Intracrystalline Organic Matrix Research Articles

Utilization of Eggshell Waste Calcite as a Sorbent for Rare Earth Element Recovery.

The green energy transition requires rare earth elements (REE) for the permanent magnets used in electric cars and wind turbines. REE extraction and beneficiation are chemically intensive and highly damaging to the environment. We investigated the use of eggshell waste as a sustainable alternative sorbent for the capture and separation of REE from aqueous solutions. Hen eggshell calcite was placed in multi-REE (La, Nd, Dy) solutions at 25 to 205 °C for up to 3 months. A pervasive diffusion of the REE inside the eggshell calcite was observed along pathways formed by the intracrystalline organic matrix and calcite crystal boundaries. At 90 °C, kozoite (REECO3OH, orthorhombic) spherulites precipitate on the surface of the dissolving calcite. At 165 and 205 °C, an interface-coupled dissolution-precipitation mechanism is observed, resulting in the complete dissolution of the calcite shell and its pseudomorphic replacement by polycrystalline kozoite. At 205 °C, kozoite is slowly replaced by hydroxylbastnäsite (REECO3OH, hexagonal), the stable form of the rare earth hydroxycarbonate polymorphs. Our results demonstrate two potential applications of eggshell waste for the uptake of rare earth elements in solution: at low temperatures, as a mixed organic-inorganic adsorbent and absorbent, given sufficient sorption time; and at higher temperatures, as an efficient sacrificial template for the precipitation of rare earth hydroxycarbonates.

Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties

Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. Statement of significanceAvian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds – it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms.

Functional analyses of chitinolytic enzymes in the formation of calcite prisms in Pinctada fucata

The mollusk shells present distinctive microstructures that are formed by small amounts of organic matrices controlling the crystal growth of calcium carbonate. The shell of Pinctada fucata has the prismatic layer consisting of prisms of single calcite crystals and the nacreous layer consisting of aragonite tablets. The calcite crystal of prisms contains small angle grain boundaries caused by a dense intracrystalline organic matrix network to improve mechanical strength. Previously, we identified chitin and chitinolytic enzymes as components of this intracrystalline organic matrix. In this study, to reveal the function of those organic matrices in calcium carbonate crystallization, calcites synthesized in chitin gel with or without chitinolytic enzymes were analyzed by using transmission electron microscope (TEM) and atom probe tomography (APT), showing ion clusters derived from chitin inside of a calcite and small angle grain boundaries at optimal chitinolytic concentration. Furthermore, we performed the experiment in which chitinase inhibitor was injected into a living P. fucata. Nanoindentation and electron back scattered diffraction (EBSD) show that mechanical properties and crystal orientation were changed. These results suggested that chitinolytic enzymes work cooperatively with chitin to regulate the crystal growth and mechanical properties of the prismatic layer.

Impact of ocean acidification on crystallographic vital effect of the coral skeleton

Distinguishing between environmental and species-specific physiological signals, recorded in coral skeletons, is one of the fundamental challenges in their reliable use as (paleo)climate proxies. To date, characteristic biological bias in skeleton-recorded environmental signatures (vital effect) was shown in shifts in geochemical signatures. Herein, for the first time, we have assessed crystallographic parameters of bio-aragonite to study the response of the reef-building coral Stylophora pistillata to experimental seawater acidification (pH 8.2, 7.6 and 7.3). Skeletons formed under high pCO2 conditions show systematic crystallographic changes such as better constrained crystal orientation and anisotropic distortions of bio-aragonite lattice parameters due to increased amount of intracrystalline organic matrix and water content. These variations in crystallographic features that seem to reflect physiological adjustments of biomineralizing organisms to environmental change, are herein called crystallographic vital effect (CVE). CVE may register those changes in the biomineralization process that may not yet be perceived at the macromorphological skeletal level.

Single Nanogranules Preserve Intracrystalline Amorphicity in Biominerals

We revisit the ultrastructural features of different calcareous biominerals and identify remarkable similarities: taxonomically very distant species show a common nanogranular structure, even if different extracellular secretion patterns are employed or calcium carbonate polymorphs formed. By these analyses, we elucidate the locus of the small fraction of intracrystalline organic matrix revealing its intergranular character and localize the intracrystalline amorphous calcium carbonate moiety commonly found in mesocrystalline biominerals and provide a first explanation for the pathway by which it is preserved.

Coccolithogenesis In Scyphosphaera apsteinii (Prymnesiophyceae).

