Environ. Sci. Technol. 2010, 44, 4930–4935
Surface Complexation of Neptunium(V) onto Whole Cells and Cell Components of Shewanella alga: Modeling and Experimental Study RANDHIR P. DEO,† WARINTHORN SONGKASIRI,‡ B R U C E E . R I T T M A N N , * ,† A N D DONALD T. REED§ Center for Environmental Biotechnology, Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5701, National Center for Genetic Engineering and Biotechnology, Excellence Center Waste Utilization and Management, King Mongkut’s University of Technology Thonburi, Bangkok, 10150 Thailand, and Earth and Environmental Sciences Division, Carlsbad, Los Alamos National Laboratory, New Mexico 88220
Received November 24, 2009. Revised manuscript received April 20, 2010. Accepted May 7, 2010.
We systematically quantified surface complexation of Np(V) onto whole cells, cell wall, and extracellular polymeric substances (EPS) of Shewanella alga strain BrY. We first performed acid and base titrations and used the mathematical model FITEQL to estimate the concentrations and deprotonation constants of specific surface functional groups. Deprotonation constants most likely corresponded to a carboxyl group not associated with amino acids (pKa ∼ 5), a phosphoryl site (pKa ∼ 7.2), and an amine site (pKa > 10). We then carried out batch sorption experiments with Np(V) and each of the S. alga components as a function of pH. Since significant Np(V) sorption was observed on S. alga whole cells and its components in the pH range 2-5, we assumed the existence of a fourth site: a lowpKa carboxyl site (pKa ∼ 2.4) that is associated with amino acids. We used the SPECIATE submodel of the biogeochemical model CCBATCH to compute the stability constants for Np(V) complexation to each surface functional group. The stability constants were similar for each functional group on S. alga bacterial whole cells, cell walls, and EPS, and they explain the complicated sorption patterns when they are combined with the aqueous-phase speciation of Np(V). For pH < 8, the aquo NpO2+ species was the dominant form of Np(V), and its log K values for the low-pKa carboxyl, mid-pKa carboxyl, and phosphoryl groups were 1.8, 1.8, and 2.5-3.1, respectively. For pH greater than 8, the key surface ligand was amine >XNH3+, which complexed with NpO2(CO3)35-. The log K for NpO2(CO3)35complexed onto the amine groups was 3.1-3.9. All of the log K values are similar to those of Np(V) complexes with aqueous
* Corresponding author phone: (480) 727 0434; fax: (480) 727 0889; e-mail:
[email protected]. † Arizona State University. ‡ King Mongkut’s University of Technology Thonburi. § Los Alamos National Laboratory. 4930
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carboxyl and N-containing carboxyl ligands. These results help quantify the role of surface complexation in defining actinidemicrobiological interactions in the subsurface.
Introduction The presence of actinides as subsurface contaminants has become a major environmental concern due to their long half-lives, radioactivity, and chemical toxicity (1). The general objective in remediation of radioactive elements is recovery, immobilization, or isolation of the actinides from environmental receptors, including humans (2-4). The research reported here contributes to understanding actinide-microorganism interactions that may affect the fate and transport of actinides through surface complexation of neptunium(V) (Np(V)) onto bacterial surfaces. In the subsurface, neptunium predominantly exists as the neptunyl Np(V)O2+ species under a wide range of oxic and suboxic conditions (5). This species does not form hydroxyl species until pH > 8, tends to have very weak interactions, and forms very weak aqueous complexes; all of these characteristics make it very mobile in most near-surface groundwater. Anaerobic microbiological activity can cause reduction of Np(V) to Np(IV), which leads to its precipitation and immobilization (6, 7). Gorman-Lewis et al. (8) quantified Np(V) adsorption on a Gram-positive soil bacterium, Bacillus subtilis, as a function of medium pH (2.6-8), ionic strength (0.001 to 0.5 M), and Np concentration (1.29 × 10-5 to 2.57 × 10-4 M). While Gorman-Lewis et al. (8) characterized trends in Np(V) adsorption on one type of bacterium, they did not explore sorption on other types of bacteria (particularly Gramnegative bacteria) and biogenically produced extracellular polymeric substances (EPS), and they did not evaluate adsorption over an extended pH range, which is needed to observe the broader changes on the cell surface and Np(V) chemistry. Bacterial surfaces consist of heterogeneous mixtures of proton-active surface sites potentially having three or four discrete surface functional groups (9-13). Daughney et al. (11) used the computer program FITEQL (14) to determine the deprotonation constant and concentration for each surface site on cell surfaces of Bacillus licheniformis, a Grampositive bacterium. Titration data over a pH range of 3-11 were best fit with a three-site model, and the pKa values were 5.2 ( 0.3, 7.5 ( 0.4, and 10.2 ( 0.5. Based on the average pKa values for simple carboxylic (pKa ∼ 4-6), phosphoryl (∼7-8), and phenolic hydroxide (∼ 9-11), Daughney et al. (11) assumed that the pKa values corresponded to carboxyl, phosphoryl, and hydroxyl surface functional groups, respectively. Similarly, Tourney et al. (10) combined potentiometric titration and surface-complexation modeling to characterize functional groups on the surface of Bacillus licheniformis S-86: phosphodiester (pKa 3.3-3.4), carboxylic (pKa 5.3-5.4), phosphoryl (pKa 7.4-7.5), and hydroxyl/amine (pKa 9.9-10.1). While Gram-positive bacteria may have relatively higher functional-group site density than Gram-negative bacteria (12), infrared spectroscopic evidence suggests that surface functional groups between Gram-positive and Gram-negative bacterium cells are nearly identical (15). Additionally, Borrok et al. (12) and Ginn et al. (13) demonstrated that, on average, a wide range of bacteria exhibit similar proton and metal adsorption behaviors for four discrete sites. Surface-complexation models (SCMs) were used in previous studies to predict the extent of metal and radionuclide sorption by bacteria in response to changes in environmental 10.1021/es9035336
2010 American Chemical Society
Published on Web 06/03/2010
parameters such as pH, bacteria-to-metal ratio, ionic strength, and temperature (16-19). Metals in solution can form complexes with surface-functional groups in much the same way as they form complexes with analogous aqueous complexants. For Np(V), the dominant species for pH e 8 is the aquo NpO2+ species, which can complex with deprotonated surface sites. We show the complexation reaction and stability constant for NpO2+ with the carboxylic surface group as an example: 0 >X - COO- + NpO+ 2 S >X - COO(NpO2)
K)
[>X - COO(NpO2)0] [>X - COO-]{NpO+ 2}
(1)
(2)
where [ ] represents mol/L concentration, { } represents thermodynamic activity, and K is the stability constant. We use concentrations to represent surface species, since the relationship of thermodynamic activity and concentration is not established for them. At pH > 8, neptunyl-carbonate complexes predominate under many subsurface conditions, including groundwater, given carbonate’s common abundance and tendency to complex with actinides (20). These Np(V)-carbonate species most likely complex with the surface sites that become active as complexants at high pH, such as > X - NH3+: 45>X - NH+ 3 + NpO2(CO3)3 S >X - NH3(NpO2(CO3)3) (3)
K)
[>X - NH3(NpO2(CO3)3)4-] 5[>X - NH+ 3 ]{NpO2(CO3)3 }
(4)
In this study, we combined experimentation and modeling to quantify Shewanella alga surface acid/base groups active in complexing Np(V), acid/base deprotonation constants (pKa), and complexation stability constants (log K) for relevant Np(V) species. We then characterized the pH-dependent sorption of Np(V) on S. alga. Our results indicate that sorption is controlled by the speciation of Np(V) and surface sites, both of which are controlled by pH.
Experimental Section Titration Experiments. We provide details on growth of Shewanella alga strain BrY, purification of cell wall, and isolation of EPS in the Supporting Information (SI). We performed acid or base titrations to determine the density and pKa values of surface acid/base groups. Titration experiments were carried out using a potentiometric autotitrator (Model KEM AT210, Kyoto Electronics Manufacturing Co., Ltd.) with a 20-mL buret unit (Model APB118-01B). Solutions were degassed for 20 min with N2 and kept in a N2 anaerobic chamber (Plas Laboratories) when not in use to keep out O2 and CO2, which could affect the pH in the nonbuffered solution. The titrant solutions and titrant aliquot were degassed again immediately prior to titration and kept under positive pressure of N2 by allowing a gentle flow of N2 through the entire system during the titration. We used a “down-up pH’” protocol for the titration experiment. A known amount of bacterial cells was added to a 0.1 M NaNO3 solution. The solution was then divided into two 30 mL portions. The first portion was titrated with 0.1 M HNO3 to pH 4. The second portion was titrated with 0.1 M NaOH to pH 9. The overall results were combined to produce the entire pH range. The reason for using the down up pH method was to minimize alterations of the biomass by extreme pH, which was documented by the methods of the next section.
