Micron 39 (2008) 380–386 www.elsevier.com/locate/micron
Nano-structured biogenic calcite: A thermal and chemical approach to folia in oyster shell Seung Woo Lee a, Young Moon Kim b, Ryun Hwa Kim b, Cheong Song Choi b,* a
Materials & Minerals Processing Division, Korea Institute of Geoscience & Mineral Resources, Daejeon 305-350, Republic of Korea b Department of Chemical & Biomolecular Engineering, Sogang University, Seoul 121-742, Republic of Korea Received 5 February 2007; received in revised form 14 March 2007; accepted 14 March 2007
Abstract The thin sheets of calcite in oyster shell in Crassostrea gigas are termed folia and comprise much of the oyster shell. The folia are covered by a layer of discrete globules that has been proposed to consist of aggregations of an organic matrix and minerals. A continuous organic framework divides each tablet into nanograins. Their shape is globular with a mean extension from 30 to 40 nm. Chemical and thermal treatments to correlate between the organic matrix and the minerals are considered using spectrometers, thermal analyzers, and electron microscopes. After treatment, the nanograins of the foliar and organic matrix are clearly identified. The organic matrix plays a key role in the thermal stability and material properties of this biological composite. From analysis of the FT-IR results, it is identified that the organic matrix in folia is composed of proteins and polysaccharides. # 2007 Elsevier Ltd. All rights reserved. Keywords: Nanograin; Organic Matrix; Folia; Calcite; Oyster shell; Biomineralization
1. Introduction Calcium carbonate is the most widespread mineral in mollusk shell as well as invertebrate calcified tissues. The material properties (strength, hardness and etc.) and thermal properties (decomposition temperature and enthalpy) of this shell depend on the properties of the mineral component and the chemical and structural component of their organic matrix. In general, the organic matrix in the shell has been suggested to play critical role on material properties as well as polymorphism (Miyamoto et al., 1996; Sud et al., 2001; Li and Nardi, 2004). While the correlation between the organic matrix and the mineral of nacre was well investigated (Sellinger et al., 1998; Levi-Kalisman et al., 2001; Checa and Rodriguez-Navarro, 2005), little is known about the correlation of oyster shell, especially folia. In the case of adult oyster shell, which is mainly composed of calcite, the mineral even within a few millimeters of the forming edge is formed into layers of thin sheets, termed folia (Carriker and Palmer, 1979; Carriker,
* Corresponding author. E-mail address: [email protected]
(C.S. Choi). 0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2007.03.006
1980); they therefore need to have favorable mechanical properties such as tensile strength and elastic modulus. Considering the amounts of the organic matrix in the boundary surface and the value of the elastic modulus of the folia, the folia in the oyster shell of Crassostrea gigas have unique characteristics to enable any external impact to be minimized, ensuring the survival of the living tissues inside. The characteristic structures of the adult shell of the oyster, C. gigas, are shown as follows: an interior view of the left valve (Fig. 1a), cut surface of adductor muscle scar (Fig. 1b) and the schematic of biomineral structure and organic-rich area at the interface between the myostracum and folia (Fig. 1c). The myostracal prisms are 5–30 mm in length, running from the umbo direction to the adductor muscle (Fig. 1b), with a prismatic form. On the other hand, the folia is more than 100 mm thick, with each single layer being 200–300 nm in size (Fig. 1c). Each lath is typically about 1–3 mm wide and about 200–300 nm thick. Comparing the folia of oyster with nacre of abalone, the folia of oyster are very interesting layer. Both layers are in sharp contrast and similarity: the folia are composed of calcite form as opposed to aragonite in nacre; the morphological structure of both layers consists of laminated layers. The folia form the bulk of the shell. Each foliar shell is subdivided into units termed laths, with each sheet just one lath
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2. Experimental methods 2.1. Sample preparations Shells of C. gigas (Namhae in Korea) were freshly collected, soaked in 5% NaOH, lightly scrubbed, and dried at room temperature. The shells were then fractured with a hammer. The folia were separated from pigmented and stored dry in a vial. To obtain pure folia, an electric mill, cutting knife was used. To identify the structure and shape of the separated folia, optical microscope (VL-11S) were used. 2.2. Thermal treatments of decalcified folia The folia in a crucible was placed in TGA (Thermogravimetric Analyzer) furnace at the experimental temperatures and heated in nitrogen from 200 to 900 8C for 13 h. 2.3. Atomic force microscopy used by phase contrast
Fig. 1. Schematic illustration showing the structure of oyster shell (adapted from Lee and Choi, 2007): (a) schematic of inner surface of left valve (C. gigas); (b) schematic of cross-sectional surface of cutting adductor muscle scar; (c) an enlargement indicating thickness dimensions of the myostracal prism, organic rich area, and folia. The size of each layer is exaggerated for clarity. Various terms are in common use in description of the bivalve shell (Galtsoff, 1964; Carriker and Palmer, 1979; Waller, 1981). The samples including myostracum and folia under adductor muscle scar are only used in this study.
