Dataset: Incubation data for N2 fixation rates (SPIROPA, July 2019)
Deployment: TN368

Nitrogen fixation rates and hydrographic data were measured in July 2019 in conjunction with the SPIROPA (Shelfbreak Productivity Interdisciplinary Research Operation at the Pioneer Array) project cruises (Chief Scientist Dr. Dennis McGillicuddy). This case study was conducted at the Mid-Atlantic Bight shelfbreak front to the south of Massachusetts, USA to examine whether significant N2 fixation occurs in the continental shelf region. On July 5th, 2019, the vessel R/V Thomas G. Thompson (cruise TN368) departed Woods Hole, MA and performed multiple transects in the area of 70 to 71 degrees West longitude and 39 to 40.5 degrees North latitude. Sampling was restricted to waters above the 0.1% light level. 

Water was collected in 10L Niskin bottles on a 24-bottle rosette. Vertical conductivity, temperature, and depth profiles were measured concurrently using a Sea-Bird 911+ CTD profiler. Samples to determine nitrate, phosphate, and silicate concentrations were filtered (0.4 um, polycarbonate) directly from the Niskin bottles into acid-washed polyethylene bottles and stored frozen until analysis via standard colorimetric methods at the WHOI Nutrient Analytical Facility using a SEAL Analytical AA3 HR discrete chemistry analyzer. Detection limits for nitrate, phosphate, and silicate methods were 0.040, 0.030 and 0.009 μM, respectively.

Nitrogen fixation rate measurements (NFR) were quantified on repeat cross-frontal transects using the ¹⁵N₂ bubble release technique (Chang et al., 2019; Klawonn et al., 2015), which is a variant of the ¹⁵N₂ tracer method (Montoya et al., 1996) which accounts for the slow dissolution time of N₂ gas (White et al., 2020).  The particular approach used for this study have been described in Selden et al. (2019), Selden, Chappell, et al. (2021), and Selden, Mulholland, et al. (2021). To measure ¹⁵N₂ uptake, triplicate PETG bottles (0.5-2 L) were rinsed with whole water then filled, ensuring that no bubbles remained. Using a gas tight syringe (VICI Precision Sampling), ¹⁵N₂ gas (~99%, Cambridge Isotope Laboratories) was added through a flexible silicon septa. Samples were gently rocked for 15 min as described by Selden et al. (2019). Any remaining bubble was subsequently removed to ensure constant isotopic enrichment over the incubation period, and bottles were incubated on-deck for approximately 24 hours. As the vessel moved across the front, a large temperature gradient was encountered (from 16°C to 26°C). To avoid temperature-shocking the samples during transit, we maintained three on-deck incubators that recirculated water through aquaria chillers (MC-1/10HP, AquaEuroUSA) housed in custom casings (built from King Starboard plastic sheet) for protection and maintained approximate temperature conditions for shelf, frontal, and offshore waters, respectively.

At the end of the incubation period, a 6 milliliter sample aliquot was collected from each bottle and transferred to a helium-purged Exetainer^TM containing 50 μl zinc chloride (50% w/v). These samples were stored at room temperature and upside-down in 15 ml Falcon^TM tubes, submerged in ultrapure water, until analysis at the UC Davis Stable Isotope Facility. The remaining incubation volume was then filtered for initial particulate nitrogen (PN) samples. Initial and final PN isotopic composition and concentration were analyzed at Old Dominion University on a Europa 20/20 isotope ratio mass spectrometer with an automated N and carbon preparation module. Low mass samples (<10 µg N) were excluded from downstream analysis as IRMS response can become non-linear at low mass (White et al., 2020).

Specific uptake rates were calculated following Montoya et al. (1996), with equations detailed below.