Environ. Sci. Techno/. 1992, 26, 782-788
Characterization of the Carboxylate Groups on Datura innoxia Using Eu(1 I I) Luminescenc e, Edward R. Birnbaum, Dennis W. Darnall, and Gary D, Rayson" ernistry, New Mexico State University, bas Cruces, New Mexico 88003
Life Science Division (LS-31, bos Alamos National Laboratory, Los Alamos, New Mexico 87545 ~
The carboxylate groups responsible for binding of Cu(111, Eu(III), Gd(III), and Cd(I1) ions at pH 6.0 to Datura innoxia cell walls have been verified as belonging to two totally different chemical moieties containing two different kinds of carboxylate groups. One of the chemical moieties contains a single carboxylate group which binds to the metal ions in a 1:1 ratio while the other contains two carboxylate groups which bind in a 2:l ratio. The binding site composed of two carboxylate groups has a greater binding strength with a lower availability than that of the binding site composed of a single carboxylate group. No significant competition for binding of Eu(II1) between two different binding sites has been observed. Inter- and intramolecular energy-transfer processes have been successfully used to interpret the measured fluorescence lifetime data. The studies of competitive uptake by carboxylate groups between Eu(II1) and these metal ions demonstrate m affinity order of Cu(I1) > Eu(II1) = Gd(II1) > Cd(I1). U
Introduction
Recently, nonliving biomass materials have been demonstrated to be a promising industrial tool for the extraction of heavy toxic and economically important metal ions from wastewaters and mining effluents (1-5). Several different strains of algae, such as Chlorella vulgaris, Scenedesmus quadrkcauda (3),Stichococcus bacillaris (21, and Cyanidium caldarium (1) have been successfully demonstrated to bind Ag(I), Cu(II), Cd(II), Zn(II), Fe(II), A1(III?, and Au(II1). On the basis of these results, metal binding to nonliving cell walls has been postulated to occur mainly by complexation of the metal ions with biological ligands contained in or on the cell walls (1). However, the mechanism responsible for the biosorption of metal ions by these materials is still not clear. Compared with algal cell walls, little work has been published on the adsorption of metal ions by nonliving higher plant cells (6, 7). However, because the higher plants possess similar biological molecules in their cell walls, it has been suggested that the same kind of adsorption on the plant cell walls is likely to occur. Previously (8),carboxylate and sulfate groups had been demonstrated to be the dominant functional groups on the cell walls of Datura innoxia that bind Eu(II1). Binding to sulfate functionalities was observed to be the primary mechanism of Eu(II1) binding at pH 1 3 while either one or two carboxylate groups were involved in binding of pH 14. In this paper, further characterization of the binding of metal ions to carboxylate groups on Datura cell walls will be addressed. Darnall et al. (1) and Crist et ai. (9) have suggested a possible list of chemical functionalities responsible for the metal uptake on the algal cell walls to include amino, thioether, sulfhydryl, carboxylate, carbonyl, imidazole, phosphate, sulfate, phenolic, hydroxyl, and amide moieties. 782
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However, little characterization of the binding of cell walls to metal ions has been reported. The first direct investigation of the local binding formation between gold and the alga C. vulgaris was undertaken using X-ray absorption near-edge structure (XANES) and the extended X-ray absorption fine structure (EXAFS). These studies indicated that Au(1) was bound to the algal cell walls through either sulfur or nitrogen moieties (10). Recently, studies using lI3Cd NMR have indicated carboxylate functional groups on the alga S. bacillaris cell walls to be responsible for the binding of CdUI), Cu(II), Fe(II), Co(II), and Na(1) (2). Additionally, the carboxylate groups on Cy. caldarium, Eisenia bicyclis, Laminaria japonica, Spirulina platensis, and Chlorella pyrenoidosa algal cell walls have been reported by Darnall et al. (1)to be the dominant functional groups that bind to Cu(I1) and Al(II1) on the basis of a series of chemical-modification experiments in which carboxylate groups on the algal cell walls were esterified using acidic methanol (1). However, the detailed characterization concerning how many types of carboxylate groups on the biomass cell walls are present or how carboxylate groups bind to metal ions has not yet been reported. In this paper, these questions will be investigated using luminescence measurements of europium(II1) bound to D . innoxia. The unique characteristic of Eu(II1) ion is that both the ground-state (7F0)and first excited state (5D0) are nondegenerate. Because neither of these levels will be split by the crystal field exerted by the ligands, a one-to-one correspondence between the number of peaks in the excitation spectrum and the number of distinct Eu(II1) environments is expected even in the presence of low-symmetry ligand environments (11-12). Thus, interpretation of the excitation spectra associated with the electronic 7F0 5Dotransition of the Eu(II1) can be undertaken without a detailed analysis of the crystal field splitting. In this paper, the mechanism of the binding of the carboxylate group on D. innoxia cell walls to metal ions will be directly characterized by Eu(II1) luminescence. Once the mechanism of metal ion binding is more clearly understood, it may be possible to efficiently incorporate this biomass material into the development of a more cost effective method for the removal of toxic metals from water supplies.
