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Recovery of poly-3-hydroxybutyrate (PHB) in three chlorinated solvents with or without acetone pretreatment and degradation of extracted PHB (99% pure) in hot chloroform were studied. When lyophilized Alcaligenes eutrophus biomass was used, the best results were obtained with acetone pretreatment and solvent reflux for 15 min in methylene chloride or chloroform. Recovered PHB had a 95% purity and molecular weights (w) of 1,050,OOO and 930,000 g/mol respectively. Further heating resulted in a serious & loss at reflux temperatures. Degradation of extracted PHB at 110°C in chloroform was due to random and chain-end scission, the former being predominant.

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(1-hydroxybutyric-co-4-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-,l-hydroxybutyric acid (PHB) liter-' under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-' was produced. The P(HB-co-HV) contained 5.0 mol% ,-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-' in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-(l-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.

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Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h(-1) but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (mu = 0.15 h(-1)) produced 70 g l(-1) biomass containing 75% PHA. At a higher exponential feed rate (mu = 0.25 h(-1)), the overall productivity was increased but less biomass (56 g l(-1)) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.

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