Biological Control on Calcite Crystallization by Polysaccharides


Sep 12, 2008 - We chose four units from the coccolith associated polysaccharide (CAP) that is known to promote formation of vicinal faces in Emiliania...
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Biological Control on Calcite Crystallization by Polysaccharides Mingjun Yang,*,†,‡ S. L. Svane Stipp,† and John Harding‡ NanoScience Centre, UniVersity of Copenhagen, UniVersitetsparken 5, DK-2100 Copenhagen, Denmark, and Department of Engineering Materials, UniVersity of Sheffield, Sheffield, U.K.

CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 11 4066–4074

ReceiVed May 15, 2008; ReVised Manuscript ReceiVed July 7, 2008

ABSTRACT: Polysaccharides control the growth of calcite in coccoliths by adsorbing preferentially onto particular surfaces of the calcite crystal. We chose four units from the coccolith associated polysaccharide (CAP) that is known to promote formation of vicinal faces in Emiliania huxleyi, namely, galactose, mannose, xylose, and rhamnose, and used molecular dynamics simulations to calculate the absorption of oligosaccharide units onto a number of calcite surfaces. The simulations show a wide range of adsorption energies, which depend on the combination of organic molecule and surface. Oligosaccharides on polar surfaces with surplus negative charge have the strongest adsorption, while those on polar surfaces with surplus positive charge have the weakest. Acute stepped vicinal surfaces have stronger adsorption than planar surfaces, while obtuse stepped surfaces have weaker adsorption than the planar surfaces. On the basis of these simulations, the behavior of two saccharides on the calcite {1 0 1j 4} surface was observed experimentally with atomic force microscopy (AFM) and shown to be consistent with the simulations. This helps explain why the polysaccharides involved in biomineralization have the chemical composition that they do and also suggests criteria for new molecules to control calcite crystal growth. Introduction The dramatic difference between calcite crystals grown in pure solution and those grown by biomineralization in nature has attracted much interest. Synthetic calcite crystals grown in the absence of additives form perfect rhombohedra with only the stable {1 0 1j 4} faces. On the other hand, calcite crystals grown in biological systems exhibit unusual crystal surfaces and, moreover, the physical shape of the crystal may be quite different from that predicted from a Wulff construction obtained from those surfaces (with the relevant surface or interfacial energies used in the calculation). For example, the unicellular algae, Emiliania huxleyi, forms a body cover of calcified platelets called coccoliths. These coccoliths consist of interlocking calcite single crystals, each of which has a complex shape.1 All coccoliths of a species have the same highly complicated and strictly defined structure and in different parts of the disk, different crystal morphologies exist. Complex organic molecules, coccolith-associated polysaccharides (CAPs), are generally believed to control the biomineralization process.2 The mechanisms that control biomineralization are not understood in detail. However, it has been generally agreed that the planes on which biomolecules are preferentially adsorbed become expressed as stable crystal surfaces because crystal growth is inhibited on these planes. Recently it was suggested that crystal shape is controlled by step-specific interactions between biomolecules and individual step edges on pre-existing crystal surfaces and changes in the elementary step shape generate a similarly modified bulk crystal shape.3 Therefore, the key factor in the biological control of crystallization is the local interaction of biomolecules with the crystal surfaces. The role of CAPs in the development of coccoliths has been studied for more than two decades. The CAP extracted from E. huxleyi has been shown to inhibit CaCO3 precipitation in in Vitro experiments. Atomic force microscopy (AFM) has been used to investigate the local interactions of CAPs with the calcite * To whom correspondence should be addressed. E-mail: [email protected] Phone: +45 35 32 01 56. Fax: +45 35 32 02 14. † University of Copenhagen. ‡ University of Sheffield.

