Environ. Sci. Technol. 2008, 42, 7242–7247
Cd(II) Speciation in Alginate Gels T H O M A S A . D A V I S , * ,† ERWIN J. J. KALIS,† JOSE PAULO PINHEIRO,‡ RAEWYN M. TOWN,§ AND HERMAN P. VAN LEEUWEN† Laboratory of Physical Chemistry and Colloid Science, Wageningen University, P.O. Box 8038, 6700 EK Wageningen, The Netherlands, IBB Centro de Biomedicina Molecular e Estrutural, Departamento de Qu´imica, Bioquim´ica e ´ Farmacia/Faculdade de Cieˆncias e Tecnologia, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal, and Institute for Physics and Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark
Received April 17, 2008. Revised manuscript received July 28, 2008. Accepted August 04, 2008.
Polysaccharides, such as those occurring in cell walls and biofilms, play an important role in metal speciation in natural aqueous systems. This work describes the speciation of Cd(II) in alginate gels chosen as a model system for biogels. The gels are formed by bridging calcium ions at junction zones present along adjacent homopolymeric guluronic acid chain sequences. The free Cd2+ concentration in the gel phase is measured by a novel in situ microelectrode voltammetric technique that monitors the electroactive probe cation Cd2+ by its reduction at a Au-amalgam microelectrode. In situ voltammetric measurement, coupled with total Ca(II) and Cd(II) determinations, as well as potentiometric titration, permits the full reconstruction of the charging environment and the cation binding for the gel phase. Three independent combinations of measuring and modeling the charged gel layer thereby permit accurate prediction of the Donnan potential, ΨD, and the Donnan enrichment coefficient, ΠD. At an ionic strength of 10 mM, Donnan potentials in the gel ranged from approximately -10 to -20 mV, corresponding to an enhancement in the level of free Cd2+ ions in the gel phase relative to the bulk solution by a factor of approximately 3. In contrast, the total level of Cd(II) was found to be enhanced by a factor of approximately 60, resulting predominantly from the specific binding of the Cd by the uronic acids of the alginate gel. These results emphasize that large differences in Cd(II) speciation can arise due to the combination of specific and electrostatic modes of binding. The results of this speciation analysis, for charged biological gels, have important consequences for mechanistic interpretation of metal biouptake processes involved in complex media.
Introduction Polysaccharides are ever increasingly being recognized for their role in the speciation of trace metals in natural aqueous systems (1-8). This can be related to a number of factors, * To whom correspondence should be addressed. Present Address: Department of Chemistry, University of Montreal, P.O. Box 6128, succursale Centre-ville, Montreal, Quebec, Canada H3C-3J7. Fax: (514) 343-7586. E-mail:
[email protected]. † Wageningen University. ‡ Universidade do Algarve. § University of Southern Denmark. 7242
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the most important of which are (i) their abundant contribution to the total percent mass of the plant kingdom [up to 25% of NOM in freshwaters and up to 80% of organic carbon in marine systems (5)], (ii) their participation in maintaining the structural integrity of biogels such as biofilms, flocs, and cell walls, and (iii) the important subclass of charged polysaccharides (e.g., uronic acids) that provide functional group sites for metal binding. Accordingly, trace metals such as Cu, Pb, Cd, Zn, and Hg are bound specifically to plant root system cell walls (9, 10), to the outer membranes of Gram-positive bacteria (11, 12), to fresh and salt water colloids (1), to the matrix polysaccharides of macroalgal cell walls (13, 14), and to the extracellular polymeric substances (EPS) in biofilms (15). Much attention has traditionally been paid to estimation of the concentration of the free and total metal species in these polysaccharide-containing phases. This is largely due to the requirement of understanding and predicting metal speciation within the scope of ecotoxicology and the bioavailability of trace metals, whereby much emphasis is placed on understanding the mechanistic detail of the biouptake process of microorganisms. Alginate was chosen as a model polysaccharide for this study because it occurs in a number of priority systems in