No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Sampling site and sample description

A 25-cm long sediment core (949C3) was collected from a methane-seep site at the Omine Ridge, Nankai Trough, off the Kumano area, Japan (33° 7.2253′ N, 136° 28.6672′ E), 2,533 m below sea level, by the manned submersible RV Shinkai 6500 (cruise YK06-03, dive no. 6K949, 6 May 2006). The detailed sediment core sample and site information has been published previously15,51,52. Our previous geochemical and 16S rRNA gene analysis indicated that the occurrence of anaerobic oxidation of methane reactions was mediated by archaeal anaerobic methanotrophs in the sediment15,51. The SSU rRNA gene analysis also showed that the sediment contained abundant and diverse microorganisms, most of which were affiliated with uncultured microbial groups, including Asgard archaea15,51.

Culturing

The deep-sea methane-seep sediment sample was first enriched using a continuous-flow bioreactor system supplemented with methane as the major energy source. The bioreactor, called a down-flow hanging sponge (DHS) bioreactor, has been operated in our laboratory, JAMSTEC, Yokosuka Headquarters, since 28 December 2006. The detailed operation conditions for the DHS bioreactor have been described previously15. To isolate anaerobic microorganisms, including Asgard archaea, from the DHS reactor, 2-ml samples of the bioreactor enrichment sediment slurry were inoculated in 15-ml glass tubes with a simple substrate and a basal medium. The composition of the basal medium was almost similar to that used for cultivation in the DHS bioreactor15, but it did not contain sulfate (that is, Na 2 SO 4 ). The basal medium composition was as follows (per litre): 9.47 g MgCl 2 ·6H 2 O, 1.36 g CaCl 2 ·2H 2 O, 20.7 g NaCl, 0.54 g NH 4 Cl, 0.14 g KH 2 PO 4 , 2.7 g NaHCO 3 , 0.3 g Na 2 S·9H 2 O, 0.3 g cysteine·HCl, 1 ml trace element solution15, 1 ml Se/W solution, 2 ml vitamin solution15 and resazurin solution (1 mg ml−1). The medium was purged with N 2 /CO 2 gas (80:20, v/v), and the pH was adjusted to 7.5 at 25 °C. The culture tubes were sealed with butyl rubber stoppers and screw caps. Autoclaved or filter-sterilized organic substances (such as protein-derived materials, sugars and fatty acids) were added to the tubes with stock solutions before inoculation with the bioreactor-enriched community. After establishing a stable Ca. P. syntrophicum culture, cultivations were performed at 20 °C in 50-ml serum vials containing 20 ml basal medium supplemented with casamino acids (0.05%, w/v), 20 amino acids (0.1 mM each) and powdered milk (0.1%, w/v, Hohoemi, Meiji) under an atmosphere of N 2 /CO 2 (80:20, v/v) in the dark without shaking, unless mentioned otherwise. Information regarding the purity check of MK-D1 cultures, as well as additional information about cultivation, is included in the Supplementary Methods.

SSU rRNA gene-based analysis

DNA extraction and PCR mixture preparation were performed on a clean bench to reduce contamination. DNA extraction from culture samples was performed as described previously53. The concentration of extracted DNA was measured using a Quant-iT dsDNA High-Sensitivity Assay Kit (Life Technologies). PCR amplification was performed using the Takara Ex Taq (for conventional clone analysis) or Takara LA Taq (for Illumina-based amplicon sequencing (iTAG) for targeted sequencing for the SSU rRNA gene analysis) (Takara Bio), and the reaction mixtures for PCR were prepared according to the manufacturer’s instructions. For the conventional clone analysis, a universal primer pair 530F/907R51 and an archaeal primer pair 340F/932R15,54 were used for PCR amplification. For iTAG analysis, the universal primer pair 530F/907R, which contained overhang adapters at the 5′ ends, was used. The procedures used for library construction, sequencing and data analysis were described previously21,55.

Growth monitoring using qPCR

For the quantitative analysis, a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with a SYBR Premix Ex Taq II kit (TaKaRa Bio) was used. The candidate phylum Lokiarchaeota-specific primer pair MBGB525F/Ar912r was used for amplification of 16S rRNA genes. Primer MBGB525F is the complementary sequence of the MGBG525 probe17, whereas Ar912r is an archaeal universal primer that is a slightly modified version of the originally designed primer56. The detailed procedure for qPCR is described in the Supplementary Methods. The doubling times of MK-D1 were calculated based on the semi-logarithmic plot of the qPCR data.

