j DIC and H+ fluxes during active HCO3 ? uptake. uptake, modelling studies indicate that 5% of the CO2 at the cell surface is likely to be supplied by conversion of HCO3 ? to CO2, due the slow rate of the uncatalysed reaction12. CO2 supply at the cell surface is usually therefore limited by diffusion and maintaining an inward CO2 gradient across the plasma membrane is usually a much greater problem for large cells that have a significant diffusive boundary layer12C14. Large cells may overcome this diffusive limitation either by direct uptake of HCO3 ? or by using the enzyme external carbonic anhydrase (eCA) to increase the supply of CO2 at the cell surface. It is likely that many species employ both mechanisms, although the role of eCA in photosynthetic DIC uptake in marine diatoms has been much debated15,16. Improved knowledge of these cellular mechanisms is critical for our understanding of the response of diatom communities to predicted future changes in ocean carbonate chemistry. For example, experimental analyses have demonstrated that growth at elevated CO2 increases the growth rate of large diatoms by up to 30%, whereas the growth enhancement in smaller species was much more modest ( 5%)17. The significant growth enhancement of large Evatanepag diatoms may be due to the increased diffusive supply of CO2 and/or a decreased metabolic expense in the CCM components17. Future changes in ocean carbonate chemistry may therefore lead to shifts in the size and productivity of diatom communities that will have an important implication on global carbon Evatanepag cycling through their influence on the rates of carbon export from the surface ocean. It was in the beginning assumed that the primary role of eCA in marine diatoms and other algae is usually to catalyse the conversion of HCO3 ? to CO2 at the cell surface18C20. eCA would therefore be expected to be more important in larger diatom species. A survey of 17 marine diatoms indicated that there is considerable diversity in the presence of eCA activity between different species, but found no correlation Evatanepag between eCA activity and the relative C demand:supply of each species21. eCA is present in most centric diatoms, although in smaller species it is only induced and required at very low DIC concentrations15,22. Although no overall relationship was found between the contribution of eCA to photosynthesis and cell size, larger centric diatom species exhibit a requirement for eCA at ambient DIC Evatanepag concentrations, lending some support to the increased requirement for eCA in larger cells23. Hopkinson et al.15 proposed that even relatively small raises in diffusive CO2 supply due to eCA are likely to increase the efficiency of the CCM. Other lines of evidence suggest that the primary role of eCA is not to increase the supply of CO2 at the cell surface. Studies across a range of diatom species using the isotope disequilibrium technique to discriminate HB5 between CO2 and HCO3 ? uptake surprisingly revealed a positive correlation between eCA activity and the proportion of DIC taken up across the plasma membrane as HCO3 ? (indicate that diatom cells may also experience significant changes in pH, even though underlying processes have not been explored32. Measurements using pH-responsive fluorescent dyes have also exhibited significant light-dependent increases in cell surface pH in diatoms33. Photosynthetic DIC uptake could theoretically contribute to the light-dependent increases in cell surface pH in diatoms through a number of mechanisms; drawdown of CO2, conversion of HCO3 ? to CO2 at the cell surface or uptake of HCO3 ? accompanied by uptake of H+ or extrusion of a base (OH?)33. Clearly, better definition of carbonate chemistry in the microenvironment is required to understand the relative contribution of these processes to photosynthetic DIC uptake. In order to better define the mechanisms of photosynthetic DIC uptake and the functions of eCA in this process, we set out to examine.