Controls on CaCO3 Polymorphism: From Laboratory Precipitation to Biomineralization across Geological Time

 1811-5209/25/0021-0092$2.50 DOI: 10.2138/gselements.21.2.92

Keywords: calcium carbonate; polymorphism; biomineralization; organic matrix

INTRODUCTION

Calcium carbonates (CaCO3) are among the most abundant minerals in Earth’s crust and biosphere where they occur in several polymorphic forms: three anhydrous crystalline polymorphs (calcite, aragonite, and vaterite), three hydrated phases (monohydrocalcite, ikaite, and calcium carbonate hemihydrate), as well as diverse forms of non-crystalline (polyamorphic) calcium carbonate (ACC). The most common crystalline forms of calcium carbonate differ significantly in their physicochemical characteristics, and marine organisms can deposit one or a mixture of (tailored) polymorphs when creating their biomineral structures (Table 1).

The marine fossil record demonstrates that calcification has evolved independently several times (e.g., in foraminifera and bryozoans) and that major groups/phyla have retained a given mineralogy since their origin. This phenomenon may reflect, on one hand, the need to maintain stable biomaterial characteristics of skeletal structures and, on the other hand, the evolutionary conservatism of biominorganisms (e.g., some bivalve taxa) deposit alternate layers of calcite and aragonite, evidencing tight control on polymorph selection during biomineralization.

Organisms select their specific polymorphs in a number of ways: 1) by modifying the chemical composition of the calcifying medium (fluid), 2) by transforming amorphous or nanocrystalline metastable precursor phases, 3) by involving organic macromolecules (organic templates), or by a combination of all of these. Traditionally, two different mechanisms have been proposed for controlling polymorphism during biomineralization: the first emphasizes the involvement of external, non-biological factors on the composition of calcifying medium (such as the seawater magnesium to calcium ratio [Mg2+/Ca2+] or sulfate [SO42−] concentration), while the second highlights the strong control exerted on the biomineralization process by the organism, by e.g., secreting functional organic macromolecules. In this review, we focus on these two controls while highlighting the synergistic influence of physicochemical factors, complex biochemical, and physiological pathways involved in the regulation of mineral deposition, and the coevolution of environments and organisms on Earth over hundreds of millions of years. All of these factors have profoundly impacted the evolution of CaCO3 biomineralization pathways in both Earth’s geologic past and the modern day.

PHYSICOCHEMICAL CONTROLS ON CACO3 POLYMORPHISM AND CRYSTALLIZATION

Inorganic precipitation experiments provide a key baseline for understanding how and which calcium carbonates form from seawater-like solutions. Important insights from these experiments include the discovery that differences in solution (major element) composition influence the nucleation, growth, and transformation of calcium carbonate phases, and how such factors lead to polymorph selection (Fig. 1). Broadly speaking, the boundary between the preferential precipitation of aragonite or calcite lies at a seawater Mg2+/Ca2+ ratio of ~2 mol/mol in abiogenic systems (Morse et al. 2007). However, while Mg/Ca is commonly interpreted to be the main factor controlling which polymorph precipitates, experiments have also shown that sulfate lowers the Mg2+/Ca2+ of this boundary (Fig. 1B; Bots et al. 2011), which is important because seawater contains a major sulphate component (~28 mM).

The mechanisms that control the nucleation, growth, and transformation of phases in the CaCO3 system are linked to whether the mineral forms via ‘classical’ or non-classical pathways. Classical growth refers to the ion-by-ion deposition from the fluid directly onto the growing crystal’s surface, whereas non-classical mechanisms include precipitation of some precursor phase(s) that then convert to the final crystalline polymorph. Classical crystal growth of biogenic CaCO3 has often been inferred from macroscopic observations; however, overwhelming evidence has accumulated for non-classical growth predominating in many inorganic and biological systems, and these non-classical pathways complicate the inorganic precipitation and growth fields summarized above. In the case of non-classical crystal growth, crystalline calcium carbonate forms from solid precursor phases that include amorphous calcium carbonate (ACC), vaterite, or calcium carbonate hemihydrate (Table 1; De Yoreo et al. 2015). These precursor solids, in turn, can be preceded by either a metastable dense liquid phase (Xu et al. 2018), or by pre-nucleation clusters (Gebauer et al. 2008), although the role of pre-nucleation clusters in calcium carbonate crystallization continues to be debated (Darkins et al. 2024). In both abiotic systems and biomineralisation, ACC usually nucleates and grows as polyamorphous metastable nanoparticles, which are the initial building blocks that then transform to stable, complex, hierarchical crystalline minerals. This transformation commonly occurs via dissolution and reprecipitation. In greater detail, the conversion of ACC to crystalline CaCO3 polymorphs follows a plethora of pathways and mechanisms that are dependent on variations in physicochemical parameters of both the solid phase and the solution in which it is formed or delivered. The factors controlling this process include supersaturation, pH, temperature, confinement (e.g., Rodriguez-Navarro et al. 2016; Meldrum and O’Shaughnessy 2020), and importantly the presence and concentration of various aqueous ions (Fig. 1) or organic macromolecules (Tester et al. 2011). All of these factors and pathways provide potential mechanisms by which organisms can tailor the polymorph of the final crystalline CaCO3 phase.

