Accumulation of zinc during E. coli biofilm mineralization: within or surrounding the hydroxyapatite?

Accumulation of zinc during E. coli biofilm mineralization: within or surrounding the hydroxyapatite? | 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 Accumulation of zinc during E. coli biofilm mineralization: within or surrounding the hydroxyapatite? Leona J. Bauer, Laura Zorzetto, Ernesto Scoppola, Yannick Wagener, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7658792/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Biofilms are biological materials that form as bacteria secrete extracellular matrix to protect themselves from environmental challenges. Specific conditions lead to biofilm mineralization, like in dental calculus or kidney stones. To understand biofilm formation and mineralization, we grew Escherichia coli on agar plates containing a nutritive and mineralizing medium. Previous studies showed that the alkaline phosphatase (ALP) present in E. coli biofilms leads to hydroxyapatite precipitation in such conditions. Here, we analyzed the composition of mineralized biofilms with X-ray fluorescence techniques in two and three dimensions. In addition to calcium and phosphate, we found that the traces of zinc introduced via the nutrients and bacteria, also accumulates in the mineralized regions. Wide-angle X-ray scattering performed on dry mineralized biofilm powder also revealed a shortening of the hydroxyapatite crystal lattice, which could indicate partial substitution of calcium by zinc. Using an abiotic model, we reproduced enzymatic hydroxyapatite mineralization with pure ALP, without bacteria nor extracellular matrix. In this model, hydroxyapatite distortion increased with zinc concentration, whereas crystal size decreased. Since zinc is a ubiquitous element that is essential to bacteria metabolism but also antibacterial, these results are fundamental for biofilm research and call for further studies on zinc management in biofilms. Biological sciences/Biotechnology Physical sciences/Materials science Biological sciences/Microbiology E. coli biofilm mineralization (confocal) micro-XRF WAXS Zn accumulation hydroxyapatite Full Text Additional Declarations No competing interests reported. Supplementary Files 20250919ZnMinBiofilmSI.docx Cite Share Download PDF Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 31 Oct, 2025 Reviews received at journal 30 Oct, 2025 Reviews received at journal 21 Oct, 2025 Reviews received at journal 20 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers invited by journal 14 Oct, 2025 Editor invited by journal 10 Oct, 2025 Editor assigned by journal 10 Oct, 2025 Submission checks completed at journal 29 Sep, 2025 First submitted to journal 29 Sep, 2025 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-7658792","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":534421445,"identity":"bbcec87c-7e56-4b69-8484-1dcf2a95ce65","order_by":0,"name":"Leona J. Bauer","email":"","orcid":"","institution":"Technical University of Berlin","correspondingAuthor":false,"prefix":"","firstName":"Leona","middleName":"J.","lastName":"Bauer","suffix":""},{"id":534421446,"identity":"c6b3204c-27ff-423a-808c-3eded856b86a","order_by":1,"name":"Laura Zorzetto","email":"","orcid":"","institution":"Max Planck Institute of Colloids and Interfaces","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Zorzetto","suffix":""},{"id":534421447,"identity":"51ae2a72-3968-4f66-8a79-62449e83814a","order_by":2,"name":"Ernesto Scoppola","email":"","orcid":"","institution":"Max Planck Institute of Colloids and Interfaces","correspondingAuthor":false,"prefix":"","firstName":"Ernesto","middleName":"","lastName":"Scoppola","suffix":""},{"id":534421448,"identity":"01eb6f1c-d047-4b0d-bfd1-2e0e660c5fd4","order_by":3,"name":"Yannick Wagener","email":"","orcid":"","institution":"Technical University of Berlin","correspondingAuthor":false,"prefix":"","firstName":"Yannick","middleName":"","lastName":"Wagener","suffix":""},{"id":534421449,"identity":"88621db6-562c-41b7-9244-1f1dc1b8330b","order_by":4,"name":"Ioanna Mantouvalou","email":"","orcid":"","institution":"Helmholtz-Zentrum Berlin für Materialien und Energie","correspondingAuthor":false,"prefix":"","firstName":"Ioanna","middleName":"","lastName":"Mantouvalou","suffix":""},{"id":534421450,"identity":"a8bc6272-4132-4424-bccd-6dfde4cf26a2","order_by":5,"name":"Cécile M. Bidan","email":"data:image/png;base64,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","orcid":"","institution":"Max Planck Institute of Colloids and Interfaces","correspondingAuthor":true,"prefix":"","firstName":"Cécile","middleName":"M.","lastName":"Bidan","suffix":""}],"badges":[],"createdAt":"2025-09-19 12:53:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7658792/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7658792/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-33942-3","type":"published","date":"2026-01-14T16:29:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94509124,"identity":"4b4f46ce-0795-466d-9bc8-53c920b74ddc","added_by":"auto","created_at":"2025-10-28 16:33:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14259509,"visible":true,"origin":"","legend":"","description":"","filename":"20250919ZnMinBiofilmmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7658792/v1/62f5d0ec3465cf5db8dc2183.pdf"},{"id":94509122,"identity":"6dd83ed3-85c8-4522-a75d-d2699369b5c9","added_by":"auto","created_at":"2025-10-28 