Coccolithophores are the most significant producers of marine biogenic calcite, although the intracellular calcification process is poorly understood. In the case of Scyphosphaera apsteinii Lohmann 1902, flat ovoid muroliths and bulky, vase-shaped lopadoliths with a range of intermediate morphologies may be produced by a single cell. This polymorphic species is within the Zygodiscales, a group that remains understudied with respect to ultrastructure and coccolith ontogeny. We therefore undertook an analysis of cell ultrastructure, morphology, and coccolithogenesis. The cell ultrastructure showed many typical haptophyte features, with calcification following a similar pattern to that described for other heterococcolith bearing species including Emiliania huxleyi. Of particular significance was the reticular body role in governing fine-scale morphology, specifically the central pore formation of the coccolith. Our observations also highlighted the essential role of the inter- and intracrystalline organic matrix in growth and arrangement of the coccolith calcite. S. apsteinii secreted mature coccoliths that attached to the plasma membrane via fibrillar material. Time-lapse light microscopy demonstrated secretion of lopadoliths occurred base first before being actively repositioned at the cell surface. Significantly, growth irradiance influenced the coccosphere composition with fewer lopadoliths being formed relative to muroliths at higher light intensities. Overall, our observations support dynamic metabolic (i.e., in response to growth irradiance), sensory and cytoskeletal control over the morphology and secretion of polymorphic heterococcoliths. With a basic understanding of calcification established, S. apsteinii could be a valuable model to further study coccolithophore calcification and cell physiological responses to ocean acidification.

Testing the limitations of artificial protein degradation kinetics using known-age massive Porites coral skeletons

High-temperature isothermal heating of biominerals has commonly been used to artificially accelerate protein degradation in order to extrapolate kinetic parameters to the lower temperatures experienced in vivo and in the burial environment. It is not easy to test the accuracy of these simulations due to the difficulty of finding samples of known age held at a known temperature. We compare protein degradation in the intra-crystalline organic matrix of heated (80 °C, 110 °C, and 140 °C) massive Porites sp. coral to that directly measured in the skeleton of colonies growing at ∼26 °C and deposited over the last five centuries. This provides the opportunity to critically evaluate the underlying assumption that high-temperature experiments accurately mimic degradation processes and kinetics occurring in a ‘naturally aged’ biomineral. In all samples the intra-crystalline protein fraction was isolated and the L- and D- concentration of multiple amino acids measured using reverse-phase high-performance liquid chromatography (RP-HPLC). There was no evidence of a failure of the closed system in the high-temperature experiments (assessed by X-ray diffraction, thermogravimetric analyses and determination of leached amino acid concentration). We compared conventional methods for estimation of kinetic parameters with a new ‘model-free’ approach that makes no assumptions regarding the underlying kinetics of the system and uses numerical optimisation to estimate relative rate differences. The ‘model-free’ approach generally produced more reliable estimates of the observed rates of racemization in ‘naturally aged’ coral, although rates of hydrolysis (as estimated from the release of free amino acids) were usually over-estimated. In the amino acids for which we were able to examine both racemization and hydrolysis (aspartic acid/asparagine, glutamic acid/glutamine and alanine), it was clear that hydrolysis was less temperature sensitive than racemization, which may account for the differences in degradation patterns observed between the ‘naturally aged’ coral and high-temperature data. It is clearly important to estimate the individual temperature dependence of each of the parallel reactions.

Synchrotron XRD Study on the Nacre of Freshwater Bivalve <I>H. cumingii</I> Lea

Synchrotron XRD was used to study internal stress of nacre and microstructure of individual nacre tablet in the shell of freshwater bivalve H. cumingii Lea. The diffraction patterns show some biopolymers (intra-crystalline organic matrix) are occluded inside nacre tablets during biomineralization, causing that nacre powder exhibits tensile- like internal stress which is strongly anisotropic along different vectors of the reciprocal lattice. The characteristic of internal stress suggests that the intra-crystalline organic matrix is most probably arranged in the specific form. And line profile analysis which is conducted with the synchrotron XRD patterns of heated and unheated nacre powder samples, further confirms that the intra-crystalline organic matrix is adsorbed on (002) atomic planes of nacre tablets. The findings will facilitate the study on the formation mechanism and the strengthening and toughening mechanism of the nacre and provide theoretical basis for designing new organic-inorganic composite with high performances and cultivating pearl.

Arrangement of intra-crystalline organic matrix in nacre from red abalone: A view by high-resolution synchrotron powder diffraction combined with SEM observation

In the present study, we combine high-resolution synchrotron powder diffraction with SEM to shed additional light on the arrangement of an intra-crystalline organic matrix in the nacre from red abalone. SEM observation and synchrotron diffraction analysis show that an intra-crystalline organic matrix exists inside nacre tablets and is adsorbed on the (002) atomic plane in an isolated form. Most probably, the intra-crystalline organic matrix is nacrein and its atomic groups are connected with the (012) atomic plane. The findings provide a new understanding of the arrangement of intra-crystalline organic matrices and may facilitate studies on the formation mechanism and the strengthening and toughening mechanism of the nacre. It is worth pointing out that the new experimental method in this present paper can be extended to nanostructural research of other biominerals.

