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 deg C and 470 umol photons/m-2/s PAR.

pCO2: 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.

Dissolution of existing coccoliths:  Coccoliths were dissolved by 1.75 M HCl to drop the pH to 5.5 for 2 min.  Following the dissolution (de-lithing), 1.75 M NaOH was added to bring the pH back to the respective starting pH.  Dissolution of coccoliths was immediately confirmed by looking at the cells under cross-polarized light microscopy to verify the absence of birefringence indicative of CaCO3.  Dissolution was further confirmed by filtering the acidified/neutralized sample onto a 0.4 um polycarbonate filter.  Filters were mounted on stubs, sputter-coated with gold using a Denton Desk IV sputter coater (Denton Vacuum, Moorestown, NJ, USA), and imaged on a Zeiss Supra25 field emission scanning electron microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).  At least 15 cells per sample were imaged and the number of coccoliths/cell was manually counted to determine the number of coccoliths that remained after the acidification/neutralization dissolution step.

24 h incubation in either light or dark conditions:  To determine the number of coccoliths formed (as a proxy for calcification rate) in 24 h in either light or dark conditions, for each pCO2 level, 15 mL of de-lithed culture were added to 8 scintillation vials.  Three vials were ‘light’ replicates, three vials were ‘dark’ replicates, and two vials were poisoned with buffered formalin to serve as a ‘light’ blank and a ‘dark’ blank.  Dark replicate and blank vials were covered in black aluminum foil and all vials were incubated together for 24 h in an incubator set at 16.5 +/- 0.5 deg C and 470 umol photons/m-2/s PAR on a 14-10 h light-dark cycle. The experiment was timed to start when the lights in the incubator turned on in the morning, thus the ‘light’ replicates were exposed to light for 14 of 24 h. 

Determination of attached coccoliths:  Coccolith formation was assessed by counting the number of coccoliths formed during the incubation period.  After the 24 h incubation period, each replicate and blank vial was filtered onto a 0.4 um polycarbonate filters.  Filters were mounted on stubs, sputter-coated with gold using a Denton Desk IV sputter coater (Denton Vacuum, Moorestown, NJ, USA), and imaged on a Zeiss Supra25 field emission SEM (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).  At least 15 cells per replicate were imaged and the number of coccoliths/cell was manually counted.  The counted coccoliths represented calcification during the 24 h incubation period and the average number of coccoliths per cell for each replicate and blank is reported.