Environ. Sci. Technol. 1997, 31, 759-764
Chemical and Spectroscopic Characterization of Algae Surfaces
Environ. Sci. Technol. 1997.31:759-764. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 01/28/19. For personal use only.
E L K E K I E F E R , † L A U R A S I G G , * ,† A N D PAUL SCHOSSELER‡ Swiss Federal Institute of Environmental Science and Technology (EAWAG) and Swiss Federal Institute of Technology (ETH), CH-8600 Du ¨ bendorf, Switzerland, and Laboratory of Physical Chemistry, Swiss Federal Institute of Technology (ETH), ETH-Zentrum, CH-8092 Zu ¨ rich, Switzerland
The surfaces of two algae species (Cyclotella cryptica, diatom; Chlamydomonas reinhardtii, green alga) were characterized with regard to their interactions with copper. Chemical and spectroscopic methods (FT-IR, continuouswave EPR, and two-pulse ESEEM) gave information about the kinds of functional groups (-NH2, R-COOH, R-OH, and SiOH) on the surfaces of the algae. Maximum proton binding capacities of 9.7 × 10-4 and 9.1 × 10-4 mol/g algae (dry weight) were determined for C. cryptica and C. reinhardtii, respectively. The maximum Cu(II) binding capacity was 7.6 × 10-7 mol/g for the diatom and 6.2 × 10-6 mol/g for the green alga with [Cu2+] ) 10-13-10-11 in solution (at pH 6.9). Less than 1% of the total proton binding sites are occupied by copper under these conditions. The high conditional stability constants (log K ) 11.9 for C. cryptica and log K ) 11.3 for C. reinhardtii, at pH 6.9) demonstrate the strong binding of copper to the algae surfaces. These results are confirmed by the CW-EPR and two-pulse ESEEM spectroscopy, which indicate binding to N-ligands, with histidine being one of the possible ligands for copper. The binding sites on algae surfaces represent a buffer capacity for Cu2+ in natural waters.
Introduction Algae play an important role in the regulation and uptake of metals in oceans and in lakes. The interactions between algae and metals involve adsorption of the metal ions on their surfaces and intracellular uptake (1-4). Algae surfaces that comprise the cell walls and the membranes are important for metal binding; they contain various functional groups with different metal affinities. The cell walls of the algae Chlamydomonas reinhardtii and Cyclotella cryptica, which are examined in this study, have been previously studied with respect to their composition (5, 6). The electrophoretic analysis of the cell walls of C. reinhardtii by Davies (5) showed that they consist of about 30% proteins with a high amount of hydroxyproline. Hecky et al. (7) investigated the cell walls for a number of diatoms including C. cryptica. These walls consist of a silica frustule encased in an organic coating. Compared to cellular proteins, cell wall proteins are enriched in serine, threonine, and glycine, while acidic, sulfur-containing, and aromatic amino acids are less present. * Corresponding author telephone: +41 1 823 5494; fax: +41 1 823 5028; e-mail:
[email protected]. † EAWAG and ETH, Du ¨ bendorf. ‡ ETH, Zu ¨ rich.
