Controlling Biomineralization: The Effect of Solution Composition on

Mar 30, 2009 - Current address: Nano-Science Center, Department of Chemistry, University of Copenhagen; e-mail: [email protected]. ... that regulate cr...
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Controlling Biomineralization: The Effect of Solution Composition on Coccolith Polysaccharide Functionality K. Henriksen* and S. L. S. Stipp# Geological Institute, UniVersity of Copenhagen, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark

CRYSTAL GROWTH & DESIGN 2009 VOL. 9, NO. 5 2088–2097

ReceiVed April 25, 2008; ReVised Manuscript ReceiVed December 20, 2008

ABSTRACT: The calcite shields produced by unicellular marine algae demonstrate the remarkable crystal control that organisms can achieve through biomineralization. Emiliania huxleyi produces complex polysaccharides (“coccolith associated polysaccharides”, CAP) that regulate crystal morphology by preferentially attaching to calcite acute step edges, thus promoting growth of the specific crystal faces required for design of that species of coccolith. However, to control crystal growth, the alga must be able to control CAP behavior at the local scale, so its functionality can be switched on and off. Here, we show that the functionality of CAP from E. huxleyi depends directly on solution composition. We used atomic force microscopy (AFM) to investigate the behavior of calcite surfaces under varying pH, in the presence of CAP and cations chosen to test the role of ionic potential, that is, charge per unit radius (K+, Na+, Sr2+, Ca2+, Mg2+, Zn2+, and Eu3+ at 1 M charge concentration). Site-specific adsorption to calcite steps, essential for regulating morphology, only occurs in neutral to acidic pH (range investigated: 3.4-7.7) and in the presence of K+, Na+, Sr2+, and Ca2+. Basic pH (range investigated: 9.9-11.3), or cations of higher ionic potential than Ca (Mg2+, Zn2+, and Eu3+), caused CAP to ignore step edges, turning off its normal functionality. We propose that complexation between cations, CAP, and the calcite surface controls CAP behavior. Thus, cations provide an on/off switch for CAP function, with the power to regulate and disturb coccolith biomineralization as well as to control calcite growth at the unblocked precipitation sites. Introduction Coccolithophores are unicellular marine algae that cover their cell walls with calcite shields. These shields or coccoliths are one example of the elaborate crystal design that is possible in biogenic crystal growth. They are only a few micrometers across. A number of them interlock to form a shell, known as a coccosphere, around the alga. Typical coccoliths are double discs consisting of many interlocking crystal elements of complex shape. Figure 1 shows a coccosphere of the species Emiliania huxleyi, the dominant coccolithophore in modern oceans. Its intricate coccoliths demonstrate how extensively a biological system can control the expression of a mineral phase through biomineralization, a process of interest to researchers in biology, mineralogy, and materials science, that is ultimately controlled by chemical processes. Coccoliths are formed inside the algal cell, in separate vesicles where chemical composition is controlled. Polysaccharides (“coccolith associated polysaccharides”, CAP) are present in the vesicle during coccolith growth and play an important role in their formation.1-7 When a coccolith is complete, it remains covered by a coating of CAP. Fichtinger-Shepman et al.3 isolated and characterized the CAP from E. huxleyi. They reported a large, branching molecule with a backbone of mannose (simple sugar, C6H12O6) and side-chains containing rhammose (C6H12O5) and other methylated sugars, xylose (C5H10O5) and polygalacturonic acid (n(C6H8O6)), as well as sulfonate groups. The basic repeating unit of the CAP molecule is shown in Figure 2, together with the structures of the dominant building blocks. The molecular weight of CAP from one strain of E. huxleyi was found to be 88.6 kDa.8 The polygalacturonic acid units contain carboxylic groups, which, when they shed a proton, * Corresponding author. Current address: Mærsk Olie og Gas AS, Esplanden 50, DK-1350 Copenhagen, Denmark. E-mail: [email protected]. # Current address: Nano-Science Center, Department of Chemistry, University of Copenhagen; e-mail: [email protected].

Figure 1. A colorized scanning electron micrograph (SEM) of an Emiliania huxleyi coccosphere; diameter is about 5 µm.

result in negatively charged carboxylate groups that are capable of binding cations such as Ca2+. In an earlier paper, we demonstrated how the E. huxleyi CAP interacts with calcite.7 In the pure H2O-CaCO3 system, calcite is characterized by rhombic morphology, with acute (78°) and obtuse (102°) corners (Figure 3a) and surface steps that have either acute or obtuse edges (Figure 3b,c). E. huxleyi CAP preferentially attaches on acute, rather than obtuse, step edges during crystal growth. Stabilization of acute steps drives the morphology away from calcite’s rhombic {101j4} faces, favoring formation of vicinal faces with lower angles to the c-axis (Figure 3d). The calcite crystallites of E. huxleyi coccolith shields are defined by such faces, with only a small facet present of the {101j4} form (Figure 3e). If a face has only acute steps, the density of these steps defines the inclination of the vicinal face. Thus, inclination varies with the amount of step-edge blocking, and therefore, it varies as a function of CAP presence in the crystallizing solution. So, if a coccolithophore can control CAP concentration in the crystallization compartment, the vesicle, it can control the geometry of the faces expressed. This discovery brought us a step closer to understanding how complex coccolith

10.1021/cg8004272 CCC: $40.75  2009 American Chemical Society Published on Web 03/30/2009

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Figure 2. E. huxleyi coccolith associated polysaccharide, CAP. (a) Structure of the basic repeating unit, redrawn from Fichtinger-Schepman et al.3 The repeat unit is shown by [ ]. A, B, and C indicate sites where there is variability; the possible attached groups at these positions are shown in the box to the lower left. Man: mannose, Xyl: xylose, Rha: rhammose, GalA: galacturonic acid, Rib: ribose, Ara: arabinose, Me: methyl group, CH3. (b) Molecular structures of the basic building blocks of CAP. • Indicates attachment of OH, | indicates attachment of H. The attachment of a methyl group is shown for xylose. The carboxylic group of galacturonic acid, which can shed H+ and become negatively charged carboxylate, is capable of binding to cations and thus is the active group for attachment to calcite as well as to cations.11

