Dataset: PUPCYCLE 2019 incubation experiments
Deployment: OC1905B

For the Fe status incubations:
Seawater was collected using trace-metal clean techniques from a depth of 90 meters (m) (corresponding to the 8.4 degree Celsius isotherm) for the wide shelf incubation and 80 m (corresponding to the 8.9 degree Celsius isotherm) for the narrow shelf incubation. Seawater from both sites was pumped directly into a positive pressure trace-metal clean plastic bubble created in the ship’s laboratory into large, 50-gallon acid-washed high-density polyethylene (HDPE) drums to homogenize the seawater using a Wilden air-operated double-diaphragm pump made of polytetrafluoroethylene and acid-washed HDPE tubing. Preparation of the cubitainers was carried out prior to the cruise and included trace-metal clean techniques. Cubitainers were initially soaked in 2% Extran detergent for 7 days, then rinsed with deionized water four times and Milli-Q water three times prior to being soaked in 10% reagent grade hydrochloric acid for 2-3 days and rinsed with Milli-Q water. Subsequently, the cubitainers were soaked in 1% trace metal grade hydrochloric acid for 7 days and rinsed with Milli-Q water, then soaked in 0.1% trace metal grade acetic acid 3-4 days before final storage in low Fe water from Station P (50 degrees North,145 degrees West). Station P water had been collected and filtered from the 2018 EXPORTS North Pacific field campaign using trace metal clean techniques and stored in the dark at 4 degrees Celsius (°C) until use. In the bubble, these triplicate acid-cleaned 10-liter (L) low-density polyethylene cubitainers were filled and then incubated in large on-deck plexiglass incubators circulated with water chilled to the temperatures at which the subsurface samples were collected using Aqua Logic Delta Star® In-Line Water Chillers. Incubators were covered with neutral density screening to achieve 30% incident irradiance. To assess the effects of Fe addition or removal on the simulated upwelled plankton communities, samples were incubated with no amendment (control), amended with 5 nanomolar (nM) of FeCl2 (Fe treatment), or amended with 200 nM desferrioxamine B (DFB), a strong Fe chelator, which inhibits dissolved Fe uptake (DFB treatment). Three cubitainers were immediately harvested for the initial time- point (T0), also referred to as deep-water or DW. The remaining cubitainers were incubated for two additional time points for a total of 18 cubitainers per incubation. Cubitainers were harvested for various biological and chemical parameters (see below) following 48 hours (T1) for both incubation experiments, and following 120 hours in the wide shelf incubation and 96 hours in the narrow shelf incubation (T2).

For the seed population incubations:
Seawater was collected using trace-metal clean techniques at 15 m depth for the surface incubations and at 90 m for the subsurface incubations (corresponding to the 8.4 °C isotherm). Seawater was pumped directly into a positive pressure trace metal clean plastic bubble created in the ship's laboratory into large, 50-gallon acid-washed high-density polyethylene (HDPE) drums to homogenize the seawater using a Wilden air-operated double-diaphragm pump made of polytetrafluoroethylene (PTFE) and acid-washed HDPE tubing. Preparation of the cubitainers was carried out prior to the cruise and included trace-metal clean techniques (Crawford et al., 2003): 10 L cubitainers were initially soaked in 2% Extran detergent for 7 days, then rinsed with deionized water four times and Milli-Q water three times prior to being soaked in 10% reagent grade hydrochloric acid for 2-3 days and rinsed with Milli-Q water. Subsequently, the cubitainers were soaked in 1% trace metal grade hydrochloric acid for 7 days and rinsed with Milli-Q water, then soaked in 0.1% trace metal grade acetic acid 3-4 days before final storage in low-Fe water from Ocean Station Papa (St. P). The St. P water had been collected and filtered from the 2018 EXPORTS North Pacific (50 °N, 145 °W) field campaign using trace metal clean techniques and stored in the dark at 4 °C until use. In the bubble, these triplicate acid-cleaned 10 L low-density polyethylene (LDPE) cubitainers were filled and then incubated in large on-deck plexiglass incubators circulated with water chilled to the temperatures at which the subsurface samples were collected using Aqua Logic Delta Star® In-Line Water Chillers. Incubators were covered with neutral density screening to achieve 30% incident irradiance. For the experimental design, a portion of the surface and subsurface collections was directly collected and denoted as the initial (T0) surface and subsurface communities, respectively. The resulting incubations (triplicates) were characterized by either the surface waters (SW) or the subsurface waters (DW) incubated for 96 hours (T1). Another portion (triplicates) was subjected to mixture with filtered waters from the respective subsurface or surface communities, such that the 96 hour incubations consisted of an equal mixture of filtered-subsurface waters and surface waters, denoted as FDW/SW (filtered deep water/surface water), an equal mixture of filtered-surface waters and subsurface waters, denoted as FSW/DW (filtered surface water/deep water), and an equal mixture of both unfiltered surface and subsurface waters, denoted as mixed (surface water/deep water). No additional macronutrients or micronutrients were added to any of the incubations. Cubitainers were harvested for various biological and chemical parameters (see below) in the initial time point and following 96 hours of incubation.