unit of up to only a few hundred nanometers in thickness. Calcite could be more advantageous than aragonite because calcite is secreted more economically (i.e., calcite fills a larger volume per mole than does aragonite) (Stenzel, 1964) and exhibits a growth rate at equal thermodynamic driving force that is more than three times higher than that of aragonite (Westin and Rasmuson, 2005). Moreover, the most oyster shell routinely contains less than a few percents and often less than 1% by weight of the shell as organic matrix (Sikes et al., 2000). The objective of this research is to understand how the folia are put together, particularly the relationships between the mineral and the organic components. Secondly, it is desired to understand how the effect of the organic matrix affects the mechanical characteristics in folia. Lastly, in terms of synthetic material synthesis, this work points to possibilities for new routes to hybrid inorganic–organic material creation.
The microscope used was a Multimode SPM connected to an electronic controller, the Nanoscope IIIaTM produced by Digital Instruments (USA). The spatial and vertical resolutions are less than 1 nm and the field is between 100 nm and 100 mm. The images recorded in this work were taken at high resolution (512 512 pixels) by using an intermittent-contact mode (called Tapping ModeTM) coupled with phase detection imaging (PDI). The tapping-mode makes it possible to minimize the interactions between the probe and the surface during acquisition and considerably improves the resolution compared to the contact mode (Aime´ et al., 2001). Coupled with PDI, this mode, in addition to the topographic images, provides a map characterizing the variations of the mechanical properties of the scanned surface (phase contrast) (Magonov et al., 1997). The probe is in Si with a round tip of between 5 and 10 nm. The work frequency, the stiffness and the amplitude of cantilever were 270 kHz, 42 N m 1 and 25 nm, respectively. The scanning rate was 1.0 mm s 1. Data were collected in air and at room temperature after polishing parallel to the nacre surface. 2.4. Nanoindentation Nanoindentation tests were performed using a Nanoindenter XP Testing System (MTS) in conjunction with the CSM (continuous stiffness measurement). The Hysitron nanoindenter monitors and records the dynamic load and displacement of the indenter, a diamond Berkovich three-sided pyramid, with a force resolution of 50 nN and displacement resolution of 0.1 nm. Hardness and elastic modulus properties were calculated from the recorded load–displacement curves. 2.5. FE-SEM (field emission scanning electron microscope) The microstructures with osmium coater (WO-001, MEIWA) were examined by a FE-SEM (JEOL JSM-6700 F).
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2.6. TG (thermogravimetric)-dTG (differential thermogravimetry) TG-dTG was performed using a LabsysTM instrument equipped with microbalance. 2.7. FT-IR (Fourier transform infrared spectroscopy) The folia (30 mg) was placed in an aluminum pan and measured against a reference empty pan. The heating was performed in a nitrogen atmosphere (50 ml min 1), with a heating rate of 10 8C min 1.The folia (approximately 1 mg) was mixed with about 10 mg of anhydrous KBr. The mixture was pressed into a 8 mm diameter pellet. The analysis was performed at 2 cm 1 resolution using a FTS-3000 (Bio-Rad). 2.8. Results In Tapping ModeTM coupled with PDI, two types of determination are simultaneously obtained: the Phase Contrast Map (Fig. 2) enables the detection of variations in composition, adhesion, friction, viscoelasticity or other properties, including the detection of the different composite components (Rousseau et al., 2005). Technically, the image (Fig. 2) is obtained by measuring the phase difference between the excitation signal and the cantilever response. This information can be related to the dissipation of energy during the interaction of the tip on the surface. This data is related to local variations of the material. The resulting phase contrasts are often difficult to interpret because of the complex interactions of chemical and physical effects (Cleveland et al., 1998; Tamayo and Garcia, 1998). Here, the interactions can be related to the drastic difference of the elastic properties of the intracrystalline organic matrix regarding those of the mineral phase. The Si tip of the AFM is
Fig. 2. AFM images of the same polished surface of folia in tapping-mode. Picture in phase contrast showing the foam-like structure of the intracrystalline organic matrix.
