In situ SAXS targets magnesium and pH role in calcium carbonate formation

Biomineralization – the process by which living organisms build mineral tissues – enables corals, molluscs, algae, sea urchins, and other marine species to assemble complex calcium carbonate structures. These biominerals begin as transient amorphous calcium carbonate (ACC) nanoparticles that assemble and form through crystallization via particle attachment (CPA) [1]. In this non-classical pathway, nearly monodisperse ACC particles aggregate into mesocrystals before transforming into the final mineral phase; high polydispersity, however, can introduce defects.

Although Mg²⁺ and pH are known to stabilize ACC, their combined effects on nucleation kinetics and particle size distribution remain unclear. Addressing this gap is crucial, especially given the threat of rising CO₂ and ocean acidification. In this study, systematic variations in pH (8.4-8.9) and Mg²⁺:Ca²⁺ ratios (20:20 to 80:20) were applied to mimic marine conditions and to quantify their impact on ACC formation dynamics and uniformity.

Time-resolved small-angle X-ray scattering (SAXS) measurements at ID02 employed a stopped-flow mixer coupled to a high-brilliance X-ray beam, capturing ACC formation from CaCl₂ and Na₂CO₃ in real time with 10 ms resolution (Figure 1a). By recording at two sample-to-detector distances, both early precursors and larger particles were tracked. Fitting the scattering profiles with analytical models allowed extraction of size, shape, and polydispersity evolution, shedding light on how subtle shifts in conditions govern early ACC formation (Figure 1b).
 

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Fig. 1: a) Schematic of the stopped-flow experimental setup and illustration of the narrowing of the ACC nanoparticle size distribution with decreasing Mg²⁺ concentration and increasing pH. A narrow size distribution is a prerequisite for the formation of transient mesocrystals in biomineral construction. b) SAXS curves of the final Mg-ACC nanoparticles formed at different pH values, with fitted radial size distributions.

The data showed that both higher pH and lower Mg²⁺ accelerate ACC formation, reducing the critical nucleation energy and resulting in smaller, more uniform nanoparticles, as predicted by classical nucleation theory (Figures 2a-b). This can be interpreted within the framework of liquid–liquid phase separation. As conditions push deeper into the metastable binodal region (Figure 2c), the energy barrier decreases until the spinodal line is reached, beyond which ACC forms spontaneously through spinodal decomposition – no longer as discrete spheres but as continuous nanostructures (Figures 2d-e).
 

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Fig. 2: a) The radius of Mg-ACC nanoparticles decreases exponentially with increasing formation kinetics, yielding smaller nanoparticles under faster conditions. b) Faster formation kinetics also result in narrower particle size distributions. c) The acceleration of formation kinetics (red arrows in (a) and (b)) with increasing pH and decreasing Mg²⁺ concentration is interpreted within the framework of liquid–liquid phase separation, corresponding to a shift deeper into the metastable binodal region (red arrow). d) Nanoparticles with the narrowest size distributions form near the spinodal boundary. e) In the spinodal region, continuous calcium carbonate structures form via spontaneous phase decomposition.

ACC nanoparticles synthesised closest to the spinodal boundary within the binodal region exhibited the narrowest size distributions - ideal for CPA. Yet this window is extremely narrow: a mere 0.1-unit drop in pH (as projected under ocean acidification) doubles particle size variability, threatening proper biomineral assembly. Because Mg²⁺ shifts the spinodal boundary’s position, however, marine organisms could counteract pH-driven heterogeneity by locally adjusting Mg²⁺ levels during ACC formation to maintain monodispersity.

By directly linking pH and magnesium conditions to nanoscale nucleation behaviour, this study reveals CPA’s vulnerability to ocean acidification and suggests that magnesium regulation may serve as a natural resilience strategy. It also underscores time-resolved SAXS at the ESRF as a powerful method for capturing early-stage nucleation dynamics, offering crucial insights into how global environmental changes might disrupt one of nature’s most fundamental mineralization pathways.

Principal publication and authors
Impact of Mg²⁺ and pH on amorphous calcium carbonate nanoparticle formation: Implications for biomineralization and ocean acidification, L. Kuhrts et al., PNAS 122, 1-9 (2025); https://doi.org/10.1073/pnas.2421961122

References
[1] R. Best et al., J. Am. Chem. Soc. 147, 1-9 (2025).