STUDY OF PARAMETERS AFFECTING POLY(3-HYDROXYBUTYRATE) QUANTIFICATION BY GAS-CHROMATOGRAPHY

Poly(3-hydroxybutyrate) (PHB) quantification has been developed mostly using acidic methanolysis followed by GC analysis of the 3-hydroxybut yrate methyl ester. However, under our experimental conditions, only 6 2% of the ester was detected by GC analysis. Following the study of th e different steps involved in this method (i.e., hydrolysis, esterific ation, and recovery of the ester), the recovery was shown to be limiti ng. Addition of water to the organic phase, required for its purificat ion before injection, led to the partition of the ester between the or ganic and the aqueous phase. The influence of the length of acidic met hanolysis time on the amount of ester detected was also investigated. NMR analysis was used to show that secondary products were absent in b oth phases, regardless of heating time. Moreover, increasing acid conc entration and the use of lyophilized cells were shown to lead to the d ecrease of the treatment time. Concerning internal standard choice, me thyl benzoate was found to meet all the requirements to correct inject ion volume errors or to follow organic phase volume changes as a funct ion of acid and water concentrations. The validity of the method was c hecked on Rhizobium meliloti M5N1 cells, which are shown to produce ab out 60% PHB (w/w) when cultivated with fructose as the carbon source. (C) 1995 Academic Press, Inc.