【作者】 陈杰；

【导师】 李元广； 魏鸿刚；

【作者基本信息】 华东理工大学 ， 生物化工， 2013， 硕士

【摘要】 海洋芽孢杆菌B-9987对细菌性土传病害病原菌以及真菌性叶部病害病原菌均具有良好的拮抗效果,有望被创制成国内外第一个海洋微生物农药。由于急需大量发酵液制备足量样品进行剂型研究及田间药效试验,所以本文首先对B-9987原有发酵工艺进行中试放大,然后针对放大过程中存在的问题对原有工艺进行优化并对优化后的工艺进行放大；此外,本文还对B-9987产新结构环脂肽抗菌物质BM-3的发酵培养基进行优化。500L及5000L发酵罐中试结果表明,原有50L罐发酵工艺基本可放大,放大后发酵液中的活菌含量、芽孢率及抑菌活性等指标与50L罐基本接近。针对放大过程中出现的发酵过程不稳定问题,进行了培养条件的系统优化,确定了如下摇瓶培养工艺：初始pH为7.0,培养温度28℃,适合菌体生长的接种量0.6-2%,适合活性物质产生的接种量1-2%,装液量50mL/250mL:此外,所确定的芽孢率测定条件为：80℃水浴处理15min。对中试放大过程中出现的两种不同菌落形态菌株进行对比分析,通过平板传代培养、不同摇瓶及不同培养基培养等方法,探讨了产生不同菌落的原因。针对放大过程中出现的起泡难以控制、培养基成本高等问题,对原有培养基BMF-1进行优化,所确定的BMF-2培养基不仅有利于B-9987菌体生长、芽孢形成及起泡控制,而且成本大幅度降低(约为BMF-1的5%),适合于大规模培养。基于BMF-2培养基及优化后培养条件的500L罐发酵结果表明,与优化前相比,发酵液中的活菌含量提高41.7%,抑菌圈增大4.5mm,起泡高峰期缩短l0h。最后,本文通过影响面法对B-9987产新结构环脂肽抗菌物质BM-3的发酵培养基(BMF-2)进行优化,确定了BMF-3培养基,5L罐的发酵结果表明,发酵过程中的最高BM-3浓度达362mg/L,较原来的282mg/L提高了28.4%。上述研究结果,不仅为海洋芽孢杆菌B-9987微生物农药的创制与产业化提供了重要的技术保障,而且为该菌产生的新结构环脂肽抗菌物质BM-3的发酵工艺优化奠定了基础。更多还原

【Abstract】 Bacillus marinus B-9987has a great antagonism effect on a wide range of plant pathogens (both bacterial soil-borne disease and fungal leaf disease), It has a promising potential for being made the first marine microbial pesticide. First, we completed the pilot research based on the original Fermentation technology because we need a large number of fermentation broth to make formulation research and field tests, then we solved problems in the process of scare up by optimizing the cultivation technology and we enlarge the new technology. Besides, we optimizted the medium for new angifungal cyclic lipopeptide BM-3produced by B. marinus B-9987.The cultivation results in500L and5000L fermenters showed that the original50L cultivation technology can be scared up. The cell density, bacteriostatic activity of fermentation broth, spore rate in500L and5000L fermenters are close to the results in50L fermentation.The cultivation conditions of B. marinus B-9987was optimized for the problems of process instability. The results are in the below:initial pH7.0, temperature:28℃, inoculum concentration:0.6-2%for cell growth,1-2%for bacteriostatic substance production, liquid volume:50mL/250mL in flask, detection conditions of spore rate:80℃water bath treated15min.We compared the difference between two different colonies strains and explored the reasons of the two colonies by subculturing two colonies in plate medium, culturing them in different media, different flasks.The original medium were optimized for the problems of high cost and foaming hard to control. Compared to the original one, the new medium BMF-2was better for cell growth, sporulation, foaming control, and the cost was decreased sharply (about5%of the medium BMF-1). The mew medium was suitable for large-scale cultivation.Compared to the original cultivation technology, cell density was increased41.7%, inhibition zone was increased4.5mm, foaming time was shortened10hours using new technology based on cultivation conditions and medium optimization in500L fermenter.Finally, response surface methodology (RSM) was employed to optimize the medium for increasing new antifungal cyclic lipopeptide BM-3concentration. The BM-3concentration was increased from282mg/L to362mg/1(increased by28.4%)in a5L fermenter.The above results not only provided important technical support for the creation and commercialization of the microbial pesticide of B.marinus B-9987, but also made a solid basis for the fermentation technology optimization of new antifungal cyclic lipopeptide BM-3concentration produced by the strain.更多还原