A simplified approach for measuring Rubisco carbon isotope fractionation and the first determination in marine haptophyte Gephyrocapsa oceanica

Abstract. Rubisco is the central photosynthetic enzyme that catalyzes the fixation of CO₂ to RuBP, initiating the most dominant carbon assimilation pathway on Earth that supports nearly all trophic chains in the biosphere. The CO₂ fixation reaction expresses a strong kinetic isotope effect, producing biomass depleted in ¹³C and leaving characteristic imprints in sediments and sedimentary rocks, which are widely used to reconstruct past biological activity and environmental conditions, including ancient atmospheric CO₂ levels. Despite its importance, carbon isotope fractionation of Rubisco (ϵ_Rubisco) has been measured in only a limited number of organisms, with most studies focusing on land plants rather than on major contributors to the sedimentary record, such as cyanobacteria and coccolithophores. This scarcity reflects the complexity of existing experimental procedures and the high cost of instrumentation. Here, we present a simplified method that overcomes these limitations, eliminating the need for complex purification protocols, specialized equipment, and experimental designs that yield little CO₂ fixation and high uncertainties. Using this protocol, we accurately determined ϵ_Rubisco for the model plant Spinacia oleracea, the cyanobacterium Synechococcus sp., and provide the first determination for the coccolithophore Gephyrocapsa oceanica. The measured values span a striking range, from 13.1 ‰ to 30 ‰, highlighting both the variability of Rubisco fractionation and the versatility of our approach for studying carbon isotope discrimination across diverse biological systems. This study establishes a method that enables reliable determination of ϵ_Rubisco across phylogenetically diverse groups, thereby supports research that provides new insights into the mechanisms of Rubisco fractionation, and improves interpretation of environmental carbon isotope records.