Abstract
The preeminent source of biological methane on Earth is methyl coenzyme M reductase (Mcr)-dependent archaeal methanogenesis. A growing body of evidence suggests a diversity of archaea possess Mcr, although experimental validation of hypothesized methane metabolisms has been missing. Here, we provide evidence of a functional Mcr-based methanogenesis pathway in a novel member of the family Archaeoglobaceae, designated Methanoglobus nevadensis, which we enriched from a terrestrial hot spring on the polysaccharide xyloglucan. Our incubation assays demonstrate methane production that is highly sensitive to the Mcr inhibitor bromoethanesulfonate, stimulated by xyloglucan and xyloglucan-derived sugars, concomitant with the consumption of molecular hydrogen, and causing a deuterium fractionation in methane characteristic of hydrogenotrophic and methylotrophic methanogens. Combined with the recovery and analysis of a high-quality M. nevadensis metagenome-assembled genome encoding a divergent Mcr and diverse potential electron and carbon transfer pathways, our observations suggest methanogenesis in M. nevadensis occurs via Mcr and is fueled by the consumption of cross-fed byproducts of xyloglucan fermentation mediated by other community members. Phylogenetic analysis shows close affiliation of the M. nevadensis Mcr with those from Korarchaeota, Nezhaarchaeota, Verstraetearchaeota, and other Archaeoglobales that are divergent from well-characterized Mcr. We propose these archaea likely also use functional Mcr complexes to generate methane on the basis of our experimental validation in M. nevadensis. Thus, divergent Mcr-encoding archaea may be underestimated sources of biological methane in terrestrial and marine hydrothermal environments.
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Introduction
Biological methane has shaped the past [1, 2] and present [3] planetary climate as an agent of atmospheric warming. However, the full diversity and abundance of microorganisms responsible for biological methane production is still not entirely understood. Earth’s major methane source is methyl coenzyme M reductase (Mcr)-dependent metabolism in anaerobic archaea. This 300 kilodalton protein complex catalyzes the terminal step in methanogenesis [4, 5] as well as the initial step in anaerobic methane oxidation [6]. Divergent homologs of the Mcr complex have been identified in metagenome-assembled genomes (MAGs) of uncultured archaea, including the Bathyarchaeota, Verstraetearchaeota, Methanonatronarchaeia, Nezhaarchaeota, Nitrososphaerales (syn. Thaumarchaeota), Hadarchaeota, Korarchaeota, Archaeoglobales, ANME-1, and GoM-Arc1 [7,8,9,10,11]). The divergence of these new members of the Mcr family appears to be associated with a transition from methane metabolism to short-chain alkanes, such as ethane [12, 13] and butane [14], leading them to be referred to as alkyl-CoM reductases (Acrs). The increased availability of MAGs from uncultured archaeal lineages containing Mcr- and Acr-encoding sequences in the last few years has revealed a large uncertainty in our understanding of methane and alkane metabolisms.
Recently, Mcr/Acr complexes have been uncovered in MAGs corresponding to members of the Archaeoglobi, cultured representatives of which are not known to catalyze methanogenesis or methanotrophy. One such genome from Candidatus Polytropus marinifundus, derived from the deep subseafloor along the Juan de Fuca Ridge [15], contained two putative Acr operons. This archaeon was hypothesized to use these enzymes for the oxidation of alkanes, similar to other Acr-encoding archaea [12,13,14]. Despite several reports of small amounts of methane (<200 µmol L–1 culture) produced by Archaeoglobus isolates [16,17,18,19], a role in methane metabolism in this organism seemed unlikely in light of the high-degree sequence divergence from experimentally validated Mcr and, moreover, methanogenesis has been suggested to have been lost in the Archaeoglobi [20]. In contrast, a survey of metagenomic data from hot springs and oil reservoirs revealed additional, phylogenetically distinct MAGs of the family Archaeoglobaceae containing canonical Mcr operons [21] and, thus, pointing toward the potential for methane metabolism in the class Archaeoglobi. However, experimental evidence of significant methane production or consumption in the Archaeoglobi in association with a functional Mcr complex has been lacking and their role in methane cycling in their thermal habitat remained unresolved. While methanogenic activity occurs in marine systems at temperatures of up to 122 °C [22], in-situ substrate abundances may limit H2-based methanogenesis in terrestrial systems [23] where it has been observed at ≤75 °C. In light of the possible substrate diversity of divergent Mcr, it may be conceivable that phyla using divergent Mcr for methane production extend terrestrial methanogenesis to higher temperatures.
We investigated methane metabolism in a member of the Archaeoglobaceae, here designated Methanoglobus nevadensis XG-1, that was enriched in a mixed, anaerobic culture from Great Boiling Spring (GBS), Nevada, while fed with the polysaccharide xyloglucan (XG). After detecting methane in the headspace of the culture, we hypothesized M. nevadensis to be responsible for methane formation and assessed effects of a suite of different substrates and inhibitors on methane production. Analysis of the medium-quality XG-1 MAG, the only Mcr-encoding member of the enrichment culture, and a conspecific high-quality MAG designated Methanoglobus nevadensis GBSTs from an in-situ anaerobic ammonia fiber expansion (AFEX)-treated corn stover enrichment in GBS, suggests an Mcr-dependent pathway with electrons sourced from organic compounds in conjunction with H2. Our study demonstrates Mcr-based methane production in a member of the Archaeoglobaceae, a group previously unknown to include true methanogens, describes a plausible ecology and physiology, and provides a link to biological methane production in a terrestrial hydrothermal setting.
