Biological nitrogen (N₂) fixation is the main input of fixed nitrogen to ecosystems on Earth. Nitrogen isotope fractionation during this process is a key parameter for understanding the nitrogen cycle, however, relatively little is known about its regulatory mechanisms. Here we examine the effects of varying CO₂ concentrations on biomass δ¹⁵N signatures of the cyanobacterial diazotrophs Trichodesmium erythraeum and Crocosphaera watsonii. We show that these organisms produce biomass up to ~3‰ lower in δ¹⁵N under either decreased (~180 µatm) or elevated (~1400 µatm) CO₂concentrations in comparison to modern levels (~380 µatm). Our results pointed towards changes in nitrogenase enzyme efficiency in response to CO₂ perturbations impacting isotopic fractionation during N₂ fixation and thus the biomass δ¹⁵N. This study contributes to an improved interpretation of the observed fluctuations in the δ¹⁵N records, and thus the past nitrogen cycle on Earth.

N2 fixation,Ocean acidification,N isotopic composition,diazotroph