Stable Isotope Probing Experiments

For stable isotope probing experiments, samples were collected from one culture replicate from each condition. Incubations were performed by sub-sampling cultures into 6 mL gas-tight vials and amending the ASP2 medium with NaH¹³CO₃ (all cultures) and either ¹⁵N₂ (N₂ and SC-N₂ cultures) or Na¹⁵NO₃ (NO₃ culture). Vials were incubated under light and temperature conditions that were equivalent to culture conditions. Incubation durations were 2 h in the morning, 2.5 h during the day, and 10 h during the night, with shorter incubations in early night (3 h) and late night (5 h). Isotope labeling was calculated from the known amounts of label added to the incubation medium and measured initial concentrations of unlabeled substrate in the bioreactors (Supplementary Table 1). The ¹⁵N₂ enriched stock was prepared by injecting 10 mL of ¹⁵N₂ gas into 43 mL of the ASP2 medium, followed by an equilibration for >24 h. The ¹⁵N-N₂ atom fraction in the incubation medium was calculated assuming that ¹⁵N₂ was fully equilibrated with the stock solution. Since this may lead to an underestimation of N₂ fixation rates (Mohr et al., 2010), we refrain from comparisons of N assimilation rates between N treatments (N₂ vs. NO₃). However, comparisons over time and among cells within each treatment are not affected since any potential underestimation would be similar in all ¹⁵N₂ incubations. NO₃ concentration in the incubation medium was estimated by averaging NO₃ concentrations measured in the bioreactor on the respective day of the experiment. Dissolved inorganic carbon (DIC) concentration in the incubation medium was estimated by measuring DIC concentrations in the bioreactor at three time points during the day and interpolating them to the starting time points of our stable isotope incubations. Because the DIC concentration in the cultures varied depending on the time of the day, ¹³C-DIC atom fractions varied during our incubations, although the amounts of added NaH¹³CO₃ were the same (Supplementary Table 1).