Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin

Abstract Hadarchaeota inhabit subsurface and hydrothermally heated environments, but previous to this study, they had not been cultured. Based on metagenome-assembled genomes, most Hadarchaeota are heterotrophs that grow on sugars and amino acids, or oxidize carbon monoxide or reduce nitrite to ammonium. A few other metagenome-assembled genomes encode alkyl-coenzyme M reductases (Acrs), β-oxidation, and Wood-Ljungdahl pathways, pointing toward multicarbon alkane metabolism. To identify the organisms involved in thermophilic oil degradation, we established anaerobic sulfate-reducing hexadecane-degrading cultures from hydrothermally heated sediments of the Guaymas Basin. Cultures at 70°C were enriched in one Hadarchaeon that we propose as Candidatus Cerberiarchaeum oleivorans. Genomic and chemical analyses indicate that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-coenzyme M. A β-oxidation pathway and a tetrahydromethanopterin methyl branch Wood–Ljungdahl (mWL) pathway allow the complete oxidation of hexadecane to CO2. Our results suggest a syntrophic lifestyle with sulfate reducers, as Ca. C. oleivorans lacks a sulfate respiration pathway. Comparative genomics show that Acr, mWL, and β-oxidation are restricted to one family of Hadarchaeota, which we propose as Ca. Cerberiarchaeaceae. Phylogenetic analyses further indicate that the mWL pathway is basal to all Hadarchaeota. By contrast, the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex in Ca. Cerberiarchaeaceae was horizontally acquired from Bathyarchaeia. The Acr and β-oxidation genes of Ca. Cerberiarchaeaceae are highly similar to those of other alkane-oxidizing archaea such as Ca. Methanoliparia and Ca. Helarchaeales. Our results support the use of Acrs in the degradation of petroleum alkanes and suggest a role of Hadarchaeota in oil-rich environments.

have been important in the evolution of Mcr/Acr genes and alkane metabolisms in archaea [24].Yet, neither these Acrcontaining organisms, nor close relatives have been cultured.Among them, Hadarchaeota might have a globally relevant role, because of their wide distribution in subsurface environments [25].Hadarchaeota were originally described as the South African Goldmine Miscellaneous Euryarchaeal Group, which were found in alkaline sulfate-rich mining fissure waters [26].Hadarchaeota inhabit diverse anoxic subsurface habitats such as hot springs, hydrothermal sediments, deep marine sediments, aquifers, and cold seeps [23,25,[27][28][29][30].The high GC content (57%-61%) in the sequenced rRNAs suggested that Hadarchaeota are thermophiles [26].This lineage was recently proposed to be a phylum (Hadarchaeota) [31], including at least 30 MAGs from public repositories [23,25,32].Of these, some hot-spring Hadarchaeota MAGs encode an Acr related to those of Ca.Methanoliparia, but their alkane substrates have not been characterized [23].
In this study, we targeted the cultivation of long-chain alkane degrading microorganisms from hydrothermal sediments of the Guaymas Basin, located in the Gulf of California at 2000 m depth [33].Here, hydrocarbon-rich f luids diffuse from deeper layers toward the sediment surface, where they fuel the metabolism of diverse microbial communities [34][35][36].Of these, we enriched anoxic microbes with hexadecane as substrate and analyzed the microbial community and metabolism by "omics and metabolite analysis."We tested the hypothesis that members of the Hadarchaeota are involved in alkane degradation in thermophilic anoxic environments and suggest that Hadarchaeota acquired the necessary pathways for alkane metabolism via HGT from other archaea.

Sediment sampling and culture setup
For this study, we retrieved sediments from Guaymas Basin during R/V Atlantis cruises AT37-06 (December 2016) and AT42-05 (November 2018) with the submersible Alvin.On cruise AT37-06, the push cores were taken during Alvin dive 4869 (27 • 0.45 N 111 • 24.54 W, water depth 2001 m) from a site densely covered by an orange mat of large sulfur-oxidizing Beggiatoaceae bacteria.Below the mat, temperatures rapidly increased and reached 85 • C at 50 cm depth [37].On cruise AT42-05, a push core was taken during Alvin dive 4991 (27 • 0.69 N, 111 • 24.27 W, water depth 2013 m) from a site covered with orange-white Beggiatoaceae mats.Temperatures at 50 cm depth reached at least 80 • C. Both samples were immediately transferred to glass bottles sealed with butyl rubber stoppers, and the headspace was exchanged to argon.Bottled sediments were stored at room temperature until further processing.Anoxic sediment slurries were prepared as described before [38].Homogenized sediment from 2 to 10 cm was mixed with synthetic sulfate-reducer medium [38,39].This slurry was distributed into replicate cultivation vials and further diluted reaching a final density of ∼1 g sediment per 100 ml.The slurries were amended with1 ml n-hexadecane (99% purity, Sigma-Aldrich) as carbon and energy source.The vials were sealed with butyl rubber stoppers and pressured with 2 atm N 2 :CO 2 (90:10).The bottles were incubated upside down to avoid chemical reactions of the alkane substrate with the rubber stopper.Samples were incubated at 37, 50, and 70 • C with mild agitation (rotation 40 rpm).As marker for anaerobic alkane degradation, we followed the formation of sulfide using a colorimetric copper sulfate assay [40].Cultures with hexadecane at 70 • C (hexadecane70) were subsequently diluted (1,4 dilution steps) when sulfide concentrations reached between 5 and 10 mM.

