The methods below are described in Sun et al. 2011.

Cultures and growth conditions
Stock cultures of marine diatom Pseudo-nitzschia multiseries (Hasle) (CCMP 2708, originally isolated from Eastern Canada) were maintained at 17 degrees C in 0.2 um-filtered, microwave-sterilized natural seawater, enriched with levels of phosphate, nitrate, silicate, vitamins, and trace nutrients as in Price et al. (1988). Light was provided on a 12 h dark:12 h light cycle using cool white fluorescent bulbs at 120 umol photons per square meter per second. Irradiance was measured with a biospherical LICOR sensor (model LI-250).

Experimental design
Semi-continuous culturing methods were used in order to measure the effects of P availability and/or pCO2 levels during acclimated, steady-state growth. Cultures were diluted daily with medium that was previously adjusted to the appropriate temperature and pCO2. Each bottle was diluted back to the same cell density present in that bottle directly after the previous day’s dilution. Cultures were harvested following approximately 4 to 6 weeks of semi-continuous incubation when they were fully acclimated to the experimental conditions, after statistically invariant growth rates were recorded for at least 4 to 6 consecutive dilutions.

Triplicate bottles at two conditions of phosphate availability were equilibrated at three different CO2 concentrations by gentle bubbling with commercially prepared certified standard air and CO2 gas mixtures (Praxair Gas). CO2 concentrations examined included preindustrial atmospheric levels (~22 Pa), near-present day concentrations (~41 Pa), and values predicted to occur before the end of this century (~74 Pa, IPCC 2007). In-line high efficiency particulate air (HEPA) filters were used to avoid contamination from particles in the gas tanks or lines. Phosphate levels used were 20 umol per liter (P replete) and 0.5 umol per liter (P limited). A total of six different phosphate and CO2 conditions were used in this study: 20 umol per liter P and ~22 Pa CO2; 20 umol per liter P and ~41 Pa CO2; 20 umol per liter P and ~74 Pa CO2; 0.5 umol per liter P and ~22 Pa CO2; 0.5 umol per liter P and ~41 Pa CO2; and 0.5 umol per liter P and ~74 Pa CO2.

Carbonate buffer system measurements and pCO2 treatments
The pH in each bottle was monitored daily using a high sensitivity microprocessor pH-meter (Orion EA 940), calibrated with pH 4, 7 and 10 buffer solutions. The relative precision of this instrument is ~0.01 and accuracy is ~0.03 pH units. For the analysis of total dissolved inorganic carbon (DIC), DIC samples were stored in 2 mL capped borosilicate vials free of air bubbles and were preserved with 20 uL saturated HgCl2 per liter, and stored at 4 degrees C until analyzed. Total DIC was measured by acidifying 2-mL 10% of H3PO4 and quantifying the CO2 trapped in an acid sparging column (model CM 5230) with a carbon coulometer (model CM 140, UIC). Certified reference materials obtained from Andrew Dickson (University of California, San Diego, http://andrew.ucsd.edu/co2qc/index.html) were measured periodically during the run and used for calibration. pH values remained invariant before and after the dilution, suggesting that bubbling rates were sufficient to maintain the target CO2 equilibration levels in the medium, regardless of diel changes in photosynthesis and respiration. Based in the daily measurements of pH and DIC, pCO2 stabilized during the early part of the semi-continuous growth period and then remained steady throughout the latter part of the incubation period. Calculated pCO2 values (using CO2SYS; http://www.cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS ) for the three CO2 treatments in both P treatments ranged from 22-23 Pa, 39-42 Pa, and 73-75 Pa (see table below where the numbers in parenthenses are the standard deviations of triplicate samples), very close to the certified standard gas mixture values. For convenience, these values were averaged and rounded to 22 Pa, 41 Pa, and 74 Pa when referring to the three pCO2 treatments throughout the dataset and paper (Sun et al. 2011).

Treatment conditions and calculated pCO2:

Treatment	Measured pH (sd)	Measured DIC (sd); umol/L	Calculated CO2 (sd); umol/L	Calculated pCO2 (sd); Pa
P-limited, 22 Pa	8.38 (0.05)	1917 (38)	7.4 (0.6)	23 (2)
P-limited, 41 Pa	8.15 (0.02)	2029 (8)	13.9 (0.7)	42 (2)
P-limited, 74 Pa	7.94 (0.01)	2145 (9)	24.5 (0.6)	75 (2)
P-replete, 22 Pa	8.40 (0.03)	1970 (4)	7.1 (0.5)	22 (2)
P-replete, 41 Pa	8.19 (0.02)	2066 (11)	12.8 (0.8)	40 (3)
P-replete, 74 Pa	7.96 (0.01)	2177 (6)	23.9 (0.4)	73 (1)

Determination of P-E curves and primary production
Photosynthesis vs. irradiance (P-E) curves were performed by measuring 14C fixation rates at a range of light intensities using a photosynthetron (Composite High Pressure Technologies). Five mL of scintillation cocktail was added and the filters were stored in the dark overnight, and then counted using a Wallac System 1400 liquid scintillation counter.

All 14C uptake rates were corrected for dark uptake and carbon assimilation values were subsequently normalized to Chl-a. The initial slope of the P vs. E curve, i.e., the photosynthetic efficiency alpha [(mg C per mg Chl a per hour per (umol quanta per square meter per second)] and the maximum chlorophyll specific carbon fixation rate PBmax [mg C per mg Chl a per hour] were calculated from least-squares nonlinear regression using the exponential function of Platt et al. (1980). Ek (umol quanta per square meter per second), the light saturation point and index of light adaptation, was calculated as PBmax:alpha. All carbon fixation rates for PE curves were calculated using measured initial experimental DIC and Chl a concentrations for each treatment.

References:
Platt, T., C. L. Gallegos, and W. G. Harrison. 1980. Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J. Mar. Res. 38: 687-701.