Dataset: Thalassiosira pseudonana CCMP1335 in nitrate-limited and nutrient-replete cultures

The culture was grown in either a nitrate-limited or nutrient-replete continuous culture system on a 14:10 L:D cycle of illumination at temperatures of 5, 10, 15, 20, 25, 30, 31, and 32°C. The irradiance during the photoperiod was either 50 or 300 micro-mol photons m–2 s–1. 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. The CO2 concentration in the laboratory was monitored with a CO2METER model AZ-004 meter calibrated at 0 and 400 ppm CO2 with a standard gas mixture.

The system was judged to be in steady state when cell counts, measured with a Beckman Coulter model Z1 particle counter, had 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. Concentrations of particulate carbon (PC) and particulate nitrogen (PN) were determined by filtering replicate 50-mL samples from the growth chamber onto GF/F glass fiber filters followed by analysis with an Exeter Analytical model CE-440 elemental analyzer. pH was measured with a Thermo Spectronic Heios spectrophotometer, as described in SOP 6B by Dickson, et al 2007 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 2365 meq per liter. Nutrient concentrations corresponded to f/2 medium, with the exception of trace metals, which were added at the concentrations specified by Sunda and Hardison (Limnology & Oceanography 52[6]:  2496–2506 [2007]). The nitrate concentration in the nitrate-limited experiments was 20 micromolar. The medium was sterile filtered (0.2 micron) 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 2183 mL. In the first few experiments, the cells in the growth chamber were uniformly labeled with C-14 by adding 20 microcuries of C-14 bicarbonate to the nutrient reservoir to facilitate monitoring the concentration of organic carbon in the growth chamber. In those first few experiments, five-milliliter samples for C-14 activity in the organic carbon were withdrawn in triplicate from the growth chamber at two-hour intervals during the photoperiod. 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. During those first few studies, we determined that addition of C-14 in this way was unnecessary because we could adequately monitor the concentrations of PC by withdrawing samples for CHN analysis. Subsequent experiments relied entirely on CHN analyses for determination of particulate carbon and nitrogen concentrations.

Short-term (5-minute) photosynthesis-versus-irradiance curves (P-E 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 5, 10, 20, 30, 55, 80, 120, 150, 200, 250, 300, and 350 micro-mol photons per square meter per second 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, et al 2007. DIC 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 in these short-term experiments were found to be best described by a hyperbolic tangent function of the form P = Pm*tanh(E*alpha/Pm), where E is the irradiance, alpha is the initial slope of the photosynthesis-irradiance curve, and Pm is the asymptotic light-saturated photosynthetic rate. The values of Pm with units of grams carbon per gram chlorophyll a per hour and the value of alpha with units of meters squared (carbons/photon) per gram chlorophyll a were determined by least squares. 

Measurements of Fv/Fm ratios (the ratio of variable fluorescence to maximal fluorescence after dark adaptation) were made within 30 minutes of each P-E assay, using a Z985 AquaPen fluorometer (Qubit Systems). Briefly, a 4-mL aliquot of culture from the growth chamber was added to each of three plastic 1-cm cuvettes, and each cuvette was immediately wrapped in aluminum foil. The cuvettes were incubated at the growth chamber temperature for 30–40 minutes, after which the foil was removed and a single Fv/Fm measurement was made on each cuvette in a darkened room. The background-corrected Fv/Fm ratio was automatically calculated by the AquaPen software. The light intensities of the saturating pulse and measurement pulse were 2100 and 0.03 micro-mol photons per square meter per second, respectively, both at a wavelength of 450 nm.