At each titration point, the autotitrator recorded the pH of the bacterial cell suspension and the corresponding amount of acid or base added. At each point, we allowed the sample to equilibrate at a new pH for at least 3 min or once a potential stability of 0.1 mV/s had been attained. Effects of Extreme pH. To determine if pH extremes altered the reversibility of the biomass’s acid/base characteristics, we adjusted the pH of whole-cell suspensions directly to 2 or 12 with concentrated HNO3 or NaOH. The suspension at pH 2 was titrated with 0.1 M NaOH, while the suspension at pH 12 was titrated with 0.1 M HNO3 with the method described above. We saw no evidence of irreversibility under these conditions. Batch Experiments of Np(V) Surface Complexation. We provide details on preparation and analysis of NpO2+ in the SI. We performed batch experiments to establish the rate and concentration dependence of neptunium complexation onto S. alga cell surfaces. Batch experiments were carried out in 1.5 mL centrifuge tubes containing 1 mL of 0.1 M NaNO3 solution under nongrowth conditions. A known amount of biomass solid (i.e., bacterial whole cells, cell wall, or EPS) was added to the medium. The pH of the solution was adjusted prior to the experiments by adding a small amount of 0.2 µm-filtered NaOH or HNO3. Aliquots from a concentrated Np(V) stock solution were added to bring the initial neptunium concentration to approximately 6 µM. To establish the stability of NpO2+ in solution, we prepared controls with no S. alga additions. Redox stability was also established with S. alga addition, but under nongrowing aerobic condition. The oxidation state of Np in solution was established by XANES analysis (21). These synchrotron-based analyses were performed at the MR-CAT beamline of the Advanced Photon Source at Argonne National Laboratory (21) within one hour of the preparation of the samples. Bottles were gently shaken at 25 ( 2 °C throughout all sorption experiments. For each experiment, aliquots of 0.5 mL were drawn from the centrifuge tubes and analyzed for Np concentration after 120 min. This equilibration time was established based on reversibility and equilibration experiments (up to 4 h in duration) to establish the time needed for complete equilibration. No irreversibility was noted in these experiments. The absence of irreversible sorption is typically observed for NpO2+ as long as reduction to Np(IV) is suppressed. Similar equilibration times were reported by Gorman-Lewis et al. (8) and Haas et al. (22). The total neptunium concentration and the neptunium concentration after filtration through a 0.22 µm filter (Millipore UFC30GV25) were determined by R scintillation counting (Packard model 2500 TR liquid scintillation analyzer) of duplicate samples. We calculated the amount of biomass-associated with Np as the difference between the total and filtered concentrations of Np.
Results and Discussions Effects of Extreme pH on S. alga. Exposure of S. alga whole cells to extreme pH caused three effects. First, exposure to pH 2 caused the cells to visibly aggregate. Second, titration of cells exposed to pH 2 with NaOH resulted in a loss of buffering capacity, probably caused by the loss of surface functional groups, the denaturation of the cell proteins, or the loss of exposed surface site due to aggregation. Third, exposure to pH 12 resulted in a loss of optical density that was most likely due to hydrolysis of cell solids (23). The extreme-pH results demonstrate the utility of the down-up pH protocol and the need to limit the pH titrations to 4 and 9 in order to preserve the chemical properties of the cell components. Additionally, this methodology was consistent with Ojeda et al. (24) who reported that this pH range VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. The pKa Values and Specific Site Concentrations for the Best-Fit Three-Site Models for S. alga Cell Walls, EPS, and Whole Cells pKa1a
CS1b
pKa2
CS2
pKa3
CS3
∑Cs
V(Y)
cell walls
5.2
7.2
9.7
6.1
7.8
whole cells
5.1
7.1 (48.5%) 3.0 (49.3%) 4.3 (48.9%)
14.6
4.8
0.7 (5.1%) 0.9 (14.5%) 1.4 (15.9%)
10.2
EPS
6.8 (46.4%) 2.2 (35.2%) 3.1 (35.2%)
cell component
7.3 7.2
10.9 10.0
8.8
18
a pKa values for the surface functional groups correspond to the condition of zero ionic strength and zero charge coverage. b Absolute concentrations of the surface functional groups are in mmoles per gram dry weight of bacterial cell component. Percentages in parentheses give the percentages of each row total.
