Table of Contents

• Dataset Description
  • Methods & Sampling
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

15N Nitrogen Fixation, 13C Primary Production:
Standard protocols were used to measure in vitro primary productivity via the 13C radiotracer method [Lengendre et al., 1996] and N2-fixation rate measurements via a modified bubble release 15N2 method modified from Montoya et al [1996]. Briefly, water samples were collected before dawn in 4.3L polycarbonate bottles, a plug of 4ml of 15N2 gas was injected through a septa cap using a gas tight syringe. Bottles were then inverted for 15 minutes using a gas sample tumbler to assist in bringing the gas into solution. Following mixing the bubble was released and a Tinitial was collected for 15N2 enrichment. Bottles were then topped off using water for the same depth, sealed with caps fitted with septum and injected with 0.5ml of 47mM 13C bicarbonate stock and gently mixed by inversion. Samples for 13C-DIC to test the 13C enrichment concentration were taken by removing 1ml of enriched sample volume and injecting it into an evacuated exetainer pre-loaded with 85% phosphoric acid. Bottles were incubated on deck in incubators screened to 48% of surface irradiance and plumbed with flow through surface seawater for temperature regulation. All bottles were incubated in the incubator for ~ 24hr. Duplicate light bottles were paired with dark bottles and time final bottles that received no enrichment. Time zero bottles were also collected and sacrificed for each station. Natural abundance of dissolved 15N2 samples were also collected at each station. Following dawn to dawn incubations, a sample for Tpost 15N2 enrichment was collected from each enriched bottle and the remaining volume was filtered onto a combusted 25mm glass fiber filter, filters were folded in half and placed in combusted aluminum foil and stored at -80C for later analysis. Post-cruise, all filters were thawed and dried overnight at 60C. Samples were then balled into Ag and Sn boats and plated for analysis. The carbon and nitrogen isotopic composition (13C and 15N, respectively) were analyzed by continuous-flow isotope ratio mass spectrometry using a Carlo Erba elemental analyzer (EA) connected to a Thermo DeltaPlus isotope ratio mass spectrometer (IRMS). For all samples the international standards USGS40 (d13C = -26.389 permil vs VPDB, d15N = -4.52 permil vs air) and USGS41 (d13C = +37.626permil vs VPDB, d15N = +47.6 permil vs air) were used to calibrate each run of samples and the international standard IAEA-600 (d13C = -27.771 permil vs VPDB, d15N = +1.0 permil vs air) was used as a check standard (i.e., not used to calibrate data).13C-DIC samples were sent to UC Davis for analysis, resultant data was used to correct the 13C DIC addition for each bottle. 

Diazotroph Abundance:
At select stations where N2 Fixation measurements were conducted, the entire volume of a Niskin bottle at 25m was gravity filtered through a 47-mm diameter, 10 micron pore size, black polycarbonate filter with a polyester drain disk as a backing filter. If the volume was not filtered after 2 hours, filtration was terminated and the remaining volume of the carboy was measured in order to calculate volume filtered.  Following filtration, filter holders were fit with a short section of tubing and a syringe leur fitting on once side and a 2-way valve on the outflow side. For each filter, 5-ml of 2% glutaraldehyde was slowly injected onto the filter and samples were allowed to fix for 30 minutes. Fixative was drained after this time and 60ml of air was used to flush all filters. Polycarbonate filters were then mounted onto 3x2 glass slides with immersion oil, cover slides were added and the edges of each cover slip was sealed with quick dry nail polish. All slides were stored at -20C and counted within 30 days. Enumeration of diazotrophic taxa was performed using epifluorescence microscopy.The entire slide was counted for DDAs and Trichodesimum abundance. Endosymbiont bearing diatoms of the following genus were enumerated: Rhizosolenia, Hemiaulus, Climacodium and Chaetoceras. Free Richelia intracellularis were also counted. Trichodesmium filaments were counted and the length of each filament was recorded. Trichodesmium cell number was then calculated by dividing the filament length by the mean cell length (9.9 micron +/-2.5 micron). 

Flow Cytometry Bacteria:
Flow cytometry samples were collected from rosette nisken bottles at specific depths where 3ml of sample volume was pipette into cryovials, preserved by adding 60ul of 2% paraformaldehyde, allowed to sit in the dark for 10 minutes then stored in a -80C until analysis. Post cruise, the samples were thawed in the dark, a 250ul aliquot of sample was removed and added to 250ul Deionized water and 45ul 300mM Potassium citrate, 5ul of SYBR Green stain and 25ul of 1um beads were added, samples were run on a Becton Dickinson FACSCalibur flow cytometer using the methods adpated from Marie, et al 1997. All values are reported in cells/L, NaN are no data.

