Studies On Screening Of Poly-(γ-glutamic Acid) Strains And Optimization Of Fermentation Condition

The analytical method of poly-(y-glutamic acid), screening method and optimization of fermentation conditions have been studied. At the same time, the flowing field in shaker has also been simulated by CFD software (Fluent). The results are as followings:(1) The improvement on y-PGA analytical method of fermentation brothThe complicated compositions in fermentation broth and a large number of small black particles produced in acid hydrolysis process seriously affected the analysis of poly-(y-glutamic acid). The pretreatment of fermentation broth and acid hydrolysis conditions were also improved. Firstly, the fermentation broth was centrifuged and supernatant were extracted by alcohol two times. Secondly, the deposition was dissolved and filtered after dilution. The optimal conditions of acid hydrolysis were that the equal volume of 6 mol/1 HCl was added to supernatant and sealed the cover, then hydrolyzed the supernatant for 16h at 90℃.(2) The mixed mutation by UV and He-Ne laserBacillus licheniformis NXTK0001 was mutated by UV and He-Ne laser. A high y-PGA producing strain Bacillus licheniformis A91 was obtained by resistance screening. The 16 g/L y-PGA was obtained in flask and which was 2.67 times higher than that of original strain. The results showed that the strain was genetic stability.(3) The optimization of fermentation mediumIn order to improve the y-PGA production, the fermentation medium was optimized by the single factor and the orthogonal experiments. The optimal fermentation medium was (g/L):glucose 80, MgSO40.1, Yeast extract 20, NaCl 10, ammonium nitrate 4.1, iron chlorides 0.01, CaCl2 0.1, monosodium glutamate 80. The 22.5 g/L y-PGA was obtained in flask under the optimal conditions.(4) The flowing field in shaker by CFD software (fluent)The flowing field by Bacillus licheniformis in shaker has also been simulated with the CFD software (fluent). The relationship between the y-PGA production and the flask position at the shaker was studied. Finally, the optimum position was determined and located lower level, inner side.(5) The study on y-PGA separationThe effective separation method for y-PGA and biomass was build. The results were summarized as followings:Firstly, the pH of fermentation broth was adjusted to 3.0 by acid for 24h at 4℃ and biomass was obtained by centrifuging. Secondly, the supernatant was siphoned and concentrated by rotary evaporator and the crude y-PGA was obtained by alcohol-precipitated the concentrated broth. Thirdly, the small molecule impurities, pigments and proteins were removed by dialysis and activated charcoal, respectively. White y-PGA has been obtained by vacuum freeze drying and the purity was 80%.