Biomineralization of Monohydrocalcite Induced by the Halophile Halomonas Smyrnensis WMS-3

Abstract

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.

Bio-Precipitation of Carbonate and Phosphate Minerals Induced by the Bacterium Citrobacter freundii ZW123 in an Anaerobic Environment

“…As seen from Figure 7a Figure 7b) and these functional groups may be due to the amino acids and polysaccharides there. The FTIR spectra of supernatants, after the minerals were processed by thorough ultrasonic shaking, are identical with the distilled water ( Figure 7c) [11]; this indicates that the organic substances on the mineral surfaces were removed completely. Some organic functional groups within the mineral interiors at various Mg/Ca ratios, such as C-O-C, C-O, O-H, C-H and C=O, were also detected, shown in Figure 7d; most of these organic functional groups were also detected in the EPS from the ZW123 bacterium (Figure 7b).…”

“…Mg/Ca ratios do not only affect the mineral phase but also the crystallinity. In the previous studies, with increasing Mg/Ca ratios, the crystallinity of monohydrocalcite induced by Staphylococcus epidermis Y2 decreased [29], whereas that induced by the halophile Halomonas smyrnensis WMS-3 increased [11]. Other factors that have been investigated include the presence of crystal seeds and the culture conditions.…”

“…Extensive research has been undertaken on the biomineralization processes of carbonate and phosphate minerals in the past decades, and the significant roles that microorganisms play in these processes have been widely reported [1][2][3][4][5][6][7][8][9][10][11]. Many species of microorganisms have been used to induce the precipitation of carbonate and phosphate minerals, including cyanobacteria, halophiles, methanogens and sulfate-reducing bacteria [8,[11][12][13][14][15][16][17]. Among these, halophiles play an important role in the precipitation of microbially-induced minerals in various natural environments with high salinity.…”

Biomineralization of Carbonate Minerals Induced by The Moderate Halophile Staphylococcus Warneri YXY2

“…It has been reported that the peak at 284.3 eV is assigned to the C-C and C-H, originating from lipids or side chains of amino acid, and the peak at 288.97 eV can be due to the O-C=O from carboxylic acid, carboxylate, or ester [38]. The O 1s peak at 530.69 eV (Figure 6d) also indicates the presence of C=O and O-C=O from carboxylic acids, carboxylates, carbonyls and amides [26]. There was one peak at 132.78 eV (Figure 6e) and this peak could be attributed to phosphate groups [39].…”

“…The liquid culture medium inoculated with YXY2 bacteria (OD 600 = 1.0) at a volume ratio of 1:100 was set as the experimental group, and that inoculated with the same volume ratio of sterilized distilled water was set as the control group. Ammonia test of YXY2 bacteria, the concentration of ammonium ion, CA activity, concentrations of carbonate and bicarbonate ions, the calculation of pH values based on the concentration of ammonium, bicarbonate and carbonate ions were all conducted according to published papers [26][27][28][29]. ALP activities were measured according to a previous study [30] and the concentrations of phosphate ions were also measured with the molybdenum blue method [31].…”