Methods
In-situ enrichments and laboratory cultivation
The geochemistry and microbiology of Great Boiling Spring (GBS) near Gerlach, Nevada [24, 25], and Little Hot Creek (LHC) in California’s Long Valley caldera [26] have been described previously. In-situ enrichments on 1 g of ammonia-fiber expansion (AFEX) corn stover sealed in 100 µm nylon mesh bags at ~77 °C were performed in the GBS main pool from 26 October 2013 to 28 March 2014 as described in ref. [27], which enriched for Archaeoglobi before. At LHC, in-situ enrichments at ~78 °C were deployed the same way from 19 Oct 2013 to 6 April 2014. Transfer to GBS mineral salts medium (under pure N2 at 75 °C) followed an established approach [28] with the difference that 0.02% (m/v) xyloglucan served as a sole carbon source and a trace metal solution was added according to a previous protocol [29].
Genomic DNA extraction
DNA was extracted from the XG-degrading culture by bead-beating with the Fast DNA SPIN Kit for Soil (MP Biomedicals) according to a slightly modified protocol [28].
16S rRNA gene sequencing
Amplification within the V4 region of the 16S rRNA gene and sequencing on initial enrichment cultures was performed essentially as described [30]. A modified forward primer 515 (5′ GTGYCAGCMGCCGCGGTAA) with a Y instead of a C at the 4th position from the 5′ end was used in combination with reverse primer 5′ TAATCTWTGGGVHCATCAGG to increase coverage of archaea, and a corresponding change was made to the SeqRead1 primer. Sequencing (2 × 250 bp) was performed on an iSeq sequencer (Illumina) using the V2 reagent kit and reads were processed with the DADA2 pipeline using the SILVA (version 138) database for taxonomy [31] resulting in 39,305 amplicon sequence variants.
Metagenomic sequencing, assembly, analysis, and binning of MAGs
Extracted culture DNA was sequenced using the MiSeq platform (Illumina, short reads), along with Oxford Nanopore sequencing (long reads). Shotgun metagenome sequencing with MiSeq (2 × 250 bp) was performed at Argonne National Laboratories, with library preparation using the Nextera Flex DNA kit. For Oxford Nanopore sequencing, libraries were prepared following the Native Barcoding (EXP-NBD103) and Ligation Sequencing (SQK-LSK108) Kit 1D protocols according to the manufacturer’s instructions, and sequencing was performed on a MinION FLO-MIN106 flow cell (Oxford Nanopore, Oxford Science Park, UK). Reads were mapped to known Archaeoglobi reference genomes using BowTie 2 [32]. The mapped short and long reads were assembled with hybridSPAdes [33] and the resulting contigs were filtered, merged, and dereplicated. This assembly was then subjected to binning with MetaBAT2 v. 1.7 [34], as implemented in KBase [35]. Bins were refined to reduce redundancy and contamination with Anvi’o [36] and MAG quality and classification was determined using CheckM v1.4.0 [37] and the GTDB Toolkit v1.1.0 [38] in KBase. Because the Archaeoglobaceae MAG obtained from the enrichment culture was only of medium quality, we compared it to MAGs previously derived from GBS metagenomes by our group using the JSpeciesWS online server [39]. Bin 20 of metagenome 3300005298 in the JGI database was shown to belong to the same species, with an average nucleotide identity (ANI) of 98.4% and was therefore used for comparative genomics. To screen metagenomic raw reads for mcrA, GraftM version 0.13.1 [40] was trained using the 499 mcrA sequences obtained from NCBI’s PSI-Blast (default configuration) with an e-value of 10−9. The mcrA sequences were aligned with IQ-TREE [41] using 0.5 perturbation strength and 1000 iterations, and the alignment was visualized in the iTOL interface version 6 [42].
All 76 genomes from the GTDB release 207 in the class Archaeoglobi were retrieved and supplemented with four additional genomes from IMG and our incomplete MAG from the xyloglucan enrichment for 80 total genomes. All genomes were assessed for completeness and contamination using CheckM v1.4.0 implemented on the KBase platform. The concatenated, masked alignment of 43 marker genes produced by CheckM was used to generate a phylogenomic tree using RAxML v8.2.12 implemented on XSEDE via the CYPRES Science Gateway v3.3, with the following parameters: protein substitution model: PROTGAMMA, protein substitution matrix: WAG, with 100 iterations of rapid bootstrapping.
Cultures further enriched with antibiotics and methanol were sequenced with Illumina NextSeq 2000 producing 8.3 M 151 bp read pairs. Raw reads were mapped to the Archaeoglobi MAG generated in our enrichment cultures described above, bin 20 of metagenome 3300005298 (JGI), and the complete genome of Pseudothermotoga hypogea (GCA_000504105.1) using the Geneious aligner (v. 2019.2.1).
