Guillaume Borrel
email: guillaume.borrel@pasteur.fr

Panagiotis S Adam

Luke J Mckay

Lin-Xing Chen

Isabel Natalia Sierra-García

Christian M K Sieber

Quentin Letourneur

Amine Ghozlane

Gary L Andersen

Wen-Jun Li

Steven J Hallam

Gerard Muyzer

Maia Valéria

De Oliveira

William P Inskeep

Jillian F Banfield

Simonetta Gribaldo
email: simonetta.gribaldo@pasteur.fr

Wide diversity of methane and short-chain alkane metabolisms in uncultured archaea

Methanogenesis is an ancient metabolism of key ecological relevance, with direct impact on the evolution of Earth's climate. Recent results suggest that the diversity of methane metabolisms and their derivations have probably been vastly underestimated. Here, by probing thousands of publicly available metagenomes for homologues of methyl-coenzyme M reductase complex (MCR), we have obtained ten metagenome-assembled genomes

(MAGs) belonging to potential methanogenic, anaerobic methanotrophic and short-chain alkane oxidizing archaea. Five of these MAGs represent under-sampled (e.g., Verstraetearchaeota, Methanonatronarchaeia, ANME-1) or previously genomically undescribed (ANME-2c) archaeal lineages. The remaining five MAGs correspond to lineages that are only distantly related to previously known methanogens and span the entire archaeal phylogeny. Comprehensive comparative annotation significantly expands the metabolic diversity and energy conservation systems of MCR-bearing archaea. It also suggests the potential existence of a yet uncharacterized type of methanogenesis linked to short-chain alkane/fatty acid oxidation in a previously undescribed class of archaea ('Ca. Methanoliparia'). We redefine a common core of marker genes specific to methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea, and propose a possible scenario for the evolutionary and functional transitions that led to the emergence of such metabolic diversity.

Methanogenesis is an archaeal-specific metabolism of key relevance in the anaerobic degradation of organic matter and biogas production 1,[START_REF] Schink | Energetics of syntrophic cooperation in methanogenic degradation[END_REF] . It is considered one of the most ancient energetic metabolisms [START_REF] Ueno | Evidence from fluid inclusions for microbial methanogenesis in the early Archaean era[END_REF][START_REF] Sousa | Early bioenergetic evolution[END_REF] with direct impact on the evolution of the Earth's climate system [START_REF] Kasting | Life and the evolution of Earth's atmosphere[END_REF] . Methanogens have been detected in virtually all types of anaerobic environments. Until recently, all methanogens were thought to belong to two euryarchaeal clades, named Class I and Class II methanogens [START_REF] Bapteste | Higher-level classification of the Archaea: evolution of methanogenesis and methanogens[END_REF] . The majority of Class I/II methanogens can grow by reducing CO 2 into methane using H 2 as an electron donor [START_REF] Whitman | The Methanogenic Bacteria[END_REF] . Several representatives of the Methanosarcinales (Class II methanogens) use additional energetic substrates, including acetate and methylated compounds 8 . Methanosphaera spp. (Class I methanogens) are restricted to the reduction of methanol with H 2 [START_REF] Oren | The Prokaryotes: Other Major Lineages of Bacteria and The[END_REF] . Regardless of the encoded methanogenic pathway, all members of Class I/II methanogens possess the H 4 MPT methyl-branch of the Wood-Ljungdahl pathway (m-WL), the N 5 -Methyltetrahydromethanopterin:coenzyme M methyltransferase complex (MtrABCDEFGH or MTR), and the methyl-coenzyme M reductase complex (McrABG or MCR) [START_REF] Borrel | Phylogenomic data support a seventh order of methylotrophic methanogens and provide insights into the evolution of methanogenesis[END_REF] . The same enzymes are present in ANaerobic MEthanotrophic archaea (ANME) and are used in reverse to oxidize methane [START_REF] Timmers | Reverse Methanogenesis and Respiration in Methanotrophic Archaea[END_REF][START_REF] Scheller | Anaerobic Utilization of Hydrocarbons[END_REF][START_REF] Hallam | Reverse methanogenesis: Testing the hypothesis with environmental genomics[END_REF] .

Our understanding of the diversity and metabolic versatility of methanogenic archaea is undergoing a rapid transformation with the availability of additional isolates and metagenome-assembled genomes (MAGs). This has revealed additional lineages only distantly related to Class I/II methanogens [START_REF] Borrel | Methanogenesis and the Wood-Ljungdahl Pathway: An Ancient, Versatile, and Fragile Association[END_REF][START_REF] Adam | The growing tree of Archaea: New perspectives on their diversity, evolution and ecology[END_REF][START_REF] Spang | Genomic exploration of the diversity, ecology, and evolution of the archaeal domain of life[END_REF] , including Methanomassiliicoccales 10 , Methanofastidiosales [START_REF] Nobu | Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen[END_REF] , Methanonatronarchaeia [START_REF] Sorokin | Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis[END_REF] and Verstraetearchaeota [START_REF] Vanwonterghem | Methylotrophic methanogenesis discovered in the novel 14 archaeal phylum Verstraetearchaeota[END_REF] . A striking characteristic of these recently described methanogens is the absence of the MTR complex, a partial or missing m-WL pathway, and the presence of specific methyltransferases for the utilization of methylated compounds. Accordingly, they are predicted to be limited to reduce methylated compounds with H 2 for methanogenesis, which was experimentally validated for Methanomassiliicoccales 20 and Methanonatronarchaeia [START_REF] Sorokin | Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis[END_REF] . Interestingly, the implication of a divergent McrABG-like complex in the oxidation of short-chain alkanes (butane, propane) has been demonstrated in two representatives of a recently described euryarchaeal order, the 'Ca. Syntrophoarchaeales' 21 . Divergent MCR sequences were also found in two members of the Bathyarchaeota [START_REF] Evans | Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics[END_REF] (TACK superphylum), in the GoM-Arc1 (a lineage within the Methanosarcinales) [START_REF] Dombrowski | Genomic insights into potential interdependencies in microbial hydrocarbon and nutrient cycling in hydrothermal sediments[END_REF] , and from environmental samples [START_REF] Mckay | Occurrence and expression of novel methyl-coenzyme M reductase gene (mcrA) variants in hot spring sediments[END_REF] . Altogether, this suggests that methanogens, anaerobic methanotrophs and short-chain alkane oxidizers may have an even wider phylogenetic and environmental distribution than previously anticipated, provoking new questions on the diversity and evolution of these metabolisms.

Results and Discussion

Additional lineages of archaea with an MCR or MCR-like complex

To identify previously undescribed lineages of potential methanogens, anaerobic methanotrophs and short-chain alkane oxidizers, we probed available metagenomes from the JGI/IMG database for McrA homologues and identified sequences distantly related to well characterized lineages (Methods). Ten MAGs were reconstructed from the corresponding metagenomes sourced from a wide range of anoxic environments including an inland petroleum reservoir from Brazil, oil seeps from USA [START_REF] Hawley | Metagenomes from two microbial consortia associated with Santa Barbara seep oil[END_REF] , soda lake sediments from Russia, and hot-springs from China and USA (Table 1). Nine of the ten MAGs had an estimated completeness ranging from 78.4 to 94.4%, and one was only 51.5% complete. Estimated contamination (without strain heterogeneity) ranged from 0 to 3.3%.

Four MAGs represent three previously undescribed lineages only distantly related to known methanogenic/methanotrophic archaea (Fig. 1): NM1 (NM1a and NM1b MAGs) branches within the Methanotecta superclass [START_REF] Adam | The growing tree of Archaea: New perspectives on their diversity, evolution and ecology[END_REF] , between Archaeoglobales and the clade formed by 'Ca. Syntrophoarchaeales' and 'Ca. Methanophagales' [START_REF] Adam | The growing tree of Archaea: New perspectives on their diversity, evolution and ecology[END_REF] (ANME-1); NM3 branches within the Acherontia superclass [START_REF] Adam | The growing tree of Archaea: New perspectives on their diversity, evolution and ecology[END_REF] , at the base of the clade formed by the nonmethanogenic Theionarchaea 26 and 'Ca. Methanofastidiosa' (former WSA2/Arc1); NM4 branches within the TACK superphylum and is related to Korarchaeum cryptofilum. In the NM4 MAG, the markers for methane metabolism are present on two contigs with a lower coverage than the other contigs and contain few genes related to Korarchaeum. However, an independent study supports that these contigs belong to the same organism, provisionally named "Candidatus Methanodesulfokores washburnensis" for its methaneand sulfur-cycling capacities (McKay et al., submitted). One additional MAG (NM2) was too partial to assess its phylogenetic placement.

Finally, five MAGs correspond to currently under-sampled archaeal lineages: a deepbranching Verstraetearchaeota, a third Methanonatronarchaeia, a second 'Ca. Methanophagales' (ANME-1), a second GoM-Arc1 (showing a close relationship with the methanotrophic Methanoperedens), and the first representative of ANME-2c (Fig. 1).