Physiological Measurements:
For chlorophyll a measurements, 250 milliliters (mL) of seawater was gravity filtered through 5-micrometer (μm) Isopore membrane filters (47-millimeters (mm)) and subsequently vacuum filtered onto GF/F (25 mm) filters under 100 mmHg of vacuum pressure. Filters were then rinsed with 0.45 μm filtered seawater and immediately stored at -20 °C until onshore analysis in the lab. Chlorophyll a extraction was performed using a 90% acetone solution at -20 °C for 24 hours and measured on a 10-AU fluorometer (Turner Designs, San Jose, CA) using the acidification method.

Dissolved inorganic nutrients (nitrate + nitrite, phosphate, and silicic acid) were measured by filtering 30 mL of water through a 0.2 µm filter, using acid-washed syringes into an acid-cleaned polypropylene FalconTM tube. Dissolved nutrient concentrations were analyzed using an OI Analytical Flow Solutions IV auto analyzer by Wetland Biogeochemistry Analytical Services at Louisiana State University. Concentrations of nitrate measured from the discrete samples were used for the calculation of absolute nitrate uptake rates.

Samples for dissolved iron (dFe) were collected from each cubitainer within a trace-metal clean, positive pressure plastic bubble by filtering through pre-cleaned 0.2 μm pore size polyethersulfone membrane Acropak-200® capsule filters into LDPE bottles that had been rigorously cleaned as described in the GEOTRACES cookbook. Sample bottles were rinsed three times with sample before filling. Samples were acidified at sea to pH ~1.7 with optima HCl (2 mL of 12 M HCl per liter of seawater) and were analyzed after the cruise. Briefly, this method involves pre-concentration onto Nobias-chelate PA1 resin followed by analysis with a High-Resolution Inductively Coupled Plasma Mass Spectrometer. For quality control, a few samples were rerun with a flow injection analysis method. This method involves pre-concentration on toyopearl resin followed by in-line spectrophotometric analysis.

To assess the isotope uptake of DIC and NO3, a 618 mL polycarbonate bottle was filled to the top with incubation samples from each cubitainer. The subsamples were collected for dissolved inorganic carbon (DIC) and nitrate uptake rates at each respective time point, and immediately spiked with both NaH13CO3 and Na15NO3 at approximately 10% of the estimated ambient DIC concentrations and measured nitrate concentrations in a trace metal clean (TMC) space located on the ship (see below). Since there were no underway measurements of DIC, an expected ambient concentration of 2000 micromoles per liter (μmol L-1) was derived from literature values for both incubation sites. Nitrate concentrations of the deep-water and surface water in both incubation sites were measured using the Submersible Ultraviolet Nitrate Analyzer (SUNA), which quantifies dissolved nitrate concentrations by illuminating the water sample with UV and using the absorbance values to estimate nitrate concentrations from a multi-variable linear regression based on the MBARI method. After spiking with respective trace concentrations of both stable isotopes of DIC and nitrate, samples were returned to the incubator for six hours. All rate measurement incubations were initiated at approximately the same time of day (i.e., close to dawn). Following incubation, seawater was immediately filtered where cells greater than 5 μm were gravity filtered onto 5 μm Isopore membrane filters (47 mm) and then washed onto a pre-combusted (450 °C for 5 hours) GF/F filters (0.7 μm nominal porosity) by vacuum filtration using 0.2 μm filtered seawater and preserved at -20 °C. Cells smaller than 5 μm passed through the initial 5 μm Isopore membrane filters and were collected on pre-combusted GF/F filters and preserved at -20 °C. Prior to analysis, filters were dried at 60 °C for 24 hours, encapsulated in tin and pelletized. Particulate organic carbon (POC), particulate organic nitrogen (PON), and atom percentages of 13C and 15N were subsequently quantified using an isotope ratio mass spectrometer (EA-IRMS) at the UC Davis Stable Isotope Facility. For each sample, POC and PON concentrations (μmol L-1) were calculated by dividing the measured POC/PON mass (micrograms (μg)) by the respective atomic mass of carbon and nitrogen over the volume filtered (0.62 L).

For the wide shelf incubation, samples were spiked with 200 μmol L-1 of NaH13CO3 and 1 μmol L-1 of Na15NO3 according to estimated ambient DIC concentrations of 2000 μmol L-1 and measured surface nitrate concentrations of 10 μmol L-1. For the narrow shelf incubation, samples were spiked with 200 μmol L-1 of NaH13CO3 for all samples and 1.5 μmol L-1 of Na15NO3 for the deep-water triplicate samples. This amount was amended according to estimated ambient DIC concentrations of 2000 μmol L-1 and measured nitrate concentrations of 15 μmol L-1 in the deep-water. Absolute uptake rates of dissolved inorganic carbon and nitrate (ρ, DIC or NO3 taken up per unit time) were derived from the constant transport model. The 13C fraction was first calculated by dividing the measured 13C atom percentage in each sample by the natural 13C atom percentage (1.087%), over the difference between the percentage of 13C over total C in the sample and the natural 13C atom percentage. The 13C biomass was then derived by multiplying the 13C fraction by the calculated POC concentration for each sample, and 13C absolute uptake rate was measured as the 13C biomass over the incubation time (6 hours). The same calculations were applied to assess the 15N absolute uptake rates, but with the respective natural atom percentage of 15N (0.367%). Biomass-normalized uptake rates (V, DIC or NO3 taken up per unit POC or PON, respectively, per unit time) were derived from the specific uptake model, and was assessed by dividing the absolute uptake rates of DIC or NO3 by the measured POC and PON concentrations (μmol L-1), respectively. Correction for ammonium regeneration during the incubation period was not performed.