Study On The Strain Rejuvenation And Fermentation Optimization In 5000L Scale Up For L-Asparaginase

In order to solve the instability problem of the asparaginase-producing bacteria in the small-scale process,we have screened the strain used for the production.The fermentation volume was upgraded to 5000 L when the production process was also optimized.Results achieved in this study are as follows:1.The mutant FZ-1-15 with the strongest enzyme production ability was screened through mutagenesis with UV and LiCl.The enzyme activity of FZ-1-15 was 34.9 IU/mL,which was 56,5%higher than that of the original strain.2.Through the experiments of single factor,Plackett-Burman and response surface analysis,the fermentation medium was optimized as:tryptone 2.2%+ sodium glutamate 2.1%+ cornstarch 2%+ yeast 0.5%+ NaCl 0.5%,pH7.0.The medium sterilization should be at 121 ℃ for 30 min.The key parameters for process control in the first stage of seed culture were determined by the triangulation and shaker fermentation.The fermentation temperature was 37℃ when the medium dosage was 250 mL in the 1000 mL shaking flask.The inoculum amount of Erwinia was 1.5%and the rotation speed was 220 r/min for 18 h.The experimental results showed that the shearing stress had a significant negative effect on the fermentation.Factors affecting the shearing stress should be reduced by regulating the ventilation rate and the tank pressure in the process control.3.The fermentation of the rejuvenating strain FZ-1-15 was studied in a 50 L fermenter.Results showed that the optimal condition was:10%inoculation amount of Erwinia,30 L medium volume with the stirring speed of 170-210 r/min for 12 h,dissolved oxygen>20%during the logarithmic growth phase.4.On the basis of the fermentation experiments using shaking flasks and 50 L fermenters,the secondary production of seeds in the 500 L and the 5000 L volume was studied.The results were:1)The cultivation process of secondary production seeds in the 500 L tank:① Theoptimal medium formulation was:glucose 2%(W/V)+ trypton 2%(W/V)+ yeast 1%(W/V),pH7.0;②The training condition was:300 L medium with 1%inoculation amount.The culture temperature,pressure,and ventilation capacity were 37 ℃,0.06-0.09 MPa and 14～22 M3/h.The stirring speed was 170～200 r/min for 9 h to control the dissolved oxygen above 20%during the logarithmic growth phase;③ The concentration of the seed culture was OD500≥25.2)The cultivation process in the 5000 L fermenter.① The optimal medium formulation was:tryptone 2.2%+ sodium glutamate 2.1%+ cornstarch 2%+ yeast 0.5%+ NaCl 0.5%,pH=7.0;②The training condition was:3000 L fermentation medium with 10%inoculation.The culture temperature,pressure,and ventilation capacity were 37 ℃,0.06-0.09 MPa and 140～220 M3/h.The stirring speed was 170-200 r/min for 12 h to control the dissolved oxygen above 20%during the logarithmic growth phase.Finally,scale-up fermentation of the Erwinia-L-Asp was realized in the 5000 L tank.The yield of the Erwinia-L-Asp was up to 80 IU/mL.