Phylogeny and functional inference of MCR/MCR-like complexes

To investigate the evolutionary relationships and characteristics of the MCR complexes identified in this study, we built a phylogeny based on a concatenated alignment of McrABG subunits. This phylogeny is in overall agreement with recently published ones [START_REF] Spang | Genomic exploration of the diversity, ecology, and evolution of the archaeal domain of life[END_REF][START_REF] Sorokin | Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis[END_REF] . Three MAGs (NM1a, NM1b and GoM-Arc1-GOS) encode alternative McrABG-like complexes that cluster with those of 'Ca. Syntrophoarchaeum' and Bathyarchaeota (Fig. 2A, in blue). The presence of an MCR-like complex and the absence of a canonical MCR in GoM-Arc1-GOS are consistent with the recent description of a MAG of this lineage [START_REF] Dombrowski | Genomic insights into potential interdependencies in microbial hydrocarbon and nutrient cycling in hydrothermal sediments[END_REF] , and represent so far a unique feature within the Methanosarcinales.

The remaining MAGs harbour canonical MCR complexes (Fig. 2A), branching next to their closest MCR-bearing neighbors in the reference archaeal phylogeny (Fig. 1), suggesting no recent horizontal gene transfers (HGT). The clustering of NM3 with Methanofastidiosa supports an early presence of methanogenesis in the Acherontia. The clustering of NM4 with Verstraetearchaeota, support that it is a genuine methanogenic/methanotrophic representative of the TACK. The separate branching of ANME-2c from the other ANME-2 lineages suggests that anaerobic methane oxidation in Methanosarcinales emerged multiple times independently from methanogenic ancestors. Interestingly, both NM1a and NM1b encode, in addition to the McrABG-like complex, a canonical MCR complex branching at the base of Class II methanogens, consistently with the reference phylogeny. The coexistence of MCR and MCR-like complexes in the same archaeon has never been observed before and brings into question the metabolism of this lineage (see below).

It is striking to observe that most of the predicted or experimentally proven methyldependent hydrogenotrophic methanogens are closely related in the MCR tree (Fig. 2A, in red), irrespective of their placement in the reference phylogeny (Fig. 1). This might be the consequence of ancient exchanges of the MCR complex among these lineages, whose direction is hard to define. Nevertheless, some more recent transfers may be identified. For example, 'Ca. Methanophagales' (ANME-1) MCRs branch far from their Methanotecta relatives, and might have acquired their MCR complex from a methanogenic member of the Acherontia.

The clustering of MCR-like homologues belonging to distantly related lineages (Fig. 2A, in blue) is also puzzling. This might be due to HGT and/or tree reconstruction artefacts linked to their high sequence divergence with respect to canonical MCRs, exemplified by their longer-than-average branches. Such divergence is probably related to a change in function, as MCR-like complexes are involved in activating short-chain alkanes (butane and propane) in 'Ca. Syntrophoarchaeum' 21 . Accordingly, several residues playing an important role in canonical MCR, either by interacting with cofactors, forming the catalytic site cavity wall or being post-translationally modified, are not conserved in 'Ca. Syntrophoarchaeum' sequences (Fig 2B). The replacement of large aromatic residues (e.g. Phe330, Tyr333, Tyr444, Tyr446) present in the cavity wall of canonical MCR [START_REF] Ermler | Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation[END_REF] by smaller ones in 'Ca. Syntrophoarchaeum' MCR-like complexes could have occurred to accommodate butane/propane (larger substrate than methane) in the catalytic site (Fig. 2B). The presence of smaller amino acids at these positions in NM1 and Bathyarchaeota MCR-like complex suggest a similar function in short-chain alkane oxidation. Finally, the MCR-like sequences of GoM-Arc1 show fewer modifications at these sites, suggesting the utilization of a smaller alkane, possibly ethane or methane.

Expanded diversity of methyl-dependent hydrogenotrophic methanogens

The NM3 and NM4 MAGs share several similarities with the recently discovered order-level lineages of methanogens that were proposed or experimentally proven to perform methyldependent hydrogenotrophic methanogenesis [START_REF] Borrel | Phylogenomic data support a seventh order of methylotrophic methanogens and provide insights into the evolution of methanogenesis[END_REF][START_REF] Nobu | Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen[END_REF][START_REF] Sorokin | Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis[END_REF][START_REF] Vanwonterghem | Methylotrophic methanogenesis discovered in the novel 14 archaeal phylum Verstraetearchaeota[END_REF] (Fig. 3, Supplementary Table 1). First, these relatively complete MAGs (85,5% completeness) lack at least 24 genes coding for the MTR complex, H 4 MPT biosynthesis, and the H 4 MPT methyl-branch of the WL pathway, otherwise present in all Class I/II methanogens (Supplementary Table 1). Second, they encode [Ni-Fe] hydrogenases and methyltransferases with the potential to support methanogenesis from methanol (MtaABC) in NM3 and NM4 and methanethiol (MtsAB) in NM3 (Fig. 3; Supplementary Table 1). Interestingly, energy conservation complexes of NM3 are mostly similar to Methanofastidiosales 17 (Supplementary Fig. 1), their closest related methanogens in the reference phylogeny (Fig. 1). Altogether, this suggests that NM3 and NM4 rely on methyl-dependent hydrogenotrophic methanogenesis (Fig. 3; Supplementary Discussion for details on energy conservation in NM3 and NM4).

The predicted methanogenesis pathway in Verst-YHS (Verstraetearchaeota) and Mnatro-ASL (Methanonatronarchaeia) MAGs also supports methyl-dependent hydrogenotrophic methanogenesis (Fig. 3, Supplementary Table 1), as described in the first genomic assemblies for these lineages [START_REF] Sorokin | Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis[END_REF][START_REF] Vanwonterghem | Methylotrophic methanogenesis discovered in the novel 14 archaeal phylum Verstraetearchaeota[END_REF] . However, comparison of the energy conservation enzymes in the seven currently available Verstraetearchaeota (order Methanomethyliales) suggests an alternative model than previously described [START_REF] Vanwonterghem | Methylotrophic methanogenesis discovered in the novel 14 archaeal phylum Verstraetearchaeota[END_REF] (Fig. 3, Supplementary Table 1). Indeed, we found that all Methanomethyliales MAGs (95% average completeness) lack the HdrA/MvhD and possibly MvhAG subunits of the electron-bifurcating complex HdrABC/MvhADG, suggesting that this complex is absent in these archaea. In contrast, we identified in these genomes a gene cluster encoding a potential complex composed of a membrane-bound hydrogenase and of HdrBC (tentatively named Energy-converting Hydrogenase D or Ehd; Supplementary Fig. 2). We propose that this complex could be involved in a previously unreported mode of energy conservation associated with methanogenesis (Fig. 3; Supplementary Discussion).

Insights into methane and short-chain alkane oxidizers

GoM-Arc1-GOS, ANME-1-THS and ANME-2c MAGs possess a WL pathway and lack the methyltransferases and [Ni-Fe] hydrogenases required for methylotrophic and hydrogenotrophic methanogenesis, respectively (Fig. 3; Supplementary Table 1), similar to all available MAGs of methanotrophs and short-chain alkane oxidizer (Supplementary Fig. 3). Although they encode an AMP-producing acetyl-CoA synthetase (Acs) which is used for aceticlastic methanogenesis in Methanosaeta spp., they could rather use it for acetate assimilation [START_REF] Timmers | Reverse Methanogenesis and Respiration in Methanotrophic Archaea[END_REF] . Comparison with methanotrophs and short-chain alkane oxidizers also reveals a common core of enzymes for energy conservation, comprising the F 420 H 2 :quinone (or phenazine) oxidoreductase (Fqo/Fpo) and a potential electron confurcating complex (HdrABC/MvhD/FdhB 28 ) coded by a conserved gene cluster (Supplementary Fig. 4). ANME-2c and GoM-Arc1-GOS encode 17 and 10 multiheme c-type cytochromes respectively, supporting the importance of direct electron transfer to syntrophic partners in anaerobic methane [START_REF] Wegener | Intercellular wiring enables electron transfer between methanotrophic archaea and bacteria[END_REF][START_REF] Mcglynn | Single cell activity reveals direct electron transfer in methanotrophic consortia[END_REF] and short-chain alkane oxidation 21 metabolisms (Supplementary Fig. 3; Supplementary Table 1).

ANME-1-THS MAG is the first sequenced representative of a "Land clade" within the 'Ca. Methanophagales' (Supplementary Fig. 5), suggesting different adaptations to environmental conditions than members of the ANME-1b clade, which are mainly from marine methane seeps. ANME-1-THS differs from the ANME-1b MAG [START_REF] Meyerdierks | Metagenome and mRNA expression analyses of anaerobic methanotrophic archaea of the ANME-1 group[END_REF] by the presence of a bacterial-like Rnf complex that could couple the NAD:ferredoxin oxidoreduction with chemiosmotic gradient generation/utilisation (Fig. 3; Supplementary Fig. 6; Supplementary Discussion). If these genes are not in the missing region of this MAG, ANME-1-THS might also differ from the other ANMEs by the lack of multiheme c-type cytochromes to transfer electrons from methane oxidation to a syntrophic partner (Fig. 3; Supplementary Fig. 3). Alternatively, two PsrABC-like complexes, including a molybdenum/selenocysteinecontaining dehydrogenase subunit, could be involved in the reduction of inorganic compounds such as polysulfide/elemental sulfur [START_REF] Grimaldi | The prokaryotic Mo/W-bisPGD enzymes family: A catalytic workhorse in bioenergetic[END_REF][START_REF] Jormakka | Molecular mechanism of energy conservation in polysulfide respiration[END_REF] (Fig. 3). This contrasts with ANME-1b MAG which misses the membrane integral (PsrC-like) subunit needed to transfer electrons from membrane-associated electron transporters (Supplementary Fig. 3). These characteristics might indicate growth of ANME-1-THS without bacterial syntrophs.

