Linking chemical speciation to metal bioavailability

It is recognized that dissolved organic matter (DOM) plays an utmost role in aquatic ecosystems by binding trace metals and influencing their speciation, bioavailability and detrimental effects. DOM is considered to protect the aquatic microorganisms from heavy metal stress by decreasing free metal ion concentration and thus decrease their bioavailability. Nonetheless, a fundamental understanding of the relationship between trace metal speciation, bioavailability and effects is still lacking for many environmental systems, although progresses have been made in recent years. The main objective of the present thesis was to link Pb(II) and Cd(II) speciation to its bioavailability to green micro algae in presence of DOM. In particular, (i) to characterize metal binding properties of different DOM components (e.g. EPS), (ii) to determine their effect on metal bioavailability and (iii) to quantify as rigorously as possible the relationship between chemical speciation and bioavailability in presence of different DOM components. The proton, Pb2+ and Cd2+ binding capacities of extracellular polymeric substances (EPS), in particular alginate and Rhizobium meliloti exopolysaccharides were quantified by determination of complex stability constants and the concentration of binding sites using ion selective electrodes in static or dynamic titration. The influence of ionic strength, pH and metal to EPS ratio was determined over large concentration ranges. Metal binding and conditional stability constants increased with increasing pH due to a decrease of competition with the proton. In contrast, an increase in the metal to EPS ratio or ionic strength resulted in a decrease in the average conditional stability constants as could be expected by the polyelectrolytic and polyfunctional character of the EPS. A non ideal competitive adsorption isotherm (NICA) combined with a Donnan electrostatics approach previously developed for humic substances, takes into account the intrinsic polyfunctionality and polyelectrolytic properties and can reasonably account for electrostatic and competition effects. It was successfully applied to describe Pb(II) and Cd(II) complexation by EPS. Results demonstrated that EPS played a non neglected role in Pb and Cd speciation. The influence (both direct and indirect) of different DOM components on Pb bioavailability by green algae Chlorella kesslerii was studied. Results demonstrated that some of the studied EPS (e.g. alginate) may exhibit similar influences on Pb bioavailability as humic substances. DOM components had several simultaneous effects on Pb bioavailability including complexation in the medium, adsorption to algal surfaces, and modification of membrane permeability. An important discrepancy between Pb bioavailability in the presence of HS and alginates (as individual components or mixtures) and that predicted for similar free Pb ion concentrations in the presence of synthetic ligands (NTA, citrate) was obtained. In contrast, the experimental results of Cd and Cu uptake were in good agreement with that predicted from free metal concentration. An improved fit between experimental observations and Pb speciation was obtained by extending the free ion activity model (FIAM) when the formation of a ternary complex between metal, DOM and the algal surface is taken into account. The contribution of ternary complexes on metal biouptake was thus dependent on (i) the metal affinity to microorganisms, (ii) the concentration of different components that constituted DOM, (iii) their capacity to adsorb onto the surface of the microorganisms and (iv) their affinity to bind the metal. By simultaneously examining metal speciation and biological availability in the presence of different DOM components and their mixtures, this work provided new information with respect to the applicability and limitation of existing equation models. An extension of existing models will further improve a site specific predictive capacity by avoiding underestimation of metal biouptake in presence of high DOM concentration.

Comparison of Cd(II), Cu(II) and Pb(II) Biouptake by Green Algae in the Presence of Humic Acid

Cristina Lamelas

The present study examines the role of humic acid, as a representative of dissolved organic matter, in Cd(II), Cu(II) and Pb(II) speciation and biouptake by green microalgae. Cellular and intracellular metal fractions were compared in the presence of citric and humic acids. The results demonstrated that Cd and Cu uptake in the presence of 10 mg L-1 humic acid was consistent with that predicted from measured free metal concentrations, while Pb biouptake was higher. By comparing Cd, Cu and Pb cellular concentrations in the absence and presence of humic acid, it was found that the influence of the increased negative algal surface charge, resulting from humic acid adsorption, on cellular metal was negligible. Moreover, the experimental results for all three metals were in good agreement with the ternary complex hypothesis. Given that metal has much higher affinity with algal sites than humic acid adsorbed to algae, the contribution of the ternary complex to metal bioavailability was negligible in the case of Cd (II) and Cu (II). In contrast the ternary complex contributed to over 90% of total cellular metal for Pb(II), due to the comparable affinity of Pb to algal sites in comparison with humic acid adsorbed to algae. Therefore, the extension of the biotic ligand model by including the formation of the ternary complex between the metal, humic acid and algal surface would help to avoid underestimation of Pb biouptake in the presence of humic substances by green algae Chlorella kesslerii.

