Research On The Optimization Of Medium And Scale-up Fermentation Experiments Of ?-polyglutamic Acid(?-pga)

Poly-?-glutamic acid(?-PGA)is a special water-soluble anionic high molecular weight biopolymer witch was formed by D-and/or L-glutamic acid of ?-amide bonds between ?-carboxyl and ?-amino groups.?-PGA is a kind of new natural biodegradable polymer of harmless to the human body and the environment,witch has many excellent characteristics such as film forming,fiber forming,water soluble,moisture retention,biodegradability,edibility and so on.It has great economic and social value witch was widely used in many fields for instance food,cosmetics,medicine,environmental protection,agriculture and all.At present,?-PGA is mainly produced by microbial fermentation.How to achieve low-cost fermentation and improve ?-PGA production is an urgent problem to be solved.This paper focuses on the initial fermentation medium optimization of Bacillus amyloliquefaciens YP11;5 L and 500 L fermentor scale-up experiments;kinetic model establishment.The main research contents and results are as follows:Response surface method to optimize initial fermentation medium.In order to improve the production of the ?-PGA production of Bacillus amylobacillus YP11,the experiment was based on single-factor optimization experiment optimized medium with the shaking-flask culture fermentation.Using the Plackett-Burman Design in Design-Expert V8.0.6 software three factors that affected the yield of ?-PGA were selected from 9 components of the initial fermentation medium: sodium glutamate,(NH4)2SO4 and Ca Cl2.The steepest ascent experiments method was used to approach the maximum ?-poly glutamic acid production area,and then the optimization of fermentation medium concentration of sodium glutamate,(NH4)2SO4 and Ca Cl2 was obtained through Box-Behnken Design experiments of three-factor and three-level.The results showed that the best medium composition was glycerol 30 g/L,sodium glutamate 84.53 g/L,sodium citrate 12 g/L,(NH4)2SO4 21.67 g/L,Ca Cl2 0.07 g/L,Mn SO4·H2O 0.1 g/L,Mg SO4·7H2O 0.5 g/L,K2HPO4·3H2O 0.5 g/L,Fe Cl3·6H2O 0.06 g/L.In these condition,6 sets of validation experiments were carried out,and the average yield of ?-PGA was 37.048 g/L,which was 1.39 times of the pre-optimization ?-PGA yield(26.730 g/L).Scale-up batch fermentation experiments in 5 L and 500 L fermentor.On the basis of shake flask fermentation,scale-up fermentation experiments was conducted in the 5 L fermentor.The optimal medium and the initial medium were carried out respectively.It was found that the bacterial biomass and the viscosity of the fermentation broth were higher than those of the initial medium,and the ?-PGA yield of optimal medium was 35.853 g/L,which was 1.41 times of the the initial medium(25.456 g/L).On the basis of the 5 L fermentor fermentation parameters,the scale-up fermentation experiments of 500 L fermentor was carried out.Research the change of bacterial biomass,p H,?-PGA yield,sodium glutamate concentration and viscosity of the fermentation broth.Compared with the 5 L fermentor,the final bacterial biomass and the viscosity of the fermentation broth and ?-PGA yield were all higher than those of the 5 L fermentor.The highest ?-PGA yield was 36.245 g/L.In summary,this scale-up experiments has achieved a relatively better fermentation scale-up effect witch providing a reference basis for the industrial-scale ?-PGA production.Fermentation kinetics model analysis of 5 L and 500 L fermentor.The fermentation kinetics model of 5 L and 500 L fermentor to produce ?-PGA was established by analysis of the fermentation characteristics of ?-PGA produced by Bacillus amyloliquefaciens YP11,and the nonlinear fitting analysis of kinetics equation was carried out by using Origin 9.0 software.The results of fitting curve analysis of ?-PGA fermentation kinetic model show that the growth kinetics of the bacteria growth were all consistent with the Logistic equation.The kinetics of the product(?-PGA)were all consistent with the Leudeking-Piret equation.The substrate(sodium glutamate)consumption kinetics were in line with the material consumption of the material balance equation;Through fitting and analyzing the growth kinetic model of the bacteria,it can be seen that the maximum specific growth rate was less than 5 L fermentor(0.285 h-1)in the 500 L fermentation experiment(0.187 h-1).The ?-PGA formation product and substrate consumption in 5 L fermentor are faster than those in 500 L fermentor.The linear correlation R2 values of the two models are all above 0.98.It can basically reflect the trend of bacterial growth,production of ?-PGA and substrate sodium glutamate during the whole fermentation process.