environmental science and ecotoxicology. Alginic acid or alginate is the common term applied to a family of linear polysaccharides containing 1,4-linked β-D-mannuronic (M) and R-L-guluronic (G) acid residues arranged in a blockwise, nonregular order along the chain (Supporting Information Figure S1). The proportion of M and G residues and their macromolecular conformation determine the physical properties and the affinity of the alginate for heavy metals (16). Polyguluronic acid contains two diaxially (1a,4a) linked R-Lguluronic acid residues in the chair form which produce a rodlike conformation with a molecular repeat of 0.87 nm (17) (Supporting Information Figure S1b). In contrast, polymannuronic acid forms a flat ribbonlike chain; its molecular repeat is 1.04 nm, and it contains two diequatorially (1e,4e) linked β-D-mannuronic acid residues in the chair form (18). It is this difference in conformation between the two homopolymeric blocks which is believed to be chiefly responsible for their strong but variable affinity for heavy metal ions (16). A higher specificity of guluronic acid for divalent metals is explained by its “zig-zag” structure which can accommodate the Ca2+ (and other divalent cations) more easily. The alginates are thought to adopt an ordered solution network, through interchain dimerization of the polyguluronic sequences in the presence of calcium or other divalent cations similar in size (Supporting Information Figure S1d). The rodlike shape of the poly-L-guluronic sections results in an alignment of two chain sections yielding an array of coordination sites, with cavities suitable for calcium and other divalent cations because they are lined with the carboxylate and other oxygen atoms of the G residues. This description is known as the “egg-box” model (19, 20). The regions of dimerization are terminated by chain sequences of polymannuronate, which display the carboxylic groups chiefly responsible for the fixed charged groups distributed throughout the calcium cross-linked alginate gel. Recent evidence suggests that subsequent aggregation of dimers also contributes to the final structure of the gel (21) in addition to that suggested by the egg-box model. The focus of this work is to obtain physicochemical data (e.g., electrostatic Donnan potentials) directly from the charged polysaccharide gel phases that are reasonably representative (i.e., cell walls, mucosal layers) of the types of environments that typically surround microorganisms. 10.1021/es801068c CCC: $40.75
2008 American Chemical Society
Published on Web 09/05/2008
To do so, we explore the use of a recently developed microelectrode-based approach (22) applied to laboratoryprepared biogels. The technique allows for experimental determination of the Donnan potential and the Donnan enrichment factor of free metal ions in the equilibrated, traceCd-containing calcium alginate gels studied in this work. The voltammetric data are coupled to data obtained from (i) total metal determination by inductively coupled plasma atomic emission spectrometry (ICP-AES) on acid leachates of the laboratory-prepared calcium alginate gels and (ii) potentiometric titration for determining their fixed charge density, which refers to the structural charges present along the polysaccharide chain. In what follows, we demonstrate that the fixed charge environment can be reasonably reconstructed, confirming the “proof of concept” that Donnan measurements may be made on biogels by employing the novel in situ microelectrode technique.
Materials and Methods Reagents and Apparatus. All solutions and gels were prepared with distilled, deionized water from an EasyPure UV system (Barnstead, resistivity of 18.3 MΩ cm). Solutions containing Ca(NO3)2, Cd(NO3)2, and NaNO3 were prepared from solids obtained from Aldrich and J. T. Baker; KNO3 solutions were prepared from solid KNO3 (Aldrich). Sodium alginate was from Sigma (A-2033, lot 128F0050). Ca, Na, and Cd concentrations were measured by ICP-AES (Spectroflame). The quality of the measurements was routinely controlled by means of internal standards. Measurements at a constant ionic strength (10 mM) were performed in Cd(II) solutions of 1 × 10-6, 3 × 10-6, 1 × 10-5, and 3 × 10-5 M Cd(II) denoted as solutions I, II, III, and IV, respectively, that also contained 1 mM Na(NO3)2 and 3 mM Ca(NO3)2 at pH 5.6. Adjustments in pH were performed