High solids induced metabolic reprogramming of acetoclastic and hydrogenotrophic methanogenesis during anaerobic co-digestion of food waste and sewage sludge

High solids induced metabolic reprogramming of acetoclastic and hydrogenotrophic methanogenesis during anaerobic co-digestion of food waste and sewage sludge | 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 High solids induced metabolic reprogramming of acetoclastic and hydrogenotrophic methanogenesis during anaerobic co-digestion of food waste and sewage sludge Hemapriya Srinivasan, Pallavi Vadla, Sankar Ganesh Palani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9174971/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract High-solids anaerobic digestion (AD) often suffers from instability due to volatile fatty acid (VFA) accumulation, leading to reduced biogas yields. This study elucidates metabolic adaptations governing process stability under high-solids conditions (19.8–28.3% TS) and elevated organic loading (140–260 g VS/L). Anaerobic co-digestion of food waste (FW) and sewage sludge (SS) at varying ratios (100%FW, 75%FW:25%SS, 50%FW:50%SS, 25%FW:75%SS, 100%SS) identified the 50:50 ratio as optimal, achieving a maximum biogas yield of 0.37 m 3 /kg VS and 66.77% COD reduction. Process instability induced by transient pH decline triggered metabolic reprogramming, characterized by suppression of acetoclastic methanogenesis and enrichment of hydrogenotrophic pathway. These shifts were systematically evaluated using integrated indicators including CH 4 /CO 2 ratios, hydrogen dynamics, VFA speciation, and pH–pKa differentials. Multivariate analysis revealed BOD (r = 0.73) and volatile solids (r = 0.67) as key drivers of biogas production, while pKa showed a strong negative correlation (r = -0.67), indicating inhibition under high-stress conditions. Metagenomic profiling confirmed enrichment of hydrogenotrophic methanogens, particularly Methanobacterium (11.9%) and Methanoculleus (5.9%). Network analysis (216 nodes) revealed structured interactions among acidogenic (n = 51), acetogenic (n = 46), and methanogenic (n = 13) communities. These findings provide a mechanistic framework for enhancing AD stability under high organic loading. Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Food waste Sewage sludge High solids Volatile fatty acid Methane 16S rRNA Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 May, 2026 Reviews received at journal 26 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 13 Apr, 2026 Submission checks completed at journal 26 Mar, 2026 First submitted to journal 26 Mar, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9174971","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":629751480,"identity":"b5de2f42-321c-4632-a16b-7995ac34035f","order_by":0,"name":"Hemapriya Srinivasan","email":"","orcid":"","institution":"Birla Institute of Technology and Science - Hyderabad Campus","correspondingAuthor":false,"prefix":"","firstName":"Hemapriya","middleName":"","lastName":"Srinivasan","suffix":""},{"id":629751481,"identity":"fb0fcc0d-0202-4fe5-a481-9302d97c9c64","order_by":1,"name":"Pallavi Vadla","email":"","orcid":"","institution":"Birla Institute of Technology and Science - Hyderabad Campus","correspondingAuthor":false,"prefix":"","firstName":"Pallavi","middleName":"","lastName":"Vadla","suffix":""},{"id":629751484,"identity":"2c15fcb8-0d48-4b3e-a0ec-3be66857a533","order_by":2,"name":"Sankar Ganesh Palani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABWklEQVRIie2RMWvCUBDHLwTS5cWsAU3tR0gJCIWSfhXDA6dgh44t9IXAc1LXSr9ER8cnAV1iuwp2aBHEIUKkUHRQekktGCKdC82f48h73C//u3sAhQr9XV3WATAkhqFjRpESJpF8KUeRRgbxEoYovyPBIQLfNscrAaqtcbDY9F+a5ogO403fNljZb72v4bWinIwHAu5sKJXZIWKGzcZFO5zemOGc9tohtVhl4PkE5kQhzbqAIQWlIjIIuDVT5VPnaeJaoHLZYbrj+dIuwFlcU4CCodczjXWj2vmWPyNy/SFt+X2KeGtARIsQ2eUQwJ/PVC4SF1lWeZAijCSIji4SzyHmJKrJBqdOL5xbshGOLI6zPKaz6OjidCjJNeZaqyW3nc6IzqRl/9bolltvK9zYlaa5Vhx/2qfVh2xjuH89yWc/W9k/x/6IxST/NHKc2rHsrcgXFipUqNB/1RcRzIHN5k8k+gAAAABJRU5ErkJggg==","orcid":"","institution":"Birla Institute of Technology and Science - Hyderabad Campus","correspondingAuthor":true,"prefix":"","firstName":"Sankar","middleName":"Ganesh","lastName":"Palani","suffix":""}],"badges":[],"createdAt":"2026-03-20 