Award: OCE-1358041

The bioavailability of nutrients plays a critical role in ocean biological productivity and its carbon cycle. Nitrogen (N) is perhaps most important, controlling most primary production and representing the foundation for all ocean life, including ocean fisheries. Bio-available N is controlled by a complex balance of sources and removal mechanisms. One of the greatest current challenges in oceanography is to understand the cycles of ocean N, so that we may understand how changing climate may alter them. The largest pool of actively cycling organic N is present in dissolved form in ocean water at all depths, and is known as dissolved organic nitrogen (DON). While one of the most important reservoirs of N in the sea, it is also without doubt the most mysterious. Present at extremely low concentrations, most of this material resides in the deep. Its chemical structures, which would let us understand its sources, fates, and how its cycles are regulated, have remained largely unknown. The overarching goal of this project was to develop a unique new approaches to solve this problem, and to use it to tackle one of the main current theories which might explain where highly refractory organic N compounds come from, and how they survive. Our first goal was to develop a new approach to studying dissolved material. Using a series of molecular filters and chemical resins we aimed, for the first time, to selectively isolate "semi-labile" vs. "refractory" DON in very large amounts. We would then be able to use these large purified samples to study the composition of the material using an array of chemical and isotope techniques. One main aim was to test "the microbial N pump" hypothesis: simply put, the idea that bacteria are not only the main source for the refractory DON that builds up in the deep ocean, but that specific molecular structures made by bacteria are the reason that very old DON accumulates, effectively "regulating" the size and age of this key biochemical reservoir. To test this, used our new sample types, and then applied a number of chemical and isotope tools together for the first time. A main focus was to couple radiocarbon (14C) dating with a number of chemical tracers for bacteria, such as the D- enantiomers of amino acids, as well as isotope ratio patterns in amino acids. The bulk of our findings are quite technical, related to details of 15N isotope patterns in extracted amino acids and proteins, and how these can correlate with increasing 14C ages of dissolved material. However, the most important take-home result is simple: it worked. Our new approach succeeded in isolating absolutely unique samples of old and young materials for independent study. Further, our results together strongly supported the "microbial N pump" hypothesis, and also led to a number of novel and unexpected findings that greatly expand our understanding of DON sources and cycling. Among our most exciting specific results: 1. We found that the chemical form of most of the DON in the deep ocean is completely different than has been previously assumed: most appears to be composed of N heterocyclic compounds, not proteins or amino sugars as had been thought before. This major finding will open up important new areas for research. 2. We identified an entirely new suite of D-Amino Acid (AA) tracers for microbial input. Only 4 such D-AA had previously been known in ocean, however our new methods allowed us to identify 7 more! However, these new molecules are mainly concentrated in the oldest material. This suggests a new area of research for specific bacterial input, using them. 3. We made the first nitrogen isotope measurements in the DON pool, both old and young, throughout the water column. In surprising results, our data show these two ends of the spectrum have extremely different, and diagnostic, N isotope values at all depths. Together with structural data, this strongly suggests an entirely new paradigm for how DON is sourced and cycles. It was previously assumed the old material in the deep ocean was formed from younger material, and changed slowly over thousands of years. In contrast, our new results indicate both pools may have independent sources in the surface, but cycle at different rates due to their extremely different chemical structures. Overall, we believe that our results are highly significant, both within the core disciplines of organic geochemistry and chemical oceanography, but also in many related fields. For example, our project has pioneered the use of amino acid isotopes in DON, and this can now be applied to terrestrial or lake studies to investigate parallel questions about how bacteria regulate biogeochemical cycles. This research has contributed a major suite of new chemical tools and approaches toward studies of N cycling in any environment. Last Modified: 08/01/2018 Submitted by: Matthew D Mccarthy