Article pubs.acs.org/crystal
Polysaccharide Effects on Calcite Growth: The Influence of Composition and Branching J. W. Nielsen,*,† K. K. Sand,† C. S. Pedersen,† L. Z. Lakshtanov,†,‡ J. R. Winther,§ M. Willemoes̈ ,†,§ and S. L. S. Stipp† †
Nano-Science Center, Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark Institute of Experimental Mineralogy RAS, 142432 Chernogolovka, Russia § Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark ‡
ABSTRACT: Polysaccharides play a key role in the biomineralization of the elaborate and species specific calcite platelets, known as coccoliths. Coccoliths cover some species of algae and the polysaccharides serve as controlling agents, directing crystal growth. We conducted experiments of calcite growth in the presence of five well described polysaccharides to test the influence of composition and structural properties. Alginate and polygalacturonate contain a carboxylate group for every glycosyl unit, whereas the unbranched amylose and the progressively branched polysaccharides, ß-limit dextrin and amylopectin, contain only glucosyl units. Calcite growth was monitored by the consumption of Ca2+ and CO32− in a constant composition setup. Langmuir isotherms effectively describe the uptake data from experiments where polysaccharide concentration was varied. Our results demonstrate that polysaccharides containing acidic glycosyl units strongly inhibit calcite growth, compared to neutral polysaccharides, but fine structure, i.e., the anomeric configuration and/or the position of functional groups in the polymer, also plays a role, as illustrated by the difference in inhibitory properties of the acidic polysaccharides, alginate, and polygalacturonate. Of the neutral polysaccharides, the branched molecules were stronger inhibitors than the linear molecule and the longer the branches, the stronger the inhibition. Thus, amylopectin was 2 orders of magnitude more effective than ß-limit dextrin and amylose was inactive.
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INTRODUCTION Polysaccharides are thought to play a pivotal role in the biomineralization carried out by the unicellular algae known as coccolithophorids.1,2 These one celled algae cover themselves with elaborately designed calcite platelets known as coccoliths which are a few micrometers in diameter. The pattern of the discs is species dependent and there is evidence that polysaccharide composition is also species dependent, although only a few coccolith associated polysaccharides (CAP) have been characterized, and, at the level of sequence and monosaccharide composition, only the CAP from the coccolithophore Emiliania huxleyi is known in detail.2 E. huxleyi CAP (EhCAP) is an acidic polysaccharide consisting of a highly branched mannan.2 Pleurocrysis carterae is known to produce CAP that is similar but not the same, denoted PS-3 and P. carterae (strain CCMP 645) produces two different polysaccharides that have been labeled PS-1 and PS-2.3 The composition of PS-1 is known but not much is known about its © 2012 American Chemical Society
structure. The linear CAP, PS-2, is rich in acidic monosaccharide units3 and Pleurocrysis haptonemofera produces an isomer of PS-2, denoted CMAP.4 Although no conclusive evidence is available, coccoliths are assumed to be covered by polysaccharides that inhibit calcite dissolution. However, several observations suggest that organic material also plays an important role in maintaining the integrity of coccoliths under otherwise challenging conditions. The dissolution rate of coccoliths has been shown to be slower than that of inorganic calcite,5,6 and they are stable in solutions where inorganically produced calcite readily dissolves.5 Single specimens of cultured coccoliths did not dissolve in Ca2+-free artificial seawater at pH ≥ 8, but did dissolve at pH < 8.5 When cultured coccoliths are treated with oxidizing agents, their Received: June 6, 2012 Revised: August 17, 2012 Published: August 22, 2012 4906
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glucosyl units but with frequent branching through α-1,6 linkages. ß-Limit dextrin is an enzymatic product of amylopectin degradation, where the branches have been hydrolyzed from the nonreducing end, resulting in a molecule containing only very short chains extending from the branch points. These three classes of polysaccharides offer a means to assess the influence of increased polysaccharide branching and branching chain length. Two other polysaccharides, alginate and polygalacturonate, were chosen as models for evaluating the contribution of carboxylate on the inhibitory effect. Alginate and polygalacturonate both contain one carboxylate group per glycosyl unit (Figure 1d,e), but the monomeric composition and anomeric configurations are different. Alginate is formed by repeating units of ß-1,4-linked mannuronate and α-1,4-linked guluronate.16 Polygalacturonate is a polymer of α-1,4-linked galacturonate. The different structure of these two acidic polysaccharides could reveal if the orientation of the carboxylates and other functional groups contribute a steric effect to CAP inhibition.