iswithinthereversible-pHlimitsofprotonation-deprotonation processes of Aquabacterium commune, a Gram-negative bacterium. Calculating the Deprotonation Constants and Concentrations of Surface Functional Groups on S. alga Whole Cells, Cell Walls, or EPS using FITEQL. We used FITEQL version 4.0 (14) to solve for the deprotonation constants and the absolute concentrations (mol/L) of each distinct type of bacterial surface site. The methods we used to apply FITEQL are in the SI. For each titration, we fit the titration data by employing models involving one, two, or three discrete surface functional groups. FITEQL was unable solve for the parameters when we tried to use four groups, and this prevented us from testing a four-site model. We normalized the surface-site concentrations (mol/L) to the mass of biomass per liter of electrolyte, yielding specific site concentrations in moles per gram of bacterial cells or cell component suspensions. SI Figure S1 compares the best-fit modeling results with the experimental results from the titration experiment with 10.1 g of S. alga whole-cell dry weight/L. A good model fit to the experimental data was obtained only when three distinct types of surface functional groups were used. Models including only one or two distinct types of surface functional groups did not adequately fit all of the experimental data. For example, the two-site model with pKas of 6.7 and 9.5 described the titration data above pH 6, but had almost no buffering capacity for pH less than 5. The three-site model showed the proper buffering capacity for the entire pH range with best-fit pKa values of 5.1 ( 0.4, 7.2 ( 0.3, and 10.0 ( 0.3. Although direct analysis of the bacterial cell surfaces would be required to confirm the existence of these surface functional groups, we propose that the three pKa values of 5.1, 7.2, and 10.0 correspond to the carboxyl, phosphoryl, and amino groups, respectively. Similar pKa values and surface functional groups were reported for Shewanella putrefaciens, a Gram-negative bacterium (22). The corresponding concentrations for the three functional groups on S. alga were 3.1 × 10-3 moles, 1.4 × 10-3 moles, and 4.3 × 10-3 moles of the surface sites 1, 2, 3, respectively, per gram dry weight of bacteria. Similarly, deprotonation constants and concentrations of each functional group on S. alga cell walls and EPS were estimated by using the titration data and model fit with three distinct types of surface functional groups. The experimental and best-fit results are shown in SI Figure S2. Table 1 lists all the pKa values and specific site concentrations for isolated cell walls and EPS, as well as for whole cells. The pKa values were similar for whole cells, purified cell walls, and EPS: 4.8-5.2 for pKa1, 7.2-7.3 for pKa2, and 10.0-10.9 for pKa3. As observed with the pKa values, individual site concentrations were also similar to those reported by Haas et al. (22). Approximately 20 commonly occurring amino acids polymerize to form peptides and proteins. Amino and carboxyl groups on amino acids react in a head-to-tail fashion, 4932
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eliminating a water molecule and forming a peptide bond as a backbone structure of proteins. Voet and Voet (25) listed pKa values of terminal amino groups on amino acids ranging from 8.8 to 11.0, while terminal carboxylate groups have pKa values near 2.4. Several amino acidssaspartic acid, glutamic acid, histidine, arginine, lysine, and serinescontain a side chain group that also has acid/base properties. For example, aspartic acid and glutamic acid contain a side chain with a carboxyl functional group; histidine, arginine, and lysine contain a side chain with an amino functional group; and serine contains a side chain with a hydroxyl functional group. The pKa values of R groups of aspartic acid, glutamic acid, histidine, arginine, lysine, and serine are 3.9, 4.3, 6.0, 12.5, 10.5, and 13, respectively. Thus, proteins possess reactive amino and carboxyl termini, and they further display characteristics of the side chain groups that have