Figure 1. Monosaccharide and trisaccharide units.

surface during dissolution, precipitation, and dynamic equilibrium. The AFM experiments demonstrate that CAPs interact preferentially with surface sites defined by acute, rather than obtuse, angles and it blocks acute sites during dissolution and growth.4 The polysaccharides from coccoliths have also been used to study the influence on the crystallization of calcium oxalate monohydrate crystals, and the results indicate that the inhibitory effect proceeds through a monolayer type of adsorption onto the crystal surface.5 The basic monosaccharide units that make up CAPs are mainly rhamnose/ribose, xylose, mannose, galactose, and others.6 Structures for these units are presented in Figure 1. Molecular simulation is being increasingly used to study the crystal form of calcite in contact with water and the growth or inhibition of the mineral. Two stepped {1 0 1j 4} surfaces of calcite (corresponding to the standard acute and obtuse steps) have been investigated by de Leeuw and colleagues,7 and the results indicate both that water can stabilize calcite surfaces and that dissolution of calcite tends to occur preferentially from the obtuse step, in agreement with experimental observations.8 The competitive adsorption among organic molecules with different functional groups on calcite surfaces has been studied with a combination of potential models and results suggest that some

10.1021/cg800508t CCC: $40.75  2008 American Chemical Society Published on Web 09/12/2008

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Figure 2. Calcite surfaces simulated.

organic molecules are able to block some specific surfaces and slow calcite growth significantly.9 Calculations of the interfacial energies of calcite interacting with arrays of organic molecules have shown that both local epitaxial matching and electrostatic interactions are important in controlling the orientation of calcite nucleation.10 In this study we used classical molecular dynamics (MD) simulations to obtain detailed information about the local interactions between calcite surfaces and four representatives of the CAP side chains using a newly developed set of force fields. Then we used AFM to test if there was a difference in behavior of the calcite {1 0 1j 4} cleavage face when exposed to the most and least adsorbing of the units. The objective was to understand the underlying molecular mechanism of biological control of crystallization, which may suggest routes to synthesize novel biomaterials. Modeling Methods and Procedures. We chose to represent the CAP side chains with linear homosaccharides of galactose, mannose, xylose, and rhamnose, with three units joined by a 1,4-linkage (as shown in Figure 1). Because the number of atoms and monomers used is small (about 60 atoms per molecule, corresponding to three monosaccharides), they are better described as oligosaccharides than polysaccharides. The term polysaccharide is usually reserved for polymers containing hundreds of units (and therefore thousands of atoms). Our simulations should therefore be construed as describing the local behavior of a small block of a full polysaccharide molecule. For convenience, we refer to our model molecules as polysaccharides. They were built with the AMBER 9.0 software.11 Calcite has a rhombohedral crystal structure,12 space groupR3jc, and a ) b ) 4.988 Å, c ) 17.061 Å, R ) β ) 90°, and γ ) 120°. We simulated the planar{1 0 1j 4}, {1 0 1j 0} surfaces; the acute stepped {1 0 1j 0} and {3 1 4j 8} surfaces; the obtuse

Figure 3. Starting configuration (the image was produced with VMD 1.8.616).

stepped {2 1 3j 4} and {3 1 4j 16} surfaces; and the polar {0 0 0 1} surfaces terminated with either CO32-or Ca2+ (Figure 2). All the surfaces were built with Materials Studio 4.2.13 The creation of a calcite surface from bulk calcite is specified by the orientation of the bulk cleavage defined by Miller indices and the displacement of the plane relative to the unit cell origin. At the calcite {0 0 0 1} surface, there are two specific displacements that lead to two distinct surfaces terminated with Ca2+ or CO32-. For the polar surfaces, we reduced the charge density of the terminating planes by omitting half of the rows of CO32- or Ca2+ at regular intervals to remove the macroscopic dipole.14 The thickness of the mineral layers that we modeled varied from 12 to 14 Å, for example, we used five layers for {1 0 1j 4} and four layers for{1 0 1j 0}. On the mineral surfaces, we put water and one polysaccharide molecule. The initial water

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Figure 4. Configuration of the adsorption energy calculation.

