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 > 9 generations before experiments were performed. Cultures were maintained in an incubator at 16.5 +/- 0.5 degrees C and 470 umol photons/m-2/s PAR on a 14-10 light-dark cycle.
pCO2 Treatments: Carbonate chemistry was manipulated by bubbling cultures and prepared media with 500 mL/min 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. pCO2 values of the cultures may be different than the target levels due to biological activity.
Growth rate measurements: At the same time each day, the cell density of each pCO2 treatment culture was measured in order to calculate the growth rate. The data analyzed represent three consecutive growth cycles.
Cell density: Culture density was measured using a Moxi Z mini automated cell counter (ORFLO Technologies, Ketchum, ID, USA), which has a coefficient of variation of 4%.
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