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.
Growth Cycle (14 d) Culture Dynamics Monitoring: 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 pCO2 treatment every day at approximately the same time of day for one full growth cycle. 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.
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 weekly 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 using the corresponding cell density measurements.
Particulate Organic Carbon: To 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. 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. Bulk culture POC measurements were corrected to POC/cell using the corresponding cell density measurements.
Nutrients: 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), 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).
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).
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