Batch incubation experiments
Batch incubations were performed under static conditions for a maximum of 4 weeks at 75 °C, except the temperature variation series, and in replicates of 2–4 (Fig. 1). At the beginning of stationary phase, 100 µL of growing culture was transferred to fresh anoxic GBS mineral salts media [28] amended with filter-sterilized, anoxic stock solutions added with a flushed syringe. All substrate stocks were prepared using molecular grade reagents and the final concentrations were the following: 0.02% (w/v) xyloglucan, 0.05% (w/v) of each monosaccharide (L-fucose, D-galactose, D-xylose), 500 µM of each organic acid (acetate, pyruvate, malate, dimethylsulfoniopropionate), 1 mM 2-methoxybenzoate, and 500 µM trimethylamine. Xyloglucan was added as powder directly to the media. The H2/CO2 (80:20) mix was added to reach 0.7 bar overpressure. The antibiotic treatment consisted of 100 µg mL–1 streptomycin and 100 µg mL–1 carbenicillin. The inhibitor bromoethanesulfonate (BES) was added to a final concentration of 10 mM and coenzyme M was added at 140 mg L–1. The base medium contained 3.1 mM sulfate and traces of sulfate in a trace metal solution, which was all left out in the (-)SO42– treatment.
A Community composition (%) in anaerobic enrichments (maintained over multiple years, 2 weeks after the last transfer) from Great Boiling Spring (GBS, the XG-degrading culture) and Little Hot Creek (LHC) based on 16S rRNA genes. Taxa are identified at the lowest named rank according to Silva. All Archaeoglobaceae sequences detected belong to M. nevadensis. B Headspace methane concentration over time (n = 2–4). The second y-axis shows the relative amount of methane derived from the organic carbon added. The antibiotics mix consisted of streptomycin and carbenicillin to specifically target bacteria. Approximate range in values from four GBS cultures is colored in gray. SO42–, sulfate. C Headspace methane concentrations in duplicate treatments on day 15. If not specifically indicated, incubation temperature was maintained at 75 °C. MBA Methoxybenzoate, TMA Trimethylamine, DMSP Dimethylsulfoniopropionate.
To enrich the culture further we removed the complex carbon substrate XG, added 100 mM methanol as the main carbon substrate and added 30 psi H2/CO2 (80:20) to the headspace. Tetracycline, Ampicillin, Kanamycin were added to final concentrations of 100 µg mL–1, 1 mg mL–1 and 50 µg mL–1, respectively, in order to help inhibit the growth of bacteria. Other components of this enrichment medium were (per liter): KCl (0.15 g), MgCl2*6H2O (0.102 g), Na2SO4 (0.3 g), CaCl2*2H2O (0.14 g), K2HPO4 (0.14 g), NaCl (3 g), Yeast Extract (0.2 g), Peptone (0.2 g), Sodium acetate (0.6 g), Sodium pyruvate (0.5 g), NaHCO3 (5 g), NH4Cl (1 g), L-cysteine HCl (0.5 g), Na2S*9H2O (0.5 g), 1.36 mL 0.1% w/v solution FeSO4*7H2O, 1.6 mL 0.1% w/v NiSO4*6H2O, 38 µL 0.1% w/v Na2WO4*2H2O, 10 mL trace element mix, 10 mL vitamin mix. Trace element mix composition (per liter): trisodium nitrilotriacetic acid*H2O (1.5 g), Fe(NH4)2(SO4)2 (0.8 g), Na2SeO3 (0.2 g), CoCl2*6H2O (0.1 g), MnSO4*H2O (0.1 g), Na2MoO4*2H2O (0.1 g), Na2WO4*2H2O (0.1 g), ZnSO4*7H2O (0.1 g), NiCl2*6H2O (0.1 g), H3BO3 (0.01 g), CuSO4*5H2O (0.01 g). Vitamin mix composition (per liter): p-Aminobenzoic acid (10 mg), Nicotinic acid (10 mg), Ca panthotenate (10 mg), Pyridoxine HCl (10 mg), Riboflavin (10 mg), Thiamine HCl (10 mg), Biotin (5 mg), Folic Acid (5 mg), α-Lipoic Acid (5 mg), Vitamin B12 (5 mg). Growth in Balch-type anaerobic tubes were monitored by optical density measurements at 600 nm (OD600) with a UV–Vis Spectrophotometer (Gensys 50, Thermo Fisher Scientific). To test methanogenic basis for this growth, BES was added to final concentration of 200 μM.
Microscopy
Enrichment cultures supplemented with antibiotics and methanol were monitored by differential interference contrast and epifluorescence microscopy with a ZEISS Axio Imager M2 microscope. Putative cofactor F420 fluorescence was detected using a DAPI filter set: Ex 350/50, Em 460/50.
Methane and hydrogen gas measurements
Culture headspace was sampled with flushed syringes into 12 mL exetainers by replacement with N2. Using a xyzTek Bandolero auto sampler, gas from 12 mL exetainers was drawn into a gas chromatograph (Shimadzu GC-2014) equipped with a flame-ionization detector (FID) kept at 250 °C. A 1 m HayeSep N pre-column was serially connected to a 5 m HayeSep-D column conditioned at 75 °C oven temperature. Nitrogen gas (UHP grade 99.999%, Praxair Inc.) served as carrier with 21 mL min–1 flow rate. Methane concentration measurements were calibrated with customized standard mixtures (Scott Specialty Gases, accuracy ±5%). Gas phase concentrations were corrected to account for dilution using Henry’s law with the dimensionless concentration constant KHcc(CH4) = 0.0249 for gas dispersion into the aqueous phase at 73 °C [43]. Cell-specific methane production rates were approximated based on the methane production in XG-amended culture, the total number of cells approximated by direct microscope cell counts with a Petroff-Hausser chamber, and the relative proportion of M. nevadensis based on 16S rRNA gene abundances.
