Fed-batch Culture And Physiological Funcction Of Nitrate Reduction By Bacillus Licheniformis

The polyγ-glutamic acid (γ-PGA) is a kind of water-soluble and biodegradable amino acid polymer. Multifarious applications ofγ-PGA have been developed in food, cosmetics, medicinal industries and agriculture. A high level of fermentation technology is the key point for efficient production ofγ-PGA, reducing its costs and expansion of its application. But the problem of the low fermentation level, the dissolved oxygen limit and the high viscosity of fermentation broth was need resolved imminently in fermentation process. In this study, the fed-batch process for the production ofγ-PGA by Bacillus licheniformis WX-02 was optimized, and the effect of nitrate onγ-PGA fermentation, which was the potential substitute of oxygen electron receptor, was investigated.By flask-shaking fed-batch fermentation research, it suggested that the optimum proposal of initial glucose concentration was 70 g/L, and the optimum glucose feeding method forγ-PGA fermentation by Bacillus licheniformis WX-02 was as follows:the initial concentration of glucose was 70 g/L, and then 15 g/L of glucose was added at 15th h of fermentation. Furthermore, the optimum proposal was carried out in a 3L fermenter. The results showed that the biomass, the yield ofγ-PGA were 3.9 g/L and 44.5 g/L, increased by 56% and 50% respectively compared to the control; the utilization rate of glutamic acid was 45.7%, compare with 30% of control.The optimal nitrate concentrations in the medium was 10 g/L, the biomass and the yield ofγ-PGA were 3.3 g/L and 31.0 g/L. The optimum proposal of feeding nitrate was as follows:the initial concentration of nitrate was 10 g/L, and then 5 g/L of nitrate was added at 15th of fermentation. Furthermore, the optimum proposal was carried out in a 3L fermenter. The results showed that the biomass and the yield ofγ-PGA were 4.5 g/L and 38.7 g/L, increased by 50% and 15% respectively compared to the control.The addition of metabolic intermediate enhanced the synthesis ofγ-PGA. The supplement of glycerol(20 g/L), lactic acid(5 g/L)and succinic acid(2.5 g/L),increased the production ofγ-PGA by 49%,38% and 35.5% respectively compared to the control.The physiological function of nitrate reduction inγ-PGA fermentation by B. licheniformis WX-02 was completed. It was found that the biomass(OD600) in the stable phase and the viscosity of the broth were increased by 1.9 and 3 times respectively with nitrate in the culture. The apparent utilization rate of glutamic acid was 80%, comparing with 55% of control and the yield ofγ-PGA increased by 130%. The addition of nitrate enhanced glycolysis and TCA circulation, it resulted in the significant increase of the concentration of pyruvate. The concentration of formic acid, acetic acid and lactic acid increased by 2.20,1.12 and 0.23 times respectively compared to the control. The pH value of the culture with nitrate was slightly higher than that without nitrate, although organic acid in the culture with nitrate generated in a great quantity. It revealed the regulating effection of nitrate reduction for y-PGA fermentation. The analyse of enzyme activities showed that nitrate reductase was expressed naturally in B.licheniformis WX-02 during the fermentation process and nitrate enhanced the activity of nitrate reductase.