Figure 3. (a) Calcite rhomb, the most stable crystal form in the pure H2O-Ca2+-CO2 system. (b) A rhomb with surface steps showing the obtuse (o) and acute (a) sides of the crystal. (c) Cross-sections through the crystal in b). (d) A plane consisting solely of acute steps is parallel to the c-axis and forms a prism face, whereas a set of acute steps of unequal lengths produces a viscinal plane (gray line) that slopes toward the c-axis direction. (e) Schematic cross-section through an E. huxleyi coccolith element, which is a single calcite crystal, showing its relationship with the calcite rhomb (gray). The single crystal has three extensions, all close to parallel to the c-axis and consisting of acute steps (after Henriksen et al.7).

crystals are formed by the organism, but questions about the particular E. huxleyi CAP control mechanism remain.

Most necessary is a switch for turning on, and off, the activity of the growth controlling molecule. To attain the elaborate morphologies of coccolith crystals, the organism must be able to control the functionality of CAP very precisely. Controlled variability of CAP functionality in vivo is required to explain how coccolith crystals are able to simultaneously express stable, rhombohedral calcite faces, known from inorganic crystal growth, as well as the highly controlled surfaces observed only in biogenic systems. In some species, rhombic faces cover large portions of the surface, such as for Coccolithus pelagicus.9,10 In others, such as E. huxleyi, they are present as only small facets (Figure 3e). These features cannot be explained by a simple model assuming growth in a compartment containing a solution of a given CAP concentration. Nor can they be explained by a system controlled only by the presence of inorganic trace components. In these cases, symmetrical crystals with one type of faces would be formed. Therefore, coccolithophores must have a means to alter the functionality of CAP on a local scale and as a function of location inside the vesicle and of time during development of the shield. Such modification could result from interplay between CAP and specific cations, thus linking the macromolecule to the surface. Local control of pH would cause local differences in CAP protonation/deprotonation and alter the surface properties of the calcite. Borman et al.11 reported a difference of inhibitory effect of CAP in solutions where Na or Ca dominated, possibly as a result of a conformational change induced by attachment of the two cations. To investigate this possibility and to attain a more sophisticated understanding of the mechanisms of CAP functionality, we examined CAP behavior as a function of solution composition. The purpose of this study was to investigate the interaction of E. huxleyi CAP with the calcite surface, (i) in the presence of a selection of cations (K+, Na+, Sr2+, Ca2+, Mg2+, Zn2+, and Eu3+) that would test system response to differences in ionic potential; (ii) in solutions of higher pH (9.92-11.30) containing K2CO3, Na2CO3, NaOH, and Ca(OH)2, that would test anion

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Figure 4. By varying the tip-surface force, AFM resolves crystal morphology and CAP attachment. (a) Freshly cleaved calcite surface exposed to 10-5 M CAP showing irregular steps and flat terraces. The step edges facing mostly west in the images make acute angles with the terraces above and below (marked a), whereas the steps facing mostly south form obtuse angles (marked o) to the terraces. Tip-surface force moderate so calcite step edges are visible. (b) Image taken immediately after (a) showing the same area with low tip-surface force. CAP attachment on the steps is clearly visible.

effects; and (iii) in solutions of lower pH (3.4) containing HCl and HNO3 to test proton effects. Cations were selected to cover a range from mono- to trivalent, where size and ionic bonding character were compatible for substitution into calcite. We also chose ions that are present in seawater and that could affect coccolith biomineralization. Materials and Methods Samples were cleaved from single crystals of optical quality Iceland spar, by the method described by Stipp and Hochella,12 immediately before each experiment. To avoid the spontaneous surface alteration that occurs on calcite in air,13 we immediately put cleaved samples under solutions of water equilibrated with CaCO3 that had been filtered through a 0.1 µm mesh. The cleaved surfaces were imaged with AFM in a fluid cell until conditions were stable. Then the liquid was replaced with an experimental solution containing CAP. The CAP was isolated from cultures of E. huxleyi by the method described for Pleurochrysis carterae in Marsh et al.4 The molar mass of CAP is 88.6 kDa.8 We used a Digital Instruments Multimode IIIa atomic force microscope (AFM) running in contact mode using sharpened tips of Si3N4 with a spring constant of about 0.6 nN. The samples were imaged in a glass fluid cell. By varying the set-point on the photodiode detector, we controlled the force acting between the tip and the surface during scanning, to either slightly push or pull on the cantilever, by the method developed for study of poly(acrylic acid) adsorption by Stipp.14 Images recorded using moderate repulsive (push) force showed the calcite surface exclusively (Figure 4a), whereas decreasing the set-point to the state just before tip withdrawal resulted in imaging of the attractive properties of the surface (pull) so the soft organic CAP was visible (Figure 4b). One can measure the height and width of micro- and nanoscale features using AFM, but aspects of the tip/sample interaction can affect the measurement. If the tip presses, soft adsorbed materials are pushed down, making them seem thinner, or they can be swept away. Imaging with a force light enough to result in a net pull makes the soft material appear thicker. Tip width convolutes imaged features. The smallest tips we have been able to find are about 20-25 nm in diameter at the apex,15 so features that are about the same size as the tip, such as CAP molecules, appear wider than they really are. Because the Si3N4 tips have an oxidized layer of SiO2, they are negatively charged at pH > 2, so if organic material is not firmly attached at the surface and it is attracted to the tip, it can be pulled free of the surface, causing streaks in the x direction. Sometimes tips become covered by organic material, making imaging impossible. To be sure that the behavior we saw at one site was representative and to minimize the possibility of artifacts on our interpretations, we collected many images and verified that observations of CAP were

