Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

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).

No data values are represented by blank cells in the data table. These values indicate that no sample was collected at the given time point. 

Data were generated from the laboratory experiments were processed using Microsoft Excel.

NSF Award Abstract:
The nitrogen (N) cycle in the marine environment is controlled by biological processes. Unfortunately, quantifying these processes and assessing their effect on the N cycle is difficult by direct measurements because of large spatial and temporal differences. Isotopic composition measurements of N provide a means to constrain these processes indirectly; however, there is still a great deal to be understood about isotope fractionation of recycled nitrogen through biological processes, which has made interpretation of novel nitrogen isotope data difficult. A researcher from the University of Connecticut plans to determine the influence of biological consumption and production on the isotope fractionation in ammonium. By helping to understand the processes surrounding fractionation of recycled ammonium at the organism level, this research will create a basis for which future researchers can better interpret isotope composition data to infer nitrogen cycle dynamics. A graduate student, a postdoctoral fellow, and two or more undergraduate students will be involved in the research. The researcher plans to integrate science with community-engaged learning by developing an undergraduate field and laboratory course that will require the students to present their research to stakeholders in the community. There will be a manual created for this course that will be disseminated in open-access forums for teachers hoping to develop similar courses.

Biological nitrogen isotope fractionation associated with nitrogen recycling remains poorly constrained despite the advent of a variety of new techniques to analyze nitrogen isotopes in recent years. The use of isotopic composition data can be incredibly useful to interpreting nitrogen cycle processes in the ocean that are difficult to measure directly, which makes it crucial to further understand the processes behind fractionation to catch up with the advancement of the datasets available to researchers. This research will characterize the isotope fractionation dynamics of ammonium during biological consumption and production. The researchers will investigate whether the characteristic low concentrations of ammonium in the surface ocean affect isotope fractionation when the ammonium is recycled and whether there is a trophic isotope effect associated with ammonium recycling by protozoan grazers. With this research, there will be a baseline from which researchers can interpret recycled nitrogen dynamics from ammonium isotope datasets. The methods of comparing nitrogen cycling studies will become significantly clearer with such a standard making interpretation uniform by removing significant uncertainties.