Gc 2014 gas chromatography

After shake-flask growth, cells were harvested by centrifugation (CR21 GIII, Hitachi, Japan) at 8000 × g for 10 min, washed twice with distilled water, and placed in −80 °C for 4 h. After cells lyophilizing for 12 h, their intracellular PHA was extracted using a Soxtec 2050 Soxhlet extractor (Foss, Denmark). PHA polymers were purified by re-dissolving the extracted materials in chloroform, then precipitated using absolute ethanol, centrifuged at 12,000 × g for 10 min to harvest the purified PHA materials, followed by lyophilization again to remove residual ethanol.
The dry cell mass was determined by weighing 30 mL lyophilized centrifuged bacterial cells. For the esterification reaction, 40–60 mg lyophilized cells were placed into a 2 mL esterification reagent containing 3% sulfuric acid and 0.1% benzoic acid, both dissolved in absolute methanol. After adding 2 mL chloroform, the whole reaction was performed at 100 °C for 4 h. PHA contents were determined by GC-2014 gas chromatography (Shimadzu, Japan). P3HB (99.9%, Sigma-Aldrich, Germany) was used as standards, respectively. The peak of benzoic acid was regarded as the internal standard.

The oxidation reactions were carried with catalyst (25 mg), 1-hexene (5 mmol), and H₂O₂ (5 mmol) in methanol (5 mL) in a 20 mL glass reactor with 60 °C oil bath with stirring for 2 h. After reaction, the mixture was analyzed using Shimadzu GC-2014 gas chromatography (Kyoto, Japan) with a 30 m TC-1 capillary column and a flame ionization detector. Internal standards of cyclohexanone were used to calculate the mole of the reactants and epoxides. The H₂O₂ was determined with Ce(SO₄)₂ solution (0.1 M). The conversion and selectivity of epoxide (S_Epoxide) and efficiency of H₂O₂ (S_H2O2) were calculated by the following equations:


i and f represent initial and final molar value, respectively.

During the experiment, pH was monitored for each anaerobic culture tube using a Senseline pH meter F410 (ProSense, Oosterhout, Netherlands). Samples were harvested as described above and used to quantify the amounts of short chain fatty acids (SCFA) and lactate. SCFAs were extracted with diethyl ether and analyzed using a GC-2014 gas chromatography (Shimadzu) instrument as described previously using a 1 μL injection volume (32 (link)). A commercially available kit (R-Biopharm, Darmstadt, Germany) was used to determine lactate levels following the manufacturer’s guidelines. SCFAs detected were acetate, propionate, butyrate, valerate, and branched chain SCFAs (BCSCFAs), isobutyrate, isovalerate, and isocaproate. Total SCFAs were calculated as the sum of all SCFAs listed.