2007

Effect of humic acid on Cd(II), Cu(II) and Pb(II) uptake by freshwater algae: kinetic and cell wall speciation considerations

Cristina Lamelas

The present study examines the effect of humic acid on the uptake kinetics of Cd(II), Cu(II), and Pb(II) by the freshwater alga Chlorella kesslerii. The results demonstrated that the relative proportion of Pb in the cell wall layer is greater than that of the internalized Pb, while internalized Cd and Cu were comparable or greater than the adsorbed metal concentration. In the presence of 10 mg L-1 humic acid (HA), Cd and Cu uptake kinetics were consistent with that predicted by measured free metal concentrations. For Pb, the uptake flux and amount of lead bound to internalization and adsorption sites were an order of magnitude higher than those found at the same free lead ion concentrations in the presence of citric acid. Chemodynamic modeling suggested that the enhancement of the Pb uptake flux in the presence of HA originates from an increasing amount of Pb bound to the internalization sites through a ternary complex formation between lead-humic acid complex and internalization sites. Cell wall speciation calculations indicated that the lead-humic acid complex is the predominant species in the cell wall layer, while for Cu(II) and Cd(II) metal bound to the internalization (Cu) and adsorption (Cd) sites significantly dominated over the M-HA complex. The findings of the work show the relevance of the cell wall layer concentration and speciation and its key role in defining the local equilibrium conditions between metal and internalizations sites. The results of the present kinetic study have important consequences for improvement of the mechanistic understanding of the role of dissolved organic matter in metal uptake in phytoplankton and biogeochemical cycling of metals in the surface waters.

2009

Toxic metal interactions with soil bacterium "Cupriavidus metallidurans"

Rita Hajdu Schenk

Living bacteria offer an ecological and cost-effective alternative to the classical physical and chemical techniques for the treatment of environmental contaminations. Although their increasing use in toxic metal removal, systematic studies on the influence of physicochemistry of the medium on metal uptake and thus on their metal removal efficiency are very limited, even though it would deserve greater environmental interest. The overall goal of the present work is to improve the mechanistic understanding of the interactions between toxic metals and microorganisms. The emphasis is on the effect of different site-specific characteristics e.g. the presence and concentrations of different complexing agents, metal mixtures and pH- on the Cd and Pb uptake by a metal-resistant bacterium (Cupriavidus metallidurans CH34). Conversely, the influence of bacteria on metal speciation and mobility in the surrounding environment is also addressed. Different processes involved in the overall metal uptake, such as the diffusion of metal species, adsorption, internalization and efflux, were quantified and described with a consistent dynamic model for a range of metal concentrations representing slightly to heavily polluted environments. Obtained results demonstrated that at 10-8 and 10-6 M of initial Cd concentrations, the bulk Cd depletion determined both adsorbed and intracellular Cd amount. Cd efflux affected Cd internalization over the whole concentration range. The findings of the present study demonstrated the key role of metal depletion in the bulk medium and the efflux on the overall metal uptake by C. metallidurans. Therefore, also the need to account for these processes in order to improve the efficiency of metal removal from contaminated environments. The relationship between Cd and Pb speciation and the metal uptake was also explored. The presence of citric, nitrilotriacetic and humic acids decreased both intracellular and total cellular metal contents. Intracellular and total cellular metal contents were directly related to the experimentally determined free cadmium and lead ion concentrations rather than the total metal concentrations, emphasizing the important role of metal speciation in uptake. Furthermore, the influence of the major cations, such as Ca and Mg, trace metals, such as Zn, Cu, Co, Mn and pH on Cd and Pb uptake were studied. Increasing concentrations of Ca, Mg, Zn or protons decreased both intracellular and total cellular Cd contents. The intracellular and total cellular Pb contents decreased significantly in the presence of Ca, while Mg decreased only total cellular metal content. In order to enhance even more the environmental relevance of the studied conditions, both metal speciation and Cd and Pb uptake by bacteria were determined in six metal contaminated soil solutions. A Michaelis-Menten based equation taking into account both speciation and competition effects was used to predict metal uptake in the soil solutions. Predictions were usually within the same order of magnitude as the experimentally measured values in the soil solutions. Cd uptake was stronger affected by the competition of other metals than Pb. In contrast Pb uptake was stronger influenced by the changes in chemical speciation than Cd, as Pb usually forms stronger complexes with dissolved organic matter. These results suggest that neglecting of metal speciation and competition effects will result in overestimation of the metal removal capacity of bacteria. Metal uptake and removal will depend on the nature and concentration of the metal of interest and ligands but also the presence and concentration of other metals. Bacteria can influence the speciation of the metals by producing exopolymeric substances (EPSs). The EPSs excreted by C. metallidurans contained components of proteinaceous and polysaccharide nature with a wide molar mass distribution. The excreted EPS formed stronger complexes with Pb than with Cd, though these complexes were up to two orders of magnitude weaker than those found with humic acids. Coupling of asymmetrical flow-field flow fractionation system to inductively coupled plasma mass spectrometer revealed that Cd bind preferentially to lower molar mass fractions, while Pb associates to both lower and higher molar mass fractions. The preference of metals to bind to different molar masse/size biopolymers is of environmental importance as it determines the mobility and fate of the metals. The results obtained in the present work emphasize the importance of taking into account chemical site-specificity of soil solutions and water, including dissolved organic ligands, pH, the presence of other metals, as well the ability of bacteria to influence metal speciation, when developing metal removal technologies by live bacteria.

EPFL2009

Toxic metal interactions with soil bacterium "Cupriavidus metallidurans"

Rita Hajdu Schenk

EPFL2009