Size-Dependent Dynamics of the Internal Carbon Pool Drive Isotopic Vital Effects in Calcifying Phytoplankton

Isotopic offsets in biogenic calcite from equilibrium values can provide unique insights into the physiology and mechanisms of carbon regulation in calcifying phytoplankton. This study examines the impact of varying CO2 (controlled via pH) on five coccolithophore species chosen for varied cell sizes, physiology, and calcification. The study investigates isotopic offsets in coccolith calcite and organic matter, in relation to carbon demand and supply (µ/CO2). Species-specific CO2/pH optima for growth (µopt) were derived from variations in growth rates with varying CO2 concentrations. Growth rates for all species declined with rising CO2 (decreasing pH) due to H+-driven inhibition. C. braarudii and C. leptoporus exhibited high µopt values (suggesting high carbon-demand) and limited growth under low CO2 (high pH) suggesting carbon limitation. Under low CO2 supply, when growth rates were CO2-limited, both species exhibited coincident isotopic depletion in calcite and organic matter as a consequence of CO2 diffusion into the cell that experienced no equilibration as a result of a highly depleted internal carbon pool. In these two species, isotopic values in calcite  remained unaffected by growth rates and CO2 concentration (µ/CO2) when CO2 was sufficient for optimal growth. G. huxleyi and G. oceanica displayed optima for growth (µopt) at low CO2 concentrations and showed no growth limitation under low CO2 (indicating low carbon-demand). Both species experienced depleted (negative) vital effects caused by an excess of CO2 diffusion into the small internal carbon pool of the cell when diffusive carbon supply outpaced low demand (low µ/CO2). Enriched (positive) vital effects were observed under low carbon supply and high demand, likely due to diffusive loss of CO2 from the cell's intracellular carbon pool due to a lower intracellular pH. C. carterae exhibited an intermediate µopt and isotopically equilibrated intracellular carbon pool such that δ13C values in calcite and organic matter suggested a shared carbon pool. This study illustrates that pH-CO2 driven vital effects and fractionation into organic matter indicate the residence time for carbon in the intracellular carbon pool, and the size of this pool is proportional to cell size. C. leptoporus and C. carterae can elevate intracellular pH to minimise CO2 leakage, whereas vital effects in G. huxleyi and G. oceanica are caused by diffusive CO2 due to the small size of their internal carbon pool.