Dataset: Data Set 3A: Utilization of dissolved organic carbon by a natural bacterial community as a function of pCO2
Deployment: KM1416

TOC measurements:

The procedures used to set up each experiment (inoculum filtration and dilution with 0.2 um filtrate) removed the majority of particulate organic carbon such that changes in bacterioplankton carbon production and DOC removal were mainly a function of the growth of the inoculum. Ideally, samples collected for organic carbon would be filtered in order to directly assess DOC removal separate from bacterioplankton carbon production over the course of the incubations. However, sample handling during filtration can result in contamination that obscures changes in DOC on the scale of a few micro-molar C. To avoid contamination, seawater samples from the incubation experiments were not filtered. Therefore, measured values of organic carbon include both DOC and bacterioplankton carbon and are considered total organic carbon (TOC).

TOC samples were collected into 60 mL high-density polyethylene bottles (Sargasso Sea and South Pacific Subtropical Gyre) or in combusted 40 mL glass EPA vials with Teflon coated silicone septa (Santa Barbara Channel). All TOC samples were frozen at -20 C until analysis. Samples were analyzed via high temperature combustion method on a modified Shimadzu TOC-V or Shimadzu TOC-L using the standardization and referencing approaches described in Carlson et al. 2010.

Bacterioplankton abundance measurement – Samples for bacterioplankton abundance were analyzed by epifluorescence microscopy with 0, 6-diamidino -2-phenyl dihydrochloride (5ug/mL, DAPI, SIGMA-Aldrich, St. Louis, MO, USA) according to Porter and Feig 1980, or by Flow Cytometry (FCM) on an LSR II with SYBR Green I according to Nelson et al. 2011. See Parsons et al. 2014 and Nelson et al. 2011 regarding sample preparation and instrument settings for epifluorescence microscopy and FCM analyses, respectively. DAPI direct counts and FCM analysis enumerate total prokaryotic abundance. We were not able to differentiate between bacterial and archaeal domains and refer to the combined cell densities as bacterioplankton abundance (Glockner et al. 1999).

Water sources:

Experiment OA11 was conducted on board a research cruise R/V Kilo Moana KM1416. The Sargasso Sea experiments were conducted at the Bermuda Institute for Ocean Sciences (BIOS) with water was collected via the R/V Atlantic Explorer. The Santa Barbara Channel experiments were conducted with water collected near-shore via a pier near the UCSB campus.

Experimental design:

At all three study sites, experiments consisted of 0.2 um-filtered (0.2 um GSWP, Millipore, Billerica, MA) seawater or 0.2 um-filtered phytoplankton exudate that was inoculated with natural bacterial communities. The inoculum of natural bacterial communities consisted of either unfiltered whole seawater (Sargasso Sea and South Pacific Subtropical Gyre experiments) or 1.2 um filtrate (Santa Barbara Channel experiments; 1.2 um RAWP, Millipore, Billerica, MA). Particulate organic carbon concentration in oligotrophic gyres is low (1-3 umol L-1) so to avoid filtration artifacts such as reduced bacterial production (unpublished data) and contamination of DOC due to handling, the inoculum was not pre-filtered for the experiments conducted in oligotrophic waters. Because particulate organic carbon concentration can be much greater in coastal upwelling systems it was necessary to remove large particles and organisms from the inoculum. Inoculum was added at 25 – 30% of final volume, effectively diluting grazer concentrations and grazing pressure. All filters were pre-rinsed with ~2 L of deionized distilled water and sample water prior to use in order to remove organic contaminants from the filters.

Four types of DOC treatments were used and are described in the data as "doc_additions":

1. None: unamended seawater, which provided naturally occurring DOC.
2. CNP: Naturally occurring DOC amended with glucose (~10 uM C) plus NH4 Cl (1uM) and K2HPO4 (0.1uM) (CNP)
3. Species name + " exudate": phytoplankton exudate
4. Species name + " lysate": naturally occurring DOC amended with phytoplankton lysate (~10 uM C L-1; labeled by phytoplankton species used).

The various treatments were generated by inoculating the 0.2 um pre-filtered seawater or exudate with the microbial community; this solution was then divided into two polycarbonate (PC) containers to adjust pCO2. pCO2 levels were adjusted via chemical additions (Sargasso Sea experiment) or by bubbling with CO2-mixed air (Santa Barbara Channel and South Pacific Subtropical Gyre experiments). Adjusted seawater incubations were then transferred into new PC carboys and CNP or lysate was added, if appropriate. A very small volume of lysate (1.2 mL to 11.5 L of experimental volume) or CNP (12 mL to 10 L of experimental water for the Sargasso Sea experiment; 0.28 mL to 10 L of experimental volume for the Santa Barbara Channel experiment) was added to minimize perturbing the carbonate chemistry. All experiments were conducted in duplicate, at in situ temperatures, and in the dark to eliminate photoautotrophic production. All PC bottles had been acid-washed (5 % or 10 % HCL) and rinsed with deionized distilled water and sample water before use.