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Experimental Section
Materials and Methads. Europium(II1) and gadolinium(II1) chloride solutions, prepared from Eu203and Gd,O, (Molycorp), were standardized with an ethylenediaminetetraacetic acid (EDTA) solution at pH 6 by the method described elsewhere (13-17). A 0.1 M 2-(Nmorpho1ino)ethanesulfonic acid (MES) (Sigma Chemical Co.) was used as a noncomplexing buffer. Hydrochloric acid (Baker, Inc.) and sodium hydroxide (Mallinckrodt) were used to adjust solutions to the desired pH. Distilled deionized water was used throughout the work.
00 14-936X/92/0926-0782$Q3.0010
0 1992 American Chemical Society
Table I. Fitted Parameters of Excitation Spectra of Eu(II1)-Datura in Figure 1
Eu(II1) concn, pg mL-I
first intensity"
first wavelength, nm
first line width, nm
second intensity"
second wavelength, nm
second line width, nm
base line intensity"
150 100 70 50 30 20
2604 2107 1964 1182 768 457
579.09 579.08 579.09 579.06 579.04 579.03
0.68 0.70 0.68 0.73 0.70 0.64
1385 1519 1381 913 895 648
579.39 579.38 579.38 579.37 579.33 579.37
0.41 0.45 0.41 0.43 0.42 0.45
128 241 67 384 622 697
Arbitrary units.
Table 11. Fitted Parameters of Excitation Spectra of Gd(II1)-Doped Eu(II1)-Datura in Figure 2 metal concn, r g mL-' ELI Gd 152.0 121.6 91.2 60.8 30.4 15.2
31.4 62.8 94.2 125.6 141.3
first intensity"
first wavelength, nm
first line width, nm
second intensity"
second wavelength, nm
second line width, nm
base line intensity"
1833 1513 1269 1071 454 214
579.09 579.09 579.08 579.09 579.09 579.02
0.68 0.66 0.68 0.67 0.65 0.68
1319 1000 1036 737 362 240
579.39 579.39 579.39 579.39 579.39 579.34
0.42 0.44 0.46 0.43 0.42 0.46
289 243 169 216 162 352
aArbitrary units.
The procedures used to grow and wash Datura innoxia biomass materials have been described in our previous paper (8). For Eu(II1)-binding experiments, a series of solutions containing different Eu(II1) concentrations varying from 20 to 150 pg mL-l were prepared by adding the stock europium(II1) chloride solution to 0.1 M MES buffer solutions at pH 6.0. A 5000 pg mL-l D. innonia sample was suspended in each of these Eu(II1) solutions. After a contact time of 1 h to establish equilibrium, the solutions were centrifuged. The solid Eu(II1)-Datura samples were dried with acetone and stored for spectral analysis. It should be noted that there was no detectable difference of the Eu(II1) excitation spectra between the acetone (Le., trace amount) and air-dried Eu(II1)-Datura samples. In gadolinium-doped Eu(II1)-Datura experiments, a series of solutions containing different mole ratios of Eu(111) and Gd(II1) were prepared by mixing Eu(l1I) and Gd(II1) with 5000 pg mL-' Datura in 0.1 M MES buffer solutions at pH 6.0. The total Eu(II1) and Gd(II1) concentrations were fixed at 1 mM. In the competitive binding experiments, the solutions containing 1mM Eu(111) (i.e., 152 pug mL-l) with 5000 pg mL-l Datura in 0.1 M MES buffer and various concentrations of Cu(II), Gd(111),or Cd(I1) were prepared by directly mixing the europium(II1) chloride solution with CuSO, (Aldrich), GdCl,, or CdS04 (Mallinckrodt) solution. All solutions were allowed to interact with the Datura cells for l h before centrifugation. The solid metal-containing Datura samples were dried with acetone and stored for spectral analysis. Excitation spectra of the electronic 7F0 5D0 transition of the Eu(II1) ions were acquired with the laser spectrofluorometer previously described (8, 13-1 7). Solid EuDatura samples (5000 pg) were glued on a thread using Glueit (Yasutomo Co.) and suspended in a cylindrical quartz cuvette (NSG Precision Cells). Emission was monitored a t 615 nm with an effective bandwidth of 1.67 nm. Luminescence spectral deconvolution was performed using a nonlinear least squares regression routine. The spectra were fitted to a sum of peaks having the conventional Lorentzian line shape, 1/[[2(x - W)/LI2+ 11, where I is the maximal intensity, W the location of the peak maximum, and L the line width. These fitted parameters
