H-1 NMR spectroscopic determination of poly 3-hydroxybutyrate extracted from microbial biomass

H-1 NMR analysis was performed in order to identify the nature of the poly 3-hydroxyalkanoate (PHA) accumulated by Rhizobium meliloti M5N1 and determine its concentration within the cells. Since the PNA was identified as being poly 3-hydroxybutyrate (PHB), the method of quantification was previously tested on standard PHB. The use of either methanol or benzene as the internal reference showed good accuracy. The extraction of PHB was carried our using dispersions of sodium hypochlorite and chloroform. A treatment time of 1 h with a 10% concentration of hypochlorite allowed for total recovery of PHB. After extraction of different amounts of PHB, aliquots were submitted to H-1 NMR analysis. Methanol used as the internal reference gave an overvaluation of the PHB content. However, benzene met all the requirements of an internal reference due to its chemical inertia in our experimental conditions. NMR data obtained using benzene as the internal reference are in good agreement with corresponding data obtained by gas chromatographic (GC) analysis.