H2 gas concentration in 12 mL exetainer samples was determined with a reactive mercury bed reduced gas analyzer (Peak Laboratories). Gas samples (250 µL) were injected with a gas-tight syringe (VICI) into an N2 carrier gas stream through a Unibeads 1S and a Molecular Sieve 13X column conditioned at 104 °C. The retention time for H2 was 51 s. Blank controls, consisting of uninoculated media with xyloglucan, never produced H2 above the detection limit. The analyzer was calibrated using a 100 ppm H2 standard (Scott Specialty Gases, accuracy ±5%) that was serially diluted with nitrogen gas.
In-situ incubations and gas sampling
To determine methane production rates in the source pool of GBS, as well as nearby source pools G04b and SSW, hot spring sediment was transferred into a glass flask that was continuously flushed with N2. Fresh hot spring water was N2-sparged and used to dilute the sediment 1:4. The sediment slurry was distributed into pre-flushed vials with a 18G needle. In-situ incubations did not receive substrates and the closed, anoxic vials were submerged into the spring (~77 °C). Methane production rates were calculated from triplicates based on the headspace accumulation of methane sampled at 4 time points over 3 days with N2-flushed syringes by N2 replacement. Gas for stable isotope analysis was extracted from water collected with a 60 mL syringe at ~15 cm depth. Half the syringe was filled with sample water after which the other half was filled with N2. The dissolved gas was equilibrated with the N2 by shaking the syringe for 10 min. The gas phase was then injected into evacuated 12 mL exetainers.
Stable isotope analysis
Isotopes of C and H in methane gas were analyzed using a Thermo Scientific GasBench-Precon concentration unit interfaced to a Thermo Scientific Delta V Plus isotope ratio mass spectrometer (IRMS) at the UC Davis Stable Isotope Facility and data collection followed the method by Yarnes [44].
Results
Methane formation in the xyloglucan-degrading culture
After screening diverse thermophilic enrichments for methanogenic activity, we detected methane in the headspace of an anaerobic culture derived from Great Boiling Spring (GBS), Nevada. Another enrichment maintained under identical conditions derived from Little Hot Creek (LHC), California, did not show detectable methane. Both cultures were supplied with XG. The cultures contained comparable abundances of Candidatus Caldatribacterium species (11.1–13.9%) and Pseudothermotoga (6.4–12.3%) but differed particularly in one member belonging to the family Archaeoglobaceae, here introduced as Methanoglobus nevadensis. Although at a low proportion (0.1%), M. nevadensis was present in the GBS culture but not in the LHC culture (Fig. 1A). No amplicon sequence variant (ASV) was classified to a canonical methanogen in either culture.
After transfers into fresh media, we measured methane production in microcosms and assessed the influence of potential stimulants and inhibitors. The XG enrichment from LHC did not produce methane at any time during the experiment, while the GBS culture reached 104–158 µmol methane L–1 after two weeks of incubation at 75 °C, which was within the optimal temperature range for methane production (Fig. 1B, C). At that time, there were 1.2–2.0 × 108 cells mL–1 culture based on direct total cell counts. Methane formation at 80 °C was not significantly lower than that at 75 °C (Student’s t test, p < 0.05) and we also observed methane in cultures grown at 85 °C (Fig. 1C). Methanogenesis in the Archaeoglobi may, thus, increase the known temperature limit of terrestrial methanogenesis by 5–10 °C [23]. We henceforth refer to the culture from GBS (and not LHC) as XG-degrading culture. The omission of sulfate as a potential oxidant for anaerobic respiration and the addition of antibiotics targeting bacteria resulted in only minor differences in methane formation (Fig. 1B). Amendment with coenzyme M (CoM), an essential nutrient for some methanogens [45] and one species of non-methanogenic Archaeoglobus [17], did not increase activity or growth. In contrast, addition of bromoethanesulfonate (BES), a structural analog to methyl-CoM and commonly used methanogenesis inhibitor specifically targeting Mcr, halted methane production completely (Fig. 1C). Because methane production was not substrate limited, generally channeling ~10–16% of the added XG-carbon into methane (Fig. 1B), we were able to compare the effect of monosaccharide products of XG breakdown, specifically fucose, xylose, or galactose, on methane production. Less than half the amount of methane was produced by any of these substrates individually (with the exception of one xylose replicate reaching ~85 µmol methane L–1). Incubations with methoxybenzoate (MBA) supported 14–19 µmol methane L–1, suggesting the capacity for methoxydotrophic methanogenesis [46]. Other organic acids and well-known methanogenic substrates (H2/CO2, TMA, DMSP) resulted in low methane formation (<10 µmol methane L–1), with ambiguous results from pyruvate incubations (Fig. 1C). Media with methanol, hydrogen gas and antibiotics resulted in an enrichment culture where M. nevadensis accounted for the majority (68%) of the metagenome reads and BES amendment of that highly enriched culture exhibited strong growth dependence on Mcr (Fig. S1). Cell morphology in M. nevadensis-dominated cultures resembled irregular cocci of 1–2 µm (Fig. S1).
