Bacterial protein production on particles obtained by gravity filtration of water collected on R/V Endeavor EN556 (Patterns of activities project) from 2015-04-27 to 2015-05-02 (NCEI Accession 0278168)

Acquisition Description:
Seawater was transferred to 20 L carboys that were rinsed three times with water from the sampling depth. Experiments on (operationally defined) particles were carried out by gravity-filtering water through 3 um pore size filters. Fractions of the filters (1/8 of each filter) was incubated in autoclaved seawater from the same depth/station; bacterial protein production was calculated to account for the volume of seawater that had passed through the filter.

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 uL 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 uL 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 uL of L-[3,4,5-3H(N)]-Leucine radioactivity, divided by incubation time.