with either HNO3 (LPS Benelux) or NaOH (Merck). The equilibrated gel/solution systems were purged overnight in the equilibrating stock solutions with oxygen-free nitrogen. They were then transferred into the electrochemical cell which was purged between measurements and in which a positive argon pressure was continually maintained. Calcium Alginate Gel Preparation. Homogeneous alginate gels were prepared according to the method of Draget et al. (23). The gels were prepared by internal liberation of calcium ions after CaCO3 particles had been mixed with the slowly hydrolyzing proton donor D-glucono-β-lactone (GDL) in sodium alginate solutions. Briefly, 1.0% sodium alginate solutions were prepared and left stirring overnight. Gelation was achieved with 15 mM CaCO3 and 30 mM GDL. Following complete dissolution of the sodium alginate, CaCO3 particles were dispersed into the viscous aqueous phase. The resulting suspension was then degassed using a vacuum pump, and the freshly made GDL solution was added as the final step. The suspensions were briefly mixed, then poured into cylindrical well plates, and left to solidify overnight. The individual gels of calcium alginate, cylindrically shaped, were transferred into a gel-setting solution containing 20 mM NaNO3 and 50 mM Ca(NO3)2 for 2 days. Finally, the gels were transferred into the final equilibrating stock solutions containing the electroactive probe ion Cd2+. These solutions were refreshed a minimum of six times over a period of 8 weeks prior to measurement. Additional information about the gel preparation and macromolecular structure as determined by 1H nuclear magnetic resonance (NMR) spectroscopy can be found in the Supporting Information. Gel Volume, Total Metal Determination, and Potentiometric Titration. Digital calipers were used to measure the diameter and height of all gel cylinders to calculate the volume. The mean volume of the gel cylinders prior to setting (syneresis or shrinking) was 3.34 ( 0.01 cm3 (n ) 32). Following setting, the gels shrank (syneresis) to 1.14 ( 0.01
cm3, or by a factor of 3. Gels equilibrated with solutions I-IV were cut in half to permit total metal determination as well as voltammetric analysis. Volume measurements of the gels were taken again prior to leaching of the metals in 0.1 M HNO3 for 4 days. The final solution volume was corrected for the volume of the gels, and the total metal content of gels was determined by ICP-AES. Characterization of the fixed charge density of the gels was conducted by potentiometric pH titration. For each titration, the sample, which consisted of five individual gels that were pre-equilibrated in 3 mM Ca(NO3)2 and 1 mM NaNO3 (pH 6), was brought to a pH of 9. A total of 25 mL (including the volume of the gels) was placed in an automated pH titrator once the individual weight and volume of the gels were measured. Gels and solution were purged of CO2 with nitrogen for 10 h. The titrations were performed with 0.1 M HCl from pH 9 to 2. After each aliquot, the solution was stirred while being purged for 15 min. pH measurements were required to be stable (maximum drift of pH of 0.01 unit/min) before the next aliquot was added. Following titration of the gels, a blank solution of 25 mL of 3 mM Ca(NO3)2 and 1 mM NaNO3 (pH 9) was titrated for subtraction from the gel titration curve. The difference in proton consumption between the solution-gel curves and the blank curves was divided by the sum of the gel’s volume for the five gels that were titrated. The titrations were performed in triplicate. Electrochemical Apparatus and Parameters. An Ecochemie µAutolab type II potentiostat was used together with a Metrohm 663 VA stand. The working microelectrode was constructed in laboratory according to the approach of Brendel and Luther (24) with a diameter (12.5 µm) smaller than that described by Xu et al. and Dexter and Luther (25, 26). A platinum counter electrode was used in conjunction with a Ag/AgCl/3 M KCl microreference electrode with a flexible barrel (MI-402, Microelectrodes, Inc.) that was encased in a 0.25 mol/L KNO3 jacket (see Supporting Information Figure S2). Mercury was plated onto the Au substrate at -0.4 V (vs Ag/AgCl/3 M KCl/0.25 M KNO3) in a deoxygenated 5 mM Hg(CH3COO)2 and 0.1 M HClO4 solution. Deposition was performed until a charge of 60-70 µC accumulated, for the first deposition. Subsequent mercury