Characterization of Lanthanide Ions Binding Sites in the Cell Wall of

Earlier studies have shown that Pseudomonas aeruginosa can adsorb selectively La3+, Eu3+, and Yb3+ from aqueous solution. These bacterial cells may fi...
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Environ. Sci. Technol. 2000, 34, 610-615

Characterization of Lanthanide Ions Binding Sites in the Cell Wall of Pseudomonas aeruginosa A N N E - C L A I R E T E X I E R , † Y V E S A N D R EÅ S , ‡ MYRIAM ILLEMASSENE,§ AND P I E R R E L E C L O I R E C * ,† De´partement Syste`mes Energe´tiques et Environnement, Subatech UMR 6457, Ecole des Mines de Nantes, 4 rue Alfred Kastler, BP 20722 44307 Nantes Cedex 03, France, and Institut de Physique Nucle´aire - Orsay, 91406 Orsay Cedex, France

Earlier studies have shown that Pseudomonas aeruginosa can adsorb selectively La3+, Eu3+, and Yb3+ from aqueous solution. These bacterial cells may find promising applications for removal and separation of lanthanide ions from contaminated effluents. In this work, potentiometric titrations and time-resolved laser-induced fluorescence spectroscopy were used to determine the binding sites of the biomass and, consequently, to elucidate the underlying mechanisms involved in the biosorption of lanthanide ions. Around 90 ( 5% of the adsorbed lanthanum was easily desorbed with an EDTA 0.1 M solution. In most instances, lanthanides seemed to concentrate extracellularly. The diversity of potential metal-binding groups was revealed by potentiometric titrations of the biomass. The amount of strong and weaker acidic functional groups in the wet biomass was estimated at 0.24 ( 0.04 and 0.86 ( 0.02 mequiv/ g, respectively. Time-resolved laser-induced fluorescence spectroscopy on europium-loaded P. aeruginosa biomass suggests that europium binding occurs mostly through carboxyl and phosphate groups.

Introduction Among the considerable diversity of biomass available, Pseudomonas aeruginosa has already proved to be very promising for heavy metal recovery (1-8). The lanthanides binding performance and selectivity of this bacterium have been demonstrated in previous works (9), but responsible mechanisms are still poorly understood. The use of cells from P. aeruginosa can be intended in potential applications for the removal and separation of lanthanide ions from aqueous effluents. It may be interesting to amplify investigations in order to determine the active binding sites usable to adsorb lanthanide ions. This kind of work can be the first necessary step in the attempt to elucidate the underlying mechanisms involved in the biosorption of lanthanide elements. A multitude of chemical microenvironments are present on the bacterial surface. These include phosphate, carboxyl, hydroxyl, and amino functional groups, among others (10). Various methods have been investigated to identify the bacterial surface functional groups involved in metal uptake. A first approach consists of performing metal binding studies * Corresponding author phone: 33 (0) 2 51 85 82 50; fax: 33 (0) 2 51 85 82 99; e-mail: [email protected]. † De ´ partement Syste`mes Energe´tiques et Environnement. ‡ Subatech UMR 6457. § Institut de Physique Nucle ´ aire. 610

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on extracted cell wall polymers, such as peptidoglycan and teichoic acid, to determine the types of cell wall components responsible for metal binding (11). In addition, selective chemical modifications of the various functional groups have been carried out to appreciate their contribution to the metal uptake (12, 13). The major inconvenience in the use of this kind of technique is a rather heavy experimental protocol which does not allow the study of intact cells for adsorption investigations. The potentiometric titration technique provides a simple and efficient method to measure and determine the different functional groups available to bind metallic ions. Consequently, the use of this method is interesting for the surface characterization of algae (14-16), fungi (17), and bacteria (18). Furthermore, various spectroscopic methods, including IR spectroscopy, XANES spectroscopy (X-ray absorption near-edge structure), EXAFS spectroscopy (extended X-ray absorption fine structure), and NMR spectroscopy, among many others, can provide information about the chemical environment of the sorbed metallic ions on biological material. Until recently, the emphasis has been placed on the use of such spectroscopic methods to characterize the surfaces of algae (19), bacteria (20), fungi (21), and plant cells (22, 23). Drake et al. (24) investigated the binding of europium to a biomaterial derived from the plant Datura innoxia and characterized functional groups answerable for metal ion uptake with the help of laser-induced spectrofluorometry. A simultaneous determination of emission wavelength and fluorescence lifetime provides two-dimensional information of fluorescing ions. Europium ion, which presents a strong fluorescence, is particularly useful for such investigations (25). The aim of the present study is to investigate qualitatively and quantitatively the potential ligands present in the biomass from P. aeruginosa. First, it was verified that lanthanide ions were essentially bound in the bacterial cell wall. Then, two main strategies were employed in order to achieve characterization of lanthanide ions binding sites: potentiometric titrations and time-resolved laser-induced fluorescence spectroscopy (TRLFS).