Figure 5. Sequential images of a surface of calcite in the presence of Na2CO3, decorated by CAP. The series demonstrates reproducibility in the experiments, showing that CAP is well attached, unaffected by the scanning tip. (a) Initial image, (b) the same area 4 min later, (c) zoom on a particular CAP structure after 7 min, (d) a further zoom after 9 min. The morphology and site of CAP adsorption remains constant with time. reproducible through time, at different sites, at different scan speeds, in different areas analyzed, with a variety of directions of scanning and with old and new tips. Although these precautions do not exclude imaging artifacts, they provide comparisons with which to judge the quality of the images. Figure 5 shows how CAP structures on the calcite surface remain stable through time during scanning, and demonstrates the reproducibility of sequential images at the low forces that we used in these experiments. In some experiments, the soft material was dragged over the surface, even under very light forces, indicating weak attachment; for these experiments we have been tentative in our interpretation of CAP morphology. In all experiments, imaging commenced within 30 min after exposure of fresh calcite surfaces to solution. The samples showed very little change with time, except for a general increase in the amount of CAP attached at the surface. The range of image variability from three sites on a sample that had been exposed to CAP-HCl solution is illustrated in Figure 6. On a freshly cleaved calcite surface, the number and direction of steps vary. These differences are carried through when solution is added. Dissolution etch pit size, depth, density, and distribution are also controlled by the original calcite crystal surface, which is relatively inhomogeneous.13,16 Regardless of the variability of the surface, the behavior of the CAP is relatively constant on the three surfaces. The amount of attached CAP varied from one site to another, but sparse aggregation on terraces and preferential attachment to steps were the same. To investigate the effects of cation size and charge on CAP attachment, we prepared solutions with 10-5 M CAP and the salts listed in Table 1. CAP concentration was defined as the number of moles of uronate, that is, the concentration of carboxylate groups per liter. To focus on cation behavior, rather than on total charge, we used solutions with 2 M positive charge for each cation (Table 1) resulting in an overall 1 M charge when mixed 1:1 with the CAP solutions. These high concentrations were chosen to ensure cation equilibration with CAP. Our final experimental solutions (CAP + additive) had a CAP concentration of 10-5.3 M and a total ionic strength ranging from 1.0 to 2.0 M for the monovalent cation to trivalent cation-CAP solutions. At higher ionic strength the electrical double layer on surfaces condenses but a factor of 2 is not expected to have a significant effect on the adsorption behavior of CAP or the cations. Ionic strength within a coccolith vesicle in vivo is unknown, but it would certainly be higher than the surrounding cellular fluid (cytosol), which has been measured as 0.41 M (salinity 20 psu17). For comparison, seawater is 0.7 M. The pH of the original salt solutions ranged from acidic to neutral

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Figure 6. Images from different sites on the same sample exposed to CAP solution adjusted to pH 3.4 with HCl. The images demonstrate the range of variability typical in our experiments. This variability results from inhomogeneity of the initial calcite surface, not the CAP. (a) Shallow, irregular etch pits. CAP sits on all steps and terraces as rounded bumps and short bands. (b) Numerous steps sloping down to the left. CAP lines all steps and sits on terraces in structures with the same morphologies as in a). The bands look different because the tip is broader and probably doubled, blurring the features. (c) The density of steps and the type of dissolution features are different on the underlying calcite, but CAP attachment on the steps edges and terraces is the same as on a). The small white figure on the lower right corner of panel (a) shows the orientation of the crystal; (o) indicates the direction of the planes that make an obtuse angle with the surface and (a) shows the side of the etch pits where the edges form an acute angle with the surface. The arrow indicates the c-axis direction. Table 1. The Experimental Solutions Were 1:1 Mixtures of 10-5 M CAP with the Compounds Shown in the Column to the Lefta salt solutions for mixing 1:1 with 10-5 M CAP compound deionized water NaNO3 KNO3 Mg(NO3)2 · 6H2O Ca(NO3)2 · 4H2O Sr(NO3)2 Zn(NO3)2 · 6H2O Eu(NO3)3 · 6H2O Na2CO3 K2CO3 NaOH Ca(OH)2 HCl HNO3 a

concentration

pH

2M 2M 1M 1M 1M 1M 0.67 M 1M 1M 0.0028 M 0.0014 M

5.6 6.31 6.64 6.54 6.17 6.80 3.67 4.91 11.45 11.58 11.45 11.45

pH of experimental solution (1:1 CAP + salt solution) 7.77 6.90 6.78 6.98 6.83 6.98 5.45 5.01 11.16 11.30 9.92 10.04 3.4 3.4

pH of experimental solution (1:1 CAP + salt solution) equilibrated with CaCO3 8.24 8.18 8.26 7.37 6.87 6.84 5.77 5.33 10.99 11.12 8.34 8.30

Resulting CAP concentration in the experiments was 10-5.3 M.

(3.67-6.64), but mixing with CAP (pH ) 7.7) moved them into the slightly acidic range (5.01-6.98). To investigate the effect of pH over a wider range, we made 1:1 solutions of 10-5 M CAP with 1 M K2CO3 and 1 M Na2CO3, as well as NaOH and Ca(OH)2 with the same pH as the Na2CO3 solution (11.45). The aim was to test whether behavior depended on pH or on the nature of the base or cation-base combination. Similarly, we tested the influence of 10-5 M CAP adjusted to pH of 3.4 with HCl and HNO3. Just before scanning, two or three droplets of the experimental solution were put on the calcite surface. The approach to equilibrium was visible by AFM, as steps roughened, retreated, and etch pits formed. Equilibrium was defined as the time when step retreat ceased. We measured the pH of the experimental solutions after equilibration with the Iceland spar using a microelectrode, at the completion of the experiment (Table 1), but for the acid solutions, too little fluid was available for measuring.

