Razvoj in optimizacija materiala za 3D tisk na osnovi bakterijsko inducirane precipitacije kalcijevega karbonata

Jakob, Sašo (author), Kopač, Tilen (mentor)

Mikrobno inducirana precipitacija kalcijevega karbonata se v naravi pojavlja v mnogih različnih oblikah in ima na področju inženirstva materialov velik potencial. Predstavlja lahko energijsko varčnejšo in kemijsko milejšo pot strjevanja. V interdisciplinarni magistrski nalogi smo razvili in testirali material na osnovi alginata, ki se zaradi ureolitične aktivnosti bakterij Sporosarcina pasteurii po ekstruziji in inkubaciji v mineralizacijski raztopini spremeni v trden izdelek. Ureazno aktivnost bakterij smo potrdili z visokozmogljivostnim ureaznim testom in s konduktometričnim pristopom. V začetnem preizkusu biomineralizacije smo potrdili mineralizacijo z alginatom zamrežene bakterijske kulture po inkubaciji v mineralizacijski raztopini. Z infrardečo spektroskopijo s Fourierjevo transformacijo smo v enem vzorcu potrdili prisotnost karbonata, z rentgensko difrakcijo pa v drugem vzorcu prisotnost kalcita in vaterita. Reološke parametre formulacij materiala z različno viskoznimi alginatnimi osnovami ter različno vsebnostjo kalcijevega karbonata in kalcijevih ionov smo primerjali s prileganjem teoretičnih modelov Carreau-Yasuda in Roberts-Barnes-Carew rezultatom tokovnih testov. S testi smo potrdili izrazito strižno redčenje pri vseh formulacijah, razlike med njimi pa prikazali s parametri teoretičnih modelov. Tako smo iz nabora izbrali dva materiala, ki sta se izkazala za najprimernejša za 3D-tisk in mineralizacijo. Vsebnost bakterij v materialu se je izkazala za enega najpomembnejših dejavnikov pri formuliranju materiala. Pri uporabi liofilizata z vsebnostjo 3,3 × 10⁷ CFU/mg smo določili, da ga je v 40 mL formulacije potrebno dodati vsaj 180 mg. Pri nižjih vsebnostih je alginat mineraliziral prepočasi, nabreknil ter razpadel zaradi povišanja pH. Vsebnost bakterij v materialu je v 19 dneh padla za en velikostni razred, v liofilizatu pa je bila po začetnem intenzivnem padcu stabilnejša. Za izboljšanje procesa bi bilo ključno optimizirati metodologijo določevanja števila bakterij v vzorcih in podrobno raziskati dinamiko mineralizacije v odvisnosti od števila bakterij, pH, časa inkubacije ter razmerja med volumnom mineralizacijske raztopine in površino ter volumnom materiala.

• Language: Slovenian
• Keywords: Sporosarcina pasteurii, alginat, 3D-biotisk, biomineralizacija, reologija
• Work type: Master's thesis/paper
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2026
• PID: 20.500.12556/RUL-190632
• Publication date in RUL: 30.10.2026

Secondary language

• Language: English
• Title: Development and optimization of a 3D printing material based on bacterially induced precipitation of calcium carbonate

Abstract

Microbially induced calcium carbonate precipitation (MICP) offers a route to materials that consolidate through biological activity rather than through energy-intensive processing. This thesis develops and characterises an extrudable, alginate-based material for 3D printing that hardens after extrusion through MICP driven by the ureolytic activity of lyophilised Sporosarcina pasteurii. The work is interdisciplinary, combining materials engineering, microbiology and biochemistry. Ureolytic activity of the bacteria was confirmed by a high-throughput quantitative urease assay and by conductometry. An initial biomineralisation test showed that a bacterial culture crosslinked with alginate mineralises during incubation in a mineralisation solution. Fourier-transform infrared spectroscopy confirmed the presence of carbonate in one sample, while X-ray diffraction confirmed the presence of calcite and vaterite in another sample. The rheological parameters of four formulations, differing in alginate viscosity grade, pre-crosslinking with calcium ions and addition of CaCO₃ filler, were compared. Flow curves were fitted with the Carreau-Yasuda and Roberts-Barnes-Carew models, confirming pronounced shear thinning in all four and quantifying the differences in zero-shear viscosity, relaxation time and transition sharpness. Two materials were thus selected from the set as most suitable for printing and subsequent mineralisation. The bacterial content of the material proved to be the decisive parameter for mineralisation. Using a lyophilisate of 3.3 × 10⁷ CFU/mg, mineralisation occurred only above 180 mg per 40 mL of material. At lower contents the elevated pH of the mineralisation solution caused swelling and disintegration of the alginate before mineralisation could stabilise the structure. Viable counts within the material fell by one order of magnitude over 19 days. Viable counts within the lyophilisate were more stable after a steep initial decline. To further improve the process of making and storing the material, the methodology for determining bacterial counts in samples would need to be refined and detailed research into mineralisation dynamics, depending on pH, bacterial count, incubation time and the volume-to-surface and volume-to-volume ratios between the material and mineralising solution would need to be carried out.

• Keywords: Sporosarcina pasteurii, alginate, 3D bioprinting, biomineralisation, rheology