2.4 Quantification of free fatty acids
All strains were precultured in YPD medium for 24 h and then transferred
to Delft minimal medium containing 20 g/L glucose and specific amino
acids or nucleotides at 30℃, 220 rpm. After cultivation for 48 h, FFA
were determined according to the previous method[19]. In brief, 100 μL sample was taken for FFA
extraction. Then, the 100 μL ddH2O, 10 μL 40%
tetrabutylammonium hydroxide and 200 μL dichloromethane (200 mM methyl
iodide as methyl donor and 100 mg/L pentadecanoic acid as an internal
standard) were successively added to the sample. The mixtures were
shaken for 30 minutes at 1,600 rpm in whirlpool mixer, and
centrifugation was performed at 3,000 g for 10 minutes to promote phase
separation. The 200 μL dichloromethane was transferred to GC bottles
containing glass insert. The samples were dried at room temperature for
4 h, and re-suspended with 200 μL N-hexane. Finally, the samples were
analyzed by gas chromatography. Specific procedures is shown by
predecessors [19].