3H-leucine and thymidine incorporation of North Atlantic subseafloor sediments from cruise KN223 on R/V Knorr in the North and West Atlantic Ocean in November 2014 (NCEI Accession 0277955)

Acquisition Description:
All samples used in this work were collected as part of the North Atlantic long-coring expedition in Oct.-Dec. 2014 (R/V Knorr, Cruise KN223); this project focuses on sediments from 4 sites (2, 3, 11, 12) exhibiting variations in the depth to which oxygen penetrates. The sediment subsamples were collected from long piston cores or shorter gravity cores. While oxygen penetrates through the full long core depth at sites 11 and 12, oxygen was consumed in the sediment column at site 3 and especially at site 2. All samples were collected anaerobically in order to perform on-board culture enrichments via the most probable number (MPN) method. Sediments were placed in sterile serum vials, capped with butyl rubber stoppers and flushed with N2 for 2 min and maintained at 4 degrees C for immediate shipboard MPN inoculation work (see MPN dataset ). Parallel samples were similarly collected from these and additional core sections and maintained at 4 degrees C for later determination of microbial production rates (this dataset).

We assessed microbial production on selected core sections at sites 11 and 12 using proxies for DNA synthesis (incorporation of methyl-3H thymidine) and protein synthesis (incorporation of 4,5-3H leucine). Core material was retained at 4 degrees C under an N2 atmosphere prior to slurry preparation. Aerobic slurry was prepared 1:1 by volume with 0.2 um-filtered deep seawater and incubations began immediately thereafter. Incubations (n=4 live treatments, n=4 TCA-killed controls) of 0.5 ml slurry each were conducted in sterile microfuge tubes for each label addition. Seawater-only blanks incubated and processed along with samples exhibited near background levels of activity. 50 ul of working 3H-Thy or 3H-Leu stock was added at time zero. This equates to 3.75 uCi Leu or 4.4375 uCi Thy per sample at concentrations of appx. 114 nmol label compound per liter slurry final. Incubations were carried out at 4 degrees C in the dark.

Incubations were terminated at 18-24 hr; a time-course experiment confirmed linearity of incorporation out to at least 24 hr. Live incubations were terminated with TCA and an extraction protocol modified from Dixon & Turley (2001, Microb. Ecol. 42:549) was used to isolate the protein + DNA fraction, which was analyzed by liquid scintillation counting for 3H-Leu incorporation; the DNA fraction alone was isolated and similarly analyzed for 3H-Thy incorporation. Rates are reported as pmol leucine or thymidine incorporated per ml of sediment per day (based on mean treatment minus mean control). Errors were calculated by propagating the standard deviations of treatments and controls.