Research On The Key Technology Of γ-polyglutamic Acid Fermentation

γ-Polyglutamic acid(γ-PGA)is an extracellular macromolecular peptide which composed of glutamic acids through γ-glutamyl bond.γ-PGA has many excellent properties such as nontoxicity,degradability,biocompatibility and outstanding solubility in water,and so on.Due to these,γ-PGA is widely used in food,environmental protection,agriculture and other fields.The paper researches on the mutation breeding of strain L13 and fermentation process optimization to improve γ-PGA yield.Main results are as follows:(1)A new method of ARTP was used to mutation of L13,the mutagenesis conditions were as follows: treatment distance 2 mm,the optimum treatment time 300 s,sample volume 10 μL,aeration rate 10 SLM.Mutant strain ZF5 was obtained with screening criteria of no lipopeptide producing and high-producing γ-PGA.The mutant didn’t synthesize lipopeptide,and few foam generating during fermentation process in shake flask and 5 L fermentor,The yield of γ-PGA attained 26.1 g/L at 18 h in 5 L fermentor.(2)Based on the research of fermentation characteristics of Bacillus subtilis ZF5,γ-PGA productivity was improved by adding appropriate amount of metal ion or amino acids.The results showed that suitable concentrations of Ca2+ and Mo6+ could promote cell growth and synthesis of γ-PGA,the optimum concentration of 0.1 g/L and 0.06 g/L respectively.Zn2+ had no significant effects;Cu2+ and Mn2+ had certain inhibition on the cell growth,but Mn2+ could stimulate the accumulation of γ-PGA.With the supplement of 0.3 g/L Mn2+,the yield of γ-PGA attained 28.5 g/L,increased by 11.8%.L-Glu,L-Val,L-Asp,L-Gln,L-Pro could promote the synthesis of γ-PGA.Among them,the effects of L-Glu and L-Asp were more remarkable.Adding 3 g/L L-glutamic acid in the beginning,the yield of γ-PGA attained 31.5 g/L,increased by 23%.Adding 5 g/L L-asprtic acid at stationary phase,the yield attained 30.7 g/L,increased by 17.6%.(3)Through single pH experiment,the optimum conditions of cell growth was pH 6.5,the γ-PGA accumulation was pH 7.0.So two steps pH controlling strategies was used in 5 L fermentor.With pH 6.5 before 10 h,and pH 7.0 subsequently in 5 L fermentor,the cell could grow rapidly,and γ-PGA yield up to 32.6 g/L,increased by 24.9% than that of the control.(4)Through the feed-batch fermentation,the effects of glucose,yeast extract,and sodium glutamate on γ-PGA producing were studied.The optimum experimental results were as follow.The suitable initial concentrations of glucose,yeast extract,sodium glutamate were 100,10,20 g/L respectively,with adding amount of 100,10,50 g/L respectively,and final concentrations were 200,20,70 g/L respectively.The optimum feeding time of glucose and yeast extract was about 17 h at end-log phase,and the time of sodium glutamate was about 14 h at mid-log phase.The optimum yield of γ-PGA was up to 52.3 g/L at 28 h,increased by 60.4% than that of batch fermentation process.