Cruise: HYL0802, HLY0902, TN249

Contact: Evelyn Sherr
Email: sherre@coas.oregonstate.edu

Summary: Samples for analysis of bacterioplankton abundance and relative nucleic acid content were collected at 6 depths in the upper 50m from primary production CTD profiles during the three long spring BEST cruises (HLY0802, HLY0902, and TN249) from March to June in 2008, 2009, and 2010.

Three ml aliquots of seawater were pipetted into 4 ml cryovials and preserved with 0.2% (w/v) final concentration of freshly made paraformaldehyde. The samples were gently mixed and let sit in the dark at room temperature for 10 minutes before storage in a -80 oC freezer. On return of samples to the lab, the cryovials were kept at -80 oC until processing.

For analysis, samples were thawed and kept on ice prior to running on a Becton-Dickinson FACSCaliber flow cytometer with a 488nm laser, as previously described in Sherr et al. (2006). For each sample, a 250 ml aliquot was diluted with 250 ml of DiW, and stained with SYBR Green I with 25 mM potassium citrate for 15 minutes, following the protocol of Marie et al. (1997). Each sample was spiked immediately before processing with a known amount of 1.0 micron Polysciences Fluoresbrite yellow-green beads from a stock solution of beads pre-calibrated with Becton-Dickinson True-Count beads. The number of 1.0 micron beads enumerated in each sample run was used to accurately determine the sample volume processed and thus the abundances of bacteria.

Bacterial counts were made during a 3 minute sample run at low flow rate, approximately 20 ul per minute. Regions were established in cytograms of side scatter and green fluorescence to define both total bacteria and bacteria with high nucleic acid content (HNA) and low nucleic content (LNA). An analysis contour plot was used to individually inspect the data for each sample, and HNA and LNA regions moved to conform to the appropriate areas for each bacterial cytogram. Mean cell-specific SYBR fluorescence, as a proxy for relative specific nucleic acid content, was recorded for total bacteria and for HNA and LNA cells, along with abundance of cells within each group.

Total bacterial abundance in the upper 50 m during the three spring cruises ranged from 0.5 to 3.3 x 106 cells/ml, with highest abundances (1.5 – 3.3 x 106/ml) observed during the 2010 cruise, which was later in the spring bloom season (May-June) than were the 2008 and 2009 cruises (March-May). Proportions of HNA cells ranged up to > 90% of total bacteria, similar to those found for bacteria in the eutrophic upwelling system off the Oregon coast (Sherr et al. 2006). This suggests an actively growing bacterioplankton community despite the cold water temperatures during spring in the Bering Sea. For this reason, an appropriate conversion factor to estimate bacterioplankton carbon biomass would likely be 30 fgC/cell (Fukuda et al. 1998, Sherr et al. 2006), equivalent to 30 ugC/liter for 1 x 106 bacteria/ml. Fukuda, R., Ogawa, H., Nagata, T., Koike, I., 1998. Direct determination of carbon and nitrogen contents of natural bacterial assemblages in marine environments. Applied and Environmental Microbiology 64: 3352–3358. Marie, D., Partensky, F., Jacquet, S., Vaulot, D., 1997. Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Applied and Environmental Microbiology 63: 186–193. Sherr E.B., Sherr B.F., Longnecker K. 2006. Distribution of bacterial abundance and cell-specific nucleic acid content in the Northeast Pacific Ocean. Deep-Sea Research I 53 (4):713-725.