Synthesis of authentic hexadecyl-CoM standards
One equivalent (0.3 g) of sodium 2-mercaptoethanesulfonate (≥98% coenzyme M sodium salt, Sigma-Aldrich) was mixed with two equivalents (0.62 ml) of 1-bromohexadecane (97%, Sigma-Aldrich) in 2.4 ml basic ammonia solution (30% NH 4 OH pH ∼12).The mix was incubated overnight at RT with gentle shaking in a vortex (500 rpm).The aqueous phase was transferred to a new vial and pH was adjusted to 7.0 with 37% HCl.

Metabolite sample extractions
Thirty milliliters of culture were centrifuged at 4000 rpm for 30 min at RT, keeping both the pellet and the supernatant.The supernatant was removed and filtered onto polycarbonate filters (0.22 μm pore size, Merck Millipore) under gentle vacuum (>30 kPa).Filter pieces and pellet were transferred to bead beating tubes (Lysing Matrix E, MP Biomedicals) with 1 ml of acetonitrile:methanol:water (4,4,2, v,v,v).The tubes were vortexed for 10 min at maximum speed.Beads and debris were pelleted by centrifugation at 10000 rpm for 20 min at RT.The clear supernatant was transferred to glass vials and stored at 4 • C.

Liquid chromatography-mass spectrometry of metabolite extracts and standards
Chemical analysis of metabolite extracts and alkyl-CoM was done as described previously [5].Culture extracts and hexadecyl-CoM standards were analyzed via high-resolution accurate-mass mass spectrometry on a Bruker maXis plus quadrupole time-off light mass spectrometer (Bruker Daltonics, Bremen, Germany) connected to a Thermo Dionex Ultimate 3000RS UHPLC system (Thermo Fisher Scientific, Bremen, Germany) via an electrospray ionization ion source.Sample aliquots (equivalent to 20% of total extract) were evaporated under a nitrogen stream and redissolved in 10 μl of methanol:water (1:1, v:v) before injection.Separation was done on an Acclaim C30 reversed phase column (Thermo Fisher Scientific, 3.0 × 250 mm, 3 μm particle size).The column oven was set to 40 • C, and the binary pump was programmed with a f low rate of 0.35 ml/min and the following gradient of eluent A (acetonitrile:water:formic acid 5:95:0.1,v:v:v) and eluent B (2-propanol,acetonitrile,formic acid 90:10:0.1,v:v:v): 0% B at 0 min, ramp up to 100% B at 30 min, hold at 100% B until 50 min, and reequilibration at 0% B from 51 min until the end of the run at 60 min.Parameters for the electrospray ion source were set as described previously [5].The mass spectrometer was set to acquire alternating scans of full scan and broad-band collision-induced dissociation spectra (25 eV collision energy) in a mass range of m/z 50-600 in negative ionization mode.Every analysis was mass-calibrated to reach mass accuracy of 1-3 ppm by loop injection of a calibration solution containing sodium formate cluster ions at the end of the analysis during the equilibration phase and using the high-precision calibration algorithm.Extracted ion chromatograms were generated using a mass window of 0.01 Da.Data processing was performed using the Compass Data Analysis software package version 5.0 (Bruker Daltonics, Bremen, Germany).