dissolution rate in deionized water increases compared with untreated samples.6,7 Fossil coccoliths extracted from Cretaceous chalk show a similar resistance to dissolution,5 and polysaccharides with a composition similar to EhCAP have been identified in the same samples.8 This suggests that the organic material that was originally produced by the algae, and that is still associated with the coccoliths, is still active and continues to prevent coccolith dissolution. EhCAP has been shown to adsorb at the acute steps of calcite crystals, thereby blocking further growth.7 Chemically reduced EhCAP has a decreased inhibitory effect, suggesting its activity can be attributed to adsorption by carboxylate groups.9 Finally, addition of ethanol to the system decreases the inhibitory effect of EhCAP.9 Addition of ethanol can drastically change the physical properties of the polysaccharides, such as solubility,10 but the altered behavior could also result from ethanol interacting with the calcite.11,12 In general, polysaccharides isolated from coccoliths strongly inhibit calcium carbonate precipitation.13 To understand how the polysaccharides influence calcite, researchers have focused on exploring the interaction between various functional groups and the mineral surface. The calcite growth inhibiting properties of simple molecules, such as short alcohols and carboxylic acids, have been extensively studied. Ethanol binds strongly to calcite through hydrogen bonds from −OH and displaces H2O.11,12 It is also well established that carboxylate forms complexes with calcium in solution and on the surface of calcite.14,15 To determine which properties of the CAP molecule, i.e., composition or structure, exert the inhibitory effects, we chose five commercially available polysaccharides: amylose, amylopectin, ß-limit dextrin, polygalacturonate, and alginate (Figure 1). Amylopectin is highly branched where the properties of the substructure can be represented by amylose and ß-limit dextrin (Figure 1a−c). Amylose is the simplest of the neutral polysaccharides formed by α-1,4 linked glucosyl residues. Amylopectin is highly branched, mainly formed by α-1,4 linked
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MATERIALS AND METHODS
All chemicals were obtained from Sigma Aldrich except ß-limit dextrin which came from Megazyme. All polysaccharide solutions were prepared in deionized water (Milli-Q, resistivity >18.2 MΩ/cm) except the amylopectin solution, which was supplemented with 10% vol/vol dimethylsulfoxide (DMSO) and the amylose solution which was made in 100% DMSO because of its very low solubility in water. The maximum amount of DMSO added to the system was 2.0%. Experiments demonstrated that DMSO did not affect calcite growth rate at the concentrations used in these experiments. Polygalacturonic acid was titrated to neutral pH with NaOH. The Sigma calcite was aged, to remove organic compounds used during its production, during treatment in CO2 saturated MQ water overnight at 60 °C through several cycles, by a method adapted from Stipp and Hochella (1991).17 The constant composition method, described elsewhere,18 uses a solution at pH 8.3 containing CaCl2 and NaHCO3 that is slightly supersaturated with respect to calcite. As Ca2+ and CO32− are consumed during calcite growth, H+ is released according to the reaction:
HCO3− + Ca 2 + ⇄ CaCO3 + H+ A pH stat controls the addition of equal amounts of CaCl2 and Na2CO3 to the solution:
H 2O + Na 2CO3 ⇄ 2Na + + HCO3− + OH− The injected volume per unit time is recorded and is proportional to solid growth rate. In our system, ionic strength was held constant with 0.1 M NaCl and the initial CaCO3 solution composed of 1 mL of 0.1 M NaHCO3, 1 mL of 0.1 M CaCl2, and 23 mL of 0.1 M NaCl was seeded with 25 mg of treated Sigma calcite crystals. The reaction vessel was thermostatted at 25 o C and stirred with an overhead propeller through a port in the closed vessel lid. Precipitation rate was monitored for about 10 min in the pure system, before one of the polysaccharides was added. Precipitation rate was recorded continuously. Figure 2 presents an example of a typical curve obtained for the rate of solution addition, that is, calcite growth rate, as a function of time. The fractional inhibition of calcium carbonate precipitation, θ, could then be quantified by the change in growth rate, using the relationship:
Figure 1. The polysaccharides used in this study: (a) amylose composed of glucose units linked by α-1,4 glycosidic bonds; (b) amylopectin composed of (a) with additional α-1,6 linkages at branch points; (c) structure ß-limit dextrin, similar to amylopectin but with shorter branches; (d) polygalacturonic acid composed of α-1,4 linked galacturonic acid; (e) alginic acid composed of different sized blocks (m and n) of ß-mannuronic acid and α-guluronic acid attached by 1,4 linkages.
