Samples were collected on the& R/V Atlantic Explorer BATS Validation cruise #58 (BVal58) (cruise ID AE2121), which took place from October 11 to 25, 2021, and transited from St. George's, Bermuda to San Juan, Puerto Rico. The research group collecting these data disembarked on October 21, 2021.
Suspended POM Sampling in the Euphotic Zone (Depths of 5 - 120 meters):
Seawater was collected at 5, 40, 80, and 120 meters (m) deep using a CTD rosette fitted with twenty-four 10-liter (L) Niskin bottles (OTE). All sampling carboys were rinsed 3 times with collected seawater before filling to 8 L. Particulate Organic Matter (POM) was collected on 25-millimeter (mm) GF/F filters (0.7-micrometer (µm) pore size, Whatman, GE Healthcare) pre-combusted at 500° Celsius (C) for 4 hours. The 6 filters collected at each depth of each station resulted in duplicate samples for the Particulate Organic Carbon and Nitrogen (POC/N), Particulate Organic Phosphorus (POP), and Particulate Chemical Oxygen Demand (PCOD) assays. After filtering 8 L, the filters for the POP assay were rinsed with 5 milliliters (mL) of 0.17 molar (M) NaSO₄ to remove dissolved inorganic phosphorus, and the filters for the PCOD assay were rinsed with 5 mL of deionized water to remove chloride ions. All filters were folded in half after filtration, sealed inside pre-combusted aluminum foil (500°C for 4 hours), and stored at -80°C. Post-assays, we accounted for POM on blank filters by subtracting the average value of 10 dry blanks. The dry blanks were pre-combusted filters that had not been used for filtering.
Suspended POM Sampling in the Disphotic Zone (Depths of 150 - 1000 m):
McLane WTS-LV pumps were lowered to depths of 150, 200, 300, 400, 500, and 1000 m and each directly filtered 557 to 821 L of seawater through a 142 mm diameter pre-combusted GF/F filter (0.7 µm pore size, Whatman, GE Healthcare). After recovering the McLane pumps, the filters were similarly folded in half, sealed with pre-combusted aluminum foil, and stored at -80°C. Post-cruise, we hole-punched the 142 mm diameter filters in 15 places with a carbon-steel 18 mm hole-puncher that had been pre-combusted (500°C for 4 hours). Taking into account the O-ring, each hole punch equaled 3.8% of the total filter area containing POM. One to four chad pairs served as one replicate sample depending on the sensitivity of the assay. Two to four replicate samples were made for each assay. Dry blanks were prepared by hole-punching a pre-combusted, but unused 142 mm GF/F filter.
Particulate Organic Carbon and Nitrogen (POC/N) Assay:
POC/N filters were processed using a JGOFS protocol (Ducklow & Dickson, 1994). Filters for the POC/N assay were dried at 55°C for 24 hours. Filters were then placed in a desiccator with 12 M hydrochloric acid for 24 hours to remove inorganic carbonates. Filters were then re-dried for a minimum of 48 hours at 55°C. After drying, the filters were folded and pelletized into pre-combusted tin capsules (CE Elantech, Lakewood, NJ). Each tin-wrapped sample was analyzed in a FlashEA 1112 Elemental Analyzer using the NC Soils setup (Thermo Scientific, Waltham, MA). Known masses of atropine and acetanilide were used as standards for each run. The minimum detection limits for carbon and nitrogen were 2.4 micrograms (µg) and 3.0 µg respectively.
Particulate Organic Phosphorus (POP) Assay:
We quantified particulate organic phosphorus using an ash-hydrolysis method presented by Lomas et al. (2010). Filters were placed in autoclaved glass vials with 2 mL of 0.017 M MgSO₄, covered with pre-combusted aluminum foil (500°C for 4 hours), and then combusted at 500°C for 2 hours. 5 mL 0.2 M HCl was added and incubated at 80° to 90°C for 30 minutes. After cooling, the solution was poured into a glass centrifuge tube. The glass vial was rinsed with 5 mL of deionized water, which was then poured into the same centrifuge tube. A mixed reagent of 0.0243 M ammonium molybdate tetrahydrate, 5 N sulfuric acid, 0.004 M potassium antimonyl tartrate, and 0.3 M ascorbic acid (2:5:1:2) was added to each tube before being set in the dark for 30 minutes. Tubes were then centrifuged at 4000 rotations per minute (rpm) and quantified at 885 nanometers (nm) with a spectrophotometer using a potassium monobasic phosphate standard (1.0 mM-P). The minimum detection limit was 0.3 µg for phosphorus.
Particulate Chemical Oxygen Demand (PCOD) Assay:
The PCOD assay is a wastewater assay that has been modified by Moreno et al. (2020) to accurately quantify oxygen needed to fully oxidize organic carbon on GF/F filters. Note that because dichromate does not oxidize ammonium, this assay does not quantify oxygen demand for nitrification. Prior to the assay, the filters were dried at 55°C for at least 24 hours. For analysis, filters were added into HACH COD HR+ reagent vials (Product no. 2415915 containing mercuric sulfate) with 2 mL of milli-Q water. Vials were digested at 150°C for 2 hours, then 92.1 μL of 0.163 M NaCl was added to induce even precipitation of silver chloride. Vials were then inverted twice and centrifuged for 30 minutes at 2500 rpm. The absorbance of each vial was measured at 600 nm using a spectrophotometer. The oxygen demand was quantified using a standard curve made from HACH certified phthalate-based standards. The minimum detection limit was 3.1 µg for oxygen demand.
Calculating Ratios and Σ-O₂:
Elemental ratios (micromolar:micromolar (µM:µM)) were quantified using mean concentrations from the same depth and station. We quantified the total oxygen demand for complete remineralization (Σ-O₂) as PCOD plus double PON in units of micromolar.