depositions that followed a brief repolishing (30 s, 0.25 µm diamond paste) required less charge deposition (i.e., 10-20 µC). The amalgam was formed as described by Brendel and Luther (24). The following conditions were applied for acquisition of the direct current (DC) voltammograms: pretreatment, equilibration time of 5 s; measurement, interval time of 0.1 s and scan rate of 1.5 mV/s; initial and final potentials of -0.35 and -0.75 V, respectively, step potential of 0.15 mV, and standby potential of 0.1 V. Confirmation of a steady-state current was achieved by variation of the scan rate. Free Cd Determination by In Situ Gel Voltammetry. The approach used in this study has previously been described (22). Briefly, gels were pre-equilibrated with solutions of varying trace cadmium metal concentrations. Several gel disks were then stacked at the bottom of the voltammetric cell to provide a sufficiently thick yet quickly deoxygenated (i.e., overnight) thin gel layer (Supporting Information Figure S2). Furthermore, the inherent elasticity of the alginate gel ensured a direct contact between the gel and the electrode once the electrode was positioned at the surface of the gel. Multiple voltammograms were obtained from the equilibrating solution until the electrode stabilized and a reproducible signal was obtained. Typically, at least four voltammograms were recorded in the equilibrating solution prior to the microelectrode being placed in contact with the gel disk. Immediately following contact, repeated measurements were taken in the gel phase. VOL. 42, NO. 19, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Total Ca(II) and Cd(II) Leached from Calcium Alginate Gelsa solution
[Cd2+]sol (M)
Cd(II)tot in gel (mM)
Ca(II)tot in gel (mM)
ΨD(E) (mV)
ΠD(I)
ΨD(I)b (mV)
I II III IV
1.03 × 10-6 2.98 × 10-6 1.02 × 10-5 3.16 × 10-5
0.061 0.18 0.63 1.7
54.9 57.3 57.3 52.6
-16 -23 -28 -24
2.5 3.1 4.2 3.2
-12 -14 -18 -15
a
Measured ΨD(E) and ΠD(I) and calculated ΨD(I) for calcium alginate gels.
FIGURE 1. Steady-state voltammograms for a 1.0% calcium alginate gel-sol system. In solution II, [Cd2+] ) 3 × 10-6 M. The lower voltammogram depicts the solution voltammogram, vs the upper, gel voltammogram. The pH was 5.6 and the ionic strength 10 mM.
Results and Discussion Total Ca(II) and Cd(II) in the Gel. To assess the overall speciation of cadmium in the gel, both the total Ca(II) and Cd(II) and the total Na were quantified for the gels later studied by voltammetric analysis. The gels were, therefore, digested in strong acid to leach the metals that were subsequently quantified by ICP-AES. The results are presented in Table 1 for solutions I-IV from 1 × 10-6 to 3 × 10-5 M Cd(II). The average total Ca content was determined to be 55.5 mM, whereas the total Cd(II) content ranged from 0.061 to 1.7 mM (both values representing averages of the measured duplicates). The results of Cd(II) accumulation in the gel were internally consistent (linear uptake as a function of solution concentration; R2 ) 0.996) over the concentration range that was studied (Table 1). On average, total Cd(II) was enriched in the gel, relative to the solution concentration [Cd(II)gel/Cd2+sol] by a factor of approximately 60 for the 10 mM ionic strength equilibrating solutions. A small amount of Na was present due to Donnan enhancement in the gel (e.g., 1.62 mM measured vs 1.68 mM calculated; see below), in agreement with the fact that Na is not specifically bound to the carboxylate groups of the calcium alginate gel. Free Cd2+ in the Gel Phase. The experimentally observable enhancement in the concentration of free cations in a negatively charged gel is described by the Boltzmann equation [MzM]gel zM
[M ]sol
)
(
)
IL-(Cd-gel) -zMF ) exp ΨD ) ΠD IL-(Cd-sol) RT
(1)
where MzM is a metal ion with charge zM, IL is the limiting current, ΠD is the Donnan partition coefficient, and the other symbols have their usual meaning. Voltammetric measurements were performed on the calcium alginate gels in Donnan equilibrium with solutions I-IV. Figure 1 depicts typical diffusion-limited steady-state voltammograms obtained in both the solution (bottom wave) and gel (top wave) phases for calcium alginate in Donnan equilibrium with a Cd2+ concentration of 3 × 10-6 M. Inspection of the voltammetric 7244
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b
ΨD(I) is computed from ΠD(I) via eq 1.
