Investigation of the Metal-Algae Binding Site with '13Cd Nuclear Magnetic Resonance Vahld Majidi,+ David A. Laude, Jr., and James A. Hoicombe"
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712 Metal-algae interactions in a unialgal culture (Stichococcus bacillaris) are examined by l13Cd nuclear magnetic resonance (NMR) spectroscopy. In addition to a peak attributed to cadmium in solution, a second broad resonance is assigned to cadmium binding with the algal cell wall. Studies of competitive uptake with other metals permit an affinity ranking assigned as Cu, Fe > Cd, Co, Na. pH studies indicate that although the presence of protons in the solution does not chemically alter the nature of the binding sites, the excess protons will directly replace the bound Cd. An NMR chemical shift of -10 to -18 ppm for the bound cadmium indicates the functional group responsible for metal uptake is most likely a carboxylic group. The chemical shift of the resonance also suggests the carboxyl groups involved in metal adsorption may possess larger chain lengths and perhaps multiple carboxyl groups per chain.
Introduction The potential utility of algae for preconcentration, speciation, separation, and detection of metals in aqueous samples has been well established (1-8). Several pure strains of algal microorganisms have been used in a variety of analytical experiments. These include algae-paste electrodes for determination of Cu(I1) ( 4 ) , immobilized algae for use in ion-exchange columns (7)) and suspended algae in solutions for single-metal or multielemental determination (5, 6, 8). Although these microorganisms are commonly used for metal adsorption, the nature of bonding between the metal and algae remains unclear. Mahan et al. (8) have worked with three different strains of freshwater blue-green algae (Chlamydomonas reinhartii, Chlorella pyrenidosa, and Stichococcus bacillaris). These organisms were mixed with aqueous solutions containing 16 different metal ions and the adsorption profiles for each element were determined with inductively coupled plasma atomic emission spectrometry. Surprisingly, the adsorption profiles of various metals were nearly identical for all three algae strains, indicating similar chemical environment and bonding nature. The mechanisms for metal uptake by bioorganisms are energy-dependent transport and adsorption to the cell walls ( I ) . However, researchers typically utilize dead or+ Present address: Dept. of Chemistry, University of Kentucky, Lexington, KY 40506.
0013-936X/90/0924-1309$02.50/0
ganisms so that the metal uptake is only due to adsorption onto the algae through interactions with the chemical functional groups found on the cell wall. Green et al. (9) suggested a list of potential functional groups on the cell wall that may be responsible for the metal uptake. These include amines, imidazoles, hydroxyls, carboxylates, phosphates, thiols, and thioethers. Crist and co-workers (10) also suggested several functional groups possibly involved in metal adsorption. The amino and carboxyl groups, the imidazol of histidine, and the nitrogen and oxygen of peptide bonds were suspected to be involved in coordinating metallic ions, while the unprotonated carboxyl oxygen and sulfate were suggested to be the sites for electrostatically bound metal ions. Watkins et al. ( I I ) presented some of the first direct evidence of metal-algae bond formation by using X-ray adsorption near-edge structure (XANES) and the extended X-ray adsorption fine structure (EXAFS). EXAFS provided information on the type and number of atoms bound to the metal, and their distances from the metals and XANES was used in determining the oxidation state of the bound metals. For these studies various gold samples were placed in contact with a single strain of algae (Chlorella vulgaris). Watkins and co-workers reported the formation of bonds between Au(1) and sulfur and/or nitrogen contained in the algae cells. Modes of binding for Au(I) different from those for Au(1II)Cl; were also observed. The uptake of Cd by S. bacillaris is well documented (1-3). Despite the ongoing research in this area, very little is known about the type of interactions that are responsible for metal uptake by these algae. From several pH studies and with comparison of pK values, Skowronski (2) suggested that carboxyl groups may be responsible for Cd adsorption by S. bacillaris. However, to data there is no direct evidence that aids in determining the chemical nature of metal binding sites. This paper explores the algae-metal interactions for S. bacillaris with W d NMR. Currently, the observed chemical shift of Il3Cd covers a range of 850 ppm. This large sensitivity of l13Cd chemical shifts to very small differences in the bonding environment is the most noteworthy characteristic of this nucleus. In addition to excellent sensitivity, l13Cd nuclei possess a nuclear spin of ' I zand a significant natural abundance (12.26%), which make this isotope an ideal choice for these studies. We have examined the nature of metal-algae bonding, the effect of pH variations, interference from various cations,
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and the possibility of multiple adsorption sites on the algae.