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(Le., I , W, and L for each peak) were then used to plot the individual resolved peaks and the fitted spectra. Lifetimes of the europium luminescence were obtained by following excitation at 580 nm. The luminescence at 615 nm was sampled with an Applescope A/D convertor (RC Electronics, Inc.). The detailed instrumental setup and modifications have been described elsewhere (8). The luminescence decay curves were fitted either with single- or multiple-exponential functions from which the individual lifetimes were determined. The peak areas of the excitation spectra were calculated using a trapezoidal approximation to an integral. Results and Discussion Excitation Spectra of Europium(II1). The excitation spectra taken from a series of solid Eu(II1)-Datura samples containing different Eu(II1) concentrations varying from 20 to 150 pg mL-l are shown in Figure 1, and those taken from gadolinium-doped Eu(II1)-Datura solid samples are shown in Figure 2. The corresponding fitted parameters including wavelengths, line widths, and peak and base line intensities are summarized in Tables I and 11, respectively. Because only two different exponential decay curves are present at pH 6 (a), the excitation spectra in Figures 1and 2 have been fitted with two Lorentzian curves. The fitted excitation peak near 579.1 nm in Figures l a and 2a has been previously assigned to the presence of both Eu(III)-sdfate and Eu(III)-carboxylate (i.e., 1:l ratio species) on the D. innoxia cell walls. The other peak near 579.4 nm in Figures l a and 2a has been proposed to be indicative of the formation of another Eu(II1)-carboxylate (i.e., 1:2 ratio species) in which Eu(II1) is bound by two carboxylate groups (8). The excitation peaks associated with the Eu(111)-sulfate and 1:l ratio species are severely overlapped so that the line width of the 579.1-nm peak is significantly greater than that of the 579.4-nm peak. The detailed characterization regarding the binding sites on D. innoxia cell walls has been described in our previous paper (8). In this paper, our attention will be focused on the further characterization of carboxylate groups on D. innoxia cell walls. Characterization of the Carboxylate Groups from the Analysis of Excitation Spectra. Carboxylate groups Environ. Sci. Technol., Vol. 26,
No. 4, 1992 783
577.4
579.0
Wavelength (nm)
58C
577.4
579 0
580.6
Wavelength (nm)
Figure 1. Excltatlon spectra at pH 6.0 of solid Eu(II1)-Datura samples with Eu(II1) concentrations of (a) 150, (b) 100, (c) 70, (d) 50, (e) 30, and (f) 20 pg mL-l: 0 , data points; -, individual Lorentzian fitted curves; -, the sum of the individual Lorentzian fitted curves.
have been shown to bind to Eu(II1) ions in either a 1:l or 2:l ratio (8). However, one question that needs to be answered is whether these carboxylate groups are from the same moiety which binds to Eu(II1) either through one or two carboxylate groups as acetate (8) or whether they are from two totally different moieties which contain different kinds of carboxylate groups. In order to discriminate the binding sites on Datura cell walls, the two fitted excitation peaks (Le., 579.1- and 579.4-nm peaks) shown in Figure 1are separately plotted with peak intensities w Eu(II1) concentrations. As shown in Figure 3, when the Eu(II1) concentrations are greater than 70 gg mL-', curve b derived from the 1:2 ratio species is saturated (i.e., levels off) while curve a (Le., 1:l ratio species) is still increasing, suggesting that the two binding sites associated with curves a and b do not compete with each other for binding of Eu(II1) ions. This implies that the carboxylate groups forming the two binding sites on Datura cell walls are different and independent. If all the carboxylate groups on the cell walls were located in a similar chemical environment, there would be a competition between the carboxylate groups of the two binding sites for Eu(II1). Therefore, it is likely that the carboxylate groups associated with the two binding sites on Datura cell walls are located in two different chemical environments. One of the chemical environments contains one single carboxylate group which binds to Eu(II1) in a 1:l ratio while the other contains two carboxylate groups which bind 784
Environ. Scl. Technol., Vol. 26, No. 4, 1992