wave, measured in the gel phase, relative to the solution phase, reveals an enhancement in the limiting current (i.e., enhancement in the Cd2+ concentration) by a factor of 3.2 according to eq 1. This factor represents the value of ΠD(I) provided that (i) the diffusion coefficients of Cd(II) in the gel and in the bulk solution do not differ significantly (see the Supporting Information) and (ii) the gel does not contain any mobile, labile complexes of Cd(II) as is the case in this study. It should be noted that the steady-state voltammetric technique measures only the free Cd2+ in the gel phase and not the specifically bound Cd(II), because the latter is completely immobile. Preliminary transient experiments unambiguously show that these immobile complexes are not labile on the time scale of tenths of seconds and shall be fully described in our subsequent article on Cd(II) speciation in alginate gels. Hence, it does not distinguish between modes of binding or binding strength. Via application of the Boltzmann equation (eq 1) to the voltammetrically obtained ΠD(I), the Donnan potential ΨD(I) may also be calculated (Table 1). In addition, a distinctive negative shift of E1/2 (half-wave potential) is evident in comparison to the solution phase voltammogram. This shift (∆E1/2) equals the Donnan potential, ΨD(E). The various ΨD(E), ΨD(I), and ΠD(I) values for the suite of exposure solutions are collated in Table 1. From at least two replicate measurements for each solution, the mean ∆E1/2 or ΨD(E) ranged from -16 to -28 mV with the mean ΠD(I) values ranging from 2.5 to 4.2. The slight variability in these results across the four solutions is ascribed to the inherent reproducibility of the data. There was no evidence of any saturation effects: uptake in the gel is linear in this concentration range, and the microelectrode is in good working order in all cases. A full description of the microelectrode voltammetric approach is given in the Supporting Information. Additional voltammograms for the remaining solutions studied are also presented in the Supporting Information (Figures S4-S7). Calculation of the Fixed Charge Density in the Gel. Estimation of the fixed charge density which represents the structural charge of the calcium alginate gels would allow an independent computation of the Donnan potential via eqs 7-9 (Supporting Information). We approached this by measuring the concentration of titratable carboxylate groups (assumed to equal the sum of the mannuronic plus guluronic residues along the polymer chain), together with an estimation of the concentration of network-bridging Ca ions, to arrive at the number of uncompensated charge groups in the gel phase. Specifically, the titration yielded a total of 113 ( 7 mM carboxylate groups. The free Ca2+ in the gel was estimated from the Donnan enrichment factor measured by voltammetry (Table 2) and subtracted from the total Ca(II) concentration in the gel phase [determined by ICP-AES (Table 1)]. For example, for solution II, the measured mean ΠD(I) of 3.1 corresponds to a free Ca2+ concentration in the gel phase of 9.3 mM (cf. the solution concentration of 3 mM). The bound Ca(II), i.e., the concentration of network-bridging Ca ions in the syneresed gel, follows as the averaged total, minus the free Ca(II), i.e., 55.5 mM - 9.3 mM ) 46.2 mM Ca(II) for this example. To arrive at the uncompensated charge density, the equivalent charge of the bound Ca(II)
TABLE 2. Calculated Donnan Potentials Based on Titration Data and Total Ca(II) Determinations calculated ΨDa solution I II III IV a
ΠD(I)
free Ca2+ (mM) in gel due to Donnan enrichment
[COO-]Tot ) 106 mM
[COO-]Tot ) 113 mM
[COO-]Tot ) 121 mM
2.5 3.1 4.2 3.2
7.5 9.3 12.6 9.6
-10 -12 -16 -11
-15 -16 -19 -15
-19 -20 -22 -19
ΨD is computed according to eqs 7-9 (27) of the Supporting Information.