The gene content of GoM-Arc1-GOS is consistent with the recent description of the first member of the GoM-Arc1 lineage [START_REF] Dombrowski | Genomic insights into potential interdependencies in microbial hydrocarbon and nutrient cycling in hydrothermal sediments[END_REF] . While GoM-Arc1 members encode an MCR-like complex possibly involved in short-chain alkane oxidation (Fig. 3), they lack the betaoxidation pathway proposed to be involved in butane/propane utilization in 'Ca. Syntrophoarchaeales' 21 (Supplementary Fig. 3). If GoM-Arc1 members are capable of oxidizing ethane (CH 3 CH 3 ), as suggested by the fewer modifications observed in the catalytic site of its MCR-like complex relative to canonical MCRs (Fig. 2), the oxidation of the ethylgroup would lead to an acetyl-group that could directly enter the oxidative WL pathway, making the beta-oxidation pathway unnecessary (Fig. 3; Supplementary Discussion). With the presence of Fqo, HdrABC/MvhD/FdhB, multiheme c-type cytochromes, and HdrDE (Supplementary Table 1), the energy conservation system associated with this potential ethane-oxidation metabolism in GoM-Arc1 would mostly resemble that associated with methanotrophy in their closely related ANME-2 lineages (Supplementary Fig. 3). The question remains whether the MCR-like homologs of GoM-Arc1 could also be capable of methane oxidation.

A previously uncharacterised type of methanogenesis?

The two NM1 MAGs represent the first archaea predicted to encode both an MCR and an MCR-like complex (Fig. 2), suggesting that they might be potentially capable of both methane and short-chain alkane metabolisms (Fig. 4; Supplementary Table 1). Interestingly, while both NM1 MAGs encode the MTR and the m-WL pathway similarly to Class I/II methanogens, they lack the [Ni-Fe] hydrogenases (MvhA and FrhA) and methyltransferases needed for hydrogenotrophic and methylotrophic methanogenesis, respectively. They also diverge from Class I/II methanogens by the replacement of the F 420 dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) by MtdB, which relies on NAD(P) redox cofactor in Methylobacterium extorquens [START_REF] Hagemeier | Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1[END_REF] .

Beyond the presence of an MCR-like complex, the potential ability of NM1 for shortchain alkane oxidation is also suggested by the presence of a complete beta-oxidation pathway with several gene copies per step, and a complete WL pathway (including CODH/ACS) as in 'Ca. Syntrophoarchaeales' 21 . In addition, NM1 encode multiple long-chain fatty acid acyl-CoA synthases (FadD-like), not present in 'Ca. Syntrophoarchaeales'. Long chain fatty acids (LCFA) activated with these enzymes can enter the beta-oxidation pathway. NM1a and NM1b also encode multiple AMP-forming acetyl-CoA synthetase (Acd) to generate ATP from LCFA degradation. These enzymatic redundancies suggest a versatility toward substrates, as previously proposed for Syntrophus aciditrophicus [START_REF] Mcinerney | The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth[END_REF] and Archaeoglobus fulgidus [START_REF] Klenk | The complete genome sequence of the hyperthermophilic, sulphatereducing archaeon Archaeoglobus fulgidus[END_REF] . Consistently, analysis of the environmental distribution of NM1 (Supplementary Fig. 7) reveals their common association with anoxic hydrocarbon-rich environments including methane seeps and oil-rich environments, where short-chain alkanes and long-chain carboxylic acids can be present in substantial concentrations [START_REF] Milkov | Molecular and stable isotope compositions of natural gas hydrates: A revised global dataset and basic interpretations in the context of geological settings[END_REF][START_REF] Meredith | Influence of biodegradation on crude oil acidity and carboxylic acid composition[END_REF] . In particular, NM1a and NM1b originate from an enrichment culture based on petroleum fluids and from a natural oil seep.

In addition to this potential wide substrate range, NM1 also contrast with 'Ca. Syntrophoarchaeales' in terms of energy conservation by lacking homologues of the NADH/F 420 H 2 :quinone oxidoreductase (Nuo/Fqo) and multiheme c-type cytochromes (Fig. 4; Supplementary Table 1). Also, NM1 contain an Rnf complex potentially using NAD instead of menaquinone for ferredoxin oxidoreduction, similarly to ANME-1-THS (Supplementary Fig. 6; Supplementary Discussion). In the absence of membrane-bound enzymes involved in oxidoreduction of lipid-soluble electron carriers, of multiheme c-type cytochromes for direct interspecies electron transfer, of confurcating [Fe]-hydrogenase for interspecies H 2 transfer [START_REF] Sieber | Genomic Insights into Syntrophy: The Paradigm for Anaerobic Metabolic Cooperation[END_REF] , and of enzymes involved in dissimilatory reduction of inorganic compounds, the nature of the terminal electron acceptor coupled to alkane/LCFA oxidation remains elusive. Although both MAGs are mostly complete (~90%), it cannot be excluded that some of these enzymes are coded in their missing regions, or that an alternative way to transfer electrons to a terminal acceptor exists (e.g. utilisation of the assimilatory-type sulfite reductase present in both MAGs for dissimilatory reduction of sulfite, direct electron transfer not relying on cytochromes, or utilization of cytochromes produced by a syntrophic partner). Alternatively, we speculate that in NM1, methanogenesis involving the canonical MCR complex could act as a sink for the electrons produced during alkane and LCFA oxidation. Several electron-bifurcating/confurcating complexes encoded in the two NM1 MAGs (Supplementary Fig. 8) together with the Rnf complex could be involved in this metabolism. The conversion of alkane and LCFA into CH 4 and acetate is thermodynamically feasible but was only reported to occur through syntrophic partnerships between a bacterium (performing the beta-oxidation) and a H 2 -consuming methanogen [START_REF] Zengler | Methane formation from long-chain alkanes by anaerobic microorganisms[END_REF][START_REF] Stams | Role of syntrophic microbial communities in high-rate methanogenic bioreactors[END_REF] , and it thus remains to be proven experimentally whether this can occur in a single organism.

Based on the presence of methane and short-chain alkane/fatty acid-related enzymes and the preferential association with hydrocarbon-rich environments, we propose the provisional class 'Candidatus Methanoliparia', with 'Candidatus Methanoliparum thermophilum' for NM1a and 'Candidatus Methanolliviera hydrocarbonicum' for NM1b (see Supplementary Discussion for full taxonomy and nomenclature). A core of markers related to methane and short chain-alkane metabolisms A group of 38 genes present in most methanogens and absent from most other organisms, generally referred to as "methanogenesis core markers", was previously defined from Class I/II methanogen genomes [START_REF] Gao | Phylogenomic analysis of proteins that are distinctive of Archaea and its main subgroups and the origin of methanogenesis[END_REF][START_REF] Kaster | More than 200 genes required for methane formation from H2 and CO2 and energy conservation are present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus[END_REF] (Supplementary Table 2). Half of them have an unknown function. The others correspond to MCR and MTR subunits, enzymes for biosynthesis and activation of the F 430 prosthetic group of MCR, and post-translational modifications in the McrA catalytic site [START_REF] Nayak | Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic archaea[END_REF][START_REF] Lyu | Mmp10 is required for post-translational methylation of arginine at the active site of methyl-coenzyme M reductase[END_REF] . We reassessed the occurrence of these markers in the ten assembled MAGs as well as reference genomes covering all recently discovered lineages of methanogens, methanotrophs and short-chain alkane oxidizers (Table 2).

Our analysis shows that some markers are no longer universal in Class I/II methanogens (e.g. m37, 38). Also, many marker genes shared by all or most Class I/II methanogens were predicted to be nonessential in Methanococcus maripaludis S2 [START_REF] Sarmiento | Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis[END_REF] (Table 2). These non-universal and nonessential genes could possibly be involved in fine-tuning of methanogenesis (e.g. post-translational modification of MCR [START_REF] Wagner | Didehydroaspartate Modification in Methyl-Coenzyme M Reductase Catalyzing Methane Formation[END_REF] ) or in its regulation under specific environmental conditions that are not encountered by all methanogens. For example, m21 and m24 are missing in several methanogens from nutrient-rich environments, such as Methanobrevibacter, Methanosphaera and Methanocorpusculum, and could be involved in regulatory processes related to changes in substrate/nutrient availability.

All the lineages of predicted and experimentally proven methyl-dependent hydrogenotrophic methanogens [START_REF] Nobu | Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen[END_REF][START_REF] Sorokin | Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis[END_REF][START_REF] Vanwonterghem | Methylotrophic methanogenesis discovered in the novel 14 archaeal phylum Verstraetearchaeota[END_REF] lack numerous markers (Table 2), similarly to what was previously noted in Methanomassiliicoccales [START_REF] Borrel | Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine[END_REF] . These markers correspond to MTR complex subunits (m27-31), an McrA post-translational modification enzyme (m33) [START_REF] Lyu | Mmp10 is required for post-translational methylation of arginine at the active site of methyl-coenzyme M reductase[END_REF] and several uncharacterized markers that are mostly nonessential in M. maripaludis [START_REF] Sarmiento | Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis[END_REF] (Table 2). The existence of the same pattern in NM3 and NM4 supports our inference of a potential methyl-dependent hydrogenotrophic methanogenesis. Finally, Bathyarchaeota BA1 and BA2 [START_REF] Evans | Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics[END_REF] which were described as methyl-dependent hydrogenotrophic methanogens [START_REF] Evans | Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics[END_REF] but possess an MCR-like complex instead of the canonical MCR (Fig. 2), lack almost all methanogenesis markers (Table 2), questioning their actual metabolism.