acid/base properties with a range of pKa values. Based on the typical pKa values of the common functional groups discussed above, the functional groups with the pKa value of 4.8-5.2 most likely correspond to the carboxyl functional groups from polysaccharides or from side chains of amino acids and proteins, the functional groups with the pKa value of 7.2-7.3 most likely correspond to the phosphoryl functional groups from phospholipids, and the functional groups with the pKa value of 10.0-10.9 most likely correspond to the terminal amino groups or the side chain of amino acids from proteins. Tourney et al. (10) used a similar approach for assigning surface functional groups to measured pKa values. Furthermore, the identified functional groups are consistent with spectroscopic evidence of the common functional groups found on most bacterial surfaces (15, 26-29). The amino groups (Cs3), comprising about 50% of the total sites (Table 1), were the most plentiful in bacterial whole cells, cell walls, and EPS. The second largest group, from 35 to 46%, was carboxyl (Cs1) for all three components. Bacterial cell walls displayed the highest total site density (∑Cs), and the cell-wall sites were dominated by the carboxyl and amino groups, with few phosphoryl groups. Cell walls are composed mainly of polysaccharides and peptidoglycans, macromolecules that contain carboxyl and amino functional groups. Having the least phosphoryl groups indicates that cell walls have the least phospholipids, which house the phosphoryl sites. Phosphoryl groups were greatest in the whole cells, but always the group having the fewest functional groups. Redox Stability of Np(V). Prior to batch Np(V)-sorption experiments, the stability of Np(V) was established by UV-vis-NIR spectrometery (absence of S. alga) and XANES analysis for biosorbed neptunium in the presence of S. alga. No reduction of Np(V) to Np(IV) or precipitation of Np(V) occurred under the experimental conditions, that is, pH range, duration, and NP concentration, although 2-5% of Np(V) was lost by sorption onto the polypropylene vessel in the absence of S. alga. Our Np(V)-stability results in the absence of S. alga are consistent with the published solubility
FIGURE 1. Comparison of experimental (solid circles) and modeling (lines) results for Np(V) surface complexation by S. alga components as a function of pH and suspended bacterial concentrations for (a) whole cell -3.7 g/L, (b) EPS - 9.75 g/L, and (c) cell wall -0.77 g/L. Model curves were produced by plotting total Np sorbed at 1 pH intervals between 2 to 12. data for Np(V) in chloride (30) and the very high redox stability normally observed for Np(V) in aqueous systems. Moreover, Gorman-Lewis et al. (8) recently reported that stability of Np(V) in the presence of B. subtilis depends on ionic strength and pH. They suggested reduction of Np(V) to Np(IV) in high ionic strength (0.1 M) and low pH (XCNpO2 and >XCOONpO2 in the pH Range of 2-6. In the pH range 2-6, the dominant Np(V) species is the NpO2+ aquo ion (SI Figure S3), and the dominant ligand surface species is >XC-, the low-pKa carboxyl groups, with >XCOO- rising sharply at pH 6 (SI Figure S4). We used SPECIATE to calculate the best-fit stability constants for NpO2+ complexation with >XC- and >XCOO-. In a SPECIATE input file, we specified each surface site and NpO2+ as components. The metal, that is, NpO2+, complexed onto each bacterial surface site was specified as a complex or species. The components were H+, NpO2+, H2CO3, X-C-, and X-COO-. The species for the system were OH-, CO32-, HCO3-, X-CH, X-COOH, NpO2OH, NpO2CO3-, NpO2 (CO3)23-, VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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NpO2(CO3)35-, X-CNpO2, and X-COONpO2. In this pH range, the complexation of NpO2+ onto bacterial surface functional groups can be described by: 0 >X - C- + NpO+ 2 S >X - C(NpO2)
(5)
0 and > X - COO- + NpO+ 2 S >X - COO(NpO2)
(6)
