Formation Of Calcium Carbonate Crystals Research Articles

Carboxylic acid-induced freshwater acidification effects on behavior and operculum ultrastructure in the gastropod mollusc Bellamya bengalensis

ABSTRACT Global freshwater acidification, accelerated by rising pCO2, remains a critical ecological concern. Despite the prevalence of carboxylic acids like acetic acid (AA) and benzoic acid (BA) in freshwater, their potential to exacerbate acidification and impact biota is not fully understood. In this study, we examined behavioural disruptions during acute exposures (96 h) and operculum ultrastructure changes during chronic exposures (28 days) to ecologically-relevant concentrations of AA (39.7, 79.5, and 99.4 mg/L) and BA (31.4, 62.8, and 78.5 mg/L) in Bellamya bengalensis. Acute exposures revealed significant behavioural disruption, including reduced activity and impaired escape behaviour. Chronic exposures led to ultrastructural defects in the operculum, with more pronounced effects in BA-exposed groups. Acidification scenarios appeared to impact calcium carbonate crystal formation, compromising opercula structure. Our study underscores the importance of understanding both acute and chronic effects of freshwater acidification on molluscan species, emphasising the need for further research into the underlying mechanisms and thresholds of acidification tolerance.

Accelerated curing of cement mortar: In-situ carbonation utilising CO2-impregnated faujasite

This study explores the use of in-situ carbonation using CO2-impregnated faujasite (FAU) for accelerating the curing of cement mortar. Results indicate that adding 3–12 wt% CO2-impregnated FAU reduces the setting time of the mortars by 2–11% and significantly enhances the compressive strength of the mortars at 1, 7, and 28 days, compared to plain FAU. Microstructural analyses reveal that the in-situ carbonation promotes the hydration of cement by providing additional calcium carbonate nucleation sites for the C-S-H precipitation. This increases the formation of hydration products, which leads to the refinement of the pore structure. Additionally, the in-situ carbonation results in the formation of calcium carbonate crystals with a particle size of 150–200 nm at 28 days, with the calcium carbonate content increased by 1.9 wt% at an addition level of 12 wt% CO2-impregnated FAU. Overall, the in-situ carbonation technique shows great promise as an alternative method for accelerating the curing of cement-based materials, particularly for cast-in-place concrete.

Autogenous Healing in 10-Years Aged Cementitious Composites Using Microfibers and Superabsorbent Polymers

Cement-based materials are the most widely used construction materials in the world for infrastructure works. Unfortunately, they come with a high environmental burden due to carbon dioxide emissions and the need for regular maintenance and repairs. Without these, the service life can decrease. By using a self-healing approach, the service life can be extended, as well as the durability and sustainability of the building material. As the ability to self-heal depends on the age of the material, so will the potential influence of added materials to promote this healing. However, the effects of reduced healing beyond one year are not ubiquitous in the literature. In this study, specimens were studied after a decade of maturation under different storage conditions to conclude on the self-healing capabilities of the old samples. Cracks can still be partially healed after ten years, mainly due to the formation of calcium carbonate crystals, related to the observed regain in mechanical properties measured by repeated four-point bending tests. The initial addition of superabsorbent polymers to the mixture results in greater healing compared to the reference samples, making it a sustainable option for the future of cement-based composites.

Strain Screening and Particle Formation: a Lysinibacillus boronitolerans for Self-Healing Concrete.

