Direct utilization of long-chain fatty acid for methane production by thermophilic Archaeoglobi

Direct utilization of long-chain fatty acid for methane production by thermophilic Archaeoglobi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Direct utilization of long-chain fatty acid for methane production by thermophilic Archaeoglobi Fengping Wang, Tiantian Yu, Shujian Yuan, Yinzhao Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7873196/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The methanogenic degradation of long-chain fatty acids has traditionally been considered to occur through syntrophic partnerships between long-chain fatty acid degrading bacteria and methanogenic archaea. However, recent genomic evidence suggests that certain archaea may independently perform entire process. Here, we report the cultivation of an archaeon from the class Archaeoglobi, Candidatus (Ca.) Methanoglobus sphaerolipidus DLY3, from hot spring sediments in Tengchong, China. Using an integrated approach including targeted cultivation, growth experiments, microscopy, stable isotope tracing, metagenomics, and metatranscriptomics, we demonstrate that Ca. M. sphaerolipidus directly converts long-chain fatty acids to methane. This transformation involves the beta-oxidation pathway, the Wood-Ljungdahl pathway (WLP), and methanogenic methyl-coenzyme M reductase (MCR) and methyltransferases (MTR) complexes—a process we term liparotrophy. In addition to oleic acid, Ca. M. sphaerolipidus is also capable of utilizing methanol as a substrate for methanogenesis. Our findings expand the known substrate range for methanogenic archaea beyond CO₂ reduction, acetoclastic methanogenesis, methylotrophy, methyl reduction, methoxydotrophy, and the recently reported alkylotrophy. Biological sciences/Microbiology/Archaea/Archaeal biology Biological sciences/Microbiology/Environmental microbiology/Water microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 SupplementaryTable2.xlsx Supplementary Table 2 SupplementaryTable4.xlsx Supplementary Table 4 SupplementaryTable3.xlsx Supplementary Table 3 SupplementaryInformation.pdf Supplementary Information SupplementaryTable5.xlsx Supplementary Table 5 SupplementaryTable6.xlsx Supplementary Table 6 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7873196","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":533583910,"identity":"c5734200-bef8-4e5c-8439-db2c1aa9e9bf","order_by":0,"name":"Fengping Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBAC9gYQaQBhGYCFDhDQwnMApgXIMjhAvBYQkEiAqiaohb338OuCgjt2G24+f1D8sY1Bju9GAuPnAnxaeM6lWc8weJa84XaOgcHBNgZjyRsJzNIz8Gixl8gxM+YxOJxsdjuHAaQlccONBDZmHny2yL+Barl5/AFISz1hLRI8xo+BWuzMbjCAHZZgQFALT44Z8wyDwwn2Z4B+OXNOwnDmmYfN0ni1sJ8x/lzw57C9ZPvxZwYVZTbyfMeTD37GpwUI2KSBRGIDkAGMUQkgm7EBvwYGBubPQMIexHhASOkoGAWjYBSMTAAA2PVPEfOv+RAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3429-8410","institution":"Shanghai Jiao Tong University","correspondingAuthor":true,"prefix":"","firstName":"Fengping","middleName":"","lastName":"Wang","suffix":""},{"id":533583911,"identity":"93d42aba-951b-43fc-92d2-c221f82ea5e3","order_by":1,"name":"Tiantian Yu","email":"","orcid":"https://orcid.org/0000-0003-3257-612X","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Tiantian","middleName":"","lastName":"Yu","suffix":""},{"id":533583912,"identity":"af808552-46f7-43fa-ac4c-fc660257ccf4","order_by":2,"name":"Shujian Yuan","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Shujian","middleName":"","lastName":"Yuan","suffix":""},{"id":533583913,"identity":"3a6b0eb3-aef4-4455-8d63-ff70fd1907cc","order_by":3,"name":"Yinzhao Wang","email":"","orcid":"https://orcid.org/0000-0001-8715-1386","institution":"State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Yinzhao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-10-16 04:30:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7873196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7873196/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94246593,"identity":"a0506eba-fc43-4bb9-9638-10e1ed727e42","added_by":"auto","created_at":"2025-10-24 05:38:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":549766,"visible":true,"origin":"","legend":"\u003cp\u003eMethane production and microbial composition of enrichment cultures from hot spring sediments incubated with and without oleic acid. a, Methane accumulation in the headspace of cultures over 238 days. See Supplementary Table 1 for details. b, Relative abundance