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Experimental study of the effect of EDTA on Cd adsorption by Bacillus subtilis: a test of the chemical equilibrium approach
Chemical Geology 161 Ž1999. 375–383 www.elsevier.comrlocaterchemgeo
Experimental study of the effect of EDTA on Cd adsorption by Bacillus subtilis: a test of the chemical equilibrium approach Jeremy B. Fein ) , Deirdre Delea CiÕil Engineering and Geological Sciences, UniÕersity of Notre Dame, Notre Dame, IN 46556, USA Received 5 January 1998; accepted 14 September 1998
Abstract The interactions between aqueous metals, organic acids, and bacteria can significantly affect mass transport in many geologic settings. This study examines the Cd–EDTA–Bacillus subtilis system to investigate these interactions, and to test the applicability of a chemical equilibrium approach to quantify aqueous and surface complexation reactions that occur in the system. The experiments indicate that fully-protonated EDTA adsorbs onto the bacterial surface through hydrophobic interaction, and that adsorption of the deprotonated EDTA molecule is negligible. Aqueous EDTA can strongly compete with the bacterial surface for aqueous Cd, and the presence of aqueous EDTA significantly diminishes Cd adsorption onto B. subtilis. Independent of the experimental measurements, we use chemical equilibrium modeling to estimate the extent of Cd adsorption in the Cd–EDTA–bacteria systems. The observed adsorption behavior is in excellent agreement with the estimations, suggesting that chemical equilibrium modeling can successfully account for the distribution of mass in a system in which both aqueous and surface metal–organic complexation occurs. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Adsorption; Bacteria; Cadmium; Organic acids
1. Introduction Adsorption of aqueous metal cations onto bacterial surfaces can significantly affect metal speciation and transport in near-surface water–rock systems. Recently, Fein et al. Ž1997. demonstrated that a chemical equilibrium model of proton and metal adsorption onto bacterial surfaces can be used to quantify metal–bacteria adsorption reactions in simplified systems. In theory, if the chemical equilibrium model is effective, then metal speciation in a more complex multi-component system can be esti)
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mated based on equilibrium constants for each of the individual reactions that occur in the system. The objective of this study is to test the accuracy of the chemical equilibrium approach by using equilibrium constants obtained from studies of isolated reactions to estimate the extent of metal adsorption in a mixed metal–organic–bacteria system. Aqueous systems containing dissolved metals, dissolved organic acids, and bacteria are common in both natural and contaminated geologic environments. For instance, sedimentary basin diagenetic fluids can contain significant concentrations of carboxylic acid anions and dissolved rock-forming cations Že.g., MacGowan and Surdam, 1988; Land and Macpherson, 1992., and bacteria have been found
0009-2541r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 5 4 1 Ž 9 9 . 0 0 1 1 6 - 3
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J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
at depths as great as 8 km in these systems ŽGhiorse and Wobber, 1989.. Relatively shallow groundwater contamination by radionuclides and carboxylic Žand other organic. acids, including EDTA Žthe tetra-functional carboxylic acid studied here., has occurred at several DOE sites ŽRiley et al., 1992.. Dissolved organic acid anions can either augment metal mobilities through aqueous metal–organic complexation Že.g., Means et al., 1978., or they can enhance adsorption through the formation of ternary metal– organic-surface complexes Že.g., Schindler, 1990; Boily and Fein, 1996.. A chemical equilibrium model of a water–rock system can account for these effects in a wide range of fluid conditions by enabling determination of metal and organic acid speciation based on equilibrium constant values for individual reactions. In this study, we examine Cd–EDTA– bacteria interactions. To estimate Cd and EDTA adsorption onto bacterial surfaces, we must know: Ž1. the equilibrium constant values for reactions that occur between aqueous Cd and all other dissolved species, including EDTA; Ž2. equilibrium constant values for Cd–bacteria interactions and for EDTA– bacteria interactions; and Ž3. deprotonation constants for both EDTA and the individual bacterial surface functional groups. Each of the above equilibrium constant values has been previously determined, except for the one quantifying the extent of EDTA– bacteria interaction. Therefore, in this study we measure the extent of EDTA adsorption onto bacterial surfaces to obtain an equilibrium constant value for the adsorption reaction. We use the results to estimate the extent of Cd adsorption that occurs in a mixed Cd–EDTA–Bacillus subtilis system. Comparison of measured Cd adsorption onto bacterial surfaces in EDTA-bearing solutions relative to that calculated using the chemical equilibrium approach provides a test of the applicability of the approach to metal–organic–bacteria systems. If the estimated extent of adsorption is lower than that observed in the experiments, it indicates that either the thermodynamic database is incorrect or that an additional process, such as bacterial surface site blockage by adsorbed EDTA, leads to diminished Cd adsorption. Conversely, if the estimated extent of Cd adsorption in EDTA systems is significantly higher than that observed, it could be caused by the formation of an additional Cd bacterial surface complex,
likely one involving EDTA. This would not be a failure of the chemical equilibrium approach, but rather a demonstration that all important equilibria must be quantified for the approach to work. Experiments conducted as a function of EDTA:Cd ratio can constrain the stoichiometry and thermodynamic properties of a surface Cd–EDTA–bacteria complex, if such a species is found to exist. These experiments are not designed to directly simulate a specific realistic geologic system, but rather to examine the dominant interactions that occur between aqueous metals, aqueous multi-functional carboxylic acids, and bacterial surfaces. If the chemical equilibrium approach to quantifying metal and organic acid adsorption onto bacterial surfaces is successful, then estimations of realistically complex systems can be built from studies that determine equilibrium constants of each of the important reactions that occur in the system. This approach requires a large number of component studies of isolated equilibria, but the approach offers the flexibility of estimating the effects of bacteria on metal and organic transport in a wide range of fluid–rock systems of geologic and environmental interest.