FIGURE 2. Cd(II) speciation in alginate gels. Enrichment of Cd2+ relative to the bulk solution I at pH 5.6. Combined ICP-AES and voltammetric data reveal large differences in the bound and free Cd(II) concentrations. and of the bound Cd(II) is subtracted from the concentration of titratable sites (the bound Na and H can be neglected because of their insignificant contribution to the charge compensation). So, by example, for solution IV, we have 113 mM (titratable sites) - 91.8 mM [equivalent charge of bound Ca(II)] - 3.2 mM [equivalent charge of bound Cd(II); i.e., total Cd(II)gel - free Cd2+gel] ) 18 mM. Using this approach, lower and upper estimates of the Donnan potential were set by applying the results of each of the three independent titrations to yield a lower and upper bound of the uncompensated charge. These fixed charge densities are mainly attributed to the mannuronic acid residues, and we have used 106, 113, and 121 mM COO- (i.e., results of the individual titrations) as starting points for the estimation of the fixed charge densities. Results are collated in Table 2. Comparison of Estimates of the Donnan Potential. The calculated Donnan potentials (ΨD) for a total number of titratable groups ([COO-]) of 113 mM ranges from -15 to -19 mV. The range in estimates from the lower and upper bounds of fixed charge densities was calculated to be between -10 and -22 mV for all solutions. Inspection of the directly measured Donnan potentials in Table 1, for an ionic strength of 10 mM, reveals that the ΨD(E) values are slightly higher than those calculated in Table 2, while the ΨD(I) values in Table 1 are in good agreement with those computed from the fixed charge density. The average standard deviation of (6 mV for ΨD(E) places these estimates just within the range of the calculated Donnan potentials (Table 2).
We have thus achieved a self-consistent set of three independent estimates of Donnan potentials in calcium alginate gels for a fixed ionic strength of 10 mM. First, we calculated the Donnan potential by the Boltzmann equation using the measured Donnan partition coefficient, ΠD(I). Second, we performed direct measurements in the gel phase by comparing the half-wave potentials of the free Cd2+ ion in the solution and gel phases. Third, we modeled the fixed charge density by using potentiometric titration and total metal data analysis to calculate the Donnan potential using the method of Oshima and Kondo (27). The agreement between the values obtained by these approaches underscores the utility of the in situ voltammetric approach for straightforward and direct measurement of the Donnan potential in biogels. Cd(II) Speciation in the Gel and Implications for Bioavailability. The overall picture of Cd(II) speciation is summarized in Figure 2 as exemplified by the results for solution I. The total Cd(II) concentration in the gel phase is 6.1 × 10-5 M, which represents an accumulation factor of 58 as compared to the surrounding solution. This degree of sequestration is a well-characterized feature of alginates or alginate-based biomaterials and has been previously documented in the literature (14, 28) for Cd and other divalent cations (13, 29). It has also been reported (30) that the selectivity for divalent metals increases with an increase in guluronic acid content of the alginate (FGG). Calcium and cadmium have nearly the same ionic radius (i.e., 0.100 and VOL. 42, NO. 19, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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0.095 nm, respectively, for a 6-fold coordination), and their reported similar affinity under equilibrium conditions (28) can be attributed to the importance of steric placement in the alginate gel network. The cavity formed by the alignment of two chain sections of guluronic acid results in a multidentate environment that consists of the carboxylate groups themselves, the ring oxygen, and the hydroxyl groups of the G residues, all of which contribute to the binding of the divalent cations (31). In this work, a large disproportion between Ca and Cd concentrations in solution exists. Therefore, any Cd replacing Ca in juncture zones likely does not contribute significantly to gel stabilization. The fact that homopolymers of mannuronic acid and guluronic acid both bind Ca2+ with different dissociation constants of 2 × 10-4 and 1 × 10-3 M (32), respectively, indicates that it is reasonable to assume that calcium-mannuronic complexes are not relevant for the discussion. Cd(II), on the other hand, with its reportedly higher affinity (14, 28, 33) may plausibly bind to both homopolymeric regions of the alginate polymer. The voltammetric measurement of the free Cd2+ metal concentration in the gel [i.e., ΠD(I) ) 2.5 (Figure 2)] yields a [Cd2+]gel value of 2.6 × 10-6 M. This enhanced free Cd2+ concentration is distinct