S0013-936X(96)00415-4 CCC: $14.00
1997 American Chemical Society
The surface functional groups can be identified by chemical modification like esterification of COOH groups (8) or the reaction of SH groups with specific chemical reagents (9, 10). The surfaces of algae and other microorganisms have also been investigated by acid-base titrations and adsorption studies (11-21). Various spectroscopic methods can provide information about the binding sites for metals on algal surfaces (22). IR spectroscopy has been used to study the binding of Co to a marine alga (23) as well as to characterize functional groups on plant material and on biomass of a brown seaweed (24, 25). Watkins et al. (26) investigated the binding of Au to a green alga with XANES (X-ray absorption near- edge structure) and EXAFS (extended X-ray absorption fine structure) spectroscopy. Other methods, such as [113Cd]NMR (27) and [31P]NMR spectroscopy (28) were used to determine the affinity of the unicellular alga Stichococcus bacillaris for different metals (Cu, Fe > Cd, Co > Na) and to investigate the adsorption of Zn2+, Mn2+, Cd2+, and Cu2+ to the same algae. Mo¨hl et al. (29) applied magnetic resonance techniques such as CW-EPR (continuous-wave electron paramagnetic resonance), ENDOR (electron-nuclear double resonance), and two-pulse ESEEM (electron spin-echo envelope modulation) spectroscopy (30) to study the complex formation of copper with the bacterium Klebsiella pneumoniae. Many studies have been carried out at high metal concentrations; information on the more specific binding groups that may be relevant at the low metal concentrations of natural waters are however needed. Interactions of algae surfaces with Cu(II) are of special interest because of its importance as an essential and toxic element and because of its strong affinity toward organic ligands. In this work, the surfaces of two different algae species (Cyclotella cryptica and Chlamydomonas reinhardtii) and their interactions with Cu(II) are characterized using chemical and spectroscopic methods. The maximum proton binding capacity and the maximum number of binding sites for metals are determined by acid-base titrations as well as conditional acidity constants. Furthermore, the binding capacities for copper are determined at neutral pH and low Cu concentrations. The surface groups are analyzed using FT-IR (Fourier transform infrared spectroscopy) to determine the most important functional groups. Their interactions with Cu(II) are studied using CW-EPR and the complementary pulse technique, two-pulse ESEEM, that give information about the coordination sphere of Cu(II) and therefore about the Cu ligands.
Materials and Methods Cultures of the diatom Cyclotella cryptica No. 1070-1 and the green alga Chlamydomonas reinhardtii No. 11-32a were obtained from the Center of Algae Cultures of the Plant Physiological Institute of the University of Go¨ttingen, Germany. They were cultured in synthetic media at pH 7.2 under axenic conditions at room temperature and a constant irradiance of 35 µEinstein m-2 s-1. The composition of the culture media was chosen according to refs 31 and 32. pH was buffered with phosphate. EDTA (ethylenediaminetetraacetate, 2.5 × 10-5 M) was added to buffer the metal ions, with total Cu ) 1 × 10-8 M, total Zn ) 1 × 10-6 M, and total Mn ) 1 × 10-6 M. The calculated free [Cu2+] in the diatom medium was 10-13.6 M, and in the green alga medium it was 10-14.2 M [calculated with the speciation program MacMICROQL (33, 34)]. For all experiments, algae were harvested during the stationary phase. Algae dry weights were determined by filtering 5 mL of the algal suspension through 0.45-µm
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cellulose nitrate membrane filters (Sartorius GmbH) and drying at 110 °C. Acid-Base Titrations. The algae from the culture media were centrifuged (Kontron-Hermle Centrikon H-401) at 6000 rpm (3327g) for 10 min in 250-mL polypropylene beakers. They were washed three times with a sterile 0.01 M KNO3 solution at pH 7.2 to remove exudates and debris. Finally, the algae were resuspended in 100 mL of 0.01 M KNO3. A 50-mL sample of the algae suspension was pipetted into a titration beaker and de-aerated for 30 min with N2. The pH was measured after an equilibration time of 10 min with a Ross pH electrode (Orion) and a Metrohm 605 pHmeter. The titration was carried out with 0.1 M NaOH (Titrisol solution from Merck, Darmstadt) and 