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

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

PRODUCTION OF PRIMARY ALIPHATIC-ALCOHOLS WITH A RECOMBINANT PSEUDOMONAS STRAIN, ENCODING THE ALKANE HYDROXYLASE ENZYME-SYSTEM

In this paper we describe the biosynthesis of a series of 1-alkanols from the corresponding n-alkanes. To this end, we introduced the alkane hydroxylation system of Pseudomonas oleovorans into Pseudomonas putida PpS81 (alcA81). The resulting recombinant strain PpS8141 can oxidize n-alkanes to the corresponding 1-alkanols but is unable to utilize the alkanols, which therefore accumulate in the medium. PpS8141 was grown in two-liquid-phase bioreactors. The different medium-chain-length alkanes formed a bulk organic phase, which constituted 20% of the total volume. The aqueous phase contained the growth substrate citrate, octanoate, or pyruvate. PpS8141 was able to grow well in the presence of a bulk apolar phase only when pyruvate was used as the growth substrate. Hexane was toxic to this strain and did not allow growth. The strain oxidized C7-C-11 linear alkanes to 1-alkanols with production rates superior to the wild-type P. oleovorans rates. With n-octane and n-nonane as organic phase and substrate, we found the highest production rates of 1-alkanol. The growth rate in the exponential phase and the maximal 1-alkanol production rate showed an inverse relationship, which we interpret to be the result of the metabolic burden of the conversion. The results show that the recombinant strain PpS8141 may allow the biotechnological production of primary aliphatic alcohols

A biodegradable rubber by crosslinking poly(hydroxyalkanoate) from Pseudomonas oleovorans

Poly((R)-3-hydroxyalkanoate)s (PHAs) are bacterial storage polyesters, currently receiving much attention because of their potential application as biodegradable and biocompatible plastics. Among them are the PHAs from Pseudomonas oleovorans, which are semicryst. elastomers. Their applicability is seriously limited by their low melting temp. as well as by their low crystn. rate. Both problems were overcome by crosslinking of unsatd. pendent groups, which were incorporated in the polymer by tailoring the carbon source for biosynthesis. Crosslinking was established by electron-beam irradn. and resulted in a true rubber with const. properties over a large temp. range from -20 to +170 DegC. Even after crosslinking, the material was still biodegradable. To the authors' knowledge this is the first microbially produced biodegradable rubbe