Fermentation process development for the production of medium-chain-length poly(3-hydroxyalkanoates) by Pseudomonas putida KT2440

Fed-batch fermentation processes were developed for efficient and automated lab scale production of saturated and unsaturated (functionalized) medium-chain-length poly(3-hydroxyalkanoates) (MCL-PHA) by Pseudomonas putida KT2440. Four automated glucose feeding strategies were first developed for the high-cell-density cultivation of P. putida KT2440. An overall biomass productivity of 4.3 g X l-1 h -1 (highest reported for P. putida KT2440) was obtained by continuous feeding based on cumulative glucose consumption estimation. An exponential feeding strategy proved to be an effective way of applying carbon-limitation and controlling the specific growth rate of the culture while also achieving a satisfactory cell concentration. Contrary to the common literature belief, nitrogen limitation proved to be unnecessary for MCL-PHA synthesis by P. putida KT2440. Instead, MCL-PHA production was achieved by single-stage, carbon-limited, exponential nonanoic acid feeding fed-batch fermentations. This approach resulted in an overall PHA productivity of 1.4 g PHA l-1 h-1 (highest reported for PHA production using nonanoic acid as substrate) at a specific growth rate of 0.25 h-1 and a high final PHA content of 75% (highest reported final MCL-PHA content) at a specific growth rate of 0.15 h-1. By co-feeding of nonanoic acid and glucose, the nonanoic acid to PHA yield was increased up to 30% without significantly altering the composition of the PHA or compromising the overall PHA productivity (1.4 g PHA l-1 h-1). This would lead to a substantial substrate cost reduction if MCL-PHA is produced at a commercial scale. Unsaturated (functionalized) MCL-PHA was also produced at a productivity of up to 1.1 g PHA l-1 h-1 (highest reported for unsaturated MCL-PHA production) and a final content of up to 56% by using nonanoic acid with 10-undecenoic acid as a co-substrate. The molar fraction of each monomeric component remained relatively constant through such fermentations. A linear relationship between the degree of unsaturation of the PHA product and the fraction of unsaturated carbon source in the substrate mixture was demonstrated. Therefore, such a co-feeding fed-batch process can be used to produce functionalized MCL-PHA with controlled compositions.