A functional, divergent Mcr in M. nevadensis
To better understand the potential pathway of methane generation in M. nevadensis, we carried out metagenome sequencing of our enrichment cultures. We were able to recover a MAG estimated to be 63% complete with 2.4% contamination (M. nevadensis XG-1). GTDB_tk placed this MAG within the genus “WYZ-LMO2” [47], members of which were reported to contain Mcr operons and originally named Candidatus Methanoproducendum [11] or later described with the synonym Candidatus Methanomixophus [21]. Phylogenomic analysis of currently available Archaeoglobi genomes placed our MAG together with “Bin 20”, or M. nevadensis GBSTs, which was derived from an in-situ AFEX-treated corn stover enrichment (IMG: 3300005298_20) [27]. These two MAGs represent the same species, sharing 98.4% average nucleotide identity [48] with a tetra-nucleotide index of 0.996 (Table S1). Since the 1.6 Mb MAG from the in-situ enrichment was far more complete (98% and 3.7% contamination), the genomic analysis below focused on M. nevadensis GBSTs. Screening the unassembled metagenomic reads for the mcrA gene using GraftM revealed a gradual enrichment of M. nevadensis from 0.3% in GBS sediment, over 10% in the in-situ cellulolytic enrichment (corn stover bag), to 100% in the XG enrichment culture (Fig. 2A). While a diversity of Mcr-containing organisms is present in situ, M. nevadensis was the sole source of methane in our incubations. The phylogenetic placement of the recruited McrA is close to the “WYZ-LMO2” McrA on a monophyletic branch with protein sequences derived from Korarchaeota, Nezhaarchaeota, and Verstraetearchaeota [47] (Fig. 2B). Although the McrA shows affiliation with Archaea outside of the Euryarchaeota, a genome tree derived from an alignment of 43 genomic markers placed M. nevadensis among other Euryarchaeota within the family Archaeoglobaceae with strong support (Fig. 2C). Based on monophyly and ANI values >95% [49], MAGs derived from GBS belong to a single species in the genus Methanoglobus with M. nevadensis GBSTs as the nomenclatural type for the species (Fig. 2C, Table S2), whereas genomes representing other species in the genus have been recovered from Yellowstone National Park or Jinze Hot Spring in Tengchong, China [11, 21, 47]. To promote best practices in systematics, protologues for new taxonomic names are included in the Supplements, including the genus Methanoglobus and the species Methanoglobus nevadensis (type is GBSTs). These names, together with Methanoglobus hydrogenotrophicum (type is Bin16Ts) and Methanoglobus dualitatem (type is LMO3Ts), will be registered in the SeqCode Registry [50].
A Genus-level classification of mcrA gene sequences in the original GBS sediment, the in-situ cellulolytic enrichment, and culture grown on XG at 75 °C, following an enrichment trend of M. nevadensis. The McrA sequence composition of the XG-degrading culture corresponds to the 16S rRNA composition from GBS shown in Fig. 1A. Cell density data and 16S rRNA gene abundances of M. nevadensis after continued enrichment are shown in Fig. S1. B Phylogenetic relationship between M. nevadensis McrA (red star) and 499 related McrA amino acid sequences aligned by PSI-Blast. C Genome tree of the two M. nevadensis MAGs and 78 other Archaeoglobi genomes. M. nevadensis forms a distinct branch with McrA from Methanodesulfokores washburnensis.
Potential carbon and electron transfers in M. nevadensis methanogenesis
The M. nevadensis MAG encodes several methane-specific metabolic features that could support a variety of pathways for methane generation and energy conservation. Most conspicuous are the Mtr and Mcr operons, which are only known to function in methane-metabolizing organisms, catalyzing the penultimate and final steps of methanogenesis (Fig. 3). Besides these operons, the Wood-Ljungdahl (WL) pathway is largely the same as in other Archaeoglobi and could be used in either the anabolic direction for acetyl-CoA generation or in the catabolic direction for multi-carbon substrate breakdown. An important difference is absence of the main subunit of Mer (methylenetetrahydromethanopterin reductase), which carries out one of the key C1 oxidation/reduction steps in this pathway (Fig. 3). This absence was reported in a previous analysis of similar MAGs [21], but we add here that two homologs of Mer are encoded in this genome (as well as cultured Archaeoglobus strains). Mer and its homologs are part of the large luciferase-like monooxygenase family (pfam00296) and it is possible one of these paralogs is able to complete the C1 pathway between CO2 and methyl oxidation states. However, an incomplete WL pathway could explain the inability of M. nevadensis to use H2 as a sole methanogenic substrate in our experiments (Fig. 1).
Key genes encoded in the M. nevadensis GBSTs MAG are shown along with pathways for carbon and electron flow that would be consistent with our substrate incubation experiments. All genes present in the genome are shown in bolded black letters, including six of the seven traditional methanogenesis pathways (Fwd, Ftr, Mch, Mtd, Mtr, and Mcr, see Table S3). The ability to incorporate methyl groups from methanol and methoxylated compounds is enabled by the Mta and Mto complexes, respectively. Methyl groups could be used for methanogenesis on their own via methyl disproportionation (dark blue), or methyl reduction with electrons from hydrogen (light blue). An additional possibility is the degradation of more complex carbon compounds for carbon and electrons (light green). Electron flow (pink) to the CoM/CoB heterodisulfide could be supported by three HdrD homologs found in the MAG (inset, top right). The HdrD2-GlcD gene cluster is found together with the Mtr operon and associated methanogenesis genes, making this the most likely candidate. Another conspicuous electron transport gene cluster is highlighted in the top right, consisting of a NiFe hydrogenase, two HdrA homologs, and a membrane-bound cytochrome b HdrC-QmoC fusion expected to interact with the menaquinone pool. The flow of methyl groups through Mtr (depending on the direction) and electron flow through Fqo generates an ion motive force (yellow), ultimately generating ATP via ATP synthase.