thickness is about 15 Å, and the initial density is about 0.9 g/cm3, as suggested by experiment.15 The initial distance between the polysaccharide and the surface is set to about 2.5 Å. If the distance is too large, it would take a long time for the polysaccharide to adsorb. On the other hand, if the distance is too short, the polysaccharide might adsorb to the surface very quickly, without freedom to assume its most energetically favorable position. Above all the molecules is a vacuum of about 100 Å to avoid interactions between the system and its images; a three-dimensional periodic boundary condition is applied in the simulations. During the simulations, calcite atoms in the middle layers were fixed, and only the atoms at the top and the bottom of the mineral surface were mobile so that the atoms on the top could alter their position while interacting with polysaccharide and water. Because water molecules might vaporize and move away to the bottom of the calcite layer (image), which changes the surface area between calcite surfaces and water, we also put another water layer on the bottom to keep the interface area constant and therefore avoid the potential energy changes caused by such additional adsorption. The starting configurations were generated with the help of the PACMOL code.17 This code packs molecules into a cubic box, avoiding atomic overlaps that would result in excessively large potential energy. MD simulations were carried out using the DL _ POLY 2.16 code18 with a set of force fields designed for use at bio-inorganic interfaces.19 Full details about the force fields between the organic molecule and the mineral are given in that reference. The interaction potentials used for CaCO3 were those derived by Pavese et al.20 for modeling a range of properties of calcite and aragonite crystals. The potentials for CAPs were obtained from the Glycam04 force field21 in AMBER 9.0, and the flexible TIP3P potential was used for water.22 The parameters for interactions between CAPs and water were obtained from AMBER. The adhesion energy in an aqueous environment between a calcite surface and a molecule can be defined as the difference between the energy of a crystal slab with an adsorbed polysaccharide with water on the top and the energy of a calcite crystal with water on top and a polysaccharide in water.23 Given this definition, in the second system, the polysaccharide should be

Figure 5. Total potential energy during polysaccharide adsorption.

placed far from the calcite surface to make sure it has no interaction with the surface, as shown in Figure 3. The advantage of this method for slab calculations is that the vacuum/water and vacuum/calcite interfaces manifestly cancel out when the difference between simulations E(2) - E(1) is taken. However, it is inconvenient, because large simulation cells are required. We therefore use the alternative setup shown in Figure 4. Here the difference E(2) - E(3) - (E(4) - E(5)) also shows the required cancelation when periodic boundary conditions are applied. The size of the box for (5) in Figure 4 may need to be varied somewhat to ensure that the number of water molecules in the simulations balances. However, it is desirable to ensure that all simulations have similar overall box sizes to make possible the cancelation of size-dependent terms. Another concern in this kind of simulation is ensuring that the polysaccharide has found a low energy configuration with respect to the surface, while maintaining stability over the whole system. It is not possible to guarantee this completely, but the following protocol gives reasonable results. In the simulations, for every system, first we gradually increased the temperature from 0 to 300 K by steps of 10 K. At each step, a 5 ps MD simulation was used to relax the system. After the temperature reached 300 K, another 2000 ps MD simulation was carried

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Figure 6. Gyration radius of a polysaccharide during adsorption. Table 1. Adsorption Energy between Polysaccharides and Calcite Surfaces, With Reference to Water (kJ/mole) surface {0 {1 {3 {1 {1 {2 {3 {0

0 2j 1 0 0 1 1 0

0 1 4j 1j 1j 3j 4j 0

1}, CO320} 8} 0} 4} 4} 16} 1}, Ca2+

galactose

mannose

xylose

rhamnose

average

-93.7 -91.5 -41.2 -50.8 -22.9 -11.1 2.0 -10.6

-40.7 -81.6 -43.7 -19.0 -29.6 28.9 15.7 21.9

-60.4 23.2 -73.4 -81.8 -60.6 0.6 1.0 12.5

-40.8 -51.6 -36.4 -31.7 -55.5 -45.2 -24.0 9.6

-58.9 -50.4 -48.7 -45.8 -42.1 -6.7 -1.3 8.4

out and the trajectory was recorded every picosecond (ps). Statistical data were collected every 0.1 ps, and the last 1000 ps of the simulation was used for calculating the average of potential energy. For example, if we check the total potential of the system and the gyration radius of the polysaccharide during the adsorption procedure, as shown in Figures 4, 5, and 6, we can see that initially, both the potential energy and the radius change quickly as the polysaccharide molecule approaches the surface. After about 800 ps, the energy and radius become stable with minor variation over time and we can conclude that adsorption is complete. Experimental Details Once the properties of the four oligosaccharides had been predicted, we tested the behavior of the two units that would be most, and least,

