| Citric acid is a very important intermediate metabolite during the glucose metabolism in microorganisms, which is widely used in food industry, environment protecting, chemical industry, textile industry, pharmaceutical industry and other fields. High temperature resistant citric acid producing strains become an urgent need, because a lot of cooling water is needed during citric aicd fermentation in summer. To obtain a high temperature resistant strain, a citric acid producing strain W was treated by mutagen. Then the inactivated parental protoplasts fusion technology was used to further study. Moreover, the fermentation medium and culture conditions were optimized. Lastly, the citric acid fed-batch fermentation was also studied. The detail contents and results are as follows:(1) A citric acid producing strain W was treated by UV, atmospheric and room temperature plasma(ARTP), high temperature combined with ARTP mutation. The strain Y-04 with resistance to high temperature was obtained. After cultivated 84 h, the original strain could accumulate 109.5 g·L-1 acid at 35℃. While only 50.3 g·L-1 acid was produced by original strain during cultivated at 40℃. However, the strain Y-04 could accumulate 98.1 g·L-1 acid at 40℃ after cultivated 72 h. In addition, the genetic stability test indicated that the strain had good genetic stability.(2) The strain Y-04 and original strain W were fused by the inactivated parental protoplasts fusion technology. The fusion conditions were determined as follows: the time of UV inactivation time was 6 min, PEG concentration was 45%, fusion time was 10 min, fusion temperature was 35℃. After protoplast fusion, a fusion strain R502 was obtained, which could yield acid 118.7 g·L-1 during cultivated at 40℃ for 72 h. Compared with the parental strains(strain W and strain Y-04), the production of strain R502 was 10.5% and 21.0% higher, respectively. Moreover, the cultivate time decreased by 14.3% and 7.7%, respectively. And the yield of citric acid is 103.0 g·L-1. The genetic stability test indicated that the genetic stability of R502 was good.(3) The optimization of fermentation medium and conditions for the fusion strain R502 was conducted. At first, the fermentation conditions were studied by single factor experiment. And the results were as follows: seed age was 24 h, inoculated volume was 10%, rotating speed was 280 r·min-1, liquid volume was 40 m L(500 m L flask), initial p H was 5. Then the effects of initial sugar concentration, the nitrogen type and concentration on acid yielding were studied by single factor experiment. The effects of Mg SO4·7H2O, Mn SO4·4H2O, Fe SO4·7H2O and K2HPO4 on acid yielding were studied by orthogonal experimental design. Four prominent factors influencing acid production were confirmed by Plackett-Burman design, which were initial sugar concentration,(NH4)2SO4, Mg SO4·7H2O and K2HPO4. Finally, the significant factors were optimized by response surface methodology design. The optimal medium components were as follows: initial sugar concentration 181.61 g·L-1,(NH4)2SO4 2.11 g·L-1, Mg SO4·7H2O 0.51 g·L-1, Mn SO4·4H2O 0.10 g·L-1, Fe SO4·7H2O 0.10 g·L-1 and K2HPO4 1.10 g·L-1。The strain was cultivated under the optimal conditions, and its production reached 142.5±0.8 g·L-1, which is 12.0% higher than before.(4) The fed-batch fermentation of citric acid was studied. The results indicated that fermentation medium containing 3 g·L-1(NH4)2SO4 was added to broth at 24 h in two times. And the fed volume was 8% of fermentation medium. After fermentation, the acid production reached 172.5±0.7 g·L-1, which was 13.1% higher than that of no sugar added. And the yield of citric acid is 87.2% of total acid, which is about 150.4 g·L-1. |
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