In the SPECIATE calculation, the log K values of X-COONpO2 and X-CNpO2 species were adjustable parameters. We adjusted the log Ks to minimize the difference between the input experimental value for adsorbed Np(V) (i.e., total sorbed Np(V) as a function of pH) and the calculated value for sorbed Np(V) (i.e., the sum of the X-COONpO2 and X-CNpO2 concentrations). The best-fit log K stability constants for Np(V) adsorption (Figure 1) for the low-pKa carboxyl and the other carboxyl group were 1.8 and 1.8. The calculated log K values are in good agreement with the log K values for Np(V) complexation with other carboxylate-containing ligands, such as acetate (1.46), lactate (1.56), and succinate (1.72) (34), as well as with low-pKa sites L1 (1.7) and L2 (1.6) of Bacillus subtilis (8). Determining the Stability Constants for >XPO4NpO2 in the pH Range of 6-8. In the pH range 6-8, the dominant Np(V) aqueous species was still the NpO2+ aquo ion (SI Figure S3), and the significant ligand surface species were >XC-, >XCOO-, and >XPO4- (SI Figure S4). Using the log K values for the two carboxyl groups obtained from the previous step, we further used SPECIATE to determine the stability constant for >XPO4NpO2 that best-fit the experimental data in the pH range 6-8 (Figure 1). The sorption of NpO2+ onto the phosphoryl functional groups can be described by the following: 0 + >X - PO4 + NpO2 S >X - PO4(NpO2)
(7)
The additional component for SPECIATE input file was >XPO4-, and the additional species were >X-PO4H, and >XPO4NpO2. The best-fit log K value for Np surface complexation for the phosphoryl group was 2.20. The calculated log K values are in good agreement with the log K values for Np complexation with inorganic phosphate (2.54 for NpO2HPO4-) (34). Determining the Stability Constants for >XNH3NpO2(CO3), >XNH3NpO2(CO3)22-, and >XNH3NpO2(CO3)34- in the pH Range of 8-10. In the pH range 8-10, the dominant Np species shifted dramatically from NpO2+ to NpO2(CO3)-, NpO2(CO3)23-, and NpO2(CO3)35- species, with NpO2(CO3)35becoming completely dominant by pH 10 (SI Figure S3). The shift away from NpO2+ as the dominant Np(V) species resulted in loss of Np(V) sorption from the aforementioned carboxyl and phosphoryl surface groups. However, Np(V) sorption was maintained in this pH range (Figure 1), which indicates that sorption was due to Np-carbonate complexes onto >XNH3+ group. This sorption can be described by >X - NH+ 3 + NpO2CO3 S >X - NH3(NpO2CO3)
(8)
23>X - NH+ 3 + NpO2(CO3)2 S >X - NH3(NpO2(CO3)2) (9) 45>X - NH+ 3 + NpO2(CO3)3 S >X - NH3(NpO2(CO3)3) (10)
The new components for SPECIATE input file were NpO2(CO3)-, NpO2(CO3)23-, NpO2(CO3)35-, and >X-NH3+, and the additional species were >X-NH2, >X-NH3(NpO2CO3), >XNH3(NpO2(CO3)2)2-, and >X-NH3(NpO2(CO3)3)4-. 4934
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We calculated the best-fit log K values for >XNH3NpO2(CO3)34-, >XNH3NpO2(CO3)22-, and >XNH3NpO2(CO3) for the experimental data in the pH range of 8-10 (Figure 1). The best-fit log K values were 3.1, 2.7, and 1.8, respectively. >XNH3NpO2(CO3)34- was the important surface complex in that its log K value was higher than for other surface ligands that complex with NpO2+. The complexation at high pH of the Np-carbonate species was observed as long as the >XNH3+ surface ligand was present. The calculated log K values are in reasonable agreement with the log K values for Np(V) complexation with N-containing carboxylates (3.4 for alanine and 3.6 for glycine (34). SI Table S2 summarizes the Cs, pKa, and log K values for Np(V) complexation to sites on whole cells of S. alga, purified cell walls, and isolated EPS. The best-fit stability constants for Np(V) adsorption, along with Cs and pKa values, also are given for the cell components in SI Table S2. The comparisons between the modeling results and the experimental results in Figure 1 show that the model captured the trends in Np(V) complexation to whole cells for the pH range 2-10) and for the cell walls and EPS over the pH range of 2-12. For all biomass materials, Np(V) surface complexation increased nearly linearly from pH 4-8 due to >XCOOand >XPO4- ligands increasing as pH increased (SI Figure S4). For pH 8-10, Np(V) sorption reached a plateau for