Dataset: Halomethane concentrations in cell culture

Six taxonomically diverse strains of marine bacteria: Alphaproteobacteria (DFL12 and MED193), Gammaproteobacteria (AND4) and Bacteroidetes (MED134, MED152 and MED217) were grown in culture. Cultures were grown in 120 mL ZoBell marine broth medium in 250 mL glass vials (ca. 120mL headspace) at 22°C on a shaker at 10rpm and full light conditions using an artificial light source maintained at approximately 125 μmol photons m-2 s-1 until they reached stationary phase, which ranged from ca. 8 to 100 hours. Cultures reached high cell densities, typically on the order of 107 - 108 cells mL-1 during late log growth phase, and one final “late” time-point was taken 24 hours after the last experimental replicates to assess behavior in stationary phase. Duplicate culture vials were killed by acidification to ca. pH 2.0 with HCl (0.5mL 3mol L-1 solution for 125mL culture volume) HCl and refrigerated prior to analysis where they are stable for up to two weeks (EPA 1986). Samples were analyzed for dissolved halocarbon concentrations using a gas chromatography (GC) method adapted from Schall and Heumann (1993) and quantified relative to an internal standard. For cell abundance and volume determination, duplicate samples were fixed with 10% formalin (4% formaldehyde), stained with acridine orange (Hobbie et al. 1977), filtered onto pre-blackened filters and counted with epifluorescence microscopy. Purge-and-trap capillary column gas chromatography with electron capture detection (GC-ECD) was employed for dissolved halocarbon analysis (Schall and Heumann 1993). 25mL media samples were purged with ultra-high purity He for 45min at a flow rate of 60mL min-1 through an in-line K2CO3 drying tube and onto a liquid nitrogen trap. The purge vessel is rinsed with methanol and the drying trap replaced with 0.75g fresh K2CO3 between individual analyses. Cryo-concentrated samples were introduced into an Agilent 7890A GC by means of a splitless injection with sweep pressure at 50psi for 1.5min returning to analytical column pressure of 18psi 2.5min after injection. Inlet temperature was set to 60°C to facilitate cryo-focusing on the column. Initial oven temperature was 40°C for 10min increasing to 120°C by 4°C min-1 and held there for another 2min. Temperature was then ramped to a final 240°C at a rate of 5°C min-1 and held for 20min Calibration was carried out using 20µL of 0.5µg/L of tribromochloromethane as an internal standard (Gonzalez-Gago et al. 2007).