Biomimetic Mineralization of Ca-Mg Carbonates: Relevance to Microbial Cells and Extracellular Polymeric Substances.

Research on Ca-Mg carbonate crystallization and phase transition regulated by microbial cells and extracellular polymeric substances (EPS) is significant for carbon sequestration, remediation of polluted soil and water, and synthesis of functional biomaterials. This study focused on the mineralogical transformation from amorphism to crystal, and interaction among cells, EPS, and minerals. By tracing the law of mineral growth and morphological evolution, the influences of cells and EPS on mineral formation were investigated. The results revealed that calcification and the template of rod-shaped cells of strain HJ-1 were the basis for the formation of dumbbell-shaped particles, and directional aggregation and differential growth were the keys to the development and stability of dumbbells. Cell participation had a noticeable impact on mineral prototypes, instead of determining the polymorphism. EPS contributed to aragonite formation and stability. The acidic amino acids or proteins in EPS were likely to cause an increase in MgCO3 content in Mg-calcite. EPS promoted aggregation of particles and induced spherical formation. Exopolysaccharides and proteins were the main components of EPS that can affect carbonate mineralization. EPS could influence the morphology and polymorphism by providing nucleation sites, interacting with Mg2+, adsorbing or incorporating mineral lattices, and inducing particle aggregation.

Stigmasterol alleviates allergic airway inflammation and airway hyperresponsiveness in asthma mice through inhibiting substance-P receptor.

CONTEXT: Stigmasterol has significant anti-arthritis and anti-inflammatory effects, but its role in immune and inflammatory diseases is still unclear. OBJECTIVE: The potential advantages of stigmasterol in asthma were explored in IL-13-induced BEAS-2B cells and asthmatic mice. MATERIALS AND METHODS: The optimal target of stigmasterol was confirmed in asthma. After detecting the cytotoxicity of stigmasterol in BEAS-2B cells, 10 µg/mL and 20 µg/mL stigmasterol were incubated with the BEAS-2B cell model for 48 h, and anti-inflammation and antioxidative stress were verified. Asthmatic mice were induced by OVA and received 100 mg/kg stigmasterol for 7 consecutive days. After 28 days, lung tissues and BAL fluid were collected for the following study. To further verify the role of NK1-R, 0.1 µM WIN62577 (NK1-R specific antagonist), and 1 µM recombinant human NK1-R protein were applied. RESULTS: NK1-R was the potential target of stigmasterol. When the concentration of stigmasterol is 20 µg/mL, the survival rate of BEAS-2B cells is about 98.4%, which is non-toxic. Stigmasterol exerted anti-inflammation and antioxidant stress in a dose-dependent manner and decreased NK1-R expression in IL-13-induced BEAS-2B. Meanwhile, in vivo assay also indicated the anti-inflammation and antioxidant stress of stigmasterol after OVA challenge. Stigmasterol inhibited inflammation infiltration and mucus hypersecretion, and NK1-R expression. DISCUSSION AND CONCLUSIONS: The protective effect of stigmaterol on asthma and its underlying mechanism have been discussed in depth, providing a theoretical basis and more possibilities for its treatment of asthma.

The effect of Bacillus cereus LV-1 on the crystallization and polymorphs of calcium carbonate.

The study of CaCO3 polymorphism is of great significance for understanding the mechanism of carbonate mineralization induced by bacteria and the genesis of carbonate rock throughout geological history. To investigate the effect of bacteria and shear force on CaCO3 precipitation and polymorphs, biomineralization experiments with Bacillus cereus strain LV-1 were conducted under the standing and shaking conditions. The results show that LV-1 induced the formation of calcite and vaterite under the standing and shaking conditions, respectively. However, the results of mineralization in the media and the CaCl2 solution under both kinetic conditions suggest the shear force does not affect the polymorphs of calcium carbonate in abiotic systems. Further, mineralization experiments with bacterial cells and extracellular polymeric substances (EPS) were performed under the standing conditions. The results reveal that bacterial cells, bound EPS (BEPS), and soluble EPS (SEPS) are favorable to the formation of spherical, imperfect rhombohedral, and perfect rhombohedral minerals, respectively. The increase in the pH value and saturation index (SI) caused by LV-1 metabolism under the shear force played key roles in controlling vaterite precipitation, whereas bacterial cells and EPS do not play roles in promoting vaterite formation. Furthermore, we suggest that vaterite formed if pH > 8.5 and SIACC > 0.8, while calcite formed if pH was between 8.0-9.0 and SIACC < 0.8. Bacterial cells and BEPS are the main factors affecting CaCO3 morphologies in the mineralization process of LV-1. This may provide a deeper insight into the regulation mechanism of the polymorphs and morphologies during bacterially induced carbonate mineralization.

