Optimization And Omics Analysis Of γ-polyglutamic Acid Flow Plus Fermentation Process

γ-Polyglutamic acid(γ-PGA)is a kind of biopolymer widely used in food,agriculture,medicine and other fields.It has biodegradable,edible,water-soluble and other characteristics.At present,γ-PGA is mainly obtained by microbial fermentation.In this study,Bacillus Licheniformis CGMCC NO.3336 was used as the starting strain,and fermentation conditions were optimized with batch feeding on 5L fermentation tank.Amplification experiments were carried out on 200 L and 50 T fermenters.The effect of fed-batch fermentation on the synthesis of γ-PGA was analyzed by metabolomics and transcriptome.Finally,the effect of yeast extract on the synthesis of γ-PGA was investigated.The main experimental results of this paper are as follows:(1)Through batch feeding fermentation optimization on a 5L fermenter,it was determined that when glucose concentration and ammonium ion concentration in the medium were lower than 5 g/L and 0.5 g/L,respectively,glucose solution and ammonium sulfate solution were added to keep glucose concentration and ammonium ion concentration in the medium at 5-15 g/L and 0.5-1.0 g/L,respectively.And sustained for 12 h;It was determined that 30 g/L sodium glutamate was added initially,and when the glutamate concentration in the medium was lower than 3 g/L,MSG solution was added to maintain the glutamate concentration at 3-10 g/L until the end of fermentation.Under this optimization,the yield of γ-PGA was 45.54 g/L at the end of72 h,which was 25.32% higher than that of batch fermentation.The conversion rate of glutamate was 79.89%,which was 64.45% higher than that of batch fermentation.(2)The effects of five stirring speeds of 250,300,350,400 and 450 r/min on the synthesis of γ-PGA were studied in a 5L fermenter.The effects of three ventilation ratios of 1.00,1.25 and 1.50 vvm on the synthesis of γ-PGA were investigated.At last,the stirring speed of 400r/min and ventilation rate of 1.25 vvm were selected as the fermentation conditions.Finally,the fermentation was verified in a 200 L fermentation tank,and the final production of γ-PGA reached 47.09 g/L.(3)The effect of batch feeding fermentation on the synthesis of γ-PGA by Bacillus licheniformis was investigated by metabolomics and transcriptome.Based on GC-MS,it was found that fed-batch fermentation mainly promoted glycolysis,TCA cycle and amino acid metabolism.Based on transcriptome,it was found that batch feeding fermentation promoted the synthesis of L-glutamate and enhanced the energy metabolism of bacteria.(4)The scale up experiment of industrial production was carried out on 50m3 fermenter,and the parameters in the fermentation process were basically consistent with the experiment of 200 L fermenter.(5)Aiming at the problem of unstable yield in the fermentation process,the type and concentration of yeast extract were optimized,and the effective components of yeast extract were analyzed.The results showed that the total nitrogen and ammonia nitrogen contents of yeast extract were 12% and 3.5,respectively,which were most favorable for the synthesis of γ-PGA.In addition,it was found that the different contents of vitamins and amino acids in different yeast cream were also important factors affecting the stability of γ-PGA yield.