Figure 7. Polysaccharide (R-D-galactose) on a calcite surface (the CO32- terminated {0 0 0 1} surface); water molecules are included in the calculation but were made invisible in the figure; red lines represent hydrogen bonds (the image was produced with VMD 1.8.616).

Figure 8. Z-density of hydrogen atoms from the OH groups of a polysaccharide (R-D-galactose) adsorbed on the CO32- terminated {0 0 0 1} surface. likely to react with the {1 0 1j 4} cleavage terraces and steps of natural calcite crystals. We prepared fresh surfaces by the method described by Stipp and Hochella24 and exposed the samples to 10-2 and 10-3 M saccharide solutions for 5 min. Trisaccharides are not readily obtainable, so we used commercially available (reagent grade) monosaccharides. Solutions were prepared in MilliQ deionized water, immediately prior to use, to avoid growth of bacteria. When the samples were removed from the solution, the last adhering droplet was mechanically swept away with a quick jet of N2. This is a standard method shown to be effective to avoid precipitation from the remaining droplet. The calcite samples were examined in air, with contact mode AFM. We used a Digital Instruments Nanoscope Multimode IIIa with standard Si3N4 pyramidal cantilevers. Behavior of the solution-exposed samples was compared to surfaces exposed only to MilliQ water and air.

Results and Discussion The adsorption energies for the various polysaccharides and surfaces are presented in Table 1. They have been determined relative to water’s ability to adsorb. The energies vary considerably and some units fail completely to adsorb (positive energies). The pattern varies from one surface to another. For example, galactose adsorbs very strongly on the {1 2j 1 0} surface, whereas xylose does not adsorb at all. On the other hand, all the polysaccharides adsorb strongly to the polar {0 0 0 1} surface, provided that there is a carbonate termination. Thus, it is clearly possible to design a monosaccharide sequence that favors adsorption onto one calcite surface rather than another and so inhibit the growth of some surfaces. Some immediate comments can be made. The marked difference in adsorption strength between the Ca2+ and CO32terminations of the {0 0 0 1} polar surface leaves little doubt that all saccharides bind more strongly to carbonate anions. In all cases, it is necessary to recall that adsorption is reported with respect to water molecules as a reference. We do not consider the absolute ability of saccharide OH groups to bind to Ca2+ or CO32-, but their relative efficiency in comparison with water, that is, how well can they replace the surface bound water in an aqueous system. Here the point is that the OH groups of the polysaccharides are not much more efficient at completing for the Ca2+ coordination shell than water; calcium is satisfied whether the coordinating O comes from an OH in the polysaccharide, or from water (H2O). The saccharide OH groups are, however, better at binding to the carbonate ion than water (Figure 7).

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Figure 9. Z-density of hydrogen atoms from OH groups of polysaccharides on calcite surfaces.

We have also calculated the density of the saccharide as a function of distance from the surface for each case. The Z-density plot of the hydrogen atoms (Figure 8) from the OH groups in a polysaccharide demonstrates that hydrogen bonds link it to the calcite surface.