Mechanism of carbonate mineralization induced by microbes: Taking Curvibacter lanceolatus strain HJ-1 as an example.

Microbial-induced carbonate precipitation is important in the global carbon cycle, especially in fixing atmospheric CO2. Many simulation experiments have shown that microbes can induce carbonate precipitation, although there is no established understanding of the mechanism. In this study, several mineralization experiments were performed using Curvibacter lanceolatus strain HJ-1, including its secreted extracellular polymeric substances (EPS) and carbonic anhydrase (CA). We found that strain HJ-1, EPS, and CA could promote carbonate precipitation if compared with the respective control experiments (CK). Also, both HJ-1 and EPS1 experiments contained calcite and aragonite, whereas CA experiments formed calcite only. Therefore, HJ-1 and EPS is favorable for carbonate precipitation, especially for aragonite. Besides, the formation of calcite in the EPS2 experiments indicated that EPS contains a trace amount of CA, which might promote CO2 hydration and eventually lead to carbonate precipitation. It was suggested that CA only provide CO32- for the formation of carbonate minerals. In the absence of exogenous HCO3-, the optimized calcification rate followed the order: HJ-1(49.5 %) > CA(6.6 %) > EPS2(4.1 %). In addition, MICP mechanisms was studied, an increase in pH and CO2 hydration by CA play synergetic roles in providing supersaturated alkaline conditions in the system with bacteria. Finally, bacterial cells and EPS promote the formation of calcite and aragonite by acting as nucleation sites.

Comparison of carbonate precipitation induced by Curvibacter sp. HJ-1 and Arthrobacter sp. MF-2: Further insight into the biomineralization process.

Microorganisms are generally involved in the nucleation, growth and phase transformation of carbonate minerals, and influence the development of their morphology and polymorphism. However, understanding of the process of microbially induced carbonate precipitation (MICP) remains limited. Herein, MICP experiments were carried out using Curvibacter sp. HJ-1 and Arthrobacter sp. MF-2 in M2 medium, and the processes of MICP were monitored. Bacterial cells induced the precipitation of carbonate by creating favorable physicochemical conditions and acting as nucleation templates for carbonate particles and thereby, markedly influenced the morphology and growth of the carbonate structure. The extracellular polymeric substance (EPS) secreted by the bacteria was readily absorbed by the precipitated carbonate, which modified its crystal growth orientation. The MgCO3 content of Mg-calcite, induced by MF-2, was dramatically higher than that induced by HJ-1; HJ-1 promoted the formation and stability of aragonite. Multiple formation mechanisms coexisted during the evolution process of the mineral morphologies in the presence of the bacteria. The spherulites observed mainly evolved from dumbbell-like precursors in the presence of MF-2, whereas aggregate growth was the main formation mechanism of radial spherulites in the presence of HJ-1.

Controlled Crystallization and Transformation of Carbonate Minerals with Dumbbell-like Morphologies on Bacterial Cell Templates.

Research on the biogenic-specific polymorphism and morphology of carbonate has been gaining momentum in the fields of biomineralization and industrial engineering in recent years. We report the nucleation of carbonate particles on bacterial cell templates to produce a novel dumbbell-like morphology which was assembled by needle-like crystals of magnesium calcite or aragonite radiating out from both ends of the template bacterium. Mature dumbbell-like structures had a tendency to break apart in the central template region, which was made up mostly of weak amorphous carbonate. Further crystal growth, especially at the template region, transformed the broken pieces into spherulites. Rod-like cell templates were essential for the formation of dumbbell-like morphologies, and we propose a possible formation mechanism of the dumbbell-like morphology. Our findings provide new perspectives on the morphological formation mechanism in biomineralization systems and may have a potential significance in assembling composite materials suitable for industrial applications.

Nucleation and Growth of Mg-Calcite Spherulites Induced by the Bacterium Curvibacter lanceolatus Strain HJ-1.

