Seawater samples were collected aboard the R/V S.A. Agulhas II (from CTD Niskin bottles and the underway intake) and immediately frozen at −20°C until analysis. Hydrographic data for each profile station were acquired from a conductivity-temperature depth (CTD) sensor mounted on the Niskin bottle rosette.

An aliquot of each seawater sample was treated with sulfamic acid to remove nitrite (Granger & Sigman, 2009). Both nitrate+nitrite and nitrate-only samples were measured for concentration by chemiluminescence (Braman & Hendrix, 1989) and isotope composition using the denitrifier method (Sigman et al., 2001). Briefly, sample nitrate or nitrate+nitrite is converted to N₂O gas by denitrifying bacteria and measured for N isotope composition by GC-IRMS with an on-line N₂O extraction and purification system (Sigman et al., 2001; Casciotti et al., 2002; Weigand et al., 2016). The pooled standard deviations (1σ) of replicate denitrifier measurements was 0.09‰ for nitrate+nitrite δ¹⁵N and 0.06‰ for nitrate-only δ¹⁵N.

Hydrographic profiles were acquired by a Sea-Bird conductivity-temperature depth sensor during each cast. The isotopic composition of N₂O was measured at Princeton University by GC-IRMS using a Thermo MAT 253 mass spectrometer with a purpose-built on-line N₂O extraction and purification system.

See methods section of Smart et al. (2020) for more detail.