Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Sample Collection. Samples were collected on the Antarctic continental shelf and slope west of the Antarctic Peninsula within the PAL-LTER sampling domain (http://pal.lternet.edu/) during summer (cruise dates 30 Dec 2017 through 12 Feb 2018; sampling dates 5 Jan to 4 Feb 2018) from the ARSV Laurence M Gould (LMG 1801, PAL-LTER cruise 26, DOI: 10.7284/907858). Sampling focused on three or 4 depths at each station chosen to represent the Antarctic Surface Water (ASW, 0 -34 m depth), the Winter Water (WW, the water column temperature minimum, generally between 35 and 174 m) the Circumpolar Deep Water (CDW, 175-1000 m) and slope water (SLOPE, >1000 m, generally ~10 m above the bottom at deep stations on the slope, 2500-3048m). Water samples were collected from Niskin bottles (General Oceanics Inc., Miami, FL, USA) into opaque 2 L HDPE plastic bottles or into aged, acid-washed, sample-rinsed 250 ml polycarbonate bottles (Nalge) completely filled (~270 mL) directly from Niskin bottles as soon as possible after the rosette was secured on deck. Subsequent processing took place in an adjacent laboratory.

Samples for DNA analysis were taken from the 2 L opaque HDPE bottles and were filtered under pressure through 0.22 um pore size Sterivex GVWP filters (EMD Millipore, Billerica, MA, USA) using a peristaltic pump. Residual seawater was expelled from the filter using a syringe filled with air, then ~1.8 ml of lysis buffer (0.75 M sucrose, 40 mM EDTA, 50 mM Tris, pH 8.3) was added to the filter capsule, which was capped and placed in a -20 °C freezer. The frozen samples were aggregated into Ziploc Freezer Bags and transferred to a -80 °C freezer for the remainder of the cruise and for shipping to the laboratory.

Two samples of the Sterivex filtrate (40 mL each into new 50 mL disposable centrifuge tubes, VWR, rinsed 3x with sample) were frozen immediately at -20 °C, then aggregated into Ziploc Freezer Bags and transferred to a -80 ° freezer for the remainder of the cruise and for shipping to the laboratory. These were used for subsequent determination of 1) urea concentration and 2) the natural abundance of ¹⁵N in the nitrite plus nitrate pools (¹⁵NOₓ hereinafter). An additional sample of the Sterivex filtrate was stored in a polycarbonate bottle at 4 °C for subsequent onboard determination of ammonia concentration by the Holmes et al (1999) o-phthaldialdehyde method and nitrite concentration by the diazo-coupling method (Strickland and Parsons 1972). Technical difficulties encountered during onboard analysis resulted in the loss of ammonium and nitrite data for some samples.

Samples for DNA and chemical analyses were shipped on dry ice from Punta Arenas, Chile to the Hollibaugh laboratory at the University of Georgia. Upon arrival they were stored in a -80 °C freezer until analyzed. Samples for ¹⁵N analysis were shipped on dry ice from Punta Arenas, Chile to the Popp laboratory at the University of Hawaii. Upon arrival they were stored in a -40 °C freezer until analyzed.

Chemical analyses. Urea content was determined by the diacetyl monoxime method (Rahmatullah and Boyde 1980, Mulvenna and Savidge 1992). Subsamples from samples that were also used to determine oxidation of ¹⁵N supplied as putrescine were sent to Dr. X. Mou's laboratory at Kent State University where they were analyzed to determine polyamine and DFAA content as described previously (Lu et al 2014).