from the level of Cd(II) bound to the alginate polymer chains. A comparison of the total bound Cd(II) to the free Cd2+ in the gel phase [Cd(II)total/Cd(II)free] yields a ratio of 23 for solution I (Figure 2). That is, we can distinguish Cd(II) speciation in the gel phase, comprising ions that are bound and possibly biologically inert, and those which are free and mobile in the gel phase. This important finding has obvious consequences for translations of metal speciation measured in an exposure medium, under conditions prevailing within a biogel. In this context, let us now briefly extrapolate our results to a medium containing metal complex species. We select an exposure solution composition comprising a total Cd(II) concentration of 1 × 10-5 M and a complexing agent Y4- such that only 1% of Cd(II), i.e., 10-7 M, is in the free metal ion form whereas 99%, i.e., 0.99 × 10-5 M, is present as the CdY2- complex (the ligand Y could be any common multidentate ligand, e.g., EDTA). For a cell wall with a Donnan potential of approximately -30 mV (34), this would imply that Cd2+ would be enriched by a factor of ∼10, i.e., to 10-6 M, whereas the CdY2- concentration in the cell wall would be electrostatically reduced by a factor of 10; that is, its concentration would also be approximately 10-6 M. For conditions under which the Free Ion Activity Model (35) applies, this would mean that the negative cell wall enhances the bioavailable free Cd2+ by a factor of 10 and hence may increase its biouptake rate (36). The differences in free Cd2+ concentration between the bulk solution and cell wall turn bioaffinities, derived on the basis of solution concentrations, into apparent ones. For example, in the linear regime of the Michaelis-Menten uptake rate equation, the real bioaffinity of Cd2+ would be a factor of 10 lower than its apparent value as related to the Cd2+ concentration in solution. We assume that the CdY2complex is not bioactive itself and thus can only contribute to biouptake via dissociation into free Cd2+ inside the diffusion layer, provided it is sufficiently labile. This indirect bioavailability of CdY2- is diminished by the largely reduced concentration level of approximately 10-6 M inside the cell wall but possibly enhanced by the increased lability. This simple numerical example clearly demonstrates that the speciation and bioavailability of metal species within a given exposure medium can be dramatically dependent on local conditions in the cell wall. With this in mind, translation of speciation in the medium outside the cell wall into estimates of bioavailability should be done with great care. Clearly, more information and further study are required to further characterize the various types of polysaccharide gels which form the protective layers of microorganisms. In 7246
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particular, estimates of the Donnan potentials and their effects on the enrichment of bioavailable species must be conducted with the full spectrum of contributing variables such as pH, ionic strength, and, eventually, complexing ligands. The possibility of examining such systems in situ for a wide variety of polysaccharide gels opens up new avenues in the determination and investigation of the underlying mechanisms that govern the biouptake processes of microorganisms and plants.
Acknowledgments This work was made possible by funding in the form of a postdoctoral fellowship awarded to T.A.D. by “Le Fond Que´be´cois de la Recherche sur la nature et les Technologies”, a granting agency of the Province of Que´bec, Canada. Additional funding was provided within the framework of European Commission Projects BIOSPEC (Contract EVK1CT-2001-00086) and ECODIS (Contract 518043). We extend our gratitude to Profs. Bjorn Sundby and George W. Luther, III, and Ce´dric Magen for introducing T.A.D. to the application of gold amalgam microelectrodes, including their construction, their maintenance, and stimulating discussions. W. F. Threels and R. A. J. Wegh of Wageningen University are thanked for their technical assistance and expertise. Dr. L. Yezek is thanked for scientific discussions on the topic. We are also grateful to Dr. Synnøve Holtan, for instruction in the preparation of the homogeneous calcium alginate gels as well as discussions with Dr. Kurt I. Draget and Prof. Olav Smidsrød (Department of Biotechnology, NTNU, Trondheim, Norway).
Supporting Information Available Details of the alginate gel preparation and the voltammetric measurement of the Donnan potential and Donnan partition coefficient, calculation of the Donnan potential for the alginate gels characterized in this work, and representative potentiometric titration results. This material is available free of charge via the Internet at http://pubs.acs.org.
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