of Ca. M. sphaerolipidus in the before incubation slurry and in enrichments after 238 days of incubation, as determined by metagenomic sequencing (MG). Other, including the unmapped reads and MAGs with relative abundance \u0026lt; 5%. See Supplementary Table 2 for details. r1 and r2 represent two replicates. c, HCR-FISH analysis of the enrichment culture. Cells were hybridized with probes: Arc915 (targeting archaea, green), a specific probe Sphaero-910 (targeting Ca. M. sphaerolipidus, green), and EUB338 (targeting bacteria, red). Subsequently, all cells were counterstained with 4',6-Diamidino-2-Phenylindole (DAPI, blue). Scale bar, 2 µm. d, Cell morphology in the enrichment culture as observed by transmission electron microscopy (TEM). Scale bar, 2 µm.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7873196/v1/70f26cd6014edb5dca1a1a78.jpg"},{"id":94246596,"identity":"18a008dc-9e7e-40b9-910f-c2a1b5ac5b5a","added_by":"auto","created_at":"2025-10-24 05:38:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":657321,"visible":true,"origin":"","legend":"\u003cp\u003eMethane production and community composition of the methanogeni enrichment cultures containing Ca. M. sphaerolipidus with oleic acid as the substrate. a, methane and carbon dioxide content, and 13 C-labled methane and carbon dioxide content in the cultures amended with 12 C-oleic acid or 13 C-oleic acid. See Supplementary Table 1 for details. b, community composition of the culture based on the relative abundance of the Ca. M. sphaerolipidus genome and other MAGs recovered from metagenomic and metatranscriptomic sequencing (MG+MT). Other, including the unmapped reads and MAGs with relative abundance \u0026lt; 5%. r1 and r2 represent two replicates. See Supplementary Table 2 for details.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7873196/v1/847f55280bf80e226d2aa685.jpg"},{"id":94245956,"identity":"07bb49da-7d41-4a42-a7b9-b55dc60f3dcc","added_by":"auto","created_at":"2025-10-24 05:30:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":641589,"visible":true,"origin":"","legend":"\u003cp\u003eMethane production and community composition of the methanogenic enrichment cultures containing Ca. M. sphaerolipidus with methanol as the substrate. a, methane and carbon dioxide content, and 13 C-labled methane and carbon dioxide content in the cultures amended with 12 C-methanol or 13 C-methanol. See Supplementary Table 1 for details. b, community composition of the culture based on the relative abundance of the Ca. M. sphaerolipidus genome and other MAGs recovered from metagenomic and metatranscriptomic sequencing (MG+MT). Other, including the unmapped reads and MAGs with relative abundance \u0026lt; 5%. r1 and r2 represent two replicates. See Supplementary Table 2 for details.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7873196/v1/ff8484e6a792094096264a3a.jpg"},{"id":94245938,"identity":"7a028744-56ca-4906-8c55-a67e165fdf49","added_by":"auto","created_at":"2025-10-24 05:30:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3433289,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic model of Ca. M. sphaerolipidus. a, genome-inferred model for oleic acid degradation and methane production. b, genome-inferred model for methanol degradation and methane production. The names of genes not found in the genome were shown in brown font. Fdred, reduced ferredoxins; Fdox, oxidized ferredoxins. c, Gene expression of Ca. M. sphaerolipidus during growth on oleic acid and methanol; for enzymes comprising multiple subunits, the sum of FPKM value representing the transcribed enzymes is used. The dots represent significant differences in gene expression levels were observed (p value \u0026lt;0.05; t-test). The steps involving beta- oxidation, WLP, methanogenesis, methyltransferase, energy metabolism, gluconeogenesis, Pentose Phosphate Pathway (PPP) and TCA cycle. List of related genes and gene transcription levels was showed in Supplementary Table 4.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7873196/v1/80ecd90506030dcdeb5237bc.jpg"},{"id":94245943,"identity":"850d47dc-c901-40a1-a4e1-6a98ee20f1ca","added_by":"auto","created_at":"2025-10-24 05:30:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1646139,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenomic affiliation of the AcrAs and McrAs. a, the tree branches were classified into three distinct groups: conventional MCR clade, the TACK clade, and the ACR clade. b, shows MAGs of genus Ca. Methanoglobus. Maximum-likelihood tree, alignments were generated using MAFFT46 and then filtered with trimAl47 , and the trees were built by the IQ-Tree48 method with the model LG + C60 + F + G with 1,000 bootstrap replicates. Bootstrap values \u0026gt;90% shown in black dots.