Environ. Sci. Technol. 2010, 44, 1644–1649
Investigation of the Ionic Strength Dependence of Ulva lactuca Acid Functional Group pKas by Manual Alkalimetric Titrations J O H A N S C H I J F * ,† A N D A L I N A M . E B L I N G ‡,§ Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, P.O. Box 38, Solomons, Maryland 20688, and Kutztown University of Pennsylvania, P.O. Box 730, Kutztown, Pennsylvania 19530
Received September 29, 2009. Revised manuscript received January 16, 2010. Accepted January 19, 2010.
We performed a series of manual alkalimetric titrations in NaCl solutions (0.01-5.0 M) at T ) 25 °C on both fresh and dehydrated samples of the marine chlorophyte Ulva lactuca (sea lettuce), a strong metal accumulator holding considerable promise in biosorbent and biomonitor applications. Functional groups were characterized in terms of their number, site densities, and acid dissociation constants (pKas). FITEQL4.0 modeling shows that, at any ionic strength, titration curves for dehydrated biomass in the pH range 2-10 are adequately described by three functional groups with remarkably uniform site densities of about 5 × 10-4 mol/g. Lower site densities for fresh U. lactuca are consistent with ∼87% water content. The pKas display pronounced ionic strength dependent behavior obeying an extended Debye-Hu¨ckel relation. Extrapolation to I ) 0 yields values of 4.26 ( 0.04, 6.44 ( 0.02, and 9.56 ( 0.04. This information by itself is insufficient to unambiguously identify the groups. Similar site densities suggest that all three are linked to major molecular building blocks of the cell material, pointing to carboxylic acids, phosphate esters, and amines as likely candidates. Highly acidic sulfate esters, not detected in our titrations, may also play a role in trace metal adsorption on U. lactuca.
Introduction The environmental mobility and bioavailability of trace metals is largely controlled by interactions with solid particles and colloids. In natural waters, mineral surfaces normally acquire an organic coating that can substantially alter their reactivity toward trace metals (1). While oxide minerals are structurally intricate, their affinity for trace metals is mediated mainly by hydroxyl groups, which may be uncharged or even positively charged at circum-neutral pH. Organic matter, on the other hand, is both structurally and chemically intricate, containing numerous functional groups that run the gamut from strong to very weak acids (2). Many of these deprotonate * Corresponding author phone: (410) 326-7387; fax: (410) 3267341; e-mail:
[email protected]. † University of Maryland Center for Environmental Science. ‡ Kutztown University of Pennsylvania. § Present address: Department of Oceanography, Florida State University, P.O. Box 3064320, Tallahassee, Florida 32306. 1644
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above pH ∼4-5, creating negatively charged surfaces prone to sequester metal cations by coordination or electrostatic attraction. Recent investigations of trace metal adsorption on organic matter have increasingly shifted to whole organisms, especially bacteria, fungi, and vascular plants that favor terrestrial habitats (3-5). Studies of marine organisms are less common, probably due in part to the chemical complexity of seawater. One exception is Ulva lactuca L., or sea lettuce, a green macroalga (class Ulvophyceae) ubiquitous in coastal areas of all oceans, from the tropics to as far north as Alaska. It thrives in warm, eutrophic waters where nuisance blooms may smother benthic communities, causing hypoxia and noxious odors when large amounts of the decaying seaweed wash ashore (6). A notable tendency to accumulate heavy metals marks U. lactuca as a potentially inexpensive biosorbent for use in wastewater cleanup (7-9). Linear correlations between metal contents of live U. lactuca and the surrounding seawater (10) have inspired suggestions that it may also be an ideal biomonitor of heavy metal contaminants in littoral ecosystems (11). Heavy metal uptake is a unique concern in impoverished regions of the world where seaweed harvested for consumption by humans and livestock may grow in severely polluted waters (12). Despite a pervasive interest in U. lactuca, surprisingly little progress has been made elucidating the microscopic mechanisms that underlie its affinity for various metals, partly because prior studies have focused instead on macroscopic equilibrium partitioning or reaction kinetics (13-16). Webster et al. (17) determined that oxygen-containing groups are responsible for Cd binding on U. lactuca, but could not distinguish carboxyl from sulfate or phosphate and reported no thermodynamic data. Schiewer and Wong (18) conducted alkalimetric titrations on U. fasciata biomass at two ionic strengths (∼0-0.1 M), but used only a one-site model to interpret their data. Turner et al. (19) required one low-affinity and one high-affinity site to describe Pd, Cd, Hg, and Pb adsorption on U. lactuca as a function of salinity, but obtained no acid dissociation constants (pKas) as their experiments were done at constant pH. This work is the initial step in a larger endeavor to expand our knowledge of metal adsorption on U. lactuca through a combination of spectroscopic techniques (e.g, EXAFS, NMR) and traditional batch experiments. Its goal was to set past and future studies on firmer quantitative footing by characterizing, for the first time, all acid functional groups and by deriving equations to calculate their pKas at any ionic strength (e5.0 M NaCl). Although we recognize that there may be other modes of metal binding on U. lactuca (15), these titrations are aimed specifically at understanding reversible cation-exchange. Our results thus apply to situations wherein the adsorbing cells are metabolically inactive and should aid environmental engineering efforts to develop a cheap, yet effective, U. lactuca-based biosorbent. More fundamentally, our data reaffirm that functional group properties are conserved across a dazzling array of marine and terrestrial taxa, which has important implications for a unified representation of biogenic organic matter in computer codes capable of modeling metal transport in a variety of natural waters.
