Hard X-rays help to understand the formation of the pearl oyster shell

A team of scientists has uncovered important new pieces of information on the structure of amorphous calcium carbonate precursors in the mineralisation of the pearl oyster Pinctada margaritifera. Valued for its lustrous pearls, Pinctada margaritifera also serves as an important model organism for calcareous biomineralisation, helping us to understand how Nature forms complex nanostructures at ambient pressure and temperature.

Biomineralisation is a strategy widely employed by animals to build functional skeletons. This remarkable process is able to fulfil the many requirements of various organisms by choosing the appropriate nanostructural arrangement [1] and the crystalline polymorph for the task. A general feature of biomineralisation is the presence of an amorphous calcium carbonate (ACC) precursor phase [2], whose roles include lowering the energy barrier required for crystallisation, pre-moulding shapes of crystallising units and acting as a reservoir for Ca and CO₃^2-, all the while transforming into crystalline calcite. Crucially, biogenic ACC is often found in the final mineralised state, keeping a record of the intermediate stages of the biomineralisation process [3]. Pinctada margaritifera (Figure 1a) is an example that has an easily accessible growth zone, in which isolated, disc-like units form a densely tessellated prismatic layer during growth (Figure 1b), often containing residual amorphous calcium carbonates [2,3] as shown by the red zones in Figure 1c compared with crystalline calcite (blue).

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Fig. 1: Hard X-rays help to understand the formation of the pearl oyster shell. a) Micrograph of a juvenile Pinctada margaritifera specimen. The black rectangle indicates the investigated shell growth zone. b) Scanning electron microscopy image of the shell edge, showing the isolated, disc-like early mineralisation units at the top and the more developed prism structure that is slowly forming on the periostracum, an organic membrane. c) Stimulated Raman Scattering map, showing the symmetric contrast between ACC (red) and calcite (blue). The presence of more ACC in young disc-like units and the accumulation of ACC in the inter-prismatic space is noteworthy.

While the mature prismatic layer, made of crystalline calcite, is comparatively well characterised, the early, mostly amorphous stages in Pinctada margaritifera have escaped quantitative description, particularly as to the structure and spatial distribution of ACC. This gap stems from the difficulty in characterising the ACC phase in the presence of a crystalline signal, to which structural probes are far more sensitive. Researchers from the Institut Fresnel in Marseille and ESRF beamline ID15A teamed up to develop a novel, high-energy X-ray total scattering approach as part of the ERC-funded research project 3D-Biomat (No 724881).

Firstly, the signals from the crystalline and amorphous phases have different distribution patterns in the reciprocal space and so can be separated by using filtering methods, provided that the X-ray scattering data cover a large portion of the reciprocal space. Secondly, to resolve the distribution of ACC with sub-micron resolution, the researchers scanned a focused beam the size of 500 nm across early mineralising units in a specimen of the Pinctada margaritifera shell. The combination of the two methods gave a picture of the atomic arrangement of the amorphous compounds in the shell through a function known as pair distribution function, from which one can build a quantitative model of the structure in the absence of long-range order.

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Fig. 2: a) X-ray fluorescence image indicating the location of the scan presented (red line). b) Principal components analysis of the PDF data, showing the presence of three, spatially separated contributions. The red lines indicate the border of the disc-like unit. c) Ca-O pair distance extracted from the structural model, showing the contraction of the Ca-O pair towards the centre of the disc. The green lines indicate the border of the disc-like unit.

One of these scans is indicated by a red line in the X-ray Ca fluorescence map shown in Figure 2a. Data analysis based on principal component analysis and reverse Monte Carlo atomistic modelling identified three distinct states of ACC (Figure 2b), differentiating the young and mature prismatic components from the periostracum, the organic membrane supporting the shell growth. The 3D structural models show that the structural fluctuation in the Pinctada margaritifera shell happens mostly at the level of Ca-O first-neighbours, whose mean distance varies between 2.35 Å and 2.42 Å across the early mineralising unit (Figure 2c). With the help of element-specific techniques like coherent Raman spectroscopy (Figure 1c) [2,3] and energy-dispersive X-ray spectroscopy, the structural fluctuation could finally be explained by the local concentration of magnesium in certain structural units of the oyster shell. Therefore, among several plausible mechanisms, this analysis singled out the key role that magnesium plays in the transition between different amorphous phases.

As this study sheds light on the fundamental biomineralisation processes by giving a spatially resolved picture of the first states of ACC, it offers tools that enable further studies on other similarly complex amorphous/crystalline systems, helping to entangle the complex pathways in non-classical crystallisation processes.

Principal publication and authors
Structure of an amorphous calcium carbonate phase involved in the formation of Pinctada margaritifera shells, T.A. Grünewald (a)*, S. Checchia (b)*, H. Dicko (a), G. Le Moullac (c), M. Sham Koua (c), J. Vidal-Dupiol (d), J. Duboisset (a), J. Nouet (e), O. Grauby (f), M. Di Michiel (b), V. Chamard (a), Proc. Natl. Acad. Sci. 119, 45, 2212616119 (2022); https://doi.org/10.1073/pnas.2212616119
* TAG and SC contributed equally to this work
(a) Aix-Marseille Univ, CNRS, Centrale Marseille, Institut Fresnel, Marseille (France)
(b) ESRF
(c) Ifremer, ILM, IRD, Univ Polynésie française, EIO, Taravao, Tahiti, French Polynesia (France)
(d) IHPE, Univ Montpellier, CNRS, IFREMER, Univ Perpignan Via Domitia, Montpellier (France)
(e) GEOPS, Univ. Paris-Sud, CNRS, Université Paris-Saclay, Orsay (France)
(f) Aix-Marseille Univ, CNRS, CINaM, Campus Luminy, Marseille (France)

References
[1] F. Mastropietro et al., Nat. Mater. 16 (4), 946-952 (2017).
[2] J. Duboisset et al., Acta Biomater. 142, 194-207 (2022).
[3] H. Dicko et al., J. Struct. Biol. in press.

Top image: Polarised light image of a pearl oyster shell, Pinctada margaritifera, showing differently oriented, birefringent calcite crystals.