PHYSIOLOGY AND POLYESTER FORMATION OF PSEUDOMONAS-OLEOVORANS IN CONTINUOUS 2-LIQUID-PHASE CULTURES

Pseudomonas oleovorans is able to grow on linear aliphatic hydrocarbons of medium chain length as sole energy and carbon source. When nitrogen, sulfur, or magnesium is limiting, P. oleovorans produces an intracellular polyester poly(beta-hydroxyalkanoate) (PHA) from the excess alkanoic acid formed from the alkanes supplied in the medium. To study the effect of growth rate and exposure to bulk amounts of n-octane on the physiology and morphology of P. oleovorans, we have established continuous cultures of this organism in two-liquid phase media containing about 15% (v/v) n-octane. P. oleovorans was grown in an ammonium-limited single-stage chemostat at growth rates varying from D = 0.05 to D = 0.46 h-1. In contrast to batch cultures of P. oleovorans grown on n-octane, both rapidly and slowly growing cells remained fully viable during the entire continuous culture experiments, which typically lasted 200-300 h. The cellular morphology of these cells was studied as a function of time by freeze-fracture electron microscopy, which provided information on changes in membrane ultrastructure and revealed large and small PHA granules in slowly and rapidly growing cells, respectively. The cell density, cellular protein content, and PHA content were determined as a function of growth rate. The cell density decreased from 2.25 to 1.32 mg ml-1, while the PHA content of the cells decreased from 46.7% to 8.3% of the total cell dry weight when the dilution rate (= growth rate) increased from 0.09 to 0.46 h-1. The rest biomass concentration, defined as the difference between total biomass and PHA, was almost independent of the cellular growth rate. The cellular protein content relative to the rest biomass increased from 24% to 46% when the growth rate increased from 0.09 to 0.46 h-1, indicating that rapidly growing cells contain more protein than slowly growing cells, which correlates well with the qualitative data of the electron micrographs.