Materials and Methods Titrations were conducted in a 1 L Teflon FEP wide-mouth bottle, fitted with a custom Teflon PTFE cap containing two ports for the pH electrode and N2 gas line, plus a smaller one 10.1021/es9029667
2010 American Chemical Society
Published on Web 02/02/2010
for adding the titrants. Constant temperature was established by placing this reaction vessel inside a jacketed beaker connected to a recirculating water bath (LAUDA-Brinkmann RE-106). The contents of the vessel were stirred with a Tefloncoated magnetic “floating” stir bar that does not grind the sample. Dissolved inorganic carbon (DIC) was purged from the experimental solutions at low pH, because its presence interferes with the titration. To prevent DIC incursion at high pH, the vessel headspace was continuously flushed with UHPgrade N2 passed through an in-line trap that captures any residual CO2 (Supelco). Evaporation was suppressed by prehumidifying the N2 in a bubbler with Milli-Q water (18 MΩ · cm) from a Millipore Direct-Q 3UV purification system. Solution pH was measured on the free hydrogen ion concentration scale with an Orion combination electrode (no. 810200) connected to a PerpHecT 350 millivolt meter. The electrode was checked regularly for proper Nernstian slope (59.16 mV/pH at 25 °C) by titrating a 0.5 M NaCl solution with 1 M HCl. The internal electrolyte (3 M NaCl) was replaced prior to each titration. In concentrated NaCl solutions, even the best glass pH electrodes suffer from Na+ interference. A manufacturer-supplied nomograph that allows errors to be estimated as a function of Na+ concentration, temperature, and pH, indicates that the Orion electrode is off by no more than 0.03 pH units at the highest [Na+]/[H+] ratio encountered in this study (5 M NaCl, pH 10). Such deviations generally fall within the observed reproducibility of our model-derived pKa values and were deemed negligible. Most titrations were performed with ∼1 g of Certified Reference Material BCR-279 (“Trace Elements in Sea Lettuce”), purchased from Fluka and stored in a desiccator cabinet (Secador, Bel-Art). It is issued by the Institute for Reference Materials and Measurements (Geel, Belgium) as a dehydrated, homogenized powder. Fresh U. lactuca samples were also titrated for comparison. Specimens collected around Chesapeake Bay were propagated indoors (8 h/16 h light/dark cycle) in Patuxent River estuarine water (salinity ∼11) sterilized by triple in-line filtration (10-0.45-0.2 µm) and replaced every 2-3 days. Fresh samples comprised as many 5-10 cm2 pieces as would fit in the reaction vessel without perturbing the pH electrode (∼2-3 g wet weight). A coarse Teflon-mesh barrier was placed inside the vessel to keep pieces of alga from getting tangled in the stir bar. Thalli were manually cleaned of epibiota, rinsed in Milli-Q water to remove contaminant cations and adhering debris, carefully blotted with Kimwipe, and weighed. Fein et al. (20) demonstrated that even prolonged washing in dilute nitric acid does not affect the buffer intensity of bacterial cells. Nonetheless, we held rinsing to a minimum and promptly transferred samples to the experimental solutions. A 5 M NaCl stock solution was made from ReagentPlus grade salt (Sigma-Aldrich) in a PMP volumetric flask and its density determined to verify the concentration (21). Titrations were performed in 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 M NaCl solutions, prepared by gravimetrically diluting the stock solution with Milli-Q water to the desired molarity. Additional titrations were performed at the ionic strength of seawater (0.7 M NaCl). NaCl solutions were acidified to pH 2.00 with 1 M HCl, except for the 0.01 and 0.05 M solutions which were acidified to pH 3.00 to maintain the correct ionic strength. While functional groups may continue to reversibly protonate well below pH 2, erratic behavior of the glass electrode tends to preclude reproducible results in more acidic solutions (20). The reaction vessel was filled with 1000 mL of acidified NaCl solution at T ) (25.0 ( 0.1)°C and stirred for 24-48 h under N2 atmosphere to eliminate DIC and to condition the electrode until it attained a stable potential. This pH standard was used to construct a 1-point calibration curve appropriate for the subsequent titration, assuming Nernstian slope. A
biomass sample, weighed to the nearest 0.1 mg, was then introduced under continued stirring and the “pH of immersion” measured following 15-20 min of equilibration. Solution pH, elevated due to proton adsorption on the alga, was lowered back to 2.00 (or 3.00) by adding 1.0011 M HCl from a 2 mL Gilmont micrometer buret via a Teflon needle. The dispensed volume was recorded to the nearest 0.1 µL. The actual titration was started by slowly adding 1.0012 M NaOH from a 2 mL micrometer buret over a period of 5-10 min, until an electrode potential corresponding to a 0.1-0.2 unit pH increment was achieved as calculated from the calibration curve. The stable electrode potential was recorded along with the dispensed volume. In some cases a 0.2 mL buret was also used where the titration curve is particularly flat. Titrations were ended at pH 10, before the onset of lysis (20). Each was completed in a single consecutive session of 5-6 h, with one 20-30 min break during which the electrode typically drifted by no more than (0.02 pH units. These small jumps were accounted for in the data analysis. A rapid cyclic titration of BCR-279 on an automatic titrator (pH 2-10-2) revealed no hysteresis that might be a sign of permanent cell damage, within analytical uncertainty.
