Abstract
Methanogenic archaea are main contributors to methane emissions, and have a crucial role in carbon cycling and global warming. Until recently, methanogens were confined to Euryarchaeota, but metagenomic studies revealed the presence of genes encoding the methyl coenzyme M reductase complex in other archaeal clades1,2,3,4, thereby opening up the premise that methanogenesis is taxonomically more widespread. Nevertheless, laboratory cultivation of these non-euryarchaeal methanogens was lacking to corroborate their potential methanogenic ability and physiology. Here we report the isolation of a thermophilic archaeon LWZ-6 from an oil field. This archaeon belongs to the class Methanosuratincolia (originally affiliated with ‘Candidatus Verstraetearchaeota’) in the phylum Thermoproteota. Methanosuratincola petrocarbonis LWZ-6 is a strict hydrogen-dependent methylotrophic methanogen. Although previous metagenomic studies speculated on the fermentative potential of Methanosuratincolia members, strain LWZ-6 does not ferment sugars, peptides or amino acids. Its energy metabolism is linked only to methanogenesis, with methanol and monomethylamine as electron acceptors and hydrogen as an electron donor. Comparative (meta)genome analysis confirmed that hydrogen-dependent methylotrophic methanogenesis is a widespread trait among Methanosuratincolia. Our findings confirm that the diversity of methanogens expands beyond the classical Euryarchaeota and imply the importance of hydrogen-dependent methylotrophic methanogenesis in global methane emissions and carbon cycle.
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Data availability
The 16S rRNA gene amplicon sequences, metagenomic, genomic and transcriptome data generated in the current study are publicly available under BioProject accession number PRJNA992765 and at the NODE database (https://www.biosino.org/node/project/detail/OEP003742). Further details are provided in Supplementary Table 7. All other data are presented in the Article and its Supplementary Information. Source data are provided with this paper.
Code availability
The codes and programs used for analyses are mentioned in the Methods, and they are available at GitHub (https://github.com/zhuozhou1993/Methanosuratus/blob/main/code).
Change history
02 September 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41586-024-08000-z
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Acknowledgements
We thank P. Geesink for performing Nanopore sequencing of strain LWZ-6; W. B. Whitman for comments on the manuscript; L.-r. Dai, M. Yang and L. Fu for assisting in cultivation and experiments; Z. Zhou for technical support; and P. Vandamme for advice on the etymology of the new names proposed. This study was supported by National Natural Science Foundation of China (92051108, 92351301, 31970066, 42203080 and 42207167), Agricultural Science and Technology Innovation Project of the Chinese Academy of Agriculture Science (CAAS-ASTIP-2021-BIOMA-01), CAAS Center for Science in Agricultural Green and Low Carbon (CAAS-CSGLCA-202301), Sichuan Science and Technology program (2024NSFTD0093), Netherlands Ministry of Education, Culture and Science (project 024.002.002: Soehngen Institute of Anaerobic Microbiology), European Research Council (grant 817834), the Dutch Research Council (grant VI.C.192.016), the Central Public-interest Scientific Institution Basal Research Fund (1610012017002_05103), the Stable Support Plan Program of Shenzhen Natural Science Fund (20200925173954005) and the Shenzhen Key Laboratory of Marine Archaea Geo-Omics, Southern University of Science and Technology (ZDSYS201802081843490). The Article is dedicated to our co-author W.-H.H.
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Contributions
L.C. initiated the study. L.C., D.Z.S., K.-j.W., T.J.G.E. and L.Z. designed the research. L.-y.L. performed the initial cultivation. K.-j.W., L.Z., L.-y.L. and J.L. performed the isolation process and physiological experiments. G.T. and W.-h.H. performed all bioinformatics analyses. C.-p.D. performed CARD-FISH. J.-c.Z. performed NanoSIMS. F.-f.Z. and C.-l.Z. performed lipid-SIP. L.F. and L.-p.B. performed biochemical analysis. K.-j.W. and L.Z. analysed data. X.-z.D. provided constructive suggestions on the isolation process. K.-j.W., L.C., X.-z.D., T.J.G.E. and D.Z.S. wrote the manuscript with the contributions from all of the authors.
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Extended data figures and tables
Extended Data Fig. 1 Methanosuratincolia communities were detected in numerous enrichments.
The enrichments were initially established from oily sludge or oil-produced water and incubated with various substrates such as acetate, propionate, butyrate, alkanes, and oil at temperatures ranging from 25 °C to 75 °C. Alkanes mix includes n-docosane, n-hexadecylcyclohexane, and n-hexadecylbenzene. The blank represents no enrichments performed on these conditions. Abundance 20, 40, and 60 show the relative abundance of Methanosuratincolia populations in total archaea by 16S rRNA gene amplicon sequencing.
Extended Data Fig. 2 Consecutive subcultures amended with methanol as a substrate to eliminate Methanoculleus recptaculi (Mcl).
a, CH4 production, b, the copy numbers of LWZ-6, c, the copy numbers of Mcl. Culture-1,2,3 represent triplicate tubes used for subcultures. The arrows indicate the subculture points, the subculture dilution varied from 0.1% to 10%. Copy numbers of LWZ-6 and Mcl were determined by qPCR with the primers mcr4F/mcr4R and ZC2F/ZC2R targeting their mcrA and 16S rRNA genes.
