 21, 23, 80]. Notably, Ca. Korarchaeia were present at high relative sequence abundance (16%) in LCB003.1. Anaerobic methane-oxidizing archaea (ANME-1) as well as Ca. Methanofastidiosales were detected only with low relative sequence abundance (<2% and <4%, respectively; SI Data 1).

Overall, this survey of mcrA genes indicated that taxonomically diverse mcrA-containing archaea exist across a wide range of physicochemical regimes in the geothermal environments of YNP, particularly in the LCB geothermal area. As primer-based diversity surveys are inherently biased, we note that the primer set used in this study has historically been widely applied to amplify mcrA genes of Euryarchaeota origin [47]. While these primers bind to currently known mcrA genes of Ca. Methanomethylicia and Ca. Korarchaeia without mismatches, multiple mismatches to mcrA genes of other lineages exist (e.g., Ca. Nezhaarchaeia). Consequently, our amplicon-based gene survey likely underrepresented certain mcrA genes and underestimated mcrA gene diversity. To further investigate the methanogenic communities in the LCB, metagenomics and mesocosm experiments were conducted with material from hot springs LCB003, LCB019, and LCB024 characterized by elevated temperatures (47–73 °C) and circumneutral pH (3.0–7.8; SI Tables 13, Fig. 1B–D).

mcrA gene diversity and mcr-containing MAGs recovered from the LCB

In the hot springs selected as main study sites, abundant mcrA ASVs were related to confirmed methanogens in LCB019 and LCB003, and archaea with proposed methane/alkane metabolism in LCB024 and LCB003 (SI Table 6, SI Data 1). Environmental metagenomics recovered ten medium and two high quality MAGs [81] encoding McrA and a complete/near-complete MCR complex, which ranged in size from 0.73–1.78 Mbp and estimated completeness from 72 to 100% (Table 1). According to phylogenomic analysis of 18 archaeal single copy genes (SI Table 4), four MAGs belonged to lineages of previously cultured methanogens: Methanothermobacter (LCB019-055), Methanomassiliicoccales (LCB019-061), Methanothrix (LCB019-064), and Methanolinea (LCB019-065) while eight MAGs belonged to lineages of proposed methanogens or methane/alkane-oxidizing archaea: Ca. Methanomethylicia (LCB019-004, −019, −026, LCB024-024, −038, LCB003-007), Archaeoglobi (LCB024-003), and Ca. Hadesarchaeia (LCB024-034) (Fig. 3, SI Fig. 3). Interestingly, Ca. Methanomethylicia related MAGs were recovered from all three hot spring metagenomes indicating that members of this lineage can inhabit a wide range of physicochemical conditions. In contrast, MAGs affiliated with lineages of confirmed methanogens were only identified in LCB019, as initially reflected by mcrA amplicons. In total, 12 near-complete (≥500 aa) and 24 partial (100–499 aa) McrA sequences were recovered from the metagenome assemblies, suggesting that the Mcr-encoding MAGs reconstructed here do not reflect the full diversity and metabolic potential of the Mcr-encoding populations present. According to phylogenetic analysis, McrA proteins were categorized into MCR-type and ACR-type [21, 82] and affiliated with McrA of confirmed methanogens (group I), proposed methanogens (group II), or with McrA-like proteins of proposed alkane-metabolizing archaea (group III) [22, 27] (Fig. 3). Overall, the mcrA genes and Mcr-encoding MAGs recovered via metagenome sequencing confirmed the diversity of mcrA-containing archaea detected via amplicon sequencing and extended it by detecting Methanomassiliicoccales, Ca. Nezhaarchaeia, and Ca. Hadesarchaeia (Fig. 3).

