Characterization and genome analysis of Neobacillus mesonae NS-6, a ureolysis-driven strain inducing calcium carbonate precipitation
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ABSTRACT:
SUBMITTER: Xu R
PROVIDER: S-EPMC10646308 | biostudies-literature | 2023 Jan
REPOSITORIES: biostudies-literature
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Project description:Urease containing nickel cofactor is crucial for urea-hydrolytic induced calcium carbonate (CaCO3) precipitation (UICP). However, limited information exists regarding the influence of amino acid residues interacting with nickel ions in its structure on induced CaCO3 mineralization. Herein, RT-qPCR was used to demonstrate that the addition of NiCl2 dramatically upregulated the expression of urease structural gene ureC that was correlated with nickel binding in Neobacillus mesonae strain NS-6. Homology modeling and molecular docking were employed to construct the three-dimensional structure of urease and seek the key residues involved in nickel binding process, and virtual mutation technology was adopted to inform three key residues coordinated with nickel ions and urea, His249, His275, and Asp363. Four metrics, including root mean square deviation values for mutations of those key residues in urease-urea complexes severally and wild-type, were calculated by molecular dynamics simulations when they were mutated into alanine, respectively. Subsequently, the mutations of H249A, H275A, and D363A were characterized using western blotting to reveal a decrease in the relative expression and activity of urease, along with a corresponding reduction in CaCO3 precipitation. Ultimately, the mutations also exhibited that they had lower substrate affinity and catalytic efficiency for urea through enzymatic properties analysis. The findings suggested that those residues played a pivotal role in UICP of strain NS-6, which would expand the theoretical basis for modulating urease activity.IMPORTANCEUrease-producing bacterium is of great importance in diverse application fields, such as environmental remediation, due to its key driving characteristics in catalyzing urea hydrolysis via urea-hydrolytic induced CaCO3 precipitation (UICP). As essential cofactors of urease, nickel ions play a crucial role in regulating urease catalysis and maintaining structural stability. Numerous investigations have emphasized the impact of nickel ions on urease activity in recent years, to our best knowledge, only a few literatures have studied the molecular-level regulation of nickel-ligand residues. This study focused on the highly urease-producing bacterial Neobacillus mesonae NS-6 to explore the effects of specific nickel-ligand residues on the urease-aided CaCO3 mineralization process using molecular simulation predictions and targeted mutation experiments. The aim was to provide a molecular-level understanding of the interactive effects between urea and critical residues associated with the urease active center, as well as propose an effective modification strategy to enhance the application of UICP in future environmental areas.
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Project description:During a study of ureolytic microbial calcium carbonate (CaCO(3)) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO(3) crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K(m)) and maximum hydrolysis rates (V(max)) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.
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Project description:Calcium carbonate is an important component in exoskeletons of many organisms. The synthesis of calcium carbonate was performed by mixing dimethyl carbonate and an aqueous solution of calcium chloride dihydrate. The precipitation product was characterized by means of scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) measurements. In addition, the turbidity of the reaction solution was acquired to monitor the kinetics of the calcium carbonate structure's growth in the investigated system. In this study, samples of CaCO₃ particles obtained with individual proteins, such as ovalbumin, lysozyme, and a mixture of the proteins, were characterized and compared with a control sample, i.e., synthesized without proteins. The obtained data indicated that the addition of ovalbumin to the reaction changed the morphology of crystals from rhombohedral to 'stack-like' structures. Lysozyme, however, did not affect the morphology of calcium carbonate, yet the presence of the protein mixture led to the creation of more complex composites in which the calcium carbonate crystals were constructed in protein matrices formed by the ovalbumin-lysozyme interaction. It was also observed that in the protein mixture, ovalbumin has a major influence on the CaCO₃ formation through a strong interaction with calcium ions, which leads to the coalescence and creation of a steric barrier reducing particle growth. The authors proposed a mechanism of calcium carbonate grain growth in the presence of both proteins, taking into account the interaction of calcium ions with the protein.
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Project description:Patterned calcium carbonate materials with controlled morphologies have broad applications in both environmental and engineering fields. However, how to fabricate such materials through environmental-friendly methods under ambient conditions is still challenging. Here, we report a green approach for fabricating patterned calcium carbonate materials. This eco-friendly approach is based on template-assisted microbially induced calcium carbonate precipitation. As a proof of concept, by varying the templates and optimizing fabrication parameters, different patterned calcium carbonate materials were obtained. The optimized parameters include C Ca2+ = 80 mM, T i = 15 °C, and templates made of small-sized CaCO3 particles with a concentration of 1.5 mg mL-1, under which better and more sharp patterns were obtained. Materials with periodic patterns were also fabricated through a periodic template, showing good scalability of this approach. The results of this study could mean great potential in applications where spatially controlled calcium carbonate depositions with user-designed patterns are needed.
