Dataset: Phytoplankton Nitrogen isotope fractionation

Phytoplankton cultures

Two strains of marine algae, the diatom Thalassiosira weissflogii (actin) and the prasinophyte Tetraselmis sp. were grown in batch cultures in sterile, acid-washed borosilicate glass or polycarbonate bottles using artificial seawater medium in an environmental chamber at 180C, illuminated with fluorescent light (40 μmol photons m-2 s-1 photosynthetically available radiation) on a 12-hour light and 12-hour dark cycle. The artificial seawater medium was prepared from low nutrient Instant Ocean™ salt dissolved in Milli-Q water and filtered through a 47 mm Whatman GF/F glass microfiber filter (0.7 μm nominal pore-size) and sterilized by autoclaving at 1000C via Pasteurization cycle in PRIMUS steam sterilizer, then supplemented with filter-sterilized 100 – 250 μM NH4+, 10 μM phosphate, 100 μM silicic acid (only for T. weissflogii cultures), and f/2 trace metals and vitamins (Guillard and Ryther 1962). The batch cultures were initiated from inocula of approximately 1,000 cells mL-1, and cell densities monitored daily using Multisizer 4 Beckman Coulter counter. Media subsamples were collected during exponential growth for analyses of NH4+ concentration and N isotope ratios. Subsamples were filtered through a 0.45 μm pore-size polyether-sulfone (PES) syringe filters and collected into acid-washed High Density Poly-Ethylene (HDPE) bottles, solution pH adjusted to ca. 4.5 with dilute hydrochloric acid in order to minimize ammonia volatilization during storage, and samples stored at –200C pending analysis. Particulate organic nitrogen (PON) was also sampled during the exponential growth phase by filtering aqueous subsamples onto a pre-combusted 25 mm Whatman GF/F glass microfiber filters (0.7 μm pore-size) and stored in pre-combusted aluminum foils at –200C pending analysis of N isotope ratio analysis. To capture lower NH4+ concentrations, short-term NH4+ uptake experiments with T. weissflogii and Tetraselmis sp. were conducted. A first set of experiments was conducted with T. weissflogii and Tetraselmis sp. cells in early stationary phase, wherein NH4+ was exhausted from the medium. The cells were collected by gentle filtration onto a 5 μm pore-size 47 mm IsoporeTM polycarbonate membrane filter and resuspended into fresh medium containing ~60 μM NH4+ for T. weissflogii and 20 μM NH4+ for Tetraselmis sp. Aqueous subsamples were collected at regular time intervals until NH4+ in the medium was exhausted. A second set of experiments was conducted with N-replete (cells in exponential growth phase) and N-starved cells (cells two days into stationary phase) of T. weissflogii and Tetraselmis sp. Cell cultures were either diluted into fresh medium containing ~20 µM NH4+, or gently filtered onto a polycarbonate membrane filter and resuspended into said medium. Short-term incubations occurred largely under constant illumination, although some Tetraselmis sp. uptake experiments were inadvertently subject to light-dark conditions.

Determination of NH4+ concentration

Ammonium concentrations at or above 50 μM were measured fluorometrically following derivatization with o-phthalaldehyde (OPA; Holmes et al. 1999) while concentrations < 50 μM were analyzed with the indophenol method (Solórzano 1969).

Analyses of N isotopes of NH4+ and PON

Ammonium samples were diluted to 5 µM or 1 µM with deep Atlantic seawater and N isotope ratios determined using the hypobromite-azide method (Zhang et al. 2007), wherein NH4+ is first oxidized to nitrite by hypobromite, after which nitrite is converted to a nitrous oxide gas analyte by reacting with azide. The N isotopic composition of the nitrous oxide product was analyzed using a continuous flow purge and dual cryogenic trap system coupled to a custom-modified Gas Bench II device interfaced with a Thermo Delta V gas chromatograph isotope ratio mass spectrometer (GC-IRMS; see Casciotti et al. 2002; McIlvin and Casciotti 2011). Calibration to reference (dinitrogen gas in air) was achieved from parallel reactions of NH4+ reference materials IAEA-N1 and IAEA-N2 diluted in deep Atlantic seawater (5 µM or 1 µM solutions), with respective assigned δ15N values of 0.43‰ and 20.3‰ vs. air (Böhlke et al. 1993). To analyze N isotopes of PON, frozen glass microfiber filters were lyophilized for 24 hours using an Edwards Super Modulyo freeze-dryer. The filters were packed into tin capsules and analyzed by combustion to dinitrogen gas on a Costech ECS 4010 Elemental Analyzer followed by N isotope ratio analysis of the resulting dinitrogen gas on a Thermo Delta V isotope ratio mass spectrometer. Samples were calibrated with corresponding aliquots of L-glutamic acid reference materials USGS-40 and USGS-41, with δ15N values of –4.52 and 47.57‰ vs. air, respectively (Qi et al. 2003).