Results For all experiments, we could see CAP adhering on the calcite surface when imaging force was minimized. Too much force caused the tip to sweep the surface clean of the soft macromolecules and the images showed only the crystal surface beneath (such as Figure 4). Pure Iceland Spar. Under a solution of pure water preequilibrated with CaCO3, the surface of calcite has straight cleavage steps and broad terraces, as shown in Figure 7a. Steps are commonly one or two molecular units high, 3 or 6 Å. Control System. The behavior of pure CAP on calcite was studied in detail previously.7 For comparison is the example in Figure 7b. The system components are a freshly cleaved calcite

surface such as the one shown in Figure 7a, water containing CAP, as well as Ca2+ and carbonate species that have come from equilibration of the solution with the solid. The approach to equilibrium removes material from step edges and forms etch pits on the surface (remnant of pit marked by arrow on Figure 7b), leading to surface roughening. CAP is visible as isolated, rounded spots arranged on terraces and along step edges. Attachment is favored on acute, rather than obtuse steps. KNO3. Figure 7c shows a sample exposed to KNO3-CAP solution. Steps have roughened and irregular etch pits have appeared. CAP sits on all step edges, along their full length, resulting in elongated bands that widen and narrow to give a “string of pearls” appearance (zoom, Figure 7d). The bands were commonly 1-1.5 nm tall and 15-35 nm wide, following steps over distances of many microns. Because of the convoluting effect of tip’s diameter, the width of the CAP is probably no more than about 1 to 15 nm. Some CAP forms isolated bumps on terraces (arrow in Figure 7c), but these are less well-defined in the presence of KNO3 than when the solution contains only Ca. The white streaks visible on this image and others are an artifact caused by loosely attached CAP that is dragged or rolled along the x-direction by the tip during scanning. Material attached on terraces is dragged much more by the tip than CAP adsorbed at edges, consistent with CAP being attached more weakly on terraces than at edges. Overall behavior is very similar to that of the control system. NaNO3. Initially, dissolution was apparent and step edges retreated. Figure 7e, taken 34 min after exposure, shows that

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Figure 7. Atomic force microscopy images of freshly cleaved calcite (a) under solution equilibrated only with CaCO3 without added salt; (b) control system, 10-5 M CAP. Surface steps are roughened by removal of material; arrow shows the remnant of an etch pit. CAP is visible as bands of material lining all step edges. (c-i) exposed to solutions of 10-5.3 M CAP containing (c) 1 M KNO3. The surface shows signs of dissolution, with irregular etch pits. CAP lines all step edges and a small amount sits as islands on terraces (arrow). (d) Zoom on (c) at position indicated by square, showing detailed CAP morphology. (e) 1 M NaNO3. The surface is dissolving. CAP sits on isolated spots on steps and these resist dissolution. Etching of the step between such islands causes it to curve. (f) Image taken 23 min later on the same sample (e). CAP now decorates the full length of all step edges and sits as rounded spots on crystal terraces. (g) 0.5 M Sr(NO3)2. Behavior is similar to NaNO3. (h) 0.5 M Ca(NO3)2. CAP is strongly aggregated, sitting in linear arrays of rounded structures along step edges. (i) Zoom on (h), at position indicated by the square, showing detailed CAP morphology. Crystal orientation symbols: arrow shows c-axis direction, (o) indicates the directions of the two crystal edges that slope away from the surface with obtuse angles, (a) indicates the directions of the two edges that slope away with acute angles.

CAP sits on steps as isolated islands that inhibit dissolution; between such pinned sites the steps dissolve so overall, the edges curve. Figure 7f shows the surface 57 min after exposure. More CAP has attached to terraces and now the steps are completely decorated by ribbons about 1 nm tall and 30 nm wide, very similar to those found with KNO3 present. The main difference between the two systems is that with NaNO3, more CAP is bound to the terraces as rounded bumps. Sr(NO3)2. Figure 7g shows the Sr(NO3)2-CAP-CaCO3 system. Dissolution features are less obvious, but otherwise, behavior is very similar to Na and K, with strings of CAP, approximately 1 nm tall and 20-40 nm wide (probably 1-20 nm true width), lining step edges. Much CAP sits on terraces, as isolated spots, but it also associates in long curving bands. Ca(NO3)2. Adding solutions of Ca(NO3)2 and E. huxleyi CAP promoted behavior different than the other solutions (Figure 7h,i). Polymer clusters are visible on surfaces imaged under low to moderate force, as elongated misty patches of approximate dimensions 10 × 5 × 0.5 nm. These associate into rounded, disk-like accumulations, approximately 40-60 µm wide and 0.5-1.5 nm tall. The rounded accumulations sit side by side along straight lines, following cleavage directions. By increasing force on the tip, we pushed through the material and imaged the mineral surface alone, confirming that the CAP is positioned on crystal step-edges, with some isolated spots occurring on flat terraces. The tendency to adhere to step edges

and the morphology of the CAP is similar to that found for pure CAP on calcite (Figure 7b), except that with the higher concentrations of added Ca2+, aggregation is much more extensive. Mg(NO3)2. Figure 8a shows calcite in the presence of CAP-Mg(NO3)2. Signs of dissolution are clear as many shallow etch pits with roughly rhombohedral shape. This system is different than those described previously; there is no evidence of CAP adhering to step edges. Instead, isolated aggregates 40-50 nm across are distributed randomly over the surface and streaked out by the scanning tip, indicating loose attachment. In this system, we see the first example of interconnected ribbons that are not associated directly with a step edge. In Mg solutions, ribbons are 40-100 nm wide and as much as several micrometers long. Zn(NO3)2. Figure 8b shows Iceland spar exposed to Zn(NO3)2-CAP solution. The calcite surface shows pronounced dissolution with formation of irregular-shaped pits. CAP sits on terraces in isolated, rounded islands, 0.8-1.1 nm tall and 30-40 nm wide and in ribbons 20-50 nm wide and up to a few micrometers long. They adhere in straight segments but do not align themselves along step edges or along the crystallographic planes of the calcite. Eu(NO3)3. Figure 8c shows a calcite surface exposed to CAP-Eu(NO3)3. Dissolution is visible as rounded, irregularly