Growth test with multiple substrates

To examine the effect of the presence of other substances on the growth of MK-D1, medium containing casamino acids, 20 amino acids, powdered milk and supplemented with an individual substrate (Extended Data Table 3) was prepared, followed by qPCR and iTAG analyses. Each cultivation condition was set in duplicate; however, the H 2 -fed culture was prepared in triplicate because a previous study7 reported that a Lokiarchaeum has potential to grow with hydrogen based on a comparative genome analysis. Detailed culture liquid sampling and the subsequent qPCR and iTAG analyses are described in the Supplementary Information.

Evaluation of growth temperature

The test was performed using a basal medium containing casamino acids and powdered milk, with a pure co-culture of MK-D1 and Methanogenium as the inoculum (20%, v/v). The cultures were incubated at 4, 10, 15, 20, 25, 30, 37 and 40 °C. All incubations for the test were performed in triplicate. After 100 days of incubation, 16S rRNA gene copy numbers of MK-D1 were evaluated using qPCR.

FISH

Fixation of microbial cells, storage of the fixed cells and standard FISH were performed in accordance with a previously described protocol21. The 16S rRNA-targeted oligonucleotide probes used in this study are listed in Supplementary Table 10. The design of MK-D1-specific probes is described in the Supplementary Methods. As clear fluorescent signals were not obtained using the standard FISH technique, we used an in situ DNA-hybridization chain reaction (HCR) technique57. The FISH samples were observed using epifluorescence microscopes (BX51 or BX53, Olympus) and a confocal laser scanning microscope (Nikon A1RMP, Nikon Instech).

SEM

Microbial cells were fixed overnight in 2.5% (w/v) glutaraldehyde in the casamino acids–20 amino acid medium at 20 °C. The sample preparation procedure has been described previously58. The cell samples were observed using field emission-SEM (JSM-6700F, JEOL) or extreme high-resolution FIB-SEM (Helios G4 UX, ThermoFisher Scientific).

Ultrathin sectioning and TEM

Cells were prefixed with 2.5% (w/v) glutaraldehyde for 2 h. The specimens were frozen in a high-pressure freezing apparatus (EM-PACT2, Leica)59. The frozen samples were substituted with 2% OsO 4 in acetone for 3–4 days at −80 °C, and the samples were warmed gradually to room temperature, rinsed with acetone embedded in epoxy resin (TAAB). Thin sections (70 nm) were cut with am ultramicrotome (EM-UC7, Leica). Ultrathin sections of the cells were stained with 2% uranyl acetate and lead-stained solution (0.3% lead nitrate and 0.3% lead acetate, Sigma-Aldrich), and were observed using TEM (Tecnai 20, FEI) at an acceleration voltage of 120 kV.

Cryo-EM

Owing to the low cell yield culture, 400 ml of the culture of MK-D1 was prepared and concentrated to about 5 ml using a 0.22-μm-pore-size polyethersulfone filter unit (Corning) in an anaerobic chamber (95:5 (v/v) N 2 :H 2 atmosphere; COY Laboratory Products). The concentrated culture liquid was placed in a glass vial in the anaerobic chamber. After that, the head space of the glass vial was replaced by N 2 /CO 2 gas (80:20, v/v). Immediately before the observation using electron microscopy, the glass vial was opened, and the liquid culture was concentrated to about 200 μl by centrifugation at 20,400g for 10 min at 20 °C. Subsequently, 3 μl of the concentrated liquid culture was applied onto a Quantifoil Mo grid R1.2/1.3 (Quantifoil MicroTools) pretreated with glow-discharge, and was plunged-frozen in liquid ethane using a Vitrobot Mark IV (FEI Company) at 4 °C and 95% humidity.

The frozen grid was mounted onto a 914 liquid-nitrogen cryo-specimen holder (Gatan) and loaded into a JEM2200FS electron microscope (JEOL) equipped with a field emission electron source operating at 200 kV and an omega-type in-column energy filter (slit width: 20 eV). The images were recorded on a DE-20 direct detector camera (Direct Electron) at a nominal magnification of 15,000×, which resulted in an imaging resolution of 3.66 Å per pixel, with the total dose under 20 electrons per Å2 using a low-dose system. For electron tomography, tilt series images were collected manually in a range of approximately ±62° at 2° increments. The total electron dose on the specimen per tilt series was kept under 100 electrons per Å2 to minimize radiation damage. The tilt series were aligned using gold fiducials and tomograms were reconstructed using filtered back projection or SIRT in the IMOD software60 with an image binning of 5.

Lipid analysis

About 120 ml of a highly purified culture sample was concentrated using the same method as described above, except that the filtration concentration procedure was performed on a clean bench instead of the anaerobic chamber. After cell collection, the cells were washed with the anaerobic basal medium to eliminate the interfering matrix. Subsequently, lipid analysis was conducted for the collected cells after the improved method61. For precise qualitative liquid analysis, GC–MS was conducted on the 7890 system (Agilent Technologies) to compare the retention time and mass fragmentation signatures.