0.1 M HNO3 (Titrisol solution from Merck, Darmstadt) respectively. Algae concentrations were about 2 g dry weight/L. The total proton exchange capacity was determined by titrating the algae with an excess of 0.1 M HNO3. Adsorption of Copper. The cultured algae were harvested by centrifugation, washed, and resuspended in a sterile 0.01 M KNO3 solution. For each data point, two batch samples were prepared. A short reaction time (10 min) was used in order to distinguish between adsorption and intracellular uptake of Cu; this reaction time is a compromise that should keep uptake into the cells to a minimum (35). NTA solution (3 × 10-3 M, nitrilotriacetic acid disodium salt, Aldrich Chemicals, p.a.) and between 0.2 and 2.5 mL of a 10-4 M CuSO4‚5H2O solution were pipetted in 50-mL polypropylene bottles. A 20-mL sample of the algal suspension (2.5 g algae dry weight/L) was then added, and the volume was adjusted to 25 mL with 0.01 M KNO3. The pH was adjusted to 6.9 with 0.1 M NaOH or 0.1 M HNO3. The final NTA concentration was 1.6 × 10-5 M, and the total copper concentrations ranged between 10-5 and 8 × 10-7 M, which corresponded to calculated free copper ion concentrations between 4.4 × 10-11 and 2.6 × 10-13 M Cu2+. After a reaction time of 10 min, the suspensions were filtered, and the filter papers were dried in the desiccator. Before use, the 0.45-µm cellulose nitrate membrane filters were cleaned in hot 0.2 M HNO3 for 2 h and rinsed with nanopure water. The filters with the dried algae were digested with 65% HNO3 and 30% H2O2 in PTFE-PFE beakers (SV-140, Mikrowellen-Labor System GmbH) in a microwave oven (MLS-1200, Mikrowellen-Labor System GmbH). Copper was measured with a graphite tube-AAS (Varian AA-875 with GTA 95). The copper blanks of the digestion beakers were between 1.6 × 10-9 and 4.7 × 10-9 M (0.1-0.3 µg/L), and the copper blanks of the filter papers were between 1.6 × 10-9 and 6 × 10-9 M (0.1-0.4 µg/L). FT-IR Spectroscopy. The algae from the culture media were centrifuged and washed two times with a 0.2 M NaCl solution followed by resuspension in a NaCl solution to obtain a final concentration of 20-50 mg of algae/mL. The sample was replaced in the attenuated total reflectance (ATR) accessory sample boat in the FT-IR sample chamber. The spectra were recorded with 2000 scans and a resolution of 8 cm-1 using a BioRad FTS-45 spectrometer with a 3200 data station. CW-EPR and Two-Pulse ESEEM Spectroscopy. The EPR spectrum gives information about the ligands present in the coordination sphere of a Cu(II) ion with the spectroscopic tensor g, for which the values of its components (g| and g⊥) depend on the type of ligands and on the geometric arrangement of the complex. The coupling of the electron spin with the nuclear spin I ) 3/2 of copper gives the hyperfine structure characterized by A|. The ESEEM spectrum gives additional information about the molecular environment within about 0.5 nm of a paramagnetic species by resolving weak hyperfine interactions. The signal obtained is sensitive to the type and number of nearest-neighbor nuclei and to their distance from the paramagnetic species (29, 30).
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FIGURE 1. Titration curves of viable Cyclotella cryptica (b) and of Chlamydomonas reinhardtii (O) (2 g dry weight/L of viable algae, 0.01 M KNO3). The algae were harvested, centrifuged, and washed with 0.01 M KNO3. After resuspending the algae in 45 mL of 0.01 M KNO3 solution, 5 mL of 10-3 M CuSO4‚5H2O solution was added. The pH was adjusted to 7.2 with 0.1 M NaOH. After 3 h, the algae were centrifuged and dried in a desiccator. The CW-EPR spectra were recorded at room temperature on a Varian E 9 spectrometer. The spectra were measured at a microwave frequency of 9.50 GHz with a microwave power of 5 mW, a modulation amplitude of 0.125 mT, and a modulation frequency of 100 kHz. The ESEEM spectra were measured at 10 K on a home-built spectrometer, equipped with a helium gas cooling system (36). Two-pulse echo sequences with pulse lengths of 20/40 ns were used. The time increment between the two pulses was 10 ns, and the repetition rate was 500 Hz. The time signals were converted to frequency spectra by Fourier transformation after subtraction of the unmodulated part of the echo envelope, apodization with an exponential window function, and zero-filling to 1024 points. Magnitude spectra were calculated because of the spectrometer dead time.
