CRYSTAL GROWTH & DESIGN
Structure of Biogenic Aragonite (CaCO3) B. Pokroy,† A. N. Fitch,‡ and E. Zolotoyabko*,† Department of Materials Engineering, TechnionsIsrael Institute of Technology, Haifa 32000, Israel, and European Synchrotron Radiation Facility, B. P. 220, 38043 Grenoble Cedex, France
2007 VOL. 7, NO. 9 1580-1583
ReceiVed NoVember 26, 2006; ReVised Manuscript ReceiVed June 5, 2007
ABSTRACT: By using high-resolution X-ray powder diffraction at a dedicated synchrotron beam line, ID-31, of the European Synchrotron Radiation Facility (ESRF, Grenoble, France), we studied structural and microstructural modifications in biogenic aragonite crystals, obtained from mollusk shells, and subjected to heat treatments at elevated temperatures. All investigated shells revealed anisotropic lattice distortions of the orthorhombic unit cell as compared to geological aragonite. Annealing at temperatures above 150-200 °C led to pronounced lattice relaxation which is accompanied by a substantial reduction of crystallite sizes and related growth of microstrain fluctuations. These findings indicate that biogenic aragonite crystals are strained, apparently, as a result of the amorphous/crystalline phase transformation, which proceeds within the supporting network of oriented biomacromolecules at early stages of biomineralization. Introduction The growth of biogenic crystals, i.e., crystals produced by organisms, is extensively studied by numerous research groups since these crystals often demonstrate superior characteristics as compared to their counterparts of non-biogenic origin.1-4 Particular consideration has been given to mollusk shells, built of calcite and/or aragonite, because of their fascinating mechanical properties (see e.g., ref 5). For years it has been known that biogenic crystals are, in fact, bio-nanocomposites which comprise an organic matrix and a ceramic (mineral) phase. The mineral growth is strongly affected by organic macromolecules that lead to complicated hierarchical microstructures on various scales from nanometer to millimeter (see, e.g., refs 1-3 and 6-8). The ability of organisms to control polymorphism and crystal morphology on a nanometer scale is amazing.1,2 Surprisingly, our recent findings9 indicated that even the atomic structures of biogenic and geological crystals of the same kind are slightly different, and this we believe is also due to organic macromolecules which became trapped within crystallites during biomineralization. By using high-resolution X-ray powder diffraction at dedicated synchrotron beam lines, we discovered anisotropic lattice distortions (of about 0.2% in maximum) in biogenic aragonite and calcite (both obtained from mollusk shells), as compared to their geological counterparts.9-11 This phenomenon was found to be widespread in the mollusk phylum.10,11 In fact, very similar lattice distortions were observed in mollusk shells belonging to different classes (bivalves, gastropods, and cephalopods) and taken from different habitat origins (sea, freshwater, and land). Lattice distortions are present in crossed-lamellar, prismatic, and nacre layers. Very recently, practically the same lattice distortions were detected in the scleractinian coral biominerals.12 It is reasonable to assume that some common mechanism is responsible for this universal behavior. By analyzing the complete set of experimental findings, we conclude that biogenic crystals are mechanically stressed/strained and the intracrystalline organic molecules (i.e., confined within individual crys* To whom the correspondence should be addressed. E-mail: zloto@ tx.technion.ac.il. † Technion. ‡ European Synchrotron Radiation Facility.
Figure 1. SEM micrograph revealing a well-developed nacre layer at a depth of 1.5 mm beneath the shiny surface of the Unio treminalis shell.
tallites)13,14 play an important role in this process. In more detail, the concept of strained biogenic crystals will be elaborated below. Strong support for the above conclusion is given by the results of structural measurements in samples subjected to mild shortperiod annealing at low temperatures of about 150-200 °C. At these temperatures, pronounced lattice relaxation due to the heatinduced degradation of organic macromolecules occurs.10,11 Besides that, when studying calcitic shells, we also found that lattice relaxation is accompanied by a drastic reduction of the crystallite sizes “visible” by X-rays.15 Such correlation between strain relaxation and subdivision of single crystals into smaller coherently scattered blocks under annealing is very typical for strained heterostructures used for optoelectronic and microelectronic applications.16 Conceptually, it is important to prove that the effect of crystal size reduction under heat treatment is also common for various biogenic crystals, as strain relaxation. In this paper, we report on this effect in aragonitic shells that strengthens the universality of our findings and concepts. Experimental Procedures High-resolution X-ray powder diffraction measurements were carried out at the dedicated beam line, ID-31, of the European Synchrotron
10.1021/cg060842v CCC: $37.00 © 2007 American Chemical Society Published on Web 07/06/2007
Structure of Biogenic Aragonite (CaCO)3
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Figure 2. Lattice relaxation along the c-axis in the Unio treminalis (squares) and Nautilus macrophalus (circles) shells under annealing at elevated temperatures. The c-values measured as a function of annealing temperature in geological aragonite (triangles) are also given for comparison.
Figure 3. Reduction of the crystallite size, L (nm), in the Unio treminalis (upper panel) and Nautilus macrophalus (lower panel), as a result of annealing at elevated temperatures.