Analysis of gene abundance. DNA was recovered from Sterivex filters using a lysozyme and proteinase K digestion, followed by purification using a phenol–chloroform extraction following Tolar et al. (2013). Abundances of Archaea and Bacteria genes were determined by quantitative PCR (qPCR) used an iCycler CFX Connect Real-Time PCR Detection System (BioRad) using either SYBR Green I dye (BioRad, Hercules, CA, USA) or TaqMan (Applied Biosystems, Carlsbad, CA, USA) chemistries following published protocols (Kalanetra et al., 2009; Tolar et al., 2013) and the primers and probes listed in the attached Supplemental Files ("Primer_Specs.pdf" or "Primer_Specs.xlsx"). Reactions were set up in triplicate and analyzed against a range of standards (10¹–10⁷ copies per ul) as described in Tolar et al. (2013). qPCR conditions for Archaea amoA, ammonia-oxidizing Betaproteobacteria amoA and Bacteria 16S rRNA (rrs) genes have been described previously (Kalanetra et al., 2009; Tolar et al., 2013). Thaumarchaeota ureC genes were quantified under the same conditions as amoA, with an annealing temperature of 53 °C (from Alonso-Sáez et al., 2012). Nitrospina rrs genes were quantified as in Mincer et al. (2007). Raw abundance data (copies per ul of DNA extract) were converted to concentrations of genes (copies per liter of seawater) using the volume filtered and the extract volume and assuming 100% extraction efficiency as in Tolar et al. (2013). The percent of total prokaryotes represented by Thaumarchaeota was calculated using rrs gene abundance (Bacteria plus Thaumarchaeota) determined by qPCR and corrected using an average of 1.8 Bacteria rrs genes per genome (Biers et al., 2009) and 1.0 Thaumarchaeota rrs gene per genome, as described previously (Kalanetra et al., 2009). Prokaryote abundance determined by qPCR correlated well with total prokaryote counts made with a flow cytometer by Palmer LTER personnel during LMG 18-01 (model II regression: n=78, r² = 0.45, p<<0.0001; slope = 0.65, 95% CL = 0.49 – 0.82; intercept = 0.36, 95% CL = 0.31 - 0.41 * 10⁹ cells L⁻¹).

- replaced 'NAN' with 'nd' as missing data identifier;
- renamed fields to comply with BCO-DMO naming conventions;
- corrected dates where month and day were reversed;
- converted cast start date/time field to ISO8601 format;
- 2021-03-17: revised/updated the Methods & Sampling section of the metadata;
- 2021-04-08: replaced data file with copy received on 2021-03-18.

NSF Award Abstract:
Part 1: The project addresses fundamental questions regarding the role of nitrification (the conversion of ammonium to nitrate by a two-step process involving two different guilds of microorganisms: ammonia- and nitrite-oxidizers) in the Antarctic marine ecosystem. Specifically, the project seeks to evaluate the contribution of primary production supported by the energy in nitrogen compounds to the overall supply of organic carbon to the food web of the Southern Ocean. Previous measurements indicate that nitrification could contribute about 9% to primary production supporting the Antarctic food web on an annual basis, but those measurements did not include the additional production associated with nitrite oxidation. Additionally, the project will aim to determine the significance of the contribution of other sources of nitrogen, (specifically organic nitrogen and urea released by other organisms) to nitrification because these contributions may not be assessed by standard protocols. Such work will aid in better understanding the basis of the energy for the Antarctic marine ecosystems on an annual basis as well as better aid in understanding the energetics of the ecosystem in times and places where primary production based on light energy is limited (i.e. during the polar night or under sea ice cover).

This project will result in training a postdoctoral researcher and provide undergraduate students opportunities to gain hand-on experience with research on microbial geochemistry. The Palmer Long Term Ecological Research (LTER) activities have focused largely on the interaction between ocean climate and the marine food web affecting top predators. Relatively little effort has been devoted to studying processes related to the microbial geochemistry of nitrogen cycling, yet these are a major themes at other LTER sites. This work will contribute substantially to understanding an important aspect of nitrogen cycling and bacterioplankton production in the study area. The team will be working synergistically and be participating fully in the education and outreach efforts of the Palmer LTER, including making highlights of the findings available for posting to their project web site and participating in any special efforts they have in the area of outreach.

Part 2: The proposed work will quantify oxidation rates of 15N supplied as ammonium, urea and nitrite, allowing the estimation of the contribution of urea-derived N and complete nitrification (ammonia to nitrate) to chemoautotrophy and bacterioplankton production in Antarctic coastal waters. The project will compare these estimates to direct measurements of the incorporation of 14C into organic matter in the dark for an independent estimate of chemoautotrophy. The team aims to collect samples spanning the water column: from surface water (~10 m), winter water (50-100 m) and circumpolar deep water (>150 m); on a cruise surveying the continental shelf and slope west of the Antarctic Peninsula in the austral summer of 2018. Other samples will be taken to measure the concentrations of nitrate, nitrite, ammonia and urea, for qPCR analysis of the abundance of relevant microorganisms, and for studies of related processes. The project will rely on collaboration with the existing Palmer LTER to ensure that ancillary data (bacterioplankton abundance and production, chlorophyll, physical and chemical variables) will be available. The synergistic activities of this project along with the LTER activities will provide a unique opportunity to assess chemoautotrophy in context of the overall ecosystem's dynamics- including both primary and secondary production processes.