Results and Discussion Results of the FITEQL Regressions. Full details of the regression protocol, including definitions of all model parameters and terms, are given in the Supporting Information (SI). A blank titration in 0.5 M NaCl was fit with H2O/ OH- as the only acid-base pair, and KW and [H+]T0 as the only adjustable parameters. Whereas the quality-of-fit was relatively poor (WSOS/DF ∼55), the regression gave pKW ) 13.57, close to the accepted value of 13.71 (SI Table S2), as well as a low value of [H+]T0 ∼-58 µM. Efforts to improve the fit by including a second acid-base pair (e.g., HCO3-/CO32-) were unsuccessful, indicating that there were no other protonactive species in the NaCl medium and that DIC was adequately removed. A total of 14 manual titrations was performed with the U. lactuca standard BCR-279 in 0.01-5.0 M NaCl solutions. This material was chosen to ensure consistent sample composition and because environmental engineering studies (e.g., ref 7-9) commonly employ dehydrated biomass. In addition, two titrations were performed with fresh U. lactuca samples in 0.5 M NaCl. FITEQL models with 3, 4, or 5 acid functional groups were fit to the data. The five-site model would not always converge from arbitrary initial guesses of the pKas and sometimes needed many iterations. About half the regressions were under-constrained (WSOS/DF < 0.1), while site densities were not constant nor did pKa values show a meaningful dependence on ionic strength. This model was therefore rejected and results are not reported here. The four-site model performed better, producing excellent fits (0.1 < WSOS/DF < 1) in nearly all cases (SI Table S3). However, whereas the acid dissociation constants and site densities of L1 and L4 are rather consistent, those of the two intermediate groups display large, seemingly random variations. The three-site model (Figure 1) does not at first appear to have a marked advantage, producing slightly inferior fits (WSOS/DF ∼1-2) albeit from fewer iterations (SI Table S2). Whenever two models produce statistically equivalent outcomes, preference should be given to the simpler, that is, the more constrained one. Pagnanelli et al. (22) called this “the principle of maximum parsimony”. Indeed, a comparison of SI Tables S2 and S3 shows the three-site model to be clearly superior. The most compelling evidence is the pronounced ionic strength dependence that emerges when the pKa(Lx) are plotted vs I as in Figure 2, where each is fit with an extended Debye-Hu ¨ ckel relation (SI eq S3). Extrapolation to I ) 0 yields values of 4.26 ( 0.04, 6.44 ( 0.02, and 9.56 ( 0.04, respectively. The fit is best for pKa(L2), being the farthest VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Examples of manual titrations of ∼1 g/L BCR-279 in 0.05 M, 0.5 M, and 5.0 M NaCl. Symbols represent total hydrogen concentrations in molar units, HT(M), calculated from titrant volumes, shown as a function of free hydrogen ion concentrations in solution (pH ) -log[H+]), measured with a glass combination electrode. The titration in 0.05 M NaCl was started at pH 3 to maintain the correct ionic strength. Solid curves are FITEQL4.0 regressions using the constant ionic strength three-site model. Gray symbols are a blank titration (without U. lactuca biomass) in 0.5 M NaCl. from the edges of the pH window. This effect is also borne out by replicate titrations conducted at several ionic strengths (Table 1). Error bars for pKa(L2) are small everywhere (Figure 2), yet those for pKa(L1) increase when approaching the lower pH boundary (intermediate I) while those for pKa(L3) increase when approaching the upper pH boundary (low I). Maximum uncertainties are of the same order as the pH resolution of the titrations (∼0.2 units), which could not be enhanced due to time limitations. A strong ionic strength dependence of proton adsorption on organic matter was not found in some studies that were conducted over a much narrower ionic strength range (4, 20). It is moreover possible that results from variable ionic strength FITEQL models have occasionally been misconstrued as signifying a lack of ionic strength dependence. Parameters produced by such models apply to I ) 0, regardless of the