The Skeletal Organic Matrix from Mediterranean Coral Balanophyllia europaea Influences Calcium Carbonate Precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions

Resolution revival technique for subwavelength imaging

<p>Crystallization experiments of calcium carbonate from 10 mM CaCl₂ solution with a Mg/Ca molar ratio equal to 5 (Ctrl5) and in the presence of soluble organic matrix (SOM), insoluble organic matrix (IOM) or both additives (IOM+SOM). The direction of the crystallographic <i>c</i>-axis of aragonite is indicated. <i>left</i> Scanning electron microscope images, the insets show samples details <i>right</i> FTIR spectra of the precipitated. The main absorption bands of calcium carbonate are indicated.</p

Skeleton of the coral <i>Balanophyllia europaea</i>.

<p>(A) Digital camera picture of the skeleton of the coral <i>Balanophyllia europaea</i> after digestion in a sodium hypochlorite solution to remove the soft organic tissues. (B) X-ray powder diffraction pattern from a powdered coral skeleton sample. Only the characteristic diffraction peaks from aragonite are observable. The main diffraction peaks of the Miller index are indicated according to the reference pattern PDF 98-006-0908 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022338#pone.0022338-Pilati1" target="_blank">[60]</a>. (C) FTIR spectrum from a powdered coral skeleton sample. Only the typical absorption bands from aragonite were detected. They were assigned as ν₂ = 858 cm⁻¹; ν₃ = 1470 cm⁻¹; ν₄ = 713 cm⁻¹<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022338#pone.0022338-White1" target="_blank">[42]</a>.</p

Amino acid compositions (relative mol %) of proteins extracted from the soluble (SOM) and insoluble (IOM) fractions of the <i>Balanophyllia europaea</i> skeleton intra-skeletal organic matrix.

<p>*- indicates a not detectable amount.</p

Calcium carbonate overgrowth experiments.

<p>(A–D) SEM pictures of a fragment of coral skeleton after the calcium carbonate overgrowth experiment. The overgrowth of calcite and aragonite crystals was observed as shown in (B). In (B) the arrow indicates an aggregate of crystals of aragonite and the dashed square the area illustrated at higher magnification in the inset. The crystals of aragonite appear clustered in bunch of fibers (C), which locally exhibited preferential orientation (arrows in the inset in C). Organic matrix is visible in contact with the crystals of aragonite (arrows in inset in B and in C). The crystals of calcite showed an additional group of {hk0} faces other than the {104} set (D). These new faces showed as smooth surface, typical of interaction with macromolecules (inset in D).</p

OM characterization.

<p>SDS-polyacrylamide gels electrophoresis of intra-skeletal insoluble (IOM) and soluble (SOM) organic matrix extracted from the <i>Balanophyllia europaea</i> skeleton. In the first lane the markers are reported. The arrows indicate the major proteic bands.</p

OM characterization.

<p>FTIR spectra of intra-skeletal soluble (SOM) and insoluble (IOM) organic matrix from the <i>Balanophyllia europaea</i> aragonitic skeleton. Typical absorption bands from protein molecules (around 1600 cm⁻¹), polysaccharides (around 1000 cm⁻¹) and lipids (around 2900 cm⁻¹) are indicated. The absorption due to the polysaccharidic regions appeared stronger than the one due to the proteic regions in both the IOM and SOM spectra.</p

Calcium carbonate crystallization experiments.

<p>Crystallization experiments of calcium carbonate from 10 mM CaCl₂ solution with a Mg/Ca molar ratio equal to 5 (Ctrl5) and in the presence of soluble organic matrix (SOM), insoluble organic matrix (IOM) or both additives (IOM+SOM). The direction of the crystallographic <i>c</i>-axis of aragonite is indicated. <i>left</i> Scanning electron microscope images, the insets show samples details <i>right</i> FTIR spectra of the precipitated. The main absorption bands of calcium carbonate are indicated.</p