Several homologues of the methanogenesis markers are also known to be present in non-methanogenic archaea. This is the case of the MCR/MCR-like (m1-3) and MTR (m27-31) complexes in archaeal methanotrophs [START_REF] Whitman | The Methanogenic Bacteria[END_REF] and GoM-Arc1 [START_REF] Dombrowski | Genomic insights into potential interdependencies in microbial hydrocarbon and nutrient cycling in hydrothermal sediments[END_REF] , as well as the MCR-like complex in 'Ca. Syntrophoarchaeales' 21 . Based on our analysis, archaeal methanotrophs and short-chain alkane oxidizers also appear to possess numerous markers previously exclusively associated with methanogenesis (Table 2), supporting the common origin and functional links of these metabolisms.

In addition to the MCR/MCR-like complex subunits, the most specific and conserved markers in all lineages of methanogens, methanotrophs and short-chain alkane oxidizers appear to be the genes involved in the biosynthesis (nflD/cfbD, murD/cfbE and possibly mcrD [START_REF] Zheng | The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea[END_REF] ) and activation (atwA and possibly mcrC [START_REF] Prakash | Elucidating the process of activation of methyl-coenzyme M reductase[END_REF] ) of the F 430 prosthetic group of MCR, along with six genes encoding uncharacterized proteins (m4 to m9) (Table 2). These six genes are co-localized in most genomes (Supplementary Fig. 9) and are among those that were predicted to be co-transcribed in Methanolobus psychrophilus R15 [START_REF] Li | Global mapping transcriptional start sites revealed both transcriptional and post-transcriptional regulation of cold adaptation in the methanogenic archaeon Methanolobus psychrophilus[END_REF] , suggesting they operate in a common process. These six marker enzymes do not co-purify with MCR [START_REF] Prakash | Elucidating the process of activation of methyl-coenzyme M reductase[END_REF] . However, their phylogeny (Supplementary Fig. 10) and their restriction to archaea having MCR or MCR-like complexes strongly suggest they are involved in essential aspects of the regulation, folding and/or function of the respective holoenzymes (Supplementary Discussion).

Finally, several markers are present in archaeal lineages without MCR/MCR-like complexes (Supplementary Table 3) and are possibly remnants of an ancestral methanemetabolism (Supplementary Fig. 111213; Supplementary Discussion).

Taken together, these observations indicate that none of the previously defined methanogenesis markers are unique to methanogens but are rather more generally indicative of metabolisms involving MCR or MRC-like complexes, including methanogenesis, methanotrophy, and short-chain alkane oxidation. Elucidating the roles of these markers (MCR-Associated Markers or MAM) will be essential not only for understanding methanogenesis, but also anaerobic methanotrophy and short-chain alkane oxidation in archaea.

Evolution of methane and short-chain alkane metabolisms

Our results significantly extend recent data by highlighting the overwhelming presence of lineages with an MCR or MCR-like complex in the Archaea (Fig. 1). This supports an early origin of methanogenesis in this domain of life, and multiple losses of this metabolism during archaeal diversification [START_REF] Sousa | Early bioenergetic evolution[END_REF][START_REF] Borrel | Methanogenesis and the Wood-Ljungdahl Pathway: An Ancient, Versatile, and Fragile Association[END_REF][START_REF] Sorokin | Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis[END_REF][START_REF] Raymann | The two-domain tree of life is linked to a new root for the Archaea[END_REF] .

The sharing of a common set of genes (Table 2) clearly indicates that methanogens, anaerobic methanotrophs and short-chain alkane oxidizers are evolutionarily linked. However, it remains unclear which type of metabolism is the most ancient, and what evolutionary and functional transitions led to such diversity [START_REF] Borrel | Methanogenesis and the Wood-Ljungdahl Pathway: An Ancient, Versatile, and Fragile Association[END_REF] . The antiquity of the WL pathway [START_REF] Weiss | The physiology and habitat of the last universal common ancestor[END_REF][START_REF] Adam | Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes[END_REF] , and the recent proposal that the root of the archaeal tree might lie in between Class I and II methanogens [START_REF] Raymann | The two-domain tree of life is linked to a new root for the Archaea[END_REF] , would suggest that CO 2 -dependent hydrogenotrophic methanogenesis is the ancestral type of methanogenesis. Nevertheless, the growing diversity of methyl-dependent hydrogenotrophic methanogens, including this work (Fig. 1 in red), indicates that this metabolism has been largely overlooked. Its origin and evolutionary relationship with CO 2 -dependent hydrogenotrophic methanogenesis remain unclear. The fact that it is a simpler metabolism, requiring fewer genes than CO 2 -dependent hydrogenotrophic methanogenesis might suggest its earliest origin. However, it may also signify that it could have emerged later through loss of the WL pathway and/or HGT, as suggested by the grouping of most archaea sharing this metabolism in the phylogenies of MCR (Fig. 2A) and of m4 to m9 markers (Supplementary Fig. 10). Also, the clustering of NM4 with Verstraetearchaeota on a separate and well-supported clade in the MCR tree (Fig. 2A) is compatible with a possible inheritance of this metabolism from the last archaeal common ancestor, even under the classical root in between Euryarchaeota and the TACK. However, the possibility of an acquisition through ancient HGT cannot be excluded at present. More insights into the ancestral type of methanogenesis might also be gained from re-examination of the root of the archaeal tree [START_REF] Raymann | The two-domain tree of life is linked to a new root for the Archaea[END_REF] including all recently discovered archaeal lineages.

The phylogenetic placement of the ANME lineages (Fig. 1), strongly suggests that the capabilities for anaerobic methanotrophy emerged multiple times independently during archaeal diversification. In the Methanosarcinales this could have occurred relatively recently and repeatedly by reversal of methanogenesis, possibly through switch of function of a resident canonical MCR, leading to the different ANME-2 (Fig. 2A) and possibly ANME-3 lineages. The pool of genes associated with energy conservation in methanogenic and methanotrophic Methanosarcinales is in fact relatively similar [START_REF] Mcglynn | Energy Metabolism during Anaerobic Methane Oxidation in ANME Archaea[END_REF] (Fig. 3 and Supplementary Fig. 3) and some methanogenic Methanosarcinales encode c-type multiheme cytochromes [START_REF] Timmers | Reverse Methanogenesis and Respiration in Methanotrophic Archaea[END_REF] providing the necessary background for electron transfer in AOM archaea.

The identification and experimental demonstration of the capacity for oxidation of short-chain fatty-acids (butane, propane) by a divergent MCR-like complex in the Synthrophoarchaeales 21 is among the most interesting findings of the recent years in the field of environmental microbiology. Our results extend the distribution of these MCR-like complexes in the archaea (Fig. 2A), and therefore also of potential short-chain alkane oxidation capabilities (Figs. 3 and4). The rapid evolutionary rates of MCR-like homologues coupled to the change of key residues (Fig. 2B) suggest that these complexes might have arisen from canonical MCRs through modifications in the catalytic site to accommodate larger hydrocarbons than methane. Transitions between anaerobic methanotrophy and short-chain alkane utilisation could have occurred in both directions as suggested by i) the close phylogenetic relationships between 'Ca. Methanophagales' and 'Ca. Syntrophoarchaeales' and the position of GoM-Arc1 within a clade comprising ANME-2a/ANME-2d (Fig. 1), ii) the proposed mechanism of alkane activation in their MCR/MCR-like complexes 21 , iii) their very similar modes of energy conservation (Supplementary Fig. 3), and iv) their numerous shared markers (Table 2). If GoM-Arc1 is a short-chain alkane oxidizer, as suggested by its MCR-like complex, this capacity could have emerged from methanotrophy. Conversely, 'Ca. Methanophagales' (ANME-1) might have shifted from short-chain alkane oxidation to methanotrophy after acquisition of their MCR through HGT (Fig. 2). Finally, the first report of co-existence of an MCR and an MCR-like complex in members of the "Ca. Methanoliparia" class opens up the possibility of an additional type of methanogenesis associated with alkane and/or LCFA oxidation.

Further exploration of archaeal lineages with an MCR/MCR-like complex and their experimental characterization will lead to a more complete understanding of methane metabolisms and their derivations, as well as their environmental impact.