Table 1 Statistics for twelve mcr-containing MAGs reconstructed from LCB metagenomes.
Fig. 3: Phylogenetic tree of Mcr-encoding MAGs and McrA.
figure 3

A Maximum-likelihood tree, inferred with IQtree and the best-fit LG + F + R10 model, using a concatenated set of 18 conserved arCOGs (SI Table 4). Squares indicate ultrafast bootstrap values of 100 (black) and 95–99 (gray). Diamonds indicate lineages with Mcr-encoding MAGs detected in this study and shown in detail. B Maximum-likelihood tree, inferred with IQtree and the LG + C60 + F + G model, from the amino acid alignment of McrA. Filled circles: McrA identified in a MAG, open circles: metagenomic McrA (unbinned, >100 aa), open squares: abundant mcrA ASVs (>1% relative sequence abundance). For details see SI Table 6. Dashed line indicates previously proposed McrA/AcrA groups [21, 82]: I) McrA from methanogens and ANME (MCR-type), II) McrA from TACK lineages (MCR-type), III) McrA-like from proposed and experimentally confirmed alkane oxidizing archaea (ACR-type). Colors: orange, LCB003, magenta, LCB019, blue, LCB024.

Potential for methane and alkane metabolism in mcr-containing MAGs

Four MAGs were affiliated with lineages of confirmed hydrogenotrophic, aceticlastic, and hydrogen-dependent methylotrophic methanogens and encoded mcrA genes related to those of cultured methanogens (group I). LCB019-065 and LCB019-055 shared amino acid identity (AAI) values of 80% and 98% with cultured representatives of the hydrogenotrophic methanogens Methanolinea and Methanothermobacter, respectively. Congruently, both MAGs encoded the genes required for generating methane from H2 and CO2, including the complete Wood-Ljungdahl Pathway (WLP), methyl-H4M(S)PT:coenzyme M methyltransferase (Mtr) complex, F420-reducing hydrogenase (Frh), methyl-viologen-reducing hydrogenase (Mvh) (incomplete in LCB019-065), and energy-converting hydrogenase (Ehb, LCB019-055; Ech, LCB019-065) (Fig. 4, SI Discussion). Additionally, a complete formate dehydrogenase complex (FdhABC) was encoded in LCB019-65 and while LCB019-055 encoded FdhAB, FdhC was not detected. Consistently, cultured representatives of Methanolinea utilize formate as a substrate for methanogenesis while those of Methanothermobacter do not [83, 84]. LCB019-064 showed AAI values of 90% to the aceticlastic methanogen Methanothrix thermoacetophila and encoded all genes necessary for aceticlastic methanogenesis including the Mtr complex and acetyl-CoA decarbonylase/synthase:CO dehydrogenase complex (ACS/CODH) (Fig. 4, SI Discussion). LCB019-061 shared AAI of 59% with cultured Methanomassiliicoccus sp., suggesting it may represent a novel lineage within the Methanomassiliicoccales. Consistent with a hydrogen-dependent methylotrophic methanogenesis lifestyle of Methanomassiliicoccales isolates, LCB019-061 encodes methyltransferases (SI Fig. 4) but lacks the WLP and a complete Mtr complex. A mtrH gene encoded in proximity to methyltransferase corrinoid activation protein (ramA) suggests LCB019-061 may reduce unknown methylated substrates to methane [19, 80].

Fig. 4: Methanogenic potential of the twelve Mcr-encoding MAGs.
figure 4

Squares indicate gene/gene set detected (filled), gene/gene set not detected (open) or gene set partially detected with the majority of genes present (half filled). * indicates only one gene in a gene set detected. Circles indicate the methane/alkane metabolism predicted for each MAG based on the gene repertoire. Colors: orange, LCB003, magenta, LCB019, blue, LCB024. A complete list of genes described in this figure and their abbreviations is reported in SI Data 3.

