Determination of the PHB content

PHB quantification was done using the procedure described by Schlebusch and Forchhammer (2010) and Taroncher-Oldenburg et al. (2000). Pre-weighed dried cells (5–10 mg) were boiled with 1 mL conc. H₂SO₄ at 100 °C on a heating block (Accublock™, Labnet, USA) for one hour to convert PHB to crotonic acid. Samples were allowed to cool down and subsequently diluted 20 times using 0.014 M H₂SO₄. Crotonic acid was determined using a high-performance liquid chromatography system (Thermo-Fischer Scientific, USA) with a Nucleosil C8 column (Macherey–Nagel, Germany) using an isocratic method. The mobile phase used was 20 mM NaH₂PO₄ buffer; pH 2.5 and acetonitrile (70:30) with a flow rate of 0.85 mL min⁻¹ and a column temperature of 30 °C. Detection of crotonic acid was done using a diode array detector (DAD) detector (Thermo-Fischer Scientific, USA) at 210 nm. For calibration, pure PHB (Sigma-Aldrich, USA) was treated accordingly and analyzed in parallel with samples. Instrument control and peak evaluation were done with Chromeleon 7.2 (Thermo-Fischer Scientific, USA). The percentage (dcw) PHB was determined with the amount of PHB obtained from HPLC analysis and the cell dry weight of biomass used for the analysis using Eq. (2):