Experimental Section Algal Preparation. A pure strain of Stichococcus bacillaris was obtained from the culture collection of algae, (UTEX no. 419, Department of Botany, The University of Texas at Austin). The algae were transferred from an agar growth medium to 1L of a growth solution containing 118 mg of Ca(NO3)-4Hz0,40 mg of MgS04.7Hz0, 60 mg of disodium glycerophosphate pentahydrates, 50 mg of KC1,4.5 mg of Na2ETDA, 582 pg of FeCl3-6H20,246 pg of MnCl2.4HzO,30 pg of ZnCl,, 12 pg of CoC12-6H20,24 pg of Na2Mo04.2Hz0,0.25 pg of biotin, and 0.15 pg of vitamin B12. The organisms were harvested after 1 week of cultivation. The algae were heat treated (killed) at 85 "C for 20 min to eliminate complexities introduced by energy-dependent metal uptake. The suspended algae were centrifuged (Beckman 52-21) at 12lOOg for 10 min. The algae were then washed with double-distilled deionized water, and the centrifugation process was repeated. The resulting biomass pellet was finally lyophilized to yield a dry algae powder. Sample Preparation. Solutions were prepared for the NMR analysis by combining 300 mg of the dried algae powder with 3 mL of 0.05 M CdS04 in a 10-mm NMR tube. Lypholization permitted storage of the algae until a sufficient amount was harvested for the experiments and ensured a relatively uniform mixture would be used in any given set. The lypholized algae also prevented active transport of the metal, which might occur with living cells. The mixture was agitated for approximately 10 min in an ultrasonic bath and stored for several hours to permit the algae to settle in the NMR tube. A pH of 4.6 was measured for the unbuffered algae suspension. The slightly acidic character of the unbuffered suspension of the algae likely reflects the partially protonated surface resulting from algal growth in a slightly acidic media. Metals ions for competitive binding studies were obtained from three separate solutions of FeS04, CuS04,and CoS04,each metal being present at 0.5 M. For pH studies, the acidity and basicity of the NMR samples were adjusted by addition of dilute HzS04 or NaOH. All NMR samples were in aqueous media. NMR Parameters. NMR experiments were performed on a Nicolet Analytical Instruments FT-360 NMR spectrometer with an 8.4-T narrow-bore superconducting magnet. A 10-mm broad-band probe was tuned to observe the l13Cd signal at 80.093 MHz. Samples were not spun or locked. Magnet shimming was accomplished by using the proton decoupler channel to measure the free induction decay. The pulse sequence consisted of a 90' pulse, data acquisition over a A10 000-Hz bandwidth with 32K double-precision data points, and a 300-ms reequilibration delay. Initially, data were acquired over a f30000-Hz bandwidth (f380 ppm); however, since the resonances observed were very near the reference l13Cd signal, the observation window was reduced to improve digital resolution. Experiment times of approximately 10 h generated acceptable signals for the broad metal-algae resonance. All experiments were performed in triplicate on different days with the exception of the competitive binding studies, which were conducted in duplicate. Results and Discussion Competitive Binding. Figure l a is the NMR spectrum of 0.05 M CdS04. This spectrum contains a single resonance for aqua complex of CdS04, which is used as the chemical shift reference for the following experiments. 1310 Environ. Sci. Technol., Vol. 24, No. 9, 1990
i
I 40 30 20
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Chermcal Shift @pm)
Figure 1. '13Cd NMR spectrum of (a) a 0.05 M CdSO, solution at pH 4.6 and (b) 300 mg of the dried algae suspended in 3 mL of a 0.05 M CdS0, solution.