ionic strength of the titration. This is illustrated in SI Figure S2, where our titrations were fit with a three-site variable ionic strength model (cf. SI Table S1): for I < 0.5, where the Davies Equation (DE) is valid, pKas at I ) 0 (Table 1, second row)sand site densities (SI Table S4)sare identical within error to the values extrapolated from Figure 2 (Table 1, top row). In contrast, at higher ionic strength the results deviate increasingly from these extrapolated values. SI Figure S2 shows that the deviations are linear and presumably caused by discrepancies in the factor β (SI eq S3) between the DE and the extended Debye-Hu ¨ ckel relations. Variable ionic strength FITEQL models are valid only for I < 0.5 and should not be used for conditions favored by marine organisms such as U. lactuca. The parameter [H+]T0 is in good agreement with values calculated from titrant volumes (SI Figure S3) and measurements of the pH of immersion match values calculated from the regression parameters. The pKa of the most acidic functional group (g3.7) implies that it is largely protonated at pH 2 hence [H+]T0 should approximate aggregate site densities, both for BCR-279 and fresh U. lactuca. Site densities are insensitive to ionic strength, as expected, varying by no 1646
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FIGURE 2. Open symbols are pKa values (molal units) of BCR-279 acid functional groups as a function of ionic strength I, fit with extended Debye-Hu¨ckel relations (SI eq S3). Closed symbols, representing fresh U. lactuca samples, were not included in the fits. For pKa(L3) the closed symbol falls outside the frame of the graph and is not shown. Probabilities (P) result from an ANOVA of the nonlinear regressions. See Table 1 for details of data and error calculations. more than 5-6% for I ) 0.1-5.6. Only at lower ionic strength are they different (SI Table S2, entries in parentheses), ostensibly because L1 is partly deprotonated at pH 3. Those data were excluded from the averages, which are (5.4 ( 0.5) × 10-4 mol/g for all three acid functional groups. Such uniformity suggests that these groups are linked, in about equal parts, to the major macromolecular building blocks of the cell material. One BCR-279 titration in 0.7 M NaCl initially gave a high {L1}T, combined with a low pKa(L1). Since the two parameters are substantially correlated, this titration was refit after fixing {L1}T at the mean of all BCR-279 samples (4.87 × 10-4 mol/g). With no discernible effect on the other adjustable parameters and just a modest increase of WSOS/ ¨ckel curve DF, the resulting pKa(L1) is closer to the Debye-Hu (Figure 2). The process was repeated for the variable ionic strength three-site model (SI Table S4), however no meaningful convergence was achieved when applying this constraint to the four-site model (SI Table S3). Titrations of fresh U. lactuca conform to a three-site model with nearly the same pKa values at I ) 0.5 as BCR-279 (Table 1), but site densities that are almost an order of magnitude lower. Assuming identical functional groups and tissue composition, the low site densities could simply reflect
TABLE 1. Acid Dissociation Constants of BCR-279 Functional Groups (L1-L3), Derived From FITEQL4.0 Regressions Using Two Different Three-Site Modelsa NaCl (m) b
0 0c 0.010 0.050 0.10 0.51 0.51d 0.71 1.0 2.1 5.6
pKa(L1) 4.26 4.11 4.15 4.14 3.97 3.71 3.75 3.69 3.71 3.74 3.78
( 0.04 ( 0.15 ( 0.01 ( ( ( (
0.06 0.08 0.04 0.02
pKa(L2) 6.44 6.34 6.32 6.29 6.15 6.00 6.10 5.93 5.93 5.94 6.12
( 0.02 ( 0.09 ( 0.06 ( ( ( (
0.01 0.02 0.01 0.02
pKa(L3) 9.56 9.63 9.43 9.44 9.48 9.40 8.99 9.33 9.32 9.34 9.43
( 0.04 ( 0.09 ( 0.06 ( ( ( (
0.04 0.21 0.01 0.01
n 8 2 1 1 4 2 2 2 1 1
a Results from n replicate titrations were averaged at each ionic strength, where m denotes molal units. Errors are equal to one standard deviation or to half the difference between duplicates (n ) 2). See SI Tables S2 and S4 for primary data. b Extrapolated from results obtained with the constant ionic strength model (I ) 0.01-5.6); see Figure 2. c Average of results obtained with the variable ionic strength model in the range where the DE is valid (I ) 0.01-0.51); see SI Figure S2. d Fresh U. lactuca sample.
