Transmission electron microscope (TEM) observations of calcite crystals synthesized in chitin gels.
We analyzed calcite crystals synthesized in the chitin gel by using transmission electron microscope (TEM). Calcite crystals was synthesized in chitin gel with yatalase, a commercially available chitinolytic enzyme, at different concentration (0, 0.12 and 1.2 mg/mL) (Fig. S1). We also prepared calcite crystals synthesized in chitin nanofiber solution which is made by chemical and physical treatment and has uniformed thickness, because we predicted the thickness of chitin was important for the formation of crystal defects. Each calcite crystal was shaped into thin cross-section with focused ion beam (FIB) for TEM analysis. TEM bright field images of the cross-sections revealed that the calcite crystal formed in the chitin gel without yatalase treatment (0 mg/mL) contained many thick chitin fibers with a bright contrast indicated by red arrows in Fig. 1a. The bright contrast means the void of organic molecules inside the calcite crystal. The contrast of typical bend contour (interference fringe of equal inclination) which was observed in the single crystal indicated by white arrows in Fig. 1a was distributed in whole area, indicating that chitin gel without chitinolytic enzyme treatment did not affect the formation of crystal defects in calcite crystals. In addition, selected area electron diffraction (SAED) patterns reflected that the calcite crystals synthesized in these conditions were single crystals (Fig. S2a).
By contrast, following 0.12 mg/mL chitinolytic enzyme treatment (Fig. 1b), slightly disordered interference fringes were partially observed around the small spherical Fresnel contrasts indicated by red arrows. The small spherical Fresnel contrasts mean the localization of thin organic molecules [14, 17]. The thin organic molecules might be a degraded chitin fiber by chitinolytic enzymes. If the sample was a perfect single crystal, equal inclination interference fringes would have shown clear straight lines. The disordered lines of interference fringes contrast showed that the small misorientations were generated. In Fig. 1b, interference fringes of equal inclination around the area of white circles were interrupted by lattice distortions generated by thin chitin fibers indicated by red arrows. These observations suggested that the positions of partial interrupted interference fringes corresponded to that of chitin fibers. SAED pattern (Fig. S2b) showed that the crystal orientation in whole area in Fig. 1b was almost uniformed, suggesting that the misorientation of crystal might be small. Thus, thin chitin fiber might induce the small angle grain boundaries in calcium carbonate crystals.
At 1.2 mg/mL chitinolytic enzyme treatment, thin chitin fibers were embedded with calcium carbonate. In Fig. 1c, the Fresnel contrasts means the position of thin chitin fibers indicated by red arrows. The interference fringes of equal inclination were completely stopped by the thin chitin fibers suggesting that the thin chitin fibers treated by 1.2 mg/mL yatalase made the perfect grain boundaries. The SAED pattern from the whole area in Fig. 1c showed the ring pattern (Fig. S2c), which means the calcite crystal containing grain boundaries was polycrystal. Since high concentration of yatalase decreased the thickness of chitin fiber, thinner chitin fiber might become the grain boundary.
Finally, we used the chitin nanofiber prepared by the physical treatments. In the chitin nanofiber solution, the interference fringes indicated by white arrows were disordered throughout the entire area (Fig. 1d). In the high magnification image, there were many Fresnel contrasts indicated by red arrows showing the distributions of chitin nanofibers. SAED pattern (Fig. S2d) showed that the crystal orientation in whole area in Fig. 1d was almost uniformed, suggesting that the misorientation of crystal might be small. This result was really similar to that of 0.12 mg/mL chitinolytic enzyme treatment. The chitin nanofibers interrupted the lines of interference fringes, suggesting that the small angle grain boundaries were generated around the areas of white circles in Fig. 1d
Based on these observations, TEM observations also supported the possibility that lattice distortion increased as the chitin fibers were degraded by chitinolytic enzymes, because the increasing surface areas of the chitin fibers strengthened the physical and/or chemical interactions between calcium carbonate and the chitin fiber. This strong interaction may have allowed chitin fibers to attach to the crystal growth front and inhibit growth of the crystal face in a random manner to induce a rounded shape.
Atom probe tomography (APT) of calcite crystals synthesized in chitin gels.
To examine the interaction of chitin with calcium carbonate in an atomic level, the synthesized calcite crystals were analyzed by atom probe tomography (APT). APT is a useful technique to determine the three-dimensional (3D) distribution of atoms or ions in the materials [19–21]. In APT analysis, the specimen shaped into a needle-like tip with a radius of 50–100 nm by a focused ion beam was ionized by field evaporation to detect atoms of ions with 2D ion distributions, followed by a 3D reconstruction. In Fig., the 3D reconstruction images show that ion clusters of COH+ (yellow) and COH2+ (pink) derived from chitin are detected within calcium carbonate ions (red). Fragments of chitin were partially detected in calcium carbonate at a concentration of 0 mg/mL (Fig. 2a). Most of signals were localized in the edge of sample. These signals might be due to the artifact from the sample preparation. On the other hand, there were few signals inside the calcium carbonate. These results suggested that no treatment of chitinolytic enzymes made no distribution of chitin signals inside the calcium carbonate.
