Cultures: The strains used in this study included Prochlorococcus MIT0915 and Prochlorococcus MIT0917. Before each experiment, all strains were assayed for heterotrophic contaminants by staining cells with SYBR green and assessing the fluorescence and light scattering properties of both stained and unstained cells using a Guava easyCyte 12HT Flow Cytometer (MilliporeSigma, Burlington, MA, USA) – cultures that did not exhibit the presence of non-photosynthetic cells in the stained samples and had a single cyanobacteria population were presumed axenic and unialgal. Before each experiment, all presumed axenic cultures were assessed for purity by confirming a lack of turbidity after inoculation into a panel of purity test broths as described previously (Berube et al., 2015).

Culture medium: The cultures were grown using AMP1-Mo-NO3 medium which is a variant of AMP1 artificial medium (Moore et al., 2007) with the following modifications: 400 µM ammonium sulfate was replaced with 800 µM sodium nitrate; the sodium molybdate concentration was increased from 0.3 nM to 100 nM; 1 mM HEPES pH=7.5 was replaced with 5 mM TAPS pH=8.2; and the concentration of sodium bicarbonate was decreased from 6 mM to 2.5 mM. The medium was filter sterilized using a 0.2 µm polyethersulfone (PES) membrane filter instead of using steam sterilization in an autoclave. A nitrogen-free variant (AMP1-Mo) was also prepared by omitting all inorganic nitrogen sources.

Methodology and sampling for light and temperature perturbations: Batch cultures were grown as triplicates in 35 mL of medium in borosilicate glass culture tubes at a temperature of 24^oC and a photon flux of 20 µmol photons m^-2 s^-1 of blue light. Cultures were acclimated for at least 10 generations prior to starting the experiment. Each biological replicate was diluted to 1 x 10⁸ cells mL¹ in a total of 50 mL of nitrogen-free AMP1-Mo supplemented with 2.5 mM sodium bicarbonate in duplicate, one to serve as a control and one to be subjected to the perturbation. Shock experiments were conducted in light- and temperature-controlled water baths. In both experiments, the control box was maintained at 23–25^oC and 21–23 µmol photons m^-2 s^-1 of blue light. In the light shock experiment, experimental cultures were exposed to 220 µmol photons m^-2 s^-1 of broad spectrum light at 23-25^oC. In the cold shock experiment, the cultures were exposed to 21–23 µmol photons m^-2 s^-1 of blue light at 14–16^oC. Prior to initiating the cold shock, nitrogen-free AMP1-Mo medium was pre-chilled to 15^oC before inoculation with the harvested biomass. Prior to spiking with nitrate, an initial set of samples were collected for flow cytometry, nitrite concentration determination, and fluorescence induction and relaxation (FIRe) fluorometry. Nitrate was then added to each tube at a final concentration of 80 µM sodium nitrate. Samples for nitrite concentration determination were collected every 30 min for 150 min by syringe filtration of 4 mL of culture through a 0.2 µm PES membrane into a 14 mL polystyrene tube and stored at -20^oC. Endpoint flow cytometry and FIRe fluormetry samples were also collect at the final time point. Media-only control tubes were prepared using the same batch of nitrogen-free AMP1-Mo medium to facilitate assessment of background signal in nitrite concentration determination; the mean background signal was equivalent to 0.050 µM nitrite (+/- 0.004) for the light shock experiment and 0.076 µM nitrite (+/- 0.012) for the cold shock experiment.