dilution with water. If so, a mean water content of ∼87% can be deduced for fresh biomass, which is typical for U. lactuca (23). The aggregate dry weight site density for BCR-279 (1.6 × 10-3 mol/g) is strikingly similar to that of 2.0 × 10-3 mol/g reported for the marine alga Dunaliella tertiolecta (24). The wet weight value for fresh U. lactuca (∼4-6 × 10-4 mol/g) likewise resembles published data for more distantly related organisms, for example, 3 × 10-4 mol/g for bacteria (3), 1.6 × 10-4 mol/g for yeast (4), and 2.4 × 10-4 mol/g for fescue grass (5). Our results may also be compared with those of Schiewer and Wong (18) who titrated dehydrated, ground biomass of U. fasciata at two unspecified ionic strengths, “high” and “low”. These authors titrated from pH 2 to 8, missing the high-pKa group, and modeled their data using a single functional group with pKa ) 5.3 (low I) and a site density of 1.1 × 10-3 mol/g. This is in good agreement with our data for the two more acidic groups: average pKa ) 5.23 and combined site density 1.0 × 10-3 mol/g. Identification of the Acid Functional Groups. Positive identification of acid functional groups solely from potentiometric titrations is a challenge that elicits two convergent approaches. One is to propose likely candidates based on the chemical composition of the investigated biomass. The second is to compare observed pKas with those of simple organic acids. The latter is more ambiguous, since pKas of functional groups depend on medium effects, the molecular scaffold to which they are linked (aliphatic, aromatic etc.), as well as the proximity, acidity, and site density of other proton-active moieties. It must be emphasized that the titrations convey little information about the location of acid functional groups, on or in the cell. Dehydrated or newly killed cells may be more permeable than those with active proton pumps, allowing some access to groups inside the plasmalemma. It is generally assumed that any groups detected in the titrations are also available for reversible cation-exchange (3). Our data have no direct bearing on alternate, irreversible modes of metal binding (15), which are beyond the scope of this paper. The chemical composition of U. lactuca has been studied in detail and is reasonably well-known. The cell material outside the plasmalemma is made up of polydisperse heteropolysaccharides that consist predominantly of the
(deoxy)hexoses rhamnose and glucose, the pentose xylose, and D-glucuronic acid. Hydroxyl groups on most rhamnose and up to 15% of the xylose units form sulfate half-esters (25). Percival (26) describes these as very strong acids, typically occupied by a variety of metal counterions. While sulfate groups are found in the cellular material of many plants, their role in proton and metal adsorption has received little attention. EXAFS experiments motivated Wang et al. (27) to attribute REE coordination by polysaccharides extracted from tea leaves to REEsOsS bonds, without providing much analytical detail. Fourest and Volesky (28) identified a strong acid functional group with a site density of ∼2.5 × 10-4 equiv/g on biomass of the brown macroalga Sargassum fluitans as sulfonate (lacking a bridging oxygen), but reported no pKa value. Commercial sulfonated polystyrene resins retain high affinity for tetra- and trivalent metal cations at pH < 1 (29). Sulfate or sulfonate groups should therefore contribute little buffer intensity within the pH window of our titrations and were probably not detected. Haug (30) proposed that the C-2 and C-3 hydroxyls of rhamnose form a cyclic borate ester that is subsequently cross-linked with Ca2+ ions into a stable gel. It appears that U. lactuca can regulate the stiffness of the gel by exerting some control over the presence of sulfate at the C-2 position, which prevents formation of the borate ester. This may help the organism avert desiccation, structural damage from wave action, and possibly even bacterial or viral attack (26, 30). Haug demonstrated that the gel dissolves in artificial seawater when either borate or Ca is omitted hence its stability clearly requires the presence of both. It was not feasible to conduct our experiments in seawater, because its alkalinity far exceeds that of the biomass. Brucite precipitation at pH ∼9 also obscures high-pKa groups like borate. Absence of the gel in our samples should only influence proton exchange on sulfate groups, which was undetected in any case. Takahashi et al. (31) stated that bacterial cell wall adsorptive properties are not modified by extracellular polymeric substances (gels). Our data indicate that U. lactuca biomass contains three distinct acid functional groups, or types of acid functional group, dissociating around pH 4, 6.5, and 9.5. Many authors have associated similar pKa distributions for bacterial biomass with two or three recurring functionalities, but their conclusions do not always agree. Ngwenya et al. (32) identified functional groups with pKas of 4.3, 6.9, and 8.9 on the Gramnegative enterobacterium Pantoea agglomerans respectively as carboxyl, phosphate, and amine or hydroxyl. They argued that Zn, Pb, and Cu adsorption occurs mostly on carboxyl groups at low pH. In a subsequent paper, batch adsorption experiments and EXAFS data revealed that REE adsorption at low pH on the same species is primarily due to phosphate groups, with carboxyl groups contributing only at higher pH and adsorption densities (33). Ngwenya et al. explain this