Project: EAGER: Unlocking the clumped isotope signatures of marine nitrate

NSF Award Abstract:

Nitrogen is a limiting nutrient for life in many marine environments. Its availability shapes the structure and productivity of marine ecosystems. To better understand the cycling of nitrogen, scientists need tools to track the processes that affect nitrogen in nature. Past research into the nitrogen and oxygen stable isotopes of nitrate has enabled pivotal insights into the global nitrogen cycle. At the same time, it has highlighted many unresolved complexities in the cycling of nitrogen. Unfortunately, classical isotope measurements are limited in capturing the processes that transform nitrogen in nature. This is because the resulting isotope signatures are always a combination of at least three factors. First, the isotopic composition of the source compound. Second, the mechanism of the process. And third, the completeness of the transformation. In this EArly-concept Grant for Exploratory Research (EAGER) project, the investigators thus propose to explore a new analytical approach. The goal of the proposed research is to measure the doubly-substituted or “clumped” isotopologs of nitrate for the first time. Clumped nitrate isotopes have the potential to record specific nitrogen transformations. The proposed work could unlock these clumped nitrate isotope signatures as a new tool to study nitrogen in the oceans. In addition to the technology itself, this project will develop open-source software tools for the analysis of isotopolog data to support future work on similar analytes of interest. The proposed project will also create an opportunity for community college students to visit participating laboratories at CU Boulder. In collaboration with the five community colleges of the Denver Metro STEM Alliance for Minority Participation, interested students will meet current CU Boulder students, learn about ongoing undergraduate research, summer research opportunities and 4-year college transfer pathways. The participating community colleges educate a large number of students from underrepresented backgrounds. Creating pathways for these students to continue on to a 4-year degree and a successful career in STEM research and industry fields is a critical piece of the puzzle for increasing diversity in STEM disciplines nationwide.

Specifically, the investigators propose to develop a new technological approach to studying the isotopes of marine nitrate by electrospray ionization (ESI)-Orbitrap mass spectrometry. This approach provides a path to quantifying up to 3 new isotopic dimensions of nitrate. These doubly substituted, “clumped” nitrate isotopologs (15N18O, 18O18O, 17O18O) have never before been analytically accessible and could fundamentally transform our understanding of how individual nitrogen cycling pathways interact to shape ocean ecosystems and the global nitrogen cycle. Because they are intramolecular isotopic dimensions, the relative abundances of these clumped nitrate species directly reflect kinetic and equilibrium isotope effects of the processes that form and destroy them. The objectives of this project are thus to 1) establish an ESI-Orbitrap methodology for the reproducible quantification of all singly and the three major doubly substituted isotopologs of nitrate; 2) develop and demonstrate scalable and easily transferable protocols for the extraction and preparation of nitrate from marine samples for ESI-Orbitrap analysis; 3) initiate a pilot study of the clumped nitrate space in marine samples. Beyond its immediate impact on research in chemical oceanography, this project will introduce the geochemistry community to a new generation of intact molecule isotope-ratio mass spectrometry that can be adapted to other oxyanions and metabolites widely studied in marine systems (e.g., sulfate, amino acids, lipids). Developing nitrate as a model for ESI-Orbitrap isotope analytics also will benefit other fields of science including agricultural research, terrestrial ecosystems science, and atmospheric chemistry.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.