Validation of thermally assisted hydrolysis and methylation-gas chromatography for rapid and direct compositional analysis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in whole bacterial cells

Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of an organic alkali was validated for the compositional analysis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] accumulated in whole bacterial cells. Recombinant Cupriavidus necator Re2058/pCB113 was grown in a batch fermentation with different concentration of palm oil and fructose in order to control the molar fraction of 3HHx in P(3HB-co-3HHx) produced in the cells. Trace amounts (30μg) of freeze-dried cells were directly subjected to THM-GC in the presence of tetramethylammonium hydroxide (TMAH) at 400°C. The obtained chromatograms clearly showed nine characteristic peaks, attributed to the THM products from 3HB and 3HHx units in the polymer chains, without any appreciable interference by the bacterial matrix components. Based on these peak intensities, the copolymer compositions were determined rapidly without using any cumbersome and lengthy sample pretreatment as in conventional GC method. Moreover, the compositions thus obtained were strongly correlated with those by NMR and conventional GC involving solvent extraction.