ENVIRONMENTAL CHEMISTRY AND
MICROBIOLOGY OF TRACE METALS:
An Archive of Past Research
Previous Research Themes and Achievements
Over the years, the research activities in the Morel group have been focused principally on the related research themes of the chemical speciation of trace metals in natural waters, the interactions between trace metals and aquatic microorganisms, and the role of trace metals in controlling the biogeochemical cycles of bioactive elements. We have addressed such questions as: What are the chemical forms (the chemical species) of various metals in natural waters? How does the speciation of the metals affect their availability to microorganisms? What metals are limiting or toxic to organisms under what conditions? How do organisms affect the chemical speciation of the metals in their external and internal milieu? How do trace metals affect or control the uptake or utilization of carbon, nitrogen and phosphorus by the planktonic biota? To what extent do trace metals control productivity and phytoplankton assemblages in oceanic waters? A central goal has been to relate the trace metal biochemistry and physiology of marine phytoplankton to the biogeochemical cycles of elements, including carbon, nitrogen and phosphorus, as well as trace metals. As an extension of this work, we have addressed the related questions of inorganic carbon acquisition by marine phytoplankton and of the role of CO2 on plankton production and ecology. Recently, we have begun to examine the interactions of trace element geochemistry and phytoplankton in the context of Earth history and evolution. We have also investigated various questions related to the biogeochemical cycling of arsenic and mercury.
I. Chemical Speciation in Natural Waters
We developed of a series of computer programs (the REDEQL and MINEQL series) to calculate chemical equilibrium in complex systems, including natural waters and man-made chemical systems. These programs, as well as a number of derivative programs (including EPA’s MINTEQ) have been widely used in industry, government and academia for a variety of applications. The conceptual architecture of these programs is based on a re-discovery of Gibbs’s notion of chemical components and a general algebraic formulation of the chemical equilibrium problem. The FITEQL program uses the same formulation to solve the opposite problem: determining equilibrium constants from experimental equilibrium data. Key publications are: Morel and Morgan ES&T 1972; Westall et al, R. M. Parsons Technical Notes #18 and 19, 1977,1978. This conceptual approach to the problem of chemical equilibrium in complex systems is also utilized in the teaching text Principles of Aquatic Chemistry, Morel, Wiley 1983, and its sequel Principles and Applications of Aquatic Chemistry, Morel and Hering, Wiley 1993. The corresponding “tableau method” which bedeviled many a graduate student was eventually adopted by other authors, including Stumm and Morgan in the third edition of Aquatic Chemistry Wiley 1996. Principles of Aquatic Chemistry and Principles and Applications of Aquatic Chemistry together have sold more than 10,000 copies. A large but unknown number of copies of MINEQL, FITEQL and derivative programs have been distributed over the years.
We critiqued existing electrochemical and sequential extraction methods for measuring metal speciation in natural waters and soils and proposed some new ones. Westall et al. Anal. Chem 1979, Waite and Morel Anal. Chem. 1983;1984; Hering et al. Marine Chemistry 1987; Nirel and Morel Water Res. 1990.
By incorporating solutes into the solid phase, adsorption controls the geochemical cycles of many trace elements and compounds. We performed experimental studies of solute adsorption on solid surfaces and implemented thermodynamic models to describe the data. The key to modeling adsorption is to quantify in a coherent thermodynamic formulation the relative roles of long-range electrostatic and short-range chemical interactions at solid surfaces. Besides providing some of the experimental data, our main contributions were to develop: 1) a method to include all sub-varieties of the so-called “Surface Complexation Model” into MINEQL (thus allowing, for example, the development of the “Triple Layer Model” used by the Stanford group in many publications); 2) the surface precipitation model (in which the surface phase is treated as a solid solution) for describing the transition between adsorption and precipitation; and 3) a coherent data base for adsorption of hydrous ferric oxide. More recently, we have extended this work to describe adsorption of solutes on permanently charged clays. Swallow et al. ES&T 1980, Farley et al. JCIS 1985, Dzombak and Morel JCIS 1986; J. Hydraulic Eng. AICHE 1987; Dzombak and Morel Surface Complexation Modeling: Hydrous Ferric Oxide; Wiley 1990; Kraepiel and Morel ES&T 1998, Kraepiel et al. JCIS 1999.
The net effect of complexation on the cycling of trace elements is opposite to that of adsorption since it increases the solubility of elements and compounds. We carried out theoretical and experimental studies of the complexation of trace metals by inorganic and natural organic ligands in aquatic systems. Our work entailed the modeling of complexation of trace metals by major anions upon mixing of freshwater with seawater, a quantitative description of metal complexation by humic substances, and the search for specific biogenic chelating agents. We showed the importance of sulfide binding upon mixing of wastewater with seawater in sewage outfalls and the role of chloride complexation of metals in estuarine systems. We also made a thorough analysis of the “polyelectrolyte effect” (i.e., the long range coulombic interactions between metal and ligands) to quantify the complexation of metals by humates and approached the problem of identifying specific biogenic chelators by using pure cultures of microorganisms. In more recent work, we quantified the role of cysteine-rich polypeptides known as phytochelatins in the intracellular binding of trace metals in marine phytoplankton and studied the relation between phytochelatin concentrations and metal exposure in seawater. Morel et al. ES&T 1975; McKnight and Morel L&O 1979, 1980, Dzombak et al. ES&T 1986, Fish et al ES&T 1986, Hering and Morel ES&T 1988, Bartschat et al. ES&T 1992; Green et al. ES&T 1992; Ahner et al. PNAS 1994; L&O 1995a&b; 1997; Kraepiel et al. GCA 1997.
