【作者】 张艳；

【导师】 刘大群； 杨文香；

【作者基本信息】 河北农业大学 ， 植物病理学， 2006， 硕士

【摘要】 链霉菌是一种来源于土壤的革兰氏阳性菌,是重要的工业微生物,可以产生各种各样的生物活性物质,被广泛的应用在生物防治植物病害的研究中。玫瑰黄链霉菌Men-myco-93-63是从土壤中分离得到对棉花黄萎病菌表现强烈抑制作用的菌株。本研究采用纸碟法,以棉花黄萎病菌为指示菌,对该菌株的培养基、摇床发酵条件进行优化,最后进行了30L发酵罐的小试试验。 本研究首先进行发酵培养基的优化。考察了碳源、氮源、无机盐对发酵的影响,然后再单因素试验的基础上进行了正交试验的优化。确定的培养基配方为:葡萄糖2.4%,淀粉0.8%,玉米浆0.8%,花生饼粉1.5%,NaCl0.4%,KH₂PO₄0.02%,CaCO₃0.3%。改良后的发酵培养基成本降低由原来2.13元/升降低到0.5元/升,降低了76%,在此培养基下发酵,抑菌圈直径比原发酵培养基提高了27%。 对于种子培养基的优化采取在发酵培养基的基础上,改变碳、氮源的含量设计正交试验,以菌体生长量为指标进行种子培养基组成试验。确定了种子培养基配方为:葡萄糖1.8%,花生饼粉0.5%,玉米浆1.4%,NaCl0.4%,CaCO₃0.3%,KH₂PO₄0.02%。 针对培养条件分别进行了种子培养和发酵培养的培养基初始pH值、摇床转速、摇瓶装液量、培养温度的研究并考察了种子培养的种龄和发酵培养接种量。确定的种子培养条件为:初始pH值7.5,装液量50mL/500mL,温度30℃,转速220 r/min,种龄30h。发酵培养基的培养条件是:接种量8%,初始pH 7.5,装液量100mL/500mL,温度30℃,转速200r/min,培养时间120h。并在摇瓶优化条件下测定了摇瓶中的代谢曲线。 在以上试验的基础上进行了30L发酵罐的小试试验。测定了包括pH值、总糖、还原糖、氨基氮、生物量、抑菌圈直径在内的代谢情况,得到了在发酵罐中生物指标和产物合成规律,为发酵放大奠定了基础。更多还原

【Abstract】 Streptomyces is a kind of Gram-positive bacteria which exist in the soil. They are a kind of important microorganisms used in industrial production. Microorganisms in this genus can produce kinds many bioactive compounds. Streptomyces roseoflavus Men-myco-93-63 is a strain isolated from soil and showed strong inhibition against Verticillium dahliae. In this research, Verticillium dahliae was used as the target pathogen to study the substrate and culturing conditions for achieving higher productivity.Study for substrate was carried out for optimizing nutritional requirements. First the concentration of glucose was tested. Then different carbon sources （starch, dextrin, sucrose, maltose） were tested as a combination with glucose. Various of nitrogen sources （peanut meal, corn slurry, yeast extract, beef extract, urea, peptone, （NH₄）₂SO₄, NH₄NO₃, NaNO₃） was studied. Similarly, optimum requirements for micronutrients were also studied by varying the concentration of FeSO₄, ZnSO₄, MgSO₄, MnCl₂, CoCl₂, KH₂PO₄, CaCO₃, NaCl. Some factors were chosen. In the last, the chosen factors were optimized by orthogonal design. The optimized fermentation medium consisted of 2.4% glucose, 0.8% starch, 0.8% corn slurry, 1.5% peanut meal, 0.4% NaCl, 0.02% KH₂PO₄,0.3% CaCO₃. Its cost reduced 76% and inhibition zone enhanced 27% compared to former medium.In optimizing seed medium, the concentration of carbon and nitrogen sources was changed in another orthogonal design. In this experiment, biomass was chosen as the index and seed substrate was got. Its components were: 1.8% glucose, 1.4% corn slurry, 0.5% peanut meal, 0.4% NaCl, 0.02% KH₂PO₄, 0.3% CaCO₃.Some fermentation control parameters including aeration, agitation, pH, temperature of incubation, fermentation cycle time, etc were studied. The germ culturing condition was as follows: the inoculums was grown in 50 ml seed medium in 500 ml Erlenmeyer flasks at 30℃ for 30 h on a rotary shaker （220 r/min）. Of this, 8% v/v seed was transferred to 100 ml production medium in 500 ml Erlenmeyerflasks. Fermentation was carried out at 30°C for 140 h. Metabolizing curve was tested in Erlenmeyer flasks.After optimizing nutritional requirements and control parameters at shake flask level, scale up studies were carried out in 30 L fermentor. The metabolizing parameters including pH, biomass, total sugar, deoxidize sugar, ammonia nitrogen and output were tested. It laid a foundation for large scale fermentation.更多还原