Microbial-induced calcite precipitation is a promising technology to solve the problem of cracks in soil concrete. The most intensively investigated microorganisms are urease-producing bacteria. Lysinibacillus that is used as urease-producing bacteria in concrete repair has rarely been reported. In this study, Lysinibacillus boronitolerans with a high urease activity was isolated from soil samples. This strain is salt- and alkali-tolerance, and at pH 13, can grow to ~OD600 2.0 after 24 h. At a salt concentration of 6%, the strain can still grow to ~OD600 1.0 after 24 h. The feasibility of using this strain in self-healing concrete was explored. The data showed that cracks within ~0.6 mm could be repaired naturally with hydration when spores and substrates were added to the concrete in an appropriate proportion. Moreover, the number and morphology of CaCO3 crystals that were produced by bacteria can be influenced by the concrete environment. An efficiency method to elucidate the process of microbial-induced calcium carbonate crystal formation was established with Particle Track G400. This study provides a template for future studies on the theory of mineralization based on microorganisms. IMPORTANCE The formation of calcium carbonate crystals in concrete by urease-producing bacteria is not understood fully. In this study, a Lysinibacillus boronitolerans strain with a high urease activity was isolated and used to analyze the counts and sizes of the crystals and the relationship with time. The data showed that the number of crystal particles increases exponentially in a short period with sufficient substrate, after which the crystals grow, precipitate or break. In concrete, the rate-limiting steps of calcium carbonate crystal accumulation are spore germination and urease production. These results provided data support for the rational design of urease-producing bacteria in concrete repair.

Insights on the biomineralisation processes and related diversity of cyanobacterial microflora in thermogenic travertine deposits in Greek hot springs (North‐West Euboea Island)

AbstractThe aim of this study is to identify the biomineralisation processes in hot springs of North‐West Euboea Island by assessing the physico‐chemical parameters of the hot water, the travertine mineralogical composition and facies, and the cyanobacterial microflora. In the studied area, the main mineral phases are calcite and aragonite, creating laminated and shrub facies of travertine deposits in close association with the cyanobacterial microflora. Microscopic analysis of fresh and cultured field samples shows the presence of 81 taxa of Cyanobacteria belonging to six orders, that is, Oscillatoriales, Synechococcales, Spirulinales, Chroococcales, Nostocales and Chroococcidiopsidales with the main factors controlling biodiversity being temperature, salinity and access to sunlight. No Cyanobacteria species were identified in areas with temperatures over 65oC. In areas with high salinity (27–37‰), the order Oscillatoriales predominates. On the other hand, in areas with high temperatures (63oC), fewer orders were observed, usually only Synechococcales and Spirulinales. In areas with lower temperatures (37oC), larger numbers of Cyanobacteria orders were identified. Additionally, salinity seems to regulate the presence of the Nostocales order. The combined geobiological study revealed the presence of four biomineralisation processes involving calcium carbonate minerals, that is, (i) filamentous Cyanobacteria and extracellular polymeric substances trapping calcium carbonate crystals, (ii) extracellular polymeric substances acting as a template favouring mineral precipitation for crystal nucleation, (iii) formation of calcified Cyanobacteria sheaths and (iv) alteration of calcium carbonate crystals by endolithic Cyanobacteria. The identified biomineralisation processes suggest that the formation of calcium carbonate crystals is due to the metabolic activity of Cyanobacteria, or that the Cyanobacteria favour the deposition or the alteration of already existing crystals. The combination of these processes and the non‐biotic (abiotic) mineralisation result in the formation of hybrid carbonates in the study area.

Calcium Carbonate Precipitation by Rock Dwelling Bacteria in Murree Hills, Lower Himalaya Range Pakistan

Calcium carbonate precipitation in the natural environment is associated with a variety of factors including type species of bacteria. In this study, we investigated calcium carbonate precipitation induced by bacteria isolated from rocks of Murree Hills, Lower Himalayan region, Pakistan. Four species; Acinetobacter N1, Acinetobacter N6, Bordetella N30, and Brevundimonas N5, having ureolytic activity, were found to precipitate calcium carbonate. FTIR analysis revealed the formation of calcium carbonate crystals confirmed by comparison to synthetically produced calcium carbonate. Morphological characterization by scanning electron microscopy showed crystals formed were mostly of the irregular and polygonal shape having sharp spike-like edges. All the four strains having urease enzymatic capability to convert urea into ammonia to produce calcium carbonate crystals in higher pH due to the production of an alkaline environment. The results of our research revealed a variety of rock bacteria capable of precipitating calcium carbonate in natural environments and are expected to have a great influence on the cycling of carbonates in these environments.

