Assessing soluble and insoluble calcium sources for growth, biofilm formation, and biomineralization in Bacillus subtilis

Biofilms formed by soil microbes hold immense potential for bioremediation, carbon dioxide sequestration, and the development of sustainable cementitious materials. However, quantifying the complex temporal coupling among bacterial growth, extracellular matrix (ECM) production, and mineralization dynamics remains a significant challenge due to the inherent nonlinearity of these processes and signal noise in high-throughput assays. To address this, we utilized an automated kinetic framework to evaluate the biomineralization competence of Bacillus subtilis under varying calcium regimes. Our results demonstrate that calcium carbonate promotes microbial growth as effectively as soluble calcium acetate, indicating that B. subtilis actively solubilizes the crystalline powder. Despite this growth efficacy, calcium carbonate was an inadequate source for macro-calcite production compared to organic salts. By quantifying expression from the sinI promoter, which initiates SinI production to activate matrix synthesis, we suggested that calcium-acetate-driven extracellular matrix (ECM) expression significantly enhances the structural template required for robust biomineralization. Kinetic expression analysis and consolidation assays indicate that overproduced ECM partially mitigates crystalline calcite growth defects, offering a baseline for utilizing mineral-rich construction waste Importance Biomineralization in B. subtilis has been studied as a potential avenue for developing engineered living materials. However, organic calcium salts, such as calcium acetate and calcium lactate, typically yield higher bioconversion efficiencies than certain inorganic sources often used in construction. This advantage has been largely attributed to the metabolic utilization of the organic anion as a carbon source, thereby promoting local alkalinity. Here, our experiments indicate that this limitation is influenced by structural and regulatory constraints rather than purely chemical barriers. Moreover, we explored a targeted synthetic biology approach to address this by genetically decoupling matrix production from native environmental sensing, thereby engineering strains with increased matrix production that can partially overcome the limitations of non-inductive substrates. This approach can contribute to the development of sustainable construction technologies, moving toward actively programmed Engineered Living Materials (ELMs).