Factors involved in the formation of amorphous and crystalline calcium carbonate: a study of an ascidian skeleton.

The majority of invertebrate skeletal tissues are composed of the most stable crystalline polymorphs of CaCO(3), calcite, and/or aragonite. Here we describe a composite skeletal tissue from an ascidian in which amorphous and crystalline calcium carbonate coexist in well-defined domains separated by an organic sheath. Each biogenic mineral phase has a characteristic Mg content (5.9 and 1.7 mol %, respectively) and concentration of intramineral proteins (0.05 and 0.01 wt %, respectively). Macromolecular extracts from various biogenic amorphous calcium carbonate (ACC) skeletons are typically glycoproteins, rich in glutamic acid and hydroxyamino acids. The proteins from the crystalline calcitic phases are aspartate-rich. Macromolecules extracted from biogenic ACC induced the formation of stabilized ACC and/or inhibited crystallization of calcite in vitro. The yield of the synthetic ACC was 15-20%. The presence of Mg facilitated the stabilization of ACC: the protein content in synthetic ACC was 0.12 wt % in the absence of Mg and 0.07 wt % in the presence of Mg (the Mg content in the precipitate was 8.5 mol %). In contrast, the macromolecules extracted from the calcitic layer induced the formation of calcite crystals with modified morphology similar to that expressed by the original biogenic calcite. We suggest that specialized macromolecules and magnesium ions may cooperate in the stabilization of intrinsically unstable amorphous calcium carbonate and in the formation of complex ACC/calcite tissues in vivo.     

High-magnesian calcite mesocrystals: a coordination chemistry approach

While biogenic calcites frequently contain appreciable levels of magnesium, the pathways leading to such high concentrations remain unclear. The production of high-magnesian calcites in vitro is highly challenging, because Mg-free aragonite, rather than calcite, is the favored product in the presence of strongly hydrated Mg(2+) ions. While nature may overcome this problem by forming a Mg-rich amorphous precursor, which directly transforms to calcite without dissolution, high Mg(2+)/Ca(2+) ratios are required synthetically to precipitate high-magnesian calcite from solution. Indeed, it is difficult to synthesize amorphous calcium carbonate (ACC) containing high levels of Mg, and the Mg is typically not preserved in the calcite product as the transformation occurs via a dissolution-reprecipitation route. We here present a novel synthetic method, which employs a strategy based on biogenic systems, to generate high-magnesian calcite. Mg-containing ACC is produced in a nonaqueous environment by reacting a mixture of Ca and Mg coordination complexes with CO(2). Control over the Mg incorporation is simply obtained by the ratio of the starting materials. Subsequent crystallization at reduced water activities in an organic solvent/water mixture precludes dissolution and reprecipitation and yields high-magnesian calcite mesocrystals with Mg contents as high as 53 mol %. This is in direct contrast with the polycrystalline materials generally observed when magnesian calcite is formed synthetically. Our findings give insight into the possible mechanisms of formation of biogenic high-magnesian calcites and indicate that precise control over the water activity may be a key element.     

High‐Magnesium Calcite Mesocrystals: Formation in Aqueous Solution under Ambient Conditions

Mesocrystals of high‐magnesian calcites are commonly found in biogenic calcites. Under ambient conditions, it remains challenging to prepare mesocrystals of high‐magnesian calcite in aqueous solution. We report that mesocrystals of calcite with magnesium content of about 20 mol % can be obtained from the phase transformation of magnesian amorphous calcium carbonate (Mg‐ACC) in lipid solution. The limited water content on the Mg‐ACC surface would reduce the extent of the dissolution–reprecipitation process and bias the phase transformation pathway toward solid‐state reaction. We infer from the selected area electron diffraction patterns and the dark‐field transmission electron microscopic images that the formation of Mg‐calcite mesocrystals occurs through solid‐state secondary nucleation, for which the phase transformation is initiated near the mineral surface and the crystalline phase propagates gradually toward the interior part of the microspheres of Mg‐ACC.     

On the structure of amorphous calcium carbonate--a detailed study by solid-state NMR spectroscopy

The calcium carbonate phases calcite, aragonite, vaterite, monohydrocalcite (calcium carbonate monohydrate), and ikaite (calcium carbonate hexahydrate) were studied by solid-state NMR spectroscopy ( (1)H and (13)C). Further model compounds were sodium hydrogencarbonate, potassium hydrogencarbonate, and calcium hydroxide. With the help of these data, the structure of synthetically prepared additive-free amorphous calcium carbonate (ACC) was analyzed. ACC contains molecular water (as H 2O), a small amount of mobile hydroxide, and no hydrogencarbonate. This supports the concept of ACC as a transient precursor in the formation of calcium carbonate biominerals.     

