Award: OCE-2023498

Nitrogen is an essential element required for all life on Earth as it is an important component of biomolecules including DNA, proteins, and lipids. The availability of nitrogen ultimately limits biomass production in both terrestrial and aquatic ecosystems and is mediated by biological, non-biological and anthropogenic forces. The nitrogen cycle is unique in that a huge reservoir of nitrogen exists in an inert form, nitrogen gas (N₂), in the atmosphere and ocean, however only specialized microorganisms, called diazotrophs, can use N₂ to meet their nitrogen needs. In the vast open ocean, the availability of nitrogen limits the growth of phytoplankton, but when diazotrophs are present they act as a biological fertilizer by converting N₂ into ammonia, the N form used by many microorganisms for growth. The importance of N₂ fixation in marine ecosystems has been well-established, but our understanding of the microorganisms that carry out this process has long been limited to those that are conspicuous (large and easily seen using microscopy) or easy to bring into culture. With recent advances in tools to study marine microbes, such as global genetic surveys, we now have a better understanding of the diversity of diazotrophs. These surveys revealed the ubiquitous presence of a group of N₂-fixing microbes - non-cyanobacterial diazotrophs (NCDs) - but prior to this study, it was uncertain whether they were actively fixing N₂ in surface ocean.    

The overarching goal of this project was to directly address whether NCDs are contributing to the fertilization of the ocean by actively fixing N₂. We applied a suite of cutting-edge cell staining techniques (immunofluorescence assays, and several different types of fluorescently labeled in-situ hybridization approaches) to visualize these tiny microorganisms and then measured the uptake of N₂ into individual cells using stable isotope tracers and a specialized instrument for measuring isotopic compositions at nanoscales (nanoscale Secondary Ion Mass Spectrometry, nanoSIMS).

Using these techniques, we were able to obtain some of the first single-cell N₂ fixation rates for marine NCDs in the North Pacific. Some of our most significant findings indicate that NCDs inhabit marine particles in both sunlit surface waters and in dim, deeper waters where light is nearly unavailable. In surface waters, we measured single-cell N₂ fixation rates from tens of thousands of mostly particle-associated microbial cells and detected active N₂ fixation by putative NCDs associated with small organic-rich particles. Deeper it the water column, we characterized the diazotroph diversity and N₂ fixation rates of putative NCDs on different types of particles — suspended, slow-sinking, and fast-sinking - and found a diverse and active community of NCDs on fast sinking particles. Collectively these findings support that particle-associated NCDs may represent a previously underappreciated but important component of pelagic nitrogen fixation in the open ocean.

Demonstrating active N₂ fixation by NCDs has been elusive until the last few years. This research quantifies the contribution of NCD-derived N₂ fixation to whole community rates, and shows that under favorable conditions, it may account for up to 10% N₂ fixed in the photic zone in oligotrophic ecosystems. We now have a basis for advancing the incorporation of this group into ecosystem models, which are used to predict the magnitude of N₂ fixation in contemporary and future oceans.

This project provided training and professional development of two postdoctoral researchers and seven undergraduate researchers at UCSC, Stanford, and UH Manoa, and two of the undergraduate researchers were able to participate in our 35-day research cruise. This project also supported two sessions focused on marine NCDs at oceanography scientific meetings. Our findings led to multiple scientific publications, a majority of which are open access, and presentations (>20) at scientific meetings. This project has also supported the creation of a publicly available pipeline and workflow for the bioinformatic analysis of nifH gene amplicons (a technique used to evaluate the presence of N₂-fixers in a sample), and a global database of nifH microdiversity (hosted on GitHub and archived on Zonodo). Data products are hosted at BCO-DMO (Project 815423).

 

Last Modified: 07/02/2025
Modified by: Kendra Antoinette Turk-Kubo