Do corals form their skeletons by precipitation from solution or by attachment of amorphous precursor particles as observed in other minerals and biominerals? The classical model assumes precipitation in contrast with observed “vital effects,” that is, deviations from elemental and isotopic compositions at thermodynamic equilibrium. Here, we show direct spectromicroscopy evidence in Stylophora pistillata corals that two amorphous precursors exist, one hydrated and one anhydrous amorphous calcium carbonate (ACC); that these are formed in the tissue as 400-nm particles; and that they attach to the surface of coral skeletons, remain amorphous for hours, and finally, crystallize into aragonite (CaCO3). We show in both coral and synthetic aragonite spherulites that crystal growth by attachment of ACC particles is more than 100 times faster than ion-by-ion growth from solution. Fast growth provides a distinct physiological advantage to corals in the rigors of the reef, a crowded and fiercely competitive ecosystem. Corals are affected by warming-induced bleaching and postmortem dissolution, but the finding here that ACC particles are formed inside tissue may make coral skeleton formation less susceptible to ocean acidification than previously assumed. If this is how other corals form their skeletons, perhaps this is how a few corals survived past CO2 increases, such as the Paleocene–Eocene Thermal Maximum that occurred 56 Mya.

Calcified skeletons are produced within complex assemblages of proteins and polysaccharides whose roles in mineralization are not well understood. Here we quantify the kinetics of calcite nucleation onto a suite of high-purity polysaccharide (PS) substrates under controlled conditions. The energy barriers to nucleation are PS-specific by a systematic relationship to PS charge density and substrate structure that is rooted in minimization of the competing substrate-crystal and substrate-liquid interfacial energies. Chitosan presents a low-energy barrier to nucleation because its near-neutral charge favors formation of a substrate-crystal interface, thus reducing substrate interactions with water. Progressively higher barriers are measured for negatively charged alginates and heparin that favor contact with the solution over the formation of new substrate-crystal interfaces. The findings support a directing role for PS in biomineral formation and demonstrate that substrate-crystal interactions are one end-member in a larger continuum of competing forces that regulate heterogeneous crystal nucleation.biomineralization | calcium carbonate | free energy | algae | crustacean E fforts to decipher patterns of biomineralization have identified proteins and polysaccharides (PSs) as major components of the organic matrices associated with sites of calcification. In mollusks and other organisms including the red algae, coccolithophores, and foraminifera (1, 2) (Table 1), calcifying macromolecules are dominated by functional groups with an acidic character-proteins that are rich in carboxylated amino acids (14) and PSs that are highly sulfated and carboxylated (15,16). Although a physical picture of these interactions is not well developed, this recurring affiliation of carboxylate and sulfate groups with zones of mineralization in organisms suggests specific roles for macromolecules in nucleation and growth. Early studies have led the biomineralization community to generally assume that charged proteins actively regulate mineralization. In contrast, PSs, such as the chitin found in the insoluble fraction of the mollusk shell, are thought to provide an inert scaffolding to support these proteins (17,18).Recent studies challenge this assumption with qualitative evidence that PSs can also promote calcium carbonate (CaCO 3 ) mineralization (19,20) (Table 1). For example, specific orientations of chitin fibers with neutral functional groups promote the templating of CaCO 3 in the lobster carapace (13, 21), crab cuticle (21), and nautilus shell (22). More generally, the monosaccharide sequences along PS chains can influence biological function and their interactions with proteins (23). This suggests PS chemistry and interactions with proteins could regulate patterns of mineralization.Anecdotal observations from in vitro studies also support the thinking that PSs influence mineral formation, but their specific effects are unclear. For example, PSs with higher carboxyl and sulfate content promote either faster (24, 25) or slower (2...