Phylogenomic and phylogenetic analyses
For archaeal phylogenomics, 289 archaeal genomes were used to build a tree based on 76 archaeal marker proteins (Supplementary Table S2) [58].To build phylogenetic trees for methyl/alkyl-coenzyme M reductase subunit A (McrA/AcrA), formylmethanofuran dehydrogenase (FwdABC), and carbon monoxide dehydrogenase (CdhABCDE), we annotated the gene sequences using PFAMs for McrA/AcrA and Fwd, and custom hidden Markov models for Cdh [59].The amino acid sequences for each gene set were aligned and concatenated with the anvi'o software [45,46], using muscle as alignment tool [60].The alignments are available as Supplementary Material (keeper.mpdl.mpg.de/library/a5b76ef5-0a9f-475e-9ed1-602c9c70ba03/Benito_Merino_ Hadarchaea/Extended_data_alignments).Maximum likelihood trees for all protein sets with 100 bootstraps were calculated using IQTree v. 2.0.3, using the -test option to estimate the best substitution model for each protein [61,62].The phylogenetic trees were visualized and edited on the Interactive Tree of Life web server [63].
Anoxic sediment slurries in sulfate-reducer medium were supplemented with hexadecane as sole energy source and incubated at 37, 50, and 70 • C. The cultures at 37 and 50 • C showed relatively slow increase of sulfide, reaching ∼5 mM after 150 days (Fig. 1B).They contained mostly sulfate-reducing Desulfobacterota (Fig. 1A) (formerly * Deltaproteobacteria).These have been described before as alkane degraders in marine hydrocarbon seeps [64,65].Also, these enrichments contained large proportions of Ca.Thermoprofundales (phylum Thermoplasmatota) and Woesearchaeales (phylum Nanoarchaeota), as well as some methyl-reducing hydrogenotrophic methanogens of the order Methanofastidiosales, all corresponding to the groups that were present in the original sediments (Fig. 1A).
The incubation at 70 • C produced 10 mM sulfide in 80 days and continued being active after a 1/5 dilution (Fig. 1B).The archaeal amplicons consisted mostly of Hadarchaeles, Archaeoglobales, and Thermoplasmatota (JdFR-43) (Fig. 1A).All three groups were rare or absent in the control incubations at 70 • C without hexadecane, or in incubations with hexadecane at lower temperatures.Archaeoglobales is an order that contains freeliving sulfate reducers.A. fulgidus was suggested to grow on hexadecane as carbon and electron source and to activate this substrate via alkylsuccinate synthases of bacterial origin [66].The JdFR-43 family has been found in hydrothermal vents and is thought to utilize proteins and peptides for growth [67].Also, at 70 • C, most of the bacterial 16S rRNA gene amplicons were different from the lower temperature enrichments and comprised Thermodesulfobacteriales (Fig. 1).Thermodesulfobacteria are often autotrophs or grow on small organic molecules and are not known to degrade hydrocarbons [68][69][70][71].However, they have been described recently as partners for thermophilic ANME-1c in the anaerobic oxidation of methane (AOM) and of Ca.Alkanophaga in the anaerobic oxidation of midchain alkanes, respectively [5,16].
After two 1/5 dilutions, the 70 • C cultures from the AT37-06 cruise were almost sediment-free.We sequenced three metagenomes at different cultivation stages to resolve its hexadecane-degrading community.Based on 16S rRNA genes recruited from the metagenomes, the cultures were dominated by Archaea (>85% relative abundance, Fig. 2).The early phase of the enrichments was characterized by dominance of Archaeoglobi (30% relative abundance), followed by Bathyarchaeia and Hadarchaeota (15%-25% relative abundance; Fig. 2).Acetothermia and Thermodesulfobacteriales sequences composed most of the bacterial fraction of the metagenome.In later culture dilutions, a shift occurred between Archaeoglobi and Hadarchaeota, with the latter becoming the most abundant group (40%-50% relative abundance), suggesting its involvement in hexadecane degradation.This enrichment consist of only two Hadarchaeota species as indicated by the 16S rRNA genes found in the metagenomes (Supplementary Fig. S2).We obtained similar results in an enrichment culture from a later cruise (AT42-05, Fig. 2).From the metagenomic coassembly, we reconstructed 39 medium-to good-quality MAGs (completeness >50%, redundancy <10%, Supplementary Table S3).A Hadarchaeota MAG recruited ∼18% of the metagenomic reads in the latest stage of the culture (91% completeness, 0% redundancy Table 1).This MAG encodes the only Acr operon present in the metagenome (Supplementary Table S4), leading to the hypothesis that these archaea may degrade hexadecane.Wang et al. described an Acr-encoding Hadarchaeota clade based on MAGs reconstructed from environmental metagenomes [23].Here, we describe a Hadarchaeota MAG in our culture affiliated with this Acr-encoding Hadarchaeota clade.The ANI between our Hadarchaeota MAG and the rest of the clade is below 75% (Supplementary Fig. S3).The ANI value of our MAG is also below 75% with the placeholder genome Ca.Hadarchaeum yellowstonense [25,72].The hexadecane70 Hadarchaeota MAG represents a novel genus.Based on its affiliation to Hadarchaeota and its metabolism (see results below), we propose the species name Ca. Cerberiarchaeum oleivorans (see Supplementary Text).

Function of Acr in Ca. Cerberiarchaeum oleivorans and Acr phylogeny
The genome of Ca. C. oleivorans harbors a single complete Acr operon.Based on the phylogenetic comparison of the catalytic alpha subunit, the Acr of Ca. C. oleivorans is closely related to those in MAGs of Bathyarchaeia, Ca.Helarchaeales, and Ca.Methanoliparia (Fig. 3A) [17-20, 22, 23].To investigate the capacity of Ca. C. oleivorans to activate hexadecane with Acr, we analyzed culture extracts from the hexadecane70 culture via liquid chromatography coupled to high-resolution mass spectrometry (Supplementary Fig. S1).A peak with the exact mass of hexadecyl-CoM eluted at the same retention time as an authentic hexadecyl-CoM standard (Fig. 3B).A second peak eluted shortly before the hexadecyl-CoM standard.We hypothesize that this second compound is the product of hexadecane activation in the second carbon (2-methyl-pentadecyl-CoM, C 2 -substituted hexadecyl), as previously described for the activation of butane (C 4 alkane) and dodecane (C 12 alkane) [4,5].In Ca. C. oleivorans, hexadecyl-CoM is the product of the activation of n-hexadecane by the Acr, as described for the anaerobic short-and midchain ANKA Ca.Ethanoperedens, Ca.Syntrophoarchaeum, and Ca.Alkanophaga [4,6,7].

