Culturing and experimental conditions
Stock cultures of the two Atlantic C. watsonii isolates used in this study were provided courtesy of Dr. Eric Webb. Both isolates were collected in March 2002, WH0401 from 6º 58.78' N, 49º 19.70' W and WH0402 from 11º 42.12S', 32º 00.64'W. Triplicate cultures were grown using a semi-continuous culturing technique (Garcia et al., 2011) at 28 degrees C in an artificial seawater medium (Chen et al., 1996). Nutrients were added to autoclaved seawater at the concentrations listed in the AQUIL recipe (Morel et al., 1979), except for nitrate, which was omitted. The growth rates of cultures were measured over 2–3 day intervals and were used to determine the dilution rate. Culture cell density was kept low (cells ml–1 = 50–500 × 103 for experiments with WH0401 and 5.0–30 × 103 for WH0402) to prevent light limitation of photosynthesis and deviation from the expected pH values for respective pCO2 culture treatments. Light was supplied with cool-white fluorescent lamps on a 12:12 h light:dark cycle and measured with a LI-250A light meter (LiCor Biosciences, light sensor serial# SPQA 4020). Because of large differences in cell size between WH0401 and WH0402, WH0401 was cultured at higher cell densities to maintain relatively equivalent levels of total culture biomass (0.1–2.5 mM particulate C for cultures of WH0401; 0.1–1.3 mM particulate C for WH0402). For CO2 experiments, media and cultures were bubbled with filtered air from the room (0.2 µm filtered, present-day pCO2 concentration of ~385 ppm) or premixed air prepared by Gilmore Liquid Air Company with certified values of 190 ppm pCO2 (last glacial maximum levels: Petit et al., 1999) and 750 or 761 ppm pCO2 (within the range predicted for the year 2100: Alley et al., 2007) for the entire term of the experiment. Cells were considered fully acclimated to treatment conditions after cultures had remained at steady-state growth for seven generations or more (unless stated otherwise). Fast growing cultures (i.e. high light cultures) were acclimated for more than ten generations while slow growing cultures (i.e. low light and low pCO2 cultures) were acclimated over two months but for fewer generations. Cultures were sampled over the period between 24 and 48 h after the preceding dilution to measure growth rates, gross and net 15N2-fixation rates, CO2-fixation rates, particulate elemental composition, and carbonate system measurements (for CO2 experiments).

CO2 experiments
To investigate variability in the effects of CO2 on growth and N2-fixation rates between strains of C. watsonii, the investigators conducted experiments with cultures of WH0401 and WH0402. They measured growth and gross and net N2-fixation rates (see N2-fixation rates) in response to three levels of CO2 (190, air, and 750 ppm) at a light intensity of 155 µmol quanta m–2 s–1. This light intensity was chosen so that growth rates in these cultures would not be limited by light.

Growth rate and cell density estimates
Growth rate was determined as an increase in culture cell density over time with the equation N_T=N₀e^µT, where N₀ and N_T are the initial and final culture cell densities, respectively, T is the time in days between culture cell density estimates, and µ is the specific growth rate. Culture cell density was determined using a haemocytometer and an Olympus BX51 microscope. Cell diameter was measured using an ocular micrometer calibrated with the same microscope. Growth rates were fitted to a Monod linear hyperbolic function of light (Monod, 1949) using Sigma Plot 10 software program. The hyperbola was fit to the data without including the origin to yield the highest r² value.

Carbonate system measurements
Culture pH was measured intermittently during the CO2 experiments with a pH meter using the NBS seawater scale (Orion 5 star Thermo Scientific, Beverly, MA, USA). Samples for total CO2 (TCO2) measurements were preserved in unfiltered water collected from cultures (5–70 ml; stored at 4°C) with a 5% HgCl2 solution (0.5% final concentration) until later analysis with a carbon coulomb meter (CM 140, UIC, Joliet, IL, USA). TCO2 was measured by acidifying a 5 ml sample with phosphoric acid (1–2% final concentration) and quantifying the CO2 trapped in an acid sparging column as described in Garcia et al. (2011). TCO2 analyses were not available in the preliminary CO2 experiments. pCO2 was calculated with the CO2sys program (Lewis & Wallace, 1998) using the NBS pH scale and K1 and K2 constants from Mehrbach et al. (1973), refit by Dickson & Millero (1987).

N2 fixation
The acetylene reduction assay described by Capone et al. (1993) was used to estimate the gross N2-fixation rate. Rate measurements were initiated at the beginning of the 12-h dark period, when C. watsonii is known to fix N2 (Mohr et al., 2010a; Saito et al., 2011). For the CO2 experiments, the acetylene assay was initiated during the seventh hour of the 12-h dark period and continued for 4 h. For this assay, two 50 ml (light and CO2–light experiments) or 60 ml (CO2 experiments) culture samples were collected from each replicate and incubated in 80-ml polycarbonate bottles at 28 degrees C. Four millilitres of acetylene were injected into the headspace ~1 h after the beginning of the dark period and samples were withdrawn from the headspace every 2–3 h to measure acetylene reduction. Gross N2-fixation rates were calculated in the same way as described in Garcia et al. (2011), using a Bunsen coefficient for ethylene of 0.082 (Breitbarth et al., 2004) and an ethylene production : N2-fixation ratio of 3:1.

