2.4. Fatty alcohol and fatty acid GC analysis

To detect and quantify both fatty alcohols and fatty acids, a 30 m, 0.32 mm, 0.25 μm DB-FATWAX-UI (Agilent) GC column was used. All equipment parameters were identical for both alcohols and acids except for the ramp rate (and consequently the total run time). The injected volume was 1 μL (split ratio of 10) with Flame Ionization Detection on a Trace 1310 Gas Chromatograph (Thermo-Fisher). The initial oven temperature was 40 °C with a ramp to 250 °C as previously described (Liu et al., 2015). For fatty alcohols, the ramp rate was 3.0^°C/min while it was 5.0 °C/min for fatty acid methyl esters.

Fatty acid samples were analyzed as previously described following a Folch extraction (Folch et al., 1957; Cordova and Alper, 2018). Cell pellets were analyzed for lipid content and nonadecanoic acid was added as an internal standard at a final concentration of 0.1 g/L. After evaporation of solvent from lipid extractions, transesterification was performed at 85 °C with acidic methanol. The resulting fatty acid methyl esters were separated using 0.9% NaCl and hexane. The hexane layer was transferred to vials for GC analysis.

Fatty alcohols were extracted with ethyl acetate using a combined supernatant and cell pellet strategy previously described (Cao et al., 2015; Wang et al., 2016b). In brief, 300 μL of cell culture were collected and vortexed in ethyl acetate for 20 min. Silica beads were added to aid in cell lysis and nonadecane was added as an internal standard (10 mg/L final concentration). Samples were centrifuged at > 23,000 g for 10 min to separate ethyl acetate phase which was transferred for GC analysis. For experiments with a dodecane overlay, samples were briefly centrifuged after collection to isolate the dodecane layer which was directly analyzed using GC.