Formation and Inhibition of Calcium Carbonate Crystals under Cathodic Polarization Conditions

The formation of CaCO3 crystals on the cathode surface and the scale-inhibition performance of scale inhibitor 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTCA) on the cathode surface were studied by methods of solution analysis, gravimetric analysis, SEM, FTIR, and XRD techniques. They were then compared with the results of the formation and suppression of CaCO3 crystals in aqueous solution. PBTCA had a good solution-scale-inhibition performance and good lattice-distortion effects on CaCO3 crystals in solution, which could change the CaCO3 from calcite to vaterite and aragonite crystals. The solution-scale-inhibition efficiency exceeded 97% when the PBTCA concentration reached 8 mg/L. Under cathodic polarization conditions, the surface-scale-inhibition efficiency of the cathode and solution-scale-inhibition efficiency near the cathode surface both exceed 97% at polarization potential of −1V. The addition of PBTCA significantly reduced the amount of CaCO3 crystals formed on the cathode surface and had good surface and solution-scale-inhibition effect. However, the lattice-distortion effect of PBTCA on CaCO3 crystals disappeared on the cathode surface, and the resulting CaCO3 contained only calcite crystals. The high-scale-inhibition effect of PBTCA under cathodic polarization was mainly due to the inhibition of the formation of calcium carbonate crystals by PBTCA, and not because of the lattice distortion of CaCO3 crystals.

Controlling the hydration and carbonation in lime-based materials: Advantage of slow carbonation in CO2 curable construction materials

In this study, the role of natural additives (sodium chloride or sugar) in aerial lime mortar was elucidated. It was found that both the hydration and carbonation were accelerated by sodium chloride but retarded by sugar. Especially, sugar prevented the rapid clogging of pores around the mortar surface, which caused the even formation of calcium carbonate crystals throughout the volume of mortar. Therefore, the strength development was significantly accelerated. The slow carbonation and resulting rapid strength development can be practically implementable since it does not rely on long term atmospheric carbonation or use of high concentration of CO2 gas.

Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis)

The α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α–CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α–CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.

Calcium Carbonate Formation in the Presence of Biopolymeric Additives.

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

Effect of pH and temperature on calcium carbonate precipitation by CO2 removal from iron‐rich water

AbstractIn the present work, the effect of temperature and solution pH on calcium carbonate precipitation from iron‐rich waters was investigated. Calcium carbonate was precipitated by CO2 removal. The increase in the temperature or the solution pH leads to the acceleration of calcium carbonate nucleation and crystal growth. Iron addition retards the formation of calcium carbonate crystals and enhanced the precipitation in the bulk solution. At high supersaturations, the inhibition effectiveness of iron is small and it could be improved by lowering the solution pH. The results of the present work show that it is possible to reduce or completely prevent scale formation in different water treatment processes by controlling the operating parameters which favourably affects the water treatment costs, increases the equipment life and allows increased product water recovery.

Chemical aspects of the process of concrete cracks elimination with the help of bacteria

The article describes the chemical processes of biogenesis of calcium carbonate for self-healing of concrete, taking into account four main factors: the concentration of calcium, the concentration of soluble inorganic carbon, the pH value, the presence of the crystallization center. A number of bacteria that can be found in soil, sand and natural minerals have the ability to release calcium carbonate, both in natural and laboratory conditions. In the laboratory, calcium lactate (CaC6H10O6) was used as a starting material for the formation of calcium carbonate. In addition, urea necessary for bacteria as a source of urease enzyme and yeast extract as a source of carbon and nitrogen were added. The resulting pH was brought to 9 to avoid possible chemical deposition of calcium carbonate. To improve the production technology of biological concrete, specially selected bacteria of the genus Bacillus with a combination of nutrients were used to create a reducing agent in concrete. With the help of such self-healing concrete by means of bacteria, cracks more than 100 µm wide can be compacted. With this approach, the bacteria in the alkaline medium convert CO2 into carbonate ions, which then interact with the Ca ions from the concrete matrix. This leads to the formation of calcium carbonate crystals. In addition, CO2 directly reacts with the calcium hydroxide matrix, which leads to the formation of calcite precipitate. The appearance of calcium carbonate crystals of large size with the participation of bacteria incorporated into the self-healing concrete provides an excellent ability to self-healing compared to traditional or developed environmentally unsafe self-healing cement materials. That is why this area of research is a promising alternative to environmentally hazardous methods of repair using cement.