Because the main interactions between polysaccharide and the calcite surface are through hydrogen bonds, the intensity of the Z-density of the hydrogen atoms can show the strength of interaction, except on surfaces with large steps. The typical length of a hydrogen bond in water is 1.97 Å, so we have only

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Table 2. Ratio of OH Bonding to the Surface (Forming Hydrogen Bond) to Total Number of OH Groups surface {0 {1 {3 {1 {1 {2 {3 {0

0 2j 1 0 0 1 1 0

0 1 4j 1j 1j 3j 4j 0

1}, CO320} 8} 0} 4} 4} 16} 1}, Ca2+

galactose

mannose

xylose

rhamnose

average

0.25 0.15 0.21 0.15 0.03 0.00 0.04 0.05

0.20 0.20 0.05 0.05 0.00 0.07 0.21 0.05

0.36 0.04 0.36 0.31 0.14 0.00 0.00 0.16

0.21 0.00 0.00 0.00 0.00 0.28 0.16 0.21

0.26 0.10 0.16 0.13 0.04 0.09 0.10 0.12

plotted Z-density near the calcite surface. Plots for the four polysaccharides on all surfaces are shown in Figure 9. Adsorption is not stable for all polysaccharides. For example, galactose is not adsorbed on the {2 1 3j 4} face, shown as a lack of Z-density in the surface region (Figure 9f). In some cases (such as Figure 9c-e,h), the presence of OH within the surface indicates formation of hydrogen bonds between the polysaccharide and calcite surface. We can further sum the Z-density along the z-axis, until z ) 2.5 Å, assuming that beyond this distance, hydrogen atoms from OH cannot form hydrogen bonds with the surface. The ability of a polysaccharide to form hydrogen bonds represents its tendency to cover certain surfaces. From Table 2, we can see that all are able to cover the polar surface {0 0 0 1} terminated with CO32-. Some polysaccharides display extremely different adsorption behavior, depending on the surface. For example, xylose is adsorbed on surfaces {1 2j 1 0},{3 1 4j 8}, {1 0 1j 0}, and {1 0 1j 4}, but not on the faces dominated by obtuse angles, {2 1 3j 4} and {3 1 4j 8}, while in comparison, rhamnose is only adsorbed on these {2 1 3j 4} and {3 1 4j 16} faces but not the others. All surfaces show clear layering effects of water caused by the presence of the calcite surface. This is affected somewhat by the presence of the polysaccharide, but there is no obvious difference between them, which can be seen from the plot of Z-density of water with/without these polysaccharides (Figure 10). However, we should recall the earlier point: what matters is not the absolute behavior of the polysaccharide, but its behavior relative to an interface where only water is adsorbed. We therefore use a difference-density Z-density plot to represent the Z-density difference with/without these polysaccharides, where the difference, ∆Z, is defined as with without ∆Z ) ∫∫(FOH (z) - FOH (z)) dSz

(1)

where FOH (z) represents the density of OH (whether from the polysaccharide or the water) at z in the presence of the polysaccharide and Fwithout (z), the density of OH when the polysacOH with

Figure 10. Z-density of water on calcite {1 0 1j 4} surface.