Because the kinetics of reactions among most solutes are relatively fast, it is generally assumed that complexation reactions are at equilibrium in natural waters. We studied the kinetics of complexation of metals by organic ligands in natural waters and demonstrated that some of these reactions are in fact exceedingly slow, despite the inherently fast kinetics of the underlying reaction steps. This counterintuitive result is explained by the rapid formation of intermediate metastable complexes that results in extremely slow approach to equilibrium. Hering and Morel ES&T 1988, 1990; GCA 1989.
Photochemical Redox Cycling of Iron
The absorption of photons by chemical compounds can promote thermodynamically unfavorable or kinetically hindered reactions. We postulated and first demonstrated the importance of the Fe photocycle in surface waters both for the dissolution of Fe oxides and for the formation of Fe(II) from Fe(III) complexes in oxic solution. Waite and Morel JCIS 1984 and ES&T 1984; Hudson et al. Mar. Chem. 1992; Voelker et al ES&T 1997. The earliest description of these process is actually given in Principles of Aquatic Chemistry 1983. Related work on the (photo)redox cycle of mercury is described below
II. Metal-Microorganism Interactions
We designed a “chemically defined” growth medium for studying the trace metal physiology of marine phytoplankton, the Aquil medium which is now widely used; see Morel et al. J. Phycol 1979; Price et al. Biolog. Oceanogr.1988/1989; Sunda et al. 2005. We also developed methods for distinguishing extracellular and intracellular concentrations of metals and for simultaneous measurement of Fe reduction and uptake in cultures: Hudson and Morel L&O 1989; Shaked et al. L&O 2004; Tang and Morel Mar. Chem. 2005.
The Free Ion Model and A Kinetic Framework
An important outcome of our early work is the conceptual development and experimental verification of the “Free Ion Activity Model” (FIAM) for the effects of metals on aquatic organisms. This model links the biological availability and effects of essential and toxic metals to their chemical speciation. It is the basis of most modern work on the interactions of trace metals and aquatic organisms and is also now incorporated in various EPA rules based on the FIAM model, or its extension the BLM (Biotic Ligand Model). Cu toxicity: Anderson and Morel L&O 1978, Morel et al 1978, Rueter and Morel L&O 1981; Zn limitation: Anderson et al. Nature 1978; Fe limitation: Anderson and Morel L&O 1982; Cd toxicity: Foster and Morel L&O 1982, Harrison and Morel J Phycol. 1984. We subsequently modified the free ion model and provided a kinetic framework for analyzing the interactions of trace metals and microorganisms. This work lead to the general hypothesis that the acquisition of essential trace elements by planktonic organisms in oligotrophic oceanic waters is limited by the kinetics of diffusion and chemical reactions with uptake molecules at the cell surface. The net result is a general hypothesis of co-limitation of growth by major and trace nutrients that is modulated by the size of the organisms. Hudson and Morel L&O 1990, DSR 1993; Morel et al. L&O 1991.
The Role of Iron in Primary Production and the Ecology of Marine Phytoplankton
Following the laboratory studies mentioned above (and others such as Rich and Morel L&O 1990), we engaged in field work, particularly to test the “Iron Hypothesis” according to which High Nutrient Low Chlorophyll (HNLC) regions of the oceans are Fe limited. We resolved the apparent conflict between the Fe hypothesis and alternative explanations by showing that in the Equatorial Pacific large diatoms are limited by Fe while cyanobacteria are controlled chiefly by grazing. This “ecumenical hypothesis” which was criticized at the time of IRONEX-1 was then confirmed by IRONEX-2 and has stood the test of time. Morel et al. Oceanography 1991; Price et al. DSR 1991, L&O 1994. Recently, we have begun reexamining the questions of the mechanism of Fe uptake and storage in marine phytoplankton and of the bioavailability of Fe compounds in the sea using new kinetic and molecular biology tools. We have established the central importance of Fe(III) reduction by cell surface enzymes for Fe uptake, characterized an Fe storage protein in Trichodesmium (the dominant N2-fixing cyanobacterium in the oceans) and shown that the Fe bound in such protein is available to marine phytoplankton. Kustka et al. L&O 2005; Shaked et al. L&O 2005; Castruita et al. AEM 2006.