Stabilization of amorphous calcium carbonate in inorganic silica-rich environments

In biomineralization, living organisms carefully control the crystallization of calcium carbonate to create functional materials and thereby often take advantage of polymorphism by stabilizing a specific phase that is most suitable for a given demand. In particular, the lifetime of usually transient amorphous calcium carbonate (ACC) seems to be thoroughly regulated by the organic matrix, so as to use it either as an intermediate storage depot or directly as a structural element in a permanently stable state. In the present study, we show that the temporal stability of ACC can be influenced in a deliberate manner also in much simpler purely abiotic systems. To illustrate this, we have monitored the progress of calcium carbonate precipitation at high pH from solutions containing different amounts of sodium silicate. It was found that growing ACC particles provoke spontaneous polymerization of silica in their vicinity, which is proposed to result from a local decrease of pH nearby the surface. This leads to the deposition of hydrated amorphous silica layers on the ACC grains, which arrest growth and alter the size of the particles. Depending on the silica concentration, these skins have different thicknesses and exhibit distinct degrees of porosity, therefore impeding to varying extents the dissolution of ACC and energetically favored transformation to calcite. Under the given conditions, crystallization of calcium carbonate was slowed down over tunable periods or completely prevented on time scales of years, even when ACC coexisted side by side with calcite in solution.     

Biomineralization of sea urchin teeth

The sea urchin tooth, which is composed almost entirely of Mg-enriched CaCO₃, is of particular interest as a model for the study of biomineralization process due to its amazing mechanical toughness and hardness. Our recent work on the formation process, the crystal composition and orientation, and the mechanical properties of sea urchin tooth are summarized in this paper. First, transmission electron microscopy images and electron diffraction patterns, as well as crystal overgrowth experiments, show that the highly convoluted primary plate-lamellar needle complex grows into a single crystal of calcite from a transient amorphous precursor phase in the sea urchin tooth. Amorphous calcium carbonate exists in the center of both the primary plates and the needles, even though the surfaces are already well crystallized. Second, X-ray photoelectron emission spectromicroscopy demonstrates that the needles, primary plates, and polycrystalline matrix crystals are all aligned. And there are two alternating crystal orientations in the stone part of the sea urchin tooth. Microbeam X-ray diffraction patterns further prove the existence of the two crystal orientations in sea urchin tooth. The c axes of calcite in the two oriented crystals are only a few degrees from each other. Third, the mechanical properties of sea urchin tooth grinding tip were studied by nanoindentation. The polycrystalline matrix has a higher elastic modulus and hardness than single crystalline needles and plates. It is proposed that the grinding capability of the tooth can be attributed to the small and uniform sizes of the polycrystalline crystals, their high Mg contents, and the two co-orientations of single crystals and polycrystalline structure. The improved understanding of the biomineralization process of sea urchin tooth and the relations between their structures and mechanical properties may shed light on the design of mechanical grinding and cutting tools with tunable properties.     

In vitro study of magnesium-calcite biomineralization in the skeletal materials of the seastar Pisaster giganteus

The mechanisms of formation of biogenic magnesium-rich calcite remain an enigma. Here we present ultrastructural and compositional details of ossicles from the seastar Pisaster giganteus (Echinodermata, Asteroidea). Powder X-ray diffraction, infrared spectroscopy and elemental analyses confirm that the ossicles are composed of magnesium-rich calcite, whilst also containing about 0.01 % (w/w) of soluble organic matrix (SOM) as an intracrystalline component. Amino acid analysis and N-terminal sequencing revealed that this mixture of intracrystalline macromolecules consists predominantly of glycine-rich polypeptides. In vitro calcium carbonate precipitation experiments indicate that the SOM accelerates the conversion of amorphous calcium carbonate (ACC) into its final crystalline product. From this observation and from the discovery of ACC in other closely related taxa, it is suggested that substitution of magnesium into the calcite lattice through a transient precursor phase may be a universal phenomenon prevalent across the phylum echinodermata.     

