Amorphous Intermediate Phases: A Major Contribution to the ‘Vital Effect’?

 1811-5209/25/0021-0118$2.50 DOI: 10.2138/gselements.21.2.118

Keywords: non-classical nucleation; amorphous calcium carbonate; proxy; biomineralisation; calcification

THE UBIQUITY OF AMORPHOUS INTERMEDIATE PHASES IN BIOMINERALISATION AND CRYSTALLISATION

Crystalline materials, both natural and artificial, occur all over the world. It is now clear that the crystallisation of amorphous phases is of great importance during the growth of biological, natural, and synthetic materials. Here, we present a case study of amorphous calcium carbonate (ACC), which is perhaps the most-studied inter mediate phase due to its great relevance for biomineralisation (together with amorphous calcium phosphate). We note, however, that amorphous intermediate phases have also been identified in non-calcium-based systems, such as amorphous iron and manganese oxides (e.g., de Yoreo et al. 2015).

AMORPHOUS CALCIUM CARBONATE (ACC)—A CASE STUDY

Calcium carbonate is the most abundant biomineral in nature, particularly in the marine environment. There are three anhydrous polymorphs of calcium carbonate: calcite is the thermodynamically most stable form (global energy minimum) under ambient conditions, aragonite is thermo dynamically less stable, and vaterite is metastable (intermediate state, local energy minimum). Some marine biogenic calcites contain up to 30 mol% magnesium cations, which is referred to as Mg-calcite. Hydrated polymorphs of calcium carbonate, such as monohydrocalcite (MHC), calcium carbonate hexahydrate (ikaite), and calcium carbonate hemihydrate (CCHH), also occur naturally. MHC and CCHH have been discovered only recently to have biological origins as inter mediates in the biomineralisation process of corals and nacre (Schmidt et al. 2024). So far, the most relevant hydrated polymorph in the context of biomineralisation is the metastable amorphous calcium carbonate (ACC, CaCO3·nH2O, n = 0–1) (Addadi et al. 2003).

Structure

ACC is an isotropic, poorly ordered material without any long-range order, which is evident from the lack of distinct peaks within its powder diffraction pattern. ACC can have, however, short-range order with a length scale of up to a maximum of 15 Å. Biogenic and synthetic forms of ACC can exhibit distinct short-range structural order, with proto-structures resembling different crystalline calcium carbonate polymorphs (proto-vaterite, -MHC, -calcite, -aragonite) (Levi-Kalisman et al. 2002)—a phenomenon termed polyamorphism (Cartwright et al. 2012). The type of protostructure, and corresponding ACC morphology, correlates with the abundance of carbonate ion in the growth medium (Mergelsberg et al. 2020).

Synthesis

ACC is typically synthesised by directly mixing calcium (CaCl2) and carbonate (NaHCO3/Na2CO3) solutions in batch reactors or by the diffusion of gaseous CO2 (e.g., decomposition of (NH4)2CO3) into the reactant solution (see overview in Blue et al. 2017). The diffusion approach has the disadvantage of unknown supersaturation and pH conditions, which is why recent studies perform the synthesis under controlled chemical conditions using titrations or reactor-based setups. The formation of ACC in solution is favoured by conditions that enhance ACC stability (see next paragraph) and higher supersaturation compared with crystalline counterparts. The resulting synthetic ACC nanoparticles typically range in size from 20 to 400 nm (Blue et al. 2017; Mergelsberg et al. 2020).

Stability

Biogenic forms of ACC may be classified according to their stability: in some organisms (e.g., crustaceans, plants, bacteria, Table 1) ‘stable ACC’ is found as a biomineral with no apparent spontaneous transformation to a crystalline phase (Addadi et al. 2003; Weiner and Dove 2003). These stable biogenic ACCs typically contain about 1 mol of structural water (CaCO3·H2O). In many other biomineralising organisms, ACC acts as a metastable intermediate phase (‘transient’ ACC) that transforms on short time scales to more stable crystalline polymorphs, such as calcite, aragonite, or vaterite. Transient ACC may be generally anhydrous and short-lived. For example, using synchrotron spectromicroscopy, Politi et al. (2008) showed that the larval spicules of sea urchins form two amorphous intermediates. The relatively stable hydrous ACC is thought to dehydrate via an energetically descending pathway to an anhydrous or less hydrous ACC that readily transforms to calcite. ACC stability is enhanced by the presence of organic macromolecules or inorganic additives (especially Mg) (Loste et al. 2003; Liu et al. 2020), the availability of water (Radha et al. 2010; Ihli et al. 2014), heterogeneous nucleators, and/ or confinement (Cavanaugh et al. 2019). Note that stabilisation by confinement has the advantage of being a fully reversible process.

