【作者】 徐凯；

【导师】 张伟国；

【作者基本信息】 江南大学 ， 发酵工程， 2015， 硕士

【摘要】 柠檬酸是微生物进行糖代谢时产生的一种重要的中间代谢产物,被普遍应用于食品、环保、化工、纺织和制药等行业中。夏季温度较高时柠檬酸发酵需要大量冷却水进行降温,因此耐高温柠檬酸生产菌株成为迫切需要。为了选育一株耐高温的柠檬酸生产菌株,本文对一株柠檬酸生产菌W进行了诱变选育,之后利用双亲灭活原生质体融合技术对出发菌W和诱变菌Y-04进行了融合。然后对发酵培养基组成和培养条件进行了优化,最后还对柠檬酸分批补料发酵进行了研究,主要结果如下:(1)对一株产柠檬酸黑曲霉菌W进行了诱变选育,通过紫外线(UV)、常压室温等离子(ARTP)及高温处理和ARTP复合诱变,选育出了一株能够耐高温的柠檬酸产生菌Y-04。其中出发菌在35℃发酵84 h可以产酸109.5 g·L-1,但是在40℃发酵84 h只可以产酸50.3 g·L-1。而菌株Y-04在40℃发酵78 h就可产酸98.1 g·L-1。通过遗传稳定性实验证明,该菌株具有良好的遗传稳定性。(2)采用双亲灭活原生质体融合技术,将菌株Y-04与出发菌W进行了融合,筛选出了一株融合菌R502。其中原生质体融合条件为:双亲株紫外灭活时间为6 min,PEG浓度45%,融合时间10 min,融合温度35℃。融合菌R502在40℃发酵72 h产酸可达118.7 g·L-1,产酸量比亲本菌株(出发菌W和诱变菌Y-04)分别提高了10.5%和21.0%,而培养时间分别减少了14.3%和7.7%,其中柠檬酸含量为103.0 g·L-1。遗传稳定性实验证明该菌株遗传稳定性良好。(3)对融合菌株R502的发酵培养基和发酵条件进行了优化。首先采用单因素实验对发酵条件进行了研究,并确定了最佳的发酵条件为:种龄24 h,接种量10%,转速280 r·min-1,500 m L三角瓶装液量40 m L,起始p H 5。其次,采用单因素实验考察了初糖浓度、氮源种类和氮源浓度对发酵产酸的影响。并采用正交实验设计考察了Mg SO4·7H2O、Mn SO4·4H2O、Fe SO4·7H2O和K2HPO4这四种无机盐对产酸的影响。然后通过Plackett-Burman实验设计选出了对产酸影响较显著的四个因素:初糖浓度、(NH4)2SO4、Mg SO4·7H2O和K2HPO4。最终通过响应面分析法进行了进一步优化,确定最佳的培养基组成为:初糖浓度181.61 g·L-1;(NH4)2SO4 2.11 g·L-1;Mg SO4·7H2O 0.51g·L-1;Mn SO4·4H2O 0.10 g·L-1;Fe SO4·7H2O 0.10 g·L-1;K2HPO4 1.10 g·L-1。在该优化条件下,进行发酵实验,最终产酸为142.5±0.8 g·L-1,比优化前提高了12.0%。(4)对柠檬酸分批补料发酵进行了研究。研究结果表明,在发酵24 h时分2次补加含有3 g·L-1(NH4)2SO4的葡萄糖溶液,补料总体积为发酵培养基的8%,可以提高产酸量,发酵结束时总酸产量高达172.5±0.7 g·L-1,比不补料发酵提高了13.1%。其中柠檬酸含量占总酸含量的87.2%,约为150.4 g·L-1。更多还原

【Abstract】 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.更多还原