16:33:44","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8175,"visible":true,"origin":"","legend":"","description":"","filename":"d69aeb9b78b94486aefb0ee74f228cfd.json","url":"https://assets-eu.researchsquare.com/files/rs-7658792/v1/3c4effede3c16e35e4369575.json"},{"id":94508973,"identity":"3e704c9e-8693-48d7-8aa1-e196f369f5e3","added_by":"auto","created_at":"2025-10-28 16:33:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11839753,"visible":true,"origin":"","legend":"","description":"","filename":"20250919ZnMinBiofilmSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7658792/v1/d4bba52cae94a3acaaa14b3b.docx"},{"id":100615917,"identity":"b1d6c363-af73-4826-a8c5-9a489c863796","added_by":"auto","created_at":"2026-01-19 17:38:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6229255,"visible":true,"origin":"","legend":"","description":"","filename":"20250919ZnMinBiofilmmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7658792/v1_covered_e089860d-8e33-4109-856e-ecbe0b6faea2.pdf"},{"id":94509075,"identity":"11644ef3-2819-403e-a48e-3f2bfb51cfd9","added_by":"auto","created_at":"2025-10-28 16:33:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11839753,"visible":true,"origin":"","legend":"","description":"","filename":"20250919ZnMinBiofilmSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7658792/v1/d6abffccaee1c2a39c3c9ff6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Accumulation of zinc during E. coli biofilm mineralization: within or surrounding the hydroxyapatite?","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"E. coli biofilm, mineralization, (confocal) micro-XRF, WAXS, Zn accumulation, hydroxyapatite","lastPublishedDoi":"10.21203/rs.3.rs-7658792/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7658792/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Biofilms are biological materials that form as bacteria secrete extracellular matrix to protect themselves from environmental challenges. Specific conditions lead to biofilm mineralization, like in dental calculus or kidney stones. To understand biofilm formation and mineralization, we grew Escherichia coli on agar plates containing a nutritive and mineralizing medium. Previous studies showed that the alkaline phosphatase (ALP) present in E. coli biofilms leads to hydroxyapatite precipitation in such conditions. Here, we analyzed the composition of mineralized biofilms with X-ray fluorescence techniques in two and three dimensions. In addition to calcium and phosphate, we found that the traces of zinc introduced via the nutrients and bacteria, also accumulates in the mineralized regions. Wide-angle X-ray scattering performed on dry mineralized biofilm powder also revealed a shortening of the hydroxyapatite crystal lattice, which could indicate partial substitution of calcium by zinc. Using an abiotic model, we reproduced enzymatic hydroxyapatite mineralization with pure ALP, without bacteria nor extracellular matrix. In this model, hydroxyapatite distortion increased with zinc concentration, whereas crystal size decreased. Since zinc is a ubiquitous element that is essential to bacteria metabolism but also antibacterial, these results are fundamental for biofilm research and call for further studies on zinc management in biofilms.","manuscriptTitle":"Accumulation of zinc during E. coli biofilm mineralization: within or surrounding the hydroxyapatite?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 19:46:12","doi":"10.21203/rs.3.rs-7658792/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-31T15:46:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T19:07:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T11:54:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T11:16:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24578829933686014915418460373466337595","date":"2025-10-19T09:56:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45073479354462824555790920697791469215","date":"2025-10-14T09:30:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54570259989619288213950484582411238553","date":"2025-10-14T09:30:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T09:22:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-10T11:05:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-10T08:56:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-29T07:13:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-29T07:06:50+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":"ca829b7d-e850-4661-8b0a-05fc46c0b26b","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56823995,"name":"Biological sciences/Biotechnology"},{"id":56823996,"name":"Physical sciences/Materials science"},{"id":56823997,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-01-19T17:02:46+00:00","versionOfRecord":{"articleIdentity":"rs-7658792","link":"https://doi.org/10.1038/s41598-025-33942-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-14 16:29:17","publishedOnDateReadable":"January 14th, 2026"},"versionCreatedAt":"2025-10-27 19:46:12","video":"","vorDoi":"10.1038/s41598-025-33942-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-33942-3","workflowStages":[]},"version":"v1","identity":"rs-7658792","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7658792","identity":"rs-7658792","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}