The Strain Breeding Of High Yield L-glutamine And Shaking Flask Fermentation Process Optimization

In the paper, we begin with the analysis of the morphological characteristics and physiological characteristics of original Corynebacterium glutamicum, determining the important factors influencing the accumulation of L-glutamine in the process of fermentation to prepare for the breeding selection.We established the L-glutamine’s qualitative and quantitative detection method in the fermented liquid and take advantage of the process of the microbial metabolic controlled fermentation. And on this basis, we study the L-glutamine bacteria breeding selection in detail and determine the L-glutamine fermentation conditions by using shake flask fermentation. The main research contents and conclusions include:(1) we set up a rapid and accuratemethod to test L-glutamine, including qualitative and quantitative test of L-glutamine by using paper chromatography and high temperature and high pressure acid solution.Based on the research of the paper chromatography, paper chromatography is chosen as the qualitative and quantitative detection method of L-glutamine. By Comparing the different effects of different exhibition layers, we find a relatively better formula (volume ratio) n-butyl alcohol: acetic acid:water=4:1:2best. Studying the characteristics under the condition of acid hydrolysis, we determine difference in volume before and after acid hydrolysis of glutamic acid under the condition of high pressure and high temperature by using biological sensor. The measuring range of the method is0.15g/L-0.5g/L,among which the decomposition rate of L-glutamine can reach100%.And therefore, we can verify it by using HPLC.(2) By using C.glutamicum SA-01as original strain, and adopting the combination of Ultraviolet mutagenesis and EMS mutagenesis, we have selected a high-yield L-glutamine bacteria which has a double resistance and a good genetic stability. Resistant mutant strains are named C.glutamicum SA-007.Its glutamine production can reach13.81g/L, which is29.8%higher than original bacterial strain and has better genetic stability.(3)Through the research of the optimization of the seed culture medium and culture conditions, it is concluded that the best composition for the seed culture medium is(g/L): glucose30,corn starch30, urea6. The culture conditions are:seed culture condition: pH=7.0;20ml liquid within a220mL triangle bottle; temperature:30℃; rotation speed:220r/min; culture time:12h. At last, through the research of the optimization of the fermentation culture medium and culture conditions, it is concluded that the best composition for the fermentation culture medium is(g/L):glucose100.5g/L, Ammonium chloride46.0g/L, corn steep liquor4.2g/L. The initial pH is7.0in the fermented liquid culture medium. The loading volume of liquid fermentation medium is20mL contained in a250mL triangle bottle. The rotation speed is220r/min.The temperature is30℃with a culture time of48h. the acid production stabilized at15.58g/L, which increased by12.8percent compared to those which were not optimized.