Cerberiarchaeum oleivorans
Following Acr-dependent hexadecane activation, a conversion of hexadecyl-CoM to a hexadecanoyl-CoA (CoA-bound fatty acid) is necessary for the complete oxidation of the alkane (Fig. 3C).This mechanism is so far unknown, but for the related short-chain alkane oxidizers, candidate enzymes have been proposed based on metagenomic and metatranscriptomic data [4,6,7].Ca.Syntrophoarchaeum butanivorans encodes a methylcobamide:CoM methyltransferase/corrinoid methyltransferase (MtaAC) that could be involved in the conversion of butyl-CoM into butyryl-CoA [4].However, Ca. C. oleivorans MAG does not have the mtaAC genes.For Ca. Ethanoperedens thermophilum, a tungsten-containing aldehyde:ferredoxin oxidoreductase (Aor) has been proposed to catalyze this conversion, based on the high expression of the gene in metatranscriptomes [6].Ca. C. oleivorans encodes three copies of tungsten-containing Aor (Supplementary Table S5).C. oleivorans activates hexadecane to hexadecyl-CoM.LC-MS analysis of hexadecane70 culture extracts shows two dominant chromatographic peaks in extracted ion chromatograms of the exact mass of hexadecyl-CoM; these peaks likely represent coenzyme M-substituted alkyls resulting from activation of the alkane in the secondary and primary position, in order of elution time [5]; (C) metabolic model for Ca. C. oleivorans; the Acr activates hexadecane to hexadecyl-CoM, which is then converted into a 16-carbon acyl-CoA (hexadecanoyl-CoA), possibly via Aor; acyl-CoA is processed to acetyl-CoA units (β-oxidation pathway); acetyl-CoA is incorporated into the downstream part of the H 4 MPT mWL via the Cdh/Acs complex; the methyl group is completely oxidized to CO 2 ; the fate of the electrons released from this metabolism is unknown; F 420 H 2 oxidation could be coupled to the production of H 2 via Frh or to the reduction of CO 2 to formate by an Fdh.
The function of these Aors in hexadecane degradation needs confirmation via metatranscriptomics.Due to low amounts of available culture, metatranscriptome analysis was impossible.
For Ca. Ethanoperedens and Ca.Syntrophoarchaeum, the formation of acyl-CoA from the corresponding fatty acid (acetate and butyrate) is not possible, because they do not encode acyl-CoA synthetases.By contrast, Ca. C. oleivorans encodes acyl-CoA synthetases (fadD) and alcohol dehydrogenases.Therefore, Ca. C. oleivorans could use long-chain fatty acids or alcohols as carbon and energy source apart from long-chain alkanes.This ability was also suggested for Ca.Polytropus marinifundus [21] and Ca.Methanoliparia [17].Furthermore, Ca. C. oleivorans encodes a complete β-oxidation pathway, with genes present in multiple copies for several of the steps of the pathway (Supplementary Table S6).The β-oxidation pathway allows the production of eight acetyl-CoA units from hexadecanoyl-CoA.Most Hadarchaea (including Ca. C. oleivorans) encode a gluconeogenesis pathway and the C 3 -module of the glycolysis pathway for synthesis of sugars and central building blocks, respectively (Supplementary Table S7).However, none of the Hadarchaea encode a complete citric acid cycle or reductive citric acid cycle (Supplementary Table S7).In Ca. C. oleivorans, acetyl-CoA can be completely oxidized to CO 2 via the CdhABCDE/acetyl-CoA synthase (Cdh/Acs) complex and the methanogenesis enzymes upstream of methyl transferase (Mtr), i.e. the tetrahydromethanopterin (H 4 MPT) methyl branch of the WL (mWL) pathway.In incubations of the enrichment cultures with 13 Clabeled hexadecane, we measured significant production of 13 CO 2 over time (Supplementary Fig. S4).In total, 16 CO 2 molecules are formed per each molecule of hexadecane, according to the following equation: The liberated electrons would reduce molecules such as coenzyme F 420 , ferredoxin, NAD + , and f lavoproteins, which need to be reoxidized in respiratory pathways, or transfer their electrons to a syntrophic partner.In our culture, alkane degradation is likely coupled to sulfate reduction.Similar to other Hadarchaea containing Acrs, Ca. C. oleivorans does not encode a sulfate reduction pathway.Other syntrophic ANKA produce large amounts of MHC that are likely mediating interspecies electron transfer (DIET) [4,6,13,14].Ca. C. oleivorans does not encode genes for MHC.Instead, it might channel the reducing equivalents in the form of molecular hydrogen produced by an F 420 -reducing NiFe-hydrogenase, or transfer small, reduced compounds like acetate and formate.Although the fermentation of hexadecane into hydrogen or acetate is unfavorable at deep-sea conditions ( G = 753.3kJ or 133.9 kJ, respectively), the reactions could become feasible if syntrophic partners keep the concentrations of these compounds at low levels [73].