θ= 4907
r0 − ri r0
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Figure 2. An example plot from a precipitation experiment. pH is held constant at 8.3 while the volume of Na2CO3 and CaCl2 added is recorded with time. r0, i.e., the slope during the initial stage of the experiment, corresponds with the precipitation rate in the pure system and ri corresponds with the rate after addition of a polysaccharide. Growth rate slowly recovers and eventually returns to the original rate. where a θ value of 0 represents no inhibition and 1 complete inhibition, r0 represents the initial growth rate in the pure system and ri represents the growth rate following polysaccharide addition. At some time later, crystal growth begins to recover and eventually the system regains its initial rate. This suggests that polysaccharides are buried under new calcite so they are incorporated into the growing calcite, as has been shown for agarose19 and alginate,18 until the solution is depleted in polysaccharides and calcite growth can continue unhindered as in the original system. The precipitation rate was proportional to the added amount of calcite seeds suggesting precipitation occurs on the seeds. On Figure 3, θ of four of the polysaccharides is plotted as a function of polysaccharide concentration, [PS]. The data are fitted with an Ltype Langmuir isotherm described by θ=A
α[PS] 1 + α[PS]
Figure 3. Langmuir isotherms. (a) Amylopectin. (b) ß-Limit dextrin. (c) Alginate. (d) Polygalacturonic acid. Error bars represent the standard deviation based on two independent measurements. Amylose has no measurable effect on calcite growth so therefore is not shown.
Table 1. Adsorption Constant, α, and Maximum Fractional Inhibition, A, Determined from the Langmuir Isotherm
(2)
where A represents the maximum fractional inhibition at infinite concentration of polysaccharide and α is the adsorption constant. This functional form suggests that inhibition is caused by adsorbed polysaccharide blocking active growth sites. For each concentration of each polysaccharide, we made two precipitation rate plots. In all cases, equilibrium concentration of the polysaccharide was assumed to be the initial concentration, neglecting the concentration change resulting from solid−solution interaction. For confirmation, we made some experiments beginning with twice the amount of calcite seed (i.e., 50 mg). The fractional inhibition, θ, did not change significantly from that determined using 25 mg of seed crystals, confirming that adsorption does not significantly deplete the amount of polysaccharide initially present in the initial phase of inhibition. Solid material for SEM investigations was obtained by repeating the experiments and taking 1 mL samples at a series of points over the precipitation sequence (Figure 2). The samples were rinsed with ethanol to prevent further precipitation and vacuum filtered within 2 min following extraction. Samples were coated with 2−3 nm of gold to minimize charging and imaged using a Quanta 200F with a field emission gun (FEG) at 2−3 keV at a working distance of 3−4 mm.
a
polysaccharide
acidity
α (mL/μg)
A
amylopectina ß-limit dextrina amyloseb alginateb polygalacturonateb
neutral
0.29 1.06 × 10−3 N.D.c 2.0 0.21
0.73 0.98 N.D. 0.99 0.70
acidic
Branched. bUnbranched. cN.D.: not detected.
Amylose did not inhibit calcite growth at concentrations of up to 0.2 mg/mL. In contrast, the adsorption constants of amylopectin and ß-limit dextrin were readily determined. Amylopectin has almost 2 orders of magnitude higher affinity for calcite than ß-limit dextrin (Table 1). Full inhibition of calcite growth could not be achieved by either polysaccharide within the tested range of polysaccharide concentration (Figure 3), suggesting that not all calcite growth sites have been blocked and full inhibition is not possible even at very high amylopectin concentrations, as indicated by the maximum fractional inhibition, A, for amylopectin. The A-value of ß-limit dextrin being so close to 1 (Table 1) suggests that complete blockage of all crystal growth sites is achievable, but the adsorption constant is rather uncertain because we do not have data for concentrations higher than 1.6 mg/mL. Although the two acidic polysaccharides are somewhat similar, in that they have one carboxylate per glycosyl unit, alginate is a much more active inhibitor than polygalacturonate (Table 1 and Figure 3c,d). Acidic polysaccharides, in contrast with neutral ones, might be stronger inhibitors of calcite growth if carboxylates bind more strongly than hydroxyls to calcite crystal surfaces. This is confirmed to some extent by the difference in calcite growth inhibition by alginate and polygalacturonate compared with that of amylose and ß-limit
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RESULTS AND DISCUSSION Growth Inhibition of Calcite by Polysaccharides. The data are presented in Figure 3. Each point represents the average of two independent experiments. The adsorption constant, α, and the maximum fractional inhibition, A, determined using eq 2 for each polysaccharide are listed in Table 1. 4908