Methods

Metagenomic database probing and contig binning

Contigs of 6108 metagenomes publicly available on the IMG/JGI database in April 2017 were screened for the presence of COG4058, corresponding to McrA, using search tools of the database. 819 contigs containing an McrA sequence with a minimal length of 750 bp were downloaded. The McrA sequences present on these contigs were aligned on those of 188 published genomes by using Mafft 56 (mafft-linsi) and were trimmed with BMGE 57 (BLOSUM30). A maximum likelihood (ML) phylogeny was calculated in IQTree [START_REF] Nguyen | IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] with the TEST option for best model selection and 100 bootstrap replicates. Metagenomes containing one or several contigs coding for an McrA homologue that was only distantly related to know lineages or belonged to undersampled lineages were downloaded from the IMG database. These metagenomes were assembled with MetaSPAdes 59 , IDBA-UD [START_REF] Peng | IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth[END_REF] and Newbler (Roche) (see Supplementary Table 4 for details). The contigs were binned with ESOM, MetaBAT 61 , ABAWACA 1.07 (http://ggkbase.berkeley.edu/), MaxBin 2.0 [START_REF] Wu | 2.0: An automated binning algorithm to recover genomes from multiple metagenomic datasets[END_REF] and CONCOT [START_REF] Alneberg | Binning metagenomic contigs by coverage and composition[END_REF] (Supplementary Table 4). An in-house pipeline (Let-it-bin, https://github.com/QuentinLetourneur/Let-it-bin) was used for read trimming, assembly, and contig binning. Two of the MAGs were refined using DAS_Tool [START_REF] Sieber | Recovery of genomes from metagenomes via a dereplication, aggregation, and scoring strategy[END_REF] . Completeness and contamination of the assembled MAGs were estimated with CheckM [START_REF] Parks | Assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes[END_REF] . Phylogenomic analyses A reference archaeal phylogeny was built from a concatenation of 40 phylogenetic markers corresponding to the 36 proteins of the Phylosift dataset [START_REF] Darling | PhyloSift: phylogenetic analysis of genomes and metagenomes[END_REF] , plus the alpha and beta subunits of the RNA polymerase and two universal ribosomal proteins (L30, S4) (Supplementary Table 5). We used a subset of the genomes available for each order/class/phylum level lineages (Supplementary Table 6) to minimize biased associated with uneven distribution of taxa among them (e.g. >100 taxa in Halobacteriales vs. 3 taxa in Methanocellales). The 147 genomes were chosen because they were the most complete and the most distant to each other within each lineage. Two phylogenies were built from a concatenation of McrABG and of six co-localized markers (m4 to m9) specific to genomes encoding an MCR/MCR-like complex. Sequences used for these trees were searched by HMM in the ten MAGs obtained in this study and in genomes present in the NCBI or IMG-databases, aligned with Mafft 56 (mafft-linsi), trimmed with BMGE 57 (BLOSUM30) and concatenated with an in-house script. Before concatenation, the genes of the two datasets (McrABG and m4 to m9) were tested for congruence using the Internode Certainty (IC) [START_REF] Kobert | Computing the Internode Certainty and Related Measures from Partial Gene Trees[END_REF] test in RaxML [START_REF] Stamatakis | RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF] . Maximum likelihood phylogenies for each gene and blind concatenations were calculated in IQTree [START_REF] Nguyen | IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] with the TEST option for best model selection and 100 bootstrap replicates. Sequences causing strong incongruences (with a bootstrap>=80%) at high taxonomic ranks (order to phylum as applicable) were removed, and the procedure was repeated until no further incongruence was found. Bayesian phylogenies were constructed in PhyloBayes [START_REF] Lartillot | PhyloBayes 3: A Bayesian software package for phylogenetic reconstruction and molecular dating[END_REF] under the CAT+GTR+Γ4 model. Four independent Markov chain Monte Carlo chains were run until convergence and checked by sampling ever two cycles with a 25% burn-in. Support at nodes was evaluated by posterior probability values. ML phylogenies were constructed in IQ-TREE [START_REF] Nguyen | IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] under the LG+C60 model.

Metabolic prediction

Gene prediction was performed using Prodigal [START_REF] Hyatt | Prodigal: Prokaryotic gene recognition and translation initiation site identification[END_REF] . All metabolic genes were identified using hidden Markov models (HMMs) searches with PFAM, TIGR and custom HMM profiles. Annotation of proteins displayed in Supplementary Table 1 were improved by inspecting the genomic context of the metabolic genes using RAST [START_REF] Aziz | The RAST Server: Rapid annotations using subsystems technology[END_REF] and SyntTax 72 (http://archaea.upsud.fr/synttax/), by phylogenetic analyses including the sequences of characterized enzymes and by identifying their conserved domains using CD-search batch [START_REF] Marchler-Bauer | CDD: NCBI's conserved domain database[END_REF] (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). Genomic context was also inspected to identify conserved patterns among multiple genomes. Identification of energy conservation systems was performed with MacSyFinder [START_REF] Abby | A program to mine genomes for molecular systems with an application to CRISPR-Cas systems[END_REF] , by defining specific and sensitive models for each system followed by manual curation. SAMN10390736, SAMN10390737, SAMN10390738, SAMN10390739. NM2 sequences corresponding to markers reported in Table 2 are deposited under MK202738 to MK202758. The data that support the findings of this study are available from the corresponding author upon request.

Table legends

Table 1: General information on the ten MAGs obtained in this study. Table 2: Occurrence in methanogens, methanotrophs and short-chain alkane users of 38 genes previously suggested as methanogenesis markers. 5 and Supplementary Table 6 for detail). Node supports refer to posterior probabilities, and for reasons of readability only values above 0.8 are shown. The tree is rooted according to Raymann et al. [START_REF] Raymann | The two-domain tree of life is linked to a new root for the Archaea[END_REF] . The scale bar represents the average number of substitutions per site. Black arrows point to the 9 obtained MAGs and accolated pie charts indicate their estimated completeness. Colors indicate that genomes of these lineages encode an MCR/MCR-like complex, Class I/II methanogens are in green, methyl-dependent hydrogenotrophic lineages are in red, methanotrophs are in orange (some being within Class II), potential or validated shot-chain alkane users are in in blue. NM1 could also have a methane metabolism (see text for discussion). 6 for details). Node supports refer to posterior probabilities, and for reasons of readability only values above 0.8 are shown. The scale bar represents the average number of substitutions per site. The color code is similar to that in Fig. 1 with the exception of NM1 which have both an MCR-like (in blue) and a canonical MCR (in purple) (see text for discussion). B) Conservation of 17 residues previously described to interact with CoM, CoB, F 430 cofactors, making part of the substrate cavity wall, or having post-translational modifications [START_REF] Ermler | Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation[END_REF][START_REF] Wagner | Didehydroaspartate Modification in Methyl-Coenzyme M Reductase Catalyzing Methane Formation[END_REF][START_REF] Kahnt | Post-translational modifications in the active site region of methylcoenzyme M reductase from methanogenic and methanotrophic archaea[END_REF] . Replacement of conserved amino acids associated to a negative value in the Blosum45 matrix are indicated by white on black background, those with a null or positive value in the Blosum45 matrix are in bold, "." indicate conserved positions and "-" indicate missing positions in the sequence due to sequencing incompleteness. Figure 3: Predicted methane and short-chain alkane metabolism of the MAGs described in this study, with the exception of NM1, which is presented in Fig. 4, and NM2, which has a low completeness. Colored arrows correspond to reactions modifying or transferring the C1 carbon group of the substrate. Details on the annotation of the enzymes are presented in Supplementary Table 1. MFR, methanofuran; H 4 MPT, tetrahydromethanopterin; Fd, ferredoxin; F 420 , coenzyme F 420 ; LCFA, Long Chain Fatty Acids; MQ, menaquinone; Mp, methanophenazine; Mhc, c-type multiheme cytochromes; DIET, Direct Interspecies Electron Transfer. Grey color indicates the absence of the enzyme, complex, reaction or compound.

Figure legends

Comparisons of with other methane-cycling or short-chain alkane oxidizers, which are discussed in the text, are presented in Supplementary Figs 1 and3. The percentages between brackets indicate the estimated completeness of the corresponding MAGs. Figure 4: Predicted methane, short-chain alkane and long/medium-chain fatty acid metabolism of the two MAGs NM1a ("Ca. Methanoliparum thermophilum" completeness of 92.5%) and NM1b ("Ca. Methanolliviera hydrocarbonicum" completeness of 90.2%) belonging to the candidate class "Ca. Methanoliparia". Colored arrows correspond to reactions present in both MAGs which modify or transfer the carbon group(s) of the substrate. Predicted possible metabolisms are the utilisation of short-chain alkanes and long/medium-chain fatty acids (L/MCFA) (blue), methanotrophy (orange) and methanogenesis from short-chain alkanes or L/MCFA (purple). Details on the annotation of the enzymes are provided in Supplementary Table 1. MFR, methanofuran; H 4 MPT, tetrahydromethanopterin; Fd, ferredoxin; F 420 , coenzyme F 420 ; L/MCFA, Long/medium chain fatty acids; MQ, menaquinone; Mhc, c-type multiheme cytochromes; DIET, Direct Interspecies Electron Transfer. Grey color indicates the absence of the corresponding enzyme, complex, reaction or compound in both MAGs. Class I / II Methanobacteriales 24 99 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0,95 0,95 0,68 1 1 0,59 1 1 1 1 1 1 1 1 1 1 1 1 1 Methanopyrales 2 100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Methanococcales 15 100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0,87 1 1 0,73 1 0,8 0,93 1 1 0,93 1 1 1 1 1 0,87 0,87 1 0,47 1 Methanosarcinales* 39 99 1 1 1 1 1 1 1 1 1 1 1 1 1 0,97 0,97 1 1 1 0,92 1 0,87 1 1 0,97 1 1 1 1 1 1 0,97 1 1 0,67 0,21 0,9 0,67 Methanomicrobiales 16 99 1 1 1 1 1 1 1 1 1 1 1 1 0,94 1 1 1 1 1 0,44 1 0,81 1 0,94 0,81 1 1 1 1 1 1 1 1 1 1 1 0 0,31 Methanocellales 3 100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0,67 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 Methanoflorentaceae 1 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 0 0 0 SCAO & AOM Msar_ANME-2a 1 99 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 Msar_ANME-2c 1 92 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 1 0 1 0 Msar_ANME-2d 2 98 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 0 0 0,5 Methanophagales (incl. ANME-1-THS)

