Culturing and experimental conditions
Experimental cultures were grown with a semi-continuous culturing method at 28 degrees C in autoclave-sterilized artificial seawater medium with nutrients added in concentrations equivalent to the recipe for the Aquil medium (except for NO3-), as in Garcia et al. (2011) and originally described by Morel et al. (1979).

Light experiment and cellular growth rates
Triplicate cultures were grown in 800 mL polystyrene flasks under 5 irradiances (18, 40, 100, 180, 300 umol quanta per m^2 per second) and diluted every 2-3 days to 10-20 x 103 cells per mL. Cells were counted microscopically in each replicate culture with a hemocytometer at the end of each dilution period, and steady state growth rates were calculated from an increase in culture cell number per unit volume between 2-3 dilution periods (4-6 days) after cultures were acclimated to treatment conditions for 7-10 generations. To calculate growth rates, the investigators used the equation N_T=N₀e^µT, where N₀ and N_T are the initial and final culture cell densities, respectively and T is the amount of time in days between culture cell number estimates. With this method, the dilution rate is determined by the growth rate of the algae as determined by the experimental treatments, rather than by controlling the growth rate through imposing a dilution rate, as one does for continuous cultures.

Cell diameters of ~12 cells from treatment replicates were measured with an ocular micrometer. In the light experiment, cells in one replicate from each light were measured treatment twice, once in the middle of the light period and once at the end of the light period on the same day.

Light was supplied on a 12:12 light:dark cycle with cool white fluorescent bulbs. The investigators terminally sampled each replicate culture 24 hours after the last dilution for N2-fixation rates and CO2-fixation rates.

Nitrogen fixation
Nitrogen-fixation rates were determined with the acetylene reduction method as described in Garcia et al. (2013). Briefly, duplicate 50 mL culture samples were collected from experimental replicates and 4 mL of acetylene was injected into 30 mL headspace at the beginning of the dark period of the light cycle. Samples were gently agitated to equilibrate gas concentrations between the headspace and culture samples after injecting acetylene and before measuring ethylene concentrations. The investigators used a Bunsen coefficient for ethylene of 0.082 (Breitbarth et al. 2004) and an ethylene production:N2 fixation rate ratio of 3:1 and N2-fixation rates were calculated over 14 h (this included the 12 h dark cycle and the first 2 h of the light cycle).

CO2 fixation
CO2-fixation rates were determined using a Multi-purpose Scintillation Counter (model: LS-6500, Beckman Coulter) similar to the method described by Garcia et al. (2011). Briefly, the investigators inoculated 40 mL samples from each treatment replicate with 0.925 KBq mL-1 H14CO3-. The concentration of H14CO3- added to the sample was negligible in comparison with the TCO2 concentration of the sample. Samples were incubated for 12 h under treatment-specific conditions of irradiance and temperature, and then filtered onto Whatman GF/F filters and rinsed 3 times with ~5 mL filtered seawater to remove extracellular H14CO3-. The incubation was initiated at the beginning of the light period and terminated at the end of the 12 h light period. Total CO2 concentrations were multiplied by the ratio of radioactivity of cellular incorporation of 14C to the total radioactivity of H14CO3-. To calculate CO2-fixation rates in the light experiment, the investigators used a mean concentration of 2061 umol L-1 TCO2 that was measured in identical non-bubbled experiments with other isolates of C. watsonii (Garcia et al. 2013). Non-photosynthetically driven 14C incorporation was determined by incubating replicate culture samples (40 mL) for 12 h during the same time period in opaque bottles at 28 degrees C with the same concentration of H14CO3-; these values were subtracted from measured total 14C incorporation to estimate photosynthetic incorporation. The total radioactivity of H14CO3- was determined by stabilizing 50 uL of the 37 MBq H14CO3- with 100 mL of a basic solution of phenylethylamine (99%) before adding 4 mL of Ultima Gold® XR (PerkinElmer).

References:
Garcia, N. S., F.-X. Fu, , C. L. Breene, P. W. Bernhardt, M. R. Mulholland, J. A. Sohm, and D. A. Hutchins. 2011. Interactive effects of irradiance and CO2 on CO2- and N2 fixation in the diazotroph Trichodesmium erythraeum (Cyanobacteria). J. Phycol. 47: 1292-1303. DOI: 10.1111/j.1529-8817.2011.01078.x

Garcia, N. S., F.-X. Fu, C. L. Breene, E. K. Yu, P. W. Bernhardt, M. R. Mulholland, and D. A. Hutchins. 2013. Combined effects of CO2 and light on large and small isolates of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii from the western tropical Atlantic Ocean. Eur. J. Phycol. 48: 128-139. DOI: 10.1080/09670262.2013.773383

Morel, F. M. M., J. G. Rueter, D. M. Anderson, and Guillard, R. R. L. 1979. Aquil: Chemically defined phytoplankton culture medium for trace metal studies. J. Phycol. 15:135-141. DOI: 10.1111/j.1529-8817.1979.tb02976.x