Stable isotope probing and NanoSIMS analysis

To confirm utilization of amino acids by MK-D1, a stable-isotope probing experiment was performed using a 13C- and 15N-labelled amino acid mixture (Cambridge Isotope Laboratories). In brief, 120 ml serum vials containing 40 ml basal medium were prepared and supplemented with the 20 stable-isotope-labelled amino acids (roughly 0.1 mM of each), casamino acids (0.05%, w/v) and non-labelled 20 amino acid mixture (0.1 mM of each). Two types of highly purified cultures of MK-D1 were used as inocula: a co-culture with Methanobacterium sp. strain MO-MB1 and a tri-culture with Halodesulfovibrio and Methanogenium. The vials were incubated at 20 °C in the dark without shaking for 120 days. A reference cultivation was also performed under the same cultivation conditions without the addition of the 20 stable-isotope-labelled amino acid mixture (Extended Data Table 2). The detailed sample preparation and analysis method using NanoSIMS is described in the Supplementary Methods.

Chemical analysis

The stable carbon isotope compositions of methane and CO 2 in the sampled gas phase were analysed as described previously62. Methane concentrations were measured by GC (GC-4000, GL Science) using a Shincarbon ST 50/80 column (1.0 m × 3.0 mm inner diameter; Shinwa Chemical Industries) and a flame ionization detector with nitrogen as a carrier gas.

Amino acid concentrations in pure co-cultures of MK-D1 and Methanogenium were quantified through a previously described method63,64. In brief, we processed the acid hydrolysis with 6 M HCl (110 °C, 12 h) for the culture liquid samples after filtration using a 0.2-μm pore-size polytetrafluoroethylene filter unit (Millipore). The amino acid fraction was derivatized to N-pivaloyl iso-propyl esters before GC using a 6890N GC instrument connected to the nitrogen phosphorus and flame ionization detectors (Agilent Technologies). For cross-validation of qualitative identification of amino acids, GC–MS on the 7890 system (Agilent Technologies) was used61.

Genome sequencing and assembly

DNA extraction was performed as described previously53. Mate-paired library with an average insert size of 3,000 bp was constructed according to the manufacturer’s instructions with Nextera Mate Pair Library Preparation kit (Illumina). Library sequencing was performed using Illumina MiSeq platform (2 × 300 bp), which resulted in 3,822,290 paired reads. The mate pair reads were processed as follows: adapters and low-quality sequences were removed using Trimmomatic v.0.3363 (ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:8:true LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:100), and the linker sequences were removed using NextClip v.1.3.165. De novo assembly was performed using SPAdes v.3.1.166 with multiple k-mer sizes (21, 33, 55, 77 and 99), which resulted in 3,487 contigs with lengths >500 bp, totalling up to 14.68 Mb. The software MyCC67 was used with default parameters for binning based on genomic signatures, marker genes and contig coverages. As heterogeneity in the sequence can cause highly fragmented or redundant contigs, the ambiguous contigs (sequence coverage <5 or a length < 1kb) and redundant contigs were discarded from binning. This resulted in the recovery of genomes related to Lokiarchaeota (that is, Ca. P. syntrophicum MK-D1, 4.46 Mb), Halodesulfovibrio (4.13 Mb) and Methanogenium (2.33 Mb). Scaffolds for each bin were constructed using SSPACE v.3.068 with mate-paired information of Illumina reads. To obtain the complete genome sequence of Ca. P. syntrophicum, the gaps were filled using Sanger sequencing. Genomes were annotated using Prokka v.1.1269 and manually curated. The curation involved functional domain analysis through CD-Search (CDD v.3.17) with its corresponding conserved domain database70,71 and InterProScan v.572; signal peptide and transmembrane domain prediction through SignalP v.4.173; carbohydrate-active enzyme, peptidase and lipase prediction through dbCAN v.5.074, MEROPS75 and lipase engineering database76; and hydrogenase annotation with assistance from HydDB77. In addition, to further verify the function, we compared the sequence similarity of each gene to enzymes found in UniProtKB/SwissProt that had experimentally verified catalytic activity and genes with extensive genetic, phylogenetic and/or genomic characterizations78,79 with a 40% amino acid similarity cut-off. For enzymes that have divergent functions even with a 40% similarity cut-off (for example, [FeFe] and [NiFe] hydrogenases, 3-oxoacid oxidoreductases, glutamate dehydrogenases and sugar kinases), phylogenetic trees were constructed with reference sequences to identify association of the query sequences to phylogenetic clusters containing enzymes with characterized catalytic activity. Publicly available metagenome-assembled genomes of Asgard archaea were annotated in the same manner.