Materials and Methods Bacterial Cultures. Pseudomonas aeruginosa (strain A 22, Institut Pasteur, Paris, France) was grown aerobically with agitation in a nutrient broth (tryptone 10 g/L, meat extract 5 g/L, and NaCl 5 g/L) at 30 °C as described previously (9). Bacteria were precultured for 12 h, and 6 mL of the growing cell preculture was aseptically transferred to 300 mL of growth medium in a 1 L flask. All bacterial cells used in the study were harvested at the stationary phase. After approximately 24 h of culture, cells were harvested by centrifugation at 10 000g for 15 min. The paste was washed three times and resuspended in its weight of NaCl 9 ‰ solution. Cell batches dried at 37 and 70 °C to constant weight and ground to a powder within a granulometric range of 500-1000 µm were also used for potentiometric titrations. Metal Sorption. A 6 mM lanthanum [La(NO3)3, 6H2O; Aldrich Chemical Co., 99.99%] cation solution was prepared in distilled water adjusted to pH 5.0 by the addition of nitric acid. Biomass was placed in contact with the metal cation solution to the extent of 0.8 g of wet biomass to 20 g of solution (4% w/w). The suspension was shaken at 500 rpm at room temperature (20 ( 5 °C) for 3 h. After centrifugation at 15 000g for 10 min, the lanthanum ion remaining in solution was determined by an energy-dispersive X-ray fluorescence apparatus (Oxford ED 2000). The initial pH gradually changed in the course of 3 h to a value near 6.0 as metal ion was 10.1021/es990668h CCC: $19.00

 2000 American Chemical Society Published on Web 01/15/2000

sorbed by the cells. pH value for the precipitation of the La3+ ions by hydroxyl ions is 7.82 (26). Consequently, the formation of lanthanum precipitates during adsorption experiments was prevented. Desorption Experiment. P. aeruginosa was grown, harvested, and loaded with lanthanum (6 mM) as described above at pH 5.0. After centrifugation at 15 000g for 10 min, bacteria were washed twice with NaCl 9 ‰ solution, resuspended in ethylenediaminetetraacetate (EDTA) 0.1 M solution adjusted to pH 6.0, and placed on a rotary shaker for 30 min at 300 rpm. The bacteria were once again separated from the solution by centrifugation, and aliquots of the supernatants were analyzed by an energy-dispersive X-ray fluorescence apparatus (Oxford ED 2000) for metal content. It was verified that the amount of lanthanum desorbed by washes in NaCl 9 ‰ solution was negligible. Potentiometric Titrations. Potentiometric titrations were performed to obtain further information on the amount of strong and weak acidic functional groups in P. aeruginosa biomass. With the aid of a titrimeter (Titrino 716 DMS), titrations were carried out on three biomass samples: the wet ones and those dried at 37 and 70 °C according to the methodology described by Fourest and Volesky (15). The bacterial cells were first rendered acidic with 0.1 M nitric acid and washed twice with distilled water. To obtain constant conductance in the experimental media, protonated biomass (1 g dry weight) was dispersed in 50 g of 1 mM sodium chloride solution prepared with deionized water. Dried biomasses (37 and 70 °C) were stirred for 4-5 h in 50 g of this NaCl solution prior to titration. Titrations were carried out by stepwise addition of 0.1 mL of 0.5 M sodium hydroxide; potential and pH measurements were performed at 2 min intervals. The same procedure was used for titration of a test medium. Each titration experiment was carried out at least in duplicate. To evaluate the amount of different moieties in the biomass, the same titration experiments as described above were performed on biomass dried at 37 °C, whereby solutions of 0.5 M base of increasing strength (NaHCO3, Na2CO3, NaOH, and NaOC2H5) were added according to the Boehm’s method (27). Base solutions were freshly prepared and maintained under a nitrogen atmosphere. During the titration, the flask was covered in order to avoid dissolution of carbon dioxide in the solution. Time-Resolved Laser-Induced Fluorescence Spectroscopy (TRLFS). Apparatus. All fluorescence experiments were carried out at room temperature at the Nuclear Physics Institute (Orsay University, France). A YAG: Nd3+ pulsed laser operating at 532 nm was used as the excitation source. The signal emitted at perpendicular angle from the sample was analyzed by monochromator (Jobin-Yvon Spex 270M) and detected by ICCD camera (1024 pixels Princeton Instrument) cooled at -30 °C. The data of the photodiode array were read out by a control unit (Princeton Instrument ST138) and stored in a PC. A programmable Pulse Generator (Princeton Instrument PG200) allowed measurements with a delay adjustable from 1 ns to 80 ms during a time of 3.5 ns-80 ms. The fluorescence lifetime measurements were made by varying the temporal delay. Eu3+/P. Aeruginosa Samples and Model Compounds Preparation. The cultivation of the P. aeruginosa cells has been described above. The biomass suspension obtained was saturated with 8 mM [Eu(NO3)3, 5H2O; EuCl3, 6H2O; Eu2(SO4)3, xH2O; Aldrich Chemical Co., 99.9%] of metal solution to the extent of 0.8 g of wet biomass to 20 g of solution (4% w/w). The suspension was shaken at 500 rpm at room temperature (20 ( 5 °C) for 3 h. After centrifugation at 15 000g for 10 min, the saturated cells were washed twice with NaCl 9 ‰ solution at pH 5.0 to remove the excess metal. Metal

