Chemostat and Modeling Investigations of Algal Photosynthetic Carbon Isotope Fractionation

Abstract

Marine eukaryotic phytoplankton produce organic matter that is depleted in 13C relative to ambient dissolved carbon dioxide. This photosynthetic carbon isotope fractionation (εP) is recorded in marine sediments and used to resolve changes in the global carbon cycle, including variations in atmospheric pCO2. These applications rely upon a coherent understanding of the environmental and physiological controls on εP. While classical models for εP are based on the balance between diffusion of CO2 and its fixation into biomass by the enzyme RubisCO, the details of phytoplankton carbon dynamics in reality are more complex. Phytoplankton employ a diversity of RubisCO types, and they also use carbon concentrating mechanisms (CCMs) that enhance intracellular CO2 concentrations. It is essential to understand the significance of these physiological features as controls on εp, as they may play important roles in explaining sedimentary archives. Here I performed CO2 and growth rate (μ) manipulation experiments with modern phytoplankton in chemostat cultures to address outstanding questions regarding the mechanistic underpinning of εP. First, I characterized the stable carbon isotope ratios of coccolith-associated polysaccharides (CAPs) and other cellular constituents (bulk biomass, coccolith calcite, and alkenones) of Emiliania huxleyi. CAPs are involved in regulating calcification and have been recovered from sediments dating back ~180 Ma. It has been proposed that the carbon isotopic contents of CAPs may be used in combination with other proxies to reconstruct ancient atmospheric pCO2 levels. I find that the CAPs are isotopically enriched relative to bulk biomass and vary with μ and CO2. These results are explained by a simple model that predicts cellular carbon allocation to major organic carbon compound classes in E. huxleyi. My findings suggest that CAPs are less sensitive than alkenones as proxies for pCO2, but that combining CAP data together with data for alkenones and calcite may help reconstruct pCO2 with fewer assumptions than current approaches. I also performed chemostat culture experiments with the dinoflagellate Alexandrium tamarense, which uses an unusual form of the carbon-fixing enzyme RubisCO (Form II). It commonly is assumed that the kinetic isotope effect associated with RubisCO establishes the theoretical maximum value of εP, which is known as εf. I found that εP values for A. tamarense varied with the ratio μ/[CO2(aq)] and approached an εf value of 27‰. This value is larger than theoretical predictions for Form II RubisCO and is not significantly different from the εf values observed for more recently-evolved taxa that employ Form ID RubisCO, including E. huxleyi. This consistency across taxa may help to explain the broad uniformity of carbon isotope fractionation between organic and inorganic pools observed throughout the Phanerozoic, and it may pave the way for new algal pCO2 proxies based on dinoflagellate biomarkers or fossil dinoflagellate cysts. My work on A. tamarense also implies that an εf value of 25-27‰ may be a universal property of red-lineage eukaryotic phytoplankton. This finding has major implications for reinterpreting the classical models for εP, because it indicates that its maximum value (εf) is unlikely to reflect the intrinsic isotope fractionation of RubisCO. By extension, this implies that RubisCO activity is not the kinetically slow step of carbon fixation in these phytoplankton, at least when cultivated in nutrient-limited chemostats. Based on this finding and other support from the literature, I propose a generalized model of carbon isotope fraction in eukaryotic phytoplankton that is able to reconcile the apparent uniformity of εf (as inferred from in vivo studies) vs. the isotopic heterogeneity of RubisCO (as inferred from in vitro studies). The model introduces a nutrient- and light-dependent step upstream of RubisCO that is proposed to be a kinetic barrier to carbon acquisition and a significant source of isotope fractionation. Together, the results of this thesis imply that the kinetics, intrinsic discrimination, and taxonomy of RubisCO may be largely irrelevant to the expression of εp under growth conditions of low nutrients and high photosynthetic activity, e.g., in the ocean gyres or away from coastal upwelling zones. Existing environmental data are consistent with this idea and suggest that alkenone and/or other organic pCO2 proxies should be reevaluated from the perspective of local nutrient dynamics and cellular growth conditions.