David Byrom

Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans

SummaryRecombinant strains of Pseudomonas oleovorans, which harbour the poly(3-hydroxybutyrate)-biosynthetic genes of Alcaligenes eutrophus, accumulated poly(hydroxyalkanoates), composed of 3-hydroxybutyrate(3HB), 3-hydroxyhexanoate (3HHx) and 3-hydroxyactanoate (3HO), up to 70% of the cell dry weight if the cells were cultivated with sodium octanoate as the carbon source. Physiological and chemical analysis revealed multiple evidence that this polymer is a blend of the homopolyester poly(3HB) and of the copolyester poly(3HHx-co-3HO) rather than a random or a block copolyester of 3HB, 3HHx and 3HO. The molar ratio between poly(3HHx-co-3HO) and poly(3HB) varied drastically during the process of fermentation. Whereas synthesis of poly(3HHx-co-3HO) started immediately after ammonium was exhausted in the medium, synthesis of poly(3HB) occurred only after a lag-phase. From freeze-dried cells poly(3HHx-co-3HO) was much more readily extracted with chloroform than was poly(3HB). The blend was fractionated into petrol-ether-insoluble poly(3HB) and petrol-ether-soluble poly(3HHx-co-3HO). The molecular weight values of these polyesters measured by gel permeation chromatography were 2.96 × 106 and 0.35 × 106 and were similar of those polymers accumulated by A. eutrophus or by wild-type P. oleovorans, respectively.

Effect of carbon source and concentration on the molecular mass of poly(3-hydroxybutyrate) produced by Methylobacterium extorquens and Alcaligenes eutrophus

In shake-flask culture, Methylobacterium extorquens accumulated poly(3-hydroxybutyrate) (PHB) possessing a substantially higher weight-average molecular mass (MW) than previously reported for this organism. The MW of PHB produced by M. extorquens was dependent on the initial concentration of methanol or sodium succinate, used as sole carbon sources. The highest MW values (0.6 and 1.7 × 106) were obtained with low initial concentrations of methanol or sodium succinate (4.0 and 3.0 g l−1, respectively) and the latter substrate always yielded PHB of higher MW than that produced from methanol. Thus PHB with an MW in the range 0.2–1.7 × 106 could be produced by selection of the carbon source and its concentration. In contrast to the findings with M. extorquens, the MW of PHB produced by Alcaligenes eutrophus was high (1.1–1.6 × 106) and generally unaffected by the choice or concentration of the carbon source. The use of glycerol as sole carbon source did, however, result in the accumulation of PHB with a markedly lower MW (5.5–8.5 × 105) than that produced from other sole carbon sources by this organism under similar conditions.