The methanogenic degradation of oil hydrocarbons can proceed through syntrophic partnerships of hydrocarbon-degrading bacteria and methanogenic archaea1,2,3. However, recent culture-independent studies have suggested that the archaeon ‘Candidatus Methanoliparum’ alone can combine the degradation of long-chain alkanes with methanogenesis4,5. Here we cultured Ca. Methanoliparum from a subsurface oil reservoir. Molecular analyses revealed that Ca. Methanoliparum contains and overexpresses genes encoding alkyl-coenzyme M reductases and methyl-coenzyme M reductases, the marker genes for archaeal multicarbon alkane and methane metabolism. Incubation experiments with different substrates and mass spectrometric detection of coenzyme-M-bound intermediates confirm that Ca. Methanoliparum thrives not only on a variety of long-chain alkanes, but also on n-alkylcyclohexanes and n-alkylbenzenes with long n-alkyl (C≥13) moieties. By contrast, short-chain alkanes (such as ethane to octane) or aromatics with short alkyl chains (C≤12) were not consumed. The wide distribution of Ca. Methanoliparum4,5,6 in oil-rich environments indicates that this alkylotrophic methanogen may have a crucial role in the transformation of hydrocarbons into methane.
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The 16S rRNA gene amplicon sequences, metagenomic and metatranscriptomic data generated in current study are available in the NODE database (http://www.biosino.org/node/project/detail/OEP001282). The data of dereplicated MAGs analysed during the current study are available in the NODE database under the accession numbers OEZ006960 and OEZ007009–OEZ007026. Further details are provided in Supplementary Table 13. All other data are available in the main text or the Supplementary Information.
The sources of the code and programs used for analyses are mentioned in the Methods, and are also available at GitHub (https://github.com/liupfskygre/Methanoliparum_MS_code/tree/main).
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We thank A. Oren (The Hebrew University of Jerusalem) for discussing the naming of the different Ca. Methanoliparum species; R. Conrad and W. B. Whitman for discussing the manuscript; K. Wrighton for providing access to the server Zenith; Q. Yuan, Y. Liu, J. Pan, M.-w. Cai and Y.-n. Tang for assisting in data analysis; L.-r. Dai, D. Zhang and L. Li for assisting in cultivation and experiments; and Z. Zhou for technical support. This study was supported by National Natural Science Foundation of China (nos 92051108, 91851105, 41802179, 31970066, 31570009 and 31970105), Agricultural Science and Technology Innovation Project of the Chinese Academy of Agriculture Science (no. CAAS-ASTIP-2016-BIOMA), the Innovation Team Project of Universities in Guangdong Province (no. 2020KCXTD023) and the Shenzhen Science and Technology Program (no. JCYJ20200109105010363), the Fundamental Research Funds for the Central Universities (LZUJBKY-2021-KB16), the Central Public-interest Scientific Institution Basal Research Fund (Y2021PT02, Y2021XK06). R.L.-P. was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy (EXC-2077-390741603) via Excellence Chair Victoria Orphan. G.W. was funded by DFG under Germany’s Excellence Strategy-EXC-2077-390741603 and the Max Planck Society.
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Molecular characterization of the methanogenic oily sludge incubated at different temperatures.
a, Accumulation of methane in the headspace of treatments at different temperatures over an incubation time of 301 days. The estimates of reported methane production rates base on the time interval for the formation of 5% and 90% of the maximum methane formation. b, Mass spectrometric analysis of extracted residual oil for n-alkanes m/z = 85, n-alkylcyclohexanes m/z = 82, n-alkylbenzenes m/z = 92. Exemplary data of the 55 °C culture is presented in Figs. 1b–1d. Data shown are mean ± standard deviation (n = 3 biologically independent replicates). c and d, Archaeal and bacterial community structure revealed by amplicon sequencing in the different temperature treatments after 204 days of incubation, respectively. Only families with relative abundances ≥ 1% are shown. “Other” indicates the sum of groups with relative abundance < 1%. Data shown are mean - standard deviation (n = 3 biologically independent replicates).
a-c, Visualization of archaea (green) and bacteria (red). d-f, Visualization of ‘Ca. Methanoliparum’ (green) and archaea (red). Hybridization of ‘Ca. Methanoliparum’ with the general archaeal probe and the specific DC06-660Mlp probe. The vast majority of archaea hybridized also with the probe for ‘Ca. Methanoliparum’. g-i, Visualization of ‘Ca. Methanoliparum’ (green) and bacteria (red). Oligonucleotide probes were ARCH-915 for archaea, EUB388 I-III for bacteria and DC06-660Mlp for ‘Ca. Methanoliparum’. Three representative recorded images from n = 3 independent samples (a-i, 9 rows of images in total) of one culture are shown. Scale bars in all images are 10 µm.