FIGURE 1. Potentiometric titrations on protonated biomass from Pseudomonas aeruginosa. biomass samples and europium salts were dried at 37 °C and run as solid powders. Several model compounds were selected to copy the chemical environments which could exist in the Eu3+/P. aeruginosa samples. A 16 mM Eu3+ stock solution was prepared from the nitrate pentahydrate (Aldrich Chemical Co., 99.9%) in distilled water adjusted to pH 5.0 by the addition of nitric acid. Each compound was prepared in distilled water adjusted to pH 5.0 from the 16 mM Eu3+ stock solution. Eu precipitates [Eu(OH)3, xH2O; Eu-Oxalate; Eu(PO4), xH2O] were washed twice in distilled water and separated from the solution by centrifugation at 15 000g for 10 min prior to be dried at 37 °C to be used as solid powders. Some Eu complexes (Eu-Asparagine and EuTETA) were obtained in blending the europium solution in an equimolar ratio with the solution containing the required ligand. After evaporation at 37 °C, dried compounds were recovered for time-resolved laserinduced fluorescence spectroscopy analysis. The Eu(III)TETA complex was prepared according to a procedure adapted from Bryden and Reilley (28). The ligand TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) was obtained commercially (Aldrich Chemical Co.). The europium complex was prepared in H2O from EuCl3, 6H2O (Aldrich Chemical Co., 99.9%) and adjusted to pH 6.57.0 with NaOH. A relative stoechiometry of 1:1 was chosen with a slight excess of TETA to avoid the presence of free EuCl3 precipitate in the sample.

Results and Discussion Desorption Experiment. The first necessary step in the attempt to characterize the lanthanide ions binding sites was to verify that metallic ions were actually bound in the cell wall of P. aeruginosa. Lanthanum was chosen as representative of the lanthanide elements which present similar chemical properties. Lanthanum loaded biomass was treated with EDTA 0.1 M solution. This complexant was chosen for its high formation constant of the species La(EDTA)- (pK ) 15.50 at 20 °C) (29). It was found that around 90 ( 5% of the biosorbed lanthanum was removed by EDTA. This result suggested that lanthanum was essentially sorbed extracellularly by binding sites located in the cell wall structure. Potentiometric Titrations. The unknown features of most biosorbents reduce their chance of being used as competitive products with respect to well-known synthetic ion exchangers (30). Potentiometric titrations of P. aeruginosa biomass were performed to obtain more information on the amount of strong and weaker acidic functional groups present. Figure 1 shows the potentiometric titration curves of protonated biomass resulting from the addition of 0.5 M NaOH. Similar curves were obtained for the three biomasses (wet and those VOL. 34, NO. 4, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Quantitative Estimations of Acidic Groups and Adsorption Capacity of Pseudomonas aeruginosa Biomass biomass dried at wet biomass

