Poly(3-hydroxybutyrate) (PHB) quantification has been developed mostly using acidic methanolysis followed by GC analysis of the 3-hydroxybutyrate methyl ester. However, under our experimental conditions, only 62% of the ester was detected by GC analysis. Following the study of the different steps involved in this method (i.e., hydrolysis, esterification, and recovery of the ester), the recovery was shown to be limiting. Addition of water to the organic phase, required for its purification before injection, led to the partition of the ester between the organic and the aqueous phase. The influence of the length of acidic methanolysis time on the amount of ester detected was also investigated. NMR analysis was used to show that secondary products were absent in both phases, regardless of heating time. Moreover, increasing acid concentration and the use of lyophilized cells were shown to lead to the decrease of the treatment time. Concerning internal standard choice, methyl benzoate was found to meet all the requirements to correct injection volume errors or to follow organic phase volume changes as a function of acid and water concentrations. The validity of the method was checked on Rhizobium meliloti M5N1 cells, which are shown to produce about 60% PHB (w/w) when cultivated with fructose as the carbon source.

Constraints created by immobilization conditions modified the physiological behavior and morphological characteristics of Gibberella fujikuroi mycelia in comparison with their development in free-cell conditions. G. fujikuroi mycelia were immobilized in different support matrices (polyurethane, carrageenan, and alginate) and showed a variety of reactions in response to the different microenvironmental factors encountered during and after immobilization. The best support with respect to gibberellic acid yield and biocatalyst stability was found to be an alginate with a high degree of polymerization. The most visible effects of immobilization included changes in growth development, morphological appearance, metabolite production, mycelial pigmentation, mycelial viability under starvation conditions, and induction of resting forms when previously immobilized mycelia were subcultured.

The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.