charide is absent. The integral is taken over a plane, Sz, at a distance, z, from the nominal surface of the mineral. Thus, ∆Z measures the effect of the presence of the polysaccharide on the density of OH as a function of distance away from the calcite surface. ∆Z is negative where the polysaccharide rings exclude water. In Figure 11, the basic shape of the curve indicates the presence of polysaccharide, and in some cases it enhances OH density close to the calcite surface. A negative ∆Z on a calcite surface means that the Z-density of OH is decreased by introducing a polysaccharide molecule, which potentially decreases the number of hydrogen bonds with the calcite surface. We focus on the change of Z-density in the surface zone. The OH density near the surface is influenced by the presence of polysaccharides and it is in this zone that hydrogen bonds form between water and the surface. For the {1 0 1j 0} and {1 0 1j 4} surfaces, introducing polysaccharides decreases the surface OH density for all except for R-L-rhamnose, which increases the density. For all the stepped surfaces, including the acute, {3 1 4j 8}, {3 1 4j 16}, and the obtuse, {1 2j 1 0} and {2 1 3j 4} faces, water and polysaccharides may sit on steps and on the terraces below, which blurs the surface boundary. For the {3 1 4j 8} and {3 1 4j 16} vicinal surfaces, which have large steps making the surface rough, there are clear differences in behavior for the various polysaccharides and these differences correspond to the strength of binding. The density of OH on the {3 1 4j 16} surface (array of obtuse steps) decreases in the presence of all of the polysaccharides. However, on the {3 1 4j 8} face (array of acute steps), mannose and rhamnose increase OH density in the step hollows, 1-2 Å below the surface. For {1 2j 1 0} and {2 1 3j 4}, which have small steps, polysaccharide generally decreases OH surface density, especially in the zone in the first 1-2 Å. On {1 2j 1 0}, xylose dramatically decreases OH density resulting in the weakest adsorption (positive adsorption energy, Table 1), while on {2 1 3j 4}, R-L-rhamnose increases OH density and it adsorbs most strongly of all the polysaccharides (adsorption energy ) -45.2 KJ/mol, Table 1). The behavior of the {0 0 0 1} surface is quite different because of its polar properties. For the {0 0 0 1} surface terminated with CO32- anions, adsorption energies are much higher than for other surfaces, mostly because of the strong attractive electrostatic interaction between CO32+ and the OH of both water and polysaccharides. For the {0 0 0 1} surface terminated by Ca2+, the adsorption energies are very much lower because there is insufficient CO32- to form a hydrogen bond with OH. There is also a repulsive electrostatic interaction between Ca2+ and OH. We tested the modeling results by experiment for two of the 32 saccharide/calcite surface systems (4 saccharides on 8 surfaces). Calcite can be cleaved easily to produce fresh {1 0 1j 4} surfaces. Although other faces are formed during crystal growth and some natural crystals have smooth, well-developed surfaces, these are not suitable for AFM experiments and they cannot be produced fresh, by cleavage. Therefore, we examined monosaccharide behavior on the rhombic {1 0 1j 4} face. We chose to examine the behavior of xylose because both its adsorption energy and its ratio of OH bonding were highest of the four saccharides, and we chose mannose because its OH bonding ratio is 0 and its adsorption energy is nearly lowest. As an indicator for adsorption, we observed differences in dissolution behavior, compared to pure calcite in pure water, and we took advantage of the tendency of calcite to recrystallize in the thin layer of water that adsorbs from air.15,25

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Figure 11. Accumulated ∆Z-density plots of OHO for the calcite surfaces studied.

Figure 12 shows a set of AFM images of calcite collected in air, fresh (a), after exposure to water (b), and dry after exposure to the humidity in air for minutes to hours (c,d). These serve as a base for comparison for samples exposed to saccharide solutions. The fresh surface immediately following cleavage has flat, smooth terraces, separated by 3 Å high steps, the height of

one molecular layer on this face of calcite (Figure 12a). The sharp-ended triangular terraces are typical of surfaces produced by cleavage using a force parallel to the crystal plane. Exposure to deionized water results in dissolution, forming rhombic etch pits (Figure 12b) that reflect the underlying crystal structure. On samples exposed to air, humidity forms a layer of water

Calcite Crystallization by Polysaccharides

Figure 12. AFM images for {1 0 1j 4} calcite surfaces in the absence of polysaccharide (a) freshly cleaved; (b) after exposure to MilliQ water; (c) after exposure to air for a few minutes; and (d) after exposure to air for more than an hour.

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Figure 13 shows the AFM images collected from surfaces of calcite exposed to monosaccharide solutions. Etch pits form and grow, but the rate of growth is slower, suggesting saccharide binding to the calcite, inhibiting the activity of water. Etch pit corners become rounded and after longer exposure, they become distorted with irregular edges (compare Figure 13a with 12b). Such distortion of the rhombic form is associated with adsorption of contaminants on the step edges, inhibiting dissolution.26,27 On samples imaged in air after removal of the saccharide solution (Figure 13b-d), recrystallization proceeds, but the rate is slower and the patches have a different form than for the pure system. After exposure to a solution of 10-2 M xylose, spots appear at step edges (arrows, Figure 13b). It was not possible to prove if these spots were monosaccharide clusters or recrystallized material, but their lack of growth with time and their ability to foul the tip are strong evidence that they were clusters of polysaccharide. The elongated patch in the middle of Figure 13b is undoubtedly recrystallized material. It grew slowly and extended along the intersection of the {1 0 1j 0} face with the {1 0 1j 4} surface. This is consistent with the calculated strong adsorption of xylose on the {1 0 1j 0} plane. In solutions of 10-3 M xylose, clusters are not observed but recrystallized patches are still elongated. In contrast, samples exposed to 10-3 M mannose have regularly formed etch pits. Recrystallized patches form but the presence of saccharide makes them grow slowly, but their form is not different from that of patches observed on clean samples. The difference in behavior is consistent with the calculated differences in adsorption energy and OH interaction for the two saccharides on the {1 0 1j 4} surface. Conclusion