Methyl groups on H4MPT or CoM could be reduced to methane in two ways. First, six electrons could be generated by oxidizing a methyl group to CO2 through the WL pathway, which could be used for the reduction of three other methyl groups to methane (Methyl disproportionation, dark blue, Fig. 3). This would rely on the distant homologs of Mer as described above. Second, electrons sourced from hydrogen could be used to reduce methyl groups to methane (Methyl reduction, light blue, Fig. 3). A third possible route of carbon into this pathway could be through the breakdown of multicarbon compounds via pyruvate and acetyl-CoA. These carbons would end up as CO2 (in oxidation reactions producing reduced Fd) and methyl groups on H4MPT (green path, Fig. 3). The methyltransferase systems MtoABCD and MtaCAB found in the MAG may enable growth on methoxylated compounds, such as 2-methoxyphenol, and methanol, respectively (Supplementary Text).
There are a variety of electron transfer complexes encoded in the M. nevadensis MAG that could support electron flow from Fd, F420H2, or H2 onto HdrD. An Fqo complex very similar to the one characterized in A. fulgidus is found in the genome and would be able to transfer F420H2 electrons onto menaquinone [51]. Fd electrons are the lowest potential, and could possibly pass to F420H2 through soluble FqoF or directly onto Fqo sans Fqof, both of which have been proposed as options in cultured methanogens [52]. A fascinating gene cluster containing the [NiFe] hydrogenase subunits MvhABG, two HdrA homologs, and a fusion of QmoC and HdrC is found in the M. nevadensis MAG and is absent in the cultivated non-methanogenic Archaeoglobi. The QmoABC complex is normally found in Archaeoglobi and other sulfate reducers and is thought to carry out electron transfer from menaquinone to AprAB during sulfate reduction [53]. AprAB is absent from M. nevadensis along with the other key sulfate reduction proteins Sat, DsrAB and DsrMKJOP. QmoABC has been proposed to bifurcate electrons [54], and in the absence of its recognized partners in QmoAB or AprAB, it is tempting to speculate this complex is involved in electron flow between H2, ferredoxin and menaquinone.
These electron flow reactions would likely be associated with the generation of a proton motive force, either through vectoral proton pumping at Fqo, or through quinol loops at the b-type cytochromes in the QmoC homologs. Also, depending on the direction of carbon flow through Mtr, additional sodium motive force maybe be generated at this step. Ion motive force ultimately would be used for the generation of ATP through ATP synthase.
XG-degrading culture consumes H2 concomitant to methane production
Because of the apparent discrepancy between the genetic potential to consume H2 for methanogenesis and the very low activity observed in H2/CO2 incubations, we tested if the enriched microbial community produces H2 and if there would be a difference in H2 with the application of BES. If Mcr-based methanogenesis consumed H2, there would be more H2 available in BES-inhibited cultures. Indeed, 12 to 20 days after XG incubation we detected H2 in the culture headspace, indicating H2 production by members of the culture. We furthermore observed more than twice the H2 accumulation when BES was added (Fig. 4). This implies H2 consumption during methanogenesis by the XG-degrading culture and suggests M. nevadensis is co-dependent on H2 and on another, unknown compound (see discussion).
H2 was significantly more abundant in +BES incubations (ANOVA, ****p < 0.0001, n = 9). Central marks in boxes indicate the median, while the bottom and top edges are the 25th and 75th percentiles. Whiskers extend to the most extreme data points not considered outliers.
Microbial origin of GBS methane?
To evaluate the influence of biological methane, possibly from M. nevadensis, on the overall methane dissolved in the hot spring water, we collected dissolved methane from seven hydrothermal pools in the Great Boiling Spring geothermal field (Fig. S2) and determined the C and H isotope composition. These values reveal a primarily volcanic/sedimentary thermogenic origin of the dissolved methane from all pools (Fig. 5A), given that lower values of δ13C and δ2H are associated with biogenic processes [55]. In contrast to microbial methanogenesis, thermogenic methane production is an abiotic process, although thermogenic methane originates from biologically produced material, i.e., high molecular weight carbon [55, 59]. However, the δ13C of methane decreases by up to ~10 ‰ in individual hot springs ranging from 48–94 °C (GBSTb, GBSTa, G04b, GBS19), suggesting the potential contribution of hydrogenotrophic methane production at these sites [60]. The more positive δ2H values in methane from spring G04c perhaps result from mixing with methane from the air due to less upwelling water movement (>1.5 days residence time in G04 springs, [25]). We complemented the isotope data with methane formation rates from in-situ anoxic sediment incubations in a subset of pools. Biogenic methane would be consistent with a significant methane accumulation over time. Reflecting the more negative δ13C results at G04b, methane production here was > 20 nmol methane g–1 sediment day–1 higher than in SSW or GBS (Fig. 5A, inset). Although this supports the idea that biological methane influences total dissolved methane abundances through mixing, a higher sample size would be needed to corroborate this trend. The measured rate in GBS also provides a baseline demonstrating increase of methane production from the native anoxic sediment to the XG-degrading culture by roughly a factor of 33. Methane produced by the XG-degrading culture was indistinguishable from GBS methane in its 13C composition but exhibited a strong depletion in 2H, conforming with its biological origin. Because of this difference, we compared δ2H values in methane from various biogenic pathways revealing a large data spread across studies (Fig. 5B). Regardless of this variability, methane putatively produced by M. nevadensis resembled most closely that originating from methylotrophic and hydrogenotrophic pathways based on δ2H. Overall, our isotopic analysis shows a dominance of abiotic methane in GBS waters, with potential contributions from methyl- and H2-dependent microbial methane production, likely based on both conventional and divergent Mcr, at some sites.