Possible sulfate-reducing partners and additional associated microorganisms
We observed a good mass balance for the coupling of CO 2 formation to sulfate reduction in the Ca. C. oleivorans culture (Supplementary Fig. S4A and Supplementary Text).We screened the other MAGs retrieved from the hexadecane70 culture with a focus on potential sulfate-reducing partner for Ca. C. oleivorans (Table 1 and Supplementary Table S3).Thermophilic ANME and their relatives Ca.Alkanophaga couple with Thermodesulfobacteria (phylum Desulfobacterota) to perform AOM and anaerobic oxidation of petroleum alkanes, respectively [5,16].Thermodesulfobacteria were present throughout the different stages of the hex-adecane70 culture (Fig. 2).We retrieved a Thermodesulfobacteriales MAG in hexadecane70 metagenomes, present in low abundances (1%-2% metagenomic reads map to the MAG, Supplementary Table S3).This MAG corresponds to the species Ca.Thermodesulfobacterium torris, described as a partner for AOM at 70 • C [16].Ca.T. torris encodes several putative MHC that may be involved in DIET with Ca. C. oleivorans, alternatively to our first Table 1.MAGs retrieved from hexadecane70 culture metagenomes (described in the main text); a complete list of MAGs can be found in Supplementary Table S3.hypothesis of H 2 transfer.Despite their low abundances in the total community metagenome, Thermodesulfobacteria are a likely partner SRB for hexadecane degradation at 70 • C. In the cultures, we visualized microbial aggregates containing Hadarchaeaota cells (Supplementary Fig. S5).This suggests that Ca. C. oleivorans relies on DIET or transfer of small molecules to a syntrophic partner for the degradation of hexadecane.In early enrichments, Archaeoglobi were highly abundant (Figs 1 and 2) and several MAGs recruit between 1% and 10% of metagenomic reads (Table 1 and Supplementary Table S3).The Archaeoglobi MAGs S5B4_HD70 and S5B11_HD70 encode a complete dissimilatory sulfate reduction pathway (Table S5) and are related to A. fulgidus (Supplementary Figs S6 and S7).The cultured species of the genus Archaeoglobus are heterotrophic or chemolithotrophic sulfate reducers [74][75][76][77][78][79][80][81][82][83].Other Archaeoglobi genera (namely Ferroglobus, Geoglobus and Ca.Polytropus) are nitrate and ferric iron reducers [21,[84][85][86].We considered whether Archaeoglobi from the hexadecane70 culture could receive reducing equivalents from Ca. C. oleivorans.However, the Archaeoglobi do not encode hydrogenases, making interspecies hydrogen exchange with Ca. C. oleivorans unlikely.They also do not code for putative MHC.We thus suggest that Archaeoglobi in our culture could be competitive hexadecane oxidizers using alkylsuccinate synthases (Ass), a bacterial mechanism for activation of alkanes [87]. A. fulgidus has been isolated from hydrothermal vents and oil reservoirs [88] and encodes a pyruvate formate lyase (Pf l) with high similarity to alkylsuccinate synthase A (AssA) and benzylsuccinate synthase A (BssA) [66].All Archaeoglobi MAGs retrieved encode proteins with high similarity to Pf l/Ass that were highly expressed in A. fulgidus during growth on hexadecane ( [66], Supplementary Fig. S8).Interestingly, one Archaeoglobus MAG (Archaeoglobi archaeon S5B9_HD70, 83% completeness, 4% contamination, Supplementary Table S3) encodes both a Pf lC/AssD and Pf lD/AssA with high sequence similarity to those of A. fulgidus and D. alkenivorans.This is the only MAG in the hexadecane70 cultures with the capacity to couple alkane oxidation to sulfate reduction within one cell (Supplementary Table S4).However, the abundance of the Archaeoglobi archaeon S5B9_HD70 MAG is below 0.1% in the later stages of cultivation (Supplementary Table S3).In summary, the Archaeoglobi of the hexadecane70 culture are unlikely to play a role as partners of Ca. C. oleivorans.Instead, they might compete for the oxidation of the hexadecane coupled to sulfate reduction, especially at the beginning of the cultivation.
The culture also contains a bacterial MAG affiliated to the phylum Bipolaricaulota (formerly * Acetothermia) that recruited 2%-6% of the metagenomic reads (Table 1).Bipolaricaulota have been found in anoxic environments such as oil reservoirs and anaerobic digesters [89,90].These bacteria are described as generalists that ferment sugars, amino acids, and peptides to acetate, formate, and hydrogen [89,91], but do not encode MHC.In the culture, we also found a Bathyarchaeia MAG (phylum Thermoproteota) that recruited 1%-3% of the metagenomic reads (Table 1).Bathyarchaeia are found in diverse environments such as deep-sea and freshwater sediments [92][93][94][95].Evans et al. described an environmental Bathyarchaeia MAG that encoded an Acr [20].To our knowledge, no other Acr-encoding Bathyarchaeia have been found since then.The Bathyarchaeia MAG present in our culture is unlikely to be involved directly in hexadecane oxidation.Furthermore, a MAG belonging to Ca. Thermoprofundales (completeness <70%) recruits 1%-2% of metagenomic reads (Supplementary Table S3).Ca.Thermoprofundales are peptidolytic organisms that have been reported from hydrothermal environments and oil reservoirs and also co-occurring with Hadarchaea [67].The Bipolaricaulota, Bathyarchaeia, and Ca.Thermoprofundales MAGs described here appear to be heterotrophic generalists that might thrive on side metabolites of the alkane-oxidizing community such as peptides, acetate, or formate [96,97].None of these MAGs encode for sulfate reduction genes, nor have they been previously reported as partners for AOM or anaerobic oxidation of alkanes.Overall, this community gives us insights on the complex metabolic networks that potentially operate in oil-rich environments.

