2.3.3. PHB Extraction

PHB was extracted from cellular biomass and quantified using gas chromatography after each of the growth phase and the PHB accumulation phase following the protocol described with some modification as follows [24]. First, 10 mL of culture media was collected and centrifuged at 8000× g for 20 min at 20 °C. Then, the supernatant was decanted. Afterward, 10 mL of 5% sodium hypochlorite was added to the pellets, vortexed, and incubated for 1 h at 40 °C in a shaker incubator. Then, the hypochlorite mixture was again centrifuged, and the supernatant was decanted. Subsequently, the previous sequence of centrifuging, decanting, adding liquid, and vortexing was repeated using 10 mL of water, acetone, and ethanol. Thereupon, boiling chloroform was added to the remaining pellets and filtered through a 0.5 µm syringe filter, followed by addition of water to collect any impurities from the sample. Then, the mixture was further incubated for 2 h at 55 °C in a shaker incubator. Finally, the chloroform clear layer was transferred to another container and kept undisturbed overnight at room temperature to evaporate. Eventually, a white powdered film of the PHB polymer was formed, which was then used for further analysis.