Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Nutrients, bacterial cell counts, and bacterial protein production as measured by ³H-leucine incorporation from the 'bulk water experiments'. Water was collected on EN556. See Niskin Bottle and Cast List EN556 to link specific casts and bottles to each experiment: https://www.bco-dmo.org/dataset/717427.

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.

BCO-DMO Processing Notes:
- added conventional header with dataset name, PI name, version date
- modified parameter names to conform with BCO-DMO naming conventions
- added columns with lat and lon in decimal degrees
- added cruise_id column
- replaced 'NA' and blank cells with 'nd' (no data)
- removed the text 'cast00-' and 'stn0-' from data records for cast and station, leaving the digits in place
- changed longitude to negative degrees, denoting West
- reduced the decimal precision of temp to 2
- removed from display the following column with no data:  Leu_3H_rep_3

NSF Award Abstract:
Heterotrophic microbial communities are key players in the marine carbon cycle, transforming and respiring organic carbon, regenerating nutrients, and acting as the final filter in sediments through which organic matter passes before long-term burial. Microbially-driven carbon cycling in the ocean profoundly affects the global carbon cycle, but key factors determining rates and locations of organic matter remineralization are unclear. In this study, researchers from the University of North Carolina at Chapel Hill will investigate the ability of pelagic microbial communities to initiate the remineralization of polysaccharides and proteins, which together constitute a major pool of organic matter in the ocean. Results from this study will be predictive on a large scale regarding the nature of the microbial response to organic matter input, and will provide a mechanistic framework for interpreting organic matter reactivity in the ocean.

Broader Impacts: This study will provide scientific training for undergraduate and graduate students from underrepresented groups. The project will also involve German colleagues, thus strengthening international scientific collaboration.