On the other hand, the yellow and pink signals of chitin increased and were distributed in wide area within calcium carbonate at higher concentrations (0.12 and 1.2 mg/ml) of chitinolytic enzymes (Fig. 2b, c). The continuous chitin signals were homogeneous. Almost all chitin signals appeared inside the calcite crystal. At 1.2 mg/ml chitinolytic enzyme treatment, the amounts of yellow and pink signals significantly increased (Fig. 2c). From these observations, these chitin signals might be derived from both chitin oligomers and chitin nanofibers degraded by chitinolytic enzymes. These APT results support the hypothesis that chitin without the treatment of chitinolytic enzymes could not give any molecules inside the calcite crystal, while high concentration of chitinolytic enzymes produced the small fragments of chitin and gave the impurities to the calcite lattice at the atomic level.
In case of the artificial chitin nanofiber produced by physical procedure, high density signals derived from chitin were detected (Fig. 2d). This result suggested that the chitin nanofibers were distributed homogeneously in calcite crystals and showed the high amounts of signals. This observation also supported the evidence that thin chitin fiber (oligomer or nanofiber) interacts with calcium carbonate inside the crystal.
Electron backscatter diffraction (EBSD) analyses of calcite prisms treated by chitinase inhibitor in vivo.
As previously mentioned, we showed that chitinolytic enzymes may play important roles in formation of the P. fucata prismatic layer. To show the importance of chitinolytic enzymes on the formation of the prismatic layer, allosamidin, an inhibitor of GH18 chitinase , was injected into a living P. fucata every 3 days for a month. P. fucata was grown in natural sea water. Measurement of chitinase activity from mantle extracts showed that allosamidin suppressed activity for 1 month, indicating that the shell was formed under conditions of low chitinase activity (Fig. S3). If our hypothesis that chitinolytic enzymes degrade chitin and affect crystal growth was correct, the prism should be affected by inhibition of activity. Figure 3 and Fig. S4 showed EBSD mapping of the surface and longitudinal cross-section of prismatic layers along the c axis, and line profiles of one prism. We chose the position of growth front in the prismatic layer to see the effect of inhibitor on the prism formation. In the normal prismatic layer without inhibitor, crystal orientation on the surface of calcite prisms were almost uniformed (Fig. S4a). The strong red color of mapping image on the surface area reflected that the c axis of calcite was almost perpendicular to the shell surface. Although the rotated c axis of calcite was observed in the part of small areas, this rotation of c axis in the prismatic layer was reported in the previous report . We also prepared the cross-section of prisms. The color of one prism was found to be uniform from mapping the image and angle spread of one prism measured from the line profile within 3 degrees; there was minimal crystal misorientation within one prism (Fig. 3a). However, in the prismatic layer treated with inhibitor, color image of the surface slightly changed. The orientations of c axis were not uniformed in various prisms (Fig. S4b). Furthermore, in the cross-section, the crystal orientation of one prism was found to be gradually changed, with the mapping image and angle spread of one prism approximately 10 degrees (Fig. 3b). The reason for the increase of angle spread was likely due to intracrystalline chitin thickening from inhibiting chitin degradation. We previously reported that thicker chitin fibers inside the calcite prism treated with allosamidin compared with those of normal calcite prism were observed using TEM. Chitin fibers that increased in thickness may have contributed to crystal misorientation.
Nanoindentation of calcite prisms treated by a chitinase inhibitor in vivo.
A previous study reported that a biotic calcite crystal with slight crystal distortion was stronger than an abiotic calcite crystal without crystal distortion because the abiotic calcite is easily cracked under high pressure . The crystal distortion blocked transmission of strength to inhibit propagation of a crack. Because allosamidin treatment caused crystal distortions in calcite prisms, we investigated how such crystal distortions influenced mechanical properties such as hardness and the elastic modulus of calcite prisms. To examine the mechanical properties of calcite prisms, nanoindentation analysis of the prism surface was conducted. A nanoindentation force-displacement curve was drawn from the applied load versus depth profile, and hardness and the elastic modulus were obtained from this curve. The force-displacement curves are shown in Fig. 4a and b. When a force was loaded up to 100 mN, the maximum displacement was approximately 1–3 µm in the non-injection treatment (measurements at eight points). However, with allosamidin treatment, the maximum displacement was approximately 1–1.5 µm (measurements at six points). In addition, the cracks caused by indentation following allosamidin treatment were blocked to a greater extent than the non-injection treatment (Fig. S5), and both hardness and the reduced elastic modulus of prisms following allosamidin treatment increased by approximately 80.6% and 105%, respectively, compared with those of the non-injection treatment (Fig. 4c). These results indicated that inhibition of chitinase activity strengthened prisms, likely because crystal defects observed by EBSD could prevent the motion of dislocation in a crystal. Crystals contain a few dislocations that exist along the end of an extra half-plane of atoms . Because the force field around dislocation creates a strain, a dislocation easily moves along the plane perpendicular to the end of an extra half-plane when a shear stress is applied. The dislocation motion causes a slip of the crystal plane. Thus, reducing the dislocation motion results in enhanced mechanical strength. A grain boundary caused by a misorientation or enclosing substance is one factor that can reduce the dislocation motion. This theory could enable us to examine why a biotic calcite containing slight crystal distortions was stronger than abiotic calcite. In the present study, because micro-EBSD mapping revealed crystal misorientation in prisms treated with an inhibitor, this crystal defect may have resulted in harder prisms than normal.