Characterisation

The instability and rapid transformation of ACC make in-depth structural characterisation of the material diffi cult. ACC is isotropic under polarised light, does not diffract X-rays, and lacks the carbonate vibration of crystalline calcite at ~714 cm−1 (υ4), which has been widely used as a diagnostic feature to detect biogenic ACC using IR and Raman spectroscopy (Addadi et al. 2003). To address this challenge, X-ray based methods (e.g., photoelectron emission spectromicroscopy (X-PEEM; Politi et al. 2008), soft x-ray tomography, or x-ray absorption spectromicros copy (Schmidt et al. 2024)) can be used to identify and study ACC transformation at high spatial resolution. The first documentation of the transformation of amorphous CaCO3 into calcite in a biological system was published in 1997 by Beniash et al. (1997), who showed by FTIR spectroscopy that the formation of sea urchin spicules begins as an accumulation of ACC that subsequently transforms to calcite. Subsequently, ACC was identified as a metastable intermediate phase in diverse organisms (Addadi et al. 2003). These often exhibit a nanoparticulate texture (Fig. 1), which increasingly correlates with ACC origins.

POTENTIAL BIOLOGICAL AND STRUCTURAL BENEFITS OF UTILISING ACC

Temporary and Recyclable Internal Ca-C Stores

The amorphous and metastable nature of ACC provides several advantages over its crystalline counterparts during biological crystallisation (Addadi et al. 2003; Weiner and Dove 2003). First, amorphous phases are significantly more soluble and, thus, easier to dissolve and reprecipitate. This property is essential for an intermediate material that needs to be stored temporarily, but subsequently transported and finally crystallised at desired locations. A stable intermediate phase thus helps organisms control the timing and location of crystallisation. In a related advantage, stabilised ACC is more easily reusable, as has been demonstrated in the cuticle of earthworms (Versteegh et al. 2017), where the ACC that is removed during moulting is partially redeposited. Independent from mineralisation processes, ACC might also be formed to transiently store calcium and/or carbon (e.g., intracellular Ca-P storage bodies in coccolithophores or ACC-containing vesicles in sea urchin larvae), thus avoiding cellular osmotic stress caused by high ionic strengths.

A Flexible and Low-Energy Intermediate that is Easy to Mould

Amorphous precursors, such as ACC, are isotropic components that, unlike crystals, do not form high-energy facets and have no preferred orientation of growth. As a result, ACC can be moulded into any complex shape prior to crystallisation with a minimal change in volume after trans formation. The attachment of preassembled oligomers or particles offers additional control at multiple levels, such as reactivity or polymorph control. However, it is not entirely clear how living organisms trigger the rapid crystallisation of the temporary precursors at the desired site of biomineral formation—the loss of final surface water has been shown to cause crystallisation in vitro (Ihli et al. 2014).

Rapid Formation of Complex and Mechanically Robust Skeletons

Biogenic ACC is commonly found in skeletal structures, where it contributes as an intrinsically isotropic building block to mechanically robust architectures. Growth by attachment of ACC particles is also significantly faster than growth by ions (see below) and enables complex geometries. In addition, when a biomineral forms by this pathway, its mechanical properties are greatly improved even after transformation to a (single-)crystalline polymorph. If the nanoparticles are coherently aligned, defects typically accumulate at the nanoparticle interfaces, thereby deflecting and dissipating cracks, which in turn strengthens the biomineral (Polishchuk et al. 2017).

TRANSFORMATION OF AMORPHOUS CALCIUM CARBONATE (ACC)

The process by which ACC transforms to calcite can be described by two contrasting mechanisms: a ‘classical’ ion attachment (IA) process involving dissolution–reprecipitation, or a ‘non-classical’ crystallisation by particle attachment (CPA) of ACC (Fig. 2). Note that both processes can occur simultaneously, and there is evidence for biomineral growth via a combination of CPA and IA in many organisms (e.g., corals and sea urchins).