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Figure 8. Atomic force microscopy images of freshly cleaved calcite exposed to solutions of 10-5.3 M CAP containing (a) 0.5 M Mg(NO3)2. CAP does not attach to step edges, but sits on flat terraces as rounded bumps and ribbons. The surface has etch pits. (b) 0.5 M Zn(NO3)2. Behavior is similar to a), but etch pits are more irregular in shape. (c) 0.33 M Eu(NO3)3. CAP is strongly polymerized into interconnected ribbons that cover most of the surface. Numerous irregular etch pits are visible beneath. (d) 0.5 M K2CO3. CAP attachment to the surface is weak. The material is associated into vague bands, with no tendency to adhere to step edges. (e) 0.5 M Na2CO3. CAP associates into long ribbons and rings, attaching to flat terraces. (f) Ca(OH)2 adjusted to pH 11.45. CAP sits on flat terraces and across cleavage steps as isolated bumps and interconnected ribbons. (g) NaOH adjusted to pH 11.45. CAP is aggregated into ribbons that adhere to flat crystal terraces. (h) Adjusted to pH 3.4 with HNO3. The surface is dominated by shallow etch pits. CAP attaches to steps, showing preferential binding to acute step edges. (i) Adjusted to pH 3.4 with HCl. Etching is observed as serration of step edges. CAP lines all cleavage steps. More images from this system are shown in Figure 5. Crystal orientation symbols: arrow shows c-axis direction, (o) indicates the directions of the two crystal edges that slope away with obtuse angles, (a) indicates the directions of the two edges that slope away with acute angles.

shaped pits and CAP is present on the surface as extensive, interconnected networks of ribbons, 20-50 nm wide and 0.5-1 nm tall. These cover most of the surface. K2CO3. In Figure 8d, we see the effects of dissolution as curved, irregular step edges. There are no isolated spots of CAP and no lining of step edges. However, CAP is visible as thin streaks extending in the x-scanning direction. Streaking results from tip drag, even at forces just below disengagement, indicating very weak attachment. The vague, wide bright band running in the y-direction at the left of the image is an accumulation of CAP, swept there by the tip. In this system, because of weak attachment, appearance and attachment patterns vary in sequential images. Na2CO3. CAP mixed with Na2CO3 behaves differently than with NaNO3. In this higher pH system, CAP adheres randomly on terraces as distinct aggregates (Figure 8e). The spots vary in size from approximately 10-50 nm across and 0.6-4.5 nm in height. CAP also forms elongated bands or rings that are not attached at step edges. Where the bands are of highest density, isolated spots are absent, suggesting self-assembly of the bands on the surface. With time, larger, web-like structures form, composed of many bands (Figures 5a and 7e, taken 34 min after exposure). These webs retain a stable morphology through time (Figure 5), but can be cut by the tip when imaging with a moderate force. There is no tendency for CAP to adhere at step edges in this solution.

The images from this system, in contrast to the others, are taken from an area of the surface where precipitation has followed after initial dissolution and equilibration between solution and surface. Dendritic growth has created a new calcite layer that is 1 monolayer (3 Å) higher than the underlying surface. It is necessary to recognize the features resulting from the precipitation of calcite and separate them from the features caused by adsorption of CAP so as not to confuse behavior typical calcite with that of the CAP. In this system, calcite growth produces irregular edges, which is typical calcite behavior when precipitation occurs in the presence of an organic component, such as when sugar or adventitious hydrocarbon is present.18 The irregular precipitate is not caused by CAP but rather the surface’s response to a solution at pH over 11. The important observation from images exposed to such solutions is that we do not see CAP lining the step edges. Ca(OH)2. Evidence of dissolution in the CAP-Ca(OH)2 system is limited to very slight roughening of step edges (Figure 8f). Bands of CAP, 20-50 nm wide and 0.5-1 nm tall, lie randomly on terraces and across steps, with no attachment at step edges. NaOH. The calcite surface exposed to CAP mixed with NaOH, adjusted to pH 11.45 is shown in Figure 8g. Signs of dissolution include a rounded pit end in the lower middle of the image and slight roughening of the steps. CAP is abundantly present on the surface, mainly in bands 80-100 nm wide, 1