Six MAGs shared high AAI values (>96%) with MAGs of Ca. Methanomethylicia and encoded an McrA affiliated with those of other Ca. Methanomethylicia MAGs (group II). Consistent with Ca. Methanomethylicia MAGs proposed to perform hydrogen-dependent methylotrophic methanogenesis [19], the six MAGs lack the WLP and a complete Mtr complex but encode a variety of methyltransferases including methanol:coenzyme M methyltransferase (mtaA), monomethylamine methyltransferase (mtmB), and dimethylamine corrinoid (mtbC) and/or trimethylamine corrinoid protein (mttC). LCB019-026 additionally encoded a trimethylamine methyltransferase (mttB). A methyltransferase subunit H of the Mtr complex, mtrH, encoded near other corrinoid protein and methyltransferase genes (SI Data 3) suggests that methane may be formed from unknown methylated substrates [19, 80]. Although methylamine-specific cobamide:coenzyme M methyltransferase (mtbA) was not identified, MtaA could substitute for the activity of MtbA (SI Fig. 4) [85]. Thus, all six Ca. Methanomethylicia MAGs contain the gene repertoire needed for hydrogen-dependent methylotrophic methanogenesis (Fig. 4, SI Fig. 4, SI Discussion). In addition, LCB019-004 encoded a second McrA, that clustered with the McrA-like proteins of ethane-oxidizing archaea Ca. Ethanoperedens and Ca. Argoarchaeum (McrA group III, ACR/ECR type) and a recently recovered MAG of Ca. Methanosuratus [25, 82] proposed to perform ethanogenesis or ethane oxidation via an unknown pathway [82]. This indicates that anaerobic methane/alkane metabolism within the Ca. Methanomethylicia may be more diverse than previously anticipated.

The Archaeoglobi affiliated MAG LCB024-003 showed low AAI values (65%) to Archaeoglobales isolates, which are all non-methanogenic sulfate-reducers. Instead, LCB024-003 shared high AAI values (>98%) to Mcr-encoding Archaeoglobi MAGs of proposed hydrogenotrophic methanogens (WYZ-LMO10, SJ34) or hydrogen-dependent methylotrophic methanogens (Ca. M. hydrogenotrophicum) [21, 33, 80]. Consistently, its two partial McrA (192 and 193 aa) cluster with McrA of other proposed methanogenic Archaeoglobi (group II) (Fig. 3) [18, 21]. LCB024-003 encodes genes required for hydrogenotrophic methanogenesis including the WLP pathway, hydrogenase Mvh, and a F420H2:quinone oxidoreductase complex (fqoDHIF) which may substitute for Frh to generate reduced F420 as previously suggested [23, 86, 87]; however, a complete Mtr complex was not detected. In contrast to Ca. M. hydrogenotrophicum, LCB024-003 encodes a truncated 5,10-methylenetetrahydromethanopterin reductase (mer) while mtaABC were not identified, suggesting it is unable to use methanol for methanogenesis [23]. Although LCB024-003 encodes the beta-oxidation pathway, other genes typically associated with short-chain alkane oxidation including an ACR-type MCR, ACS/CODH complex, and methyltransferases were absent. Hence, unlike the MAGs of Ca. Polytropus marinifundus and JdFR-42 [18, 21], LCB024-003 may not represent an anaerobic alkane oxidizer [88] and instead may utilize the beta-oxidation pathway for long chain fatty acid metabolism as has been shown for Archaeoglobus fulgidus [89]. Further, genes encoding dissimilatory sulfate reduction (sat, aprAB, dsrABC) present in some mcr-containing Archaeoglobi MAGs (Ca. M. dualitatem [23]) were not detected. Together, the genomic information from LCB024-003 suggests that this Archaeoglobi representative may live as a hydrogenotrophic methanogen (Fig. 4, SI Discussion).

LCB024-034 shared AAI values of >79% with other Mcr-encoding Hadesarchaeia [21, 80] and encoded a partial McrA (216 aa) related to the ACR-type proteins of Hadesarchaeia (group III) [27]. Congruently with the hypothesis of short-chain alkane metabolism in Ca. Hadesarchaeia, LCB024-034 encoded the beta-oxidation pathway and an ACS/CODH complex. However, most genes encoding the WLP required for oxidizing activated alkanes to CO2 were missing [27]. Thus, short-chain alkane metabolism in LCB024-034 remains speculative, awaiting further genomic and experimental data.