Results Figure 1 shows the acid-base titration curves for the diatom Cyclotella cryptica and the green alga Chlamydomonas reinhardtii. The shape of the titration curve for the green alga indicates that its surface contains a larger variety of weaker acid-base groups than the surface of the diatom. In order to interpret the acidity of the functional groups, a two-pK model was assumed, in an analogous way as used for the description of oxide surfaces (37). In the pH range between 3 and 10, the average of the intrinsic acidity constants pK sa1 and pK sa2 were obtained from the plot of pK versus surface charge Q. The surface charge Q was calculated from the experimental titration data according to
1 Q ) (Ca - Cb - [H+] + [OH-]) a
(1)
where Q is the surface charge in mol/g algae dry weight, Ca is the added acid in M, Cb is the added base in M, and a is the algae dry weight in g/L. Using these Q values and the experimentally determined maximum proton exchange capacity {>RH2+} (mol/g), the conditional acidity constants K sa1 and K sa2 were calculated for each point. For this calculation, it was assumed that Q
TABLE 1. Chemical Properties of Algae Surfaces Chlamydomonas reinhardtii (green alga)
Cyclotella cryptica (diatom)
9.1 × 10-4
9.7 × 10-4
4.9 9.0 6.2 × 10-6
3.2 9.8 7.6 × 10-7
11.3 ( 0.2b
11.9 ( 0.2b
proton exchange capacity (mol/g) acidity constants pK sa1 (intrinsic) pK sa2 (intrinsic) Cu binding capacity (mol/g)a conditional stability constant for Cu log Ksa a pH 6.9, log [Cu2+] ) -13 to -11. linearization methods.
b
Errors estimated from several
FIGURE 2. Langmuir isotherms of Cu(II) adsorbed onto viable Cyclotella cryptica (b) and onto Chlamydomonas reinhardtii (O) (pH 6.9). The theoretical curves (lines) were calculated with log Ks ) 11.87 and Cumax ) 7.6 × 10-7 mol/g (Cyclotella) and with log Ks ) 11.34 and Cumax ) 6.2 × 10-6 mol/g (Chlamydomonas). ) {>RH2+} for pH < pHzpc and Q ) {>R-} for pH > pHzpc, where >RH2+ means a protonated functional group and >Ris a deprotonated group. Linear plots were obtained for pK sa1 and pK sa2 versus surface charge Q; these plots show a relatively narrow range of pKa values. Maximum proton exchange capacities and intrinsic pKa values (extrapolated to zero surface charge) are given in Table 1. These pKa values can be compared to those of functional groups of proteins. The intrinsic pKa values for the diatom are related to terminal carboxyl groups in proteins (pK sa1 ) 3.2) and to R-amino groups of asparagine, alanine, leucine, or the phenolic OH group in the side chain of tyrosine (pK sa2 ) 9.8). For the green alga, pK sa1 ) 4.9 can be assigned COOH groups in the side chain of asparagine or glutamine and pK sa2 ) 9 corresponds to R-amino groups (38, 39). The adsorption isotherms of Cu at pH 6.9 for the diatom and the green alga are plotted in Figure 2. The complexation of copper with the algal surfaces was interpreted with the Langmuir equation at constant pH, assuming that the adsorption of Cu occurs on a single type of surface group, within the range of Cu concentrations used, and that charge effects are negligible at constant pH. Linearization of the Langmuir equation with the method of Van den Berg and Kramer (40) and Ruzic (41) permits us to calculate the maximum adsorption capacity of copper, {>R-Cu+}max and the conditional stability constant Ks (eq 2)
[Cu2+] {>R-Cu }ads +
)
1 1 + [Cu2+] + Ks{>R-Cu }max {>R-Cu+}max (2)
where {>R-Cu+}ads is the concentration of the surface-bound
FIGURE 3. FT-IR spectrum of a suspension of viable Cyclotella cryptica at pH 7.
