Continuous production of poly(3-hydroxyalkanoates) by Pseudomonas oleovorans in a high-cell-density, two-liquid-phase chemostat

Abstract

When Pseudomonas oleovorans is continuously cultured on a two-phase medium consisting of an aqueous minimal salts medium phase with growth-limiting amounts of ammonium (16.7 m m) and an n-octane phase as carbon and energy source, the cells reach a steady-state density of about 2–3 g l −1 and accumulate a storage compound, poly(3-hydroxyalkanoate) (PHA), the amount of which depends on the dilution rate used. The PHA productivity is maximal at a dilution rate of 0.20 h −1 and yields 0.17 g PHA l −1 h −1. To improve the PHA productivity of this two-liquid-phase chemostat, the density of PHA-accumulating cells growing at a rate of 0.20 h −1 was raised by increasing the concentration of the limiting nutrient ammonium in the medium feed in discrete steps from 16.7 to 116.6 m m. After medium optimization with retention of ammonium-limiting conditions and an increase of oxygen transfer rates (the stirrer speed was increased from 1000 to 1800 rev min −1 and the airflow from 200 to 550 ml min −1), the cell density could be raised to 11.6 g l −1 with a PHA productivity of 0.58 g PHA l −1 h −1. P. oleovorans remained stable with respect to PHA formation in these two-liquid phase continuous cultures for at least 1 month: cells that were isolated after 760 h of continuous growth (about 150 doublings) were phenotypically similar to the cells that were used to inoculate the long-term cultivation.