Copper uptake kinetics in diverse marine phytoplankton

We measured short-term uptake rates of copper using the gamma-emitting radioisotope 67Cu in seven algal species in natural and artificial seawater. Cellular net uptake of Cu was typically rapid over periods of 2–20 min. Net uptake ceased after about 60 min. The most copper-sensitive species examined, Synechococcus sp., exhibited 2–3 orders of magnitude higher carbon and surface-area–normalized Cu-accumulation rates (46 mmol Cu mol C21 min21 and 1,100 zmol Cu mm22 min21) (zmol 5 10221 moles) than those measured in diatoms, chlorophytes, a dinoflagellate, and a coccolithophore. Cu-accumulation rates for Thalassiosira weissflogii were three times faster in natural seawater than in EDTA-buffered artificial seawater containing an inorganic Cu concentration of 28 pmol L21. Calculations showing that the diffusive flux of inorganic Cu was insufficient to account for observed short-term uptake rates suggest that some of the Cu bound to naturally occurring organic ligands is released through the rapid dissociation of those complexes in the cell’s boundary layer. As with other metals, Cu is essential for the function of a number of enzymes involved in oxygen chemistry and redox reactions (e.g., cytochrome oxidases and superoxide dismutase), but becomes toxic when cellular concentrations are elevated (Williams and Da Silva 1996; Raven et al. 1999). Its unique chemical properties—the ability to form a reduced ion (Cu ) that forms stable complexes facilitating electron transfer—make Cu well suited for some biological functions. Over the past half billion years, dissolved Cu concentrations have increased in the ocean as the build-up of O2 has accelerated the release of Cu from otherwise insoluble CuS (Williams and Da Silva 1996). On shorter time scales (hundreds of years), there has been an increase in dissolved Cu concentrations found in coastal systems as a result of anthropogenic inputs, particularly from industry. As a result of changing ocean chemistry on a range of time scales, marine microalgae have had to find ways to cope with excess dissolved Cu in seawater (total dissolved Cu concentrations of 1029 to 1028 mol L21) relative to their biological requirements (10221 to 10218 mol Cu cell21) and maintain intracellular pools of Cu at nontoxic levels. Their ability to do so appears to be taxa specific (Brand et al. 1986). 1 Present address: Department of Marine Biology, Texas A&M University, Galveston, Texas 77551. Acknowledgments This work was supported by NSF grants OCE-0084032 to P.G.F. and OPP-9986069 to N.S.F. and a Hatch/McIntyre-Stennis grant through the New Jersey Agricultural Experiment Station to J.R. We thank S. I. Chang, Y. Zhuang, and J. Kerkfoot for laboratory assistance. P. G. Falkowski and O. Schofield are thanked for their many discussions and continued support, and to the two anonymous reviewers who provided critical comments that improved the final manuscript. Strategies evolved by phytoplankton to preserve low intracellular concentrations of metals include (i) biomethylation and transport through cell membranes of metal alkyl compounds by diffusion controlled processes, (ii) the biosynthesis of intracellular polymers that serve as traps for the removal of metal ions from solution, (iii) the sequestration of metal ions to cell surfaces, (iv) the precipitation of insoluble metal complexes (e.g., metal sulfides), and (v) metal exclusion from cells (Davis 1976; Foster 1977; Williams and Da Silva 1996; Whitfield 2001). While biomethylation appears to be limited to nonessential metals (e.g., HgII ), the other strategies are pertinent to our understanding of Cu accumulation and homeostasis in algal cells. A considerable literature exists on the toxicity and bioaccumulation of Cu in phytoplankton, but only a few studies have addressed the kinetics of Cu uptake and release in these cells (Hill et al. 1996; Knauer et al. 1997; Croot et al. 2003). Tracer studies are further limited because a long-lived Cu radioisotope or a stable isotope of low mass abundance is not readily available. Studies of Cu accumulation have revealed that Cu ions bind specialized transport ligands associated with the cell membrane. Cu uptake is light and ATP dependent (Verma and Singh 1990) as well as temperature dependent (Hill et al. 1996), and follows Michaelis–Menten kinetics for facilitated and active transport (Sunda and Huntsman 1995; Hill et al. 1996). The Cu transport rate is thought to be determined by the activity of the free metal ion in the medium and not the total complexed Cu concentration (Sunda 1994; Van Leeuwen 1999). Knauer et al. (1997) found that Cu uptake is mediated by two systems in the freshwater chlorophyte Scenedesmus subspicatus: a high-affinity and a lowaffinity system operating at pCu 14–12 and pCu ,12, respectively.