Together, the 12 mcr-containing MAGs reconstructed here reflect the potential for archaeal short-chain alkane-oxidation as well as hydrogenotrophic, aceticlastic, and hydrogen-dependent methylotrophic methanogenesis in geothermal environments of YNP. Further, these MAGs extend the genomic data available for future analysis of diversity and evolution of Mcr-encoding archaea and suggest geothermal environments are a promising source for the recovery of these archaea.

Methanogenic activity and enrichment of methanogens in mesocosms

Mesocosm experiments were performed to reveal activity and enrichment of methanogens. Methane accumulation was monitored in the headspace of mesocosms under (1) close to in situ conditions (i.e., no amendment), (2) conditions favoring methanogenesis (i.e., substrate amendment), and (3) conditions inhibiting bacterial metabolism (i.e., antibiotics treatment) (SI Fig. 5). Inhibition of bacterial metabolism may have disrupted potential symbiotic partnerships between methanogens and bacteria and/or favored substrate availability for methanogens through the limitation of competition. Mesocosms were also analyzed for enrichment in potential methanogenic populations via 16S rRNA gene amplicon sequencing. Abundant 16S rRNA gene ASVs (>1% relative sequence abundance) related to Mcr-encoding archaea amounted for ~1% in LCB019 and <1% in LCB024 and LCB003, indicating that methanogens represent a minor fraction of the in situ community (SI Figs. 6, 7). However, in mesocosms from all three hot springs, methane production was observed under close to in situ conditions with strongly varying maximum methane yields (17,000, 1900, and 150 ppm for LCB019, LCB024, and LCB003, respectively; Fig. 5, SI Fig. 5). Substrate amendment had considerably different effects on methane production and, except for LCB019, mesocosm triplicates showed strong variation and long response times (20–40 days) likely due to an uneven distribution of initially low abundant methanogen cells across replicates. For LCB024, substrate amendment (particularly H2), appeared to suppress methane production, which may indicate that either hydrogenotrophic methanogens were not present, not active, or were outcompeted by other community members considering the shift in the microbial community (SI Fig. 7). Antibiotic amendments resulted, on average across treatments, in increased methane production in mesocosms from LCB024 and LCB003, and a strong decrease in methane production in mesocosms from LCB019, indicative of substrate competition or metabolic interdependencies between methanogens and bacteria, respectively.

Fig. 5: Methane production and enrichment of Mcr-encoding archaea in mesocosms.
figure 5

A Maximum methane produced in the headspace of mesocosms. Replicates measuring <100 ppm not shown. B Enrichment of 16S rRNA gene ASVs (>3% relative sequence abundance) affiliated with Mcr-encoding archaea across treatments (bars) paired with respective headspace methane yields (circles). Circle size proportional to the log2 fold change in methane yield between treatment and control (i.e., mesocosm under close to in situ condition) for each site. Dashed lines indicate 1% methane. Solid lines indicate average methane concentration in mesocosms under close to in situ conditions (no substrate, no antibiotics) for each site. Open symbols: without antibiotics; filled symbols: with antibiotics. Colors: orange, LCB003, magenta, LCB019, blue, LCB024. Abbreviations: NON, no amendment control; ACE, acetate; MET, methanol; MMA, monomethylamine; FOR, formate; HYD, hydrogen (H2); DIC, dissolved inorganic carbon (HCO3 + CO2); BES, bromoethanesulfonate (methanogenesis inhibitor); PFA, paraformaldehyde (killed control). Replicates indicated as A-C and+, with antibiotics or -, without antibiotics. Methane curves and extended relative abundance data for all mesocosm replicates are reported in SI Figs. 57 and SI Data 4.