2. Experimental procedures We conducted batch adsorption experiments in which a known initial concentration of Cd andror EDTA was placed in contact with a known mass of bacteria in a test tube at room temperature. 0.1 M NaClO4 was used as the background electrolyte for each experiment, providing a constant ionic strength, non-complexing medium. The pH of each solution was adjusted to desired values by adding HNO 3 or NaOH. The quantities added and total concentrations attained were such that NaClO4 remained the dominant electrolyte, and therefore possible mixed electrolyte effects are neglected in our data treatment. The test tubes were shaken, and allowed to equilibrate for 60 min Žwith equilibration time determined from preliminary kinetics measurements.. After equilibration, the solution was separated from the bacteria through centrifugation and filtration through 0.45 mm membranes. The final pH was measured, and the solution was analyzed for dissolved Cd andror EDTA. The difference between the initial and the
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
final concentrations is the amount adsorbed onto the bacterial surface. Control experiments were run using bacteria-free experiments to ensure that adsorption onto test tube walls, syringe walls, filtration membranes, and sample bottle walls was negligible. The experiments were conducted with an isolated species of aerobic, gram-positive bacteria, B. subtilis. The bacteria were cultured and prepared in a manner similar to that described by Fein et al. Ž1997., except the EDTA wash was conducted for only 1 h instead of overnight. The pretreatment washing procedure ensures that cations and anions present in the growth medium are removed from the experimental systems and cannot complicate the interactions occurring during the experiments. Control desorption experiments were also conducted to ensure that EDTA from the pretreatment washing procedure was completely removed from the cell walls. The wash supernatant solutions were analyzed for EDTA after the EDTA pretreatment wash, and EDTA was not detected after the second electrolyte rinse. Our previous experiments with these techniques indicate that the wash procedures leave the cells fully intact and viable, but not undergoing active metabolism. Washed cells are whole, have not formed spores, and our previous results have demonstrated the effectiveness of the procedures in cleansing the cell surface of interfering ions. Although natural systems contain a mix of vegetative cells, spores, cell fragments, etc., we can only begin the application of surface complexation theory by studying the most simple systems possible. Metabolic processes may influence the extent of adsorption due to a proton flux through the cell wall Že.g., Urrutia Mera et al., 1992., but examination of this effect must be based on a better understanding of the purely abiotic processes involving cell wall functional groups. B. subtilis was used in the experiments because it represents a commonly-occurring type of subsurface bacteria. Gram-positive bacteria, such as B. subtilis, represent a major subgroup of bacteria, many of which are found in subsurface aquifers. Therefore, the experiments involved an important component of subsurface bacterial populations. Furthermore, the cell wall structure of B. subtilis is well-characterized through bioassay studies ŽBeveridge and Murray, 1976, 1980., and the deprotonation constants of the surface organic acid functional groups, and the corre-
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sponding surface site concentrations, have been measured ŽFein et al., 1997.. After culturing and the pretreatment rinses, the bacteria used in the experiments were separated from solution by centrifugation only, at 7500 rpm for 60 min. This is the technique used by Fein et al. Ž1997. to obtain bacterial weights to determine functional group site concentrations per gram of bacteria. If a heat-drying technique is used, a different mass of bacteria would be obtained Ždue to loss of cytoplasm fluid upon heating.. Therefore, it should be noted that the site concentration for surface functional groups is dependent on the drying procedure. We have conducted a series of tests: first centrifuging bacteria using the above technique, weighing them, then heating them to complete dryness, and reweighing the bacteria. The results of these tests indicates a centrifugedrheated weight ratio of 9.9 " 1.1. Aqueous Cd analyses were conducted using a flame atomic absorption spectrophotometer. The samples for Cd analysis were acidified with concentrated HNO 3 to a pH - 2.0, and diluted to the linear dynamic range of the instrument, usually a 15-times dilution by weight. The Cd standards were made with the same electrolyte composition and concentration as the experimental solutions to eliminate possible matrix effects on the analyses. Analytical uncertainties, determined by repeat analyses, were approximately "5%, with a detection limit of approximately 0.1 ppm. Aqueous EDTA concentrations were determined by high performance liquid chromatography, also with matrix-matched standards. Uncertainties associated with the analysis of EDTA were approximately "10%. Cd was introduced into the experimental solutions from a commercial atomic absorption aqueous standard solution. EDTA was reagent grade EDTA disodium salt. All water used in the experiments and analyses had an electrical resistance of 18 M V. Five series of experiments were conducted, and starting conditions are listed in Table 1. EDTA-only experiments were conducted to determine the extent of interaction between aqueous EDTA and the bacterial surface. Preliminary experiments indicated that minimal interaction occurs. To enhance the observed effects, these experiments used a bacterial concentration of 70 g of bacteriarl, and covered the pH range
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
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Table 1 Experimental starting conditions. NPs‘None Present’ Series
Cd molality
EDTA molality
g B. subtilisrl
1 2 3 4 5
NP 10y4 10y4 10y4 10y4
10y3 3=10y5 10y3 10y2 NP
70 10 10 10 10
of 1.5 to 10.0. Four sets of experiments were conducted at different EDTA:Cd ratios, ranging from ratios of 0 ŽCd-only experiments. to 100. These experiments were conducted at pH values below 8.0 to avoid precipitation of Cd from solution.
3. Experimental results The observed EDTA and Cd adsorption behaviors as a function of pH are depicted in Figs. 1 and 2. Series 1 results for the Cd-free system indicate that under most pH conditions, there is negligible adsorption of aqueous EDTA onto the bacterial surface. With decreasing pH below approximately pH 4.0, however, adsorption of EDTA becomes significant, increasing to at least 40% of the total EDTA being adsorbed onto the bacterial surface at pH 1.5.
Fig. 1. Results from Series 1 experiments. The dashed curve represents the best-fitting model of fully protonated EDTA molecule adsorption onto a protonated bacterial surface site Žall site types yield identical fits to the data..
Fig. 2. Results from Series 2 Žfilled squares., 3 Žfilled circles., and 4 Žopen squares. experiments. The thin solid line represents the independently estimated extent of Cd adsorption based on previously-determined stability constants for aqueous and bacterial surface complexes. The dashed curve represents the best-fitting model to the experimental data, calculated by adjusting the value of K Ž5. .
Fig. 2 illustrates a range of Cd adsorption behavior, depending on the EDTA content of the experimental solution. In solutions containing no aqueous EDTA, the observed extent of Cd adsorption onto bacterial surfaces is similar to that observed for identical Cd and bacterial concentrations in 0.1 M NaNO 3 by Fein et al. Ž1997.. That is, adsorption is minimal under low pH conditions where bacterial surface functional groups are fully protonated and neutrally-charged. With increasing pH, the functional groups deprotonate, becoming negatively-charged, and Cd adsorption increases. With increasing pH in the EDTA-free solutions, the extent of Cd adsorption under the experimental conditions reaches a plateau at nearly 100%. With increasing concentration of aqueous EDTA, however, the maximum extent of adsorption decreases. For the experiments containing 3 = 10y5 molal total EDTA, the maximum observed extent of Cd adsorption is approximately 60% of the total Cd. The experiments involving 10y3 and 10y2 molal total EDTA both exhibit insignificant Cd adsorption throughout the pH range examined, even at the highest pH conditions where the bacterial surface is highly negatively-charged due to full deprotonation of surface carboxyl and phosphate sites.
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
4. Thermodynamic analysis and discussion 4.1. EDTA adsorption EDTA exhibits adsorption behavior onto the bacterial surface that appears to be controlled by the speciation of the aqueous EDTA molecule. EDTA is a tetracarboxylic acid, with negative log dissociation constant Žp K a . values of 1.9, 2.7, 6.3, and 11.0 ŽMartell and Smith, 1982.. EDTA adsorption onto B. subtilis is likely a hydrophobic interaction between fully protonated EDTA Žwhich becomes the dominant species at pH values below 1.9. and the bacterial surface. With increasing pH and the associated development of negative charge on both the EDTA molecule and the bacterial surface, electrostatic repulsion between aqueous EDTA and the bacterial surface overcomes the hydrophobic attraction, and adsorption diminishes. This same phenomenon was observed for experiments that measured 2,4,6-trichlorophenol ŽTCP. adsorption onto B. subtilis ŽDaughney and Fein, 1998a.. A comparison between EDTA and TCP adsorption onto B. subtilis is depicted in Fig. 3. The p K a value for TCP is 6.0 ŽCallahan et al., 1979.. Fig. 3 illustrates that the
Fig. 3. Best-fitting adsorption onto the the p K a value for Daughney and Fein
models of TCP Žtop. and EDTA Žbottom. B. subtilis surface. The dashed lines indicate each organic acid. The TCP data is that of Ž1998a..