Origin of alkane metabolism in Hadarchaea
To investigate the evolutionary history of Acr-based alkane metabolism in the Hadarchaeota, we first determined the distribution of Acr, β-oxidation, and WL pathway genes in this class (Fig. 4).As previously mentioned, these pathways are necessary for short-and midchain alkane-oxidation in Ca.Syntrophoarchaeum and Ca.Alkanophaga [4,5], and longchain alkane oxidation coupled to methanogenesis in Ca.Methanoliparia [17][18][19].All Hadarchaea MAGs coding for an Acr are gathered in a single family (Fig. 4) corresponding to WYZ-LMO6 in GTDB [31], for which we propose the name Ca. Cerberiarchaeaceae.All MAGs from this family have a complete or almost complete pathway for alkane oxidation, including genes for β-oxidation, both branches of the WL pathway and HdrABC genes for the regeneration of CoM-CoB.Only two other Hadarchaeota outside of Ca.Cerbariarchaeaceae encode an almost complete WL pathway.To determine the origin of alkane oxidation in the Hadarchaea, we built a reference phylogeny of archaea and compared it to the phylogeny of enzymes of the two branches of the WL pathway (Fig. 5).In the archaeal phylogeny, Persephonarchaea and Hadarchaea form a monophyletic clade, branching next to Theionarchaea (Fig. 5).The Persephonarchaea (formerly candidate division MSBL1) [98] are an uncultured group described from hypersaline anoxic basins [99].All the Persephonarchaea MAGs have completion values of <50%.Based on our GTDB taxonomy analysis, Persephonarchaea is a sister group to the Hadarchaea, comprised within the phylum Hadarchaeaota and the class Hadarchaeia (Supplementary Table S2).Therefore, we use the term "Hadarchaea" to refer to the order Hadarchaeales excluding the Persephonarchaea.The Theionarchaea were described from estuary sediments and are affiliated with the Thermococci (Supplementary Table S2) [98,100].The phylogenies of the enzymes of the mWL pathway, i.e.FwdABC (Fig. 5B), formylmethanofuran-H 4 MPT N-formyltransferase (Ftr; Supplementary Fig. S9), and methenyl-H 4 MPT cyclohydrolase (Mch; Supplementary Fig. S9), are mostly congruent with the reference phylogeny of Archaea (Fig. 5A), supporting the results of previous phylogenies [101].In particular, the Hadarchaea, Persephonarchaea, and Theinoarchaea clades are closely related and form a separate clade from the Bathyarchaeia and Asgardarchaeota in Fwd and Mch phylogenies, similarly to the reference tree (Fig. 5 and Supplementary Fig. S9).Altogether, this indicates that the mWL pathway was likely vertically inherited in the Hadarchaeota.By contrast, for the carbonyl branch of the WL pathway (i.e. ml CdhABCDE), Ca.Cerbariarchaeaceae sequences are distantly related to those of Persephonarchaea and Theinoarchaea and branch within the Bathyarchaeia (Fig. 5C).Very similar results were obtained for phylogenies based on individual Fwd and Cdh subunits (Figs S10 and S11).Therefore, Hadarchaea most likely acquired the carbonyl branch of WL by HGT from Bathyarchaeia.The Acr from Hadarchaeota are closely related to sequences from two other groups, Bathyarchaeia and Halobacterota (Ca.Methanoliparia), suggesting HGT between these lineages, but it is not possible to conclude on the direction of the transfer.Based on BLASTp comparisons, the β-oxidation genes of Ca. C. oleivorans are also related to those of putative long-chain alkane oxidizers from phylogenetically distant lineages, such as Bathyarchaeia BA1/BA2 and Ca.Methanoliparia (Supplementary Table S6) [17,20].Similarly to Acr, this supports the existence of HGT between these lineages.