To characterize the effect of substrate amendment on methanogenic populations, we analyzed ASVs related to Mcr-encoding archaea with enrichment >3% relative sequence abundance across treatments. H2 plus DIC (HCO3 + CO2) amended mesocosms from LCB019 showed rapid methane production, with highest maximum methane concentrations (>170,000 ppm) reached within 6 days (SI Fig. 5). These mesocosms were enriched (26–35%) in ASV_5ea58, identical to the 16S rRNA gene of MAG LCB019-055 as well as Methanothermobacter thermautotrophicus, a thermophilic hydrogenotrophic methanogen isolated from YNP [40, 41]. Similarly, for LCB003, H2 plus DIC or methylated compounds resulted in the strongest stimulation of methanogenesis and most pronounced enrichment (up to 71%) of an ASV affiliated with Methanothermobacter crinale (ASV_520b7, 99.6% sequence identity). Notably, H2 amendment without DIC supply did not result in a comparable response, suggesting that in closed mesocosm systems hydrogenotrophic methanogens were limited by inorganic carbon, which unlikely occurs in situ where concentrations of aqueous CO2 were elevated (SI Table 2). For LCB019, acetate amendment resulted in elevated methane production and concomitant enrichment (3–5%) of ASV_daa7b, which shared high sequence similarity with the aceticlastic methanogen Methanothrix thermoacetophila (98%) and a 16S rRNA gene recovered from the LCB019 metagenome (100%; SI Data 4). MAG LCB019-064, related to Methanothrix thermoacetophila (89% AAI similarity) encoded the potential for methanogenesis from acetate and may represent the enriched Methanothrix sp. population. Thus, our mesocosm experiments complemented findings from metagenomics, confirming the potential for hydrogenotrophic methanogenesis by Methanothermobacter sp. and aceticlastic methanogenesis by Methanothrix sp. in LCB019 and revealing the potential for hydrogenotrophic methanogenesis by Methanothermobacter sp. in LCB003 (SI Table 6).

In addition to previously cultured methanogens, uncultured Mcr-encoding lineages were enriched. An ASV identified as Ca. Methanodesulfokores washburnensis (ASV_74dd7, 100% sequence identity) was highly abundant (25–54%) in two mesocosms from LCB003 amended with methanol, hydrogen, and antibiotics. A MAG of this Ca. Korarchaeia representative previously recovered from YNP encodes versatile metabolic capabilities including hydrogen-dependent methylotrophic methanogenesis from methanol [20]. Methane yields in these mesocosms, while comparably low after 43 days (<2000 ppm), were strongly elevated compared to the no amendment control of LCB003 (log2 fold change (FC) 3–4). mcrA and 16S rRNA genes of Ca. Methanodesulfokores washburnensis were also detected via amplicon and metagenome sequencing, confirming the presence of this lineage in LCB003 (Figs. 2, 3, SI Data 4). In one mesocosm from LCB024 amended with monomethylamine, stimulation of methanogenesis (log2 FC 4, 310,000 ppm) and enrichment (8%) of Archaeoglobi-affiliated ASV_78ad2 was observed. The Archaeoglobi MAG LCB024-003 recovered from LCB024 encoded the potential for hydrogenotrophic methanogenesis while genes required for methylotrophic methanogenesis were not detected (Fig. 4). However, potential for methylotrophic methanogenesis has been described for some Archaeoglobi MAGs and the recovery of several Archaeoglobi related mcrA and 16S rRNA genes from LCB024 suggests that diverse Archaeoglobi populations are present, possibly including methylotrophic methanogens. An enrichment of a Ca. Nezhaarchaeia related ASV (ASV_27aa3) was highest (8%) in methanol amended mesocosms from LCB003 and cooccurred with elevated methane yields (log2 FC 3.5, 85,000 ppm), confirming the persistence of a Ca. Nezhaarchaeia population detected by metagenomic 16S rRNA and mcrA genes (SI Data 2). Previously described mcr-containing Ca. Nezhaarchaeia MAGs encode the potential for hydrogenotrophic methanogenesis, and while no enrichment was detected in hydrogen amended mesocosms, microbially produced hydrogen may have facilitated limited methanogenic activity and enrichment of hydrogenotrophic methanogens in other mesocosms. Ca. Methanomethylicia related ASVs were detected in multiple mesocosms, however their enrichment remained low (<3%) (SI Data 4).