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nm tall, and a couple of hundred nanometers long. There is no attachment to step edges. The lack of binding on edges and the random association of bands with the surface are similar to observations from samples exposed to NaCO3 and Ca(OH)2, suggesting it is pH, not the nature of the anion, that controls CAP behavior. The bands observed in Figure 8g are repeated by a double tip imaging artifact. HNO3. 10-5 M CAP adjusted with HNO3 to pH 3.4 causes clear etching, producing irregular pits with roughly rhombic outline. Step edges are decorated by CAP and a clear preference for acute, rather than obtuse, steps is evident (Figure 8h). Some CAP is attached on terraces as isolated bumps, 30-50 nm across and 0.5-1 nm tall and as larger ribbons, some branching (arrow on Figure 8h). HCl. 10-5 M CAP, adjusted to pH 3.4 with hydrochloric acid, also causes the calcite surface to dissolve. Etching is less severe than under HCl without CAP.7 In CAP-HCl solutions, surfaces roughen, step edges retreat (Figure 8i), and rounded etch pits form (Figure 6). The pits are commonly shallow (Figure 6a), but we also observed a few, deeper examples, as is common on the heterogeneous calcite surface. CAP lines all step edges and sits on the flat terraces as rounded spots, approximately 45 nm wide and 1 nm tall. Overview. All experiments had a CAP concentration between 10-5 M and 10-5.3 M and in general, attachment of CAP to steps was either extensive, covering all step edges, or not observed at all. Thus, it is solution composition that provides the on/off switch for CAP attachment. In cases where not all sites were filled, CAP preferentially attached to acute, rather than obtuse step edges (Figure 8h), as seen for pure CAP on calcite (Figure 7b and ref 7). Step attachment was observed in the control system, in solutions made with nitrate salts of K+, Na+, Sr2+, and Ca2+ and in the low pH experiments with HCl and HNO3. There was no attachment at steps for solutions made with nitrate salts of Mg2+, Zn2+, and Eu3+ or in the high pH experiments containing NaOH, NaCO3, KCO3, and Ca(OH)2. For the nitrate-salt experiments, self-assembly of CAP into long chains and networks on terraces varied with solution composition, with stronger assembly in the presence of cations of higher charge. There was less assembly on terraces in solutions where CAP attached to steps, except for solutions containing Ca. In order of increasing CAP assembly, the cations can be listed as K+ < Na+ < Sr2+ < Mg2+ e Zn2+ < Eu3+, with very little difference between behavior for Mg2+ and Zn2+ (Figure 8a,b). The morphology of the CAP ribbons on terraces was variable, with broad, branching bands in the solutions with Zn2+ and Mg2+, thinner but more abundant and interwoven bands in the Eu3+ solutions (Figure 8c), and extensive, linear arrays of rounded aggregates attached to step edges in the presence of Ca2+ (Figure 7h,i). The behavior described above was consistent in multiple reproducibility experiments. Discussion The key components of our experiments are the calcite surface and CAP molecules in solution. We added cations as nitrate salts because nitrate, with its large size and low charge is one of the least complexing of all anions. The cations, chosen to cover a range of sizes, were mono- to trivalent, giving a spectrum of ionic potential (charge/unit radius, Table 2). These cations could complex both with CAP and with the surface of calcite in our experiments. Addition of acid or base provided H+, OH-, or CO32- which could likewise attach to the surface and affect the polysaccharide. Thus, to understand the results of this study, we need to consider surface complexation, CAP

Henriksen and Stipp Table 2. Ionic Potential of the Cations Used in This Studya cation

ionic radius

ionic potential (charge/radius)

+

1.38 1.02 1.18 1.00 0.74 0.72 0.95

0.73 0.98 1.7 2.0 2.7 2.8 3.2

K Na+ Sr2+ Ca2+ Zn2+ Mg2+ Eu3+

a Ionic radii from Shannon;31 cations in calcite are octahedrally coordinated.

complexation, and the interaction of the resulting CAP and surface complexes. Surface Complexation. A mineral surface is a termination of the ordered bulk structure where the surface atoms are no longer in a balanced bonding environment. The unsatisfied or dangling bonds at the surface delocalize their residual charge by restructuring, as in vacuum, or by reaction with the contacting medium.12 The charge, composition, and structure of the ordered layer at the solid/solution interface depend on atomic ordering within the bulk and the composition of the solution. For metal oxides in water, partial positive charge over the cations and partial negative charge over the oxygen atoms are satisfied by water hydrolyzed into H+ near O and OH- over the cations.19 Total surface charge therefore varies with pH. The pH at the point of zero charge (pHpzc), is where local charges balance, so the surface has no net charge; pHpzc is mineral-specific but sometimes, as in the case of calcite, it depends also on the solution in contact.20 Values ranging from about 8 to 9.5 are used for calcite pHpzc,21,22 but it is misleading to speak of pHpzc for calcite. The principal potential determining ions are Ca2+ and CO32- (or HCO3-), not H+ or OH-. Rather, one should speak of pCapzc or pCO3pzc.20,21,23 pH only helps to define calcite surface charge through its control on carbonate speciation. An important point is that if the concentration of a potential-determining ion, such as Ca2+, is constant, the calcite surface charge is also constant, regardless of pH.23 Thus, the concentration of other cations that can substitute for Ca2+ in the solid, or adsorb on the surface, help to define calcite’s surface potential. In our experiments, at neutral to low pH, where very high cation concentrations were balanced only by the large, minimally complexing NO3- groups, the surface of calcite has a net positive charge. In contrast, at higher pH, where high concentrations of either CO32- or OH- were balanced by the weakly attaching K+ and Na+, net surface charge is neutral to negative. Net surface charge attracts or repels the CAP in solution, depending on the polysaccharide’s overall charge. As a free ligand and in its deprotonated state, CAP has a net negative charge but addition of abundant complexing cations or anions would change the net charge, inducing or hindering surface attachment. CAP Complexation. Equilibrium constants describing CAP complex formation determine the extent of CAP interaction with the surface or dissolved species. Protonation constants for CAP have not been determined, but data for Ca-CAP binding were published by De Jong et al.2 CAP binds to Ca through highaffinity (dissociation constant 10-4.66) and low-affinity (dissociation constant 10-2.95) sites, in a ratio of 1:2. These constants indicate that Ca-CAP binding is strong, with only 1 free Ca2+ ion in solution for every 46000 bound to CAP or 1 for every 890 bound. Unfortunately, no data are available for CAP interaction with the other cations investigated here. It is known, however, that the active groups on CAP are the carboxylic

Controlling Biomineralization

Crystal Growth & Design, Vol. 9, No. 5, 2009 2095

Figure 9. Formation constants of complexes of the ligands: acetate, malonate, citrate, and EDTA with cations, in order of rising ionic potential (IP, charge per unit radius [Å]). Also shown is L: low affinity E. huxleyi CAP site, H: high affinity E. huxleyi CAP site, T: total constant for Ca-CAP binding. The structures of the ligands are shown on the right (data from Martell and Smith;24 Anderegg;25 De Jong et al.2).

