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 a nd determine its concentration within the cells. Since the PNA was ide ntified as being poly 3-hydroxybutyrate (PHB), the method of quantific ation was previously tested on standard PHB. The use of either methano l or benzene as the internal reference showed good accuracy. The extra ction of PHB was carried our using dispersions of sodium hypochlorite and chloroform. A treatment time of 1 h with a 10% concentration of hy pochlorite allowed for total recovery of PHB. After extraction of diff erent amounts of PHB, aliquots were submitted to H-1 NMR analysis. Met hanol used as the internal reference gave an overvaluation of the PHB content. However, benzene met all the requirements of an internal refe rence due to its chemical inertia in our experimental conditions. NMR data obtained using benzene as the internal reference are in good agre ement with corresponding data obtained by gas chromatographic (GC) ana lysis.