37 °C

70 °C

242 ( 40

228 ( 24

115 ( 15

862 ( 24

925 ( 7

978 ( 12

368 ( 64

384 ( 31

364 ( 27

equivalents of strong acidic groups (µequiv/g) equivalents of weaker acidic groups (µequiv/g) estimation of the adsorption capacity (µmol.trivalent cation/g)

FIGURE 2. Potentiometric titrations on protonated biomass from Pseudomonas aeruginosa by the addition of base solutions (0.5 M) of increasing strength. dried at 37 and 70 °C) and showed two distinct waves. This indicates the presence of at least two types of binding site, corresponding to strong and weaker binding affinity. The two waves were characterized by half-wave potentials at 150 ( 2 and -124 ( 4 mV. Quantitative estimations of adsorption capacities for the three types of biomass (wet and those dried at 37 and 70 °C) were deduced from these curves (Figure 1), considering that binding of a trivalent cation requires three equivalent acidic groups (Table 1). Free protons in solvated form were taken into account by results in test media containing 1 mM NaCl. It is evident from Table 1 that the cell wall of P. aeruginosa has substantially the same adsorption capacity for all three types of biomass used. Moreover, these results are in agreement with those obtained from the adsorption isotherms for lanthanide cations on wet biomass. Previous studies (9) showed maximum adsorption capacities of 397 µmol/g for lanthanum, 290 µmol/g for europium, and 326 µmol/g for ytterbium. pH values were determined at the twohalf-equivalence points of the titration curves, which correspond to the global pKa estimation of strong and weaker acidic groups. We found pKa1 ) 2.8 ( 0.2 and pKa2 ) 6.1 ( 0.2, respectively. A variety of potential metal-binding groups, such as phosphate, carboxyl, hydroxyl, and amino functions, occurs in the biomass (10). The envelope of Gram-negative bacteria consists of two membrane bilayers that contain a thin peptidoglycan layer between them (31). The outer membrane, characterized by a lipopolysaccharide (LPS) and phospholipids, is composed essentially of phosphate moieties that are able to bind metallic cations (11). Carboxyl moieties present, for example, in the peptidoglycan and the LPS also constitute sites of high affinity for metallic cations (12, 13). To evaluate the different functions in the biomass, the same titration experiments as above were performed on biomass dried at 37 °C, by the addition of base solutions (0.5 M) of increasing strength: NaHCO3, Na2CO3, NaOH, and NaOC2H5 (Figure 2) (27). The added NaHCO3 solution neutralizes only 612

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FIGURE 3. Fluorescence spectra of europium in Eu3+/P. aeruginosa sample and Eu3+ model compounds. Each sample was prepared from Eu(NO3)3, 5H2O (pH 5.0) and used as solid powders (37 °C). the strongest acidic groups, such as strong carboxyl or phosphate functions, whose concentration is estimated at approximately 114 ( 4 µequiv/g. The titration curve obtained by the addition of Na2CO3 solution presents two waves, showing also the presence of weaker acidic functions in the biomass (567 ( 36 µequiv/g). Results obtained by the addition of the strongest base solution, NaOC2H5, correlate very well with the NaOH titration curve. All of the acidic groups in the biomass were neutralized by NaOH and NaOC2H5 solutions, confirming the quantitative estimations of equivalents of acidic groups and adsorption capacity of P. aeruginosa biomass given in Table 1. All the strong and weaker acidic functions were estimated at 228 ( 24 and 925 ( 1 µequiv/g, respectively. From these results, it appears that the cell wall of P. aeruginosa would contain at least two types of strong acidic groups, possibly carboxyl and phosphate moieties, and two types of weaker acidic groups. The large variety of potential metal-binding groups present certainly contributes to the high adsorption capacities observed for this biomass. Time-Resolved Laser-Induced Fluorescence Spectroscopy (TRLFS). In this part, the characterization of Eu(III) binding to P. aeruginosa biomass was examined. The fluorescence properties of Eu(III) were used to study the contributions to europium ion sorption from different

