Michela Reggi

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.

Morphological and mechanical characterization of composite calcite/SWCNT–COOH single crystals

A growing number of classes of organic (macro)molecular materials have been trapped into inorganic crystalline hosts, such as calcite single crystals, without significantly disrupting their crystalline lattices. Inclusion of an organic phase plays a key role in enhancing the mechanical properties of the crystals, which are believed to share structural features with biogenic minerals. Here we report the synthesis and mechanical characterization of composite calcite/SWCNT-COOH single crystals. Once entrapped into the crystals SWCNT-COOH appeared both as aggregates of entangled bundles and nanoropes. Their observation was possible only after crystal etching, fracture or FIB (focused ion beam) cross-sectioning. SWCNT-COOHs occupied a small volume fraction and were randomly distributed into the host crystal. They did not strongly affect the crystal morphology. However, although the Youngs modulus of composite calcite/SWCNT-COOH single crystals was similar to that of pure calcite their hardness increased by about 20%. Thus, SWCNT-COOHs provide an obstacle against the dislocation-mediated propagation of plastic deformation in the crystalline slip systems, in analogy with the well-known hardness increase in fiber-reinforced composites.

Polypeptide effect on Mg2+ hydration inferred from CaCO3 formation: a biomineralization study by counter-diffusion

The use of a counter-diffusion system allows the evaluation of diverse parameters involved in a crystallization process. In this study, this tool has been used to infer the hydration status of Mg2+ during CaCO3 formation experiments in an agarose highly viscous sol entrapping charged polypeptides. The experimental data allow us to infer that the hydration status of Mg2+ is altered by the presence of poly-L-aspartate or poly-L-glutamate. This changes the CaCO3 polymorphic distribution in favor of Mg-calcite with respect to aragonite, but does not favor the isomorphic substitution of Mg2+ with Ca2+ within the calcite lattice. The latter may exclude the formation of an amorphous transient form, which leads to a high Mg-calcite, as expected when using a counter-diffusion system set up. The presence of poly-L-lysine does not affect the hydration of Mg2+, but favors the formation of aragonite with respect to calcite. In this case an inhibition of calcite formation and an alteration of the hydration sphere of Ca2+ could be invoked; both effects are able to increase CaCO3 supersaturation. In conclusion, this study reveals that charged polypeptides can orchestrate CaCO3 formation by also controlling the hydration status of cations.

Calcium carbonate crystallization in tailored constrained environments

Synthesis of inorganic particles using routes inspired by biomineralization is a goal of growing interest. Recently it was demonstrated that the size and geometry of crystallization sites are as important as the structure of charged templating surfaces to obtain particles with controlled features. Most biominerals are formed inside restricted, constrained or confined spaces where at least parts of the boundaries are cell membranes containing phospholipids. In this study, we used a gas diffusion method to determine the effect of different lecithin media on the crystallization of CaCO3 and to evaluate the influence of the spatial arrangement of lecithin molecules on templating CaCO3 crystal formation. By using inorganic synthesis, Raman spectroscopy, dynamic light scattering, electrochemical methods and scanning electron microscopy, we showed that the occurrence of surface-modified calcite crystals and diverse textured vaterite crystals reflects the geometry and spatial distribution of aqueous constrained spaces due to the lecithin assembly controlled by lecithin concentration in an ionized calcium chloride solution under a continuous CO2 diffusion atmosphere. This research shows that by tailoring the assembly of lecithin molecules, as micelles or reversed micelles, it is possible to modulate the texture, polymorphism, size and shape of calcium carbonate crystals.

Shell properties of commercial clam Chamelea gallina are influenced by temperature and solar radiation along a wide latitudinal gradient.

Phenotype can express different morphologies in response to biotic or abiotic environmental influences. Mollusks are particularly sensitive to different environmental parameters, showing macroscale shell morphology variations in response to environmental parameters. Few studies concern shell variations at the different scale levels along environmental gradients. Here, we investigate shell features at the macro, micro and nanoscale, in populations of the commercially important clam Chamelea gallina along a latitudinal gradient (~400 km) of temperature and solar radiation in the Adriatic Sea (Italian cost). Six populations of clams with shells of the same length were analyzed. Shells from the warmest and the most irradiated population were thinner, with more oval shape, more porous and lighter, showing lower load fracture. However, no variation was observed in shell CaCO3 polymorphism (100% aragonite) or in compositional and textural shell parameters, indicating no effect of the environmental parameters on the basic processes of biomineralization. Because of the importance of this species as commercial resource in the Adriatic Sea, the experimentally quantified and significant variations of mass and fracture load in C. gallina shells along the latitudinal gradient may have economic implications for fisheries producing different economical yield for fishermen and consumers along the Adriatic coastline.