FIGURE 4. FT-IR spectrum of a suspension of viable Chlamydomonas reinhardtii at pH 7. copper (mol/g of algae dry weight), {>R-Cu+}max is the maximal concentration of surface-bound copper (mol/g of algae dry weight), [Cu2+] is the free copper ion concentration (M), and Ks is the conditional stability constant. The maximum binding capacities of copper and the conditional stability constants of the surface complexes are given in Table 1. The Langmuir fits in Figure 2 are calculated with these values. A Langmuir isotherm with one type of surface groups can fit the data in this copper concentration range, although treating the data with a one-ligand model is of course a strong simplification. In both algae species, less than 1% of the total proton binding sites are complexed by copper under these conditions, but these sites form highly stable complexes. The results obtained are conditional for the conditions of the adsorption experiment, namely, pH 6.9 and low [Cu2+]. No competing cations were present in the adsorption medium. Competition by other cations (e.g., [Mn2+]) in the culture media would only influence the small concentration of Cu bound to the cells at the low end of the adsorption isotherm. The FT-IR spectra of Cyclotella cryptica (diatom) and Chlamydomonas reinhardtii (green alga) are shown in Figures 3 and 4. The wavenumbers have been assigned according to ref 42. It must be taken into account that a clear distinction between cell surfaces and the inner cell is not possible. The FT-IR beam instrument penetrates about 5 µm into the material. The diameter of the algae is, however, between 14 and 22 µm. The spectrum of the suspension of viable green algae shows dominating peaks at 1623 cm-1 (CdO stretching), 1548 cm-1 (C-N stretching, N-H deformation), and 1243 cm-1 (C-O stretching in phenols) and a double peak at 1038 and 1079 cm-1. This double peak is probably caused by C-O stretching vibrations of alcoholic groups in carbohydrates, a main component of the cell walls of the green algae. The other peaks can be assigned to functional groups mainly in proteins
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FIGURE 5. CW-EPR spectrum of Cu(II) adsorbed onto dried cells of Cyclotella cryptica. T ) 298 K, MW frequency ) 9.5 GHz.
FIGURE 7. (a) Two-pulse ESEEM pattern of copper adsorbed onto dried cells of Cyclotella cryptica. (b) Fourier transformed ESEEM magnitude spectrum.
FIGURE 6. CW-EPR spectrum of Cu(II) adsorbed onto dried cells of Chlamydomonas reinhardtii. T ) 298 K, MW frequency ) 9.5 GHz. (CdO, C-N, and N-H vibrations), which represent a significant part of the cell walls and the membranes. The spectrum of C. reinhardtii is similar to those obtained with plant material [Datura innoxia (24)] and with seaweed (25). The spectrum of the diatom shows in contrast only two peaks at wavenumbers of 1623 cm-1 (N-H deformation) and 1038 cm-1 (Si-O stretching). The intense and widened peak at 1038 cm-1 is attributed to the high silicate contents of nearly 33% in the diatoms. Figures 5 and 6 show the CW-EPR spectra of the Cu(II)treated algae C. cryptica and C. reinhardtii. The splitting into four lines arises from the coupling of the electron spin of Cu(II) with the nuclear spin I ) 3/2 of copper. In the g ) 2 region, two other EPR active species contribute to the EPR spectrum: a 6-line pattern characteristic for Mn(II) with its nuclear spin I ) 5/2 and a single sharp line, indicating the presence of a stable radical. The signals of these two species are more intense in the spectrum of the green alga than in the one of the diatom. Mn(II) was not added in preparing the algae for the EPR spectra. The observed Mn(II) must thus have been taken up from the culture media (Mn concentrations in algae grown in these media without further treatment were 0.8-1.0 × 10-6 mol/g in both algae species). The components of the spectroscopic g tensor measured in these EPR spectra are as follows: a g| value of 2.25 for Cu(II) and a giso value of 2.00 for Mn(II) for C. cryptica. The coupling constants are 17.5 mT for copper (A|) and 9.5 mT (Aiso) for manganese. The g values for C. reinhardtii are g| ) 2.24 for Cu(II) and giso ) 2.00 for Mn(II). The coupling constants are 18 mT for Cu(II) (A|) and 9.5 mT for Mn(II) (Aiso). In the spectrum of the diatom, the Cu-hyperfine structure is clearly visible, while in the spectrum of the green
