Hyperproduction of Poly-β-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD

Abstract

The transformation of Azotobacter vinelandii UW with A. vinelandii 113 DNA resulted in the formation of rifampin-resistant colonies, 13% of which also inherited a previously unrecognized mutation in the respiratory NADH oxidase. These transformants produced colonies with a white-sectored phenotype after prolonged incubation. Cells from these sectors were separated and purified by streaking and were named UWD. The dense white phenotype was due to the production of a large amount of poly-pi-hydroxybutyrate during the exponential growth of strain UWD. The polymer accounted for 65 or 75% of the cell dry weight after 24 h of incubation of cultures containing glucose and either ammonium acetate or N2, respectively, as the nitrogen source. Under the same conditions, strain UW cells contained 22 to 25% poly-j8-hydroxybutyrate, but 02-limited growth was required for these optimal production values. Polymer production was not dependent on 02 limitation in strain UWD, but the efficiency of conversion of glucose to poly- ,-hydroxybutyrate was enhanced in 02-limited cultures. Conversion efficiencies were >0.25 and 0.33 mg of poly-fi-hydroxybutyrate per mg of glucose consumed under vigorousand low-aeration conditions, respectively, compared with an efficiency of 0.05 achieved by strain UW. Strain UWD, therefore, appeared to form poly- ,-hydroxybutyrate under novel conditions, which may be useful in designing new methods for the industrial production of biodegradable plastics.

Production of polyhydroxyalkanoates by Azotobacter vinelandii UWD in beet molasses culture

“…A. tr#wlandii strain UWD (ATCC 53799) was grown in Burk's mineral salts medium containing an additional 50 ~M ferric citrate [14] and 5% w/v beet molasses (obtained from the Alberta Sugar Co., Taber, Alberta, Canada). Burk's buffer was the mineral salts medium containing ferric citrate, but no added sugar source.…”

“…Samples were collected from the fermenter at hourly intervals by an autosampler which drew 12-15-ml whole-broth samples from a side-port in the vessel wall [20]. Each sample was divided into subsamples for determination of total cell dry weight, PHA dry weight and cell protein [14], mol% HV content of the PHA [11], and polymer molecular mass [11]. Colorimetric assays were used to determine the sucrose [21], NH~ [22] and amino-N [23] remaining in the culture supernatant fluid.…”

“…At the transition to PHA formation (12 to 15 h), the cells are fixing nitrogen and start to consume sugar rapidly, necessitating the BM feed. The combination of nitrogen fixation and PHA biosynthesis in strain UWD has been shown to be a poor combination of events [14,16]. In these cells, reducing power is in demand for N 2 reduction, for respiration to produce ATP for N 2 reduction, for non-phosphorylating respiration that is used to consume 0 2 and protect the O2-1abile nitrogenase system, as well as that needed to reduce acetoacetyl-CoA in PHA synthesis [14,16].…”

“…My research has concerned the development of Azotobacter cinelandii UWD as a source of PHA [14,15]. Azotobacters are obligately aerobic, nitrogen-fixing bacteria.…”

“…Strain UWD is a stable, capsule-negative mutant that has a defect in the respiratory oxidation of NADH. It uses PHA biosynthesis as a 'quasifermentative' means of recycling NAD(P) [16] and will form 70-80% PHA per cell dry weight during exponential growth [14]. An important observation was that UWD formed PHA when grown on impure sugar sources like beet and cane molasses, malt extract or corn syrup [17].…”