Phylogenetic analysis

Phylogenomic trees of MK-D1 and select cultured archaea, eukaryotes and bacteria were calculated. Thirty-one ribosomal proteins conserved across the three domains (Supplementary Table 7) were collected from MK-D1, the organisms shown in the tree and metagenome-assembled genomes (MAGs) of uncultured archaeal lineages (Supplementary Table 8). Two alignments were performed in parallel: (1) only including sequences from cultured organisms and (2) also including MAG-derived sequences. MAFFT v.7 (--linsi) was used for alignment in both cases80. For the latter, MAG-derived sequences were included to generate an alignment that maximizes the archaeal diversity that is taken into account, but removed for subsequent tree construction to avoid any influence of contamination (that is, concatenation of sequences that do not belong to the same organism). ‘Candidatus Korarchaeum’ sequences were kept in the tree based on the cultured + uncultured alignment due to its critical position in TACK phylogeny. After removing all-gap positions and concatenation, the maximum-likelihood trees were constructed using RAxML-NG v.0.8.081 (fixed empirical substitution matrix (LG), 4 discrete GAMMA categories, empirical amino acid frequencies and 100 bootstrap replicates) and the Bayesian inference phylogenies were calculated using MrBayes v.3.2.7a82 (four chains, print/sample frequencies of 100, a relative burn-in of 25% (nchains = 4 nruns = 2 printfreq = 100 samplefreq = 100), LG model, invariable sites plus GAMMA models of rate variation across sites (prset aamodellpr = fixed(lg); lset rates = invgamma)). For 16S ribosomal RNA phylogeny, sequences were aligned using SINA83 against the Silva v.132 alignment84. The maximum-likelihood tree was calculated using RAxML85 using the same parameters as RAxML-NG.

For analysis of urocanate hydratase, serine/threonine dehydratase, succinate dehydrogenase flavoprotein, fatty-acid-CoA ligase and 3-ketoacyl-CoA thiolase, homologues were collected through BLASTp86 analysis of the Asgard archaea sequences against the UniProt database (release 2019_05). Asgard archaea protein sequences unavailable in GenBank or UniProt (that is, those without accession numbers in the trees) were predicted with Prokka v.1.1369 (--kingdom Archaea --rnammer) using the genome assemblies available in GenBank. Of homologues with sequence similarity ≥40% and overlap ≥70%, representative sequences were selected using CD-HIT v.4.8.187 with a clustering cut-off of 70% similarity (default settings otherwise). Additional homologues with verified biochemical activity, sequence similarity ≥30%, and overlap ≥70% were collected through BLASTp86 analysis of the Asgard archaea sequences against the UniProt/SwissProt database (2019_05)88. Sequences were aligned using MAFFT v.780 with default settings (or MUSCLE v.3.8.3189 where noted) and trimmed using trimAl v.1.290 (settings are specified in the caption for each corresponding phylogenetic tree). RAxML-NG81 was used for tree construction with the same parameters above (or PhyML v.3.391 with 100 bootstrap replicates, LG model and empirical amino acid frequencies where noted). For analysis of biotin ligase and biotin carboxyl carrier protein, the phylogenetic tree was constructed using FastTree92 using the LG model and 1,000 bootstrap replicates.

RNA-based sequencing analysis

To perform RNA-based sequencing analysis, 100 ml of culture liquid was prepared from 5 highly purified cultures that were incubated with casamino acids, 20 amino acids and powdered milk for about 100 days at 20 °C. Before RNA extraction, the growth of MK-D1 was confirmed using qPCR, and the cells density levels were around 105 copies ml−1 in each culture.

To collect microbial cells, the culture liquid was filtered through a 0.22-μm pore-size mixed cellulose ester membrane filter (GSWP01300, Merck MilliPore) on a clean bench. After filtration, the membrane was cut in half with sterilized scissors and then directly inserted into the PowerBiofilm bead tubes of a PowerBiofilm RNA Isolation kit (MO BIO Laboratories). The following RNA extraction procedures were performed according to the manufacturer’s instructions. The extracted RNA was applied to an RNA Clean & Concentrator Kit-5 (Zymo Research) for concentration. The obtained RNA was quantified using an Agilent 2100 Bioanalyzer system with an RNA Pico kit (Agilent Technologies) and then applied to an Ovation Universal RNA-Seq System (NuGEN Technologies) for the construction of an RNA-sequence library. At the step for Insert Dependent Adaptor Cleavage technology-mediated adaptor cleavage during the library construction, specific primers for 16S rRNA and 23S rRNA genes of MK-D1 were used to reduce rRNA gene sequences from the cDNA pool. The constructed cDNA library was sequenced using the MiSeq platform (Illumina).

The raw RNA sequencing data were trimmed by removal of the adapters and low-quality sequences using Trimmomatic v.0.3393. The expression abundance of all coding transcripts was estimated in RPKM values using EDGE-pro v.1.3.194.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.