Over the last few decades, numerous studies have demonstrated that it is possible to use ACC to produce crystalline CaCO3 polymorphs with controlled properties (Fig. 2A–2C). Indeed, the kinetics and mechanisms of ACC transforming to crystalline CaCO3 polymorphs have been studied extensively, both through molecular modelling and experimental approaches (Rodriguez-Blanco et al. 2011). These studies reveal that surface energy, surface chemistry, ‘additive’ molecules, the presence of impurities, or confinement can all be tailored to control the dissolution of ACC and guide it toward crystallizing as specific CaCO3 polymorphs and morphologies. These factors can alter surface energetics, provide nucleation sites for crystallization, and influence growth kinetics, all of which can lead to preferential growth of specific polymorphic crystal faces and different crystal morphologies (e.g., Politi et al. 2008). A factor that may be particularly important in understanding the ability of calcifying organisms to produce specific polymorphs is that these organisms typically precisely regulate the physical and chemical environment in which biomineralization occurs by doing so within confined spaces, typically at the micro- or nanoscale. Although it remains unclear how or whether confinement in biological systems leads to the formation of a particular calcium carbonate polymorph, abiotic experiments have demonstrated that the pore size of membranes used in CaCO₃ precipitation can significantly influence which CaCO3 polymorph preferentially forms (Zeng et al. 2018).

ORGANISMAL CONTROL ON CACO3 POLYMORPHISM

Although it is tempting to use abiotic crystallization models to explain the mechanisms by which living organisms select and grow their CaCO₃ polymorphs, a fundamental difference is that organisms can secrete organic components that modify the mineralizing environment and processes. The secreted organic molecules can provide a template (organic framework), facilitating the spatial control on crystallization, as well as influencing biomineral polymorphism, for example, via the presence of functional groups, which impact mineralization. However, distinguishing between biological mechanisms of polymorph selection—such as physiological modifications of the Mg²+/Ca²+ ratio in the calcifying fluid (via ion pumps) and polymorph control mediated by templating with specific macromolecules or their functional groups—remains challenging Setting aside the great challenges in precisely determining how biology controls polymorphic forms, a simple observation is that once a phylogenetic lineage has emerged, it can subsequently maintain a sole CaCO3 polymorph through time and environment, and to do so, the organism must be able to control the types of minerals that build its skeletal structures (Table 1). Moreover, many organisms form bi-mineralic structures, a fact that provides even stronger evidence that organisms can closely mediate their mineralisation (as opposed to the passive generation of conditions that would result in a single polymorph). We find evidence for this strong biological control on biomineral polymorphism across species. For example, most mollusks form shells with a nacreous (inner) layer composed of aragonite tablets, while the prismatic (outer) layer is predominantly composed of calcite. Other examples include ammonites, extinct cephalopod mollusks, which had aragonitic shells while the coverings of their lower jaws (aptychi) were composed of calcite. One of the most powerful examples comes from inner-ear carbonate structures (otoliths) in zebrafish, which are composed of aragonite (sagitta and lapillus) and vaterite (asteriscus) (Söllner et al. 2003). In this case, the organismal influence on polymorph selection was demonstrated in zebrafish by knockdown starmaker gene expression, which resulted in an instant switch from aragonite to calcite otolith mineralization (Söllner et al. 2003). The repeating series of alternating serines and aspartic acids in the Starmaker protein have therefore been proposed as a template for nucleation that selects aragonite over calcite (conversely, the absence of this protein facilitated calcite deposition).

The most extensively studied shell proteome and other shell-associated organic macromolecular are those of mollusks. The soluble proteins and their mixtures extracted from the mollusk shell matrix were shown to determine the CaCO3 polymorphs in in vitro experiments. For example, abalone nacre proteins cause nucleation and growth of aragonite crystals directly on calcite seed crystals (Belcher et al. 1996), whereas b-chitin and silk fibroin-like proteins create a microenvironment that controls the polymorph regulatory functions of proteins (Falini et al. 1996).

Indeed, the switch from calcite to aragonite polymorphs in the presence of soluble nacre proteins was directly observed at the atomic lattice scale (Thompson et al. 2000; Fig. 3A, 3B). Sharp differences in the composition of organic molecules between calcite (prismatic) and aragonite (nacreous) layers are evident, particularly in the distribution of sulfated polysaccharides, Guided formation of specific CaCO3 polymorphs via which are thought to bind to the transformation of ACC or using organic macrocomplexes, as well as calcification site chemistry (e.g., Mg2+ concentration), is required for these organisms to direct the formation of a desired CaCO3 polymorph.

A critical but poorly understood aspect of how organic molecules and their functional groups select for CaCO₃ polymorphs is the possible impact of the precipitation and subsequent crystallization of amorphous precursors (Fig. 4), given that these precursors may be present at the onset of skeletal formation. The functional groups of these components may specifically interact with mineral proto-structures during the transition from amorphous to crystalline phases, thereby influencing crystallization pathways (Heiss et al. 2003; Evans 2019). Such mechanisms could potentially explain the simultaneous formation of calcitic and aragonitic zones in the unique bi-mineralic skeleton of a modern scleractinian coral in which disordered material was reported (Stolarski et al. 2021; Fig. 3D).