FIGURE 4. Schematic diagram of a potential chemical environment for lanthanide ions bound in the peptidoglycan structure of Pseudomonas aeruginosa. This representation was made according to the peptidoglycan composition described by Quintela et al. (33). functional groups. Model compounds were chosen to represent various chemical environments which could be involved in the uptake of europium. These include Eu(OH)3 and Eu(PO4) precipitates, chosen as models respectively for hydroxyl and phosphate groups, a Eu-oxalate precipitate, and a Eu-asparagine complex, both containing carboxyl groups. First, it was verified that P. aeruginosa dried at 37 °C did not show measurable fluorescence. Figure 3 shows the fluorescence spectra obtained for Eu3+/P. aeruginosa sample and for Eu(OH)3, Eu-asparagine, Eu-oxalate, and Eu(PO4) compounds. The luminescence spectra obtained under YAG laser excitation at 532 nm are recorded in the range from 550 to 700 nm. They consist of two main lines observed around 592 and 615 nm which correspond respectively to the transition from 5D0 toward 7F1 and 7F2 multiplet. The energy of these most intense lines is reported in Table 2(a) for each compound. The weaker line at 585 nm represents the transition 5D0 f 7F0, and the broad band centered around 650 nm corresponds to the transition 5D0 f 7F3. To gain a better understanding of the contributions to europium uptake from the different functionalities, the fluorescence decay recorded at 592 and 615 nm was analyzed, and the lifetime of 5D0 emitting level was measured. Fluorescence lifetimes were determined by fitting the fluorescence decays to either a single- or biexponential function. Each distinct chemical environment for europium binding on the biomass will

TABLE 2. Main Emission Wavelengths and Lifetimes Obtained for Europium in Different Samples lifetimes of excited state 5D 0

(a)

(b) (c) (d)

samples

λemission (nm)

t1 (µs)

Eu(NO3)3 Eu(NO3)3/P. aeruginosa Eu(OH)3 Eu, asparagine Eu, oxalate Eu(PO4) Eu, TETA complex Eu2(SO4)3 Eu2(SO4)3/P.aeruginosa EuCl3 EuCl3/P. aeruginosa

592-617 592-615 593-614 592-617 593-618 592-613 588-612 593-615 592-616 593-613 592-616