Typically, the chemical shifts for l13Cd NMR experiments are assigned with respect to Cd(C104), (12). However, because the addition of Cd(C104),may interfere with the competitive binding studies (due to the added anion effect), CdS04 was selected as the reference shift. The CdS04signal appears at 2.81 ppm upfield with respect to Cd(C104)2. The NMR spectrum of CdS04 and algae mixture is shown in Figure lb. The singlet at 0.0 ppm corresponds to the residual CdS04in the solution and the much broader resonance at 10 ppm upfield is assigned to Cd adsorbed on cell walls. The broadening of the adsorbed Cd signal is expected if the analyte of interest is immobilized on a solid substrate. Similar to Figure lb, Figure 2a and c are NMR spectra of the CdS04 and algae mixture. To observe the competition for binding site(s), 100 pL of 0.5 M FeS04was added to the solution in Figure 2a with the resulting spectrum depicted in Figure 2b. As shown in this figure, despite the fact that the concentration of FeS04 in the solution is only 1/3 of the concentration of CdS04, the signal due to the adsorbed Cd is completely lost. This indicates that the Fe will selectively remove Cd from its bound state to the algae. The competitive binding experiment was repeated for CuS04 by adding 100 pL of 0.5 M CuS04 to the solution exhibiting the spectrum in Figure 2c, and the result is presented in Figure 2d. As in the previous experiment, despite an analytical Cu concentration 1/3 that of Cd, the adsorbed Cd is fully displaced. In contrast, when 100 pL of 0.5 M C0S04 was added to Cd-algae solution, it only slightly reduced the magnitude of the broad Cd peak appearing at 10 ppm upfield. Only with addition of a second 100-pL aliquot of C0S04did the adsorbed Cd signal disappear in the base-line noise. Furthermore, the presence of up to 0.1 M Na+ (with SO4,+ as the counteranion) did not alter the Cd adsorption efficiency. Therefore, the affinity of algae sites for Na and Co is less than that of Fe or Cu. It appears that the algae have different metal affinities and for this strain a qualitative ranking for metal adsorption in the order of Cu, Fe > Cd, Co > Na can be
I
a)
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')
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/A4 0 30 2 0
Chemical Shift @pm)
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AAJ
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40 3 0 Z O
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Chemical Shift @em)
Figure 3. '13Cd NMR spectrum of 300 mg of the dried algae suspended
Figure 2. '13Cd NMR spectrum of (a and c) a 0.05 M CdSO, solution at pH 4.60, (b) 0.1 mL of a 0.5 M FeSO, added to the solution in a, and (d) 0.1 ml of a 0.5 M CuSO, added to the solution in c.
in 3 mL of a 0.05 M CdSO, solution. (a) pH 2.4; (b) pH 3.3, (c) pH 3.9, (d) pH 4.3,(e) pH 5.0, (f) pH 5.8.
assigned. From the NMR spectra presented here it is evident that the metals added to the CdS0,-algae mixture will directly replace the adsorbed Cd. This implies that there is a single dominant active site responsible for the adsorption of all of the examined metals, because if different sites were responsible for the uptake of different metals, then added ions would be adsorbed onto the algae without replacing the bound Cd. Furthermore, if multiple sites having different chemical nature were involved in the metal uptake, it would be illustrated in the NMR spectrum as multiple resonances. However, because only two resonances are observed, the aqua complex of CdS0, at 0.0 ppm and the algae-bound Cd at -10 ppm, it is likely that there is only one type of chemical functional group responsible for the metal uptake by S. bacillaris. pH Studies. When the algae are suspended in water, the pH of these mixtures are generally between 4.5 and 4.7. Previous work has demonstrated that as the pH of the algae mixture is changed the metal uptake capacity is drastically altered (2,13). It is clear that the pH of the algal environment plays an important role in metal adsorption. To investigate the impact of pH variations on the chemical nature of binding site(s), the NMR samples were prepared as before and the pH of each sample was adjusted by addition of dilute H2S04or NaOH. This experiment was conducted in two ways: (1)six separate NMR sample tubes were used and the pH of each tube was adjusted separately to a given pH; and (2) a single NMR sample was titrated with dilute H,SO, or NaOH to a desired pH value and a NMR spectrum was collected. The solution pH was then altered to the next value. This process was repeated until the pH range of interest was covered. Since results obtained from these two sets of experiments were identical, only the latter data are presented in Figure 3. The NMR spectrum of six algae mixtures with varying pH values were collected over a pH range of 2.4-5.8. Below pH 2.4 the one resonance present in the spectrum is assigned to the aqua complex of CdS0,. This indicates the
chemical environment at the cell wall of the algae has changed substantially so that Cd cannot be adsorbed. As the pH of the solution increases to 3.3 the broad resonance at -10 ppm appears above the noise level. As the pH increases the relative amount of the bound Cd continues to decrease, as witnessed by its signal magnitude relative to the base-line noise level. Beyond pH 6.0 the signal is no longer detectable. This dependence on pH is probably due, in large part, to the increased loss of Cd to the Cd(OH), precipitate that forms. Interestingly, when the pH of the metal-algae mixture is changed, no significant chemical shift for either the bound Cd of aqua complex of CdS0, resonances is observed. The lack of chemical shifts for the two resonances implies that the presence of protons in the solution does not chemically alter the nature of the binding sites. Furthermore, as the pH of these solutions is decreased, the uptake affinity is also decreased. This suggests that the Cd is reversibly exchangeable with protons at the binding site(s). Hence, it is most likely that the functional groups present on the algal cell walls are acidic in nature. Chemical Nature of Binding Site. The most attractive feature of l13Cd NMR spectroscopy is the possibility of assigning the chemical functional groups that are attached to Cd from chemical shift information. Cd chemical shifts are dispersed over an 850 ppm range, which should simplify the assignment even if they are chemically analogous. An expanded and enlarged view of the Cdalgae mixture NMR spectrum at pH 4.3 is shown in Figure 4. The bound Cd resonance spans an 8 ppm range centered at a maximum of -10 ppm, with the possibility of several additional resonances in the range -15 to -30 ppm. The most conclusive piece of information available from this spectrum is the chemical shift for the bound algae at -10 ppm. Some of the functional groups on the cell wall that were suggested to be involved in metal uptake (9,10) include imidazoles, hydroxyls, phosphates, thiols, thioethers, amino groups, carboxyl groups, and the nitrogen and oxygen of peptide bonds. In the case of Cd-imidazole Environ. Sci. Technol., Vol. 24, No. 9, 1990
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take, they must be different forms of carboxyl groups. It is still possible to have various functional groups working in tandem to adsorb metal. However, at the concentration range that these studies were performed, the carboxyl groups dominate over all other functional groups to such an extent that no signal other than the Cd-carboxyl was observed. Registry No. Cu, 7440-50-8;Fe, 7439-89-6; Co, 7440-48-4;Na, 7440-23-5; Ca, 7440-43-9; water, 7732-18-5. r
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Literature Cited
Y I
10
0
-10
-20
-30
Chermcal Shift (ppm)
Figure 4. Higkesolution ‘13CdNMR spectrum of 300 mg of the dried algae suspended in 3 mL of a 0.05 M CdSO, solution.