Retardation of heat exchanger surfaces mineral fouling by water-based diethylenetriamine pentaacetate-treated CNT nanofluids

Mineral scale deposition on heat exchanging surfaces increases the thermal resistance and reduces the operating service life. The effect is usually intensified at higher temperatures due to the inverse temperature solubility characteristics of some minerals in the cooling water. Scale formation build up when dissolved salt crystallize from solution onto the heated surface, forming an adherent deposit. It is very important for heat transfer applications to cope with the fouling problems in industry. In this present study, a set of fouling experiments was conducted to evaluate the mitigation of calcium carbonate scaling by applying DTPA-treated MWCNT-based water nanofluids on heat exchanger surfaces. Investigation of additive DTPA-treated MWCNT-based water nanofluids (benign to the environment) on fouling rate of deposition was performed. 300mgL−1 of artificially-hardened calcium carbonate solution was prepared as a fouling solution for deposit analysis. Assessment of the deposition of calcium carbonate on the heat exchanger surface with respect to the inhibition of crystal growth was conducted by Scanning Electron Microscope (SEM). The results showed that the formation of calcium carbonate crystals can be retarded significantly by adding MWCNT-DTPA additives as inhibition in the solution.

The Shell of the Invasive Bivalve Species Dreissena polymorpha: Biochemical, Elemental and Textural Investigations.

The zebra mussel Dreissena polymorpha is a well-established invasive model organism. Although extensively used in environmental sciences, virtually nothing is known of the molecular process of its shell calcification. By describing the microstructure, geochemistry and biochemistry/proteomics of the shell, the present study aims at promoting this species as a model organism in biomineralization studies, in order to establish a bridge with ecotoxicology, while sketching evolutionary conclusions. The shell of D. polymorpha exhibits the classical crossed-lamellar/complex crossed lamellar combination found in several heterodont bivalves, in addition to an external thin layer, the characteristics of which differ from what was described in earlier publication. We show that the shell selectively concentrates some heavy metals, in particular uranium, which predisposes D. polymorpha to local bioremediation of this pollutant. We establish the biochemical signature of the shell matrix, demonstrating that it interacts with the in vitro precipitation of calcium carbonate and inhibits calcium carbonate crystal formation, but these two properties are not strongly expressed. This matrix, although overall weakly glycosylated, contains a set of putatively calcium-binding proteins and a set of acidic sulphated proteins. 2D-gels reveal more than fifty proteins, twenty of which we identify by MS-MS analysis. We tentatively link the shell protein profile of D. polymorpha and the peculiar recent evolution of this invasive species of Ponto-Caspian origin, which has spread all across Europe in the last three centuries.

Hemocytes participate in calcium carbonate crystal formation, transportation and shell regeneration in the pearl oyster Pinctada fucata

In this study, light microscope, scanning and transmission electron microscope, hematoxylin-eosin and fluorescent staining, and mass spectrometry methods were employed to observe the calcium carbonate (CaCO3) crystal formation, hemocyte release and transportation, and hemocyte distribution at the shell regeneration area and to analyse the proteome of hemocytes in the pearl oyster, Pinctada fucata. The results indicated that intracellular CaCO3 crystals were observed in circulating hemocytes in P.fucata, implying that there was a suitable microenvironment for crystal formation in the hemocytes. This conclusion was further supported by the proteome analysis, in which various biomineralization-related proteins were detected. The crystal-bearing hemocytes, mainly granulocytes, may be released to extrapallial fluid (EPF) by the secretory cavities distributed on the outer surface of the mantle centre. These granulocytes in the EPF and between the regenerated shells were abundant and free. In the regenerated prismatic layer, the granulocytes were fused into each column and fragmented with the duration of shell maturation, suggesting the direct involvement of hemocytes in shell regeneration. Overall, this study provided evidence that hemocytes participated in CaCO3 crystal formation, transportation and shell regeneration in the pearl oyster. These results are helpful to further understand the exact mechanism of hemocyte-mediated biomineralization in shelled molluscs.

