Biomineralization induced by microorganisms has become a hot spot in the field of carbonate sedimentology; however, the mechanisms involved still need to be explored. In this study, the bacterium Bacillus subtilis J2 (GenBank MG575432) was used to induce the precipitation of calcium carbonate minerals at Mg/Ca molar ratios of 0, 3, 6, 9, and 12. Bacillus subtilis J2 bacteria released ammonia to increase pH, but the ammonia released only made the pH increase to 8.25. Carbonic anhydrase was also produced to catalyze the hydration of carbon dioxide, and this process released carbonate and bicarbonate ions that not only increased pH but also elevated carbonate supersaturation. The biominerals formed at a Mg/Ca molar ratio of 0 were spherulitic, elongated, dumbbell-shaped, and irregularly rhombohedral calcite; at a Mg/Ca molar ratio of 3, the biominerals were calcite and aragonite, the weight ratio of calcite decreased from 26.7% to 15.6%, and that of aragonite increased from 73.3% to 84.4% with increasing incubation time. At higher Mg/Ca molar ratios, the biominerals were aragonite, and the crystallinity and thermal stability of aragonite decreased with increasing Mg/Ca molar ratios. FTIR results showed that many organic functional groups were present on/within the biominerals, such as C–O–C, N–H, C=O, O–H, and C–H. HRTEM-SAED examination of the ultra-thin slices of B. subtilis J2 bacteria showed that nano-sized minerals with poor crystal structure had grown or been adsorbed on the EPS coating. The EPS of the B. subtilis J2 strain contained abundant glutamic acid and aspartic acid, which could be deprotonated in an alkaline condition to adsorb Ca2+ and Mg2+ ions; this made EPS act as the nucleation sites. This study may provide some references for further understanding of the mechanism of biomineralization induced by microorganisms.

Biomineralization has become a research hotspot and attracted widespread attention in the field of carbonate sedimentology. In this study, precipitation of carbonate minerals was induced by Bacillus licheniformis DB1-9 bacteria, (identity confirmed with its phylogenetic tree), to further explore the biomineralization mechanisms. During experiments, lasting up to 24 days with varying Mg/Ca molar ratios and regular monitoring of conditions, ammonia and carbonic anhydrase are released by the bacteria, resulting in a pH increase. Carbonic anhydrase could have promoted carbon dioxide hydration to produce bicarbonate and carbonate ions, and so promoted supersaturation to facilitate the precipitation of carbonate minerals. These include rhombohedral, dumbbell-shaped, and elongated calcite crystals; aragonite appears in the form of mineral aggregates. In addition, spheroidal and fusiform minerals are precipitated. FTIR results show there are organic functional groups, such as C–O–C and C=O, as well as the characteristic peaks of calcite and aragonite; these indicate that there is a close relationship between the bacteria and the minerals. Ultrathin slices of the bacteria analyzed by HRTEM, SAED, EDS, and STEM show that precipitate within the extracellular polymeric substances (EPS) has a poor crystal structure, and intracellular granular areas have no crystal structure. Fluorescence intensity and STEM results show that calcium ions can be transported from the outside to the inside of the cells. This study provides further insights to our understanding of biomineralization mechanisms induced by microorganisms.

The halophilic bacterium Halomonas smyrnensis from a modern salt lake used in experiments to induce biomineralization has resulted in the precipitation of monohydrocalcite and other carbonate minerals. In this study, a Halomonas smyrnensis WMS-3 (GenBank:MH425323) strain was identified based on 16S rDNA homology comparison, and then cultured in mediums with 3% NaCl concentration to induce monohydrocalcite at different Mg/Ca molar ratios of 0, 2, 5, 7, and 9. The growth curve of WMS-3 bacteria, pH values, NH₄⁺ concentration, HCO₃⁻ and CO₃²⁻ concentration, carbonic anhydrase (CA) activity, and the changes in Ca²⁺ and Mg²⁺ ion concentration were determined to further explore the extracellular biomineralization mechanism. Moreover, the nucleation mechanism of monohydrocalcite on extracellular polymeric substances (EPS) was analyzed through studying ultrathin slices of the WMS-3 strain by High resolution transmission electron microscopy (HRTEM), Selected area election diffraction (SAED), Scanning transmission electron microscopy (STEM), and elemental mapping, besides this, amino acids in the EPS were also analyzed. The results show that pH increased to about 9.0 under the influence of ammonia and CA activity. The precipitation ratio (%, the ratio of the mass/volume concentration) of the Ca²⁺ ion was 64.32%, 62.20%, 60.22%, 59.57%, and 54.42% at Mg/Ca molar ratios of 0, 2, 5, 7, and 9, respectively, on the 21st day of the experiments, and 6.69%, 7.10%, 7.74%, 8.09% for the Mg²⁺ ion concentration at Mg/Ca molar ratios 2, 5, 7, and 9, respectively. The obtained minerals were calcite, Mg-rich calcite, aragonite, and hydromagnesite, in addition to the monohydrocalcite, as identified by X-ray diffraction (XRD) analyses. Monohydrocalcite had higher crystallinity when the Mg/Ca ratio increased from 7 to 9; thus, the stability of monohydrocalcite increased, also proven by the thermogravimetry (TG), derivative thermogravimetry (DTG) and differential scanning calorimetry (DSC) analyses. The C=O and C–O–C organic functional groups present in/on the minerals analyzed by Fourier transform infrared spectroscopy (FTIR), the various morphologies and the existence of P and S determined by scanning electron microscope-energy dispersive spectrometer (SEM-EDS), the relatively more negative stable carbon isotope values (−16.91‰ to −17.91‰) analyzed by a carbon isotope laser spectrometer, plus the typical surface chemistry by XPS, all support the biogenesis of these mineral precipitates. Moreover, Ca²⁺ ions were able to enter the bacterial cell to induce intracellular biomineralization. This study is useful to understand the mechanism of biomineralization further and may provide theoretical reference concerning the formation of monohydrocalcite in nature.