2.9. Lipid and Cellular Fatty Acid Analysis

For the analysis of intact polar lipids (IPLs) and cellular fatty acids (CFAs), triplicate cultures of strains F1^T and F21^T and a culture of K. glycovorans were grown to early stationary phase at 20 °C with glucose (10 mM) as the substrate. The biomass was harvested by centrifuging at 10,000 × g, washed twice with 1.7% sterile saline solution and freeze-dried. In order to obtain CFAs, the freeze-dried biomass was hydrolyzed and derivatized as described previously [47]. Fatty acid methyl ester (FAME) quantification was carried out on an Agilent 7890B GC (Agilent, Santa Clara, CA, USA) with an Agilent CP Sil-5 silica column (25 × 0.32 mm) with gases, flow rate and oven temperature as described previously [47]. FAME identification was carried out on an Agilent 7890A GC coupled to an Agilent 5975C VL MSD mass spectrometer (MS) operated at 70 eV, with a mass range m/z 50–800 and 3 scans per second with the same column and oven settings as for the quantification. FAMEs were identified based on literature data and library mass spectra. Double bond positions were determined using dimethyldisulfide derivatization of the FAMEs as described previously [47]. IPLs were extracted from the freeze-dried biomass using a modified Bligh–Dyer procedure and analyzed through ultra-high pressure liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) as described by Bale, et al. [47]. IPLs were quantified in terms of their MS peak area response. As different IPLs show different response behavior, the relative abundance of the peak area does not necessarily reflect the actual relative abundance of the different IPLs. However, this method allows for a comparison between the strains analyzed in this study.