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balance between electrostatic and hydrophobic interactions appears to control the adsorption behavior of these two organic acids. That is, for both organic acids, adsorption is relatively high and constant at pH values less than the p K a of the acid, and adsorption decreases significantly at pH values above the p K a. Daughney and Fein Ž1998a. model TCP adsorption onto the bacterial surface using a site-specific surface complexation model, and we apply a similar approach to the EDTA adsorption data. We use FITEQL ŽWestall, 1982. to determine the equilibrium constant for the following generic adsorption reaction: H x EDTAŽ xy4. q R-AH Ž yy1. Ž xqyy5. ym R-AH y y H x EDTA
Ž 1.
where EDTAy4 represents the fully deprotonated EDTA anion, and R-Ay represents a deprotonated bacterial surface organic acid functional group. Values for x and y in reaction Ž1. must be integers between 0 and 4, and 0 and 1, respectively. B. subtilis exhibits three proton-active surface functional groups in the pH range of the study: carboxyl Žwith a p K a of 4.8., phosphate Žwith a p K a of 6.9., and hydroxyl Žwith a p K a of 9.4. ŽFein et al., 1997.. We attempt to model the data using Žsequentially. each surface functional group type and x, y value combination, and we use FITEQL to determine the model that best-fits the data. The modeling employs stability constants for the aqueous Cd-hydroxide complexes from Baes and Mesmer Ž1976., and stability constants for other aqueous complexes in the Na–NO 3 –OH–Hq system from Wolery Ž1992.. Similar to the treatment used by Daughney and Fein Ž1998a. for TCP adsorption, we invoke a constant capacitance model, with a surface capacitance of 8.0 F my2 , to account for the bacterial surface electric field effects on adsorption. For each model considered, FITEQL calculates the goodness of fit to the data and calculates an overall variance, V Ž Y .. We use these V Ž Y . values to determine the model which best fits the data. Reaction Ž1. provides an excellent fit to the experimental data when x s 4 and y s 1. That is, the data are best characterized by adsorption of fully proto-
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J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
nated EDTA onto a protonated surface functional group. Because the only significant adsorption occurs in the pH range where all three surface functional groups are effectively fully protonated, we cannot distinguish the type of site involved in the adsorption reaction. The three different groups provide identical fits to the data. However, the data clearly indicate that EDTA adsorbs as a fully protonated molecule. V Ž Y . values decrease Žand, hence, goodness of fit increases. with increasing protonation of the EDTA molecule. For models involving H 2 EDTA, V Ž Y . are in excess of 6900; for H 3 EDTA, V Ž Y . values are approximately 1000; and for H 4 EDTA, they equal 290 for each of the different surface functional group models. For the carboxyl, phosphate, and hydroxyl models, the calculated log K value for reaction Ž1. is 2.4, 2.9, and 2.7, respectively. Although a V Ž Y . value of 290 is high compared with that obtained for models of metal adsorption onto mineral surfaces Že.g., Gunneriusson et al., 1994; Waite et al., 1994; Ali and Dzombak, 1996; Boily and Fein, 1996., it is due to the relatively large experimental and analytical uncertainties associated with these experiments. The model EDTA adsorption behavior is shown as a dashed curve in Fig. 1. The model provides excellent agreement with the observed adsorption behavior, suggesting that the chemical equilibrium approach is successful in quantifying the extent of EDTA adsorption onto the bacterial surface as a function of pH. As depicted in Fig. 3, organic acid Žat least TCP and EDTA. adsorption onto bacterial surfaces depends on the acid speciation. This relationship can be quantified by relating the acid dissociation constant Ž K a . to the K value of the adsorption reaction Žreaction 1.. A proportional correlation can be drawn between K a and K Ž1. for TCP and EDTA, but because bacterial adsorption of organic acids has been examined for TCP and EDTA only, the exact relationship between K a and K Ž1. cannot be rigorously constrained yet. However, a similar type of relationship has been documented for organic acid adsorption onto mineral surfaces ŽKummert and Stumm, 1980.. This type of correlation, once constrained, could enable estimation of K Ž1. values, and hence the extent of adsorption onto bacterial surfaces, for organic acids whose bacterial adsorption has not been studied experimentally.
4.2. Cd adsorption The presence of EDTA can significantly diminish the extent of Cd adsorption onto the B. subtilis surface. This is most likely caused by the formation of aqueous Cd–EDTA complexes, as described by the following equilibria: Cd 2qq EDTA4ym Cd Ž EDTA.
2y
Ž 2. y
Cd 2qq HEDTA3ym Cd Ž HEDTA. .
Ž 3.
These complexes have high thermodynamic stabilities ŽMartell and Smith, 1982., and serve to sequester Cd in the aqueous phase, away from surface complexation with the negatively charged bacterial surface functional groups. We can estimate the speciation and distribution of Cd between the aqueous phase and the bacterial surface if equilibrium constants are known for each important equilibrium that involves Cd. The Series 1 data constrain the value of the equilibrium constant for reaction Ž1.; K values for reactions Ž2. and Ž3. are compiled by Martell and Smith Ž1982.; and the data of Fein et al. Ž1997. yield K values for Cd adsorption onto the carboxyl and phosphate sites on the bacterial surface, as expressed by the following equilibria: Cd 2qq R-COOym R-COO Ž Cd . q
Cd 2qq R-POym R-PO Ž Cd . .
q
Ž 4. Ž 5.
Fig. 2 shows the estimated extent of Cd adsorption on the B. subtilis surface in the EDTA-bearing systems as a function of pH. For the systems with the two highest EDTA concentrations, the chemical equilibrium model predicts that over 99% of the total Cd in the system will remain in solution as either Cd 2q or one of the Cd–EDTA aqueous complexes, depending on the pH of interest. In excellent agreement with the thermodynamic model, the experiments indicate that under these conditions, virtually no Cd is adsorbed by the bacteria. At the lowest EDTA concentration studied Ž3 = 10y5 molal total EDTA., Cd–EDTA complexes dominate the EDTA budget, but there is only enough EDTA for Cd–EDTA complexes to represent at most 30% of the total Cd in the system. The chemical equilibrium model estimates that Cd adsorption will range from minimal adsorption at low pH ŽpH
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
3.0 and below., up to over 66% of the total Cd in the system at pH values of 6.0 and higher. The observed extent of Cd adsorption in the 3 = 10y5 molal total EDTA system is close to that predicted by the thermodynamic model Žsee Fig. 2 for a comparison., with slight discrepancies at pH 6.0 and higher where we observed a maximum of 60% of the total Cd adsorbed onto the bacterial surface. There are several possible causes for the discrepancy. One possibility is that the thermodynamic data upon which the chemical equilibrium model is based is inconsistent with the observed adsorption behavior. Another possibility is that the discrepancy is within the expected uncertainty of the estimation, given the uncertainties associated with the equilibrium constants that control the estimation. Alternatively, it is possible that the chemical equilibrium model fails to account for all biogeochemical processes occurring in the system, so it does not adequately characterize the ultimate fate of Cd in the experiments. Based on the reversibility of metal, proton, and organic acid uptake by bacterial surfaces ŽFein et al., 1997; Daughney and Fein, 1998a,b; Daughney et al., 1998., and the success of the chemical equilibrium approach in quantifying metal, proton, and organic acid adsorption onto bacteria, it is unlikely that the adsorption is significantly affected by additional bacterial processes. To determine if the observed discrepancy is within the uncertainty of the estimation technique, we use the data to calculate new equilibrium constant values for each important reaction involving Cd. Equilibria Ž2., Ž3., Ž4., and Ž5. have the most significant influence on Cd speciation under the experimental conditions. Of these, the interaction between Cd and the bacterial surface phosphate sites is the least well-constrained. We use the data from this study to calculate an independent value for K Ž5. , leaving equilibrium constant values for reactions Ž2., Ž3., and Ž4. fixed. This calculation yields a value of 4.8 for log K Ž5. , with a V Ž Y . value of 12.6. The value used for log K Ž5. in the original estimation was 5.4, with an uncertainty Žfactoring in the uncertainty of the acid dissociation constants of the bacterial phosphate group. of "0.8. Therefore, the discrepancy is within the uncertainty expected for the precision of the fundamental thermodynamic data. More accurate estimates of adsorption behavior, if required, would only be possible with more precise
381
determination of the stability constants for the pertinent surface complexes.
