The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Abstract

Azotobacter beijerinckii was grown in ammonia-free glucose–mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-β-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-β-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-β-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035–0.240h−1), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-β-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049–0.252h−1) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-β-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053–0.255h−1) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-β-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-β-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h−1) the oxygen uptake decreased to 100μl h−1 per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h−1). Nitrogen-limited cultures had CO2 production values in situ ranging from 660 to 1055μl h−1 per mg dry wt. at growth rates of 0.053–0.234h−1 and carbon-limited cultures exhibited a variation of CO2 production between 185 and 1328μl h−1 per mg dry wt. at growth rates between 0.035 and 0.240h−1. These findings are discussed in relation to poly-β-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-β-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.