apparent discrepancy from a heightened affinity for particular functional groups that enables certain metals to attach to them even when protonated. Depending on the regression model used, Fein et al. (20) found 2-4 acid functional groups on the Gram-positive aerobic bacterium Bacillis subtilis, with pKas in the range 3.3-3.5, 4.7-5.1, 6.8, and 8.6-8.9. Without attempting an exact identification, they point out that earlier EXAFS results (34) had attributed Cd and uranyl (UO22+) adsorption to phosphate groups at low pH and to carboxyl and phosphate groups at intermediate pH. Yee et al. (35) found three groups on the cell and sheath material of a Gram-negative cyanobacterium (Calothrix sp.) with very similar pKas (4.7-4.8, 6.5-6.6, 8.7-9.2) and identified these respectively as carboxyl, phosphate, and amine. They attributed most Cu, Pb, and Cd adsorption at near-neutral pH to the carboxyl groups. On the other hand, Gonzalez-Davila et al. (24) studied the marine alga D. tertiolecta, which is probably closer to U. lactuca in VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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composition and, finding three functional groups with pKas of 4.92, 6.28, and 10.06, associated the lowest one with carboxyl and the other two with amines. Ephraim et al. (2) invoked five functional groups to model titrations of aquatic fulvic acids in the pH window 1.7-7.0 and attributed two with pKas of 3.3 and 6.5 to phenols with either carboxyl or a second hydroxyl in ortho-position to the primary hydroxyl. Noting the great conformity in number, identities (pKas), and site densities of functional groups on both Gram-positive and Gram-negative bacteria, Ngwenya et al. (32) conjectured that “bacteria may be represented by a simple generic thermodynamic model for the purposes of modelling metal transport in natural environments” (see also ref 3). The present study of a marine macroalga corroborates evidence from recent studies of yeast (4) and land plants like red fescue grass (5) that this principle may hold for a much broader spectrum of organisms than previously realized. It seems that the highest pKa in our titrations (∼9.5) is frequently attributed to an amine, or possibly a hydroxyl. The other two pKas (∼4 and 6.5) fall in a range that covers a variety of carboxyls, in isolation or with hydroxyl in orthoposition, as well as hydroxyls with carbonyl or a second hydroxyl in ortho-position. Several authors have identified the intermediate-pKa group as phosphate, with the caveat that some metals seem to have a high affinity for phosphate groups and may thus attach to them at low pH. Phosphate is a component of cell membrane phospholipids; its pKa extends to even higher pH, although probably not as high as 9.5. There is little information about the properties of sulfate groups, yet they are a major constituent of algal biomass and their association with the lowest pKa cannot be ruled out at this point. It is however possible that these sulfate groups have pKas that fall well below the pH window of our titrations in which case they would have been missed. Resolution of these questions necessitates the application of complementary analytical techniques. Traditional batch experiments lend insight into the stoichiometry and thermodynamics of metal adsorption reactions, specifically at what pH metals attach to U. lactuca and how many moles of protons are released per mol of metal adsorbed. EXAFS spectroscopy, while difficult and laborious to perform as a function of pH (34), can supply crucial information about the coordination environment of bound metals. Both types of experiment are ongoing at CBL and will be the subject of future publications. Environmental Implications. The green macroalga U. lactuca is widely studied for its potential uses as biosorbent (e.g., refs 7-9) and biomonitor (e.g., refs 10–12). Such applications must be founded on a thorough understanding of the adsorptive properties of its biomass and how they are affected by environmental conditions like temperature, salinity (i.e., ionic strength), solution composition, etc. U. lactuca is quite tolerant of the cyclic salinity variations that occur in its estuarine and intertidal habitat (36). Figure 2 shows that these changes may have a significant effect on the protonation of functional groups and thus on their cation-exchange capacity, a fact that should clearly be accounted for when trying to relate ambient metalcontaminant levels to biomass contents. On the other hand, the persistent stability of acid functional groups from low to very high ionic strength (0.01-5.0 M) suggests that U. lactuca may be suitable as a biosorbent not just for seawater, but for aqueous solutions spanning freshwaters to brines (37). We furthermore assert that the prominent similarity of bacteria, fungi, and vascular plants in terms of functional group numbers, pKas, and site densities (3-5) extends to marine chlorophytes notwithstanding some unique chemical characteristics of their cell material. This upholds a growing notion that biogenic organic surfaces 1648
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might be parametrized in metal transport models as one archetypical cellular material with relatively few distinct moieties. Studies that yield thermodynamic pKa values (I ) 0) as well as functional relations for their accurate extrapolation to high ionic strength will help make the underlying algorithms ever more versatile and powerful.
