Dataset: [Deprecated] Thalassiosira pseudonanana in nitrate limited culture

The culture was grown in a nitrate-limited continuous culture system on a 14:10 L:D cycle of illumination at temperatures of 10, 15, 20, 25, 30, and 32°C. The irradiance during the photoperiod was 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 organic carbon (POC) 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 illero, 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 L^–1. Nutrient concentrations corresponded to f/2 medium, with the exception of nitrate, which was added at a concentration of 40 micromolar, and trace metals, which were added at the concentrations specified by Sunda and Hardison (Limnology & Oceanography 52[6]: 2496–2506 [2007]). 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. 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 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. 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 5, 10, 20, 30, 55, 80, 120, 150, 200, 250, 300, and 350 micro-mol 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, 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 as a function of irradiance were found to be best described by Hill reaction kinetics with n = 2 (Hill, A. V., J. Physiol. 40, iv-vii [1910]). For n = 2, the Hill equation takes the form

P = P_maxI^{^2}/(K_I^{^2} + I^{^2})

The light-saturated photosynthetic rate (P_max) with units of grams carbon per gram chlorophyll a per hour and the parameter K_I (Hill coefficient) with units of micro-mol photons m^–2 s^–1 were determined by least squares. 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 room temperature for 30 minutes, after which QY was measured in a darkened room.

References:

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.

Hill, A. V. 1910. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J. Physiol. 40: iv–vii.

Sunda, W. G., and D. R. Hardison. 2007. Ammonium uptake and growth limitation in marine phytoplankton. Limnol. Oceanogr. 52(6): 2496–2506.