5. Geochemical implications The experiments in this study indicate that a chemical equilibrium approach can successfully account for metal and organic acid adsorption onto bacterial surfaces. This implies that a detailed estimation of the effects of bacterial adsorption on metal and organic speciation in water–rock systems is possible, once the pertinent equilibria are defined and the equilibrium constants determined. This is a large quantity of information, but the approach offers the ability to quantify the effects of bacteria that are similar in surface properties to the Bacillus sp. studied in a wide range of geochemical conditions. For example, we can determine a detailed Cd budget for a hypothetical contaminated groundwater system. In this calculation, we calculate the distribution of Cd between several potential reservoirs: Cd adsorbed to bacteria, Cd adsorbed to mineral surfaces directly, and Cd in the aqueous phase. Further, we determine the Cd speciation within the aqueous phase, and we calculate the amount of Cd adsorbed to immobile bacteria relative to that adsorbed to bacteria that are mobile in the system. This last estimation is based on experiments that quantify the extent of bacterial adhesion to a mineral surface under similar conditions to those of the calculation ŽYee and Fein, 1999.. In these experiments, we measured a distribution ratio of 4.0, where this ratio describes the relative concentration of bacteria free in solution and bacteria attached to aluminum mineral surface sites on Al 2 O 3 . Because these are the only experiments to quantify bacterial adhesion under similar conditions, we only account for adhesion to aluminum sites in our hypothetical system, and we assume a total aluminum mineral abundance of 35 grl, or an Al surface site concentration of 10y3 molal. The data used to quantify Cd adsorption onto aluminum mineral surface sites are those of Boily and Fein Ž1996.. Clearly, a more realistic example, with other rock-forming components, must await additional data on bacteria–mineral interactions. Fig. 4 shows the results of the calculation, and it illustrates the benefits of applying the chemical equi-
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J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
highlights the need for the flexibility of the chemical equilibrium approach. Although chemical equilibrium modeling requires a large collection of thermodynamic data, most of which has not yet been obtained, no other technique enables quantitative estimations of metal and organic speciation over a wide range of conditions in complex systems.
6. Conclusions
Fig. 4. Cd distribution in a Cd–EDTA–Al 2 O 3 system, calculated with Žblack bars. and without Žgray bars. the effects of bacteria, represented in terms of the percent of the total Cd in the system present in each Cd reservoir. The abundance of aqueous Cd–EDTA complexes is independent of bacterial content in the system, so bars of the same height are depicted. Note that the bacterial reservoirs have no Cd Žand hence no gray bars. associated with them for the bacteria-free calculations.
librium approach to bacteria–water–rock systems. The calculation assumes 10y4 molal total Cd in the system, 10y5 molal total EDTA, 0.5 g bacteria Ždry weight.rl of solution, an ionic strength of 0.1 molal, and a solution pH of 7.0. The figure shows that with bacteria present Žblack bars. under the conditions of the calculation, the Cd budget is dominated by adsorption onto bacterial surfaces, and that approximately 50% of the Cd is attached to mobile Žunattached. bacteria. This example underscores the potential importance of bacteria in affecting metal Žand organic. mobilities in the subsurface. Without bacteria present in the system, more Cd would be adsorbed onto the mineral surface sites, and more would be present in solution. The bacteria-free Cd budget is shown as the gray bars in the figure. Note that the overall mobility of Cd is drastically changed by the presence of the bacteria. In the bacteria-free system, over 60% of the total Cd is attached to the mineral surface, and thereby immobilized. With bacteria present, over 68% of the Cd is mobile, either as aqueous Cd, or attached to mobile bacteria. Clearly, there are many more possible mineral surface sites and fluid composition conditions to consider when modeling realistic subsurface Cd contamination. However, it is the vast number of conditions of geologic and environmental interest that
EDTA adsorbs only weakly to the surface of B. subtilis, with adsorption driven by the hydrophobicity of the fully-protonated EDTA molecule. With the onset of deprotonation of EDTA with increasing pH above pH 1.5, EDTA adsorption diminishes significantly, and there is negligible adsorption over most of the pH range studied. Despite the hydrophobic nature of the EDTA–bacterial surface interaction, the adsorption can be described using a chemical equilibrium approach, and the extent of adsorption can be quantified through the equilibrium constant value that is determined in this study. The presence of EDTA decreases Cd adsorption onto the bacterial surface, likely through aqueous Cd–EDTA complexation. Again, the data indicate that the chemical equilibrium approach can be used to successfully estimate the distribution of Cd in the mixed Cd– EDTA–bacteria system. The experimental results from this study illustrate one of the dominant effects of strongly-complexing organic acid anions on the adsorption behavior of metals onto bacterial surfaces. Aqueous complexation between organic acid anions and dissolved metal cations directly competes with bacterial surface complexation of the cations. The chemical equilibrium approach is an effective means for quantifying this competition. The distribution of metal between the aqueous phase and the bacterial surface can successfully be described based on thermodynamic modeling if the equilibrium constants for the pertinent complexation reactions are known. Although at this point, very few of these bacterial surface stability constants have been measured, this study suggests that this approach can ultimately be applied to quantify the effects of bacterial adsorption on metal and organic mobilities in water–rock systems of geologic and environmental interest.
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
Acknowledgements Acknowledgment is made to the Donors of The Petroleum Research Fund, administered by the American Chemical Society, for support of this research. Deirdre Delea was supported by National Science Foundation ŽResearch Experience for Undergraduates. grant EEC96-19509. We thank David Fowle, Nathan Yee, and Peter Wightman for technical assistance and advice during the course of the study, and we thank an anonymous journal reviewer for carefully examining the manuscript and making helpful suggestions for improvement.
References Ali, M.A., Dzombak, D.A., 1996. Effects of simple organic acids on sorption of Cu2q and Ca2q on goethite. Geochim. Cosmochim. Acta 60, 291–304. Baes, C.F., Jr., Mesmer, R.E., 1976. The Hydrolysis of Cations. Wiley, New York. Beveridge, T.J., Murray, R.G.E., 1976. Uptake and retention of metals by cell walls of Bacillus subtilis. J. Bacteriol. 127, 1502–1518. Beveridge, T.J., Murray, R.G.E., 1980. Sites of metal deposition in the cell wall of Bacillus subtilis. J. Bacteriol. 141, 876–887. Boily, J.-F., Fein, J.B., 1996. Experimental study of cadmium– citrate co-adsorption onto a-Al 2 O 3 . Geochim. Cosmochim. Acta 60, 2929–2938. Callahan, M.A., Slimak, M.W., Gabel, N.W., May, I.P., Fowler, C., Freed, J.R., Jennings, P., Durfee, R.L., Whitmore, F.C., Maestri, B., Mabey, W.R., Holt, B.R., Gould, C., 1979. Water-Related Environmental Fate of 129 Priority Pollutants. USEPA, Vols. I and II, EPA-440r4-79-029a and EPA-440r479-029b. Daughney, C.J., Fein, J.B., 1998a. Sorption of 2,4,6-trichlorophenol by Bacillus subtilis. Environ. Sci. Technol. 32, 749–752. Daughney, C.J., Fein, J.B., 1998b. The effect of ionic strength on the adsorption of Hq, Cd 2q, Pb 2q, and Cu2q by Bacillus subtilis and Bacillus licheniformis: a surface complexation model. J. Colloid Interface Sci. 198, 53–77. Daughney, C.J., Fein, J.B., Yee, N., 1998. A comparison of the thermodynamics of metal adsorption onto two common bacteria. Chem. Geol. 144, 161–176. Fein, J.B., Daughney, C.J., Yee, N., Davis, T., 1997. A chemical
