Dataset: Thalassiosira pseudonana cyclostat

The Thalassiosira pseudonana culture was grown in a nitrate-limited or nitrate-replete continuous culture system on a 14:10 L:D cycle of illumination at temperatures of 10, 15, 20, 25, and 30°C. The irradiance during the photoperiod was either 50 or 300 mmol photons m–2 s–1. Continuous aeration of the culture was with 0.2 µm-filtered ambient air with a CO2 concentration of either 400 or 1000 ppm. Photosynthetically active radiation (400–700 nm) was measured with a Biospherical Instruments model QSL 2100 quantum sensor. Temperature was controlled to within 0.1°C by circulating water from a Haake model DC10 temperature-controlled water bath through the outer jacket of the reaction chamber. The dilution rate of the growth chamber was controlled with a peristaltic pump (Masterflex Model 77200-60) to within ± 0.002 per day. For 400 ppm CO2 treatments, the CO2 concentration in the laboratory was monitored with a nondispersive infrared absorption-based CO2 meter (AZ-0004, CO2meter.com, Ormond Beach, FL) calibrated at 0 and 400 ppm CO2 with standard gas mixture. For elevated CO2 treatments (1000 ppm), the growth chambers and media reservoirs were bubbled constantly (2–5 L/min each) with sterile-filtered ambient air amended with 100% Bone-Dry 3.0 grade CO2 (Airgas, Inc., Radnor, PA) using the system described in Fig. 1. The CO2 concentration of the amended air was monitored using a CO2 meter as for 400 ppm treatments.

See Figure 1 for Schematic of CO2 amendment and distribution. (See Supplemental Documents below)

The system was judged to be in steady state when cell counts, measured with a Beckman Coulter model Z1 particle counter, and been reproducible to within 2% for at least 4 doubling times. Chlorophyll a concentrations were determined from samples collected on glass fiber filters and extracted in methanol. The absorbances were measured at 664 and 750 nm with a Cary Model 50 spectrophotometer. Total-scale pH was measured with a Thermo Spectronic Helios spectrophotometer, as described in SOP 6B by Dickson, A.G., Sabine, C.L. and Christian, J.R. (Eds.) 2007. Guide to best practices for ocean CO2 measurements. PICES Special Publication 3, 191 pp. with minor modifications, and with a Hach SensION model PH31 pH meter calibrated with standards on the total pH scale, prepared as per Millero, F.J., et al. "The use of buffers to measure the pH of seawater." Marine Chemistry 44.2 (1993): 143-152. with minor modifications. The growth medium consisted of artificial seawater with a total alkalinity of 2.365 meq L–1. Nutrient concentrations corresponded to f/2 medium, with the exception of nitrate, which was added at a concentration of 40 mM. The medium was sterile filtered (0.2 mm) into a 40-liter glass carboy that had been previously autoclaved. The growth chamber was an autoclaved glass reaction flask with a working volume of 2143 mL. The cells in the growth chamber were uniformly labeled with C-14 by adding 20 microcuries of C-14 bicarbonate to the nutrient reservoir. Five-milliliter samples for C-14 activity in the organic carbon were withdrawn in triplicate from the growth chamber at two-hour intervals. The samples were acidified with 1 mL of 1 N HCl to drive off inorganic carbon. The activity of C-14 in the samples was then determined by counting on a Packard Tri-Carb model 3100 TR liquid scintillation counter. Short-term (5-minute) photosynthesis versus irradiance curves were measured at the start, middle, and end of the photoperiod. For these experiments, triplicate 5-mL aliquots from the growth chamber were added to liquid scintillation vials pre-inoculated with 0.85 microcuries of C-14 bicarbonate. The vials were incubated at irradiances of 0, 5, 10, 20, 30, 55, 80, 120, 150, 200, 250, 300, and 350 mmol photons m–2 s–1 for 5 minutes. Fixation was stopped by adding 0.5 mL of 1 N HCl to the vials. Total alkalinity was determined using the open cell titration method described as SOP 3B by Dickson, A.G., Sabine, C.L. and Christian, J.R. (Eds.) 2007. Guide to best practices for ocean CO2 measurements. PICES Special Publication 3, 191 pp. DIC and equilibrium CO2 concentrations were then calculated from temperature, salinity, total alkalinity, and pH using the equations in Zeebe and Wolf-Gladrow, CO2 in Seawater: Equilibrium, Kinetics, Isotopes. Photosynthetic rates as a function of irradiance were fit via least squares with the following functions: piecewise linear, simple hyperbola, hyperbolic tangent, and negative exponential. Each of these functions is described by two parameters, the light-saturated photosynthetic rate (Pmax) with units of grams carbon per gram chlorophyll a per hour and a parameter Ek with units of mmol photons m–2 s–1 that determines the characteristics of the function at irradiances sub-saturating irradiances. For example, for a simple hyperbola, Ek is the irradiance at which the photosynthetic rate equals one-half Pmax. Dark-adapted photosynthetic quantum yield (QY) was measured in triplicate for each continuous culture in steady state at mid-photoperiod. QY measurements were made with a PSI AquaPen C100 with manufacturer’s supplied plastic cuvettes containing 4 mL of culture each. Dark-adaptation of the culture samples was achieved by wrapping each of three cuvettes in aluminum foil and incubating at the treatment temperature for 30 minutes, after which QY was measured in a darkened room. 

See Figure 2 for plotted results of Thalassiosia pseudonana CO2 and light experiments. (See Supplemental Documents below)