2.6. PHA Analysis Using GC-MS and 1H-NMR

PHA was quantified and characterized using GC-MS according to a previously described method [38]. In brief, the culture broth was centrifuged and washed twice with hexane. The washed cells were transferred into a glass vial for lyophilization, and the dry cell weight was measured. Equal volumes of chloroform and 15% (v/v) H₂SO₄/85% methanol solution (2 mL total volume) were added to the glass vial, and methanolysis was performed for 2 h at 100 °C, followed by cooling to room temperature. A 1-mL aliquot of deionized water was added to the methyl ester solution, which was vortexed for 5 s. The chloroform layer was transferred into a microtube containing crystalline anhydrous Na₂SO₄ to remove the residual water. Filtered 1 μL aliquots were injected into the GC-MS (PerkinElmer, Waltham, MA, USA) equipped with triple-axis detector carrying Elite 5 ms column (30 mm length 0.25 mm internal diameter 0.25 film) [39]. For carrier gas, helium was used at 48.3 mLmin⁻¹. The temperature of injector was set at 280 °C, while the column and oven were programmed to increase 10 °C for 1 min to 120 °C at 15 °C min⁻¹, then hold for 15 min, and increase to 300 at 10 °C min⁻¹ hold for 15 min. NIST/EPA/NIH library was used to predict the methylated PHAs. Approximately 20 mg of ¹H-NMR sample was melted in 1 mL of deuterated chloroform (CDCl₃) and performed ¹H-NMR (Bruker Avance III 400 FT-NMR) (data not shown). Peaks of ¹H-NMR was compared with a previous report [15].