Overall, minor methanogenic populations, not or hardly detectable in hot springs via 16S rRNA gene or metagenome sequencing, were enriched in mesocosm experiments under selective methanogenic conditions. Specifically, acetate or hydrogen plus DIC enabled the enrichment of Methanothrix or Methanothermobacter, respectively, while methyl compounds favored the enrichment of Ca. Korarchaeia, Ca. Nezhaarchaeia, or Archaeoglobi. Further research is needed to decipher the metabolism of the here enriched populations of uncultured archaea, their proposed methanogenic capacities, and potential metabolic interdependencies with other community members.

Implications for methane cycling in YNP

We explored the potential for methanogenesis in previously uncharacterized geothermal environments of YNP, primarily the LCB, and our results warrant further research into the magnitude of biological methane production in this area. While the methanogenic communities of eight geothermal features in YNP had previously been investigated [20, 21, 24, 30, 33, 42] we detected mcrA genes across an additional 39 geothermal features indicating the wide distribution of diverse populations of Mcr-encoding archaea, including both confirmed methanogens and lineages proposed to engage in anaerobic methane/alkane cycling. The methanogenic pathways encoded across mcr-containing MAGs suggests methanogenesis in LCB hot springs could proceed from different precursors including H2/CO2, acetate, and methyl compounds plus hydrogen. The genetic potential for hydrogen-dependent methylotrophic methanogenesis was encoded by the majority of MAGs, including Ca. Methanomethylicia and Methanomassiliicoccales, and was detected in all three hot springs, possibly reflecting prevalence of this metabolism in geothermal environments as previously proposed [80]. While methanogenic populations accounted for minor fractions of the microbial community, methanogenesis may proceed in situ as it was observed in mesocosms under close to in situ conditions. The potential for hydrogenotrophic and aceticlastic methanogenesis revealed by metagenomics was confirmed by the enrichment of Methanothermobacter and Methanothrix in mesocosms under selective substrate amendment. In situ, methanogenesis in hot springs is likely constrained by physicochemical regimes, substrate availability, and metabolic interdependencies. Methanogenic precursors may be supplied from organic matter degradation as metabolic intermediates of syntrophic communities (e.g., H2, acetate), products of respiration (e.g., CO2), or through geothermal alteration from the subsurface (e.g., H2, CO2) [40, 90]. As hot springs often present dynamic systems, methanogens may frequently respond with activity and growth to favorable conditions. This may be exemplified by Methanothermobacter’s capacity to rapidly respond, resulting in high activity and fast growth upon supply of H2/CO2, which it may sporadically or consistently encounter in situ (Fig. 5, SI Table 2, SI Fig. 5).

Although methanogenic activity and isolation of Methanothermobacter thermoautotrophicus have been demonstrated [40, 41], the environmental impact of methanogens on methane emissions from YNP’s geothermal environments is not well understood. Methane is an important component of the gas flux in YNP [90,91,92] and the isotopic composition of gas emitted from geothermal features across YNP has suggested methane is primarily generated through abiogenic and/or thermogenic processes, while methanogenesis is not a significant source of methane [91]. Although we detected varying concentrations of aqueous methane in geothermal features in an area of YNP that had not been previously investigated, the source and fate of this methane is currently unknown. In general, methane emissions from terrestrial geothermal environments are not considered in estimates of the global atmospheric methane budget and little is known about their contribution to the global methane flux [1, 3, 14]. YNP contains more than 14,000 geothermal features, the largest concentration in the world, making it a superior candidate for studying CH4 flux in these environments [93,94,95].

Environmental mcrA gene surveys and metagenomics aid in identifying environments in which methanogenesis may occur. Subsequent quantification of in situ metabolic activities, including methane production rates, as well as deciphering the interplay between methanogens and methanotrophs will lead to a better understanding of the impact methanogens have on the local carbon cycle and their contribution to methane emissions from YNP’s geothermal environments.

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