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 where the lights turned on at 6 am and turned off at 8 pm.

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.

24 h Culture Dynamics Monitoring:  To understand the chemical and biological culture dynamics over a 24 h period, the investigators took measurements of one culture from each pCO2 treatment every hour for 24 h.  To reduce analytical costs, some parameters were not measured each hour (PIC, nutrients, total alkalinity).  During the dark period, a red lamp was used to limit the light that might influence the cultures.  

pH Measurements:  The pH of the cultures was measured using an OrionTM ROSSTM electrode connected to an Orion StarTM A211 Benchtop pH meter (ThermoFisher Scientific, Waltham, MA, USA), calibrated with NBS buffers (EK Industries, Inc., Joliet, IL, USA) and corrected to the total scale using spectrophotometric pH measurements of culture samples.  Spectrophotometric pH measurements of 0.2 um-filtered culture samples were made with 20 mM m-Cresol purple sodium salt indicator dye (Alfa Aesar, Ward Hill, MA, USA) using a Hitachi U-3010 spectrophotometer (Hitachi High-Technologies, Clarksburg, MD, USA) equipped with a water circulated cell holder connected to a VWR 1160 water bath (VWR, Radnor, PA, USA) set at 16.5 degrees C, holding a 1 cm quartz cell.  The method followed the procedure described by Clayton and Byrne (1993) and Dickson et al. (2007), using the refit equation of Liu et al. (2011), resulting in a resolution of +/-0.004 pH units.  

Temperature: Temperature measurements were made with an OrionTM ROSSTM electrode connected to an Orion StarTM A211 Benchtop pH meter (ThermoFisher Scientific, Waltham, MA, USA).

Salinity:  Salinity was measured using an Acorn SALT 6 handheld salinity meter (Oakton Instruments, Vernon Hills, IL, USA) with a resolution of +/- 0.1 ppt.

in vivo Fluorescence:  Fluoresence was measured using a Turner 10-AU fluorometer (Turner Designs, Sunnyvale, CA, USA).

Cell density and cell diameter: Culture density and mean cell diameter were measured using a Moxi Z mini automated cell counter (ORFLO Technologies, Ketchum, ID, USA), which has a coefficient of variation of 4%.

Particulate Inorganic Carbon:  Bulk culture PIC analyses followed the technique of Fernandez et al. (1993): 10 mL culture samples were filtered onto 0.4 um polycarbonate filters and rinsed with potassium borate buffer with the pH adjusted to 8.0 to remove seawater calcium chloride.  Filters were carefully moved to trace-metal free centrifuge tubes and digested with 5 mL of 5% nitric acid.  The calcium concentration was measured using a Jobin Yvon Ultima C inductively coupled plasma-atomic emission spectrometer (ICP-AES, HORIBA, Ltd., Kyoto, Japan).  Bulk culture PIC measurements were corrected to PIC/cell-1 using the corresponding cell density measurements.

Nutrients (measured at University of California Santa Barbara):  Culture samples were filtered to 0.2 um to remove all algal cells and coccoliths, and samples were frozen prior to analysis.  Total N (nitrate + nitrite), nitrate, phosphate, and silicate were measured by Flow Injection Analysis at the University of California, Santa Barbara, Marine Science Institute’s Analytical Lab using a QuikChem 8000 (Lachat Instruments, Loveland, CO, USA).

Nutrients (measured by Bigelow Analytical Services):  Culture samples were filtered to 0.2 um to remove all algal cells and coccoliths, and samples were frozen prior to analysis.  Nitrate and phosphate were measured by Continuous Flow Analysis by Bigelow Analytical Services using a SEAL AutoAnalyzer 3 HR (SEAL Analytical Inc., Mequon, WI, USA).

Total Alkalinity:  Culture samples were filtered to 0.2 um to remove all algal cells and coccoliths.  Total alkalinity was measured via titration with 0.01 N HCl using a Metrohm Titrando 888 controlled by Tiamo software (Metrohm, Riverview, FL, USA) to perform automated Gran titrations of 4 mL samples.  Titrations were corrected to Certified Reference Materials (supplied by the laboratory of Andrew Dickson, Scripps Institution of Oceanography, La Jolla, CA, USA).