to Eu(II1) in a 2:l ratio. The two carboxylate groups responsible for binding of Eu(II1) in a 2:l ratio could be located either adjacent to each other or linked to each other from remote locations to bridge the metal ion. Alternatively, the 2:l ratio species could also be formed if the two carboxylate groups binding to the metal ion were located in the same amino acid residue [i.e., R(CO,H),]. However, at this time, the data available are insufficient to differentiate between these possibilities. At lower Eu(II1) concentrations, ranging from 20 to 30 pg mL-', the intensities of curve b are greater than those of curve a. This result is totally different from that obtained at higher Eu(II1) concentrations (Le., 50-150 pg mL-l), where curve a has greater intensities than curve b. This might be explained by the 2:l ratio species (Le., curve b) having a greater binding strength with a lower availability than the 1:l ratio species. This would result in the Eu(II1) ions preferentially binding to the sites associated with the 2:l ratio species when the Eu(II1) concentrations are lower than 30 bg mL-l. This preferential binding behavior also suggests that the two binding sites are different and independent. In order to further characterize the two binding sites on Datura cell walls, Gd(III), which is considered to have an affinity similar to Eu(II1) to bind to the cell walls because of the similar radii and charges exhibited by both ions, was added to Eu(II1)-Datura samples to dilute the Eu(II1) concentration so that the intensities of the excitation peaks
0 . l ~ " " ' : " " ~ ' '
577.4
579.0
579.0
58( 5 577.4
Wavelength (nm)
5806
Wavelength (nm)
Flgure 2. Excitation spectra at pH 6.0 of Gd(II1)-doped solid Eu(II1)-Datura samples with Eu(II1) and Gd(II1) concentrations of (a) 152.0 pg mL-' Eu(III), (b) 121.6 pg mL-' Eu(II1) 31.4 fig mL-' Gd(III), (c) 91.2 pg mL-' Eu(II1) 62.8 pg mL-' Gd(III), (d) 60.8 pg mL-' Eu(II1) 94.2 fig mL-' Gd(III), (e) 30.4 pg mL-I Eu(II1) 4- 125.6 pg mL-' Gd(III), and (f) 15.2 pg mL-' Eu(II1) 141.3 pg mL-' Gd(II1): 0 , data points, -, individual Lorentzian fitted curves; -, sum of the individual Lorentzian fitted curves.
+
+
+
+
Table 111. Calculated Lifetimes of Eu(II1)-Datura at pH 6.0 second Eu(II1) first concn, lifetime, lifetime, Ng mL-' FS !J3
150 100 70
0
40
80
120
160
Eu(lll) Concentration (ppm)
Flgure 3. Relationship between excitation peak intensities and Eu(II1) concentrations obtained from four sets of samples (a) near 579.1 nm (in Table I) and (b) near 579.4 nm.
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associated with the 7F0 5Doelectronic transition of the Eu(II1) ions can be perturbed by Gd(II1). Since the energy difference between the 5D0state of the Eu(II1) and 6P7/a state of the Gd(II1) (Figure 4) is too large for an excitation energy transfer to take place, the relative intensity change between the two peaks near 579.1 (Le., 1:1 ratio species) and 579.4 nm (i.e., 2:l ratio species) shown in Figure 2 and Table 11will directly reflect the binding affinity difference between the Eu(II1) ions and two binding sites on the cell
15 f 1 254 f 5 15 f 1 265 f 2 15 f 1 307 f 25
Eu(II1) first second concn, lifetime, lifetime, Fg mL-' !JS J!s
50 30 20
15 f 1 16 f 1 16 f 1
413 f 17 419 f 13 571 f 35
walls. If the carboxylate groups of the two binding sites on the cell walls are located in a similar chemical environment so that the binding affinities for the two binding sites to Eu(II1) ions are the same, the excitation peak intensity ratios between the 579.1- and 579.4-nm peaks in Figure 2a-f should remain constant while the total metal ion concentrations of the Eu(II1) plus Gd(II1) are fixed (i.e., 1mM). However, as shown in Figure 2f and Table 11, the peak intensity of the 579.4-nm peak is greater than that of the 579.1-nm peak. This result is different from that shown in Figure 2a-e, where the peak intensities of the 579.1-nm peaks are greater than those of the 579.4-nm peaks. This implies that the binding affinities for the two binding sites to Eu(II1) ions are different, suggesting that the carboxylate groups responsible for the two binding sites on the cell walls are located in two different chemical Environ. Sci. Technol., Vol. 26,
No. 4, 1992 785
6D
512
P
4c
912
6I
1512 712 312
3E 6p
32
-
7-
712
2e
E m
