Batch culture and species effects modulate the decoupling between diatom frustule-bound and biomass nitrogen isotope signatures

The stable nitrogen (N) isotope composition of organic matter encapsulated in diatom silica frustules ({delta}15NDB) 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 {delta}15NDB tracks biomass {delta}15N without being affected by internal N-isotope fractionation. However, recent ground-truthing efforts have shown that {delta}15NDB can diverge from biomass {delta}15N values, though the extent and mechanisms behind this decoupling remain unclear. In this study, we cultured two freshwater and two marine diatom species in batch cultures to test whether {delta}15NDB (1) is subject to species-dependent internal 15N fractionation, and (2) reflects the {delta}15N of source nitrate to the same extent as biomass {delta}15N values, assessing potential asynchronous integration of the N isotope signal. We monitored the N-isotope systematics during diatom growth by measuring {delta}15N values of nitrate, bulk biomass and frustule-bound organic N throughout batch culture progression. We found that {delta}15NDB did not follow typical Rayleigh fractionation dynamics and remained relatively stable, while biomass {delta}15N increased predictably with progressive fractional nitrate consumption. The observed divergence could only be partially explained by asynchronous integration of source-nitrate {delta}15N values into biomass versus frustule-bound organic N (i.e., delayed incorporation into frustule-bound material), as newly formed frustules predominantly recorded the {delta}15N of 15N-labeled nitrate added during growth. This demonstrates that {delta}15NDB values 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 15N fractionation and corresponding offsets between {delta}15NDB and biomass {delta}15N. Such sensitivity to internal isotope fractionation during biosynthesis implies that the interpretation of sedimentary {delta}15NDB records is more intricate than previously assumed.