Ion Attachment (IA) and ACC Dissolution/ Reprecipitation

Ion attachment describes the ‘classical’ way crystals form, where ions undergo individual reactions to the growth surface of the forming crystal (Fig. 2A). This process is predominant when the calcifying solution has a low supersaturation with respect to the final calcium carbonate phase. Ion-attachment can also occur as the ACC under goes dissolution and reprecipitation (Fig. 2B). This process has been observed for the transformation of ACC in the presence of water (Radha et al. 2010; Ihli et al. 2014) and documented with isotope labelling (Giuffre et al. 2015). Growth rates by IA (e.g., aragonite from seawater) are typically on the order of 0.01 to 0.1 μm/day.

Crystallisation by Particle Attachment (CPA) and ACC Solid-state Transformation

Crystallisation by particle attachment (CPA) describes multiple ‘non-classical’ pathways to crystal formation (de Yoreo et al. 2015) that includes the ACC to crystal trans formation by solid-state processes involving dehydration and structural rearrangement (Fig. 2C). When ACC is attached to a crystalline ‘parent’ mineral, the parent crystal determines the orientation and polymorphism of the final mineral through epitaxial growth (i.e., the crystal lographic orientation of the growing, newly deposited layer matches the underlying substrate). Crystal growth by CPA is significantly faster than by IA. Among the fastest growing biominerals are avian eggshells, which grow by fusion of 100–300 nm size ACC particles to 300 μm per day (chicken) or even 1 mm/day (ostrich) (Gilbert et al. 2022). Minerals formed via CPA retain a characteristic nanoscale texture of the ACC nanoparticle building blocks (Fig. 1B–1E) that has been found in several calcifying organisms (Addadi et al. 2003; Gilbert et al. 2019, 2022).

IMPLICATIONS OF ACC FOR GEOCHEMICAL SIGNALS

Biomineralisation by non-classical pathways has now been recognised to have been prevalent in deep time, and Gilbert et al. (2019) showed that the nanoparticulate texture of ACC can even be preserved in fossilised structures. Evidence for ACC nanoparticles in biomineral settings was found in some of the oldest known examples of calcified skeletons: Cambrian molluscs (~500 Ma) and the Ediacaran Cloudina, one of the first animals to form a CaCO3 skeleton (~550 Ma; Fig. 1E). Evidence for the ACC-CPA mineralisation pathway was found in three phyla (cnidarians, molluscs, echinoderms) that diverged long before the emergence of calcification and is a polyphyletic trait (Gilbert et al. 2022).

Stable isotope and elemental ratios of biogenic and abiogenic CaCO3 are widely used as proxies in various environmental and palaeoclimate archives. Given the importance of these proxies as indicators of formation conditions, such as temperature or pH (see Chalk and Rollion-Bard 2025 this issue), it is crucial to understand whether non-classical mineralisation processes involving ACC influence the elemental and/or isotope ratios of the final biomineral. As illustrated in Figure 2, the multiple ACC transformation mechanisms may result in pathway dependent differences in both the isotope and elemental ratios of the resulting mineral. However, a comprehensive picture of the effects of the transformation of ACC to crystalline carbonates on the isotope and elemental composition of biominerals has not yet been established.

The CaCO3 that forms by the classical IA pathway is expected to record the composition of the individual components that are incorporated at the crystal surface. Thus, in case of formation of an ACC precursor and trans formation by dissolution and reprecipitation, the composition of the resulting biomineral will record either the conditions of the local environment (i.e., the calcifying fluid) or, in the case of localised total dissolution of ACC, the conditions at the time of crystal formation (Fig. 2A). In contrast, if mineralisation proceeds by CPA of ACC particles, the elemental and isotope ratios of the final mineral will reflect the composition of the amorphous intermediate phase, assuming no preferential rejection of ions during this process (Fig. 2C). A third endmember for the trans formation may be a combination of the two processes (IA and CPA, Fig. 2B), whereby the solid-state transformation of ACC particles is assumed to fill the space, the ions fill the inter-particle gaps, and both processes are required to achieve 100% space filling (Gilbert et al. 2022).