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equilibrium model for metal adsorption onto bacterial surfaces. Geochim. Cosmochim. Acta 61, 3319–3328. Ghiorse, W.C., Wobber, F.J., 1989. Deep subsurface microbiology. Geomicrobiol. J., Vol. 7. Crane, Russak & Co., New York, 130 pp. Gunneriusson, L., Lovgren, L., Sjoberg, S., 1994. Complexation ¨ ¨ of PbŽII. at the goethite Ž a-FeOOH.rwater interface: the influence of chloride. Geochim. Cosmochim. Acta 58, 4973– 4983. Kummert, R., Stumm, W., 1980. The surface complexation of organic acids on hydrous g-Al 2 O 3 . J. Colloid Interface Sci. 75, 373–385. Land, L.S., Macpherson, G.L., 1992. Origin of saline formation waters, Cenozoic section, Gulf of Mexico sedimentary basin. Am. Assoc. Pet. Geol. Bull. 76, 1344–1362. MacGowan, D.B., Surdam, R.C., 1988. Difunctional carboxylic acid anions in oilfield waters. Org. Geochem. 12, 245–259. Martell, A.E., Smith, R.M., 1982. Critical Stability Constants, Vol. 5: First Supplement. Plenum, New York, USA. Means, J.L., Crerar, D.A., Duguid, J.O., 1978. Migration of radioactive wastes: radionuclide mobilization by complexing agents. Science 200, 1477–1481. Riley, R.G., Zachara, J.M., Wobber, F.J., 1992. Chemical contaminants on DOE lands and selection of contaminant mixtures for subsurface science research. DOErER-0547T, U.S. Dept. of Energy. Schindler, P.W., 1990. Co-adsorption of metal ions and organic ligands: formation of ternary surface complexes. In: Hochella, M.F., Jr., White, A.R. ŽEds.., Mineral–Water Interface Geochemistry. Mineral. Soc. Am., Washington, DC, Rev. Mineral. 23, pp. 281–308. Urrutia Mera, M., Kemper, M., Doyle, R., Beveridge, T.J., 1992. The membrane-induced proton motive force influences that metal binding ability of Bacillus subtilis cell walls. Appl. Environ. Microbiol. 58, 3837–3844. Waite, T.D., Davis, J.A., Payne, T.E., Waychunas, G.A., Xu, N., 1994. Uranium ŽVI. adsorption to ferrihydrite: application of a surface complexation model. Geochim. Cosmochim. Acta 58, 5465–5478. Westall, J.C., 1982. FITEQL, A computer program for determination of chemical equilibrium constants from experimental data, Version 2.0. Report 82-02, Dept. Chem., Oregon St. Univ., Corvallis, OR, USA. Wolery, T.J., 1992. EQ3NR, A computer program for geochemical aqueous speciation-solubility calculations: theoretical manual, user’s guide, and related documentation, Version 7.0. UCRL-MA-110662-PT-III, Lawrence Livermore Nat. Lab., Livermore, CA. Yee, N., Fein, J.B., 1999. Experimental study of the pH-, ionic strength-, and reversibility behavior of bacteria–mineral adsorption. Geochim. Cosmochim. Acta, submitted.
Experimental study of the effect of EDTA on Cd adsorption by Bacillus subtilis: a test of the chemical equilibrium approach Jeremy B. Fein ) , Deirdre Delea CiÕil Engineering and Geological Sciences, UniÕersity of Notre Dame, Notre Dame, IN 46556, USA Received 5 January 1998; accepted 14 September 1998
Abstract The interactions between aqueous metals, organic acids, and bacteria can significantly affect mass transport in many geologic settings. This study examines the Cd–EDTA–Bacillus subtilis system to investigate these interactions, and to test the applicability of a chemical equilibrium approach to quantify aqueous and surface complexation reactions that occur in the system. The experiments indicate that fully-protonated EDTA adsorbs onto the bacterial surface through hydrophobic interaction, and that adsorption of the deprotonated EDTA molecule is negligible. Aqueous EDTA can strongly compete with the bacterial surface for aqueous Cd, and the presence of aqueous EDTA significantly diminishes Cd adsorption onto B. subtilis. Independent of the experimental measurements, we use chemical equilibrium modeling to estimate the extent of Cd adsorption in the Cd–EDTA–bacteria systems. The observed adsorption behavior is in excellent agreement with the estimations, suggesting that chemical equilibrium modeling can successfully account for the distribution of mass in a system in which both aqueous and surface metal–organic complexation occurs. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Adsorption; Bacteria; Cadmium; Organic acids
1. Introduction Adsorption of aqueous metal cations onto bacterial surfaces can significantly affect metal speciation and transport in near-surface water–rock systems. Recently, Fein et al. Ž1997. demonstrated that a chemical equilibrium model of proton and metal adsorption onto bacterial surfaces can be used to quantify metal–bacteria adsorption reactions in simplified systems. In theory, if the chemical equilibrium model is effective, then metal speciation in a more complex multi-component system can be esti)
Corresponding author. Tel.: q1-219-631-6101; fax: q1-219631-9236; e-mail: [email protected]
mated based on equilibrium constants for each of the individual reactions that occur in the system. The objective of this study is to test the accuracy of the chemical equilibrium approach by using equilibrium constants obtained from studies of isolated reactions to estimate the extent of metal adsorption in a mixed metal–organic–bacteria system. Aqueous systems containing dissolved metals, dissolved organic acids, and bacteria are common in both natural and contaminated geologic environments. For instance, sedimentary basin diagenetic fluids can contain significant concentrations of carboxylic acid anions and dissolved rock-forming cations Že.g., MacGowan and Surdam, 1988; Land and Macpherson, 1992., and bacteria have been found
0009-2541r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 5 4 1 Ž 9 9 . 0 0 1 1 6 - 3
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at depths as great as 8 km in these systems ŽGhiorse and Wobber, 1989.. Relatively shallow groundwater contamination by radionuclides and carboxylic Žand other organic. acids, including EDTA Žthe tetra-functional carboxylic acid studied here., has occurred at several DOE sites ŽRiley et al., 1992.. Dissolved organic acid anions can either augment metal mobilities through aqueous metal–organic complexation Že.g., Means et al., 1978., or they can enhance adsorption through the formation of ternary metal– organic-surface complexes Že.g., Schindler, 1990; Boily and Fein, 1996.. A chemical equilibrium model of a water–rock system can account for these effects in a wide range of fluid conditions by enabling determination of metal and organic acid speciation based on equilibrium constant values for individual reactions. In this study, we examine Cd–EDTA– bacteria interactions. To estimate Cd and EDTA adsorption onto bacterial surfaces, we must know: Ž1. the equilibrium constant values for reactions that occur between aqueous Cd and all other dissolved species, including EDTA; Ž2. equilibrium constant values for Cd–bacteria interactions and for EDTA– bacteria interactions; and Ž3. deprotonation constants for both EDTA and the individual bacterial surface functional groups. Each of the above equilibrium constant values has been previously determined, except for the one quantifying the extent of EDTA– bacteria interaction. Therefore, in this study we measure the extent of EDTA adsorption onto bacterial surfaces to obtain an equilibrium constant value for the adsorption reaction. We use the results to estimate the extent of Cd adsorption that occurs in a mixed Cd–EDTA–Bacillus subtilis system. Comparison of measured Cd adsorption onto bacterial surfaces in EDTA-bearing solutions relative to that calculated using the chemical equilibrium approach provides a test of the applicability of the approach to metal–organic–bacteria systems. If the estimated extent of adsorption is lower than that observed in the experiments, it indicates that either the thermodynamic database is incorrect or that an additional process, such as bacterial surface site blockage by adsorbed EDTA, leads to diminished Cd adsorption. Conversely, if the estimated extent of Cd adsorption in EDTA systems is significantly higher than that observed, it could be caused by the formation of an additional Cd bacterial surface complex,
likely one involving EDTA. This would not be a failure of the chemical equilibrium approach, but rather a demonstration that all important equilibria must be quantified for the approach to work. Experiments conducted as a function of EDTA:Cd ratio can constrain the stoichiometry and thermodynamic properties of a surface Cd–EDTA–bacteria complex, if such a species is found to exist. These experiments are not designed to directly simulate a specific realistic geologic system, but rather to examine the dominant interactions that occur between aqueous metals, aqueous multi-functional carboxylic acids, and bacterial surfaces. If the chemical equilibrium approach to quantifying metal and organic acid adsorption onto bacterial surfaces is successful, then estimations of realistically complex systems can be built from studies that determine equilibrium constants of each of the important reactions that occur in the system. This approach requires a large number of component studies of isolated equilibria, but the approach offers the flexibility of estimating the effects of bacteria on metal and organic transport in a wide range of fluid–rock systems of geologic and environmental interest.