Discussion
Before this study, Hadarchaeota was a phylum described exclusively from environmental MAGs, and no physiological studies were available to link their genomic potential with their ecological niches.Based on environmental MAGs and 16S rRNA sequences, Hadarchaea are present in a broad range of subsurface anoxic environments, and they are associated with methane seeps and oil-rich environments (Supplementary Fig. S12).The presence of genes for CO and H 2 metabolism supports their competitiveness in such environments [25,102].Furthermore, Hadarchaea are likely thermophiles, suggested, for example, by the extremely high frequency of G-quadruplex-prone regions in their DNA [103].
Predictions on optimal growth temperatures based on Hadarchaea MAGs showed that they are adapted to temperatures between 50 and 82 • C (Supplementary Table S7).Most Hadarchaea lack the WL pathway (Fig. 4).Although Ca.Cerberiarchaeaceae might use the WL pathway for carbon assimilation, other Hadarchaea might assimilate carbon via the reductive pentose phosphate cycle (Supplementary Table S7).Subsurface Hadarchaea might couple the oxidation of carbon monoxide to the reduction of H 2 O to hydrogen or to dissimilatory nitrite reduction to ammonia, as proposed for Hadarchaea MAGs from Yellowstone [25].The predominant metabolism in deep anoxic and oil-rich environments might be fermentation of organic compounds, rather than respiration [73].The metabolism of non-ANKA Hadarchaea remains poorly understood without cultured representatives and further environmental mapping.The results of our enrichment study, together with other environmental data [23], now explain their presence in oil seeps and reservoirs.The genomic data along with the analysis of metabolites suggest that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-CoM and can potentially oxidize the alkane completely to CO 2 , supporting the proposed hypothesis for Acr-based alkane oxidation in Hadarchaea [23].
This study of an enrichment culture of Ca. C. oleivorans suggests that it does not encode respiratory pathways or other electron sinking mechanisms.Syntrophic interactions based on transfer of molecular hydrogen or formate have been proposed [104].The most likely partner SRB in our culture is a Thermodesulfobacterium, as previously proposed for AOM and midchain alkane oxidation at 70 • C [5,16].Further cultivation efforts are needed to decipher this potential interaction between Hadarchaea and Thermodesulfobacteria aided by metatranscriptomics, physiological experiments, and microscopy.
In the Mcr/Acr phylogeny, we can distinguish four groups [8,105].Group I contains Mcrs from CO 2 -reducing methanogens and Group II corresponds to methyl-reducing methanogens.Groups I and II also contain Mcrs involved in AOM.Group III contains TACKlike Mcr sequences from Ca. Verstraetearchaeota, Ca.Nezharchaeota, Ca.Korarchaeota, Thaumarchaeota and Archaeoglobi [23,[106][107][108][109].Group IV is a monophyletic clade including all Acrs.As the Acr of Ca.Polytropus marinifundus is closely relate to those of short-and medium-chain alkane-oxidizing archaea [4][5][6][7], we can infer that Ca.Polytropus marinifundus uses its Acr to activate alkanes within the range C 3 -C 14 , based on Acr phylogeny (Fig. 3A) [21].Based on the results of Zhou et al. [19] and our hexadecane70 enrichments, Acrs from the Hadarchaea/Ca.Methanoliparia clade are all likely responsible for the activation of long-chain alkanes (Fig. 3A).Whether long-chain alkane Acrs are a monophyletic group should be investigated following cultivation of Bathyarchaeia and Ca.Helarchaeales from hydrocarbon-rich environments [20,22].
We investigated the occurrence of pathways for Acr-based long-chain alkane oxidation in other Hadarchaeota MAGs available in public databases.Neither the WL pathway, nor the β-oxidation pathway are present in Hadarchaeota genomes outside of the Ca.Cerberiarchaeaceae, with the exception of two MAGs that encode an almost complete WL pathway (DG-33-1 and B3_Hades, Fig. 4).In absence of the β-oxidation pathway and Acr, these two organisms might use this WL pathway for CO 2 fixation.
Previous studies suggested that Acr-based alkane-oxidation was transferred multiple times via HGTs in Archaea, but the direction of these transfers could not be resolved for most of the enzymes [24].However, we found that Ca.Cerberiarchaeaceae likely acquired the carbonyl branch of the WL pathway through HGT from Bathyarchaeota, similarly to what had already been proposed for Methanocella arvoryzae [59].Because this step is likely mandatory for the Acr-based alkane-oxidation, this transfer indicates that the last common ancestor of Hadarchaeota was likely not an ANKA and that this metabolism was acquired by HGT, at the base of the Ca.Cerberiarchaeaceae.In this context, other steps of the Acr-based alkane-oxidation could have been gained by HGT in Ca.Cerberiarchaeaceae, and in particular Acr and the β-oxidation pathway.These genes might have been horizontally acquired from Bathyarchaeia and/or Ca.Methanoliparia also dwelling in subsurface hydrothermal and oil-rich environments.By contrast, the mWL was vertically inherited from the LACA to Hadarchaeaota but was lost in members of the phylum lacking the pathway.
Overall, our study highlights the need to sample new locations and to use cultivation-based approaches to understand the extension, evolution, and physiology of Acr-based alkane metabolism in Hadarchaea and other archaea from extreme environments.
This study was funded by the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG) under Germany's Excellence Initiative/Strategy through the Cluster of Excellence "The Ocean Floor-Earth's Uncharted Interface" (EXC 2077; project no.390741603).The Guaymas Basin expeditions were supported by the National Science Foundation, Biological Oceanography (grant no.1357238) to Andreas Teske (Collaborative Research: Microbial Carbon cycling and its interactions with Sulfur and Nitrogen transformations in Guaymas Basin hydrothermal sediments).Metagenome sequencing was done in the frame of Census of Deep Life Phase 14 call (Deep Carbon Observatory) granted to David Benito Merino and Gunter Wegener.

