Cultures:\u00a0Pleurochrysis carterae\u00a0cultures 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.\u00a0 Cultures were acclimated to one of three\u00a0pCO2\u00a0treatments for > 9 generations before experiments were performed.\u00a0 Cultures were maintained in an incubator at 16.5 +/- 0.5 deg C and 470 umol photons/m-2/s\u00a0PAR.
\n
pCO2: Carbonate chemistry was manipulated by bubbling cultures and prepared media with 500 mL/min\u00a0with 0.2 um-filtered 280, 380, or 750 ppm\u00a0pCO2\u00a0air.\u00a0 The\u00a0pCO2\u00a0levels 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\u00a0(Maine Oxy, Auburn, ME, USA).\u00a0 The in-house compressed air was stripped of CO2\u00a0to less than 10 ppm CO2\u00a0using a Puregas VCD CO2\u00a0Adsorber (Puregas, LLC, Broomfield, CO, USA).\u00a0 The\u00a0pCO2\u00a0of the gas mixtures was stable to +/- 8 ppm.\u00a0\u00a0pCO2\u00a0values of the cultures may be different than the target levels due to biological activity.
\n
Dissolution of existing coccoliths:\u00a0 Coccoliths were dissolved by 1.75 M HCl to drop the pH to 5.5 for 2 min.\u00a0 Following the dissolution (de-lithing), 1.75 M NaOH was added to bring the pH back to the respective starting pH.\u00a0 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.\u00a0 Dissolution was further confirmed by filtering the acidified/neutralized sample onto a 0.4 um polycarbonate filter.\u00a0 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).\u00a0 At least 15 cells per sample were imaged and the number of coccoliths/cell\u00a0was manually counted to determine the number of coccoliths that remained after the acidification/neutralization dissolution step.
\n
24 h incubation in either light or dark conditions:\u00a0 To determine the number of coccoliths formed (as a proxy for calcification rate) in 24 h in either light or dark conditions, for each\u00a0pCO2\u00a0level, 15 mL of de-lithed culture were added to 8 scintillation vials.\u00a0 Three vials were \u2018light\u2019 replicates, three vials were \u2018dark\u2019 replicates, and two vials were poisoned with buffered formalin to serve as a \u2018light\u2019 blank and a \u2018dark\u2019 blank.\u00a0 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\u00a0PAR on a 14-10 h light-dark cycle.\u00a0The experiment was timed to start when the lights in the incubator turned on in the morning, thus the \u2018light\u2019 replicates were exposed to light for 14 of 24 h.\u00a0
\n
Determination of attached coccoliths:\u00a0\u00a0Coccolith formation was assessed by counting the number of coccoliths formed during the incubation period.\u00a0 After the 24 h incubation period, each replicate and blank vial was filtered onto a 0.4 um polycarbonate filters.\u00a0 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).\u00a0 At least 15 cells per replicate were imaged and the number of coccoliths/cell\u00a0was manually counted.\u00a0 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.
Laboratory experiment located at Bigelow Laboratory, East Boothbay, ME using Pleurochrysis carterae (NCMA strain 645), which was isolated from 41.525 deg N\u00a0\u00a070.6736 deg W\u00a0(Woods Hole, Massachusetts USA), but has been maintained in culture since 1958.
Blank correction: For each replicate, the number of coccoliths per cell determined from the corresponding blank sample (same\u00a0pCO2\u00a0and light conditions) was subtracted from the number of coccoliths per cell determined from the replicate.\u00a0 If the\u00a0blank-correction\u00a0resulted in a negative value, the value was set to 0 coccoliths per cell.
\n
Verification of de-lithing:\u00a0 The number of coccoliths per cell immediately after the de-lithing was not explicitly used in any calculations.\u00a0 However, because so few liths were formed in the \u2018dark\u2019 replicates, we performed a 2-way ANOVA to compare the number of coccoliths per cell immediately after the de-lithing with the number of coccoliths per cell in the dark incubation treatments.\u00a0 This test showed that there were significantly more coccoliths per cell for the dark incubation replicates, relative to the samples collected immediately after the de-lithing, which supports the claim that there was calcification in the dark.\u00a0 The results of this statistical test are presented in our paper, so those values are not included in the dataset.\u00a0\u00a0
\n
BCO-DMO Processing Notes:
\n- added underscores and removed spaces and units from column names
\n- changed column names to comply with BCO-DMO standards.
\n- replaced all \"n/a\" with \"nd,\" and filled in blank cells with \"nd\"