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.

Chemoautotropic production. Samples used for determining chemoautotrophic production were held in the dark at 0°C for no longer than 6 hours prior to being amended with 14C bicarbonate. Chemoautotrophy was determined by measuring the incorporation into organic matter of 14C supplied as NaH14CO₃. NaH14CO₃ (5 mCi) was diluted into 25 mL of MilliQ water made basic (pH ≈9) using NaOH. This stock solution (0.2 uCi/uL) was passed through a syringe filter (Acrodisk, 0.22 pore size) into a 30 mL polycarbonate bottle and stored at 4°C. Water from the sample depth was collected directly from the appropriate Niskin sampler into aged, acid-washed 250 mL screw-cap amber HDPE bottles (3 rinses) filled to the top (volume ~270 mL). Each experiment used two replicate treatments and a control bottle. Controls consisted of either 0.22 um filtered water from the Sterivex filtration of the same sample or of whole water that was incubated along with the 14C amended treatment, except that no 14C was added until immediately before filtering the set. Each bottle received ~ 20 uCi of NaH14CO₃ (100 uL of the working stock). Label was added in a darkened lab van illuminated with a dim, red-filtered light. Samples were mixed by inverting gently then placed in a water-ice bath contained in an ice chest wrapped in aluminum foil contained in a black 3 mil plastic garbage bag. Incubation temperature was maintained by adding ice as needed, which led to departures to above the desired incubation temperature for some stations. Water temperature in the bath was recorded at 5-minute time steps with HOBO TidBit loggers and these samples have been flagged. Lights were kept off except when working in the lab van, when red light was used to illuminate the working area.

See the attached supplemental files for detailed data on the water bath temperature. "LMG1801_Water_Bath_Temperature_Plot.pdf" contains a figure plotting the bath temperature, the measured chemoautotrophy value for each station and depth, and the intervals of the incubations by station. "Water_Bath_Temperature_Data.xlsx" contains the data used to create this plot.

At the end of the incubation (~48 hours) the bottles were removed from the ice bath, triplicate samples of 100 uL were taken from the filtered control (or from one of the treatments in later experiments) and radioassayed to verify the amount of tracer added. The remaining sample and all of the treatment samples were filtered through 25 mm diameter, 0.22 um pore size membrane filters (GSWP Millipore) under dim red light. Filters were rinsed two times with filtered seawater, removed from the filter holder into scintillation vials, then 100 uL of 10% HCl was added to each vial, soaking the filter in the process. After 24 hr in the fume hood (uncapped) to allow excess water and acid to volatilize, vials received 4 mL of Ultima Gold scintillation cocktail, then were counted in a Perkin-Elmer LSC.