EVOLUTION OF OCEAN GEOCHEMISTRY AND CALCIFYING FAUNAS: WHEN GEOLOGY MEETS BIOLOGY

As described above, a dominant driver for which calcium carbonate precipitates in non-biological systems is the concentration ratio of Mg2+ over Ca2+ in the aqueous fluid. In the modern ocean, with a Mg/Ca ratio of 5.2, high-Mg calcite precipitates along with aragonite in shallower, warmer water. However, on geological timescales, there have been substantial fluctuations in seawater Mg/Ca due to changes in e.g., tectonic activity and terrestrial weathering. In particular, the speed at which oceanic crust forms can drive changes in the Mg/Ca ratio, because the chemical composition of seawater changes as it circulates through the newly formed crust due to the simultaneous release of Ca2+ from newly formed minerals and the removal of Mg2+ by hydrothermal chemical reactions. In 1983, Sandberg proposed that the mineralogy of inorganic carbonates precipitating from seawater oscillated between calcite and aragonite throughout the Phanerozoic, resulting in periods where predominantly calcite or aragonite were formed (Sandberg 1983); these periods are now known as calcite and aragonite seas (Stanley and Hardie 1998). Since the work of Sandberg (1983), multi-proxy investigations in the geological record, utilizing the compositions of fluid inclusions, CaCO3 mineralogy, and the Mg/Ca ratios of abiogenic and biogenic carbonates, all support a switch in the dominant mineralogy of marine CaCO3 from calcite to aragonite between 1–2 mol/mol Mg2+/Ca2+.

Although many organisms are capable of exercising control on the mineralogy of their skeleton, as mentioned above, changes in seawater chemistry also appear to influence the mineralogy and polymorph of carbonate skeletons and calcification strategies on evolutionary timescales. Over the last 500 million years, the mineralogy of the major types of calcifying organisms correlates broadly with the secular trends in seawater Mg/Ca (Fig. 5). This is clearest in the case of the so-called hypercalcifying organisms, which produce large volumes of sediment, e.g., coccolithophores (Stanley and Hardie 1998), as well as reef-building organisms such as scleractinian corals. In a given calcite or aragonite sea interval within the geological record, we generally observe a rise or proliferation of organisms that produced the favored polymorph of CaCO3, and the fall or even extinction of organisms that produced the polymorph disfavored by the prevailing ocean chemistry (Stanley and Hardie 1998). As new calcifying organisms emerged, their original mineralogy seems to correspond to the favored polymorph of that time. As such, this observation can be reconciled with the conservative retention of a specific CaCO3 polymorph by phyla or broad groups across geologic time (see above) by the evolutionary cost of living in an ocean characterized by a major element chemistry that favors another polymorph (Ries 2010).

Seawater chemistry, especially the Mg/Ca ratio, is thought to impact the chemistry at the site of calcification in evolved to produce specific forms of calcium carbonate, may struggle to cope with the altered chemistry of seawater, or other additional environmental stress.

Evidence from the geological record suggests that mass extinctions have regulated long-term patterns of skeletal mineralogy by both selective recovery and extinction (Ries et al. 2010; van Dijk et al. 2016). Even though the rates of change were, in most cases, several magnitudes slower than modern ocean acidification, these findings inform our understanding of the future resilience of marine calcifying organisms, and the stability of the marine carbonate system. Ocean acidification is thought to harm many marine (calcifying) organisms (Orr et al. 2005), and the oceans will become undersaturated with aragonite sooner than for calcite, one reason that organisms producing aragonite and high-Mg calcite (scleractinian corals, gastropods, echinoderms, and some foraminiferal taxa, Table 1) are amongst those that may be most vulnerable (Orr et al. 2005). At the same time, however, the aragonite and high-Mg calcite biomineralizing taxa are supported from a solution chemistry point of view due to the current high seawater Mg/Ca.

Lastly, we emphasize that precise control over CaCO3 morphology and polymorphism is of great potential importance for understanding how to achieve desired material properties and functionalities in various practical applications. For example, calcium carbonate is widely used as a filler material in paper, plastics, building materials, textiles, cosmetics, and food. The remarkable ability of biomineralizing organisms to control the mineralogy (polymorphism), morphology, and impurities of the CaCO3 biominerals they produce offers a blueprint for engineers seeking simple and environmentally friendly solutions to tailor the properties and phase composition of manufactured CaCO3 materials, for instance, through the use of organic macromolecular additives.

ACKNOWLEDGMENTS

We sincerely thank Dorrit Jacob, an anonymous reviewer, and the issue editors—particularly David Evans—for their contributions to improving the quality of an earlier version of this manuscript. JS acknowledges support from the National Science Centre (Poland) research grant 2020/39/B/ST10/01253. LGB acknowledges support from the Helmholtz Recruiting Initiative (award no. I-044-1601). IvD acknowledges the University of Angers and French Ministry of Higher Education and Research for CPJ project CARBPAST.

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