Insight into biomolecular interaction-based non-classical crystallization of bacterial biocement

In an attempt to draw a correlation between calcium carbonate (CaCO₃) precipitation and biomacromolecules such as extracellular polymeric substances and enzyme activity in biomineralizing microbe, this report aims to elucidate the ureolytic and ammonification route in Paenibacillus alkaliterrae to explore the possible role of organic biomolecule(s) present on cell surface in mediating nucleation and crystallization of biogenic CaCO₃. After 168 h of biomineralization in ureolysis and ammonification, 2.2 g/l and 0.87 g/l of CaCO₃ precipitates were obtained, respectively. The highest carbonic anhydrase activity (31.8 µmoles/min/ml) was evidenced in ammonification as opposed to ureolysis (24.8 µmoles/min/ml). Highest urease activity reached up to 9.26 µmoles/min/ml in ureolytic pathway. Extracellular polymeric substances such as polysaccharides and proteins were found to have a vital role not only in the nucleation and crystal growth but also in addition direct polymorphic fate of CaCO₃ nanoparticles. EPS production was higher during ammonification (3.1 mg/ml) than in ureolysis (0.72 mg/ml). CaCO₃ nanoparticle-associated proteins were found to be 0.82 mg/ml in ureolysis and 0.56 mg/ml in ammonification. After 30 days of biomineralization, all the polymorphic forms stabilized to calcite in ureolysis but in ammonification vaterite predominated. In our study, we showed that organic template-mediated prokaryotic biomineralization follows the non-classical nucleation and varying proportions of these organic components causes selective polymorphism of CaCO₃ nanoparticles. Overall, the findings are expected to further the fundamental understanding of enzymes, EPS-driven non-classical nucleation of CaCO₃, and we foresee the design of fit-for-purpose futuristic biominerals arising from such renewed understanding of biomineralization. KEY POINTS: • Organic-inorganic interface of cell surface promote crystallization of biominerals • Carbohydrate and proteins in the interface results selective polymorphism of CaCO₃ • Calcite stabilized at 30 days in ureolysis, vaterite-calcite mix in ammonification.