linkage, the Cd must be attached to one of the nitrogens in the ring structure. These functional groups and others that contain nitrogen typically have chemical shifts of 1+50 ppm (12,14,15). Cd bound to a hydroxyl is shifted 1+150 ppm, and Cd coordinated with phosphates appears at 1+80 ppm. Thiols and thioethers have the largest deshielding effect. If these functional groups are attached to Cd, the chemical shifts would be 1+500 ppm. Our initial bandwidth of +550 to -210 ppm therefore does not rule out possible linkage to sulfur for S. bacillaris. Functional groups known to shield Cd and yield negative chemical shifts are carboxyl groups and nitrates. Because nitrate is not known to play an important role as a functional group on the cell wall, the likely assignment for the metal adsorption is the carboxylic functionality. Moreover, small-chain carboxyl groups (e.g., acetates and formates) will only shield Cd to -1 ppm. Therefore, the carboxyl groups involved in metal uptakes likely possess a larger chain length and perhaps multiple carboxyl groups per chain. For example, Cd(02CCHCHC02)has a chemical shift of -7 ppm (15) with respect to Cd(C104)2. One final question to be addressed is the possibility of algae-metal interaction through multiple functional groups. According to the NMR spectrum, if there are several different functional groups involved in metal up-
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(1) Skowronski, T. Chemosphere 1984,13, 1385. (2) Skowronski, T. Chemosphere 1986,15,69. (3) Skowronski, T.; Przytocka-Jusiak, M. Chemosphere 1986, 15,77. (4) Gardea-Torresdey, J.; Darnall, D.; Wang, J. Anal. Chem. 1988,60,72. (5) Majidi, V.; Holcombe, J. A. Spectrochim. Acta 1988,43B, 1423. (6) Zimnik, P.R.; Sneddon, J. Anal. Lett. 1988,21,1283. (7) Kubiak, W. W.; Darnall, D.; Wang, J. Anal. Chem. 1989, 61,466. (8) Mahan, C. M.; Majidi, V.; Holcombe, J. A. Anal. Chem. 1989,61,624. (9) Green, B.; Hosea, M.; McPhenon, R.; Henzl, M.; Alexander, M. D.; Darnall, D. W. Enuiron. Sci. Technol. 1986,20,627. (10) Crist, R.H.; Oberholser, K.; Shank, N.; Nguyen, M. Environ. Sci. Technol. 1981,15, 1212. (11) Watkins, W., 11; Elder, R. C.; Green, B.; Darnall, D. W. Inorg. Chem. 1987,26,1147. (12) Ellis, P. D. In The Multinuclear Approach t o N M R Spectroscopy; Lambert, J. B., Riddell, F. G., Eds.; NATO AS1 Series 103; D. Reidel Publishing Co: Boston, MA, 1982; p 457. (13) Skowronski, T. Eur. J . Appl. Microbiol. Biotechnol. 1986, 24,423. (14) Mennitt, P. G.; Shatlock, M. P.; Bartuska, V. J.; Maciel, G. E. J . Phys. Chem. 1981,85,2087. (15) Armitage, I. M.; Boulanger, Y. In NMR of Newly Accessible Nuclei; Laszlo, P., Ed.; Academic Press: New York, 1983; VOl. 2, p 337.
Received for review July 26,1989.Revised manuscript received February 15, 1990. Accepted March 26, 1990.