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alga only two hyperfine transitions are visible. The EPR parameters g| and A| are a sensitive measure of surface complex formation of Cu(II) ions as indicated by a decrease in the g| values as compared to the free aquoion, g| ) 2.44 (43). Similar decreases of g| values have been observed for the complexation of Cu(II) on various types of surfaces (43). The values obtained here are similar to those measured for Cu(II) bound to a bacterial surface (29). Figures 7 and 8 show the two-pulse ESEEM patterns and the Fourier transformed ESEEM magnitude spectra recorded at g ) 2.05 and g ) 2.02 positions, respectively. The resonance frequencies at 0.4, 1.6, and 3.7 MHz in the FT-ESEEM spectrum of C. cryptica can be related to weakly coupled, remote nitrogen atoms in ligands of Cu(II) complexes (29), whereas N atoms directly bound to Cu cannot be observed in this experiment. The frequencies correspond to the value of not directly bound nitrogen in Cu(His)2 complexes as determined by Mims and Peisach (44, 45). The peaks at 13.6 and 27.3 MHz are the proton resonance υH and the sum frequency 2υH. These two frequencies can be assigned to protons in water molecules in the surrounding of the paramagnetic center (46). The sum peak at 27.3 MHz corresponds to protons of axially bound and remote H2O molecules. The sum peak of protons in equatorially bound H2O molecules is shifted to 28.5 MHz. The transition frequencies in the spectrum of C. reinhardtii are 0.6, 1.9, and 3.8 MHz. The free proton frequency υH is 14 MHz, and the sum frequency 2υH is 27.8 MHz. The peak at 29.5 MHz corresponds to the sum frequency of protons in equatorially bound water molecules.
Discussion The potentiometric titration and the spectroscopic investigations demonstrate the possibilities to characterize the different surface functional groups of algae species and show that different groups are sensed, depending on the method used. The FT-IR spectra indicate a wide variety of functional groups,
FIGURE 8. (a) Two-pulse ESEEM pattern of copper adsorbed onto dried cells of Chlamydomonas reinhardtii. (b) Fourier transformed ESEEM magnitude spectrum. of which some, like the -OH groups of carbohydrates, are not relevant for proton and copper binding. They also point to the importance of N-containing groups. The data treatment used for the acidity constants involves the dependence of the pKa values on the surface charge. A relatively narrow range of proton affinities is obtained here. The variations in pKa values may be alternatively interpreted as due to the presence of a number of different surface groups with a range of pKa values (39, 47). It is therefore a strong simplification to assign only two intrinsic pKa values. The values obtained are however in a reasonable range characteristic of carboxylic and amino groups. The pKa values may be assigned to functional groups in proteins and related to the known composition of the diatom (7) and the green alga (5, 6). These pKa values are similar to those found for the marine phytoplankton species Dunaliella tertiolecta (21); the total proton binding capacity observed in ref 21 is also comparable (2 × 10-3 mol/g) to our values. Similar proton binding capacities (1-2 × 10-3 mol/g) have also been observed in Vaucheria cell walls (13) and in seaweed biomass (25). The higher copper binding capacity of C. reinhardtii in comparison with C. cryptica is compatible with the shape of the titration curve, which indicates a higher number of various types of functional groups. The results obtained here with low [Cu2+] in solution indicate that the complexation refers to a small number of ligands with a high selectivity for copper (high conditional stability constants), which represent only less than 1% of the total proton binding sites. A large part of the proton binding sites may be carboxylate groups that can also bind Cu, but with smaller affinities and selectivities, and are therefore not efficient at low [Cu2+]. The selective ligands are likely to contain N-donor atom or S-donor atoms (48), which form stronger complexes with copper than carboxylic groups. The CW-EPR spectra also indicate strong binding of Cu(II), and additionally the two-pulse ESEEM spectra confirm
the binding of copper to ligands with N-donor atoms that have a high affinity for copper. Although these N-ligands cannot be clearly identified, a comparison with the investigations of Cu/His complexes (45) and the spectra of bacteria (29) leads to the assumption that in the algae Cu(II) may also be bound to histidine residues. A possible structure may be a bidentate complex of copper with histidine and H2O molecules in the remaining equatorial and axial positions. Copper-histidine complexes are quite stable in solution [log KCu-His ) 10.2 (49)]; very high stability constants are also found for complexes of Cu(II) with peptides including histidine (49). The spectroscopic evidence confirms the conclusion from the adsorption isotherms, regarding the type of ligands involved. The conditions of the treatment of the algae with Cu are not exactly the same, more Cu being added to the algae for the spectroscopic studies. The