G A I G S Q L A G T I V G I G I S A S K A A H W G Q H P F A G T I V G V G E L S A S K V

A W G Q H P F A M G W V A S H Q S R S F I G Q A A T A Q T H W A Q I S A Q T H W A Q I T S T H W A Q L S A G T H G A G Q L A M W H Q A W H M H V K V F K V F I I I F A A D S T F M F F F F F F F F F F . . . . . .
H 2 + CoM-SH + HS-CoB 2H₂ 4H⁺ n H ⁺ / n N a ⁺ n H ⁺ / n N a ⁺ n H ⁺ / n N a ⁺ n H ⁺ / n N a ⁺ n H ⁺ / n N a ⁺ n H ⁺ / n N a ⁺

A T P s y n t h a s e A T P s y n t h a s e A T P s y n t h a s e A T P s y n t h a s e A T P s y n t h a s e A T P s y n t h a s e

2 79 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 0,5 0 1 1 1 1 0 0,5 0,5 0 1 1 1 1 1 0,5 0 0,5 0,5 0 1 0 NM1 § ‡ 2 91 1 1 1 1 1 0,5 0,5 1 1 1 1 1 1 1 0 1 0 0,5 1 0,5 1 1 1 0 1 1 1 1 1 1 1 1 0 0 1 1 0 Msar_GoM-Arc1 (incl. GoM-Arc1-GOS) § 2 78 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0,5 0,5 0,5 0,5 0,5 0,5 0,5 1 1 1 1 1 1 1 1 1 1 0,5 1 1 1 0 0 Syntrophoarchaeales 2 93 1 1 1 1 0,5 1 1 1 1 1 0,5 1 1 1 1 1 1 1 1 0 0,5 0,5 0 0 0,5 0 1 0 0 0 1 0 0 0 0 0,50 0 CH 3 -dep hydro Methanomethyliales (incl. Verst-YHS) § 7 95 1 1 1 1 0,86 1 0,86 1 1 1 1 1 0,86 0,71 0,71 1 0,71 1 0,86 0,86 1 1 1 0,14 0 0 0 0 0 0 0 0 0 0 0 0 0 Methanomassiliicoccales 11 99 1 1 1 1 1 1 1 1 1 1 1 1 0,91 0,91 1 1 1 0,91 0,27 1 0,09 1 0 1 0 0 0,09 0 0 0 0 0 0 0 0 0 0 8 86 1 1 1 0,75 1 1 1 0,88 1 1 1 1 1 1 1 1 0,63 0,63 0,88 1 0,88 0 1 0 0,5 0 0 0 0 0 0 0 0 0 0 0 0 NM3 § 1 86 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 NM4 § 1 86 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Methanonatronarchaeia (incl. Mnat-ASL) 3 97 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0,67 0,33 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NM2 § 1 52 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 0 1 1 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 Bathyarchaeota BA1/BA2 2 92 1 1 1 0,5 0,5 0,5 0,5 0,5 0,5 1 0,5 0,5 0 0 0 0,5 0 1 0,5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Essen ality index in M. maripaludis 0 0 0 0 3 2,5 0,5 0,5 3,5 0 0 0 0 0 0 9,5 0,5 3 0,5 1 5,5 16 Table 2: Occurrence in methanogens, methanotrophs and short-chain alkane users of 38 genes previously suggested as methanogenesis markers.

-

Figure 1 :

 1 Figure1: Placement of nine MAGs described in this study in the reference phylogeny of Archaea. NM2 was not included due to low completeness. Bayesian phylogeny (PhyloBayes, CAT+GTR+ Γ4) based on concatenation of 40 conserved phylogenetic markers (8,564 amino acid positions) and 156 genomes/MAGs (see Supplementary Table5and Supplementary Table6for detail). Node supports refer to posterior probabilities, and for reasons of readability only values above 0.8 are shown. The tree is rooted according to Raymann et al.[START_REF] Raymann | The two-domain tree of life is linked to a new root for the Archaea[END_REF] . The scale bar represents the average number of substitutions per site. Black arrows point to the 9 obtained MAGs and accolated pie charts indicate their estimated completeness. Colors indicate that genomes of these lineages encode an MCR/MCR-like complex, Class I/II methanogens are in green, methyl-dependent hydrogenotrophic lineages are in red, methanotrophs are in orange (some being within Class II), potential or validated shot-chain alkane users are in in blue. NM1 could also have a methane metabolism (see text for discussion). Figure 2: Phylogeny of the MCR/MCR-like complex and conservation of important positions in the catalytic site. A) Unrooted Bayesian phylogeny (CAT+GTR+Γ4) based on a concatenation of McrABG/McrABG-like subunits (1,187 amino acid positions) from 109 genomes/MAGs (see Supplementary Table6for details). Node supports refer to posterior probabilities, and for reasons of readability only values above 0.8 are shown. The scale bar represents the average number of substitutions per site. The color code is similar to that in Fig.1with the exception of NM1 which have both an MCR-like (in blue) and a canonical MCR (in purple) (see text for discussion). B) Conservation of 17 residues previously described to interact with CoM, CoB, F 430 cofactors, making part of the substrate cavity wall, or having post-translational modifications[START_REF] Ermler | Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation[END_REF][START_REF] Wagner | Didehydroaspartate Modification in Methyl-Coenzyme M Reductase Catalyzing Methane Formation[END_REF][START_REF] Kahnt | Post-translational modifications in the active site region of methylcoenzyme M reductase from methanogenic and methanotrophic archaea[END_REF] . Replacement of conserved amino acids associated to a negative value in the Blosum45 matrix are indicated by white on black background, those with a null or positive value in the Blosum45 matrix are in bold, "." indicate conserved positions and "-" indicate missing positions in the sequence due to sequencing incompleteness. Figure3: Predicted methane and short-chain alkane metabolism of the MAGs described in this study, with the exception of NM1, which is presented in Fig.4, and NM2, which has a low completeness. Colored arrows correspond to reactions modifying or transferring the C1 carbon group of the substrate. Details on the annotation of the enzymes are presented in Supplementary Table1. MFR, methanofuran; H 4 MPT, tetrahydromethanopterin; Fd, ferredoxin; F 420 , coenzyme F 420 ; LCFA, Long Chain Fatty Acids; MQ, menaquinone; Mp, methanophenazine; Mhc, c-type multiheme cytochromes; DIET, Direct Interspecies Electron Transfer. Grey color indicates the absence of the enzyme, complex, reaction or compound.

Figure 2 :

 2 Figure1: Placement of nine MAGs described in this study in the reference phylogeny of Archaea. NM2 was not included due to low completeness. Bayesian phylogeny (PhyloBayes, CAT+GTR+ Γ4) based on concatenation of 40 conserved phylogenetic markers (8,564 amino acid positions) and 156 genomes/MAGs (see Supplementary Table5and Supplementary Table6for detail). Node supports refer to posterior probabilities, and for reasons of readability only values above 0.8 are shown. The tree is rooted according to Raymann et al.[START_REF] Raymann | The two-domain tree of life is linked to a new root for the Archaea[END_REF] . The scale bar represents the average number of substitutions per site. Black arrows point to the 9 obtained MAGs and accolated pie charts indicate their estimated completeness. Colors indicate that genomes of these lineages encode an MCR/MCR-like complex, Class I/II methanogens are in green, methyl-dependent hydrogenotrophic lineages are in red, methanotrophs are in orange (some being within Class II), potential or validated shot-chain alkane users are in in blue. NM1 could also have a methane metabolism (see text for discussion). Figure 2: Phylogeny of the MCR/MCR-like complex and conservation of important positions in the catalytic site. A) Unrooted Bayesian phylogeny (CAT+GTR+Γ4) based on a concatenation of McrABG/McrABG-like subunits (1,187 amino acid positions) from 109 genomes/MAGs (see Supplementary Table6for details). Node supports refer to posterior probabilities, and for reasons of readability only values above 0.8 are shown. The scale bar represents the average number of substitutions per site. The color code is similar to that in Fig.1with the exception of NM1 which have both an MCR-like (in blue) and a canonical MCR (in purple) (see text for discussion). B) Conservation of 17 residues previously described to interact with CoM, CoB, F 430 cofactors, making part of the substrate cavity wall, or having post-translational modifications[START_REF] Ermler | Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation[END_REF][START_REF] Wagner | Didehydroaspartate Modification in Methyl-Coenzyme M Reductase Catalyzing Methane Formation[END_REF][START_REF] Kahnt | Post-translational modifications in the active site region of methylcoenzyme M reductase from methanogenic and methanotrophic archaea[END_REF] . Replacement of conserved amino acids associated to a negative value in the Blosum45 matrix are indicated by white on black background, those with a null or positive value in the Blosum45 matrix are in bold, "." indicate conserved positions and "-" indicate missing positions in the sequence due to sequencing incompleteness. Figure3: Predicted methane and short-chain alkane metabolism of the MAGs described in this study, with the exception of NM1, which is presented in Fig.4, and NM2, which has a low completeness. Colored arrows correspond to reactions modifying or transferring the C1 carbon group of the substrate. Details on the annotation of the enzymes are presented in Supplementary Table1. MFR, methanofuran; H 4 MPT, tetrahydromethanopterin; Fd, ferredoxin; F 420 , coenzyme F 420 ; LCFA, Long Chain Fatty Acids; MQ, menaquinone; Mp, methanophenazine; Mhc, c-type multiheme cytochromes; DIET, Direct Interspecies Electron Transfer. Grey color indicates the absence of the enzyme, complex, reaction or compound.

  hydro, methyl-dependent hydrogenotrophic methanogenesis; MCR, methyl-coenzyme M 430 ‡ NM1 MAGs encode for a SCAO metabolism and possibly for methanogenesis or methanotrophy. § Metabolic complexes.

