Decoupling between diatom frustule-bound and biomass nitrogen isotope signatures is modulated by batch culture and species effects

The stable nitrogen (N) isotope composition of organic matter encapsulated in diatom silica frustules (δ 15 N DB ) from sedimentary records has been used as a proxy for reconstructing N consumption dynamics in the ocean over geologic timescales. This proxy relies on the assumption that δ 15 N DB tracks biomass δ 15 N without being affected by internal N-isotope fractionation. However, recent ground-truthing efforts have shown that δ 15 N DB can diverge from biomass δ 15 N values, yet the extent of this decoupling and its drivers remain unclear. In this study, we cultured two freshwater and two marine diatom species in batch cultures to test whether δ 15 N DB (1) is subject to species-dependent internal 15 N fractionation, and (2) reflects the δ 15 N of source nitrate to the same extent as biomass δ 15 N values, assessing potential asynchronous integration of the N isotope signal. We monitored the N-isotope systematics during diatom growth by measuring δ 15 N values of nitrate, bulk biomass and frustule-bound organic N throughout batch culture progression. We found that δ 15 N DB did not follow typical Rayleigh fractionation dynamics and remained relatively stable, while biomass δ 15 N increased predictably with progressive fractional nitrate consumption. The observed divergence could only be partially explained by asynchronous integration of source-nitrate δ 15 N values into biomass versus frustule-bound organic N (i.e., delayed incorporation into frustule-bound material), as newly formed frustules predominantly recorded the δ 15 N of 15 N-labeled nitrate added during growth. This demonstrates that δ 15 N DB values primarily capture the isotopic signature of newly assimilated nitrate rather than N derived from internal, or legacy, pools. We hypothesize that shifts in growth conditions during batch culture progression alter the coupling between carbon and nitrogen metabolism, leading to physiologically driven variation in internal 15 N fractionation and corresponding offsets between δ 15 N DB and biomass δ 15 N. Such sensitivity to internal isotope fractionation during biosynthesis implies that interpretation of sedimentary δ 15 N DB records should not only focus on changes in source inorganic δ 15 N, but also consider the 15 N-fractionating effects related to diatom physiology and metabolism.