05:08:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9174971/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9174971/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108182478,"identity":"03ca6d8a-0727-4dc3-a091-03b782665366","added_by":"auto","created_at":"2026-04-30 08:59:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2375062,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9174971/v1_covered_baaf8833-813d-48c1-8c06-50e9531358a4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High solids induced metabolic reprogramming of acetoclastic and hydrogenotrophic methanogenesis during anaerobic co-digestion of food waste and sewage sludge","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Food waste, Sewage sludge, High solids, Volatile fatty acid, Methane, 16S rRNA","lastPublishedDoi":"10.21203/rs.3.rs-9174971/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9174971/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh-solids anaerobic digestion (AD) often suffers from instability due to volatile fatty acid (VFA) accumulation, leading to reduced biogas yields. This study elucidates metabolic adaptations governing process stability under high-solids conditions (19.8\u0026ndash;28.3% TS) and elevated organic loading (140\u0026ndash;260 g VS/L). Anaerobic co-digestion of food waste (FW) and sewage sludge (SS) at varying ratios (100%FW, 75%FW:25%SS, 50%FW:50%SS, 25%FW:75%SS, 100%SS) identified the 50:50 ratio as optimal, achieving a maximum biogas yield of 0.37 m\u003csup\u003e3\u003c/sup\u003e/kg VS and 66.77% COD reduction. Process instability induced by transient pH decline triggered metabolic reprogramming, characterized by suppression of acetoclastic methanogenesis and enrichment of hydrogenotrophic pathway. These shifts were systematically evaluated using integrated indicators including CH\u003csub\u003e4\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e ratios, hydrogen dynamics, VFA speciation, and pH\u0026ndash;pKa differentials. Multivariate analysis revealed BOD (r\u0026thinsp;=\u0026thinsp;0.73) and volatile solids (r\u0026thinsp;=\u0026thinsp;0.67) as key drivers of biogas production, while pKa showed a strong negative correlation (r = -0.67), indicating inhibition under high-stress conditions. Metagenomic profiling confirmed enrichment of hydrogenotrophic methanogens, particularly \u003cem\u003eMethanobacterium\u003c/em\u003e (11.9%) and \u003cem\u003eMethanoculleus\u003c/em\u003e (5.9%). Network analysis (216 nodes) revealed structured interactions among acidogenic (n\u0026thinsp;=\u0026thinsp;51), acetogenic (n\u0026thinsp;=\u0026thinsp;46), and methanogenic (n\u0026thinsp;=\u0026thinsp;13) communities. These findings provide a mechanistic framework for enhancing AD stability under high organic loading.\u003c/p\u003e","manuscriptTitle":"High solids induced metabolic reprogramming of acetoclastic and hydrogenotrophic methanogenesis during anaerobic co-digestion of food waste and sewage sludge","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 20:06:55","doi":"10.21203/rs.3.rs-9174971/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-13T12:57:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T13:34:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103967227774225913755474118646205763235","date":"2026-04-23T05:07:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206245605595593004794926898143479973804","date":"2026-04-22T10:05:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26989685889657467551337710274293498147","date":"2026-04-21T15:18:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T14:12:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T03:57:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T16:53:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-26T16:46:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ed648fb-e2f4-4e02-9cfd-50a812168414","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-13T12:57:35+00:00","index":16,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67027848,"name":"Earth and environmental sciences/Environmental sciences"},{"id":67027849,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-29T20:06:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 20:06:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9174971","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9174971","identity":"rs-9174971","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}