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7873196/v1/665b4bfce1103b5379b513b2.jpg"},{"id":94245917,"identity":"7c7532b6-f7c1-4fb3-bf58-f83aa328624f","added_by":"auto","created_at":"2025-10-24 05:30:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1081519,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenomic affiliation and proposed methane production pathway of mcr-carrying MAGs in genus Ca. Methanoglobus. a, phylogenomic affiliation of the Ca. Methanoglobus MAGs based on 37 conserved protein sequences and using 107 representative archaeal genomes. Maximum-likelihood tree, alignments were generated using MAFFT46 and then filtered with trimAl47 , and the trees were built by the IQ- Tree48 method with the model LG + C60 + F + G with 1,000 bootstrap replicates. Bootstrap values \u0026gt;90% shown in black dots. The mcr-carrying MAGs shown in red, the number in parentheses following each species identifier indicates the count of mcr- carrying MAGs within that species and the total number of MAGs for the species. b, proposed methane production pathway of mcr-carrying MAGs in genus Ca. Methanoglobus. Presence, partial and absence of key metabolic genes and methanogenesis marker proteins. See Supplementary Table 6 for details.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7873196/v1/175a7fda6b984e63d85fb846.jpg"},{"id":94247244,"identity":"8b2b7cf4-42e1-499d-9f4b-1d3ddcc9ec81","added_by":"auto","created_at":"2025-10-24 05:46:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8446826,"visible":true,"origin":"","legend":"Article File","description":"","filename":"manuscript20251016.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7873196/v1_covered_67a67716-2d09-4591-9b63-3b84129467d7.pdf"},{"id":94245950,"identity":"b2eca612-018c-4513-86b6-045952fe4226","added_by":"auto","created_at":"2025-10-24 05:30:03","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19598,"visible":true,"origin":"","legend":"Supplementary Table 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However, recent genomic evidence suggests that certain archaea may independently perform entire process. Here, we report the cultivation of an archaeon from the class Archaeoglobi, Candidatus (Ca.) Methanoglobus sphaerolipidus DLY3, from hot spring sediments in Tengchong, China. Using an integrated approach including targeted cultivation, growth experiments, microscopy, stable isotope tracing, metagenomics, and metatranscriptomics, we demonstrate that Ca. M. sphaerolipidus directly converts long-chain fatty acids to methane. This transformation involves the beta-oxidation pathway, the Wood-Ljungdahl pathway (WLP), and methanogenic methyl-coenzyme M reductase (MCR) and methyltransferases (MTR) complexes—a process we term liparotrophy. In addition to oleic acid, Ca. M. sphaerolipidus is also capable of utilizing methanol as a substrate for methanogenesis. Our findings expand the known substrate range for methanogenic archaea beyond CO₂ reduction, acetoclastic methanogenesis, methylotrophy, methyl reduction, methoxydotrophy, and the recently reported alkylotrophy.","manuscriptTitle":"Direct utilization of long-chain fatty acid for methane production by thermophilic Archaeoglobi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 05:29:56","doi":"10.21203/rs.3.rs-7873196/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-microbiology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmicrobiol","sideBox":"Learn more about [Nature Microbiology](http://www.nature.com/nmicrobiol/)","snPcode":"","submissionUrl":"","title":"Nature Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2ebde675-ffdb-48e7-a8df-88baf8e05515","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56732455,"name":"Biological sciences/Microbiology/Archaea/Archaeal biology"},{"id":56732456,"name":"Biological sciences/Microbiology/Environmental microbiology/Water microbiology"}],"tags":[],"updatedAt":"2026-04-05T02:00:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-24 05:29:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7873196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7873196","identity":"rs-7873196","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}