Cultures: Pleurochrysis carterae cultures were maintained in exponential growth phase under axenic conditions in semi-continuous batch culture using L1-Si media prepared on 0.2 um-filtered, UV-sterilized, autoclaved seawater. Cultures were acclimated to one of three pCO2 treatments for over 9 generations before experiments were performed.

pCO2: Carbonate chemistry was manipulated by bubbling cultures and prepared media with 500 mL/min^-1 with 0.2 um-filtered 280, 380, or 750 ppm pCO2 air. The pCO2 levels of the treatment air were established using two mass flow controllers (Aalborg, Orangeburg, NY, USA) for each treatment to precisely mix in-house compressed air and pure CO2 (Maine Oxy, Auburn, ME, USA). The in-house compressed air was stripped of CO2 to less than 10 ppm CO2 using a Puregas VCD CO2 Adsorber (Puregas, LLC, Broomfield, CO, USA). The pCO2 of the gas mixtures was stable to 8 ppm.

Length of Incubation (hours): Number of hours that algae were incubated with 14C-bicarbonate.

Cell density (cells/mL-1): Culture density measured using a Moxi Z mini automated cell counter (ORFLO Technologies, Ketchum, ID, USA), which has a coefficient of variation of 4%.

Photosynthetic rate (ug C/L^-1 d^-1) and Calcification rate (ug C/L^-1 d^-1): Triplicate samples from each pCO2 treatment were spiked with 14C-HCO3- and incubated at 16.5 +/- 0.5 degrees Celsius and 415 umol photons/m^-2/s^-1 PAR for 3 h. At the end of the incubation, the cells from each replicate, along with triplicate formalin-killed blanks for each pCO2 treatment, were filtered onto 0.4 um polycarbonate filters and their carbon was partitioned into organic and inorganic fractions by acidification and subsequent capture of 14CO2 (from PIC) in a trap containing a Whatman GFA filter presoaked with 0.2 mL phenethylamine (see Balch et al. 2000 for detailed methodology). The radioactivity of each fraction was measured on a Packard Tri-Carb 2750 LL scintillation counter (acquired by Perkin Elmer, Waltham, MA, USA) and the photosynthetic rate and calcification rate were calculated from the organic carbon and inorganic carbon fractions, respectively, using the equation on p. 118 from Parsons et al. (1984) and applying appropriate unit conversions:

Photosynthesis OR Calcification (mg C • (m^3)^-1 d^-1) = [(Rs – Rb) * W * 1.05]/(R * t)

Where R is the total activity (dpm) of bicarbonate added; t is the length of incubation (in days); Rs is the sample count (dpm), Rb is the formalin blank count (dpm), and W is the weight of total carbon dioxide present in mg C • (m^3)^-1, determined from the expression W=12,000*TC, where TC is the total carbon dioxide, which can be approximated from salinity, as described in Parsons et al. (1984). The factor of 1.05 accounts for preferential uptake of 12C. The calculation is the same for photosynthetic rate and calcification rate – the difference is using the sample count from either the organic fraction or the inorganic fraction, respectively.