Sorvall rc 5c plus

The growth medium contained (g L⁻¹): 10.0 yeast extract (Bacto USA), 15.0 nutrient broth (Merck, Germany) and 5.0 ammonium sulfate (Sigma Aldrich, Germany). The stock inoculum was aseptically introduced into this medium at a concentration of 3% (v/v) and incubated at 30 °C, 250 rpm for 24 h in a shaker incubator (Daihan LabTech^®, Korea). Cell biomass was harvested at 4 °C, 9000 × g for 10 min using low temperature centrifuge (Sorvall RC-5C Plus, Thermo Scientific Germany). The harvested biomass was then used to seed the minimal medium.

The accumulated PHA was extracted according to previously reported literature (Gumel et al., 2012a (link)). PHA accumulated biomass was harvested by centrifugation at 9000 × g (Sorvall RC-5C Plus, Thermo Scientific Germany) for 10 min. The supernatant was discarded while 10% (v/v) n-hexane in distilled water was used to wash the pellets three times to remove residual fatty acids. Later, the biomass was dried in vacuo in the presence of phosphorus pentaoxide using vacuum drying oven VC-6020 (Constance, Germany) at 45 °C for 24 h. About 10% (w/v) dried biomass was suspended in chloroform and refluxed at 80 °C for 4 h, after which Buchner flask equipped with 0.2 μm PTFE filter paper was used to filter the reflux mixture under vacuum. The filtrate was concentrated in rotary evaporator (Yamato RE300; Yamato, Japan) at 4 °C under reduced pressure until about 5% of the original volume. The polymer was then precipitated in cold methanol while being gently stirred. The PHA solution was allowed to settle under gravity for 24 h after which the supernatant was decanted leaving behind a layer of PHA film. This film was air-dried before the purification steps were repeated by chloroform dissolution and methanol extraction. The PHA film was dried in vacuo prior to characterization and quantification.