Acknowledgments This study was conducted in summer 2008 while A.M.E. was an REU student at CBL. The UMCES REU program is funded by NSF (OCE-0754609) and administered by Maryland Sea Grant (MSGP). Its great success is largely due to the energetic efforts of Fredrika Moser. J.S. was funded by MSGP (Rp/TX-197) and NSF (OCE-0745881). REU student Lauren Hunker performed many ancillary experiments. Jeremy Fein kindly hosted J.S. at Notre Dame University to share his expertise in automated dynamic titrations and FITEQL modeling. We are grateful for the constructive comments of four anonymous reviewers and for the interest shown by Associate Editor David Dzombak. This is UMCES Contribution No. 4356.
Supporting Information Available Technical details of the FITEQL protocol, including A and B matrix templates (Table S1) and input parameters (Figure S1). Tables S2-S4 list the primary data. Figures S2 and S3 highlight some properties of the regression results. This material is available free of charge via the Internet at http:// pubs.acs.org.
Literature Cited (1) Loder, T. C.; Liss, P. S. Control by organic coatings of the surface charge of estuarine suspended particles. Limnol. Oceanogr. 1985, 30, 418–421. (2) Ephraim, J. H.; Bore´n, H.; Pettersson, C.; Arsenie, I.; Allard, B. A novel description of the acid-base properties of an aquatic fulvic acid. Environ. Sci. Technol. 1989, 23, 356–362. (3) Ginn, B. R.; Fein, J. B. The effect of species diversity on metal adsorption onto bacteria. Geochim. Cosmochim. Acta 2008, 72, 3939–3948. (4) Naeem, A.; Woertz, J. R.; Fein, J. B. Experimental measurement of proton, Cd, Pb, Sr, and Zn adsorption onto the fungal species Saccharomyces cerevisiae. Environ. Sci. Technol. 2006, 40, 5724– 5729. (5) Ginn, B. R.; Szymanowski, J. S.; Fein, J. B. Metal and proton binding onto the roots of Fescue rubra (sic). Chem. Geol. 2008, 253, 130–135. (6) Sawyer, C. N. The sea lettuce problem in Boston Harbor. J. Water Pollut. Control Fed. 1965, 37, 1122–1133. (7) Hamdy, A. A. Removal of Pb2+ by biomass of marine algae. Curr. Microbiol. 2000, 41, 239–245. (8) Suzuki, Y.; Kametani, T.; Maruyama, T. Removal of heavy metals from aqueous solution by nonliving Ulva seaweed as biosorbent. Water Res. 2005, 39, 1803–1808. (9) El-Sikaily, A.; El Nemr, A.; Khaled, A.; Abdelwehab, O. Removal of toxic chromium from wastewater using green alga Ulva lactuca and its activated carbon. J. Hazard. Mater. 2007, 148, 216–228. (10) Seeliger, U.; Edwards, P. Correlation coefficients and concentration factors of copper and lead in seawater and benthic algae. Mar. Pollut. Bull. 1977, 8, 16–19. (11) Phillips, D. J. H. Macrophytes as biomonitors of trace metals. In Biomonitoring of Coastal Waters and Estuaries; Kramer, K. J. M. Ed.; CRC Press: Boca Raton, FL, 1994; pp85-106. (12) Phaneuf, D.; Coˆte´, I.; Dumas, P.; Ferron, L. A.; LeBlanc, A. Evaluation of the contamination of marine algae (seaweed) from the St. Lawrence River and likely to be consumed by humans. Environ. Res. A 1999, 80, S175–S182. (13) Gutknecht, J. Mechanism of radioactive zinc uptake by Ulva lactuca. Limnol. Oceanogr. 1961, 6, 426–431. (14) Stanley, J. K., Jr.; Byrne, R. H. The influence of solution chemistry on REE uptake by Ulva lactuca L. in seawater. Geochim. Cosmochim. Acta 1990, 54, 1587–1595.
