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 degrees C and 470 umol photons/m-2/s\u00a0PAR on a 14-10 light-dark cycle where the lights turned on at 6 am and turned off at 8 pm.
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pCO2\u00a0Treatments: 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.
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Growth Cycle (14 d) Culture Dynamics Monitoring:\u00a0 To understand the chemical and biological culture dynamics over an entire growth cycle (14 days, where days 1-9 represented exponential growth), the investigators took measurements of one culture from each\u00a0pCO2\u00a0treatment every day at approximately the same time of day for one full growth cycle.\u00a0 Our pH electrode malfunctioned on days 7 and 8, and the total alkalinity sample from the 750 ppm treatment on day 8 was accidentally dropped and broken, so calculations of full carbonate chemistry parameters for days 7 and 8 were not possible.
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pH Measurements:\u00a0 The pH of the cultures was measured using an OrionTM\u00a0ROSSTM\u00a0electrode connected to an Orion StarTM\u00a0A211 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 weekly spectrophotometric pH measurements of culture samples.\u00a0 Spectrophotometric pH measurements of 0.2 um-filtered culture samples were made with 20 mM\u00a0m-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.\u00a0 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. \u00a0
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Temperature:\u00a0Temperature measurements were made with an OrionTM\u00a0ROSSTM\u00a0electrode connected to an Orion StarTM\u00a0A211 Benchtop pH meter (ThermoFisher Scientific, Waltham, MA, USA).
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Salinity:\u00a0 Salinity was measured using an Acorn SALT 6 handheld salinity meter (Oakton Instruments, Vernon Hills, IL, USA) with a resolution of +/- 0.1 ppt.
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in vivo Fluorescence:\u00a0 Fluoresence was measured using a Turner 10-AU fluorometer (Turner Designs, Sunnyvale, CA, USA).
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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%.
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Particulate Inorganic Carbon:\u00a0 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.\u00a0 Filters were carefully moved to trace-metal free centrifuge tubes and digested with 5 mL of 5% nitric acid.\u00a0 The calcium concentration was measured using a Jobin Yvon Ultima C inductively coupled plasma-atomic emission spectrometer (ICP-AES, HORIBA, Ltd., Kyoto, Japan).\u00a0 Bulk culture PIC measurements were corrected to PIC/cell\u00a0using the corresponding cell density measurements.
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Particulate Organic Carbon:\u00a0\u00a0To determine the bulk culture POC concentration, 10 mL of culture were filtered onto a pre-combusted Whatman GF/F filter, which was then fumed in 10 % HCl to remove inorganic carbonates.\u00a0 The dried filters were then analyzed on an ECS 4010 CHNSO Analyzer (Costech Analytical Technologies, Valencia, CA, USA) by Bigelow Analytical Services, East Boothbay, ME, USA.\u00a0 Bulk culture POC measurements were corrected to POC/cell\u00a0using the corresponding cell density measurements.
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Nutrients:\u00a0 Culture samples were filtered to 0.2 um to remove all algal cells and coccoliths, and samples were frozen prior to analysis.\u00a0 Total N (nitrate + nitrite), nitrite, phosphate, and silicate were measured by Continuous Flow Analysis by Bigelow Analytical Services using a SEAL AutoAnalyzer 3 HR (SEAL Analytical Inc., Mequon, WI, USA). \u00a0
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Total Alkalinity:\u00a0 Culture samples were filtered to 0.2 um to remove all algal cells and coccoliths.\u00a0 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.\u00a0 Titrations were corrected to Certified Reference Materials (supplied by the laboratory of Andrew Dickson, Scripps Institution of Oceanography, La Jolla, CA, USA).
Nitrate:\u00a0\u00a0To determine the nitrate concentration of our cultures, Total N (nitrate+nitrite) and nitrite concentrations were measured and the nitrate concentration was calculated as:
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Nitrate = Total N - Nitrite
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Carbonate Chemistry Calculations:\u00a0 Using the measured values of pH (total scale), total alkalinity, temperature, salinity, phosphate, and silicate, the investigators used CO2SYS software (Pierrot et al. 2006) to calculate dissolved inorganic carbon (DIC),\u00a0pCO2, [HCO3-], [CO32-], [CO2], and\u00a0calcite\u00a0using the first and second dissociation constants (K1\u00a0and K2) of carbonic acid in seawater from Mehrbach et al. (1973), refit by Dickson and Millero (1987); KHSO4\u00a0from Dickson (1990); and [B]T\u00a0from Uppstrom et al. (1974).\u00a0 Full carbonate chemistry could not be calculated for days 7 and\u00a08,\u00a0since the pH meter malfunctioned.
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DMO 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 \"na\" with \"nd\"