Nitrogen (N2) fixation is a key biogeochemical process providing new N for the marine ecosystem. Current estimates of the global rates suggest that the inputs and outputs of N are not in balance, leaving an apparent deficiency in N2 fixation in the oceanic N budget. Based on recovery of N2 fixation gene fragments, however, numerous potential N2-fixing microbes are present in the euphotic layer and below, yet evidence for their contribution to N2-fixation is lacking. The goal of this study is to identify and determine the importance of potentially diazotrophic, heterotrophic prokaryotes occupying a previously unstudied niche in the upper ocean.

This project PIs hypothesize that N2 fixation in zooplankton-associated microbial communities is an important source of new N in the open ocean contributing to the oceanic N budget. In oligotrophic, N-limited open ocean waters, zooplankton are faced with the same low N availability in their diets that limits growth of primary producers and heterotrophic microbes in the open ocean. Zooplankton guts would be a particularly suitable environment for heterotrophic N2-fixers as the nitrogenase enzyme is inhibited by oxygen. In many terrestrial insects, N2 fixation by gut microbes serves as a source of N. The aim of this study is to investigate whether similar associations occur in marine zooplankton that live on N-limited phytoplankton. The PIs will investigate specific associations between copepods from N-limited and non-N limited oceanic waters with microorganisms that are potentially or actively fixing N. They will also investigate the fate of this N to study whether the new N is incorporated into the zooplankton host. The main research objectives are to determine whether:

1. diazotrophic microflora exist in copepods and what is their seasonal and spatial variability, 2. microbial communities associated with copepods are actively fixing N2, 3. diazotroph communities contribute to the N nutrition of their zooplankton hosts.

Monthly samples will be collected in the oligotrophic Sargasso Sea, in association with BATS sampling cruises. In addition, experiments and field studies will be conducted in Bermuda during the summer. For comparison to a site that is not severely N limited, and for methods development, additional studies will be carried out in coastal Gulf of Maine waters. Selected copepod species and their fecal pellets will be analyzed for the presence of nifH genes and their expression using cloning and sequencing and Reverse Transcriptase-PCR. Quantitative PCR will be employed to investigate the seasonal distributions of dominant phylotypes. Anaerobic cultivation will be used for the enrichment of the gut microflora. Zooplankton will be incubated with 15N2 to quantify microbial N2 fixation activity and its fate of N through zooplankton overall 15N content and nanoSIMS determination of 15N localization. Products of this study will include novel microbial isolates or consortia, evidence regarding taxa-specific expression of N2 fixation in these microhabitats, and a first estimate for the importance of this process for the oceanic N budget. Broader impacts

This project addresses fundamental issues regarding the sources of oceanic nitrogen and will test hypotheses regarding previously overlooked diazotrophic niches. As open ocean productivity is frequently N limited, characterization of new sources of N will have global impacts. The PIs will engage students from K-12 to graduate levels in this research. Graduate student at the UMD will develop their dissertation research within the framework of the study, and several undergraduate students will participate in field and lab components at UMD. Marine microbiology course content will be built into the K-12 science curriculum of the Ocean Explorium of the city of New Bedford, Massachusetts. Part of the work will be performed as a collaborative project with L. Riemann, Univ. of Copenhagen, thus strengthening international connections.

National Science Foundation (NSF)
Division of Ocean Sciences (OCE)
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David L. Garrison
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University of Massachusetts, Dartmouth
North Dartmouth
United States
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