High cell density fermentations of Pseudomonas oleovorans for the production of mcl-PHAs in two-liquid phase media

Abstract

Pseudomonas oleovorans is able to produce medium-chain length poly(3-hydroxyalkanoates) (mcl-PHA) in continuous and fed-batch two-liquid phase fermentations using n-octane as a sole carbon and energy source. We have previously shown that it is possible to increase the volumetric productivity of such a system by increasing the concentration of cells and PHA in the fermentor with maximal production limited by the oxygen transfer rate to the cultures in our bioreactor systems and by complex effects of metal ions on biomass yields, leading to a maximal biomass concentration of 37 g l −1. This paper describes further improvements in the cultivation process of P. oleovorans for the production of mcl-PHA in two-liquid phase fermentations that have led to a threefold higher final cell density. In order to further increase cell densities, we determined the growth yields for each of the metal ions and developed an optimized feed of metals. Using a bioreactor with better oxygen transfer capabilities, we were able to increase the final cell density in fed-batch cultivations up to 90 g biomass l −1. By applying a computer-controlled exponential nitrogen feed in combination with the feeding of various metal ions, a cell density of 112 g l −1 was obtained. The PHA content of these cells decreased as the cell density increased above 40–50 g l −1, thus negatively affecting overall PHA yields and productivities. Possible approaches to reducing these PHA losses are discussed.