Environ. Sci. Technol. 2005, 39, 3040-3047
Cadmium Uptake by a Green Alga Can Be Predicted by Equilibrium Modelling
flux or accumulated metal concentrations (eq 3; for reviews see refs 1-3).
H E L I A N A K O L A †,‡ A N D K E V I N J . W I L K I N S O N * ,† Analytical and Biophysical Environmental Chemistry (CABE), University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland, and Environmental and Analytical Chemistry, Battelle Geneva Research Center, 7 route de Drize, 1227-Carouge, Switzerland
Mz+ + Rcell a M-Rcell (adsorption to transport sites, Rcell, at the cell surface) (2)
Short-term uptake of cadmium by a wild-type (2137) and a cell wall-less strain (CW-2) of Chlamydomonas reinhardtii was examined as a function of Cd speciation in a welldefined, aqueous medium. Internalization fluxes were determined for free cadmium concentrations ranging from 5 × 10-10 M to 5 × 10-4 M in the presence of ligands forming both labile and inert hydrophilic complexes. A firstorder biological internalization, as predicted by the free ion activity model (FIAM), was observed for both strains. The maximum Cd internalization flux, Jmax, for the wild-type strain was 5-fold higher (1.3 × 10-11 mol cm-2 min-1) than for the CW-2 strain (2.3 × 10-12 mol cm-2 min-1) and was not influenced by the presence of competitors such as Ca in the experimental solution. The conditional stability constant for the adsorption of Cd to transport sites of the CW-2 strain was 5-fold higher (106.7 M-1) than for the wildtype strain (106 M-1). Competition experiments demonstrated that Mo, Mn, Cu, Co, Zn, Ni, Ca, and Pb inhibited, at least partially, Cd uptake, while no inhibition was observed for similar concentrations of Mg and Fe. The stability constant for the competitive binding of Ca to the Cd transport site was determined to be 104.5 M-1. Cu and Zn competed with Cd uptake sites with stability constants of 105.6 and 105.2 M-1, respectively. Protons also appeared to compete with Cd uptake sites as uptake could generally be predicted quantitatively in their presence. Finally, in the presence of low concentrations (25 min (Figure 1a), suggesting that Cd was released into the bulk solution as a result of cellular efflux or the release of a Cd-cell wall complex (35) (discussed below). In the presence of Cd complexes that ranged from inert (i.e., NTA: L < 1) to very labile (i.e., chloride: L .1) (eq 6), Cd internalization fluxes by the wild-type (WT) and cell wallless (CW-2) strains were linearly related to time ([Cd2+] < 5 × 10-7 M), i.e. a slope of unity was observed in the logarithmic plot of Jint vs [Cd2+], as would be expected under steady-state conditions (Figure 2). This result is also consistent with calculations of the maximum theoretical diffusive flux of Cd2+ (Figure 2a,b: dashed line) that demonstrated that uptake was not mass transport limited, implying that the supply of Cd2+ to C. reinhardtii was sufficient to sustain uptake in the absence of the complexes. In other words, the turnover rate of the Cd transport sites was so slow that even though the labile complexes could theoretically contribute to Cd uptake, they were not required. The observed first-order uptake and single saturation plateaus for the WT and CW-2 strains
FIGURE 3. Intracellular (circles) and dissolved (triangles) Cd after resuspension in 10-2 M MOPS (open symbols) or in 10-2 M MOPS/ 10-3 M EDTA (filled symbols) as a function of contact time. Under these conditions, effluxes corresponded to the following: (O) 2.5 × 10-14 mol cm-2 min-1; (3) 2.0 × 10-14 mol cm-2 min-1; (b) 6.8 × 10-14 mol cm-2 min-1; (1) 7.2 × 10-14 mol cm-2 min-1.
FIGURE 2. Logarithmic representation of Cd internalization fluxes as a function of [Cd2+] in the absence (O) or presence of citric acid (b), diglycolic acid (9), chlorides (1) and NTA (2) for C. reinhardtii wild-type (a) and cell wall-less strains (b). The long dashed line represents the calculated maximal diffusive flux for Cd2+ (discussed later in paper). The solid line represents a Michaelis-Menten plot for KM ) (0.9 ( 0.1) × 10-7 M and Jmax ) (1.3 ( 0.1) × 10-11 mol cm-2 min-1. The short dashed line represents the Michaelis-Menten plot for KM ) (1.9 ( 0.2) × 10-7 M and Jmax ) (2.3 ( 0.6) × 10-12 mol cm-2 min-1. (c) Logarithmic representation of the intercepts in the Cdint vs time plots as a function of [Cd2+] in the absence (O) or presence of citric acid (b), diglycolic acid (9) and NTA (2) for the wild-type C. reinhardtii. The dotted line represents a Langmuir isotherm for log Kads ) 106.2 ( 0.1 M-1 and Rmax ) (3.2 ( 0.1) × 10-11 M mol cm-2. The intercepts have been previously postulated to represent metal bound to the transport sites (36). Standard deviations are given when larger than the symbol size (n ) 2 to 6). A regression of the calculated versus experimental data gave significant r2 values of (a) 0.98, (b) 0.87 and (c) 0.90. suggested that cadmium internalization occurred by a single transport site in both strains. The WT strain of C. reinhardtii accumulated cadmium more rapidly than the CW-2 strain in the short-term experiments. For the wild-type strain, a Michaelis-Menten plot of the data (Figure 2a: solid line) gave an apparent half saturation constant, KM, of (9.9 ( 0.1) × 10-7 M and a maximal internalization flux, Jmax, of (1.3 ( 0.1) × 10-11 mol cm-2 min-1. The maximum internalization flux for the CW-2 strain was 5-fold lower than that observed for the wild-type strain with values of KM of (1.9 ( 0.2) × 10-7 M and a Jmax of (2.3 ( 0.6) × 10-12 mol cm-2 min-1 (Figure 2b: short dashed line). Under steady-state conditions, the apparent stability constants of the metal with the transporter (KM-Rcell) can be determined from the value of KM-1. A value of 106.7 M-1 obtained for the CW-2 strain indicated that transport was of slightly higher affinity than for the WT strain (KCd-Rcell ) 106.0 M-1). Despite the slightly higher affinity of the CW-2 strain for Cd, internalization fluxes were similar at low [Cd2+] suggesting
that the number of transport sites was greater for the WT strain. Macfie and Welbourn (28) have reached similar conclusions following a 24-h exposure to Cd in which the WT strain internalized 40% more Cd than the cell wall less strain. A comparative study of Cd, Ni, Cu and Co toxicity to the walled and wall-less strains of C. reinhardtii has previously concluded that the cell wall played a role in conferring metal tolerance and that the wall-less strain was more sensitive to Cd than the WT strain (27). In this study, internalization fluxes were shown to be very similar for low [Cd2+] (below KM), whereas at high [Cd2+], the wild-type strain accumulated significantly more Cd. Moreover, measured Cd internalization fluxes were an order of magnitude below the predicted diffusive flux of the free ion (long dashed line in Figure 2a,b), i.e., due to the limiting nature of Cd internalization, small (