2. Experimental procedures We conducted batch adsorption experiments in which a known initial concentration of Cd andror EDTA was placed in contact with a known mass of bacteria in a test tube at room temperature. 0.1 M NaClO4 was used as the background electrolyte for each experiment, providing a constant ionic strength, non-complexing medium. The pH of each solution was adjusted to desired values by adding HNO 3 or NaOH. The quantities added and total concentrations attained were such that NaClO4 remained the dominant electrolyte, and therefore possible mixed electrolyte effects are neglected in our data treatment. The test tubes were shaken, and allowed to equilibrate for 60 min Žwith equilibration time determined from preliminary kinetics measurements.. After equilibration, the solution was separated from the bacteria through centrifugation and filtration through 0.45 mm membranes. The final pH was measured, and the solution was analyzed for dissolved Cd andror EDTA. The difference between the initial and the
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
final concentrations is the amount adsorbed onto the bacterial surface. Control experiments were run using bacteria-free experiments to ensure that adsorption onto test tube walls, syringe walls, filtration membranes, and sample bottle walls was negligible. The experiments were conducted with an isolated species of aerobic, gram-positive bacteria, B. subtilis. The bacteria were cultured and prepared in a manner similar to that described by Fein et al. Ž1997., except the EDTA wash was conducted for only 1 h instead of overnight. The pretreatment washing procedure ensures that cations and anions present in the growth medium are removed from the experimental systems and cannot complicate the interactions occurring during the experiments. Control desorption experiments were also conducted to ensure that EDTA from the pretreatment washing procedure was completely removed from the cell walls. The wash supernatant solutions were analyzed for EDTA after the EDTA pretreatment wash, and EDTA was not detected after the second electrolyte rinse. Our previous experiments with these techniques indicate that the wash procedures leave the cells fully intact and viable, but not undergoing active metabolism. Washed cells are whole, have not formed spores, and our previous results have demonstrated the effectiveness of the procedures in cleansing the cell surface of interfering ions. Although natural systems contain a mix of vegetative cells, spores, cell fragments, etc., we can only begin the application of surface complexation theory by studying the most simple systems possible. Metabolic processes may influence the extent of adsorption due to a proton flux through the cell wall Že.g., Urrutia Mera et al., 1992., but examination of this effect must be based on a better understanding of the purely abiotic processes involving cell wall functional groups. B. subtilis was used in the experiments because it represents a commonly-occurring type of subsurface bacteria. Gram-positive bacteria, such as B. subtilis, represent a major subgroup of bacteria, many of which are found in subsurface aquifers. Therefore, the experiments involved an important component of subsurface bacterial populations. Furthermore, the cell wall structure of B. subtilis is well-characterized through bioassay studies ŽBeveridge and Murray, 1976, 1980., and the deprotonation constants of the surface organic acid functional groups, and the corre-
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sponding surface site concentrations, have been measured ŽFein et al., 1997.. After culturing and the pretreatment rinses, the bacteria used in the experiments were separated from solution by centrifugation only, at 7500 rpm for 60 min. This is the technique used by Fein et al. Ž1997. to obtain bacterial weights to determine functional group site concentrations per gram of bacteria. If a heat-drying technique is used, a different mass of bacteria would be obtained Ždue to loss of cytoplasm fluid upon heating.. Therefore, it should be noted that the site concentration for surface functional groups is dependent on the drying procedure. We have conducted a series of tests: first centrifuging bacteria using the above technique, weighing them, then heating them to complete dryness, and reweighing the bacteria. The results of these tests indicates a centrifugedrheated weight ratio of 9.9 " 1.1. Aqueous Cd analyses were conducted using a flame atomic absorption spectrophotometer. The samples for Cd analysis were acidified with concentrated HNO 3 to a pH - 2.0, and diluted to the linear dynamic range of the instrument, usually a 15-times dilution by weight. The Cd standards were made with the same electrolyte composition and concentration as the experimental solutions to eliminate possible matrix effects on the analyses. Analytical uncertainties, determined by repeat analyses, were approximately "5%, with a detection limit of approximately 0.1 ppm. Aqueous EDTA concentrations were determined by high performance liquid chromatography, also with matrix-matched standards. Uncertainties associated with the analysis of EDTA were approximately "10%. Cd was introduced into the experimental solutions from a commercial atomic absorption aqueous standard solution. EDTA was reagent grade EDTA disodium salt. All water used in the experiments and analyses had an electrical resistance of 18 M V. Five series of experiments were conducted, and starting conditions are listed in Table 1. EDTA-only experiments were conducted to determine the extent of interaction between aqueous EDTA and the bacterial surface. Preliminary experiments indicated that minimal interaction occurs. To enhance the observed effects, these experiments used a bacterial concentration of 70 g of bacteriarl, and covered the pH range
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
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Table 1 Experimental starting conditions. NPs‘None Present’ Series
Cd molality
EDTA molality
g B. subtilisrl
1 2 3 4 5
NP 10y4 10y4 10y4 10y4
10y3 3=10y5 10y3 10y2 NP
70 10 10 10 10
of 1.5 to 10.0. Four sets of experiments were conducted at different EDTA:Cd ratios, ranging from ratios of 0 ŽCd-only experiments. to 100. These experiments were conducted at pH values below 8.0 to avoid precipitation of Cd from solution.
3. Experimental results The observed EDTA and Cd adsorption behaviors as a function of pH are depicted in Figs. 1 and 2. Series 1 results for the Cd-free system indicate that under most pH conditions, there is negligible adsorption of aqueous EDTA onto the bacterial surface. With decreasing pH below approximately pH 4.0, however, adsorption of EDTA becomes significant, increasing to at least 40% of the total EDTA being adsorbed onto the bacterial surface at pH 1.5.
Fig. 1. Results from Series 1 experiments. The dashed curve represents the best-fitting model of fully protonated EDTA molecule adsorption onto a protonated bacterial surface site Žall site types yield identical fits to the data..
Fig. 2. Results from Series 2 Žfilled squares., 3 Žfilled circles., and 4 Žopen squares. experiments. The thin solid line represents the independently estimated extent of Cd adsorption based on previously-determined stability constants for aqueous and bacterial surface complexes. The dashed curve represents the best-fitting model to the experimental data, calculated by adjusting the value of K Ž5. .
Fig. 2 illustrates a range of Cd adsorption behavior, depending on the EDTA content of the experimental solution. In solutions containing no aqueous EDTA, the observed extent of Cd adsorption onto bacterial surfaces is similar to that observed for identical Cd and bacterial concentrations in 0.1 M NaNO 3 by Fein et al. Ž1997.. That is, adsorption is minimal under low pH conditions where bacterial surface functional groups are fully protonated and neutrally-charged. With increasing pH, the functional groups deprotonate, becoming negatively-charged, and Cd adsorption increases. With increasing pH in the EDTA-free solutions, the extent of Cd adsorption under the experimental conditions reaches a plateau at nearly 100%. With increasing concentration of aqueous EDTA, however, the maximum extent of adsorption decreases. For the experiments containing 3 = 10y5 molal total EDTA, the maximum observed extent of Cd adsorption is approximately 60% of the total Cd. The experiments involving 10y3 and 10y2 molal total EDTA both exhibit insignificant Cd adsorption throughout the pH range examined, even at the highest pH conditions where the bacterial surface is highly negatively-charged due to full deprotonation of surface carboxyl and phosphate sites.