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Project description:Common adhesives for nonstructural applications are manufactured using petrochemicals and synthetic solvents. These adhesives are associated with environmental and health concerns because of their release of volatile organic compounds (VOCs). Biopolymer adhesives are an attractive alternative because of lower VOC emissions, but their strength is often insufficient. Existing mineral fillers can improve the strength of biopolymer adhesives but require the use of crosslinkers that lower process sustainability. This work introduces a novel approach to strengthen biopolymer adhesives through calcium carbonate biomineralization, which avoids the need for crosslinkers. Biomineral fillers produced by either microbially or enzymatically induced calcium carbonate precipitation (MICP and EICP, respectively) were precipitated within guar gum and soy protein biopolymers. Both, MICP and EICP, increased the strength of the biopolymer adhesives. The strength was further improved by optimizing the concentrations of bacteria, urease enzyme, and calcium. The highest strengths achieved were on par with current commercially available nonstructural adhesives. This study demonstrates the feasibility of using calcium carbonate biomineralization to improve the properties of biopolymer adhesives, which increases their potential viability as more sustainable adhesives.
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Project description:The use of additives has generated significant attention due to their extensive application in the microbially induced calcium carbonate precipitation (MICP) process. This study aims to discuss the effects of Na-montmorillonite (Na-MMT) on CaCO3 crystallization and sandy soil consolidation through the MICP process. Compared with the traditional MICP method, a larger amount of CaCO3 precipitate was obtained. Moreover, the reaction of Ca2+ ions was accelerated, and bacteria were absorbed by a small amount of Na-MMT. Meanwhile, an increase in the total cementing solution (TCS) was not conducive to the previous reaction. This problem was solved by conducting the reaction with Na-MMT. The polymorphs and morphologies of the CaCO3 precipitates were tested by using X-ray diffraction and scanning electron microscopy. Further, when Na-MMT was used, the morphology of CaCO3 changed from an individual precipitate to agglomerations of the precipitate. Compared to the experiments without Na-MMT in the MICP process, the addition of Na-MMT significantly reduced the hydraulic conductivity (HC) of sandy soil consolidated.
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Project description:Bacteria that are resistant to high temperatures and alkaline environments are essential for the biological repair of damaged concrete. Alkaliphilic and halotolerant Bacillus sp. AK13 was isolated from the rhizosphere of Miscanthus sacchariflorus. Unlike other tested Bacillus species, the AK13 strain grows at pH 13 and withstands 11% (w/v) NaCl. Growth of the AK13 strain at elevated pH without urea promoted calcium carbonate (CaCO3) formation. Irregular vateritelike CaCO3 minerals that were tightly attached to cells were observed using field-emission scanning electron microscopy. Energy-dispersive X-ray spectrometry, confocal laser scanning microscopy, and X-ray diffraction analyses confirmed the presence of CaCO3 around the cell. Isotope ration mass spectrometry analysis confirmed that the majority of CO32- ions in the CaCO3 were produced by cellular respiration rather than being derived from atmospheric carbon dioxide. The minerals produced from calcium acetate-added growth medium formed smaller crystals than those formed in calcium lactate-added medium. Strain AK13 appears to heal cracks on mortar specimens when applied as a pelletized spore powder. Alkaliphilic Bacillus sp. AK13 is a promising candidate for self-healing agents in concrete.
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Project description:Our previous study reported that Saccharomyces cerevisiae could induce calcium carbonate (CaCO3) precipitation, but the associated mechanism was unclear. In the present study, Saccharomyces cerevisiae was cultured under various conditions, including the presence of different organic acids and initial pH, and the yields of CaCO3 formation induced by the different organic acids were compared. The metabolism of organic acid by the metabolites of S. cerevisiae was also assessed in vitro. The SEM-EDS and XRD results showed that only acetate acid, pyruvic acid, and α-ketoglutaric acid could induce CaCO3 formation, and the weight order of the produced CaCO3 was pyruvic acid, acetate acid, α-ketoglutaric acid. In addition, the presence of only yeast metabolites and the initial neutral or alkaline environment also limited the CaCO3 formation. These results illustrated that organic acid oxidation intracellularly, especially the tricarboxylic acid cycle, was the major mechanism, and the CaCO3 yield was related to the amount of CO2 produced by the metabolism of organic acids. These findings will deepen the knowledge of the mineralization capacity of S. cerevisiae and provide a theoretical basis for the future application of yeast as an alternative microorganism in MICP.
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Project description:The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (i) an organic ECM, which facilitates cell-cell and cell-substrate adhesion; (ii) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (iii) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an in vitro culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate in vitro excretion of ECM by primary (nondividing) tissue cultures of both soft (Xenia elongata) and hard (Montipora digitata) corals. There are structural differences between the ECM produced by X. elongata cell cultures and that of M. digitata, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate in vitro production of SOM and extracellular mineralized particles in cell cultures of M. digitata. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite. De novo calcification was confirmed by following the incorporation of (45)Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization.
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