signals sensed with the EPR spectra are however also those from stable complexes; more weakly bound Cu may not be sensed, respectively may not be distinguishable from Cu aquo complexes. The occurrence of Mn(II) in the EPR spectra indicates the presence of Mn(II) in this algal material, bound probably inside the cells. Competition of Cu(II) with Mn(II) may have occurred in the culture media, where it affects the concentrations taken up by the cells (50, 51). In cells grown in these media (with lower [Cu2+] than in the adsorption experiments), Mn was present in excess of Cu, probably mostly inside the cells. In similar experiments with another green alga (52), Mn(II) bound to surfaces was found to be negligible in this [Mn2+] range. The competition between Mn(II) and Cu(II) for surface groups in the adsorption experiments is thus probably not significant. In order to compare these results with those obtained by other authors, it is important to consider the free Cu2+ concentrations as well as the pH values used here. A number of studies were carried out at generally much higher concentrations of Cu and of other metals. Crist et al. (13, 14) carried out experiments at pH 4.5 with total copper concentrations in the millimolar range and obtained relatively small binding constants, which mean a weak complexation with copper, as expected at low pH values and high Cu concentrations. Conditional binding constants obtained for Cu(II) on a marine alga (21) are somewhat smaller (log K ) 8.5-9.5, pH 8.2) than those obtained here; these values were measured at somewhat higher Cu2+ concentrations (about 0.2-6 × 10-7 M) than those used in our experiments. Adsorption of cadmium and lead on biomass of a brown seaweed (25) were also carried out at high concentrations of these metals. In this case, the binding capacities obtained for these metals are close to the maximum proton binding capacities. The comparison with various studies indicates thus that different functional groups are important under different conditions. Carboxylate groups represent a major part of proton binding sites; their role in binding metal ions, as shown for example in refs 24, 25, and 53 is important at high metal concentrations, under which most of the available groups become saturated. At low metal concentrations however, more specific and selective groups, which are far less abundant, are significant. Generally complexation takes place at high copper concentrations with low affinity ligands, weak electrostatic bonds, and small stability constants. On the other hand, the complexation reactions at low copper concentrations occur with high affinity ligands, strong covalent bonds, and high stability constants. The conditions used in the present study favor binding to the highly selective binding sites. For comparison with a natural system, we consider Cu concentrations that have been measured in Lake Greifen, a small eutrophic lake. In this lake, the dissolved Cu concentrations are about 5-20 nM, whereas the free Cu2+ concentrations are very low, namely, in the range 10-14-10-16 M
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(54). Under these conditions, only small amounts of Cu would be adsorbed to the algae surfaces, since these Cu2+ concentrations are lower than the low end of the adsorption isotherm, and binding to the very selective functional groups only is relevant. Uptake of copper by the algae would therefore probably be more important than binding to surfaces. The binding capacities of the algae surfaces are however significant under the conditions of the lake in comparison to dissolved Cu. Assuming that algae may amount to about 1-2 mg dry weight per liter in the productive layer, binding capacities for Cu of 0.7-1.5 nM in the case of C. cryptica and of 6-12 nM in the case of C. reinhardtii are obtained, taking into account only the specific Cu binding groups. These capacities would only be saturated at high concentrations of Cu in solution, but they represent a high buffer capacity for Cu2+ in solution. Binding to algae surfaces and inside their cells may thus affect the dissolved Cu concentrations, and the sedimentation of algae contributes to the sedimentation of Cu.
Acknowledgments The authors are greatly indebted to Prof. A. Schweiger and his research group, Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, Zu ¨ rich, Switzerland, for the CW-EPR and ESEEM measurements. Furthermore, we would like to thank Stefan Hug for his introduction to the FT-IR spectrometer and Hanbin Xue for discussions of this work.
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Received for review May 13, 1996. Revised manuscript received October 16, 1996. Accepted October 23, 1996.X ES960415D X
Abstract published in Advance ACS Abstracts, January 1, 1997.
