The Study Of Glutamicum Acid Producing Strain Breeded By Carbon Ion Irradiation

Corynebacterium glutamicum was chose as research material in this thesis, and it was irradiated by 80 MeV/u 12C6+ heavy ion beam in HIRFL, Heavy Ion research facility in Lanzhou. High concentration of glucose, high concentration of glutamate, glutamine, and succinic-acid were used as only carbon source in selective medium and high potential fermentation quality of glutamic acid mutant strains were screened. Then 24-well plate was used as a high throughput screening tool, and shaking flasks fermentation was carried out to select excellent mutant strains which had high glutamic acid production. Genetic stability experiments was also carried out to guarantee the genetic stability of mutation strains. Molecular biology methods were used to compare key genes between wild type and mutant strains expression in real-time fermentation, and the metabolic mechanism of glutamic acid fermentation production was discussed to provide a basis for the study of increasing glutamic acid yield. We hope to find high glutamic acid production stains which were difficult to be obtained by other mutation methods, and also solve the problem that low rate of sugar-acid conversion and high rate of food consumption in glutamic acid industries.Through experiments in this thesis, preliminary results were obtained as following:1. The best irradiation period is in middle or later stage of logarithmic growing period when strains were cultured about 8-9 h in complete medium. In this period, strains' activity and reproductive capacity were strong, and the cell concentration was high. It was indicated that Corynebacterium glutamicum was sensitive to heavy ions radiation, and its survival rate was dropped significantly after heavy-ion irradiation, which the lethality rate was up to 90% when the dose was 600 Gy.2. Using the selective medium to screen special resistance mutant strains, it could improve the efficiency and reduce lots of workload. In this method, we got varies strains altogether 146 strains, which could resist high glucose concerntration, high fermatation temperatation, and most of strains were obtained in 200-400 doses.3. Preliminary experiments suggested that 24-well plates were practicable to produce glutamic acid by microscale fermatation, and this method could improve shaking flasks screening to enhance the screening efficiency. It was suggested that 1.2 mL was the optimum volume for strain cultures in 24-well plates, 8-9 h was the optimum time for seed fermentation. This method was applied for preliminary screening of glutamic acid fermentation had good results, and 15 strains were obtained which the production of glutamic acid increased significantly than the wild type. The strain was named GM001 which had a highest glutamic acid production by shaking flake, reached 42.83 g/L and higher than control group 10.38 %. After fermatation stability experiments, it was proved that GM001 had stable genetic traits.The heavy ions radiation is a very effective method for industrial microbial mutation.4. The optimization research was investigated that several important factors influencd the glutamic acid production(glucose, corn pulp concentration, initial urea amount, culture temperature, dissolved oxygen and pH), and the orthogonal experiment was proved that the best culture medium and fermentation conditions were: glucose 200 g/L, corn pulp concentration 6 g/L, initial urea amount 3 g/L, culture temperature 32-38 ?, rotary shaker speed 120-200 r/min, initial pH 6.5-8.0.5. The growth curve of the GM001 representing the high glutamic acid production was improved that logarithmic phase of fermentation was in advance, and it had a positive effect for shorting fermentation period. After 38 h, the rate of glutamic acid production was rised to 121.1 g/L, which was increased by 10.09 % contrast to the wild type, and the rate of glucose utilization was also rised to 94.7%. After 5 times fermentation of GM001, the glutamic acid production became stable from 119.8 g/L to 121.1 g/L, which was increased about 10 % contrast to the wild type. It suggested that GM001 was a promising industrial strain.6. Through investigating the key enzymes expression of glutamic acid production in different corn slurry concentration, we found that corn slurry had great influence on the key enzymes. The corn slurry concentration had different influences on the expression of the key enzymes in growth and acid producing period. For instance, in the growth period, the expression of isocitrate dehydrogenase was decreasing with the increasing of the concentration of corn slurry, while in the acid producing period, the expression of isocitrate dehydrogenase was increasing with the increasing of the corn slurry concentration which was in the low or middle concentration. Therefore, it was indirect proved that 6 g/L of corn slurry had a positive effect on the accumulation of glutamic acid.