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
4. Thermodynamic analysis and discussion 4.1. EDTA adsorption EDTA exhibits adsorption behavior onto the bacterial surface that appears to be controlled by the speciation of the aqueous EDTA molecule. EDTA is a tetracarboxylic acid, with negative log dissociation constant Žp K a . values of 1.9, 2.7, 6.3, and 11.0 ŽMartell and Smith, 1982.. EDTA adsorption onto B. subtilis is likely a hydrophobic interaction between fully protonated EDTA Žwhich becomes the dominant species at pH values below 1.9. and the bacterial surface. With increasing pH and the associated development of negative charge on both the EDTA molecule and the bacterial surface, electrostatic repulsion between aqueous EDTA and the bacterial surface overcomes the hydrophobic attraction, and adsorption diminishes. This same phenomenon was observed for experiments that measured 2,4,6-trichlorophenol ŽTCP. adsorption onto B. subtilis ŽDaughney and Fein, 1998a.. A comparison between EDTA and TCP adsorption onto B. subtilis is depicted in Fig. 3. The p K a value for TCP is 6.0 ŽCallahan et al., 1979.. Fig. 3 illustrates that the
Fig. 3. Best-fitting adsorption onto the the p K a value for Daughney and Fein
models of TCP Žtop. and EDTA Žbottom. B. subtilis surface. The dashed lines indicate each organic acid. The TCP data is that of Ž1998a..
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balance between electrostatic and hydrophobic interactions appears to control the adsorption behavior of these two organic acids. That is, for both organic acids, adsorption is relatively high and constant at pH values less than the p K a of the acid, and adsorption decreases significantly at pH values above the p K a. Daughney and Fein Ž1998a. model TCP adsorption onto the bacterial surface using a site-specific surface complexation model, and we apply a similar approach to the EDTA adsorption data. We use FITEQL ŽWestall, 1982. to determine the equilibrium constant for the following generic adsorption reaction: H x EDTAŽ xy4. q R-AH Ž yy1. Ž xqyy5. ym R-AH y y H x EDTA
Ž 1.
where EDTAy4 represents the fully deprotonated EDTA anion, and R-Ay represents a deprotonated bacterial surface organic acid functional group. Values for x and y in reaction Ž1. must be integers between 0 and 4, and 0 and 1, respectively. B. subtilis exhibits three proton-active surface functional groups in the pH range of the study: carboxyl Žwith a p K a of 4.8., phosphate Žwith a p K a of 6.9., and hydroxyl Žwith a p K a of 9.4. ŽFein et al., 1997.. We attempt to model the data using Žsequentially. each surface functional group type and x, y value combination, and we use FITEQL to determine the model that best-fits the data. The modeling employs stability constants for the aqueous Cd-hydroxide complexes from Baes and Mesmer Ž1976., and stability constants for other aqueous complexes in the Na–NO 3 –OH–Hq system from Wolery Ž1992.. Similar to the treatment used by Daughney and Fein Ž1998a. for TCP adsorption, we invoke a constant capacitance model, with a surface capacitance of 8.0 F my2 , to account for the bacterial surface electric field effects on adsorption. For each model considered, FITEQL calculates the goodness of fit to the data and calculates an overall variance, V Ž Y .. We use these V Ž Y . values to determine the model which best fits the data. Reaction Ž1. provides an excellent fit to the experimental data when x s 4 and y s 1. That is, the data are best characterized by adsorption of fully proto-
380
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
nated EDTA onto a protonated surface functional group. Because the only significant adsorption occurs in the pH range where all three surface functional groups are effectively fully protonated, we cannot distinguish the type of site involved in the adsorption reaction. The three different groups provide identical fits to the data. However, the data clearly indicate that EDTA adsorbs as a fully protonated molecule. V Ž Y . values decrease Žand, hence, goodness of fit increases. with increasing protonation of the EDTA molecule. For models involving H 2 EDTA, V Ž Y . are in excess of 6900; for H 3 EDTA, V Ž Y . values are approximately 1000; and for H 4 EDTA, they equal 290 for each of the different surface functional group models. For the carboxyl, phosphate, and hydroxyl models, the calculated log K value for reaction Ž1. is 2.4, 2.9, and 2.7, respectively. Although a V Ž Y . value of 290 is high compared with that obtained for models of metal adsorption onto mineral surfaces Že.g., Gunneriusson et al., 1994; Waite et al., 1994; Ali and Dzombak, 1996; Boily and Fein, 1996., it is due to the relatively large experimental and analytical uncertainties associated with these experiments. The model EDTA adsorption behavior is shown as a dashed curve in Fig. 1. The model provides excellent agreement with the observed adsorption behavior, suggesting that the chemical equilibrium approach is successful in quantifying the extent of EDTA adsorption onto the bacterial surface as a function of pH. As depicted in Fig. 3, organic acid Žat least TCP and EDTA. adsorption onto bacterial surfaces depends on the acid speciation. This relationship can be quantified by relating the acid dissociation constant Ž K a . to the K value of the adsorption reaction Žreaction 1.. A proportional correlation can be drawn between K a and K Ž1. for TCP and EDTA, but because bacterial adsorption of organic acids has been examined for TCP and EDTA only, the exact relationship between K a and K Ž1. cannot be rigorously constrained yet. However, a similar type of relationship has been documented for organic acid adsorption onto mineral surfaces ŽKummert and Stumm, 1980.. This type of correlation, once constrained, could enable estimation of K Ž1. values, and hence the extent of adsorption onto bacterial surfaces, for organic acids whose bacterial adsorption has not been studied experimentally.
4.2. Cd adsorption The presence of EDTA can significantly diminish the extent of Cd adsorption onto the B. subtilis surface. This is most likely caused by the formation of aqueous Cd–EDTA complexes, as described by the following equilibria: Cd 2qq EDTA4ym Cd Ž EDTA.
2y
Ž 2. y
Cd 2qq HEDTA3ym Cd Ž HEDTA. .
Ž 3.
These complexes have high thermodynamic stabilities ŽMartell and Smith, 1982., and serve to sequester Cd in the aqueous phase, away from surface complexation with the negatively charged bacterial surface functional groups. We can estimate the speciation and distribution of Cd between the aqueous phase and the bacterial surface if equilibrium constants are known for each important equilibrium that involves Cd. The Series 1 data constrain the value of the equilibrium constant for reaction Ž1.; K values for reactions Ž2. and Ž3. are compiled by Martell and Smith Ž1982.; and the data of Fein et al. Ž1997. yield K values for Cd adsorption onto the carboxyl and phosphate sites on the bacterial surface, as expressed by the following equilibria: Cd 2qq R-COOym R-COO Ž Cd . q
Cd 2qq R-POym R-PO Ž Cd . .
q
Ž 4. Ž 5.
