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 real-time kinetic analysis framework integrating connectivity-based segmentation, automated baseline alignment, and robust sliding-window algorithms to quantify the biomineralization competence of Bacillus subtilis under varying calcium regimes. Crucially, our results demonstrate that calcium carbonate promotes microbial growth as effectively as the highly soluble calcium acetate, providing strong evidence that B. subtilis actively solubilizes this crystalline powder to facilitate its metabolic requirements. Despite this growth efficacy, we found that calcium carbonate is an inadequate source for macro-calcite production compared to organic salts. By quantifying the expression efficiency of the sinI reporter gene, we determined that calcium-acetate-driven ECM expression significantly enhances the structural compatibility required for robust biomineralization. Furthermore, kinetic modeling suggests that ECM overproduction can partially compensate for defects in crystal growth-when provided crystalline calcium carbonate powder. These findings, enabled by high-resolution automated signal processing, underscore the critical role of self-mediated carbonate supply and present new engineering pathways for upcycling mineral-rich construction waste.