Seawater was transferred to a 20 L carboy that were rinsed three times with water from the sampling depth and then filled with seawater from three Niskin bottles, using silicone tubing that had been acid washed then rinsed with distilled water prior to use. From each carboy, water was dispensed into smaller glass containers that were cleaned and pre-rinsed three times with water from the carboy prior to dispensing. This water was used to measure cell counts, bacterial productivity, and the activities of polysaccharide hydrolases, peptidases, and glucosidases. A separate glass Duran bottle was filled with seawater from the carboy and sterilized in an autoclave for 20-30 minutes to serve as a killed control for microbial activity measurements.

Bacterial cells were counted by flow cytometry following the procedure described in Gasol and Del Giorgio [2000]. Water samples (2 mL) were fixed with 0.1% glutaraldehyde (final concentration) for 10 min at room temperature in the dark, and stored frozen at -80°C. Prior to analysis, thawed samples were pipetted through a cell strainer (Flowmi, 70 m porosity) and stained with SYBR Green I for 15 min on ice in the dark. Counts were performed on a FACSCalibur flow cytometer (Becton-Dickson) using fluorescent microspheres (Molecular Probes) of 1 µm in diameter as internal size standard. Cells were enumerated according to their green fluorescence and right angle scatter using the FloJo 7.6.1 software.

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).

Following DOC sampling, nutrient samples were analyzed for colorimetric determination of ammonia, nitrate, nitrite, and soluble reactive phosphorous [Grasshoff and Kremling, 1999]. Samples were syringe filtered using a 60 cc syringe through 0.2 μm syringe filters (Sartorius Minisart 17823) into 50 mL Falcon tubes and immediately frozen at -20°C aboard ship. A 15 mL subsample was sent to the LSU-College of the Coast & Environment, Wetland, Biogeochemistry, Analytical Services (WBAS) facility for analysis. 

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.