Table of Contents

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

Sampling and analytical procedures:


1. Analysis of NO3- N and O isotope ratios with the denitrifier method

The denitrifying bacteria strains Pseudomonas chlororaphis f. sp. aureofaciens (ATCC 13985, Manassas, VA, USA) and Pseudomonas. chlororaphis (ATCC 43928, Manassas, VA, USA) were used. Cultures were inoculated from cryo-preserved aliquots (Weigand et al., 2011) into sterile growth media prepared as originally described (Sigman et al., 2001; Casciotti et al., 2002) in 700 mL glass bottles containing 600 mL of medium, then sealed with gas-tight lids. Cells were cultured for 7-10 days at 20˚C on a rotary shaker table. Cultures were harvested by centrifugation and resuspended into 220 mL of fresh medium without potassium nitrate addition, achieving ca. 3-fold concentration of the bacteria. Two mL of the cell concentrates were added to respective 20-mL headspace glass vials, capped with pre-rinsed butyl rubber septa and crimp-seals (Mcllvin and Casciotti, 2011). Vials were sparged with a water-scrubbed N2 gas stream for 6 hours to remove any N2O produced from the residual NO3- in the medium. NO3- samples were then injected into each vial to achieve a final sample size of 10 nmoles of N. Vials were incubated inverted in order to prevent potential N2O leakage. Following overnight incubation in the dark, ca. 0.1 ml of 10 mol L-1 NaOH was injected into each vial to kill the cultures and sequester CO2 into carbonate species. The N2O gas in the vials was extracted, purified and analyzed with a Delta V Advantage continuous flow gas chromatograph isotope ratio mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) interfaced with a modified Thermo Fisher Scientific Gas Bench sample preparation device fronted by dual cold traps (Casciotti et al., 2002) and a GC Pal autosampler (CTC Analytics, Zwingen, Switzerland). Samples were referenced to pure N2O injections from a common reference gas cylinder.

2. Demonstration of volume effects in analyzes of NO3- reference materials

The reference solutions were prepared from salts into primary stocks at 200 µmol L-1 in deionized water (DIW) from a Milli‐QTM water purification system (EMD Millipore, Burlington, MA, USA), and stored frozen. Primary stocks of NO3- reference materials (IAEA-NO3 and USGS-34) were diluted in NO3--deplete surface Sargasso seawater or in aged DIW to concentrations of 1, 5 and 20 µmol L-1, corresponding to respective injection volumes of 10, 2 and 0.5 mL, in order to aliquot 10 nmoles of N analyte. The NO3- aliquots were injected into the sparged bacterial concentrates of either P. chlororaphis or P. aureofaciens. Following bacterial conversion, the resulting N2O in the reaction vials was extracted, purified and analyzed on the isotope ratio mass spectrometer.

Processing notes from submitter:

• Data were processed using Microsoft Excel 

BCO-DMO processing notes

• Date formats were changed from mm/dd/yy to yyyy-mm-dd
• Spaces and units removed from column headers

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.