High cell density cultivation of Pseudomonas oleovorans:Growth and production of poly (3-hydroxyalkanoates) in two-liquid phase batch and fed-batch systems

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g . L-1 dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30-degrees-18-degrees-C, cell growth continued to a final cell density of 35 g . L-1 due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s-1 at maximum airflow and stirrer speed of 2 L . min-1 and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L-1, after which a nutrient feed, consisting of (NH4)2SO4 and MgSO4, was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH4+ per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L-1 was obtained. The cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L-1 and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour.