Quantification of PHA by Gas Chromatography-Flame Ionization Detector (GC-FID)

The amount of PHA in bacterial pellets was quantified by GC-FID after methanolysis in a 1:1 (vol/vol) mixture of chloroform and 15% concentrated sulfuric acid dissolved in methanol. Approximately 15–30 mg of lyophilized cells were added to 3 mL of the methanolysis mixture containing 0.2% benzoic acid as internal standard, and reacted for 12 h at 100 °C. PHBV (Sigma-Aldrich) was used as external standard. After the reaction, 1.5 mL of Milli-Q water was added to separate the phases followed by careful transfer of the organic phase into GC analysis vials. The samples were analyzed by an Agilent 7820A GC-FID system (Agilent Technologies) using a 30 m long by 0.32 mm i.d. DB-5MS column with 0.25 μm film thickness. The injector temperature was set to 250 °C with a split ratio of 10:1 and the injection volume was 1 μL. The temperature program was set to 50 °C with hold for 5 min, then a linear increase to 250 °C at a rate of 15 °C/min, followed by increase to 310 °C at a rate of 30 °C/min. Hydrogen was used as carrier gas at 0.8 mL/min flow with a column head overpressure of 37 kPa. At least two technical replicates for each PHA extraction were injected.