187 254 144 239 269 745 1049 212 133 131 98

t2 (µs) 677

567 534

exhibit only one fluorescence lifetime. A comparison of the results is shown in Table 2(a). As model compounds are relatively simple systems, consisting of only one functional group, they are characterized by a unique lifetime. When europium was sorbed as the nitrate salt on bacterial cell wall, the luminescence decay could not be fitted by a monoexponential function, and the two different time components were longer than that of VOL. 34, NO. 4, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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unsorbed Eu(NO3)3, revealing the more complex P. aeruginosa sample contacted with Eu3+. The excited state of Eu(NO3)3/ P. aeruginosa was found to decay through a biexponential function with two lifetime components, t1 ) 254 µs ( 10% and t2 ) 677 µs ( 10%. This suggests that two distinct chemical environments would contribute to the europium uptake on biomass. The different lifetime values between Eu(OH)3 precipitate and Eu(NO3)3/P. aeruginosa sample lead us to discard the assumption of a significant precipitation of europium on the bacterial surfaces in the form of hydroxides. The first lifetime of Eu(NO3)3/P. aeruginosa sample (t1 ) 254 µs ( 10%) can be compared with those of Eu-oxalate and Eu-asparagine compounds, both containing carboxyl moieties, with t ) 269 µs ( 5% and t ) 239 µs ( 5%, respectively. These results are in agreement with previous assumptions: carboxyl groups would be responsible for the Eu3+ ion uptake. The second lifetime of 677 µs ( 10% indicates the presence of another chemical environment for europium binding. For a better understanding of this longer lifetime, the laserinduced spectrofluorometry study was investigated for a Eu(III) complex of TETA, a rigid macrocycle with amine and carboxylic functions. Fluorescence decay analysis on EuTETA complex resulted in a unique lifetime value of 1049 µs ( 5% (Table 2(b)). As expected, the europium lifetime in the EuTETA complex was longer than those of the previous studied compounds. The cavity of the TETA ligand can easily accommodate the Eu(III) ion, allowing nine coordinated ligands to wrap themselves around the ion. This structure constitutes a close shell arrangement with few coordinated water molecules (28) and a small vibrational amplitude (32) that limits the nonradiative desexcitation of the europium. This rigid chemical structure leads to a long-lived fluorescence. So, it can be supposed that the longest lifetime of Eu(NO3)3/P. aeruginosa would characterize a second type of ligand which would be located in a chemical environment implementing more stable interactions with the Eu3+ ion than those developed in the case of the first type of ligand. Moreover, if we consider the deviation percentage of the lifetime measurements, the longest lifetime obtained for the Eu(NO3)3/P. aeruginosa sample can be compared to that obtained for the Eu(PO4) precipitate, with t ) 677 µs ( 10% and t ) 745 µs ( 5%, respectively (Table 2(a)). It appears that the phosphate moieties could intervene in europium binding on biomass. Table 2(c,d) presents the fluorescence lifetimes obtained for europium used as sulfate or chloride salts. For the Eu(NO3)3/P. aeruginosa sample, the fluorescence decay of Eu2(SO4)3/P. aeruginosa and EuCl3/P. aeruginosa samples can be fitted by a biexponential function, implying two distinct types of potential ligands for europium sorption on biomass. However, different lifetime values were obtained for the three samples: Eu(NO3)3/P. aeruginosa, Eu2(SO4)3/P. aeruginosa, and EuCl3/P. aeruginosa. It seems that coanions, such as nitrate, chloride, and sulfate ions, could be involved in the chemical environment available for the europium uptake. However, earlier results (9) did not show a significant effect of these anions on the biosorption capacity of P. aeruginosa for lanthanide ions. This complementary information may contribute to shed further light on the mechanisms involved in lanthanide ions binding on bacterial cell wall. Adsorption studies have allowed us to show that the coanions NO3-, Cl-, and SO42-, applied as europium salts, did not inhibit quantitatively the metal binding. However, time-resolved laser-induced fluorescence spectroscopy studies have provided evidence for the presence of these anions in the chemical structure surrounding the Eu-ligand complex in the bacterial cell wall. Approach of Binding Sites Characterization. Potentiometric titrations and time-resolved laser-induced fluorescence spectroscopy have led to similar and/or complemen614

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tary results. They suggest that carboxyl and phosphate groups play an important role in europium sorption on P. aeruginosa biomass. The use of independent methods provides validation of such techniques for the characterization of bacterial surfaces. The schematic diagram presented in Figure 4 illustrates one of the numerous potential chemical environments for binding lanthanide ions in the cell wall of P. aeruginosa. The peptidoglycan structure was chosen because it is very similar in all bacteria. Most species of Gram-negative bacteria have mureins of the A1 g chemotype build up from N-acetylglucosamine-(β1f 4)-N-acetyl-muramyl-L-Ala-D-Glu-mA2pmD-Ala monomeric subunits (33). The peptidoglycan is a rigid, porous, and amorphous material with a cross-linkage of 37.2% for P. aeruginosa. Its three-dimensional network structure can offer accessible binding sites for metallic cations. As described in the Figure 4, various interactions can be developed for warranting the metal stability in the bacterial cell wall.

Acknowledgments The authors wish to thank Professor E. Simoni for making access to the TRLFS apparatus at the Nuclear Physics Institute (Orsay University, France) and for helpful discussions.

