Nitrogen is an important macronutrient for biological growth and while research has been traditionally focused on the dissolved inorganic nitrogen pool, dissolved organic nitrogen (DON) is an important, but historically less understood source of nitrogen. This work focused on creatine, a DON metabolite recognized in human nutrition that has not been heavily studied in aquatic systems. Our research investigates the extent and rate at which aquatic organisms may uptake (use) creatine and compares those rates to other nitrogen compounds including ammonium, nitrate, urea, and amino acids. Samples were collected between 2017 and 2019 and cover three North American regions. These include the polar Arctic Ocean, the California Coastal Current (Baja), and a Chesapeake Bay estuary (York River). Included in this dataset are ambient physiochemical parameters for these systems, along with measured specific uptake rates.

Table of Contents

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

Site water for incubation experiments and for the analysis of ambient nutrients was collected slightly differently for each of the sampling regions. In the Arctic, site water for incubation experiments was collected on the R/V Ukpik and R/V Sikuliaq (cruise ID number SK201712S) during the summer of 2017 using a submersible pump and CTD rosette, respectively. In Baja, sampling occurred twice on the R/V Robert Gordon Sproul during both May and October 2017 (cruise ID numbers SP1714 and SP1727), and site water was collected using a CTD rosette (Turk-Kubo et al. 2021). In Virginia, surface sites in the York River were sampled by directly filling an acid-washed PETG carboy over the side. Filtrations for sample filtrate and filters also varied. These York Rivers samples were obtained during day trips aboard a variety of small powerboats (<21 feet), every other month from June 2018 to July 2019. Powerboats are housed out of the Virginia Institute of Marine Science.

In the Arctic, filtrations were conducted in parallel, which separately passed site water through a larger Nucleopore Membrane filter (3.0 micrometer (μm) nominal pore size) and a smaller Whatman GF-75 filter (nominal pore size 0.3 μm). This same combination of filters was used in Baja in October 2017. In May 2017, Baja sampling used the same filters, but filtered sequentially, meaning that site water was first passed through the Nucleopore filter before passing through the GF-75. The York River site samples were only filtered using the GF-75. Filtrate for all regions was collected from the GF-75 filtration and kept for later nutrient analyses and filters were used for chlorophyll a analysis.

Pigments (chlorophyll a and phaeopigments) were measured after extraction with 90% acetone overnight (Parsons et al. 1984; Arar and Collins 1997). Concentrations of ammonium were analyzed using the Koroleff (1983) method and amino acids were measured as dissolved primary amines (DPA; Parsons et al. 1984). Concentrations of nitrate, nitrite, phosphate, and silica were measured using a Lachat 8500 Quickchem autoanalyzer. Urea was analyzed using the monoxime method (Price and Harrison 1987). Concentrations of total dissolved nitrogen and dissolved organic carbon were assessed using a Shimadzu TOC-V TNM (Hansell 1993). Dissolved organic nitrogen is calculated as the difference between total dissolved nitrogen and inorganic nitrogen. 

Nitrogen uptakes were measured after incubation for a set time (1 to 24 hours) in either 0.5 or 1-liter PETG bottles. Stable isotope tracer methods were used according to those described in Baer et al (2017). Uptake rates for inorganic and organic nitrogen were measured by incubating water with 15N- ammonium, nitrate, creatine, urea, and/or an amino acid mixture under in situ light and temperature conditions in a flow-through system on deck or in a cold room set to ambient site water temperatures. Rate incubation experiments were terminated with the same filtration methods as collection of site water ambients with the exception that a Sterlitech silver filter was used in lieu of the Nucleopore Membrane filter. The exact combination of nitrogen substrates varied between sites and regions. All nitrogen uptake samples were analyzed on a Sercon Integra 2 isotope ratio mass spectrometer.

Known Issues/Problems:
Note that for the October sampling in the Baja region, chlorophyll a concentrations for the nutrient size fractions are unavailable. Instead, chlorophyll a concentrations for the >0.7 μm size fraction are available thanks to the Arrigo and Zehr dataset (listed under "Related Datasets").

Data Processing:
It is important to note that potential specific uptake rates are reported based on sampled size fraction (i.e. >3.0 or >0.3 μm). When applicable, the rates of different size fractions were combined for the final reported specific uptake rate (i.e >3.0 and 0.3 - 3.0 μm fractions added to report a >0.3 μm rate). Rates of ammonium-specific uptake were corrected for isotope dilution when possible. These rates were corrected according to Gilbert et al. (1982).

BCO-DMO Processing:
- removed "NA" and "ND" (missing data values are blank/empty in the final .csv data file);
- converted the Date column to YYYY-mm-dd format;
- converted the Time column to HH:MM format;
- created the ISO_DateTime_UTC column, which presents date and time in UTC in ISO 8601 format;
- renamed fields to comply with BCO-DMO naming conventions in which the only allowed characters are letters (A-Z, a-z), numbers (0-9), and underscores.

Coverage: Atlantic bight

NSF Award Abstract:
High rates of dissolved organic nitrogen (DON) production and utilization in aquatic systems are typically attributed to microbial activity. Though it is known that there is a tight coupling between the production and consumption of biologically available DON, the composition, dynamics, and ecological significance of this rapidly cycled DON pool are less well understood. This proposal focuses on a component of the DON pool, creatine, which is historically understood as a product of metazoan activity, but appears to be both produced by phytoplankton and consumed by marine bacteria. Creatine is present in seawater in measurable quantities, which led to the hypothesis that creatine may be a significant component of the marine DON cycle. DON cycling likely has a bearing on fundamental marine ecosystem processes with large implications for carbon and nitrogen turnover on a global scale. Broader impacts of this project will include outreach that focuses on connecting scientists with K-12 students through research experiences for teachers and lesson development in collaboration with the K20 Center for Educational and Community Renewal, a statewide education research and development center at the University of Oklahoma. The project will integrate the research with inquiry-based teaching of rural secondary science teachers through Authentic Research Experiences in oceanographic science and microbial ecology. The K20 network includes 96% of Oklahoma schools, providing a unique opportunity to impact STEM education in Oklahoma.

The results of this project will help develop a better understanding of DON cycling, the ecological context of creatine uptake activity, and identify both creatine-producing and consuming organisms in the marine environment. The importance of creatine cycling will be assessed via 15N tracer studies along the natural coastal-to-offshore productivity gradient observed in the North Atlantic. Tracer and molecular approaches will be used to investigate the importance of phytoplankton vs. bacteria in creatine uptake and, the taxonomic identities of creatine-utilizing bacteria will be interrogated via molecular, stable isotope probing (SIP), and RT-qPCR approaches.