A MAIN COMPONENT OF THE VITAL EFFECT?

The elemental and isotope ratios of marine and terrestrial CaCO3 are widely used for palaeoenvironmental recon structions. However, the composition of the calcifying fluid and—consequently—the composition of the resulting biomineral may be modified by physiological processes, the so-called ‘vital effects’ (Urey et al. 1951; Weiner and Dove 2003), which need to be addressed to infer past conditions from biomineral chemistry. In this context, the term ‘vital effects’ summarises a variety of factors that may affect the elemental and isotope ratios of the final biomineral, such as ion transporters, carbonic anhydrase activity, calcifying fluid composition, precipitation rate, or non-classical crystallisation. To decipher these processes and their effect on the composition of the resulting biominerals, we need— among other things—a process-based understanding of ACC transformation, leading us to the question: Is the transformation of ACC a major component of the vital effect? So far, only a few studies have explored the role of amorphous intermediate phases and their effect on the isotope and elemental ratios of the resulting minerals.

Synthetic ACC has been used to study CaCO3 formation via amorphous intermediates outside of a cellular context. This approach is essential to disentangle the purely inorganic contribution from the biochemical cellular processes as parts of the vital effect. Because of the importance of elements such as Mg for biomineralisation, the effect of ACC on elemental ratios is much better understood than for isotope ratios. In the following, we discuss how in vitro ACC transformation experiments can contribute to our understanding of biomineral proxies.

Trace and Minor Elements

Carbonate minerals host a variety of elements as major and minor impurities. The amount of substitution of calcium by other divalent cations (e.g., Mg, Sr, Ba) in the crystal lattice of crystalline polymorphs is influenced by various parameters, such as crystal structure, size of the foreign element, concentration in the parent solution, growth rate, and temperature. For example, elements with a smaller atomic radius than calcium, such as Mg2+ ions, are more readily substituted into calcite than larger elements, such as Sr2+, which is more compatible in aragonite. The elemental composition of biogenic carbonates typically differs from their abiogenic counterparts (e.g., B, Mg, Sr, Ba in molluscs, coccolithophores, corals, foraminifera). Although there are only a handful of in vitro studies, these experiments generally show that crystallisation using ACC as a metastable phase allows the incorporation of higher concentrations of foreign elements into the final CaCO3 crystal than takes place by classical crystallisation pathways (Blue et al. 2017; Littlewood et al. 2017; Evans et al. 2020).

Transient ACC is difficult to sample due to its instability and short-lived nature. However, ACC is stabilised by the presence of magnesium and is commonly found in biogenic ACC. Thus, most in vitro studies have used Mg-rich ACC (also referred to as amorphous calcium magnesium carbonate, ACMC) to slow crystallisation and stabilise the amorphous phase to enable sampling and isotopic analysis (Loste et al. 2003; Giuffre et al. 2015; Blue et al. 2017; Mavromatis et al. 2017; Dietzel et al. 2020; Liu et al. 2020; Lucarelli et al. 2023). ACMC is an example of how the pathway may influence the extent of elemental incorporation into the calcite lattice. Via this pathway, calcite can be produced with a Mg content far exceeding that obtainable by classical crystal growth (Blue et al. 2017). In case of the classical pathway, high Mg/Ca ratios usually favour the formation of the aragonite polymorph. The Mg concentration of the ACC also affects the nature of its transformation: Mg2+ ions introduce additional (structural) water into the amorphous phase triggering a direct transformation and thus preserving the ‘complex’ morphology of the precursor ACC phase (Liu et al. 2020).

Blue et al. (2017) investigated the influence of the Mg/Ca ratio of the parent solution on the initial polymorph, as well as the evolution of solution and solids under stirred or quiescent conditions. Under quiescent conditions, high-Mg calcite with up to 26 mol% Mg could be achieved via the ACC pathway. In their experiments, the composition of the initial polymorph depended only on ion activities (aMg2+/ aCa2+ and aCO32−/aCa2). Similarly, the composition of the final calcite product was also established by the initial Mg/ Ca ratio of the aqueous solution. In both cases, the Mg concentration was far from equilibrium with the solution from which it formed. This suggests all calcites formed in their laboratory experiments, independent of the growth pathway, recorded non-equilibrium compositions. The authors also highlight the presence of MHC as a transient phase along the pathway from ACC to Mg-calcite.

