2.3. Analysis of PHA/PHB content
Cultured biomass of 0.020 g dry cell weight was completely
mixed with 2.0 mL methanol (in 1% (w/w) sulfuric acid) and 2.0 mL
chloroform, and then the mixture was stored in well-sealed test
tubes. Test-tube mixtures were incubated in 100 ◦C oven for 6 h
reaction to dehydrate intracellular poly-3-hydroxybutyrate (PHB)
as methyl(R)-3-hydroxybutyrate (MHB) monomers. After complete
reaction, the mixture was cooled down to room temperature. Then,
1 mL 1 N sodium chloride was added to salt-out residual MHB still
contained in aqueous phase. For calibration of measurement of
PHB content, 350 L internal standard (1% (w/w) benzoic acid dissolved
in chloroform) was added. After static incubation, samples
in organic phase of the mixed solutions were chosen for quantitative
assessment of PHB. Detailed determination of PHA contents
was described in Lo et al.