Properties of amorphous calcium carbonate and the template action of vaterite spheres

The fast mixing of aqueous solutions of calcium chloride and sodium carbonate could immediately result in amorphous calcium carbonate (ACC). Under vigorous stirring, the formed ACC in the precipitation system will dissolve first and, then, transform within minutes to produce crystalline forms of vaterite and calcite. After that, the solution-mediated mechanism dominates the transformation of the thermodynamically unstable vaterite into the thermodynamically stable calcite. Although ACC is the least stable form of the six anhydrous phases of calcium carbonate (CaCO(3)), it could be, however, produced and stabilized by a variety of organisms. To better understand the formation-transformation mechanism of ACC and vaterite into calcite, ex-situ methods (i.e., scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction spectroscopy) were used to characterize the formation-transformation process of ACC and vaterite in aqueous systems without organic additives, showing that ACC sampled at different conditions has different properties (i.e., lifetime, morphology, and spectrum characterization). It is also very interesting to capture the obviously polycrystalline particles of CaCO(3) during the transformation process from vaterite to calcite, which suggests the formation mechanism for the calcite superstructure with multidimensional morphology.     

Evolution Mechanism of Calcium Carbonate in Solution

Calcium carbonate was synthesized in a CaCl₂/NaCO₃ mixed solution by using ethylenedi-aminetetraacetic acid (EDTA) as an additive. The thermodynamics and kinetics analyses indicate that although the driving force of amorphous calcium carbonate (ACC) precipi-tation is always less than that of calcite and vaterite precipitation, the nucleation rate of ACC is greater than that of calcite and vaterite at the initial stage of the precipitation reaction. With the increasing incubation time, vaterite and calcite particles nucleate het-erogeneously by using the as-formed particles as active sites. Scanning electron microscopyimages indicate that the transformation mechanism of ACC and vaterite to calcite is the dissolution-recrystallisation reaction. The presence of EDTA not only improves the stabil-ities of ACC and vaterite, but also leads to forming enlongated, connected rhombohedralcalcite crystals after incubation 7 days in solutions. The ACC and vaterite are stabler in air than in solutions at room temperature, although the dissolution-recrystallisation reaction occurs on the surface.     

Calcium carbonate phase transformations during the carbonation reaction of calcium heavy alkylbenzene sulfonate overbased nanodetergents preparation

The preparation and application of overbased nanodetergents with excess alkaline calcium carbonate is a good example of nanotechnology in practice. The phase transformation of calcium carbonate is of extensive concern since CaCO(3) serves both as an important industrial filling material and as the most abundant biomineral in nature. Industrially valuable overbased nanodetergents have been prepared based on calcium salts of heavy alkylbenzene sulfonate by a one-step process under ambient pressure, the carbonation reaction has been monitored by the instantaneous temperature changes and total base number (TBN). A number of analytical techniques such as TGA, DLS, SLS, TEM, FTIR, and XRD have been utilized to explore the carbonation reaction process and phase transformation mechanism of calcium carbonate. An enhanced understanding on the phase transformation of calcium carbonate involved in calcium sulfonate nanodetergents has been achieved and it has been unambiguously demonstrated that amorphous calcium carbonate (ACC) transforms into the vaterite polymorph rather than calcite, which would be of crucial importance for the preparation and quality control of lubricant additives and greases. Our results also show that a certain amount of residual Ca(OH)(2) prevents the phase transformation from ACC to crystalline polymorphs. Moreover, a vaterite nanodetergent has been prepared for the first time with low viscosity, high base number, and uniform particle size, nevertheless a notable improvement on its thermal stability is required for potential applications.     

A quasi-time-resolved CryoTEM study of the nucleation of CaCO3 under langmuir monolayers