Extended Data Fig. 3 Phylogenetic analyses of MAGs and 16S rRNA gene sequences of ‘Ca. Methanoliparia’.
a, Phylogenomic analyses of ‘Ca. Methanoliparia’ MAGs based on the concatenated alignments of 16 ribosomal proteins67. Bootstrap values > 0.95 are marked with grey dots, ‘Ca. Bathyarchaeota’ set as outgroup. The maximum-likelihood tree was constructed by using the IQ-TREE software with the parameters ‘-m WAG -bb 1000’. b, Phylogenetic analysis of 16S rRNA gene sequences retrieved from all ‘Ca. Methanoliparia’ MAGs. For MAG- derived sequences source information is given: i.e., T55 indicates temperature of the culture (55 °C) and after the MAGs (bin) number the substrate used is indicated (e.g., n-hexadecane). The asterisk (*) marking ‘Ca. M. whitmanii’ sequence identifiers indicates 16S rRNA genes that were truncated during assembly. In these cases, the longest partial sequence was used for the phylogenetic analyses. The 16S rRNA gene sequences were added to the consensus tree with ‘quick add’ option, thus no bootstrap values are available.
Extended Data Fig. 4 Identities between ‘Ca. Methanoliparum’ clusters and phylogenetic analysis of their AcrA and McrA protein sequences.
a, Identities of the 16S rRNA gene. b, Genome based average Amino Acid Identity (AAI). c, Genome based Average Nucleotide Identity (ANI). d, Identity based on the percentage of conserved proteins (POCP). All matrices consistently showed that all ‘Ca. Methanoliparia’ MAGs from this study grouped into four species-level clusters within the genus ‘Ca. Methanoliparum’. In the box plots the central line represents the median; the lower and upper box limits correspond to the 25th and 75th percentiles, respectively; Numbers represent the times of pairwise comparisons of MAGs between two groups. Cluster 1 (C1): ‘Ca. M. thermophilum’; Clusters 2 (C2): ‘Ca. M. widdelii’; Cluster 3 (C3): ‘Ca. M. whitmanii’; Cluster 4 (C4): ‘Ca. M. zhangii’. Mv indicates the genomes of the sister marine clade ‘Ca. Methanolliviera’. e, Maximum-likelihood tree of the protein sequences of AcrA and McrA present in ‘Ca. Methanoliparum’ MAGs retrieved in the present studied. Different colours indicate the different ‘Ca. Methanoliparum’ species. Numbers in parenthesis indicate the number of acrA/mcrA sequences detected in the different metagenomes. In each MAG, maximum one acr and one mcr were detected. Trees were constructed by using IQ-TREE with the parameters ‘-m WAG, -bb 1000’, with bootstrap values >0.95 shown in grey dots.
Extended Data Fig. 5 Gene clusters associated with the alkane degradation and methanogenesis pathways detected in the representative MAGs of the four ‘Ca. Methanoliparum’ species.
Several copies of fadD and ACADM were detected and only copies with the highest transcript abundances are shown. In orange, alkane activation and conversion to a fatty acid; in blue, beta oxidation pathway and in red, the ACS/CODH complex and the methanogenesis pathway. Details of all copies are included in Supplementary Table 6.
Extended Data Fig. 6 Relative transcript abundances of alkane-degrading and methane-producing pathways coding genes.
The colour code shows the log2(FPKM) values of each gene. For enzymes or subunits with several putative coding genes, only the ones with the highest level of log2(FPKM) are shown here. Two samples were taken for cultures with n-hexadecane addition (Hex.) at day 31 and 55, while sampling at one time point (day 55) with 3 replicates (designated as r1-r3) was performed for control cultures without n-hexadecane amendment (Con.). Grey cells indicate that the corresponding genes were not found in the MAGs. Details of all copies are included in Supplementary Table 7.