Extended Data Fig. 3 The microbial community compositions of the co-culture during 5 consecutive transfer incubations.
The relative abundance of strain LWZ-6 and CY-2 was determined by 16S rRNA gene amplicon sequencing using the primer 515FmodF/806RmodR66. The samples in each generation were collected from the cultures grown at the exponential phase of methane production.
Extended Data Fig. 4 Consecutive subcultures amended with antibiotics to eliminate Acetomicrobium sp. CY-2.
a, CH4 production, b, CO2 change, c, the copy numbers of LWZ-6 determined at the endpoint of each generation, d, the copy numbers of CY-2 determined at the endpoint of each generation, ND means the copy numbers are under detectable level, the subculture dilution varied from 1%-10%, e, the proportion of CY-2 in the total bacteria and archaea, f, gel electrophoresis of PCR products using 27F/1492R61 targeting bacteria, PC: positive control of coculture, NC: negative control of ddH2O. Samples were run on one gel and the lanes in images are representative blot of n = 3 biological replicates.
Extended Data Fig. 5 Microscopy observation of the Methanosuratincolia co-culture (strain LWZ-6/CY-2).
a, d, CARD-FISH showing cells hybridized with nucleotide probes that target archaea ARCH-915 (green) and bacteria EUB-338 (red). b, e, Fluorescence images hybridized with nucleotide probes that target archaea ARCH-915 (green), representative images are from 18 recorded images of n = 3 biological replicates. c, f, fluorescence images hybridized with nucleotide probes that target bacteria EUB-338 (red). g, h, TEM showing ultrathin section of the co-culture, the black arrows are strain LWZ-6, representative images are from 8 recorded images of n = 2 biological replicates. Scale bars: 10 μm in (a-c), 2 μm (d-f), 200 nm (g-h).
Extended Data Fig. 6 Methane activity and growth dynamics of the Methanosuratincolia co-culture (strain LWZ-6/CY-2).
a, the CH4 production, b, methanol consumption, c, H2 change, d, copies numbers of strain LWZ-6, e, copies numbers of bacteria in the 50 ml fresh medium. Three groups with different substrates were set up: 0.5 g l−1 yeast extract (YE); 0.5 g l−1 YE, 10 mM methanol and 10 kPa H2 (YE + methanol + H2); without substrates addition, YE (-). All symbols represent means of three individual incubations; error bars represent SD of triplicates; the invisible error bars are smaller than symbols.
Extended Data Fig. 7 The changes of the carbon isotope composition of CH4 (a) and CO2 (b).
The Methanosuratincolia co-culture was incubated with initial different ratios of (1.76 ± 0.01)%, (2.3 ± 0.10)%, (3.65 ± 0.08)%, and (5.98 ± 0.79)%.
Extended Data Fig. 8 The physiological properties of strain LWZ-6.
a, C1-methylated compounds utilization, b, growth factors, c, Temperature, d, pH, e, NaCl tolerance concentration. µ represents the maximum specific growth rate of strain LWZ-6 or the specific CH4 production. MeOH: methanol, MMA: monomethylamine, DMA: dimethylamine, TMA: trimethylamine, MeSH: methanethiol, YE: yeast extract, CA: casamino acids, ND indicates no growth of strain LWZ-6 or CH4 detected. Data in a and b are mean ± standard deviation of triplicates. All symbols represent means of three individual incubations, error bars represent SD of triplicates, the invisible error bars are smaller than symbols.
Extended Data Fig. 9 Global distribution of Methanosuratincolia in different biotopes.
The bold circles represent the sampling locations of Methanosuratincolia MAGs. The numbers of the genomes in environments are anaerobic digester: 6, groundwater: 3, freshwater/sediment: 2, hot spring: 36, hydrothermal sediment: 10, petroleum field: 3. The triangles represent Methanosuratincolales 16S rRNA gene sequence derived from IMNGS (Detailed information was listed in Supplementary Table 5).
Supplementary information
Supplementary Information
Supplementary Figs. 1–3.
Supplementary Table 1
Genome annotation of M. petrocarbonis LWZ-6.
Supplementary Table 2
Transcript of M. petrocarbonis LWZ-6 grown on methanol and H2.
Supplementary Table 3
General information on the 23 Methanosuratincolia genomes obtained in our study.
Supplementary Table 4
Methanogenetic features and energy mechanism of five Methanosuratincolia clusters in our study, other Methanosuratincolales, ‘Ca. Nezhaarchaeales’ and ‘Ca. Culexarchaeales’ genomes and other H2-dependent methylotrophic methanogens in Euryarchaeota. 1, present; 0, absent.
Supplementary Table 5
Global distribution of 16S rRNA gene sequences and MAGs of Methanosuratincolia in different biotopes.
Supplementary Table 6
Primers and probes used in this study.
Supplementary Table 7
Summary of sequencing data submitted to the NCBI and The National Omics Data Encyclopedia.
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Wu, K., Zhou, L., Tahon, G. et al. Isolation of a methyl-reducing methanogen outside the Euryarchaeota. Nature 632, 1124–1130 (2024). https://doi.org/10.1038/s41586-024-07728-y
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DOI: https://doi.org/10.1038/s41586-024-07728-y
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