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).
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