Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
  • Problem Description
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Samples were taken at Ocean Station Papa, Subarctic North Pacific Ocean.  Sampling depth during cruise RR1813 was 3 m. The culture was isolated from a sample taken at approximate location (lat:50, lon:-145). Bathycoccus cells were isolated using a 10 mL pipette after whole seawater sampling from a trace metal clean rosette aboard the R/V Revelle.  Cultures were transported back to the University of North Carolina at Chapel Hill and stored in Aquil media under constant 120-150 µmol photons m-2 d-1 light in a 12ºC incubator.

Cultures were grown with either 1.37x10-6 M total Fe ("Fe+")  or 3.10x10-9 M total Fe ("Fe-") media.  Upon reaching exponential phase growth (based on trends in relative fluorescence units as measured by a Turner fluorometer),  cultures were filtered for chlorophyll a concentration, particulate carbon/particulate nitrogen, and RNA. Additional samples for cell count were collected and fixed with a 10% addition of paraformaldehyde. Measurements of the maximum quantum yield of photochemistry in photosystem II (Fv/Fm) and the functional absorption cross section of photosystem II (sigma) were collected. Filters used in chlorophyll and carbon/nitrogen sampling were uncombusted and combusted Whatman 0.7µm glass fiber filters, respectively.

For RNA sampling, Isopore 0.4 µm 47 mm polycarbonate filters were used. A RNAqueous-4PCR kit and RNeasy MinElute Clean-up kit were used for RNA extraction and clean-up. 

Chlorophyll was analyzed at the University of North Carolina on a Turner 10AU fluorometer following the protocol of Graff and Rynearson (2011). Particulate carbon/particulate nitrogen was analyzed on an elemental analyzer at the University of Maryland Center for Environmental Science Application Laboratory.  Cell counts were measured at North Carolina State University on a Guava easyCyte flow cytometer equipped with a red laser. RNA was analyzed at the Azenta Genewiz Sequencing Facility via Illumina high throughput sequencing with 350M raw paired-end reads per lane and single index, 2 x 150 bp per lane. 

RNA transcripts were processed according to the pipeline outlined in https://github.com/omtorano/Exports-Metatranscriptomics (Author: Olivia Torano). Programs for processing include Trim_galore (v.0.6.2), FastQC (v.0.11.9), Trinity (v.2.8.6), cdhit (v.4.8.1), Salmon (v.10.9.1), tximport, and the DESeq2 package in RStudio. Sequences were blasted to the Kyoto Encyclopedia of Genes and Genomes (Release 88.2). 

* Renamed parameters to comply with database requirements
* Added cruise ID and apporximate sampling location to data

NSF abstract:
The subtropical oceans in the middle latitudes are “ocean deserts,” where there are relatively little nutrients in the surface waters. Nitrogen is an important nutrient that is particularly scarce. Nitrogen in the ocean occurs as nitrate or ammonium. As a result of limited nutrients in the subtropical gyres, marine algae or phytoplankton there grow slowly. In deeper waters below the surface, there are more nutrients. However, it is not well understood how phytoplankton living in surface waters in summer can access the nutrients in the deeper waters. This award will investigate how phytoplankton access subsurface nitrogen at Station ALOHA in the North Pacific Subtropical Gyre (NPSG). Phytoplankton will be sorted by size and pigment composition. The researchers will make measurements of the stable nitrogen isotope ratios of sorted populations and determine where phytoplankton are getting the needed nutrients, either nitrate from the subsurface or ammonium from the surface waters. The researchers will use metatranscriptomic analysis to investigate the physiological pathways that different phytoplankton populations use to take up the needed nitrogen. The metatranscriptomic analysis involves analyzing the RNA sequences transcribed by plankton, comparing the sequence to documented gene sequences and mapped to documented physiological pathways. The researchers will assess which taxonomic groups are using nitrate versus recycled sources of nitrogen. They will test the hypothesis that eukaryotic phytoplankton primarily use nitrate whereas prokaryotic phytoplankton mostly use recycled sources of nitrogen. With these measurements, the researchers will help the society anticipate climate impacts on the productivity of subtropical gyres. This project will support two graduate students and a post-doctoral researcher in laboratory and ship-based research. Principal Investigator (PI) Granger in Connecticut will engage high school students and sponsor graduate students from underrepresented groups via AGU’s Bridge Partner Program. PI Marchetti in North Carolina will sponsor the internship of a high school student from a local minority county. PI White in Hawaii will engage with Hawaiian high school students though the Indigenous Partnership for Ocean Monitoring program to mentor and teach essential research skills.

The researchers of this project will investigate the drivers of productivity at station ALOHA in NPSG. They will exploit a high-sensitivity method for natural abundance N isotope analysis (the ‘persulphate-denitrifier’ method) to evaluate the extent to which taxonomically distinct components of the plankton sorted by flow cytometry rely on nitrate vs. reduced N sources in surface waters. We will also query physiological nitrogen pathways of the plankton with metatranscriptomic analysis to infer which clades are reliant on nitrate. We will quantify the fraction of primary production fueled by nitrate to characterize seasonal trends and explore potential mechanisms of nitrate supply to the euphotic zone and its mixed layer. The work proposed here will resolve the relative contribution of nitrate mixed from the ocean interior to surface production at station ALOHA, to better define seasonal dynamics of the biological pump in the NPSG. We will identify plankton groups that contribute dominantly to nitrate-based production and resolve eco-physiological strategies that confer fitness in a severely N-limited environment. Seasonal trends in the contributions of nitrate to total nitrogen production will be evaluated considering incident hydrography to infer potential mechanisms of nitrate supply.