  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

	Interac ons in the cataly c site	F430 axial ligant	CoB	CoB	CoB	CoB	CoB	Cavity wall	CoM / Cavity wall		CoM / Cavity wall	Cavity wall		Cavity wall			CoB	CoB
	Posi ons in Ermler et al., 1997	Gln147	Arg225	Lys256	His257*	Arg270	Arg271*	Phe330	Tyr333	Gln400*	Phe443	Tyr444	Gly445*	Tyr446	Asp450*	Cys452*	Asn481	Val482
		. . . . . .				.				.		.
		. . . . . .								.
		. . . .								.
		. . . . . .
		. . . .					.			.
				. . . .			. . .						.
		. .		.		. .			.		.
		. . . .		. .					.		.
		. . . .		. .					.		.			A
		. . . .		. .					.		.
		. . . .		. .			--------
		. . . . . .			. .		.		.		.
			-----		. . . . .		.		. .
		.		. . . .		. . . . .		.			.
		. . . . . . . . . . . . . . . . .
		. . . . . . . .		. . . . . . . .
		. . . .		. . .		.		. . . . . .
		. . . .		. . .		.		. . . . . .
		. . . . . . . . . . . . . .		. .
		. . . . . . . . . . . . . .		. .
		. . . . . . . . . . . . . .		. .
		. . . . . . . . . . . . . . . . .
		. . . . . . . . . . . .		. . . .
		. . . . . . . . . . . . . .		. .
		. . . . . . . . . . . . . .		. .
		. . . . . . . . . . . . . . . . .
	-	. . . . . . .		. . . . . .		.
		. . . . . . . . . .		. . .		. .
		. . . . . . . . . . . . . . . . .
		. . . . . . . . . .		. . . . . .
		. . . . . . . . . .		. . . . . .
		. . . . . . . . . . . . . . . . .
		. . . . . . . . . .		. . . . . .
		. . . . . . . . . .		. . . . . .
	-	. . . . . . . . . .		. . . . . .
		. . . . . . . . . .		. . . . . .
		. . . . . . . . . .		.		. . . .
	McrABG	. . . . . . . . . .		. . . . . .
		. . . . . . . . . .

.

Table 1 - MCR

 1MCR

		Genome	Origin	"6 marker cluster"	Scaffold (nbr) F 430 bioS and		Size (Mb)	Genes (nbr)	GC (%)	Compl. (%)	Cont. (%)		Strain hetero.* MTR	Cont. exd. strain hetero. (%) .
		NM1a NM1b NM2 NM3 NM4 Verst-YHS Mnatro-ASL ANME-1-THS Tibetan Hot Spring sediment, China Enrichment culture (50°C) from petroleum sample, Brazil °12 Santa Barbara Channel oil seeps, USA 183 Santa Barbara sediments, USA 210 Enrichment culture (40°C) from petroleum sample, Brazil °26 Yellowstone sulfidic hot spring, USA 122 Yellowstone sulfidic Hot Spring, USA 46 Altai Soda Lake sediments, Russia 73 181 GoM-Arc1-GOS Gulf of Mexico natural Oil Seep, USA 119 ANME-2c Gulf of Mexico natural oil seep, USA 249 Nbr of genomes / lineage Avg genome completness (%) m1_mcrA m2_mcrB m3_mcrG m4_Predicted rotamase m5_DUF2102 m6_DUF2112 m7_YjiL-like m8_DUF2113 m9_arCOG03226 m10_atwA m11_mcrC m12_mcrD	m13_c D	m14_c E	1.26 1.66 1.03 1.49 1.42 1.05 1.34 2.03 1.46 2.66 m15_mtxX	m16_AIR synthase-related	1388 1860 1254 1578 1603 1220 1451 2155 1623 2867 m17_Zn-ribbon protein m18_DUF2099	m19_DUF2117	35.7 92.5 43.8 90.2 41.8 51.5 55.2 85.5 43.4 85.5 28.4 94.4 42.2 93.3 48.7 78.4 41.0 91.2 48.5 92 m21_DUF2124 m22_DUF2098 m23_Soluble P-type ATPase	m24_DUF2111	1.3 3.6 2 2.9 1.8 0 1.3 4.9 0 2 m25_arCOG04853	m26_DUF2114	0 66.7 0 0 66.7 0 0 33 0 0 m27_mtrA m28_mtrB	m29_mtrC	1.3 1.2 2 2.9 0.6 0 1.3 3.3 0 2 m30_mtrD m31_mtrE	m32_arCOG04885	m33_Arg-methyltransf. (McrA PTM)	m34_DUF1894	m35_DUF1890	m36_DUF2115	m37_DUF2119	m38_DUF2121
														CaiA
			Nuo/
			Fqo/	X ox
			Fpo	X red
					2H +
		2H +	MQ	MQH 2	NADH									FadB
	e -	Mhc DIET Mhc												AtoB		Acetate ADP ATP
																			Acd
										CoM-S-S-CoB	CoM-SH + CoB-SH	2H 2
												Fd red Fd ox MvhD HdrC HdrA / HdrB Hdr	GltD-like MvhG MvhA 2NADH ? 4H +
																			2NAD + + 2H +
		n H + / n N a +

Acknowledgements

We thank Rudolf Thauer for feedback on an earlier version of the manuscript. G.B. acknowledges support from the Institut Pasteur through a Roux-Cantarini fellowship. P.S.A. is supported by a PhD fellowship from Paris Diderot University and by funds from the PhD Program "Frontières du Vivant (FdV)-Programme Bettencourt". S.G. acknowledges funding from the French National Agency for Research Grant ArchEvol (ANR-16-CE02-0005-01). This work used the computational and storage services (TARS cluster) provided by the IT department at Institut Pasteur, Paris. S.J.H. acknowledges support from the US Department of Energy (DOE) JGI supported by the Office of Science of US DOE Contract DE-AC02-05CH11231, the Natural Sciences and Engineering Research Council (NSERC) of Canada, Genome British Columbia, Genome Canada, Canada Foundation for Innovation (CFI), and the Tula Foundation. I.N.S.G. and V.M.O. are grateful to São Paulo Research Foundation -FAPESP (process numbers 2011/14501-6 and 2013/20436-8) and Petrobras for financial support and to Dr. Neil Gray and Dr. Ian Head from the School of Civil Engineering and Geosciences at Newcastle University for lab facilities. W-J.L. was supported by Key Projects of Ministry of Science and Technology (MOST) (Nos. 2013DFA31980, 2015FY110100). G.M. was supported by the ERC Advanced Grant PARASOL (No. 322551). L.J.M. appreciates funding from the NASA Postdoctoral Program through the NASA Astrobiology Institute and W.P.I. was supported by the Montana Agricultural Experiment Station (Project 911300).

Data availability

MAG sequences are available in the BioProject PRJNA472146 and Biosamples SAMN10387997, SAMN10390728, SAMN10390732, SAMN10390733, SAMN10390735,

Author contributions

Competing interests

The authors declare no competing interests.

Hydrothermarchaea


T Baumard

G Menary

F Schmidt

P Martin

Coupled Heating-Forming Simulation of the Thermoforming of Thermoplastic Composites

Keywords: Forming, Thermoplastic Composites, Infrared Heating, Process Simulation, Thermomechanical

A strategy for the simulation of the whole thermoforming process, from the infrared heating to the stamping, is presented here. Two loosely coupled simulation tools are being developed: the first one computes a realistic 3D, transient temperature field of the composite stack inside an infrared oven, considering the radiative, conductive and convective heat transfers; the temperature distribution is used as an input for the second that aims at simulating the thermomechanical behaviour of the composite during the forming step via a non-orthogonal constitutive model. The steps for the identification of the model parameters are introduced. Initial validation tests show realistic results in term of shear angle distribution.

Introduction

Thermoplastic composites structural parts have recently started to make their way into the transportation sector [START_REF] Stodolsky | Technology and future prospects for lightweight plastic vehicle structures[END_REF], but the use of composites in the automotive industry is currently mostly limited to low volume production parts for luxury cars, due to high manufacturing costs and cycle time.

Manufacturing processes such as thermoforming seem well adapted for high volume parts, but to reach the production rates required by the automotive industry, those processing techniques must be optimized to understand and avoid the apparition of defects. In order to avoid expensive trial-and-error procedures after the mould fabrication, robust virtual manufacturing schemes are needed to efficiently find the best process parameters (pre-heat temperature, punch speed, consolidation time …), and predict the resulting mechanical properties of the part [START_REF] Lee | Applications of virtual manufacturing in materials processing[END_REF].

Fig. 1: Thermoforming process steps: (a) infrared heating, (b)-(c) forming and consolidation, (d) demoulding

The thermoforming process consists in heating a laminate above the melting temperature of the thermoplastic matrix in an oven; it is then transferred to a press where it is formed and cooled down before demoulding. The sequential steps are presented in Fig. 1. Originally developed for thermoplastic sheets, it is now used for manufacturing thermoplastic composites parts as high production volumes are in demand.

