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.
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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\u00b0C. 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 \u00b5m in diameter as internal size standard. Cells were enumerated according to their green fluorescence and right angle scatter using\u00a0the FloJo 7.6.1\u00a0software.
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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 \u03bcL of 50% phosphoric acid then immediately frozen at -20\u00b0C.\u00a0 DOC samples were analyzed by high-temperature catalytic oxidation (HTCO) using a Shimadzu Total Organic Carbon analyzer (TOC-8000A/5050A).
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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 \u03bcm syringe filters (Sartorius Minisart 17823) into 50 mL Falcon tubes and immediately frozen at -20\u00b0C aboard ship. A 15 mL subsample was sent to the LSU-College of the Coast & Environment, Wetland, Biogeochemistry, Analytical Services (WBAS) facility for analysis.\u00a0
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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 \u03bcL 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 \u03bcL of 100% (w/v) TCA and centrifuged (10,000 rpm at 4\u00b0C for 10 min) to pelletize cell material.\u00a0 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.\u00a0 Finally, the supernatant liquid was removed and each sample was dried overnight.\u00a0 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 \u03bcL of L-[3,4,5-3H(N)]-Leucine radioactivity, divided by incubation time.
BCO-DMO Processing Notes:
\n- added conventional header with dataset name, PI name, version date
\n- modified parameter names to conform with BCO-DMO naming conventions
\n- added columns with\u00a0lat and lon in decimal degrees
\n- added cruise_id column
\n- replaced 'NA' and blank cells with 'nd' (no data)
\n- removed the text 'cast00-' and 'stn0-' from data records for cast and station, leaving the digits in place
\n-\u00a0changed longitude to negative degrees, denoting West
\n-\u00a0reduced\u00a0the decimal\u00a0precision of temp to 2
\n-\u00a0removed from display the following column with no data:\u00a0 Leu_3H_rep_3