Calcite and other crystalline polymorphs of CaCO 3 can form by pathways involving amorphous calcium carbonate (ACC). Previous studies of ACC provide important insights, but apparent inconsistencies in the literature indicate the relationships between ACC composition, local conditions, and the subsequent crystalline polymorphs are not yet established. This experimental study quantifies the control of solution composition on the transformation of ACC into crystalline polymorphs in the presence of magnesium. Using a mixed flow reactor to control solution chemistry, ACC was synthesized with variable Mg contents by tuning input pH, Mg/Ca, and total carbonate concentration. ACC products were allowed to transform within the output suspension under stirred or quiescent conditions while characterizing the evolving solutions and solids. As the ACC transforms into a crystalline phase, the solutions record a polymorph-specific evolution of pH and Mg/Ca. The data provide a quantitative framework for predicting the initial polymorph that forms from ACC based upon the solution aMg 2+ /aCa 2+ and aCO 3 2-/aCa 2+ and stirring versus quiescent conditions. This model reconciles apparent discrepancies among previous studies that report on the nature of the polymorphs produced from ACC and supports the previous claim that monohydrocalcite may be an important, but overlooked, transient phase on the way to forming some aragonite and calcite deposits. By this construct, organic additives and extreme pH are not required to tune the composition and nature of the polymorph that forms. Our measurements show that the Mg content of ACC is recorded in the resulting calcite with a ≈1:1 dependence. By correlating the composition of these calcite products with the Mg tot /Ca tot of the initial solutions, we find a ≈3:1 dependence that is approximately linear and general to whether the calcite is formed via an ACC pathway or by the classical step-propagation process. Comparisons to calcite grown in synthetic seawater show a ≈1:1 dependence. The relationships suggest that the local Mg 2+ /Ca 2+ at the time of precipitation determines the calcite composition, independent of whether growth occurs via an amorphous intermediate or classical pathway for a range of supersaturations and pH conditions. The findings reiterate the need to revisit the traditional picture of chemical and physical controls on CaCO 3 polymorph selection. Mineralization by pathways involving ACC can lead to the formation of crystalline phases whose polymorphs and compositions are out of equilibrium with the local growth media. As such, classical thermodynamic equilibria may not provide a reliable predictor of observed compositions.

The physical basis for how macromolecules regulate the onset of mineral formation in calcifying tissues is not well established. A popular conceptual model assumes the organic matrix provides a stereochemical match during cooperative organization of solute ions. In contrast, another uses simple binding assays to identify good promoters of nucleation. Here, we reconcile these two views and provide a mechanistic explanation for template-directed nucleation by correlating heterogeneous nucleation barriers with crystal-substrate-binding free energies. We first measure the kinetics of calcite nucleation onto model substrates that present different functional group chemistries (carboxyl, thiol, phosphate, and hydroxyl) and conformations (C11 and C16 chain lengths). We find rates are substrate-specific and obey predictions of classical nucleation theory at supersaturations that extend above the solubility of amorphous calcium carbonate. Analysis of the kinetic data shows the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy of the system, γ. We then use dynamic force spectroscopy to independently measure calcitesubstrate-binding free energies, ΔG b . Moreover, we show that within the classical theory of nucleation, γ and ΔG b should be linearly related. The results bear out this prediction and demonstrate that low-energy barriers to nucleation correlate with strong crystal-substrate binding. This relationship is general to all functional group chemistries and conformations. These findings provide a physical model that reconciles the long-standing concept of templated nucleation through stereochemical matching with the conventional wisdom that good binders are good nucleators. The alternative perspectives become internally consistent when viewed through the lens of crystal-substrate binding.B iological systems are unique in their ability to organize minerals into functional materials with complex patterns and architectures. A substantial body of evidence suggests specialized macromolecules, particularly proteins (1, 2) and carbohydrates (3, 4), provide preferential sites for nucleation to direct the placement, timing, and orientation of crystals (5), both intra-and extracellular. Within the biomineralization community, the conventional view of biologically directed nucleation is that macromolecular matrices present an interfacial match to the crystal lattice that assists in forming the crystal nucleus. This cooperative view of directed nucleation is rooted in the collective action of multiple residues that guide the organization of ions into a configuration defining the energetic minimum for the system. A series of in vitro observations have reinforced this picture by showing that highly ordered organic monolayers can control the location and orientation of calcite crystals precipitated from solution (6). In this approach, good templates are revealed through a direct functional assay, i.e., nucleation. Over the years, this view of mineralization, both in the context of natural stru...

In contrast to synthetic materials, materials produced by organisms are formed in ambient conditions and with a limited selection of elements. Nevertheless, living organisms reveal elegant strategies for achieving specific functions, ranging from skeletal support to mastication, from sensors and defensive tools to optical function. Using state-of-the-art characterization techniques, we present a biostrategy for strengthening and toughening the otherwise brittle calcite optical lenses found in the brittlestar This intriguing process uses coherent nanoprecipitates to induce compressive stresses on the host matrix, functionally resembling the Guinier-Preston zones known in classical metallurgy. We believe that these calcitic nanoparticles, being rich in magnesium, segregate during or just after transformation from amorphous to crystalline phase, similarly to segregation behavior from a supersaturated quenched alloy.