Inspired from ACC inclusions with high Sr/Ca and Ba/Ca ratios in cyanobacteria, Cam et al. (2015) synthesised Mg-, Sr-, and Ba-containing ACC in vitro. Sr and Ba were found to increase the lifetime of ACC in a similar manner as Mg, but they could not reproduce the fluid/solid partitioning of Sr, Ba, and Ca in cyanobacteria. This suggests that our understanding is incomplete of the compositional and biological processes that alter these organism’s calcification sites. Littlewood et al. (2017) also investigated the incorporation of Sr into calcite via ACC. In their batch reactor experiments, they showed that an enhanced uptake of Sr2+ ions can be achieved by calcite precipitation via ACC. Interestingly, the Sr distribution coefficients (KD) were higher in the presence of Mg ions in the parental solution when compared to the Mg-free pathway that involves vaterite. The presence of the smaller Mg2+ ions is assumed to compensate for the addition of the larger Sr2+ ions in the calcite lattice.

As the composition of calcifying fluids within biomineralising organisms is the result of endocytosis or ion trans port from seawater, Evans et al. (2020) studied in vitro ACC formation from seawater. They systematically analysed the partitioning of several trace elements (Li, B, Na, Mg, Mn, Sr, Ba, U) between seawater and ACC under a variety of dissolved inorganic carbon (DIC) conditions and element/ Ca ratios. Their study provides a unique insight into the cumulative effect of multiple competing elements. Notably, the presence of Mg in ACC influenced the distribution coefficients of some other elements.

In summary, crystallisation via ACC may have a substantial effect on the element/Ca ratio of both biogenic and abiogenic CaCO3, which should be considered for palaeo climate proxy development and environmental reconstructions based on elemental ratios. To improve the accuracy of such reconstructions, a deeper understanding of the contribution of intermediate phases is essential.

Stable Isotope Fractionation

Stable isotope ratios of carbon and oxygen are routinely applied as palaeoenvironmental proxies in various archives. With the continued advancement of analytical methods, the measurement of non-traditional stable and clumped isotopes has now also become feasible.

Traditional Stable Isotopes (δ13C & δ18O)

To date, only a few studies have investigated the stable isotope composition of ACC. Schmidt et al. (2005) analysed the formation pathways of dolomite in vitro. Their synthetic approach resulted in precipitation of significant amounts of Mg-containing ACC together with dolomite. The initial ACC and the corresponding bulk samples (a mix of ACC and dolomite) had lower δ18O values than expected for the dolomite-water oxygen isotope equilibrium. For larger amounts of ACC, a larger deviation was observed.

Carbon isotope fractionation of biogenic, stable ACC in earthworms was investigated in cultivation experiments by Versteegh et al. (2017). ACC microspheres were found in the ‘milky fluid’ of one life stage of the organisms. Later, these microspheres coalesced and crystallised to form calcite. During the transformation from ACC to calcite, the δ13C values became significantly more negative. The observed fractionation factor (εcalcite-ACC = −1.20 ± 0.52 ‰) agrees well with dissolution and reprecipitation (IA) rather than solid state transformation (CPA).

ACC later transforming to calcite has also been observed in speleothems (Demény et al. 2016), and the δ18O values of the ACC prior to the transformation were lower by 2.4 ± 0.8 ‰ than those of the final calcite. Demény et al. (2016) concluded that this may have serious consequences for speleothem-based fluid inclusion research, as closed system transformation of ACC to calcite may induce a negative oxygen isotope shift on the inclusion water.