Calcite spherulites have been observed in many laboratory experiments with different bacteria, and spherulitic growth has received much interest in mineralogy research. However, the nucleation and growth mechanism, as well as geological significance of calcite spherulites in solution with bacteria is still unclear. Herein, spherulites composed of an amorphous core, a Mg-calcite body and an organic film were precipitated by the Curvibacter lanceolatus HJ-1 bacterial strain in a solution with a molar Mg/Ca ratio of 3. Based on the results, we provide a possible mechanism for the biomineralization of Mg-calcite spherulites. First, amorphous calcium carbonate particles are deposited and aggregated into a stable sphere-like core in combination with organic molecules. The core then acts as the nucleus of spherulitic radial growth. Finally, the organic film grows on the surface of Mg-calcite spherulites as a result of bacterial metabolism and calcification. These findings provide insight into the growth mode and crystallization of biogenic spherulites during biomineralization, and are of significance in the application of novel biological materials.

Vaterite induced by Lysinibacillus sp. GW-2 strain and its stability.

Studies on the formation and stability of vaterite by bacteria in experimental systems are of great importance for understanding the mechanism by which microbes contribute to carbonate mineralization. In this study, mineralization experiments using Lysinibacillus sp. strain GW-2 were carried out for 72h under shaking conditions and aging experiments using biotic and chemically synthesized vaterite were performed for 60days in distilled water and air. Our results indicate that Lysinibacillus sp. strain GW-2 can induce the formation of vaterite with spherical morphology from an amorphous calcium carbonate precursor. Biogenic vaterite was more stable than chemically synthesized vaterite in distilled water, perhaps due to organic matter secreted by bacteria that enwrapped the vaterite and prevented it from transforming into more stable phases. Infrared spectrophotometry of biogenic and chemically synthesized vaterite confirmed the presence of organic matter in biogenic vaterite.

[Curvibacter sp. strain HJ-1 induced the formation of aragonite under the condition of low Mg/Ca ratio].

Objective: To study the effects of bacteria on the species and morphology of carbonate minerals. Methods: We conducted a series of cultural experiments in the medium with initial Mg/Ca ratio of 2 but without carbonate ion using Curvibacter sp. strain HJ-1 for 50 days. During the cultivation, bacterial density, precipitate quantities, calcium and magnesium concentration were determined. The morphologies of precipitated carbonates were observed using scanning electron microscopy, and mineral species of carbonate were determined by X-ray diffraction. Results: Strain HJ-1 could induce the precipitation of carbonate minerals, the quality of carbonate gradually increased with the incubation time. XRD patterns showed that the mineral precipitates consisted of high-Mg calcite and aragonite. The percentage of aragonite in the precipitates was up to 86%. The morphology of carbonate minerals was multiform, including rod-shaped, dumbbell-shaped, spherical, tabular, as well as irregular and flake. Conclusion: The formation of aragonite under the condition of low Mg/Ca ratio has a close correlation with extracellular polysaccharide secreted by Curvibacter sp. strain HJ-1.

[Formation of huntite by Lysinibacillus sp. GW-2 strain].

OBJECTIVE: We studied the formation of carbonate minerals induced by microorganism to explore the possibility of mineral capture. METHODS: Culture experiments of carbonate precipitation were done using B4 medium with 6:1 molar ration of Mg/Ca for 50 days. The same medium without inoculation was used as the control. During the cultivation, bacterial density, precipitate quantities, pH and conductivity of the medium, calciumand magnesium concentration were determined. The morphologies of precipitated carbonates were observed using scanning electron microscopy, and mineral species of carbonate were determined by X-ray diffraction. RESULTS: The main results were: (1) In the inoculation process of the Lysinibacillus sp. (GW-2 strain), we found that precipitate quantities were gradually increased with time, while precipitate was not collected in the aseptic experiments; (2) There were significant positive correlations between bacterial density and average precipitation rate (r = 0. 67, P < 0.05), precipitate quantities and pH value (r = 0.79, P < 0.05); (3) Precipitate quantities negatively correlated with conductivity, Ca2+ and Mg2+ concentration with correlation coefficients r of 0.89, 0.93, 0.98 (P < 0.001), respectively; (4) The three carbonate minerals by Lysinibacillus sp. formed according to following trend: amorphous calcium carbonate --> Huntite --> High-Mg calcite. CONCLUSIONS: The main conclusions were: (1) Lysinibacillus sp. (GW-2 strain) might induce the formation of carbonate minerals precipitation; (2) The bacterial density directly affected the precipitation of carbonate minerals, whereas pH value indirectly controlled the precipitation of carbonate minerals; (3) Decreased of conductivity, calcium and magnesium concentration of the medium could indirectly indicate the occurrence of carbonate precipitate; (4) Huntite might be formed through ageing of amorphous calcium carbonate, whereas high-Mg calcite might be formed through demagnesium of the huntite.