(15) Wang, W.-X.; Dei, R. C. H. Kinetic measurements of metal accumulation in two marine macroalgae. Mar. Biol. 1999, 135, 11–23. (16) Cosden, J. M.; Schijf, J.; Byrne, R. H. Fractionation of platinum group elements in aqueous systems: Comparative kinetics of palladium and platinum removal from seawater by Ulva lactuca L. Environ. Sci. Technol. 2003, 37, 555–560. (17) Webster, E. A.; Murphy, A. J.; Chudek, J. A.; Gadd, G. M. Metabolism-independent binding of toxic metals by Ulva lactuca: cadmium binds to oxygen-containing groups, as determined by NMR. BioMetals 1997, 10, 105–117. (18) Schiewer, S.; Wong, M. H. Ionic strength effects in biosorption of metals by marine algae. Chemosphere 2000, 41, 271–282. (19) Turner, A.; Pedroso, S. S.; Brown, M. T. Influence of salinity and humic substances on the uptake of trace metals by the marine macroalga, Ulva lactuca: Experimental observations and modelling using WHAM. Mar. Chem. 2008, 110, 176– 184. (20) Fein, J. B.; Boily, J.-F.; Yee, N.; Gorman-Lewis, D.; Turner, B. F. Potentiometric titration of Bacillus subtilis cells to low pH and a comparison of modeling approaches. Geochim. Cosmochim. Acta 2005, 69, 1123–1132. (21) Weast, R. C.; Astle, M. J. CRC Handbook of Chemistry and Physics, 63rd ed.; CRC Press: Boca Raton, FL, 1982. (22) Pagnanelli, F.; Bornoroni, L.; Toro, L. Proton binding onto soil by nonelectrostatic models: Isolation and identification of mineral contributions. Environ. Sci. Technol. 2004, 38, 5443–5449. (23) Robertson-Andersson, D. V.; Potgieter, M.; Hansen, J.; Bolton, J. J.; Troell, M.; Anderson, R. J.; Halling, C.; Probyn, T. Integrated seaweed cultivation on an abalone farm in South Africa. J. Appl. Phycol. 2008, 20, 579–595. (24) Gonzalez-Davila, M.; Santana-Casiano, J. M.; Perez-Pen ˜ a, J.; Millero, F. J. Binding of Cu(II) to the surface and exudates of the alga Dunaliella tertiolecta in seawater. Environ. Sci. Technol. 1995, 29, 289–301. (25) Percival, E.; Wold, J. K. The acid polysaccharide from the green seaweed Ulva lactuca. Part II. The site of the ester sulphate. J. Chem. Soc. 1963, 5459–5468. (26) Percival, E. The polysaccharides of green, red and brown seaweeds: Their basic structure, biosynthesis and function. Br. Phycol. J. 1979, 14, 103–117.
(27) Wang, D.; Wang, C.; Zhao, G.; Wei, Z.; Tao, Y.; Liang, X. Composition, characteristic and activity of rare earth elementbound polysaccharide from tea. Biosci. Biotechnol. Biochem. 2001, 65, 1987–1992. (28) Fourest, E.; Volesky, B. Contribution of sulfonate groups and alginate to heavy metal biosorption by the dry biomass of Sargassum fluitans. Environ. Sci. Technol. 1996, 30, 277–282. (29) Strelow, F. W. E.; Rethemeyer, R.; Bothma, C. J. C. Ion exchange selectivity scales for cations in nitric acid and sulfuric acid media with a sulfonated polystyrene resin. Anal. Chem. 1965, 37, 106– 111. (30) Haug, A. The influence of borate and calcium on the gel formation of a sulfated polysaccharide from Ulva lactuca. Acta Chem. Scand. B 1976, 30, 562–566. (31) Takahashi, Y.; Chaˆtellier, X.; Hattori, K. H.; Kato, K.; Fortin, D. Adsorption of rare earth elements onto bacterial cell walls and its implication for REE sorption onto natural microbial mats. Chem. Geol. 2005, 219, 53–67. (32) Ngwenya, B. T.; Sutherland, I. W.; Kennedy, L. Comparison of the acid-base behaviour and metal adsorption characteristics of a gram-negative bacterium with other strains. Appl. Geochem. 2003, 18, 527–538. (33) Ngwenya, B. T.; Mosselmans, J. F. W.; Magennis, M.; Atkinson, K. D.; Tourney, J.; Olive, V.; Ellam, R. M. Macroscopic and spectroscopic analysis of lanthanide adsorption to bacterial cells. Geochim. Cosmochim. Acta 2009, 73, 3134–3147. (34) Boyanov, M. I.; Kelly, S. D.; Kemner, K. M.; Bunker, B. A.; Fein, J. B.; Fowle, D. A. Adsorption of cadmium to Bacillis subtilis bacterial cell walls: A pH-dependent X-ray absorption fine structure spectroscopy study. Geochim. Cosmochim. Acta 2003, 67, 3299–3311. (35) Yee, N.; Benning, L. G.; Phoenix, V. R.; Ferris, F. G. Characterization of metal-cyanobacteria sorption reactions: A combined macroscopic and infrared spectroscopic investigation. Environ. Sci. Technol. 2004, 38, 775–782. (36) Dickson, D. M. J.; Wyn Jones, R. G.; Davenport, J. Osmotic adaptation in Ulva lactuca under fluctuating salinity regimes. Planta 1982, 155, 409–415. (37) Premuzic, E. T.; Lin, M. S.; Jin, J.-Z.; Hamilton, K. Geothermal waste treatment biotechnology. Energy Sources 1997, 19, 9–17.
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