Literature Cited (1) Strandberg, G. W.; Shumate, S. E., II; Parrott, J. R., Jr. Appl. Environ. Microbiol. 1981, 41, 237-245. (2) Panchanadikar, V. V.; Das, R. P. Intern. J. Environ. Studies 1993, 44, 251-257. (3) Panchanadikar, V. V.; Das, R. P. Intern. J. Environ. Studies 1994, 46, 243-250. (4) Shumate, S. E., II; Strandberg, G. W.; Parrott, J. R., Jr. Biotechnol. Bioeng. Symp. 1978, 8, 13-20. (5) Asthana, R. K.; Chatterjee, S.; Singh, S. P. Proc. Biochemistry 1995, 30 (8), 729-734. (6) Chang, J.-S.; Hong, J. Biotechnol. Bioeng. 1994, 44, 999-1006. (7) Hu, M. Z.-C.; Norman, J. M.; Faison, B. D.; Reeves, M. E. Biotechnol. Bioeng. 1996, 51, 237-247. (8) Texier, A.-C.; Andre`s, Y.; Le Cloirec, P. Environ. Technol. 1997, 18, 835-841. (9) Texier, A.-C.; Andre`s, Y.; Le Cloirec, P. Environ. Sci. Technol. 1999, 33, 489-495. (10) Tobin, J. M.; Cooper, D. G.; Neufeld, R. J. Enzymol. Microb. Technol. August 1990, 12. (11) Beveridge, T. J.; Fyfe, W. S. Can. J. Earth Sci. 1985, 22, 18931898. (12) Beveridge, T. J.; Murray, R. G. E. J. Bacteriol. 1980, 141(2), 876887. (13) Doyle, R. J.; Matthews, T. H.; Streips, U. N. J. Bacteriol. 1980, 143(1), 471-480. (14) Gonzalez-Davila, M.; Santana-Casiano, J. M.; Perez-Pen ˜ a, J.; Millero, F. J. Environ. Sci. Technol. 1995, 29, 9(2), 289-301. (15) Fourest, E.; Volesky, B. Environ. Sci. Technol. 1996, 30, 277282. (16) Wang, X.; Ma, Y.; Su, Y. Chemosphere 1997, 35(5), 1131-1141. (17) Deneux-Mustin, S.; Rouiller, J.; Durecu, S.; Munier-Lamy, C.; Berthelin, J. C. R. Acad. Sci. Paris 1994, 319(II), 1057-1062. (18) Van der Wal, A.; Norde, W.; Zehnder, A. J. B.; Lyklema, J. Colloids Surf. B: Biointerfaces 1997, 9, 81-100. (19) Kiefer, E.; Sigg, L.; Schosseler, P. Environ. Sci. Technol. 1997, 31, 759-764. (20) Schweiger, A. Angew. Chem. 1991, 103, 223-250. (21) Sarret, G.; Manceau, A.; Spadini, L.; Roux, J.-C.; Hazemann, J.-L.; Soldo, Y.; Eybert-Be´rard, L.; Menthonnex, J.-J. Environ. Sci. Technol. 1998, 32, 1648-1655. (22) Tiemann, K. J.; Gardea-Torresdey, J. L.; Gamez, G.; Dokken, K.; Sias, S.; Renner, M. W.; Furenlid, L. R. Environ. Sci. Technol. 1999, 33, 150-154. (23) Salt, D. E.; Prince, R. C.; Baker, A. J. M.; Raskin, I.; Pickering, I. J. Environ. Sci. Technol. 1999, 33, 713-717. (24) Drake, L. R.; Hensman, C. E.; Lin, S.; Rayson, G. D.; Jackson, P. J. Appl. Spectrosc. 1997, 51 (10), 1476-1483. (25) Berthoud, T.; Decambox, P.; Kirsch, B.; Mauchien, P.; Moulin, C. Anal. Chem. Acta 1989, 220, 235-241. (26) Vickery, R. C. Chemistry of the Lanthanons; Butterworth Scientific Publication: London, 1953.

(27) Boehm, H. P. Adv. Catal. 1966, 16, 179. (28) Bryden, C. C.; Reilley, C. N. Anal. Chem. 1982, 54, 610-615. (29) Callow, R. J. The Industrial Chemistry of the Lanthanons, Yttrium, Thorium and Uranium; Pergamon Press: Oxford, 1967; p 135. (30) Brierley, C. L. Geomicrobiol. J. 1990, 8, 201-223. (31) Beveridge, T. J. Intern. Rev. Cytology 1981, 72, 229-317. (32) Spirlet, M.-R.; Rebizant, J.; Loncin, M.-F.; Desreux, J. F. Inorg. Chem. 1984, 23(25), 4278-4283.

(33) Quintela, J. C.; Caparro´s, M.; de Pedro, M. A. FEMS Microbiol. Lett. 1995, 125, 95-100.

Received for review June 15, 1999. Revised manuscript received November 29, 1999. Accepted December 1, 1999. ES990668H

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