Mechanical properties of modern calcite- (Mergerlia truncata) and phosphate-shelled brachiopods (Discradisca stella and Lingula anatina) determined by nanoindentation

We measured distribution patterns of hardness and elastic modulus by nanoindentation on shells of the rhynchonelliform brachiopod Mergerlia truncata and the linguliform brachiopods Discradisca stella and Lingula anatina. The rhynchonelliformea produce calcitic shells while the linguliformea produce chitinophosphatic shells. Dorsal and ventral valves, commissure and hinge of the calcitic shell of M. truncata show different nanohardness values (from 2.3 to 4.6 GPa) and E-modulus (from 52 to 76 GPa). The hardness of the biocalcite is always increased compared to inorganic calcite. We attribute the effects to different amounts of inter- and intracrystalline organic matrix. Profiles parallel to the radius of curvature of the valves cutting through the different layers of shell material surprisingly show quite uniform values of nanohardness and modulus of elasticity. Nanoindentation tests on the chitinophosphatic brachiopods D. stella and L. anatina reflect the hierarchical structure composed of laminae with varying degree of mineralization. As a result of the two-phase composite of biopolymer nanofibrils reinforced with Ca-phosphate nanoparticles, nanohardness, and E-modulus correlate almost linearly from (H=0.25 GPa, E=2.5 GPa) to (H=2.5 GPa, E=50 GPa). The mineral provides stiffness and hardness, the biopolymer provides flexibility; and the composite provides fracture toughness. Gradients in the degree of mineralization reduce potential stress concentrations at the interface between stiff mineralized and soft non-mineralized laminae. For the epibenthic chitinophosphatic D. stella the lamination is also present but less pronounced than for the infaunal L. anatina, and the overall distribution of material strength in the cross-sectional profile shows a maximum in the center and a decrease towards the inner and outer shell margins (modulus of elasticity from 30 to 12 GPa, hardness from 1.7 to 0.5 GPa). Accordingly, the two epibenthic forms, calcitic M. truncata and chitinophosphatic D. stella display fairly bulky (homogeneous) nanomechanical properties of their shell materials, while the burrowing infaunal L. anatina is distinctively laminated. The strongly mineralized laminae, which provide the strength to the shell, are also brittle, but keeping them as thin as possible, allows some bending flexibility. This flexibility is not required for the epibenthic life style.

Ultrastructure of avian eggshell during resorption following egg fertilization

For skeletal mineralization, the avian embryo mobilizes calcium from its calcitic eggshell. This occurs through dissolution of specific interior regions of the shell in a process that also weakens the shell to allow hatching. Here, we have examined eggshell ultrastructure during dissolution occurring between laying of a fertilized egg (with incubation) and hatching of the chick (Gallus gallus). We have focused on changes in shell mammillae where the majority of dissolution takes place. Using scanning electron microscopy, we describe differences in matrix-mineral structure and relationships not observed in unfertilized eggs (unresorbed eggshell). We document changes in the calcium reserve body - an essential sub-compartment of mammillae - consistent with it being an early, primary source of calcium essential for embryonic skeletal growth. Dissolution events occurring in the calcium reserve sac and in the base plate of the calcium reserve body, and similar changes in surrounding bulk mammillae structure, all correlate with advancing skeletal embryonic calcification. The changes in mammillae sub-structures can generally be characterized as mineral dissolutions revealing fine surface topographies on remaining mineral surfaces and the exposure of an extensive, intracrystalline (occluded) organic matrix network. We propose that this mineral-occluded network regulates how shell mineral is dissolved by providing dissolution channels facilitating calcium release for the embryonic skeleton.

Asprich mollusk shell protein: in vitro experiments aimed at elucidating function in CaCO3 crystallization

Acidic proteins are key components of the organic matrix of many biologically formed minerals and are therefore thought to play an important role in their formation. Here we study the effect of one unusually acidic protein of the Asprich family, associated with mollusk shell prismatic layer, on the precipitation of CaCO3in vitro. We show that Asprich induces and transiently stabilizes the deposition of amorphous calcium carbonate (ACC). Asprich also induces the formation of ACC when adsorbed onto chitin, a major component of the intracrystalline organic matrix of the prismatic layer. Based on this evidence, combined with previous studies on the forming prisms in the shell layer, we suggest that the in vivo function of Asprich is inducing and stabilizing ACC particles and inhibiting their uncontrolled crystallization until they undergo secondary nucleation on the growing prisms.

Fine structure of normal and regenerated shell of Helix.

Fine structure of the normal and the regenerated shell of Helix has been studied by thin sectioning, replica technique, and scanning electron microscopy. Normal shell consists of four calcareous layers: innermost nacreous, two cross lamellar, and outermost prismatic. Crystals of the shell are well defined and are surrounded by intercrystalline organic matrix. Intracrystalline organic matrix is recognized, particularly in decalcified sections. Interrelationships between the organic and inorganic components have been studied in decalcified thin sections. Regenerated shell appears similar to nacreous layer of the normal shell. Crystals are large and stacked like bricks. Intracrystalline organic matrix is very prominent. Electron diffraction of the crystals of the regenerated shell generally gives calcite pattern whereas the normal shell gives aragonite. Surface topography of the normal and regenerated shell has been compared by replica techniques.