Several simplified steady-state models of trace metal bioavailability have been proposed to consider speciation and metals reacting with the biological binding sites. These include the Windermere Humic Aqueous Model (WHAM: Tipping 1993; 1998), the Free Ion Activity Model (FIAM: Morel 1983) and the Biotic Ligand Model (BLM: Pagenkopf 1983; Playle 1998). The BLM and the FIAM are commonly used because they link the chemistry of trace metals with their bioavailability (or reactivity). This link is important: it is the scarcity of the bioavailable fraction that affects biology, a requisite to underpin the effect of Zn co-limitation or limitation in marine systems. The BLM can take implicitly competitive effects into account through direct correlation of metal binding to sensitive sites of the cell‘s surface with bioavailability (Di Toro et al. 2001; Heijerick et al. 2002). Unlike the BLM, in the FIAM, competition must be taken into account explicitly by using equilibrium constants for each of the competition ions, including the proton.
47 The FIAM and BLM models consider the chemical speciation in solution, surface complexation with transport sites and uptake of the metal inside the organism (Fig. 5). This approach is valid if transport across the biological membrane is rate-limiting (Sunda and Huntsman 1998; Hassler et al. 2004). In that case, and below saturation of the uptake sites, biological uptake fluxes will be directly proportional to any of the metal species in equilibrium. Anderson and Morel (1978) have found that Zn uptake was proportional to [Zn2+] in the bulk solution and in qualitative accordance with the FIAM. They also noted that specific growth rate of the diatom T. weissflogii was dependent on log [Zn2+] in the bulk solution. Therefore, several authors have observed Zn uptake saturation and plotted their data with a Michaelis-Menten saturation equation. In the case where biological internalisation is rapid (compared to either the physical transport or chemical reactivity of the metal; Hudson 1998), biological uptake fluxes will be better related to some other fraction of the metal species in solution (i.e., mobile or labile species; Hudson and Morel 1993). Two papers have reviewed and analysed assumptions lying behind the application of the FIAM and the BLM (Hassler and Wilkinson 2003; Hassler et al. 2004). Both showed that these assumptions, although sometimes reasonable, clearly demonstrate the potential problems in applying models such as BLM or FIAM to Zn internalisation fluxes by phytoplankton. These arise from physiological effects on binding sites, multiple transport routes and varying acclimation conditions. Sunda and Huntsman (1992) showed that the cellular Zn content appeared to be regulated for [Zn2+] in the bulk solution ranging from 10-8.5 to 10-10.5 M. At these concentrations, transport kinetics approached the physical limits permitted by diffusion, as was observed for Fe. This implies that kinetics becomes important as
48 labile complexes could dissociate and contribute to the bioavailable pool. As a consequence, the definition of the bioavailability of trace elements needs to be rigorously established (Hassler et al. in press).
Although biological impacts are important, they are not the only reason for these models to improperly portray metal uptake by phytoplankton. Indeed, dynamics of chemical reactions have been shown to play an important role (van Leeuween et al. 2005) in the speciation and bioavailability of those metal species. The interplay of biological and chemical conditions imply predicting speciation and uptake of different species in order to better understand the behaviour of trace elements in marine systems.
Figure 5: Conceptual interactions taken into account in the FIAM and the BLM models including: (1) mass transport of the free metal and complex in solution; (2) dissociation and complexation with a ligand in solution; (3) rapid and irreversible adsorption to membrane binding sites, specific or not; (4) irreversible and rate-limiting internalisation of metal-carrier complex, characterised by an internalisation constant (kint); and (5) consequent internal
49 2.7 Conclusions and perspectives:
To get a better comprehension of marine ecosystems, interactions between Fe and microorganisms have been studied in detail. However, with recent advances in oceanographic research leading to an improved understanding of marine biogeochemistry, other trace elements, such as Zn, Co and Cd, also appear to mediate primary productivity or phytoplankton community structure. Laboratory studies have shown the importance of Fe for phytoplankton growth and the mechanisms they develop to maintain their optimal requirements. In a same way, the importance of Zn for marine microorganisms is well established through its different biological roles.
The obvious complexity of zinc's potential role as a limiting, or co- limiting, trace element in seawater is complicated by the fact that (i) Cd and Co can contribute to the biological requirement for Zn and (ii) that Fe is limiting, too, in most regions where low Zn concentrations are found (Croot et al. 2011). The uncertainties lying behind the proposal of limitation or co-limitation show our understanding of the role and importance of Zn in seawater needs further study. This must encompass the different responses Zn species can elicit in a phytoplankton community. Emerging results are suggesting that Zn affects oceanic productivity when its concentrations are extremely low, but in association with other biotic or abiotic factors, such as light, carbon or other nutrients (Muller
et al. 2003; Sunda and Hunstman 2005). For a general understanding of zinc and
its role in biological marine processes further research can be foreseen. For instance, synchrotron techniques (e.g. synchrotron-based microfocussed X-ray absorption fine structure) could be used to determine zinc location within cells or organelles, as it has been demonstrated in related work by Twining et al. (2008).
50 A second avenue of investigation would use molecular techniques, such as proteomics and transcriptomics, to reveal the physiology of Zn within the cell along the line of the selenium example (Zhang et al. 2005).
The diversity of zinc's roles in phytoplankton growth, and possible oceanic co-limitations, emphasises the need to bring together chemistry and biology in order to include this important trace metal in considerations of ocean surface fertility and the community structure of micro-organisms (phytoplankton, microbes, etc.). Owing to zinc‘s high susceptibility to contamination during sampling and analysis, determination of accurate Zn concentrations and speciation in seawater remains an open field of research: with the aim of arriving at the best technique to accomplish the task under clean conditions and for a wide range of marine systems. As a result, there is still little detailed information on its distribution, speciation and biological requirements, but thanks to the GEOTRACES research program (www.geotraces.org), new results are already emerging for its speciation in regions such as the Atlantic sector of the Southern Ocean (Baars and Croot 2011, Croot et al. 2011). High and low latitudes exhibit differences in bioavailable inorganic Zn in surface waters. Even if the nature of ligands that bind Zn is yet to be determined, these new findings are providing a new research base for Zn biogeochemistry cycling in the oceans. GEOTRACES places a high priority on isotope studies to track zinc sources and isotopic signature in biological processes in which zinc is involved.
The comprehensive picture of its cycle is yet to be completed, but recent studies, new techniques, combined with more intensive application of traditional oceanographic and laboratory studies, and new approaches from the scientific community are greatly enhancing knowledge on the role of Zn in marine systems
51 and provide exciting research outcomes. They have the potential to finally resolve the ‗zinc hypothesis‘ and reveal the potency of zinc as a micronutrient regulating communities of phytoplankton and associated micro-organisms.