A Known ranges of values [55] may indicate the origin of methane at GBS. Atmospheric methane is indicated by a red star as reference. Rates in the inset were derived by hot spring in-situ incubations. See Fig. S3 for location information and basic geochemical data on individual hot springs. B δ 2H of methane from culture headspace and reference data from hydrogenotrophic, methylotrophic, acetoclastic [56], methylphosphonate (MPn)-derived [57], and nitrogenase (Nif)-derived [58] methane production pathways. Colored vertical lines indicate means in δ 2H of individual pathways. VSMOW, Vienna Standard Mean Ocean Water reference. VPDB, Vienna Peedee Belemnite reference.
Discussion
We present evidence that a previously uncultured member of the Archaeoglobaceae, M. nevadensis, harbors a divergent Mcr and is capable of methane production (Figs. 1, 2, S1). Our calculated cell-specific methanogenesis rates of 78.9–176 fmol cell–1 day–1 reside between 0.5 fmol cell–1 day–1 [61] derived from cold sediments of the Baltic Sea, and 576–43,200 fmol cell–1 day–1 [62,63,64,65] derived from pure cultures of mesophilic (lower boundary) and hyperthermophilic (upper boundary) deep-sea methanogens. Our obtained rate values are closer to those from pure cultures than to natural environments and are competitive with rates mediated by other Euryarchaeota. Based on the cultivation experiments, we speculate about four possible modes of methane formation in the XG-degrading culture (Fig. 6):
Fermenters other than those depicted here are possible (see text). Mechanisms in (C) and (D) are considered more likely than those in (A) and (B), explaining genomic and isotopic data most parsimoniously. CODH, carbon monoxide dehydrogenase.
First, as one explanation for our observations, M. nevadensis could directly depolymerize XG and mineralize the resulting sugars into methane gas (Fig. 6, panel A). Methane accumulation in the presence of bacterial antibiotics and favorability of XG over all other substrates tested would suggest this mode, however, the M. nevadensis genome does not encode any genes typically associated with XG breakdown, including glycoside hydrolases [66], rendering direct XG metabolism unlikely, although we cannot rule out the presence of unknown XG degradation pathways.
Second, as another explanation for our results, the observed methane could be produced as an accidental byproduct of other biochemical processes, also described as “mini-methanogenesis” (Fig. 6, panel B). Several isolated Archaeoglobus strains are known to produce methane in an Mcr-independent process. Sulfate-reducing strains of Archaeoglobus fulgidus are capable of making methane at < 200 µmol L–1 culture [18, 19] and the sulfite-reducing Archaeoglobus veneficus produces methane at 0.6–2 µmol L–1 culture [16]. Archaeoglobus infectus, isolated from an active submarine volcano in the Western Pacific, also reduces sulfite and generates methane at ~5 µmol L–1 culture [17]. The source of the trace amounts of methane in these pure cultures is thought to be a spurious side-reaction of carbon monoxide (CO) dehydrogenase [67, 68]. Methane production in these Archaeoglobus cultures may serve to export excess reducing equivalents rather than for conserving energy. The complete Mcr gene cluster encoded by M. nevadensis (Fig. 3) and the observation that methane production is blocked by BES addition (Fig. 1C) strongly suggests that Mcr-independent mini-methanogenesis is not responsible for methane production in M. nevadensis.
Third, in an alternative scenario, we postulate M. nevadensis to live in a syntrophic relationship with partner bacteria or archaea (Fig. 6, panel C). Ca. Caldatribacterium, Ca. Fervidibacter, Fervidobacterium, Dictyoglomus, or Desulfurococcaceae, each genomically predicted or known to depolymerize polysaccharides, could degrade XG to simple organics (XG monomers) and H2 that is recycled to methane by M. nevadensis. This mode would make XG degradation more energetically favorable when electron acceptors with higher redox potential (e.g., O2) are missing. The 30% lower but sustained methane production in the presence of antibiotics argues against strict reliance on bacterial partners and suggests an additional archaeal-archaeal partnership with yet-uncultivated Desulfurococcaceae [69]. The fact that the XG monomers fucose, xylose, and galactose stimulated methane production (Fig. 1C), albeit not to the level of XG, is also supportive of that proposed mode. In addition to using sugars and organic acids to fuel methanogenesis, our experimental (methane production with MBA addition) and genomic data (a complete MtoABCD complex) suggest M. nevadensis is capable of methoxydotrophic methanogenesis [46], a methanogenic pathway not previously demonstrated in hot spring thermophiles. Cultured members of the Archaeoglobaceae metabolize a much broader range of carbon substrates than cultured methanogens. There is no methanogen known that can grow by fermenting pyruvate or glucose, for example. Our genome analysis (Fig. 3) highlights multiple potential pathways for energy conservation from methanogenesis in M. nevadensis by using a variety of methylated compounds on their own or with electrons sourced from H2 or the fermentation of more complex carbon compounds. Comparing the amount of methane produced (Fig. 1A) to the amount of H2 consumed (Fig. 4) by the enrichment culture, we note that the molar H2/methane ratio we measure (~1.2) deviates from the theoretical stoichiometric ratio (4:1) expected if methanogenesis is strictly hydrogenotrophic. The measured ratio corresponds more to the conversion of methanol to methane (1:1). Considering that H2 might be a “universal energy source” [70], this could be an additional argument in favor of an H2-consuming methylotrophic reaction. In support of this model, cultures amended with H2, methanol and antibiotics were able to grow robustly and became dominated by M. nevadensis (Fig S1). Methanol is an end product during the demethylation of methoxy groups from aromatic rings [71]. Alternatively, demethylation could be carried out by other organisms, e.g., ones that cleave and breakdown aromatic rings, and M. nevadensis may simply exploit intermediates, such as methanol. It is possible that methanogenic Archaeoglobi occupy a more metabolically versatile niche as compared to traditional methanogens, where they can utilize a wide range of carbon substrates generated from complex carbon breakdown supplemented with H2 consumption.
