Methyl r 3 hydroxybutyrate

All chemical reagents, unless stated otherwise, were purchased as an analytical grade. Poly(3‐hydroxybutyric acid‐co‐3‐hydroxyvaleric acid) of natural origin (8 mol% 3HV), methyl (R)‐3‐hydroxybutyrate (99%), and methyl (R)‐3‐hydroxyvalerate (≥98.0%) were purchased from Sigma‐Aldrich. Monodisperse polystyrene standards (PStQuick C) for size exclusion analysis were purchased from TOSOH Corporation. Yeast extract and glucose were purchased from Oxoid.

For sample preparation the method described by (Braunegg et al., 1978 (link)) was followed. 2 mL of sample were centrifuged, and the supernatant discarded. The pellet was then washed 3 times with 0.1% w/v sodium dodecyl sulphate (SDS) (Sigma Aldrich) solution before a final wash with distilled water. The pellet was then transferred to a pressure tube and left to dry. Once dried, the sample was treated at 95°C for 140 min with a chloroform:methanol:sulfuric acid solution (2:1.85:0.15 v/v) supplemented with 5 g/L of methyl benzoate as an internal standard. Following this procedure, 2 mL of water were added, and the mix was vortexed and left to settle. After settling the lower layer (organic phase) was extracted with the use of a syringe, filtered (0.45 μm PTFE filter) and transferred to a GC vial.
Gas chromatography with a flame ionization detector (GC-FID), a 30 m × 0.25 mm × 0.25 µm Zebron ZB-SemiVolatiles Capillary GC column capillary column (SGE Analytical Science) was used to analyse the PHA concentration. The method consisted of an initial temperature of 100°C for 3 min, followed by a ramp of 25°C/min to 200°C, followed by a second ramp of 30°C/min to 220°C, maintaining this temperature for 2 min. Helium was used as carrier gas. Standards were prepared from Methyl-(R)-3-hydroxybutyrate and Methyl-(R)-3-hydroxyvalerate (Sigma Aldrich) to create calibration curves.