Figure 1 .
Figure 1.Community composition and sulfide formation in sediment slurries incubated with hexadecane; the results correspond to cruise AT37-06; (A) community composition based on archaeal and bacterial 16S rRNA gene amplicons; (B) sulfide production in early enrichments and control incubations; control incubations did not show significant sulfide production over time; enrichments with hexadecane at 37 • C and 50 • C showed slow activities; enrichments at 70 • C grew faster and were diluted after 90 days of incubation.

Figure 2 .
Figure 2. Community composition in Hexadecane70 cultures from AT37-06 to AT42-05; relative abundance of microbial taxa based on 16S rRNA gene fragments recruited from the metagenome; archaea dominate thermophilic alkane-degrading enrichments; Archaeoglobales were abundant in early sediment enrichments from the AT37-06 cruise (February 2018), and Hadarchaea became more dominant in later stages (September 2018, March 2020); the communities include heterotrophic Bathyarchaeia and Acetothermia, and sulfate-reducing Thermodesulfobacteriales; a second enrichment attempt from cruise AT42-05 showed similar results.

Figure 3 .
Figure 3. Methyl−/alkyl-coenzyme M reductase phylogeny, hexadecane activation by Acr, and proposed metabolism for Ca. C. oleivorans; (A) ML likelihood tree of McrA/AcrA alignment with 100 bootstraps; white circles and gray circles show bootstrap values of >70% and >90%, respectively; the clades shaded in gray include all Acr sequences (Mcr Group IV) [105]; the clade of putative long-chain alkane Acrs includes the Acr of Ca. C. oleivorans; (B) Ca.C. oleivorans activates hexadecane to hexadecyl-CoM.LC-MS analysis of hexadecane70 culture extracts shows two dominant chromatographic peaks in extracted ion chromatograms of the exact mass of hexadecyl-CoM; these peaks likely represent coenzyme M-substituted alkyls resulting from activation of the alkane in the secondary and primary position, in order of elution time[5]; (C) metabolic model for Ca. C. oleivorans; the Acr activates hexadecane to hexadecyl-CoM, which is then converted into a 16-carbon acyl-CoA (hexadecanoyl-CoA), possibly via Aor; acyl-CoA is processed to acetyl-CoA units (β-oxidation pathway); acetyl-CoA is incorporated into the downstream part of the H 4 MPT mWL via the Cdh/Acs complex; the methyl group is completely oxidized to CO 2 ; the fate of the electrons released from this metabolism is unknown; F 420 H 2 oxidation could be coupled to the production of H 2 via Frh or to the reduction of CO 2 to formate by an Fdh.

Figure 5 .
Figure 5. Placement of Hadarchaea, Persephonarchaea, and Bathyarchaea in species genome tree, FwdABC phylogeny, and CdhABCDE/acetyl-CoA synthase complex (Cdh) phylogeny; maximum-likelihood phylogenetic trees with 100 bootstraps based on concatenated alignment of 38 archaeal marker genes, FwdABC, and CdhABCDE protein sequences; (A) Hadarchaea and Persephonarchaea form a clade next to Theionarchaea; (B) Hadarchaea Fwd sequences form a branch with the Persephonarchaea 2 sequences; (C) Cdh sequences from the alkane-oxidizing Hadarchaea clade cluster together and branch from Bathyarchaea sequences, probably as a consequence of an event of lateral gene transfer between subsurface alkane-oxidizing archaea.