whole cells and EPS due to the decline in NpO2+, which was compensated by the increases in NpO2(CO3)-, NpO2(CO3)23-, and NpO2(CO3)35-, which complexed with the >XNH3+ surface ligand. Cell walls did not show the plateau, because they have much less of the phosphoryl surface groups to complex with NpO2+ (SI Table S2). The strong drop off of Np(V) sorption for pH > 10 with EPS and cell walls was due to the decrease in >XNH3+ surface ligand. Quantitative Comparison of Np(V) Adsorption onto Surfaces of S. alga Whole Cell, Cell Wall, And EPS. Visual inspection of Np(V) adsorption onto S. alga cell and its components in Figure 1 reveals that Np(V) adsorption on EPS (Figure 1(b)) is most significant, compared to adsorption onto whole cell and cell wall, for the entire pH range studied. However, when normalized against the biomass components used, Np(V) adsorption onto cell wall is the most significant. For example, at pH 8, normalized comparison shows that Np(V) adsorption onto cell wall is ∼3.25 µmol/g, which is much higher than that adsorbed onto whole cell (∼0.95 µmol/ g) and EPS (∼0.46 µmol/g). Although a previous massnormalized comparison revealed that the extent of metal binding between EPS and cell wall was similar (35), our findings differ because the calculated total site density (Table 1) was highest for bacterial cell wall (14.6 mmol/g), compared to whole cell (8.8 mmol/g) and EPS (6.1 mmol/g). Adding the low-pKa group did not alter the order of total site density. In summary, we characterized Np(V)-complexation onto surfaces of S. alga whole cells, cell walls, and EPS by systematically combining experiments and modeling. The pKa values estimated from acid/base titrations and Np(V) sorption experiments allow us to propose the following surface functional groups with pKa values: mid-pKa carboxyl, 4.8; phosphoryl, 7.2-7.3; and amine, 10.0-10.9. Np-sorption experiments identified an additional site, which we interpreted as low-pKa carboxyl, 2.4. Stability constants (log K) estimated from NpO2+ speciation and adsorption experiments were 1.8, 1.8, and 2.2 to 3.1, respectively, for low-pKa carboxyl, mid-pKa carboxyl, and phosphoryl groups. Similarly, log K for NpO2(CO3)-, NpO2(CO3)23-, and NpO2(CO3)35complexed onto the amino groups was 1.8-2.0, 2.7-3.0, and 3.1-3.9, respectively. Our results indicate that Np(V) surface complexation can be best explained by pH-dependent shifts in Np(V) speciation (from NpO2+ to NpO2(CO3)35- as pH increases) and changes in available surface functional groups (from carboxyl to amine as pH increases). These results have
important implications for understanding and quantifying key actinide-microbiological interactions in the subsurface.
Acknowledgments The experimental work was performed in the Chemical Technology division at Argonne National Laboratory. XANES analyses were performed at the Advanced Photon Source which is supported by the DOE office of Science. The research was supported, in part, by the Nuclear Energy Research Initiative (NERI), the Environmental Remediation Sciences Program (ERSP) of the United States Department of Energy, and the National Center for Genetic Engineering and Biotechnology, Thailand, for partial financial support.
Supporting Information Available Tables showing basic input parameters (Table S1), and Cs, pKa, and log K values for Np(V) complexation to four sites on whole cells of S. alga, purified cell walls, and isolated EPS (Table S1). Figures comparing experimental to best-fit modeling results from titration experiments on S. alga whole cell (Figure S1), purified cell wall (Figure S2(a)) and isolated EPS (Figure S2(b)), and Figures for pH-dependent speciation of Np(V) (Figure S3) and surface functional groups on bacterial whole cell (Figure S4). Also included are details on growth of Shewanella alga strain BrY, purification of cell wall, isolation of EPS, preparation and analysis of NpO2+, and descriptions of models used, i.e., FITEQL and CCBATCH. This material is available free of charge via the Internet at http://pubs.acs.org.
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