Non-traditional Stable Isotopes (25/26Mg, 43/44Ca, 88Sr, and 137Ba)

Giuffre et al. (2015) used stable isotope labelling to study the transformation of ACC into calcite. First, ACC was synthesised from natural solutions. In a second step, the synthesised ACC was transformed into calcite using a spiked solution containing 43Ca and 25Mg in a closed reactor. In the presence of excess water (i.e., for a high fluid/solid ratio), the 43Ca/40Ca and 25Mg/24Mg isotope ratios of the crystalline calcite product changed with the ratio of ACC to the surrounding spike solution, indicating the transformation is best explained by a dissolution–reprecipitation process. The isotope ratio of small amounts of ACC added to a larger spike solution was almost completely replaced by the composition of the surrounding solution, while with increasing amounts of ACC, the isotope ratio of the final calcite mineral showed a mixture between the ACC and the solution signatures. No direct solid-state transformation was observed. Instead, the conditions at the time of ACC transformation, rather than ACC formation, deter mined the isotope ratios. Consequently, when bulk water is present, the isotope ratio of the resulting calcite reflects the evolution of the local solution conditions during the transformation period.

Mavromatis et al. (2017) studied the evolution of Mg isotopes during the transformation process. They compared the evolution of the 26Mg/24Mg isotope ratio in both the solid phase and the solution during the formation of crystalline Mg-calcite with and without the involvement of ACC. They found that the different pathways had a profound effect on the temporal evolution of the Mg content and isotopic composition of the reacting fluids and the newly formed solids. In the absence of an ACC precursor, the changes in Mg concentration and Mg isotope fractionation between calcite and fluid were only minor after titration stopped. In contrast, the Mg-ACC precursor showed a profound evolution of the Mg composition and Mg isotope fractionation. The initially formed ACC had slightly lower δ-values (−1.0 ‰) than the reactive fluid, but this fractionation was not preserved during the transformation into Mg-calcite— which again indicates a fast dissolution–reprecipitation mechanism.

Based on this evolution of Mg isotope fractionation, the initial ACC was characterised as a hydrated, nanoporous solid containing Mg bicarbonate/carbonate species and hydrated magnesium. After transformation, a more negative fractionation was observed (−3.0 to −3.6 ‰) compared with calcite precipitated without ACC, which showed a fractionation closer to isotopic equilibrium (−1.8 to −2.0 ‰). The was interpreted as a rapid transformation to Mg-rich calcite by dissolution/precipitation through percolating channels filled with the reacting fluid.

The study of stable, biogenic ACC is a unique source for understanding the signatures of a non-transformed ACC phase formed in a biological compartment prior to crystal lisation. In such an effort, the 44Ca, 88Sr, and 137Ba isotope fractionation in stable ACC formed by cyanobacteria was recently assessed by Mehta et al. (2023). The bacterial cells showed lower δ-values compared to the initial solution for both 137Ba and 88Sr, which was hypothesised to result from dissolution of ACC inclusions.

Clumped Isotopes (Δ47 and Δ48)

Clumped isotopes are molecules that contain more than one heavy (low abundance) isotope. For carbonates, the most common multiply substituted carbonate isotopo logues are Δ47 (13C18O16O) and Δ48 (12C18O18O). The thermo dynamic properties and kinetics of clumped isotopes are distinctively different from ‘traditional’ stable isotope systems. For example, the clumped isotope composition only depends on temperature and is independent from the isotopic composition of the calcifying solution.

Dietzel et al. (2020) analysed stable (δ18O) and clumped isotope values (Δ47, Δ48) of synthetic ACMC before and after transformation into high Mg-calcite in an open reservoir. ACMC showed elevated δ18O values (+3.2‰), reflecting the isotopic composition of the aqueous (bi)carbonate complexes at the time of formation. After transformation, the δ18O values approached equilibrium (+0.5‰) without any ‘isotopic memory’ of the initial ACMC. In contrast, the clumped isotope values remained constant, suggesting Δ47 as a potentially more useful, conservative temperature proxy that is independent of the involved DIC species.

In Dietzel et al. (2020)’s approach, the amorphous phase was fully transformed (in the absence of water) into high Mg-calcite prior to analysis. In contrast, Lucarelli et al. (2023) directly measured both clumped (Δ47 and Δ48) and stable isotopes (δ13C, δ18O) from the onset of ACMC precipitation and during transformation in a closed reactor. Unlike Dietzel et al. (2020), all isotope values showed an evolution during the transformation, indicating dissolution–reprecipitation. Over a one-year period, the clumped isotope values reached a non-equilibrium steady state, but oxygen isotope equilibrium was not achieved. This disequilibrium arises from progressive ACC dissolution creating a highly dynamic, local non-equilibrium environment. The forming crystal then captures the degree of isotopic disequilibrium in the DIC pool, and its isotope values may be heterogeneous and unrepresentative of the formation temperature.