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Coral skeletons were long assumed to have a spherulitic structure, that is, a radial distribution of acicular aragonite (CaCO 3 ) crystals with their c-axes radiating from series of points, termed centers of calcification (CoCs). This assumption was based on morphology alone, not on crystallography. Here we measure the orientation of crystals and nanocrystals and confirm that corals grow their skeletons in bundles of aragonite crystals, with their caxes and long axes oriented radially and at an angle from the CoCs, thus precisely as expected for feather-like or "plumose" spherulites. Furthermore, we find that in both synthetic and coral aragonite spherulites at the nanoscale adjacent crystals have similar but not identical orientations, thus demonstrating by direct observation that even at nanoscale the mechanism of spherulite formation is non-crystallographic branching (NCB), as predicted by theory. Finally, synthetic aragonite spherulites and coral skeletons have similar angle spreads, and angular distances of adjacent crystals, further confirming that coral skeletons are spherulites. This is important because aragonite grows anisotropically, 10 times faster along the c-axis than along the a-axis direction, and spherulites fill space with crystals growing almost exclusively along the c-axis, thus they can fill space faster than any other aragonite growth geometry, and create isotropic materials from anisotropic crystals. Greater space filling rate and isotropic mechanical behavior are key to the skeleton's supporting function and therefore to its evolutionary success. In this sense, spherulitic growth is Nature's 3D printing. KEYWORDS: Ion attachment, crystallization by particle attachment, CPA, biomineralization, PEEM, PIC-mapping, mesocrystal S pherulites are polycrystalline structures in which acicular crystals radiate from a common center and grow approximately synchronously so the final shape of a spherulite resembles a sphere. 1−7 Figure 1 shows two types of spherulites: "spherical spherulite", in which the crystal fibers start from a point, or "plumose spherulite", in which crystal fibers radiate at an angle from the line. 7 Spherulites start forming as an aggregate of parallel acicular crystals termed "fibers", then form a "sheaf of wheat" structure, and with the growth of more fibers eventually become complete spheres. 8 In an ideal spherulite, fibers radiate from the center and contain all possible orientations within the sphere. Since the fast-growing axis in aragonite (CaCO 3 ) is the c-axis, 9 in spherulites each fiber elongation direction coincides with the crystalline c-axis. 10,11 In real spherulites, biogenic 12 and synthetic, 7 the crystal orientations are not perfectly radial nor continuously varying with angle, they deviate slightly from radial and exhibit small but abrupt changes in orientation, termed "branching". In Figure 1 branching angles are smaller than 30°in direction and in crystal lattice orientation. This is distinct from crystallographic branching, which occurs in sno...

Please cite this article as: Giuffre, A.J., Gagnon, A.C., De Yoreo, J.J., Dove, P.M., Isotopic tracer evidence for the amorphous calcium carbonate to calcite transformation by dissolution-reprecipitation, Geochimica et Cosmochimica Acta (2015), doi: http://dx. AbstractObservations that some biogenic and sedimentary calcites grow from amorphous calcium carbonate (ACC) raise the question of how this mineralization process influences composition. However, the detailed pathway and geochemical consequences of the ACC to calcite transformation are poorly constrained. This experimental study investigated the formation of calcite from ACC by using magnesium and calcium stable isotope labeling to directly probe the transformation pathway and controls on composition. Four processes were considered: dissolution-reprecipitation, solid state transformation, and combinations of these end-members. To distinguish between these scenarios, ACC was synthesized from natural isotope abundance solutions and subsequently transferred to spiked solutions that were enriched in 43 Ca and 25 Mg for the transformation to calcite.Isotope measurements by NanoSIMS determined the 43 Ca/ 40 Ca, and 25 Mg/ 24 Mg ratios of the resulting calcite crystals.Analysis of the data shows the transformation is best explained by a dissolutionreprecipitation process. We find that when a small amount of ACC is transferred, the isotopic signals in the resulting calcite are largely replaced by the composition of the surrounding spiked solution. When larger amounts of ACC are transferred, calcite compositions reflect a mixture between the ACC and initial solution end-member.Comparisons of the measurements to the predictions of a simple mixing model indicate that calcite compositions 1) are sensitive to relative amounts of ACC and the surrounding solution reservoir and 2) are primarily governed by the conditions at the time of ACC transformation rather than the initial ACC formation. Shifts in calcite composition over the duration of the transformation period reflect the progressive evolution of the local solution conditions. This dependence suggests the extent to which there is water available would change the end point composition on the mixing line. While these findings have significant geochemical implications, the question remains whether this transformation pathway is generally followed when biomineralization involves ACC or is particular to these inorganic experiments. Insights from this study nonetheless suggest that some types of compositional variability, such as 'vital effects', may be explained inpart by a co-evolution of reservoir and products over the duration of the transformation.3