Fourth, as a last possible explanation for our findings, M. nevadensis could metabolize fermentation products derived from XG degradation but is also dependent on other compounds provided by the microbial community, such as vitamins and cofactors (Fig. 6, panel D). This mode would explain our observation that culture-derived H2 was consumed by methanogenesis, whereas exogenous H2 in the absence of XG or other organic carbon was not (Fig. 4 versus Fig. 1C). As a refinement of the third mode, the methanogen could still live in syntrophy with another archaeal species. CoM may be such a cofactor obligately required for methane metabolism [17, 72], together with vitamins such as folate [73] or amino acids. Indeed, neither of the known pathways for CoM biosynthesis in methanogens are encoded in the M. nevadensis genome. However, in our experiments, CoM addition to XG-grown cultures did not stimulate methanogenesis, suggesting additional dependence on an essential compound and/or an already saturated supply of CoM by other members of the community.
Concerning the question about the origin of methane at GBS, our data suggest that it is primarily thermogenic (abiotic), in line with methane fluxes correlating with temperature in GBS pools [74]. The range in δ13C of our measurements (Fig. 5A) is congruent with data collected in pyrolysis experiments at 400 and 500 °C involving montmorillonite clays [75]. As a reaction mechanism, carbon bond cleavage via an acid-activated carbonium ion at the clay mineral surface has been proposed [76]. Such a mineral-catalyzed mechanism would be feasible within the sedimentary deposits at GBS that are composed of clays from the Granite Range and Eastern Sierra Nevada [77]. The location of the culture methane in δ13C- δ2H space extends the coverage of biogenic methane, and we propose that methane retrieved from thermal systems exhibiting similar δ13C- δ2H values to be considered biogenic in origin. Comparing our δ2H data to reference data derived from methane produced by well-characterized thermophilic methanogens (Fig. 5B) supports the use of methyl groups and H2 by M. nevadensis as inferred from our genomic analysis.
Conclusion
We conclude that M. nevadensis is a methanogen with a H2-consuming, methylotrophic lifestyle in syntrophic partnership with carbohydrate-degrading bacteria and archaea. Because of the close relationship between the M. nevadensis Mcr complex and others from diverse uncultivated archaea (Fig. 2B), we propose that the similarly divergent Mcr complexes in Korarchaeota, Nezhaarchaeota, Verstraetearchaeota, and other Archaeoglobi are also functional and likely generate methane [10, 78, 79]. Our findings may also expand methane metabolism to a wider range of hot spring chemistries [80]. Taken together, our study represents a first step to experimentally verify the genomic potential contemplated for a variety of Mcr-encoding archaea. Future studies will further unravel the significance of such lineages in the evolution of methane metabolism and as a source of biological methane on Earth.
Data availability
Metagenome and 16S rRNA gene sequence data were deposited in the NCBI database as Bioproject PRJNA956542.
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Acknowledgements
We thank Marike Palmer for advice on systematics, Alma Parada for discussions about the computational approach, Alexander Jaffe for feedback on the manuscript, David and Sandy Jamieson for access to Great Boiling Spring, Alexander Manriquez for help with sampling at GBS, and Michael Morikone for initial metagenome analysis of the xyloglucan culture. We appreciate the CSUSB Genomics course of Spring 2018 for assistance with xyloglucan culture DNA extraction and long read sequencing.
Funding
Funding was provided by the National Science Foundation (NSF) grant DEB 1557042 and National Aeronautics and Space Administration (NASA) grant 80NNSC17KO548 and 80NSSC19M0150. SB was further supported by the NASA Postdoctoral Program (section Astrobiology), administered by Oak Ridge Associated Universities under contract with NASA. MEQ received critical funding from Stanford’s IntroSems Plus program for undergraduate students. GLC was supported by the Miller Institute for Basic Research in Science at U.C. Berkeley. Additional funding for metagenomes was provided by the U.S. Department of Energy’s Joint Genome Institute (DOE-JGI) under project 10.46936/10.25585/60007294 and DOE grant DE-EE0000716; the Nevada Renewable Energy Consortium, funded by the DOE.
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SB and AED developed the experimental design with input from BPH and JAD. SB conducted the experiments with assistance from MEQ. JAD achieved enrichment of M. nevadensis and has maintained the XG-degrading culture long-term. Metagenomic work was done by SB and GLC. GLC performed the metabolic inference and further enrichment of M. nevadensis. SB and JAD conducted field work at GBS. SB wrote the paper with contributions from GLC, BPH, and AED, and all authors edited the final version.
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Buessecker, S., Chadwick, G.L., Quan, M.E. et al. Mcr-dependent methanogenesis in Archaeoglobaceae enriched from a terrestrial hot spring. ISME J 17, 1649–1659 (2023). https://doi.org/10.1038/s41396-023-01472-3
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DOI: https://doi.org/10.1038/s41396-023-01472-3
