Dataset: EN556 LV incubations: DOC, bacterial cell concentration, and bacterial production
Deployment: EN556

For mesocosm (large volume) incubation experiments (referred to as “LV” incubations), a 30L Niskin bottle rosette was used to collect the water. From each depth, 20L seawater from single Niskin bottles was dispensed using cleaned silicon tubing into a single carboy. Prior to filling, carboys were rinsed 3x with water from the same Niskin bottle used to fill the carboy. Six carboys were filled at each depth. Triplicate 20L carboys were amended with ca. 500 mg (exact mass was recorded for each addition) of HMW Thalassiosira; unamended triplicate carboys were used for controls. All mesocosms were incubated in the dark at near in-situ temperatures. Mesocosms were sub-sampled at the start of incubation, and then after 2.5 d, 8d, 15d, 28d, and 69d for the following assays: bacterial production using 3H-Leucine, dissolved organic carbon (DOC), nutrients, bacterial cell counts, peptidase, and glucosidase activity measurements. At the 15d timepoint, sub-samples were also taken to measure polysaccharide hydrolase activities.

Samples were analyzed for nutrients and DOC content modified after Grasshoff and Kremling [1999]. Clean and acid washed syringes, tubing, and filter holders were used for each sampling. Duplicate DOC samples were filtered using the same 60 cc syringe through combusted glass fiber filters (Whatman 1825-025) secured within a polycarbonate filter holder into two combusted 20 mL scintillation vials and acidified using 100 μL of 50% phosphoric acid then immediately frozen at -20°C.  DOC samples were analyzed by high-temperature catalytic oxidation (HTCO) using a Shimadzu Total Organic Carbon analyzer (TOC-8000A/5050A).

Bacterial protein production was measured from 3H-leucine incorporation by heterotrophic bacteria using the cold trichloroacetic acid (TCA) and microcentrifuge extraction method [as in Kirchman, 2001]. All work was performed aboard ship. In brief, triplicate live samples of 1.5 mL seawater as well as one 100% (w/v) TCA-killed control were incubated with 23 μL of L-[3,4,5-3H(N)]-Leucine (PerkinElmer, NET460250UC) for between 4 and 24 hours in the dark at as close to in situ temperature as possible. Live samples were then killed with 89 μL of 100% (w/v) TCA and centrifuged (10,000 rpm at 4°C for 10 min) to pelletize cell material.  The supernatant liquid was removed and 1 mL of 5% (w/v) TCA solution was added, followed by vortex mixing and centrifugation. Supernatant removal, mixing, and centrifugation were repeated using 1 mL of 80% ethanol solution.  Finally, the supernatant liquid was removed and each sample was dried overnight.  After drying, 1 mL of scintillation cocktail (ScintiSafe 30% Cocktail, Fisher SX23-5) was added and incorporated radioactivity was measured using an LSA scintillation counter (PerkinElmer Tri-Carb 2910TR). Leucine incorporation rate was calculated from the incorporated radioactivity, compared to 1 mL of scintillation cocktail spiked with 23 μL of L-[3,4,5-3H(N)]-Leucine radioactivity, divided by incubation time.