Net N2-fixation rates were measured using the 15N2 isotope tracer method (Mulholland & Bernhardt, 2005; Mulholland et al., 2004). Samples were prepared the same way as described in Garcia et al. (2011). Briefly, 169 ml of each experimental replicate was inoculated with 169 µl of 99% doubly labelled 15N2 gas and incubated at 28 degrees C in complete darkness for 12 h during the dark period. The incubation was then terminated by filtering the entire volume onto precombusted (450 degrees C, 4 h) GF/F filters for the analysis of particulate 15N, total particulate N, and total particulate C. Filters were dried at 80–90 degrees C, pelleted, and combusted in a quartz column with chromium oxide and silver wool at 1000 degrees C. For this analysis, ammonium sulphate and sucrose were used as standards. At the time these experiments were conducted, the investigators were not aware of the criticisms of the 15N2 uptake method that have been discussed by Mohr et al. (2010b). Thus, for another independent estimate of net N2 fixation, the investigators calculated a particulate N (PN) accumulation rate in cultures over time (deltaPN = PNfinal - PNinitial) by using estimates of particulate N. Particulate N was measured in subsamples of experimental replicates that were incubated with 15N2 at the end of the dark period and used as the end-period PN measurement (PNfinal). Because only one sample of PN was collected, the investigators back-calculated an estimate of PNinitial based on their measurements of cellular growth rate using the equation: growth rate (d–1) = [ln(PNfinal)–ln(PNinitial)]/(t2–t1), where t1 is the initial time and t2 is the final time. Based on their measurements of growth rates, the investigators assumed that PN per cell was in a daily steady state. The gross N2-fixation rate:PN-accumulation rate ratio (hereafter the gross:PN accumulation ratio) was then calculated and compared with the ratio of gross N2-fixation rate:net 15N2-fixation rate ratio (gross:net), which is a proxy for cellular N retention (Mulholland et al., 2004; Mulholland, 2007).

References:
ALLEY, R.B., BERNTSEN, T., BINDOFF, N.L., CHEN, Z., CHIDTHAISONG, A., FRIEDLINGSTEIN, P., GREGORY, J.M., HEGERL, G.C., HEIMANN, M., HEWITSON, B., HOSKINS, B.J., JOOS, F., JOUZEL, J., KATTSOV, V., LOHMANN, U., MANNING, M., MATSUNO, T., MOLINA, M., NICHOLLS, N., OVERPECK, J., QIN, D., RAGA, G., RAMASWAMY, V., REN, J., RUSTICUCCI, M., SOLOMON, S., SOMERVILLE, R., STOCKER, T.F., STOTT, P.A., STOUFFER, R.J., WHETTON, P., WOOD, R.A. & WRATT, D. (2007). Summary for policymakers. In Climate change 2007: The physical science basis. Contribution of Working Group I to the fourth assessment report of the Intergovernmental Panel on Climate Change (Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M. & Miller, H.L., editors), 1–18. Cambridge University Press, Cambridge and New York.

BREITBARTH, E., MILLS, M.M., FRIEDRICHS, G. & LAROCHE, J. (2004). The Bunsen gas solubility coefficient of ethylene as a function of temperature and salinity and its importance for nitrogen fixation assays. Limnology and Oceanography: Methods, 2: 282–288. DOI: 10.4319/lom.2004.2.282

CHEN, Y.B., ZEHR, J.P. & MELLON, M. (1996). Growth and nitrogen fixation of the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. IMS101 in defined media: Evidence for a circadian rhythm. Journal of Phycology, 32: 916-923. DOI: 10.1111/j.0022-3646.1996.00916.x

DICKSON, A.G. & MILLERO, F.J. (1987). A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep-Sea Research, 34: 1733–1743.

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

LEWIS, E. & WALLACE, D.W.R. (1998). Program developed for CO2 System calculations. ORNL/CDIAC-105. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee. Available at: http://cdiac.ornl.gov/oceans/co2rprt.html

MEHRBACH, Y., CULBERSON, C., HAWLEY, J. & PYTKOVICS, R. (1973). Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnology and Oceanography, 18: 897–907.

MONOD, J. (1949). The growth of bacterial cultures. Annual Review of Microbiology, 3: 371–394.

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.

MULHOLLAND, M.R. (2007). The fate of nitrogen fixed by diazotrophs in the ocean. Biogeosciences 4: 37–51. DOI: 10.5194/bg-4-37-2007

MULHOLLAND, M.R. & BERNHARDT, P.W. (2005). The effect of growth rate, phosphorus concentration and temperature on N2-fixation, carbon fixation, and nitrogen release in continuous cultures of Trichodesmium IMS101. Limnology and Oceanography, 50: 839–849. DOI: 10.4319/lo.2005.50.3.0839

MULHOLLAND, M.R., BRONK, D.A. & CAPONE, D.G. (2004). N2 fixation and regeneration of NH4+ and dissolved organic N by Trichodesmium IMS101. Aquatic Microbial Ecology, 37: 85–94. DOI: 10.3354/ame037085

C_watsonii_CO2_expt.csv