Calcium carbonate biomineralization uses complex assemblies of macromolecules that control the nucleation, growth, and positioning of the mineral with great detail. To investigate the mechanisms involved in these processes, for many years Langmuir monolayers have been used as model systems. Here, we descibe the use of cryogenic transmission electron microscopy in combination with selected area electron diffraction as a quasi-time-resolved technique to study the very early stages of this process. In this way, we assess the evolution of morphology, polymorphic type, and crystallographic orientation of the calcium carbonate formed. For this, we used a self-assembled Langmuir monolayer of a valine-based bisureido surfactant (1) spread on a CaCl2-containing subphase and deposited on a holey carbon TEM grid. In a controlled environment, the grid is exposed to an atmosphere containing NH3 and CO2 (the (NH4)2CO3 diffusion method) for precisely determined periods of time (reaction times 30-1800 s) before it was plunged into melting ethane. This procedure allows us to observe amorphous calcium carbonate (ACC) particles growing from a few tens of nanometers to hundreds of nanometers and then crystallizing to form [00.1] oriented vaterite. The vaterite in turn transforms to yield [10.0] oriented calcite. We also performed the reaction in the absence of monolayer or in the presence of a nondirective monolayer of surfactant containing an oligo(ethylene oxide) 2 head group. Both experiments also showed the formation of a transient amorphous phase followed by a direct conversion into randomly oriented calcite crystals. These results imply the specific though temporary stabilization of the (00.1) vaterite by the monolayer. However, experiments performed at higher CaCl2 concentrations show the direct conversion of ACC into [10.0] oriented calcite. Moreover, prolonged exposure to the electron beam shows that this transformation can take place as a topotactic process. The formation of the (100) calcite as final product under different conditions shows that the surfactant is very effective in directing the formation of this crystal plane. In addition, we present evidence that more than one type of ACC is involved in the processes described.     

Structural development of mercaptophenol self-assembled monolayers and the overlying mineral phase during templated CaCO3 crystallization from a transient amorphous film

Formation of biomineral structures is increasingly attributed to directed growth of a mineral phase from an amorphous precursor on an organic matrix. While many in vitro studies have used calcite formation on organothiol self-assembled monolayers (SAMs) as a model system to investigate this process, they have generally focused on the stability of amorphous calcium carbonate (ACC) or maximizing control over the order of the final mineral phase. Little is known about the early stages of mineral formation, particularly the structural evolution of the SAM and mineral. Here we use near-edge X-ray absorption spectroscopy (NEXAFS), photoemission spectroscopy (PES), X-ray diffraction (XRD), and scanning electron microscopy (SEM) to address this gap in knowledge by examining the changes in order and bonding of mercaptophenol (MP) SAMs on Au(111) during the initial stages of mineral formation as well as the mechanism of ACC to calcite transformation during template-directed crystallization. We demonstrate that formation of ACC on the MP SAMs brings about a profound change in the morphology of the monolayers: although the as-prepared MP SAMs are composed of monomers with well-defined orientations, precipitation of the amorphous mineral phase results in substantial structural disorder within the monolayers. Significantly, a preferential face of nucleation is observed for crystallization of calcite from ACC on the SAM surfaces despite this static disorder.     

Capillarity creates single-crystal calcite nanowires from amorphous calcium carbonate

Single-crystal calcite nanowires are formed by crystallization of morphologically equivalent amorphous calcium carbonate (ACC) particles within the pores of track etch membranes. The polyaspartic acid stabilized ACC is drawn into the membrane pores by capillary action, and the single-crystal nature of the nanowires is attributed to the limited contact of the intramembrane ACC particle with the bulk solution. The reaction environment then supports transformation to a single-crystal product.     

A carbonate controlled-addition method for amorphous calcium carbonate spheres stabilized by poly(acrylic acid)s

Stable amorphous calcium carbonate (ACC) composite particle with a size-controlled monodispersed sphere was obtained by a new simple carbonate controlled-addition method by using poly(acrylic acid) (PAA) (Mw = 5000), in which an aqueous ammonium carbonate solution was added into an aqueous solution of PAA and CaCl2 with a different time period. The obtained ACC composite products consist of about 50 wt % of ACC, 30 wt % of PAA, and H2O. Average particle sizes of the ACC spheres increased from (1.8 +/- 0.4) x 102 to (5.5 +/- 1.2) x 102 nm with an increase of the complexation time of the PAA-CaCl2 solution from 3 min to 24 h, respectively. The ACC formed from the complexation time for 3 min was stable for 10 days with gentle stirring as well as 3 months under a quiescent condition in the aqueous solution. Moreover, the ACC was also stable at 400 degrees C. Stability of the amorphous phase decreased with an increase of the complexation time of the PAA-CaCl2 solution. No ACC was obtained when the lower molar mass PAAs (Mw = 1200 and 2100) were used. In the higher molar mass case (Mw = 25 000), a mixture of the amorphous phase and vaterite and calcite crystalline product was produced. The present results demonstrate that the interaction and the reaction kinetics of the PAA-Ca2+-H2O complex play an important role in the mineralization of CaCO3.     