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Figure 4. Increase of the microstrain fluctuations, S, in the Unio treminalis (upper panel) and Nautilus macrophalus (lower panel), as a result of annealing at elevated temperatures. Radiation Facility (ESRF, Grenoble, France) equipped with a crystalmonochromator and crystal-analyzer optical elements.17 X-rays from the synchrotron storage ring were monochromatized by a liquid nitrogen cooled, double-crystal silicon monochromator. The optics of the diffracted beam consisted of nine (111) Si crystal analyzers. In this setup the instrumental contribution to the X-ray profile broadening is reduced to 0.003°.17 Shell powders were loaded into 0.5-1 mm borosilicate glass capillaries. To avoid intensity spikes from the individual crystallites subjected to quasi-parallel synchrotron beam irradiation, the samples were rotated during measurements at a rate of 60 rps. Calibration of the instrument and refinement of the X-ray wavelength was performed with silicon standard samples from the National Institute of Standards and Technology (NIST). Geological aragonite from Sefrou (Morocco), practically free of impurities, was used as a control sample for comparison. Here we present experimental data collected with two aragonitic shells, Unio treminalis (U. teminalis) and Nautilus macrophalus (N. macrophalus), belonging to different classes and also having dissimilar habitat origins. U. treminalis is a freshwater bivalve shell, while N. macrophalus is a cephalopod seashell. Their microstructures were thoroughly studied (see e.g., refs 18 and 19). Both shells possess a well-developed nacre layer which can reach a few millimeters in thickness.20,21 As an example, we show in Figure 1 the micrograph of the nacre structure in U. treminalis taken at a depth of 1.5 mm beneath the inner shell surface adjacent to the mollusk mantle. In this image, well-developed aragonite lamellae having preferred crystallographic
orientation of the [001] type (i.e., perpendicular to the c-axis of the orthorhombic unit cell) are clearly seen. The lamellae are on average about 800 nm thick. Shell pieces were cleaned by sonication in methanol and doubledistilled water and then air-dried. After that, they were crushed to fine powders using a mortar and pestle, followed by sieving through a 25 µm sieve. The measured X-ray powder diffraction profiles were treated with the aid of the Rietveld refinement within the GSAS program22 and the EXPGUI interface.23
Results and Discussion Comparison between lattice parameters of biogenic and geological aragonite revealed anisotropic lattice distortions of the unit cell which in all cases remained orthorhombic. The highest tensilelike distortion (of about 0.1-0.2%) is always along the c-axis. As was mentioned in the Introduction, mild heat treatments at temperatures of about 150-200 °C led to pronounced lattice relaxation. This is illustrated in Figure 2, in which the evolution of the lattice parameter along the c-axis in the two aragonitic shells subjected to isochronous annealing for 30 min at elevated temperatures is shown. In fact, an effective lattice relaxation is observed after annealing at 150 °C for N. macrophalus and 200 °C for U. treminalis. At higher temper-
Structure of Biogenic Aragonite (CaCO)3
atures (about 350 °C) the lattice parameters in both shells approach the value of the geological sample. The latter practically does not change upon annealing (see Figure 2), which clearly indicates structural distinctions between biogenic and geological crystals. When measuring annealed biogenic samples, we found substantial broadening effect of diffraction peaks due to modifications in the crystallite size and microstrain fluctuations. These two contributions to the diffraction peak width are described mathematically by different functions (Lorentzian and Gaussian for crystallite size and microstrain fluctuations, respectively) and can be separated by using the Voigt function analysis of diffraction profiles (see, e.g., ref 24). The results of this analysis are shown in Figures 3 and 4 for crystallite size (“viewed” by X-ray diffraction) and microstrain fluctuations, respectively. Concerning the crystallite size (i.e., the size of crystal block which participates in coherent X-ray scattering), we note that it is strongly anisotropic. In both shells before annealing, the smallest crystallite size, of about 300 nm, is along the [211] vector of the reciprocal lattice, while the largest one, which is about 500 nm, is along the [111], [012], and [002] vectors of the reciprocal lattice. The latter value is somewhat lower than the thickness of [001]-oriented lamellae (of about 800 nm) visible in the scanning electron microscopy (SEM) cross-section in Figure 1. This difference is most probably influenced by the presence of intracrystalline organic molecules within crystallites, part of them limiting the effective size of crystal blocks which coherently scatter X-rays. It is clearly seen that annealing at temperatures above 200 °C results in considerable reduction of crystallite sizes (see Figure 3). For both shells the strongest effect (reduction from 500 to nearly 300 nm) is observed in the [002] direction. The decrease of the crystallite size is accompanied by increasing the microstrain fluctuations (see Figure 