N2 fixation rates

N₂ fixation rates were measured using the ¹⁵N₂ gas dissolution method⁵⁶. Briefly, 0.22 µm-filtered surface seawater was degassed as described in Shiozaki et al.⁵⁷. After that, 10 mL 98% pure ¹⁵N₂ gas (Cambridge Isotope Laboratories) was injected into a gas-tight plastic bag containing 1 L of the degassed seawater and allowed to fully equilibrate before use. The percentage of ¹⁵N₂ in the ¹⁵N₂-enriched seawater was validated using a GasBench-IRMS⁵⁸. Following 1–3 days of incubation, a sub-sample from each incubation carboy was transferred into a polycarbonate bottle, and then ¹⁵N₂-enriched seawater was added with the enriched water constituting approximately 2.6% of the total sample volume. Bottles were then returned to the flow-through incubators under the same irradiance of each experiment and incubated for 24 h. After the incubation, particulate matter in seawater from each bottle was filtered onto 25 mm pre-combusted GF/F filters (Millipore). Particulate organic matters collected from the same incubation carboy but without ¹⁵N₂ enrichment were filtered to determine ¹⁵N-PON natural abundance. All filter samples were stored at −20 °C immediately after collection. In the shore-based laboratory, sample filters were dried and analyzed using a Flash HT 2000 elemental analyzer coupled with a Thermo Finnigan Delta V Plus isotope ratio mass spectrometer. The rates of N₂ fixation were calculated according to Mohr et al.⁵⁶.