moieties of the polygalacturonic acid side chains.11 Thus we can use data collected for carboxylate-metal binding by ligands with single (acetate, C2H3O2-), two (malonate, C3H2O42-), three (citrate, C6H5O73-), and four (EDTA, C10N2H10O84-) carboxylate groups as models for CAP interaction. Figure 9 shows a plot of the equilibrium constants for pair formation of these ligands with each of the cations studied here (data from Martell and Smith24 and Anderegg25). Complexation strength increases with ionic potential (IP, Table 2). The De Jong et al.2 data for the two CAP sites, as well as a total Ca-CAP formation constant, are also shown for comparison. Because CAP interacts with Ca through numerous carboxylate groups,11 it is likely to follow the same trend observed for the model ligands (Figure 9). The active sites on CAP are attractive to cations, and it is logical that two or more active sites might share a higher ionic potential cation. Such polydentate sharing would induce the formation of CAP clusters and ribbons. CAP tendency to assemble into ribbons and networks on terraces increases on our AFM images in the order: K+ < Na+ < Sr2+ < Mg2+ e Zn2+ < Eu3+. The degree of CAP self-assembly parallels the trend of increasing cation complexation and ionic potential shown by Figure 9. We can write the relevant reactions for cation attachment to CAP as

CAP-COO- + Men+ T CAP-COOMe(n-1)+ -

CAP-COO + CAP-COOMe T CAP-COOMeCOO-CAP(n-2)+ n+

(1)

(n-1)+

-

(2)

where Me represents a cation and CAP-COO , a CAP carboxylate group. The higher the IP of the cation, the stronger the interaction between the cation and CAP, the higher the complex formation constant and the better the cation is at promoting CAP assembly. Ca2+ is the only ion showing both strong aggregation and step attachment. Ions with lower IP than Ca2+ (such as Na, K, and Sr) cause CAP to attach to steps, whereas ions of higher IP than Ca2+ (such as Mg, Zn, and Eu) cause CAP to aggregate as ribbons and networks and ignore step edges. Thus, Ca2+ is the pivot point for an on/off switch for CAP functionality. The control experiments, with 10-5.3 M CAP on a clean calcite surface, displays step attachment and weak terrace aggregation. This aggregation might be induced by the Ca2+ ions that enter solution from equilibration with CaCO3. However, CAP self-assembly is more extensive in the system where Ca2+ concentration is higher (also relative to carbonate), so not

surprisingly, aggregation depends on the nature and concentration of the cations present. For the large and singly charged K+, aggregation is weak. Bidentate complexing is not favored, so even with the high concentrations used in this study, assembly is minimal. The CAP-cation solutions were mixed and left to equilibrate overnight before they were put on the calcite surface, to allow time for formation of CAP-cation complexes. On the basis of the data of Figure 9, CAP is likely bound to Mg2+, Zn2+, or Eu3+. Equations 1 and 2 show that CAP-cation complexes would have portions with positive and neutral net charge. CAP complexes with monovalent K+ and Na+ would have neutral and negative portions. The data of Figure 9 show that CAP binding to K+ and Na+ is weaker by several orders of magnitude than for Mg2+, Zn2+, and Eu3+. Furthermore, the equilibrium condition for eqs 1 and 2 is pushed further toward the products, resulting in a higher proportion of unbound CAP-COO-. From the relative ligand effectiveness (Figure 8), Sr2+ and Ca2+ are predicted to be intermediate in their complexing behavior. CAP-Surface Interaction. We can roughly divide the observed CAP-surface interaction into three behavior patterns: (1) The calcite surface is positively charged from adsorption of cations or low pH, while CAP is weakly complexed and negative, such as for nitrate solutions of K+, Na+, Sr2+, possibly Ca2+, HCl, and HNO3. (2) The calcite surface is positively charged from adsorption of cations or low pH, while CAP is strongly complexed with neutral and positive net charge, such as for nitrate solutions of Mg2+, Zn2+, Eu3+, and possibly Ca2+. (3) The calcite surface is negatively charged from adsorption of CO32- and/or OH- at high pH, while CAP is negatively charged and weakly complexed such as in the basic solutions NaCO3, KCO3, NaOH, Ca(OH)2. Schematic representation of these three cases is shown in Figure 10. It is only in solutions corresponding to case 1 that we see CAP attaching to steps, suggesting electrostatic attachment. A calcite step edge is a defect where charge distribution is different than on a terrace because the atoms exposed at the step have an unsatisfied charge in two of their six possible directions. Thus, adsorption of dissolved species through electrostatic interaction is stronger on step edges than on terraces, which is why minerals grow by progressive expansion of steps. The calcite steps present alternating ≡Caδ+ and ≡CO3δsurface groups to solution species, where ≡ represents the surface and δ, the partial charge (δ < 1). For cases 1 and 2, the dominant anion is NO3-. Its large size and small charge make its attachment to ≡Caδ+ weak. Thus steps and surfaces have an overall positive charge because of stronger adsorption of cations to ≡CO32-, but the partial charge at steps is higher than on terraces. n+ ≡COδT ≡CO3Me(n-δ)+ 3 + Me

(3)

When CAP approaches a surface, its charge determines whether it attaches to the reactive step edges. For case 1, CAP complexes are negatively charged and attach, outcompeting NO3-. For case 2, CAP complexes are neutral or positively charged, and therefore not attracted to the step edges. This fits with the experiments showing clear signs of dissolution as CAP blocking of steps would inhibit etching. For case 3, solutions are basic, so CO32- or OH- dominate the surface and steps, imparting negative charge:

≡Caδ++ OH- T ≡CaOH(δ-1)-

(4)