Most of the current simulations of the thermoforming process consider only the mechanical draping of the part, while the effect is rarely investigated. It has been evidenced in recent research [3] [4] that the forming cannot be considered adiabatic : when the laminate contacts the mould, its temperature rapidly decreases which induces local rigidification that can lead to wrinkles. These heat transfer effects need to be studied as they represent a limit of the stamping process. The control of the laminate temperature during the entire process is therefore critical to ensure a good part quality. This work adopts a comprehensive approach for the simulation of the thermoforming of thermoplastic preimpregnated composites. Two coupled simulation tools are being developed for the preheating phase and the forming phase respectively.

Heating simulation

The first stage of the thermoforming process aims at bringing the composite laminate to the processing temperature in an oven. Infrared oven are favored in industrial composite applications for their fast heat-up

ITHEC 2016, MESSE BREMEN 2/4
time and the good absorptivity of polymers to infrared radiations.

Most of the thermomechanical simulations of the forming assume an homogeneous temperature distribution in the preform at the beginning of the stamping stage [START_REF] Wang | Thermomechanical analysis of thermoplastic composite prepregs using bias-extension test[END_REF]; this is however experimentally difficult to achieve as infrared heating can produce non-uniform temperature distributions with localized hot or cold spots. The aim of the heating simulation developed is to predict a realistic 3D temperature distribution in the composite stack during the preheating stage to be used as an input of the forming simulation; this tool also allows to optimize an oven configuration for a required temperature.

The simulation, developed with Comsol Multiphysics finite elements software, solves the 3D heat equation:

𝜌𝜌𝜌𝜌𝐶𝐶𝐶𝐶 𝜕𝜕𝜕𝜕𝑇𝑇𝑇𝑇 𝜕𝜕𝜕𝜕𝜕𝜕𝜕𝜕 = ∇. 𝜅𝜅𝜅𝜅∇𝑇𝑇𝑇𝑇 + 𝑞𝑞𝑞𝑞 + 𝑞𝑞𝑞𝑞 (1)
Where q r and q v are the radiative source term and convective losses respectively. The radiative term is treated as a view factor problem. Computation is realized using the hemicube method for the determination of the view factors. The entire environment of the oven (preform, lamps and reflectors) is modelled, and the convective losses are considered via a constant transfer coefficient.

Solving this equation requires the determination of the thermal properties of the lamps and composite, both in its non-consolidated and consolidated state . The considered material is a glass fabric/polyamide 6.6 thermoplastic composite developed by Solvay. It is supplied as a twill 2/2 woven fabric, powderimpregnated on both sides. Consolidated plates were manufactured using a in-house thermocompression machine.

This study was conducted at Institut Clément Ader, using a novel method based on infrared thermography and inverse analysis to identify the temperatures of the lamps and thermal conductivity tensor of the material [START_REF] Baumard | Determination of thermal contact conductance in thermoplastic prepreg stacks using infrared thermography[END_REF]. This experimental setup also allowed to characterize the inter-ply thermal contact resistances in a non-consolidated composite stack.

Fig. 2: Experimental 1 lamp heating test bench

The heat capacity and optical properties (reflectivity, emissivity) of the prepreg and consolidated composite plates were determined using DSC and infrared spectroscopy respectively. The material was shown to be opaque in the range of emission of the lamps, which conforts the choice of view factor over ray tracing strategy for computing the radiative heat transfer, as it allows for a faster computation while accurately representing the physics.

The modelisation of the lamps is based on the work of Monteix: only the tungsten filament is modelled as a cylinder of equivalent diameter taking into account the emissive surface of the spires. The temperature dependent emissivity and input powertemperature relation are considered for the tungsten.

After validation on the experimental set-up presented in Fig. 2, the ability of the model to optimize an infrared oven to minimize the temperature variation on a composite stack is investigated. An oven with 10 lamps disposed in two banks shifted by 4cm, with a center-to-center distance of 8cm between each lamp in the same bank. The oven configuration, viewed from the top, is shown in Fig. 3. On each bank (left and right), the lamps are numbered from the closest to the furthest of the plate symmetry axis.

Fig. 3: Oven configuration

The composite stack is heated from the top only. In a first step, only the input power of the lamps is considered as a variable. The cost function to be minimized is therefore:

min 𝑓𝑓𝑓𝑓 𝑷𝑷𝑷𝑷 , 𝑓𝑓𝑓𝑓 𝑷𝑷𝑷𝑷 = 𝑇𝑇𝑇𝑇 𝒙𝒙𝒙𝒙 -𝑇𝑇𝑇𝑇 ²𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 (2)
With P a vector containing the individual input power values of the lamps. Given the symmetry of the oven, the dimension of P is reduced to 5 (i.e., P(L i )= P(R i ) ). The objective temperature is in this case 295°C, which is approximately 30° above the melting temperature of the thermoplastic matrix. Optimization is conducted in steady-state, using a Levenberg-Marquardt algorithm. The lamp input power is constrained between 20% and 100% of the nominal power to avoid under voltage. The temperature on the The dashed blue line shows the temperature distribution along the profile with all lamps at the same input power (50% of nominal power). The solid red line corresponds to the temperature field after optimization, and exhibits a much flatter profile: the central hot spot is avoided.

The transfer phase between the infrared oven and the mold and die in the actual process allows considering a loose coupling between the two simulation tools: the three dimensional temperature field that results from the heating simulation will be used as an input for the forming model.

Forming simulation

The forming simulation is based on a non-orthogonal constitutive model based on the work of Khan [START_REF] Khan | Numerical and experimental analyses of woven composite reinforcement forming using a hypoelastic behaviour[END_REF] and Pierce [START_REF] Pierce | Implementation of a nonorthogonal constitutive model for the finite element simulation of textile composite draping[END_REF]. This model considers the composite as a continuous, anisotropic media, and tracks the orientation of the yarns, thus allowing the prediction of possible defect locations by visualizing the shear angle distribution in the part. This is done via a material user subroutine VUMAT implemented in Abaqus/Explicit. The subroutine works by calculating the fibre directions from the deformation gradient; the incremental strain is rotated from the Green-Naghdi frame used by Abaqus to the fibre frame; the incremental stress is computed in this frame from the constitutive law and converted back to the Green-Naghdi frame. The constitutive tensor assumes the following form in the fiber frame:

𝐶𝐶𝐶𝐶 = 𝐸𝐸𝐸𝐸 0 0 0 0 0 0 0 𝐺𝐺𝐺𝐺 , 𝐶𝐶𝐶𝐶 = 0 0 0 0 𝐸𝐸𝐸𝐸 0 0 0 𝐺𝐺𝐺𝐺 (3)
The superscripts 1, 2 represents the fiber directions, E the Young's modulus and G 12 the shear modulus of the material. The advantages of this approach are the relative ease of implementation in commercial finite element codes, and the simplicity of the constitutive material tensor as the computation is conducted in the material frame.

The model, originally developed for draping simulations of dry fabric, is currently being extended to take into account the temperature dependent behaviour of the resin through its contribution to the shear modulus of the material. Identification of the shear behaviour is done via bias-extension tests, to allow an easy inclusion of the setup in an environmental chamber for experiments around the processing temperature. An example of the shape of the fabric after deformation by the punch is shown in Fig. 6, with the camera direction aligned with the warp yarns.

The first validation simulations (see Fig. 7) show a good qualitative agreement with the experiments in terms of shear angle distribution and draw-in profiles, with the zones of maximum shear appearing at a 45° angle from the principal fiber directions.

The hemispherical punch will be used coupled with an infrared oven as a complete, instrumented labscale thermoforming setup to investigate the effect of temperature on the type and onset of defects generated. A separate study to understand the plyply and ply-mould frictional behaviour is also planned.

Conclusion

Understanding the thermomechanical behaviour of thermoplastic composites during the thermoforming process is critical to accurately predict the defects that can appear during the manufacturing.

In this article, the strategy for the development of a predictive, virtual manufacturing tool that relies on two coupled simulations of the infrared heating and forming steps has been presented. The material characterization procedure has been introduced for the thermophysical and mechanical properties, and is currently underway for high temperatures.

The first validation test cases used for the simulations show the pertinence of the approach with promising qualitative results; these will be confirmed with the complete thermoforming setup being finalized.

Fig. 4 :

 4 Fig. 4: Temperature along horizontal profile, dashed line: before optimization, solid line: optimized input power

Fig. 5

 5 Fig.5shows a fabric sample undergoing bias extension at room temperature. The evolution of the shear angle is observed by digital image correlation, and direct optical measurements.

Fig. 5 :

 5 Fig. 5: Bias extension test on twill 2/2 glass fabricAn experimental hemispherical punch apparatus is developed at Queen's University Belfast to validate the model formulation with stamping experiments. The fabric is held into place by the mean of a springactuated clamp to easily vary the clamping force, while the punch itself is mounted on a 60kN Promess press unit.

Fig. 6 :

 6 Fig. 6: Forming experiment, hemispherical punch on glass fabric

Acknowledgements

The work was conducted at Institut Clément Ader, Ecole des Mines d'Albi and at the School of Mechanical and Aerospace Engineering, Queen's University Belfast. The authors would like to thank Solvay for supporting this study and providing the test materials.

Fig. 7: Shear angle distribution after stamping, left: isometric view, right: bottom view Powered by TCPDF (www.tcpdf.org) Powered by TCPDF (www.tcpdf.org) Powered by TCPDF (www.tcpdf.org) Powered by TCPDF (www.tcpdf.org)