Optimization of Vaccine Production Processes of Actinobacillus pleuropneumoniae serotype2

Actinobacillus pleuropneumoniae(APP) is the etiologic agent of porcine pleuropneumonia, a worldwide endemic and highly contagious disease with high economic impact. There are 2 biovars and 15 different known serotypes. APP that require nicotinamide adenine dinucleotide (NAD) for growth are designated as biovar 1 while APP isolates that are NAD independent are designated as biovar 2. In this study, the fed-batch fermentation process was optimized for mass-production of APP serotype2 vaccine.

The initial culture was grown on TSB agar medium containing 50 ug/ml NAD. The seed culture was prepared in TSB broth containing 50 ug/ml NAD. The production culture medium composition was 10 g/L sucrose, 5 g/L yeast extract, 13.5 g/L potassium phosphate monobasic, 1.4 g/L magnesium sulfate heptahydrate, 50 ug/ml NAD, pH 7.0.

Batch culture was carried out in 5 L jar fermenter containing 3.6 L of production medium. The pH was controlled at 7.0 by automatic addition of 5 N NaOH or 1 N HCl. The temperature was maintained at 37 ℃. Dissolved oxygen (DO) was controlled at 20 %. The aeration rate was 1.0 vvm.

The optical density was measured at 600 nm using a spectrophotometer. Cell concentration was determined by measuring dry cell weight (DCW). The concentration of carbon source was analyzed according to the 3,5-dinitrosalicylic acid (DNS) method.

Various carbon sources were used to glucose, galactose, sucrose, fructose and lactose for carbon source optimization. Sucrose as carbon source showed high cell mass production. Culture sample of used to sucrose was 2.8 g dcw/L.

The batch fermentation was completed in 5 hours with a final cell mass of 4.7 g dcw/L. The residual sucrose concentration rapidly fell down after 2 hours of fermentation due to the initiation of cell growth. The residual sucrose concentration went down to 0.6 g/L after 5 hours.

The feeding solution composition was 400 g/L sucrose, 130 g/L yeast, 50 ug/ml NAD. Feeding was started at 3 hours in response to sucrose level dropping to 5 g/L. The feeding pump controlled the feed rate to maintain DO level at 20 % air saturation. The maximum cell density reached to 10.4 g dcw/L in 8 hours. The cell density was higher than batch fermentation.

Fermentation conditions were optimized to increase the concentration of cell mass. Mass production through optimized fed-batch fermentation process could provide the basic for the industrial production of APP2 vaccine.

Disclosure of Interest: None Declared.