CONTINUOUS PRODUCTION OF POLY(3-HYDROXYALKANOATES) BY PSEUDOMONAS-OLEOVORANS IN A HIGH-CELL-DENSITY, 2-LIQUID-PHASE CHEMOSTAT

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 mM) 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 mM. 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.