In summary, the few available isotope studies show that the crystallisation pathway—as observed for elemental ratios—may substantially influence the isotope ratios of both biogenic and abiogenic CaCO3. This needs to be addressed to reconstruct palaeoenvironments accurately from isotope-based records. Non-traditional stable and clumped isotopes show great potential for investigating crystallisation pathways and their effects on CaCO3 proxy data.

The Overall Picture and How it Relates to Natural Systems

In summary, in vitro experiments with ACMC paint the following picture:

i) The initial ACC phase precipitates rapidly, capturing solution species at the time of formation, and may show isotope/elemental ratios different from equilibrium values.

ii) In the presence of bulk water, transformation is dominated by dissolution–reprecipitation. In the small number of lab experiments conducted so far, this also involves MHC as an intermediate.
iii) The composition of the final crystalline products may strongly depend on the local fluid/solid ratio during transformation and the degree of mixing (Fig. 3).

The isotopic evidence thus supports a dissolution–reprecipitation mechanism for the transformation of ACC into crystalline CaCO3 at high fluid/solid ratios, which is in good agreement with theoretical calculations. MHC is probably an overlooked intermediate in the transformation of ACMC to calcite or aragonite (Blue et al. 2017). This observation has now been confirmed in biogenic systems, where additional crystalline intermediates, namely MHC and CCHH, must be added to the transformation sequence of some organisms, such as corals and nacre (Schmidt et al. 2024). How these additional steps in the energy landscape of calcium carbonate affect mineral composition remains an open question.

A major conclusion from the in vitro studies of ACC is that the isotope and elemental composition of the final crystal line CaCO3 is a result of the fluid/solid ratio and the degree of closed versus open system at the time of transformation (Fig. 3). In case of an open system (with a high fluid/solid ratio), the initial potentially non-equilibrium signature of the ACC can be modified depending on the conditions of the transformation reaction. An ongoing question is whether the few in vitro studies adequately represent the conditions of biogenic mineral formation. In organisms, the calcification site is typically a separated compartment (‘privileged space’), e.g., in a semi-open or closed confinement, and, thus, must be considered as a closed system. For the analysis of biominerals, these considerations are therefore highly relevant. In highly confined calcifying spaces (e.g., in specialised vesicles) the fluid phase is limited (=low fluid/solid ratio). Under such conditions, the final calcite mineral may completely, or at least partially, record the isotope and elemental composition of the ACC.

LOOKING AHEAD

Over the last two decades, the importance of amorphous intermediate phases has now been widely recognised as a prevalent part of biomineralisation processes. Acceptance of this pathway for mineralisation fascinates biologists and materials scientists alike. However, the importance of amorphous intermediates, such as ACC, has received much less attention in the context of mineral formation in geological environments. Establishing quantitative relationships between the (calcifying) fluid, the (amorphous) intermediates, and the final crystalline (bio) mineral based on in vitro experiments, biomineralising organisms, and natural minerals (in monitoring programs), will surely become important as the origins of compositions and textures that deviate from traditional crystal growth models are revisited.

Looking ahead, further work is needed to constrain elemental and isotopic exchanges between the amorphous solid, the aqueous solution, and the final mineral. In partic ular, conditions relevant to mineral formation in natural systems need to be addressed, such as the simultaneous interaction of multiple elements and stabilising factors in the calcifying fluid, the presence of a short-lived Mg-free ACC, the relevance of prenucleation clusters or liquid precursor phases, and the precise role of confinement. By gaining a deeper understanding of element and isotope fractionation via ACC transformation, we will be able to use biomineral compositions to understand the open/closed system behaviour and constrain estimates of the fluid and element fluxes. However, to completely understand vital effects, inorganic ACC transformation is just one part of the story. For predictive biomineralisation models, it will also be necessary to quantify the fractionation associated with the major biochemical processes that modify the calcifying fluid—a monumental but achievable task with the potential to greatly advance our understanding of biological mineralisation processes.

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