chemically non-reactive: chemical interactions with the sample are more than likely negligible with respect to the contrast expected with the elastic modulus variations between mineral and organic phases. It has long been demonstrated that an intracyrtalline matrix is present within the tablets (Sikes et al., 1998; Beniash et al., 1999) but its relationship with the mineral has never been shown at the micrometer scale. According to observations of a section of fractured folia by FE-SEM (Fig. 3) after thermal treatment (400 and 450 8C), pores of a fixed size that could be seen within the surface of the folia (black arrow in Fig. 3b) between intracrystallines and nanograins in the globular form were found within the lath, with the laths in the rod form which constituted folia disappearing within the interface as the temperature rose to 450 8C (Fig. 3d). The difference of height between the interface and the surface of the laths is identified (Fig. 3c and d). The FT-IR spectra show the differences of organic matrix of untreated and decalcified folia with EDTA (Fig. 4). All of them are verified by numerous bands from 4000 to 500 cm 1. The OH and/or NH stretching modes are found in the region 3000– 3500 cm 1, while the C–H stretching modes are assigned to the 2800–3000 cm 1 region. The carbonate ions in the mineral are demonstrated by the internal vibration modes of the CO32 ions, 713 cm 1, 700 (y4)–864 cm 1, 844 (y2)–1090 (y1) cm 1 and 1490 (y3) cm 1. The strong band detected at 1792 cm 1 could also be attributed to the C O groups of the carbonated ions. The single peak of y4 is characteristic of calcite structure. The IR band at 1660 cm 1 is attributed to the amide I (C–O bond) and/or amide II (C–N bond) groups of the organic matrix proteins. Moreover, the IR band at 1158 cm 1 is attributed to the C–C bond. Comparing the untreated folia with calcite, a OH and/or NH stretching mode has been detected in the region 3000–3500 cm 1, and IR bands at 1660 cm 1, 1158 cm 1 has been assigned to C O stretching (amide I), C–C stretching groups. Moreover, the rapid decreases of EDTA decalcified folia layer at 1660 cm 1, 1158 cm 1 has been verified. To deeply understand the correlation between organic matrix and folia, the folia definitely by chemical treatment was analyzed. Likewise, the characteristic spectra (3450 cm 1 O–H or N–H stretching, 1660 cm 1 amide I, 1158 cm 1 C–C stretching) of organic matrix in folia used by FT-IR show that the shell of C. gigas is not a simple combination of minerals, but a complex of inorganic and organic matrices. Especially, the decreases of C O stretching (amide I) and the C–C stretching by chemical and thermal treatment show that the bands of the C O stretching and the C–C stretching could be existing on the surface or interface area in folia as a soluble protein (Fig. 4c). The results from TGA and dTG show the loss of weight and decomposition temperature of the folia (Fig. 5). There is a minute weight loss (about 2 wt%) of the untreated folia layer around 600 8C (Fig. 5a, TG line). The decomposition temperatures of untreated folia and thermal treated folia at 450 8C are 829.9 and 851.2 8C. The untreated folia (Fig. 5a) is followed by a major weight loss (44 wt%) near to 850 8C, while the thermal treated folia (Fig. 5b) at 450 8C over 13 h is followed by major weight loss (42 wt%) near to 900 8C. Moreover, the enthalpy of untreated folia is 800 mV s/mg,
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Fig. 3. Field emission-scanning electron microscope (FE-SEM) shows that structures of the nanograins in thermal treated foliated laths. The sample was pyrolyzed in TG furnace at 400 8C (a and b) and 450 8C (c and d) for 3 h (scale bar: 1 mm (a) and (c), 100 nm (b) and (d)). (b) and (d) Are enlargement of inset in (a) and (c), respectively. The white arrow in (b) and (d) indicates the interface between foliated laths.
while the enthalpy of thermal treated folia at 400 8C for 13 h is 1100 mV s/mg. Wang et al. (1997) reported that the microhardness of sea urchin teeth composed of calcitic fibers ranges from a low value of 140 kg mm 2 to a high value of 360 kg mm 2. They explain that the reason for the higher values of microhardness in urchin
Fig. 4. FT-IR spectra of folia: (a) chemical treated folia by 0.1 M EDTA for 3 h, (b) distilled water for 3 h, and (c) thermal treated folia at 450 8C for 3 h. (i) –OH or/and –NH bond, (ii) amide I, and (iii) C–C bond.
