BACKGROUND Many biominerals form from amorphous calcium carbonate (ACC), but this phase is highly unstable when synthesised in its pure form inorganically. Several species of earthworm secrete calcium carbonate granules which contain highly stable ACC. We analysed the milky fluid from which granules form and solid granules for amino acid (by liquid chromatography) and functional group (by Fourier transform infrared (FTIR) spectroscopy) compositions. Granule elemental composition was determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) and electron microprobe analysis (EMPA). Mass of ACC present in solid granules was quantified using FTIR and compared to granule elemental and amino acid compositions. Bulk analysis of granules was of powdered bulk material. Spatially resolved analysis was of thin sections of granules using synchrotron-based μ-FTIR and EMPA electron microprobe analysis. RESULTS The milky fluid from which granules form is amino acid-rich (≤ 136 ± 3 nmol mg-1 (n = 3; ± std dev) per individual amino acid); the CaCO3 phase present is ACC. Even four years after production, granules contain ACC. No correlation exists between mass of ACC present and granule elemental composition. Granule amino acid concentrations correlate well with ACC content (r ≥ 0.7, p ≤ 0.05) consistent with a role for amino acids (or the proteins they make up) in ACC stabilisation. Intra-granule variation in ACC (RSD = 16%) and amino acid concentration (RSD = 22-35%) was high for granules produced by the same earthworm. Maps of ACC distribution produced using synchrotron-based μ-FTIR mapping of granule thin sections and the relative intensity of the ν2: ν4 peak ratio, cluster analysis and component regression using ACC and calcite standards showed similar spatial distributions of likely ACC-rich and calcite-rich areas. We could not identify organic peaks in the μ-FTIR spectra and thus could not determine whether ACC-rich domains also had relatively high amino acid concentrations. No correlation exists between ACC distribution and elemental concentrations determined by EMPA. CONCLUSIONS ACC present in earthworm CaCO3 granules is highly stable. Our results suggest a role for amino acids (or proteins) in this stability. We see no evidence for stabilisation of ACC by incorporation of inorganic components. Graphical abstractSynchrotron-based μ-FTIR mapping was used to determine the spatial distribution of amorphous calcium carbonate in earthworm-produced CaCO3 granules.

中文翻译：

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生物矿化-对无定形碳酸钙的稳定性和分布的研究。

背景技术许多生物矿物由无定形碳酸钙（ACC）形成，但是当以其纯净形式无机合成时，该相高度不稳定。几种species分泌的碳酸钙颗粒含有高度稳定的ACC。我们分析了形成颗粒的乳状液体和固体颗粒的氨基酸（通过液相色谱）和官能团（通过傅里叶变换红外（FTIR）光谱）组成。使用电感耦合等离子体发射光谱法（ICP-OES）和电子微探针分析（EMPA）确定颗粒元素的组成。使用FTIR对存在于固体颗粒中的ACC的质量进行定量，并将其与颗粒的元素和氨基酸组成进行比较。颗粒的散装分析是粉末状散装材料。使用基于同步加速器的μ-FTIR和EMPA电子微探针分析对颗粒的薄片进行空间分辨分析。结果形成颗粒状的乳状液富含氨基酸（每个氨基酸≤136±3 nmol mg-1（n = 3；±std dev）。存在的CaCO3相为ACC。生产后甚至四年，颗粒中都含有ACC。存在的ACC质量与颗粒元素组成之间不存在相关性。颗粒氨基酸浓度与ACC含量（r≥0.7，p≤0.05）具有很好的相关性，与氨基酸（或它们组成的蛋白质）在ACC稳定化中的作用一致。同一earth生产的颗粒的ACC颗粒内变化（RSD = 16％）和氨基酸浓度（RSD = 22-35％）高。使用基于同步加速器的颗粒薄层的μ-FTIR映射绘制的ACC分布图以及ν2：ν4峰比的相对强度，使用ACC和方解石标准品进行的聚类分析和成分回归显示可能富ACC和方解石的空间分布相似丰富的地区。我们无法确定μ-FTIR光谱中的有机峰，因此无法确定富含ACC的域是否也具有相对较高的氨基酸浓度。ACC分布与通过EMPA确定的元素浓度之间不存在相关性。结论Ca CaCO3颗粒中存在的ACC是高度稳定的。我们的结果表明氨基酸（或蛋白质）在这种稳定性中的作用。我们没有看到通过掺入无机成分来稳定ACC的证据。