4). In fact, as shown in ref 24, these two effects are closely related, viz., the reduction of crystallite size causes the growth of microstrain fluctuations due to increasing number of intercrystalline boundaries. Note that in geological aragonite the crystallite size as well as microstrain fluctuations practically do not reveal any changes upon annealing. We stress that these two phenomena, drastic reduction of the crystallite size and relaxation of lattice parameters mentioned above, are well-correlated on the temperature scale. We found similar correlations for all investigated shells (including those made of calcite),15 which implies that some universal mechanism must stay behind this. To understand the significance of our experimental findings, we recall that biomineralization of mollusk shells proceeds within the framework of spatially organized and oriented organic macromolecules.25 These molecules serve as nucleation centers for calcium carbonate. In the most plausible scenario, calcium carbonate initially appears in the form of amorphous precipitates.26 The amorphous phase is structurally more flexible than the crystalline one, and in such a way the strain energy caused by the forces at the organic-inorganic interfaces is reduced. Crystallization of amorphous calcium carbonate leads to the reduction of the specific volume per molecule (mineral shrinkage), which in turn results in increasing forces imposed by organic molecules on the atomic groups of a mineral. These forces produce lattice strains which are revealed in our highresolution diffraction measurements, as some “memory” of the initial amorphous state. Annealing at 150-200 °C leads to the degradation of the network of organic macromolecules supporting the strained mineral lattice. As a consequence, one
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observes lattice relaxation which is also accompanied by subdivision of crystallites into smaller blocks (“viewed” by coherent X-ray scattering). Conclusions Anisotropic lattice strains are very common in biogenic calcite and aragonite crystals. Most probably, these strains are caused by the forces acting between the network of organic macromolecules and ceramic crystallites growing within it. According to our current understanding, the strain state arises predominantly at the stage of amorphous/crystalline phase transformation. This is the reason that the strains are of the same order of magnitude in all the investigated shells. Mild annealing of biogenic crystals destroys the macromolecular network supporting the strained mineral lattice, which results in strain relaxation and related reduction of crystallite sizes. Further studies of the nucleation and growth of minerals within macromolecular networks will aid in deeper understanding of the biomineralization problem and the development, on this basis, of new approaches toward fabricating superior natureinspired composite materials. Acknowledgment. E.Z. acknowledges the partial financial support of this work by the Technion Research Funds and the Israel Science Foundation founded by the Israel Academy of Science and Humanities. References (1) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. (2) Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry; Oxford University Press: Oxford, U.K., 2001. (3) Weiner, S.; Addadi, L. J. Mater. Chem. 1997, 7, 689. (4) Almquist, N.; Thomson, N. H.; Smith, B. L.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Mater. Sci. Eng., C 1999, 7, 37. (5) Kamat, S.; Su, X.; Ballarini, R.; Heuer, A. H. Nature 2000, 405, 1036. (6) Meldrum, F. C. Int. Mater. ReV. 2003, 48, 187. (7) Chateigner, D.; Hedegaard, C.; Wenk, H. R. J. Struct. Geol. 2000, 22, 1723. (8) Pokroy, B.; Zolotoyabko, E. J. Mater. Chem. 2003, 13, 682. (9) Pokroy, B.; Quintana, J. P.; Caspi, E. N.; Berner, A.; Zolotoyabko, E. Nat. Mater. 2004, 3, 900. (10) Pokroy, B.; Fitch, A. N.; Lee, P. L.; Quintana, J. P.; Caspi, E. N.; Zolotoyabko, E. J. Struct. Biol. 2006, 153, 145. (11) Pokroy, B.; Fitch, A. N.; Marin, F.; Kapon, M.; Adir, N.; Zolotoyabko, E. J. Struct. Biol. 2006, 155, 96. (12) Stolarski, J.; Przenioslo, R.; Mazur, M.; Brunelli, M. J. Appl. Crystallogr. 2007, 40, 2. (13) Berman, A.; Addadi, L.; Weiner, S. Nature 1988, 331, 546. (14) Berman, A.; Addadi, L.; Kvick, A.; Leiserowitz, L.; Nelson, M.; Weiner. S. Science 1990, 250, 664. (15) Pokroy, B.; Fitch, A. N.; Zolotoyabko, E. AdV. Mater. 2006, 18, 2363. (16) Timbrell, P. Y.; Baribeau, J. M.; Lockwood, D. J.; McCaffey, J. P. J. Appl. Phys. 1990, 67, 6292. (17) Fitch, A. N. J. Res. Natl. Inst. Stand. Technol. 2004, 109, 133. (18) Checa, A. Tissue Cell 2000, 32, 405. (19) Velazguez-Castillo, R.; Reyes-Gasga, J.; Garcia-Gutierrez, D. I.; JoseYacaman, M. J. Mater. Res. 2006, 21, 1484. (20) Mutvei, H. Calcif. Tissue Res. 1977, 24, 11. (21) Dauphin, Y. Zoology 2006, 109, 85. (22) Larson, C.; von Dreele, R. B. Los Alamos National Laboratory Report, No. LAUR 86-748; Los Alamos National Laboratory: Los Alamos, NM, 2004. (23) Toby, B. J. Appl. Crystallogr. 2001, 34, 210. (24) Zolotoyabko, E.; Quintana, J. P. J. Appl. Crystallogr. 2002, 35, 594. (25) Weiner, S.; Traub, W. Philos. Trans. R. Soc. London, Ser. B 1984, 304, 425. (26) Addadi, L.; Raz, S.; Weiner, S. AdV. Mater. 2003, 15, 959.
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