teeth is due to magnesium occlusion. The occlusion of magnesium into the calcite crystal lattice causes lattice distortion, which in turn increases the sliding resistance of dislocations and the deformation resistance of the crystals. However, these urchin teeth show a high level of microhardness but with a large variation in hardness (average hardness: 220 kg mm 2) that changes depending upon the region and the magnesium concentration. By comparison of the hardness of folia (Fig. 6a and c) with the hardness of urchin teeth, the characteristics of folia can be identified as follows: uniformity in consisting only of calcite (Fig. 4); a consistency of hardness according to location (Fig. 6c, open circles); high value of hardness (3.2 GPa is the same as 325 kgf mm 2). To identify the role of the organic matrix on the mechanical properties in folia, a section of folia was analyzed for physical properties using a nanoindenter before and after thermal treatment (Fig. 6). As can be seen in the figure, folia which went through thermal treatment showed a decrease in hardness (by 27% compared with folia) and elastic modulus (by 21%). It was found that folia which went through no thermal treatment were uniform in the value of hardness and elastic modulus by position, while those which went through thermal treatment showed relatively significant variations in the value of hardness and elastic modulus by position.
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Fig. 5. TGA and dTG curves of (a) untreated folia and (b) thermal treated folia at 450 8C for 3 h before thermal analysis.
3. Discussion The results from phase contrast AFM (Fig. 2) show mineral nanograins surrounded by an organic phase, owing to the strong elastic modulus contrast between proteins and calcite. Fig. 2 is another phase-contrast image recorded on a narrower field of 200 nm 200 nm. The organic matrix is organized in the form of a ‘foam’ with very thin walls and closed cells. The mineral nanograins of the folia are thus encapsulated inside the organic framework. Post-processing treatment was applied to the
different images (grain analysis using the software EyeViewAnalyzer). This makes it possible to determine the morphology (size and shape) of the nanocrystals. A mean nanograin size of 33 (16) nm was found. Finally, the AFM images (Figs. 1b and 2) clearly show that the organic framework is continuous within the tablet; there is no evidence that the mineral is also continuous. Comparing the nanograins of folia (Fig. 2a, b, and d) to previous researchers’ results on the shell formation of molluscs (Sikes et al., 1998; Rousseau et al., 2005), it is confirmed that
Fig. 6. Results from material testing and analysis performed on polished shell samples. Hardness and elastic modulus values obtained from nanoindentations indicate uniform composition and nano-structure at (a) untreated folia and (b) thermal treated (450 8C, 3 h) folia. The measured hardness of (c) the untreated folia and (d) thermal treated folia was 3.29 0.1 GPa (Fig. 5c open circle), 2.39 0.2 GPa (Fig. 5d open circle) and the elastic modulus was 73 3.3 GPa (Fig. 5c full circle), 58 3.2 GPa (Fig. 5d full circle), respectively.