Roles of Zn, Co and Cd in Phytoplankton Physiology and Ecology
We demonstrated that Co and Cd can replace Zn as essential elements for the growth of marine phytoplankton, and showed that these metals can all serve as catalytic centers in carbonic anhydrase (CA). This enzyme catalyses the reversible transformation of CO2 into bicarbonate and is important in inorganic carbon acquisition for photosynthesis. This led to the discovery and characterization of two novel classes of carbonic anhydrases: the delta class of CAs which contain either Zn or Co as their metal center and the zeta class of CAs which are the first and only known cadmium enzymes. We have now shown that cadmium CAs are ubiquitous in marine waters. Despite the extremely fast kinetics of CAs, these enzymes are needed at high cellular concentration because of their very low affinity for their inorganic carbon substrate. As a result CAs represent a major metal requirement in marine phytoplankton. This work provides an explanation for the nutrient-like behavior of Cd in seawater. We also showed that the silica frustule of diatoms serves as a proton buffer for their external CA, thus establishing a biochemical role for silica in phytoplankton. Price and Morel Nature 1990; Morel et al. Nature 1994; Lee et al L&O 1995; Lee and Morel MEPS 1995; Yee and Morel L&O 1996; Roberts et al. J. Phycol 1997; Cullen et al Nature 1999; Cox et al. Biochemistry 2000; Lane and Morel PNAS 2000; Milligan and Morel Nature 2002; Lane et al. Nature 2005; Park et al. Env. Microbiol. 2007.
C4 Photosynthesis in Diatoms and Role of CO2 in Phytoplankton Ecology
Our work on Zn, Co, Cd in phytoplankton lead us to study the function of carbonic anhydrases (CA) and the mechanism of inorganic carbon acquisition in these organisms. We have discovered that diatoms employ a C4 photosynthetic pathway. This is a remarkable result since diatoms evolved some 150 Myr ago while C4 photosynthesis in higher plants is thought to have appeared much later. The implications is that diatoms have evolved in response to the long term decrease in pCO2 in the Earth’s atmosphere and probably contributed to it, and that inorganic carbon availability may be limiting their rate of photosynthesis. Our field studies have confirmed the importance of pCO2 in the growth and ecology of marine phytoplankton. Because of the possible importance of a feedback of marine primary production on the present increase of atmospheric CO2 the continuation of this work is a major ongoing research theme in the group. Tortell et al. Nature 1997; Reinfelder et al. Nature 2000; Lane and Morel Plant. Phys, 2000; Riebesell et al. Nature 2000; Tortell and Morel L&O 2002; Tortell et al. L&O 2000, MEPS 2002; Morel et al. Funct. Plant Biol. 2002; Reinfelder et al. Plant Phys. 2004.
We documented the activity of extracellular metal reductases in marine phytoplankton. This work which received only modest attention at the time is now the basis of new work on the mechanisms of Fe acquisition by phytoplankton (see above). Jones et al. J. Phycol 1987; Jones and Morel Plant Phys. 1988.
We also discovered extracellular amine and amino acid reductases, which provide NH4+ for uptake and produce H2O2. Palenik et al. L&O 1987; Palenik and Morel L&O 1988; 1990; MEPS 1990; AEM 1991. In this work as well as in other work on urease (Price and Morel L&O 1991), we demonstrated that trace metals are important in the nitrogen nutrition of marine phytoplankton.
Recently we have examined the question of acquisition of phosphate from organic compounds by phytoplankton --particularly coccolithophorids-- and the role of metals in this process. This work has lead to the discovery of a novel alkaline phosphatase (a zinc enzyme that hydrolyses the phosphate group from organic moieties) with no homology to other known phosphatases. As a result of the high affinity of this enzyme for its organic substrates and its fast kinetics the corresponding zinc requirements are quite low and there is no evidence for replacement by another metal. Shaked et al. 2005; Xu et al. 2006.
We discovered, and studied in cultures and in the field the respiration of arsenate by bacteria. Ahmann et al. Nature 1994, ES&T 1997; Newman et al. AEM 1997, Arch. Microb. 1997, Geomicrobio. J. 1998
Metal-Microorganism Interactions: The global Geochemical Cycle of Mercury
We have modeled the global cycle of mercury and studied some key transformations such as photoreduction of Hg(II), oxidation of Hg(0) and microbial methylation of Hg(II). In addition, on the basis of new data showing no change in Hg concentration in Pacific tuna over thirty years, we have hypothesized that methylmercury in the open oceans may be formed at the ocean bottom and may not be influenced by pollution. We are presently studying the possible importance of hydrothermal inputs of methylmercury in the oceans. Mason et al. GCA 1994; Water Air Soil Pollut. 1995a&b; ES&T 1996; Amyot et al. ES&T 1997; Morel et al;. Annual Review Ecol. and Systematics 1998; Jay et al. ES&T 2000; Lalonde et al. ES&T 2001; Ekstrom et al. AEM 2003; Kraepiel et al. ES&T 2003: Lalonde et al. 2004; Amyot et al. 2005.