Extended Data Fig. 7 Identification of coenzyme M derivatives in cultures by HPLC-MS/MS based on the corresponding retention times.
a and b, hexadecyl-CoM and the corresponding 3 characterized fragments (in blue) in cell extracts from cultures with hexadecane (C16H34) addition. c and d, eicosyl-CoM and 3 characterized fragments (in blue) in cell extracts from cultures with eicosane (C20H42) additions. Standard appears in black primary anions and second anions (produced by fragmentation) detected in hexadecane and eicosane cultures showed the same retention time as the synthetic standards of hexadecyl-CoM and eicosyl-CoM, respectively.
Extended Data Fig. 8 Identification of coenzyme M derivatives in cultures incubated with specific hydrocarbons.
a, Scheme for the activation of long-chain alkanes and alkyl-substituted compounds as CoM thioethers in ACR, and their expected fragmentation patterns. The residual ‘R-’ describes a methyl-, cyclohexane- or aromatic unit with an alkyl chain CnH2n+1 for n ≥ 13. Dash arrows and numbers above indicate the fragmentation positions. b and c, QE Plus-Orbitrap MS analyses of cultures supplemented with eicosane resulted in a mass peak of eicosyl-CoM (C20H41-SC2H4SO3− at m/z = 421.28162 and the fragments eicosyl-thiol (C20H33S-, m/z = 313.29373), ethenesulfonate (C2H3SO3−, m/z = 106.98092) and bisulfite (HSO3−, m/z = 80.96519). All peaks match those of an eicoysl-CoM standard. d-i, QE Plus-Orbitrap MS analyses of cultures supplemented with a mixture of n-docosane (C22H46), n-hexadecyl benzene (C22H38) and n-hexadecyl cyclohexane (C22H44) as substrates, and detection of d and e docosyl-CoM (C24H49S2O3−, m/z = 449.31134) with the fragment C22H45S− (m/z = 341.32495); of f and g n-hexadecyl benzene coenzyme M (C24H41S2O3−, m/z = 441.25064) with the predicted fragment C22H37S− (m/z = 333.26212) and of h and i n-hexadecyl cyclohexane CoM (C24H47S2O3−, m/z = 447.29730) with the fragment C22H43S− (m/z = 339.30939). The mass error for all mass peaks shown here are < 5 p.p.m.
Microorganisms were cultured using a mixture of n-docosane, n-hexadecyl benzene, n-hexadecyl cyclohexane as substrate. The culture was transferred when 15 to 20 mmol of methane were formed, and 30% to 50% of the culture were transferred. Displayed are transfers 3 to 6. a, Methane formation in the headspace. Grey arrows indicate transfer events. b and c, Abundance of 16S rRNA gene of ‘Ca. Methanoliparum’ and bacteria as determined by qPCR, respectively. d, Relative abundance of main archaeal groups determined by 16S rRNA gene sequencing with archaeal primer set Arch519F/Arch915R.
Extended Data Fig. 10 Proposed metabolic pathway and related gene clusters for benzene-CoA degradation in ‘Ca. Methanoliparum’.
a, Gene clusters found in the four representative MAGs with potential for benzoyl-CoA degradation. Numbers in the gene clusters indicate kilobases. b, Annotations and Locus tag for the corresponding genes shown in panel a that are found in the representative MAG of ‘Ca. M. thermophilum’ (XY_C20_T55_P2_bin.5 of Cluster 1). c, Proposed pathway for the degradation of benzoyl-CoA based on the pairwise comparison of the candidate genes of ‘Ca. Methanoliparum’ (red) with the genes involved in benzoyl-CoA degradation in the model organisms Thauera aromatica (green) and Rhodopseudomonas palustris (blue). The letters for candidate genes of ‘Ca. Methanoliparum’ refer to the letters indicate in the panel a (see Supplementary Table 10 for more details).
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Zhou, Z., Zhang, Cj., Liu, Pf. et al. Non-syntrophic methanogenic hydrocarbon degradation by an archaeal species. Nature 601, 257–262 (2022). https://doi.org/10.1038/s41586-021-04235-2
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