Figure 13. AFM images for calcite cleavage surfaces after exposure to (a) 10-3 M xylose solution; (b) 10-2 M xylose, then imaged in air; (c) 10-3 M xylose, then imaged in air; and (d) 10-3 M mannose, imaged in air.

about 15 Å thick.15 This water reaches equilibrium with the surface and begins to dissolve the calcite left as small particles after cleavage. The solution reaches saturation and precipitates calcite as islands on the surface in a recrystallization process known as Ostwald ripening. Sometimes recrystallized material is added at step edges, in ordered, single-monolayer growth, but more often the new material forms as rounded or dendritic islands (Figure 12c,d and elsewhere25,15). Eventually the islands grow and coalesce so the entire surface is covered.

We have performed a series of simulations on four oligosaccharides, chosen to represent the active side chains of CAPs, known to control calcite growth of E. huxleyi coccoliths. The purpose was to investigate the efficiency of the different monosaccharide units in binding to calcite surfaces and thus their effectiveness in inhibiting crystal growth in particular directions. The strength of the inhibition effect obviously depends on the strength of the binding (and hence surfaces that are weakly bound might be expected to grow out). The majority of saccharides bind more strongly to the acute stepped {1 2j 1 0} and {3 1 4j 8} surfaces than the planar {1 0 1j 0} and {1 0 1j 4} surfaces while the majority of saccharides bind more weakly to the obtuse stepped {3 1 4j 8} and {2 1 3j 4} surfaces. Binding to the polar surface depends considerably on the termination: strong for CO32-, weak for Ca2+. This suggests why the coccoliths have the shape that they do, with vicinal faces oriented at lower angles to the c-axis. The extreme example is the prism face {1 2j 1 0}. The results also suggest that considerable variation in crystal morphology is possible, even with only a few polysaccharide side chains, since it is clear that the effects of inhibition are considerably different, depending on the saccharide units chosen to make up the polymer. Apart from the characteristics of polysaccharides and calcite surfaces, the stereochemical match between these polysaccharides and calcite surfaces is also very important as pointed by De Yoreo et al.28 From the investigation of Z-density of OH groups on the surfaces, we can also see the influence of polysaccharides on the hydrogen bond formed between solution and surfaces. In some case, the interactions between polysaccharides and surfaces can block the water and disrupt its ordering. This suggests that polysaccharides may block other cation and anion bonding, such as calcium and carbonate, and

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thereby inhibit growth on those surfaces. The experiments have confirmed the selective adsorption of saccharides on the calcite cleavage surface. Both the simulations and experiments indicate one saccharide is more effective than another as an inhibitor during calcite crystallization, and this mechanism also explain the biological control in biomineralization such as in formation of coccoliths. Acknowledgment. We acknowledge funding from EPSRC under Grant GR/S80103/01 and Mærsk Olie og Gas. We also thank Colin L. Freeman (University of Sheffield, U.K.) and David Cooke (University of Huddersfield, U.K.) for the potentials, Dorothy M. Duffy (University College London, U.K.) for advice about simulations and access to the computation facility at the Rutherford-Appleton Laboratory (Mott2) under EPSRC Grant GR/S84415 and Karen Henriksen for advice about the biogeochemical system during the project design stage.

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