Fig. 2 shows the estimated extent of Cd adsorption on the B. subtilis surface in the EDTA-bearing systems as a function of pH. For the systems with the two highest EDTA concentrations, the chemical equilibrium model predicts that over 99% of the total Cd in the system will remain in solution as either Cd 2q or one of the Cd–EDTA aqueous complexes, depending on the pH of interest. In excellent agreement with the thermodynamic model, the experiments indicate that under these conditions, virtually no Cd is adsorbed by the bacteria. At the lowest EDTA concentration studied Ž3 = 10y5 molal total EDTA., Cd–EDTA complexes dominate the EDTA budget, but there is only enough EDTA for Cd–EDTA complexes to represent at most 30% of the total Cd in the system. The chemical equilibrium model estimates that Cd adsorption will range from minimal adsorption at low pH ŽpH
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
3.0 and below., up to over 66% of the total Cd in the system at pH values of 6.0 and higher. The observed extent of Cd adsorption in the 3 = 10y5 molal total EDTA system is close to that predicted by the thermodynamic model Žsee Fig. 2 for a comparison., with slight discrepancies at pH 6.0 and higher where we observed a maximum of 60% of the total Cd adsorbed onto the bacterial surface. There are several possible causes for the discrepancy. One possibility is that the thermodynamic data upon which the chemical equilibrium model is based is inconsistent with the observed adsorption behavior. Another possibility is that the discrepancy is within the expected uncertainty of the estimation, given the uncertainties associated with the equilibrium constants that control the estimation. Alternatively, it is possible that the chemical equilibrium model fails to account for all biogeochemical processes occurring in the system, so it does not adequately characterize the ultimate fate of Cd in the experiments. Based on the reversibility of metal, proton, and organic acid uptake by bacterial surfaces ŽFein et al., 1997; Daughney and Fein, 1998a,b; Daughney et al., 1998., and the success of the chemical equilibrium approach in quantifying metal, proton, and organic acid adsorption onto bacteria, it is unlikely that the adsorption is significantly affected by additional bacterial processes. To determine if the observed discrepancy is within the uncertainty of the estimation technique, we use the data to calculate new equilibrium constant values for each important reaction involving Cd. Equilibria Ž2., Ž3., Ž4., and Ž5. have the most significant influence on Cd speciation under the experimental conditions. Of these, the interaction between Cd and the bacterial surface phosphate sites is the least well-constrained. We use the data from this study to calculate an independent value for K Ž5. , leaving equilibrium constant values for reactions Ž2., Ž3., and Ž4. fixed. This calculation yields a value of 4.8 for log K Ž5. , with a V Ž Y . value of 12.6. The value used for log K Ž5. in the original estimation was 5.4, with an uncertainty Žfactoring in the uncertainty of the acid dissociation constants of the bacterial phosphate group. of "0.8. Therefore, the discrepancy is within the uncertainty expected for the precision of the fundamental thermodynamic data. More accurate estimates of adsorption behavior, if required, would only be possible with more precise
381
determination of the stability constants for the pertinent surface complexes.
5. Geochemical implications The experiments in this study indicate that a chemical equilibrium approach can successfully account for metal and organic acid adsorption onto bacterial surfaces. This implies that a detailed estimation of the effects of bacterial adsorption on metal and organic speciation in water–rock systems is possible, once the pertinent equilibria are defined and the equilibrium constants determined. This is a large quantity of information, but the approach offers the ability to quantify the effects of bacteria that are similar in surface properties to the Bacillus sp. studied in a wide range of geochemical conditions. For example, we can determine a detailed Cd budget for a hypothetical contaminated groundwater system. In this calculation, we calculate the distribution of Cd between several potential reservoirs: Cd adsorbed to bacteria, Cd adsorbed to mineral surfaces directly, and Cd in the aqueous phase. Further, we determine the Cd speciation within the aqueous phase, and we calculate the amount of Cd adsorbed to immobile bacteria relative to that adsorbed to bacteria that are mobile in the system. This last estimation is based on experiments that quantify the extent of bacterial adhesion to a mineral surface under similar conditions to those of the calculation ŽYee and Fein, 1999.. In these experiments, we measured a distribution ratio of 4.0, where this ratio describes the relative concentration of bacteria free in solution and bacteria attached to aluminum mineral surface sites on Al 2 O 3 . Because these are the only experiments to quantify bacterial adhesion under similar conditions, we only account for adhesion to aluminum sites in our hypothetical system, and we assume a total aluminum mineral abundance of 35 grl, or an Al surface site concentration of 10y3 molal. The data used to quantify Cd adsorption onto aluminum mineral surface sites are those of Boily and Fein Ž1996.. Clearly, a more realistic example, with other rock-forming components, must await additional data on bacteria–mineral interactions. Fig. 4 shows the results of the calculation, and it illustrates the benefits of applying the chemical equi-
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J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
highlights the need for the flexibility of the chemical equilibrium approach. Although chemical equilibrium modeling requires a large collection of thermodynamic data, most of which has not yet been obtained, no other technique enables quantitative estimations of metal and organic speciation over a wide range of conditions in complex systems.
6. Conclusions
Fig. 4. Cd distribution in a Cd–EDTA–Al 2 O 3 system, calculated with Žblack bars. and without Žgray bars. the effects of bacteria, represented in terms of the percent of the total Cd in the system present in each Cd reservoir. The abundance of aqueous Cd–EDTA complexes is independent of bacterial content in the system, so bars of the same height are depicted. Note that the bacterial reservoirs have no Cd Žand hence no gray bars. associated with them for the bacteria-free calculations.
librium approach to bacteria–water–rock systems. The calculation assumes 10y4 molal total Cd in the system, 10y5 molal total EDTA, 0.5 g bacteria Ždry weight.rl of solution, an ionic strength of 0.1 molal, and a solution pH of 7.0. The figure shows that with bacteria present Žblack bars. under the conditions of the calculation, the Cd budget is dominated by adsorption onto bacterial surfaces, and that approximately 50% of the Cd is attached to mobile Žunattached. bacteria. This example underscores the potential importance of bacteria in affecting metal Žand organic. mobilities in the subsurface. Without bacteria present in the system, more Cd would be adsorbed onto the mineral surface sites, and more would be present in solution. The bacteria-free Cd budget is shown as the gray bars in the figure. Note that the overall mobility of Cd is drastically changed by the presence of the bacteria. In the bacteria-free system, over 60% of the total Cd is attached to the mineral surface, and thereby immobilized. With bacteria present, over 68% of the Cd is mobile, either as aqueous Cd, or attached to mobile bacteria. Clearly, there are many more possible mineral surface sites and fluid composition conditions to consider when modeling realistic subsurface Cd contamination. However, it is the vast number of conditions of geologic and environmental interest that
EDTA adsorbs only weakly to the surface of B. subtilis, with adsorption driven by the hydrophobicity of the fully-protonated EDTA molecule. With the onset of deprotonation of EDTA with increasing pH above pH 1.5, EDTA adsorption diminishes significantly, and there is negligible adsorption over most of the pH range studied. Despite the hydrophobic nature of the EDTA–bacterial surface interaction, the adsorption can be described using a chemical equilibrium approach, and the extent of adsorption can be quantified through the equilibrium constant value that is determined in this study. The presence of EDTA decreases Cd adsorption onto the bacterial surface, likely through aqueous Cd–EDTA complexation. Again, the data indicate that the chemical equilibrium approach can be used to successfully estimate the distribution of Cd in the mixed Cd– EDTA–bacteria system. The experimental results from this study illustrate one of the dominant effects of strongly-complexing organic acid anions on the adsorption behavior of metals onto bacterial surfaces. Aqueous complexation between organic acid anions and dissolved metal cations directly competes with bacterial surface complexation of the cations. The chemical equilibrium approach is an effective means for quantifying this competition. The distribution of metal between the aqueous phase and the bacterial surface can successfully be described based on thermodynamic modeling if the equilibrium constants for the pertinent complexation reactions are known. Although at this point, very few of these bacterial surface stability constants have been measured, this study suggests that this approach can ultimately be applied to quantify the effects of bacterial adsorption on metal and organic mobilities in water–rock systems of geologic and environmental interest.
J.B. Fein, D. Delea r Chemical Geology 161 (1999) 375–383
Acknowledgements Acknowledgment is made to the Donors of The Petroleum Research Fund, administered by the American Chemical Society, for support of this research. Deirdre Delea was supported by National Science Foundation ŽResearch Experience for Undergraduates. grant EEC96-19509. We thank David Fowle, Nathan Yee, and Peter Wightman for technical assistance and advice during the course of the study, and we thank an anonymous journal reviewer for carefully examining the manuscript and making helpful suggestions for improvement.
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