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morphologically there is the same type of globules in different species (Pinctada maxima) as well as in the same species (Crassostrea virginica). Especially, in disregarding the differences of polymorphism in the shells (the nacre of P. maxima, aragonite; the folia of C. gigas, calcite), we can analogize the similarity of the shell formation mechanism at the point of the physiology between the folia of oyster and nacre. Generally, it is known that most molluscs in the larval stage have the same polymorphism and structure (Stenzel, 1964). Recent results (Mount et al., 2004) indicate that mineralization in adult oyster shells is mediated by granulocytic hemocytes containing calcium carbonate. Mount et al. proposed that hemocytes, after a transeptithelial migration through the mantle, are a source of mineral, which is deposited as very small calcite crystals on the shell matrix, and is subsequently remodeled into the shell. Layers as calcite within the adult oyster shell can be divided into prismatic layers, folia, and chalky layers by morphology. The outer prismatic layer initiates with spheruliths near the base of a periostracum, the spheruliths grow inwards and extend beyond the inner boundary of the periostracum (Checa, 2000). In the case of the chalky layer, it has been reported as an indication of shell disease (Carriker, 1996). It is therefore assumed that hemocyte (Mount et al., 2004) secreted from within an epithelial cell can more strongly be correlated with the process of forming folia than any other layer composed of calcite. Comparing the surface image of folia after chemical treatment (Fig. 2) and thermal treatment (Fig. 3), it can be concluded that the basic size of the nanograins composed of folia is 30–50 nm. Balmain et al. (1999) reported that the organic matrix of nacre is destroyed around 500 8C, the same temperature as the calcite to CaO transformation, revealing the great thermal stability of the organic matrix and the organic–mineral bonding. In the case of folia, the destruction of organic matrices (amide I, –OH and/or NH, C–H, and C–C bonds) is identified at a similar temperature compared to that of nacre (Fig. 4), but the temperature of the calcite to CaO transformation is verified at above 600 8C (data not shown). Thus, we can estimate the difference in the correlation between organics and biominerals of oyster shell and nacre. It is of course possible to say that the formation of folia comes from the combined functions of organic matrixes forming interfaces for nanograins, those forming a boundary layer for folia, and those forming templates. After thermal treatment, the results of the FE-SEM analysis (Fig. 3) and FT-IR analysis (Fig. 4) enable us to recall an important fact: organic matrixes with a relatively low thermal stability rather than those with a high thermal stability (e.g. insoluble proteins or polysaccharides) are widely distributed on the surface of folia and on the interface for folia rods. Sikes et al. (1994, 1998) reported that the specific lowering of the activation energy for nucleation in organic matrixmediated biomineralization is thought to involve a number of different types of interfacial interactions between ions of the mineral phase and functional groups on the macromolecular surface. As mentioned earlier, one indicator of the organic matrix in folia is the band at 2800–3000 cm 1, characteristic of the C–H bond. This band is modified as the temperature
increases, but is still present at 450 8C (Fig. 4). Compared to amide I, a characteristic in the protein spectrum, it can be inferred that C–H bonds are related to polysaccharides, templates of the organic matrix, rather than soluble proteins. Moreover, the mechanical properties of folia, organic– inorganic biocomposites, were significantly affected by the loss (less than 3 wt%; Fig. 5a, TG data) of the organic matrix by thermal treatment at 450 8C (Fig. 6). Generally, CaCO3 decomposition actively occurs at a temperature of around 850 8C; it is reported that, for limestone containing SiO2, Fe2O3 and Al2O3, the temperature for CaCO3 decomposition decreases to approximately 810 8C during the process of firing raw materials (Wilburn et al., 1991). This means that the decomposition temperature is affected by ingredients other than calcium carbonate; the results from the thermal analysis (Fig. 5) show that organic matrixes contained in a shell may also affect the decomposition temperature of calcium carbonate. Furthermore, the result of the TGA-DTG analysis (Fig. 5) shows that the organic matrix in folia also accompanies changes of decomposition temperature and enthalpy. What is more important is that such organisms exert great effects on the physical properties and are of great bulk, although they account for an extremely low percentage (less than 3 wt%) of the whole. The spectrometry results (Fig. 4) of the folia show that the organic matrix in folia consists of proteins and polysaccharides, and the thermal behavior (Figs. 5 and 6c and d) also shows that the organic matrix incorporated in folia acts not only as an important factor in the characteristics of mechanical properties, but also as an important factor that determines thermal stability. 4. Conclusions The results of this study suggest that it should be possible to exclude calcium carbonate in a globular shape and nanosize by letting a shell of bivalvia go through low-temperature thermal treatment and chemical treatment. Finally, the following conclusions can be drawn. First of all, the folia of the giant pacific oyster are composed of single crystals of calcium carbonate in 30–50 mm globular particles. Nanograins of the folia could consist of stacks and the spaces in the grains are filled with organic matrices with thermal stability. Secondly, the nanograins comprise folia in oyster shell; the biocrystal does diffract as a single crystal but is made up of a continuous organic matrix (intracrystalline organic matrix), which breaks the mineral up into coherent nanograins (45 nm mean size, flat-on) which share the same crystallographic orientation. Thirdly, the nanograins surrounded by organic matrixes in folia may have great influence on the mechanical and material properties as well as on the thermal stability of folia. Finally, this study provides interesting information to show how the nanograins first grow with a spherical shape and then turn into rod-like folia when contacting each other. The existence of nanograins encapsulated in an organic matrix raises the hypothesis of an aggregation-like control for the extension of the nanograins by the organic template and not directly at the atomic level. It is expected to exploit this information to synthesize other nano-structured materials.
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