bioRxiv2020
To date, the mechanisms of CaCO3 nucleus formation and crystal growth induced by bacterial cells still remain debatable. Here, an insight on the role of planktonic cells of Bacillus licheniformis DSMZ 8782 in the biomineralization is presented. We showed that during 14-days bacterial growth in a liquid urea/Ca2+-containing medium the transformation of CaCO3 polymorphs followed the classical pathway "ACC-vaterite-calcite/aragonite". By microscopic techniques, we detected the formation of extracellular matrix (ECM) around the cells at the stage of exponential growth and appearance of electron-dense inclusions at 24 h after the inoculation. The cells formed filaments and created a network, the nodes of which served as sites for further crystal growth. The ECM formation accompanied with the expression of proteins required for biofilm formation, the aldehyde/alcohol dehydrogenase, stress-associated Clp family proteins, and a porin family protein (ompA ortholog) associated with bacterial extracellular vesicles. We demonstrated that urea and CaCl2 acted as denaturing agents causing matrix formation in addition to their traditional role as a source of carbonate and Ca2+ ions. We showed that CaCO3 nucleation occured inside B. licheniformis cells and further crystal growth and polymorphic transformations took place in the extracellular matrix without attaching to the cell surface. The spatial arrangement of the cells was important for the active crystal growth and dependent on environmental factors. The extracellular matrix played a double role being formed as a stress response and providing a favorable microenvironment for biomineralization (a high concentration of ions necessary for CaCO3 crystal aggregation, fixation and stabilization).