GROWTH OF AZOTOBACTER-VINELANDII UWD IN FISH PEPTONE MEDIUM AND SIMPLIFIED EXTRACTION OF POLY-BETA-HYDROXYBUTYRATE

Azotobacter vinelandii UWD was grown in a fermentor with glucose mediu m with and without 0.1% fish peptone (FP) in batch and fed-batch cultu res for the production of the natural bioplastic poly-beta-hydroxybuty rate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH4+, but PHB synthesis stopped when NH4 was depleted and nitrogen fixation started. When FP was added to the same medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%, and PHB synthesis was unaffected by NH4 depletion. Thus, FP appeared to be used as a nitrogen source by these nitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. The addition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive ce lls that demonstrated impaired growth and partial lysis, with the leak age of DNA into the culture fluid, but these cells were still able to synthesize PHB at elevated rates and efficiency. When FP was continuou sly present in fed-batch culture, the yield in grams of polymer per gr am of glucose consumed was calculated to range from 0.43 g/g, characte ristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation o f an FP-free growth phase from an FP-containing growth phase in fed-ba tch culture resulted in better growth of these pleomorphic cells and g ood production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simple procedure for the extraction of high-molecul ar-weight PHB. The cells were treated with 1 N aqueous NH3 (pH 11.4) a t 45-degrees-C for 10 min. This treatment removed about 10% of the non -PHB mass from the pellet, of which 60 to 77% was protein. The final p roduct consisted of 94% PHB, 2% protein, and 4% nonprotein residual ma ss. The polymer molecular weight (1.7 x 10(6) to 2.0 x 10(6)) and disp ersity (1.0 to 1.9) were not significantly affected (P = 0.05) by this treatment. In addition, the NH3 extraction waste could be recycled in the fermentation as a nitrogen source, but it did not promote PHB pro duction like FP. A scheme for improved downstream extraction of PHB as well as the merits of using pleomorphic cells in the production of bi oplastics is discussed.