【作者】 张家学；

【导师】 陈守文； 孙明；

【作者基本信息】 华中农业大学 ， 微生物学， 2004， 硕士

【摘要】 为提高苏云金芽胞杆菌工程菌BMB005的发酵水平，本文系统研究了发酵温度、发酵过程pH值的控制以及碳氮源补料种类、时间、浓度和方式等对该菌株发酵的综合影响。通过工艺优化，以H₉培养基作为发酵培养基，30℃发酵，在对数生长期末期（第10～12h）和稳定期前期（第14～16h）分别恒速连续补加1％葡萄糖，可以使最终发酵液中杀虫晶体蛋白含量和生物效价分别提高74.4％和61.0%即7.85mg／mL和5403IU／μL（棉铃虫为供试虫靶）。 温度实验表明：在发酵罐中，与30℃培养相比，28℃培养会使杀虫晶体蛋白含量提高18.6％，但生物效价没有明显变化，且发酵周期延长5h；变温培养（在前16h，30℃培养；之后28℃培养）使杀虫晶体蛋白含量提高10.5％，生物效价也没有明显变化，发酵周期延长2h。 通过补加酸碱使发酵全程的pH值维持在6.8至7.2之间，可使发酵液中杀虫晶体蛋白含量和生物效价比对照分别提高6.7％和10.1％；若仅控制发酵中后期pH值，可使杀虫晶体蛋白含量和生物效价分别提高9.8％和16.1％。在培养基中添加1‰氯化铵对发酵前期pH值没有明显影响，但可降低发酵中后期发酵液pH值，并稳定在6.6至7.2之间，最终杀虫晶体蛋白含量和生物效价分别被提高20％和29％；稳定期前期恒速补加1％葡萄糖也可以适当降低发酵中后期的pH值，最终发酵液中杀虫晶体蛋白含量和生物效价分别提高33％和47％。 补加不同碳源的实验结果表明：发酵过程中补加葡萄糖效果最好。不同阶段补加不同浓度的葡萄糖的实验表明：在对数生长期术期（第10～12h）恒速补加1％葡萄糖可使生物效价提高39％，在稳定期前期（第14～16h）恒速补加1％葡萄糖可以使生物效价提高47％；在对数生长期术期或稳定期前册叵速补加20%葡萄糖均会使碳源对产物的转化率和发酵同步率降低。进一步实验表明：对数生长期末期补加葡萄糖是通过提高生物量进而提高发酵水平，而稳定期前期补加葡萄糖则可能是通过改善发酵中后期的pH环境和为菌体提供部分能量而提高发酵水平。通过考察补加方式发现：恒速连续补加方式比批次补加的效果好，镜检发现发酵过程中批次补加容易导致菌体形态异常。进一步实验发现：与不补加葡萄糖相比，在对数生长期木期和稳定期前期均恒速连续补加1％葡萄糖所得到的发酵效果较好，杀虫晶体蛋白含量和生物效价分别提高74.4％和61.0％；在第10～18h恒速连续补加2％葡萄糖可以使杀虫晶体蛋白含量提高82.2％，但生物效价仅提高49％。 摇瓶中氮源补加实验结果表明：稳定期前期批次补加蛋白胨不能显著地促进杀虫晶体蛋白的合成；对数生长期中期批次补加氨基酸的效果优于稳定期前期补加的效果；对数生长期中期补加0.15‰谷氨酸钠可使杀虫品体蛋白含量提高5.8％，补加0.15‰赖氨酸可以使晶体含量提高8．2％，对数生长期中期同时补加五种类型氨基酸（每种氨基酸的终浓度均为0.15‰）的效果最好，可以使晶体含量提高42％；补加氯化铵的实验发现，在起始培养基中添加1‰氯化铵时发酵效果最好。更多还原

【Abstract】 In this paper, fed-batch fermentation technology of Bacillus thuringiensis BMB005 was researched to enhance the titer of the culture. The impact of temperature, pH regulation, as well as the types, time, concentration and means of feeding carbon and nitrogen sources on yield of insecticidal crystal proteins (ICPs) and biological titer were investigated. By adopting the optimal technology, feeding 1% glucose in constant speed at both the latter exponential phase (the 10th ~ 12th h) and the fore-stationary phase (the 14th -16th h) in H9 medium at 30, the concentration of insecticidal crystal proteins and the titer increased 74.4% and 61.0% respectively to 7.85 mg/ml and 5403 IU/(j.l (Heliothis armigera to be tested).The results of fermentation temperature trials showed that the titer of the culture varied insignificantly when fermentation temperature were controlled at 28 , 30 and a altering temperature (30 for 16 h and then 28 ). The content of insecticidal crystal proteins increased 17.8% and the fermentation cycle lagged 5 hours when the fermentation temperature was done at 28 in contrast with 30. At the same time the content of insecticidal crystal proteins increased 10.5% and the fermentation cycle lagged 2 hours when the fermentation was done under the shirking tempreture (30癈 for 16 h and then 28).By feeding HC1 and NaOH solution to maintain the pH value of broth between 6.8 and 7.2 during the process of fermentation, the content of insecticidal crystal proteins and the titer increased 6.7% and 10.3% respectively. Then by controlling the pH value during the spore-forming phase and the spore-shedding phase, the yield of insecticidal crystal proteins and the titer increased 9.8% and 16.1%. The curve of pH showed that pH was not affected markedly during the lag and exponential phase by feeding l%o NFLiCl, though decreased a little during the mid-latter phase of fermentation. When l%o NtLjCl was fed in the medium, the yield of insecticidal crystal proteins and the titer increased 20% and 29% respectively. When 1% glucose was fed in constand speed at the stationary phase, higher yield content of insecticidal crystal proteins and titer were obtained than feeding l%o NHUCl.The results of experiments on choosing carbon resource showed that feeding glucose was most effective. When 1% glucose was fed in constand speed at the latter exponential and fore-stationary phase respectively, the titer increased 47% and 39%. The good result was owed to the increase of biomass when glucose was fed at the latter exponential phase, while the latter was owed to adaptive pH and more energy offered to synthesize product. Compared with feeding 1% glucose, feeding 2% glucose decreased the transformation efficiency of total sugar into products, and the phenomenon was observed by microscope that cell growth consistency decreased. As far as the feeding mode was concerned, constant-speed feed wasmore effective than batch-wise feed. The abnormal cellular form was often observed when glucose was batch-wise fed. Further experiment showed that better result was obtained when 1% glucose was fed constant-speedly at both the exponential phase (the 10th ~ 12th h) and the stationary phase (the 14th -16th h), and compared with the control, the yield of insecticidal crystal proteins and the titer increased 74.4% and 61.0% respectively; when 2% glucose was fed constant-speedly from the 10th h to the 18th h, the concentration of insecticidal crystal proteins increased 82.2% , while the titer increased only 49.0%.The concentration of insecticidal crystal proteins varied insignificantly when peptone was fed. In flask culture better result was obtained when amino acid was fed at the exponential phase than at the stationary phase. The yield of insecticidal crystal proteins increased 5.8% and 8.2% respectively when 0.15%o glutamate sodium and lysine were fed at the exponential phase. The yield of insecticidal crystal proteins increasing 42% was obtained when five different sorts of amino acid were fed simultaneously at the exponential phase. The res更多还原