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dextrin. Alginate is a strong inhibitor and can induce almost full inhibition, whereas polygalacturonate has an adsorption constant and maximum fractional inhibition comparable to those for amylopectin (Table 1). This implies that functional groups, such as carboxylate, are not decisive in the activity of calcite growth inhibitors and that the detailed structure such as anomeric configuration and position of functional groups of the polysaccharide is probably responsible for the difference. The polysaccharide isolated from E. huxleyi, EhCAP, contains a significant portion of acidic glycosyl units, and there is good evidence that it is branched2 and strongly inhibits calcium carbonate precipitation.9 Our observations of the model polysaccharides are consistent, i.e., that highly branched, acidic polysaccharides are stronger inhibitors than linear, short chain branched or neutral molecules. We also tested whether inhibition of calcite growth could be attributed only to the monomers of which the polysaccharides are composed. We checked the inhibitory effect of glucose and galacturonate at a concentration (40 μg/mL) similar to that tested for amylopectin and polygalacturonate, but no inhibition could be detected. Thus we conclude that although there is evidence that monosaccharides adsorb on calcite surfaces,20 they do not inhibit calcite growth at the macroscopic scale, at least not at these concentrations. A likely explanation is that monomers are simply not strongly bound enough and do not cover enough of the growth surface to prevent Ca2+ and CO32− adsorption. Scanning Electron Microscopy (SEM). Calcite samples that had been removed periodically during the experiments were examined with SEM to observe how exposure to polysaccharides affected crystal morphology. The images of the seed crystals grown in the presence of the various polysaccharides demonstrate that crystal growth is changed by the presence of polysaccharide in the growth solutions. Samples of calcite seed, such as that used to initiate precipitation, are shown in Figure 3a,b. These serve as a reference for the material sampled during the course of the experiments. The seed material has smooth terraces and relatively even and sharp edges and corners. After ∼10 min of growth in the pure solution, the edges of the crystals had become slightly less regular, but there was clear evidence of layer by layer accumulation (Figure 4c). Addition of polysaccharide initiated much less ordered growth. Crystal terraces, edges, and corners roughened and steps were pinned (Figure 4d−h), which demonstrated that active sites were blocked. With time, crystal growth recovered and growth rates approached those of the initial, polysaccharide free experiments but the irregularities in morphology did not disappear. Crystals grew but surfaces were irregular, with indentations, and growth progressed dendritically, i.e., in fingers (Figure 4i). This indicates that polysaccharides remain adsorbed on active sites, hindering growth at those locations, while the supersaturation of the solution drives growth continuously at new sites, i.e., the branch ends of the dentrites. Thus, growth is not hindered in the macroscopic sense but the presence of polysaccharide prevents growth at the specific sites where it is adsorbed. Inhibitors could change the mechanism of growth as suggested by Lakshtanov et al (2011).18 Because of step pinning, crystal growth requires surface nucleation, explaining the various unusual patterns (Figure 4).
Figure 4. SEM images of calcite crystals sampled from the reaction vessel before and during the experiments: (a, b) initial calcite seed crystals; (c) after ∼10 min of growth from a pure CaCO3 solution; (d) surfaces showing signs of inhibition after addition of amylopectin; (e, f) from the alginate experiments; (g, h) with polygalacturonate; and (i) after growth had recovered, in the presence of polygalacturonate.
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CONCLUSIONS The inhibitory effects of branched, linear, neutral, and acidic polysaccharides on crystal growth were compared where calcite was grown in a constant composition set up. Acidic polysaccharides are known to be strong inhibitors of calcite growth, which was confirmed in this work, but the polysaccharide composition, structure, and degree of branching play a large role in determining inhibitor effectiveness. The highly branched, neutral polysaccharide, amylopectin, is as active an inhibitor of crystal growth as polygalacturonate. Inhibition decreases with a decrease in the extent of branching, as demonstrated by comparing the effects of amylopectin and amylose. Amylose, with no branches, has no measurable effect on calcite growth. SEM images, taken from calcite grown in the presence of polysaccharides, show disruption in crystal morphology. After some time, growth recovers but the effects on terraces, steps, and edges remain after the rate of ion uptake from the solution returns to its preinhibition levels. Our results suggest that for polysaccharides to inhibit calcite crystal growth optimally they must contain acidic monosaccharides and the molecules must be branched. This is consistent with the known behavior of the branched and acidic polysaccharide that has been isolated from E. huxleyi, which is a strong inhibitor of calcium carbonate precipitation.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 4909
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ACKNOWLEDGMENTS We sincerely thank Keld West, Jeanne Olsen, Anna Johnsson, and the NanoGeoScience group for help and discussion. We are grateful to Finn Engstrøm and Karen Henriksen from Maersk Oil and Gas for encouragement and challenging questions. Funding was provided by the Nano-Chalk Venture, supported by the Danish National Advanced Technology Foundation (HTF), Maersk Oil and Gas A/S, and the University of Copenhagen and supplemented by the UK (EPSRC) Engineering and Physical Sciences Research Council Frame Grant called MIB.
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