Screening The High L-lactic Acid Production Strain And Researching Its Fermentation Conditions

This dissertation focused on the screening of L-lactic acid producing bacteria by chemical mutation breeding, and investigating the fermentation conditions and metabolic flux distributions of the original strain and mutant strain. The main contents and results are as follows:⑴The strain Lactobacillus casei G-02 was treated with DES and NTG, and a mutant strain of G-04 was screened by high temperature and concentrations of glucose. In the initial fermentation condition, the production of L-lactic acid was rasied from 98.6g/L to 142.6g/L.⑵The combination of carbon-nitrogen source of seed media was studied using orthogonal experimental design. The result show that the best medium components were glucose 30g/L, corn steep liquor 30g/L, tryptone 10g/L, yeast extract 5g/L. The effects of temperature, seed age, inoculation volume, carbon source, nitrogen source on L-lactic acid production were investigated by single factor experiments. Three most important factors influencing the lactic acid production were identified using Plackett-Burman design. The optimum levels of these significant factors were determined by the response surface Box-Behnken design. Ultimately, the optimal composition of culture medium and fermentation conditions were obtained: glucose 169.2g/L, corn steep liquor 40.1g/L, yeast extract 5.2g/L, MgSO4·7H2O 0.3g/L, MnSO4·4H2O 0.05g/L, Tween-80 1g/L, CaCO3 90g/L, seed age 10h, inoculation volume 8%, liquid volume 50mL/250mL, temperature of 41℃. Under the fermentation conditions above, the yeild of L-lactic acid reached 155.0g/L.⑶According to the optimal results obtained above, the effects of neutralizers, ventilation conditions and fed-batch culture on L-lactic acid production were investigated with 7L fermentor. The results showed that: the optimal neutralizer was the mixture of NaOH and CaCO3; and maintaining the 0.25VVM ventilation in fermentation process was the best; also the fed-batch culture is better than the batch fermentation. Finally, L-lactic acid concentration reached 180.5g/L in 7L fermentor.⑷The metabolic flux distribution of G-02 and G-04 at different fermentation time was analysed. Compared with original strain G-02, the flux distibution of G-04 was summaried as follows: the flux of the glycolysis resembles the original strain, and the fluxs of acetic acid and malic acid decreased significantly. Meanwhile, the flux of L-lactic acid at 30h increased significantly than at 12h, and the glucose consumption rate was improved obviously. It was also found that the pyruvate node was an easy control node.