Fouling mitigation on heat exchanger surfaces by EDTA-treated MWCNT-based water nanofluids

Fouling can be defined as adherent deposits of unwanted compounds that are formed by the precipitation of soluble salts from water and crystal growth on the surfaces of processing equipment. The deposition layer becomes an insulating layer that deteriorates the heat transfer efficiency and shortens the service life. The development of functionalized nanomaterial leads to multi-functionality, such as the ability to adsorb scaling cations with high thermal conductivity, which is very important for heat transfer applications to manage the fouling problems. The purpose of this study was to evaluate the mitigation of calcium carbonate scaling by applying EDTA-treated MWCNT-based water nanofluids on heat exchanger surfaces. A set of fouling experiments was conducted by using additive EDTA-treated MWCNT-based water nanofluids (benign to the environment) to verify the additives’ retardation of the fouling rate of deposition. Fouling solution for deposit analysis was prepared by using 300 mg/L of artificially-hardened calcium carbonate solution. Also the assessment of the deposition of calcium carbonate on the heat exchanger surface with respect to the inhibition of crystal growth was conducted by Scanning Electron Microscope (SEM) and Energy Dispersive X-ray Spectrometry (EDS). The results showed that the formation of calcium carbonate crystals can be retarded significantly by adding MWCNT-EDTA additives to the solution.

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Strength characteristics of microbial cement mortars treated in different calcium sources

Microbially induced calcium carbonate precipitation is a method for the protection of cement-based materials. This paper deals with the strength characteristics of microbial cement mortars, which are treated by the Enterobacter sp. FJ 973550 microorganism in different calcium sources (calcium hydroxide, calcium acetate, calcium chloride and calcium oxide). The crystalline phases of calcium carbonate crystal formation and the surface morphology of cement mortar are investigated by X-ray diffraction and scanning electron microscopy. Microbial cement mortar specimens treated in calcium hydroxide source show higher compressive strength (∼29%) and tensile strength (∼47%) compared to control specimens. Surface treatment of specimens with bacteria resulted in an approximately 56% decrease in water absorption and increased the resistance to water and hazard material penetration. This biological surface treatment shows promising prospects for increasing strength and durability aspects of cement mortar specimens.

Intrinsically disordered and pliable Starmaker-like protein from medaka (Oryzias latipes) controls the formation of calcium carbonate crystals.

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

Characterization of bacteria isolated from palaeoproterozoic metasediments for sequestration of carbon dioxide and formation of calcium carbonate.

Bacterial community of palaeoproterozoic metasediments was enriched in the chemostat in the presence of different concentrations of NaHCO3. Six bacterial isolates were isolated from the chemostat on nutrient agar plates on the basis of distinct morphology. Denaturing gradient gel electrophoresis (DGGE) proved the presence of six operational taxonomic units (OTUs) at 50 and 100 mM NaHCO3. The OTU was reduced to three and one at enrichment concentration of 150 and 200 mM NaHCO3 respectively. These six isolates were tested for sequestration of carbon dioxide by (14)C metabolic labeling of NaH(14)CO3. Among the six isolates, one of the bacterium showed better potency to fix radiolabeled NaH(14)CO3. The isolate (ISTD04) was identified as Serratia sp. by 16S ribosomal RNA (16S rRNA) sequence analysis and was found to be same as the DGGE OTU sequence at 200-mM NaHCO3 concentration. The bacterium was tested for product formation in form of calcite crystals in presence of 5 % CO2. Scanning electron microscopy (SEM) of product formed by the bacterium revealed defined faceted rhombohedral structure which resembled calcite and vaterite phases of the crystal. Formation of calcium carbonate crystals was further confirmed by Fourier transform infrared (FTIR) spectroscopy as carbonate group showing strong vibration at 1,456 cm(-1). Major calcite phase diffraction peaks were determined by X-ray diffraction (XRD) analysis, and energy-dispersive X-ray (EDX) analysis showed the presence of CaO (72 %) and carbon (18 %). Bacterium use bicarbonate as carbon source for their growth as well as by-product formation in form of calcite shows carbon circulation and storage.