Negatively charged step edges are not attractive for negatively

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Henriksen and Stipp

Figure 10. Schematic representations of the three behavior patterns for CAP interactions with the calcite surface, CAP shown in gray. (a) Situation 1. The calcite surface is positive from adsorption of H+ and/or other cations, the CAP is weakly complexed and negative. CAP is attached to step edges and some to terraces. This represents nitrate solutions of K+, Na+, Sr2+, and Ca2+, HCl and HNO3. (b) Situation 2. The calcite surface is positive as a result of adsorption of H+ and/or other cations, the CAP is strongly complexed and neutral or positive. CAP is strongly aggregated and sits on flat terraces. It does not attach to step edges, and the surface dissolves. This represents nitrate solutions of Mg2+, Zn2+, Eu3+. (c) Situation 3. The calcite surface is negative from adsorption of OH- and/or CO32- at high pH. CAP is weakly complexed and negative. CAP does not attach to step edges, but sits on terraces. There are only very slight signs of surface dissolution. This represents solutions of NaCO3, KCO3, NaOH, Ca(OH)2.

charged CAP, and we see no attachment on them. Because CAP behaves similarly in CO32- and OH- systems where pH is high, attachment must be controlled by surface charge, rather than the nature of the base. Regardless of the charge of the surface or the CAP complex, we always observe some degree of CAP attachment to terraces, even in cases where composition implies electrostatic repulsion between the two. The cleaved surface of Iceland spar is heterogeneous in terms of pit nucleation sites.26 A slightly different character at the site of a defect may be enough to enhance bonding between CAP and the surface. Alternatively, a nonelectrostatic, hydrophobic driving force may enhance attachment. Although CAP has numerous hydroxyl and carboxyl groups, and therefore is considered water soluble, it also has hydrophobic portions where carbon is bound to hydrogen (Figure 2). Carbon-containing molecules coil rather than remaining extended in solution, so CAP adhesion on terraces as an aggregate may simply be a reflection of its desire to be out of solution. Support for CAP hydrophobic behavior comes from precipitation experiments done by Borman et al.,11 who showed that E. huxleyi CAP significantly retards calcite precipitation through binding to crystal nuclei. Addition of ethanol to the solution decreased the extent of this retardation dramatically. The authors proposed that in the presence of alcohol, CAP changed conformation, binding less effectively to the nuclei. Regardless of the cause of the attachment, CAP clearly has an affinity for the calcite surface that is independent of net surface charge. Implications for Biomineralization. When CAP attaches to steps, it favors acute, rather than obtuse, sites. This is consistent with behavior in the pure CAP-calcite system.7 Preferential attachment to steps of a particular geometry blocks crystal growth in that direction, modifying crystal morphology. Thus, CAP attachment to specific steps is important for regulating morphology during biomineralization. The dependence of CAP step attachment on solution composition indicates that growth modulation can be controlled. By limiting or allowing entry of specific components in the intravesicle fluid, the algae can turn off or on the function of the polysaccharide by small changes in the composition of the crystallizing fluid. Coccolithophores pump inorganic carbon for coccolith growth, as HCO3-, into the calcifying vesicle. Ca2+ is transported via ion channels in the plasma membrane, through the endoplasmic reticulum and the reticular body.27 Other cations such as Sr and Mg are transported into the vesicle by the same pathway. The coccolith vesicle has relatively high concentrations of Ca and Sr and pH of approximately 7.1.17 Under these conditions, our

results show that CAP attaches predominantly to steps, where it must sit if it is to control morphology. However, more basic conditions or the presence of Mg, Zn, Eu or other cations of high ionic potential alter CAP function by hindering or preventing CAP step attachment. Our results show that when CAP does not sit on steps, calcite growth control would be disturbed. This would result in malformation of coccoliths. Herfort et al.28 examined E. huxleyi calcification under varied Mg concentration and demonstrated that perfect coccoliths were formed only when the Mg concentration in the growth solution was the same as that of seawater. Both lower and higher concentrations resulted in malformation, with effects becoming more severe for higher concentrations. In a similar study of the effects of Sr, Langer et al.29 showed that E. huxleyi morphology and calcification rate were unaffected by altered concentration. These results match ours precisely: CAP behavior in the presence or absence of Sr is the same as in the control experiments, whereas when Mg varies, CAP functionality changes. During calcite crystal growth, the product of the activities (effective concentrations) of calcium and carbonate must be slightly higher than the equilibrium constant. The ion activity product may be satisfied by any combination of approximately equal activities of both Ca2+ and CO32-, or by one higher and the other lower. However, for coccolith calcification, our results imply that CAP functionality changes depending on whether saturation is achieved by relatively more abundant calcium or high (bi)carbonate. High concentrations of calcium and presence of HCO3 (neutral pH) lead to CAP step attachment and move crystal morphology away from the rhomb and toward faces with lower angles to the c-axis. The result is viscinal faces analogous to those preferred by E. huxleyi.7 However, at high concentrations of carbonate or high pH, CAP-step edge activity is lacking, preventing an influence on morphology and thereby promoting rhombic crystal morphology. Thus, we propose that by varying the individual supplies of Ca and CO3 species, calcite producing organisms regulate crystallization. The initial crystal nuclei of the E. huxleyi shields are equant calcite rhombs.30 It is only later that the crystals extend to produce faces subparallel with the c-axis. Relatively equal calcium-to-carbonate ratios at the initial stage in the vesicle would allow nucleation of calcite rhombs. Changing the balance by allowing more Ca into the vesicle would switch on CAP control, producing the elongated outgrowths that form the E. huxleyi spokes. Finally, when the coccolith reaches the sizelimits of the vesicle, changed solution concentration would switch off CAP function.

Controlling Biomineralization

Acknowledgment. We wish to thank Mary Marsh, Jeremy Young, Vagn Moser, Tonci Balic-Zunic, Knud Dideriksen, Bo Christiansen, Markus Geisen, and Gerald Langer. Funding was provided by the Danish National Research Council FNU for the initial work; it was finished under support from the Danish Advanced Technology Foundation and Maersk Oil. We thank three anonymous reviewers for helpful suggestions.

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