Structure and Function of Stony Coral Intraskeletal Polysaccharides

Polysaccharides represent a main weight fraction of the intraskeletal organic matrix of corals, but their structure, as well as their function in the calcification process, has been poorly investigated. This communication shows by a combination of techniques (nuclear magnetic resonance, Fourier transform infrared, and monosaccharide composition) that their key component is a 1→3 β-d glucuronic acid polymer and evidences its influence in vitro in the calcification process.

Effects of magnesium and temperature control on aragonite crystal aggregation and morphology

In this study, the influence of Mg2+ on the aggregation and morphology of precipitated aragonite crystals is investigated at different temperatures. Different from some investigations described in the literature, aragonite precipitates in chemical systems in which Mg2+ acts specifically as crystal habit modifiers and not as polymorphic selectors. The results show that at increased Mg2+ concentration and temperature the aragonite crystals are less aggregated and that the amount of crystals sharing {110} faces and having larger extension of {001} faces increases. These outcomes may be relevant in biological, geochemical and technological contexts.

Isotropic microscale mechanical properties of coral skeletons

Scleractinian corals are a major source of biogenic calcium carbonate, yet the relationship between their skeletal microstructure and mechanical properties has been scarcely studied. In this work, the skeletons of two coral species: solitary Balanophyllia europaea and colonial Stylophora pistillata, were investigated by nanoindentation. The hardness HIT and Youngs modulus EIT were determined from the analysis of several load–depth data on two perpendicular sections of the skeletons: longitudinal (parallel to the main growth axis) and transverse. Within the experimental and statistical uncertainty, the average values of the mechanical parameters are independent on the sections orientation. The hydration state of the skeletons did not affect the mechanical properties. The measured values, EIT in the 76–77 GPa range, and HIT in the 4.9–5.1 GPa range, are close to the ones expected for polycrystalline pure aragonite. Notably, a small difference in HIT is observed between the species. Different from corals, single-crystal aragonite and the nacreous layer of the seashell Atrina rigida exhibit clearly orientation-dependent mechanical properties. The homogeneous and isotropic mechanical behaviour of the coral skeletons at the microscale is correlated with the microstructure, observed by electron microscopy and atomic force microscopy, and with the X-ray diffraction patterns of the longitudinal and transverse sections.

Ecological relevance of skeletal fatty acid concentration and composition in Mediterranean scleractinian corals

The intra-skeletal fatty acid concentration and composition of four Mediterranean coral species, namely Cladocora caespitosa, Balanophyllia europaea, Astroides calycularis and Leptopsammia pruvoti, were examined in young and old individuals living in three different locations of the Mediterranean Sea. These species are characterized by diverse levels of organization (solitary or colonial) and trophic strategies (symbiotic or non-symbiotic). Fatty acids have manifold fundamental roles comprehensive of membrane structure fluidity, cell signaling and energy storage. For all species, except for B. europaea, the intra-skeletal fatty acid concentration was significantly higher in young individuals than in old ones. Moreover, fatty acid concentration was higher in colonial corals than in solitary ones and in the symbiotic corals compared to non-symbiotic ones. Analysis by gas chromatography-mass spectrometry (GC-MS) revealed that palmitic acid (16:0) was the most abundant fatty acid, followed by stearic (18:0) in order of concentration. Oleic acid (18:1) was detected as the third main component only in skeletons from symbiotic corals. These results suggest that, in the limits of the studied species, intra-skeletal fatty acid composition and concentration may be used for specific cases as a proxy of level of organization and trophic strategy, and eventually coral age.

Influence of intra-skeletal coral lipids on calcium carbonate precipitation

Recent research studies have shown that the intra-skeletal organic matrix of corals contains lipids. This communication reports their characterization and their influence on calcium carbonate precipitation. In addition, their potential role in corals biomineralization is discussed.