Quantitative analysis of synthetic calcium carbonate polymorphs using FT-IR spectroscopy

Fourier Transform Infrared Spectroscopy (FT-IR) was used successfully for the simultaneous quantitative analysis of calcium carbonate phases (calcite, aragonite, vaterite) in ternary mixtures. From the FT-IR spectra of pure calcite, aragonite and vaterite powders with KBr, the absorptivities, α, of the absorption bands at 713 cm⁻¹ for calcite, 745 cm⁻¹ for vaterite, 713 and 700 cm⁻¹ for aragonite, were determined. In order to overcome the absorption band overlapping a set of equations based on Beer's law was developed. The detection limits were also established and found to be 1.1×10⁻⁴ mg calcite per mm² of pellet at 713 cm⁻¹, 3.6×10⁻⁴ mg aragonite per mm² of pellet at 700 cm⁻¹, 1.8×10⁻⁴ mg aragonite per mm² of pellet at 713 cm⁻¹ and 3.1×10⁻⁴ mg vaterite per mm² of pellet at 745 cm⁻¹. Analysis of a known ternary mixture of calcium carbonate polymorphs tested the validity of the method.     

Water as the Key to Proto‐Aragonite Amorphous CaCO3

Temperature and pH value can affect the short‐range order of proto‐structured and additive‐free amorphous calcium carbonates (ACCs). Whereas a distinct change occurs in proto‐vaterite (pv) ACC above 45 °C at pH 9.80, proto‐calcite (pc) ACC (pH 8.75) is unaffected within the investigated range of temperatures (7–65 °C). IR and NMR spectroscopic studies together with EXAFS analysis showed that the temperature‐induced change is related to the formation of proto‐aragonite (pa) ACC. The data strongly suggest that the binding of water molecules induces dipole moments across the carbonate ions in pa‐ACC as in aragonite, where the dipole moments are due to the symmetry of the crystal structure. Altogether, a (pseudo‐)phase diagram of the CaCO₃ polyamorphism in which water plays a key role can be formulated based on variables of state, such as the temperature, and solution parameters, such as the pH value.     

The mineral phase in the cuticles of two species of Crustacea consists of magnesium calcite, amorphous calcium carbonate, and amorphous calcium phosphate

The cuticules (shells) of the woodlice Porcellio scaber and Armadillidium vulgare were analysed with respect to their content of inorganic material. It was found that the cuticles consist of crystalline magnesium calcite, amorphous calcium carbonate (ACC), and amorphous calcium phosphate (ACP), besides small amounts of water and an organic matrix. It is concluded that the cuticle, which constitutes a mineralized protective organ, is chemically adapted to the biological requirements by this combination of different materials.     

Mineralization of CaCO3 in the presence of egg white lysozyme

The influence of egg white lysozyme on the size, shape, crystallography, and chemical composition of amorphous calcium carbonate (ACC) particles obtained from aqueous CaCl2-dimethyl carbonate (DMC)-NaOH solutions was studied. At the onset of precipitation, the presence of lysozyme led to much smaller particles (50-400 nm spherical amorphous lysozyme-calcium carbonate particles (Ly-ACC)) than those obtained from lysozyme-free solution. The nanospheres were in some cases aggregated and in addition embedded in a faint network. Their size and interconnection depended on the concentration of egg white lysozyme. When the Ly-ACC particles were left in contact with the mother liquor (CaCl2/DMC/NaOH/lysozyme solution) for 24 h, they transformed directly and exclusively into crystalline calcite. The observed results may be of relevance for a better understanding of the role of lysozyme in the process of eggshell mineralization.     

High-resolution Fourier-transform infrared spectroscopy of the Coriolis coupled ground state and ν7 mode of ketenimine

High resolution FTIR spectra of the short lived species ketenimine have been recorded in the regions 390-1300 cm(-1) and 20-110 cm(-1) using synchrotron radiation. Two thousand six hundred sixty transitions of the ν(7) band centered at 693 cm(-1) and 126 far-IR rotational transitions have been assigned. Rotational and centrifugal distortion parameters for the ν(7) mode were determined and local Fermi and b-axis Coriolis interactions with 2ν(12) are treated. A further refinement of the ground state, ν(12) and ν(8) parameters was also achieved, including the treatment of previously unrecognized ac-axis and ab-axis second order perturbations to the ground state.     

Probing the organic-mineral interface at the molecular level in model biominerals

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.