Flow Cytometry-Phytoplankton:
Flow cytometry samples were collected from rosette nisken bottles at specific depths where 3ml of sample volume was pipette into cryovials, preserved by adding 60ul of 2% paraformaldehyde, allowed to sit in the dark for 10 minutes then stored in a -80C until analysis. Post cruise, the samples were thawed in the dark, a 500ul aliquot was removed and 3um beads were added post run, samples were run for 3mins on a Becton Dickinson FACSCalibur flow cytometer using the methods outlined in Sherr, et al 2002. 

Fluorometric Chlorophyll:
Fluorometric Chlorophyll were collected from rosette nisken bottles at specific depths and filtered at low vacuum through a glass fiber filter. Samples were then flash frozen in liquid nitrogen and stored in a histoprep capsule at -80C until analysis. Post cruise, the filters were extracted using 90% acetone for a total of 48 hours in a -20C freezer, covered to prevent the photodegradation of pigments. Upon extraction samples were centrifuged to settle filter particulates and gently poured into a glass cuvette. Samples were then run on a Turner 10-AU Fluorometer. Data was corrected for average glass fiber filter blank values and where duplicate samples were taken the average and standard deviation are reported. NaN are no data, duplicates not taken.

Particulate Carbon (PC) and Particulate Nitrogen (PN):
The carbon, and nitrogen content of particulate material (PC, PN) was collected from rosette nisken bottles at specific depths and filtered at low vacuum through a combusted glass fiber filter (PCPN). Following filtration, filters for PCPN were folded in half and placed in combusted aluminum foil and stored at -80C for later analysis. Post-cruise, all filters were thawed, acidified for a minimum of 12 hours and dried overnight at 60C. Samples were then balled into Ag and Sn boats and plated for analysis. Organic carbon and total nitrogen content was analyzed using high-temperature combustion (1020 C) on a ThermoQuest NC 2500 elemental analyzer. Three standards with known %C and %N were weighed, folded in tin boats, and analyzed with the samples to develop the calibration equation for each run. The calibration equation was then used to calculate the mass (mg) of carbon and nitrogen in each sample using EAS Clarity Chromatography Station software. The standards are Cystine (Elemental Microanalysis, B2035, 11.66 %TN, 29.99 %TC), Atropine (Elemental Microanalysis, B2002, 4.84 %TN, 70.56%TC), and GoniLab Secondary Standard (low %C)-A1 (an internal standard of sediment from the Gulf of Mexico, MV0803, with low carbon - 0.12 %N, 1.3 %C). 

NSF Award Abstract:
The availability of nitrogen is required to support the growth and production of organisms living in the surface of our global ocean. This element can be scarce. To alleviate this scarcity, a special class of bacteria and archaea, called nitrogen fixers, can derive the nitrogen needed for growth from nitrogen gas. This project would carefully examine one specific method for measuring nitrogen fixation that has been used recently to suggest the occurrence of small amounts of nitrogen fixation in subsurface ocean waters. If these reports are verified, then a revision of our understanding of the marine nitrogen cycle may be needed. The Ocean Carbon and Biogeochemistry program will be used as a platform to develop community consensus for best practices in nitrogen fixation measurements and detection of diversity, activity, and abundances of the organisms responsible. In addition, a session will be organized in a future national/international conference to communicate with the broader scientific community while developing these best practices.

The goal of this study is to conduct a thorough examination of potential experimental and analytical errors inherent to the 15N2-tracer nitrogen fixation method, in tandem with comprehensive molecular measurements, in mesopelagic ocean waters. Samples will be collected and experimental work conducted on a cruise transect in the North Atlantic Ocean, followed by analytical work in the laboratory. The specific aims of this study are to (1) determine the minimum quantifiable rates of 15N2 fixation based on incubations of mesopelagic waters via characterization of sources of experimental and analytical error, and (2) seek evidence of presence and expression of nitrogen fixation genes via comprehensive molecular approaches on corresponding samples. The range of detectable rates and diazotroph activity from the measurements made in this study will be informative for the understanding of the importance of nitrogen fixation in the oceanic nitrogen budget.