腐熟用枯草芽孢杆菌菌剂的研制

发布时间：2018-02-12 21:20

  本文关键词： 枯草芽孢杆菌 芽孢 发酵工艺 微生物菌剂 制备　出处：《河北科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：腐熟菌剂主要是指针对畜禽粪便,植物残体以及一些生活废弃物等难分解的有机物进行处理而研究生产出的一类微生物菌剂,广泛应用农作物秸秆、畜禽粪便的资源化和无害化处理。枯草芽孢杆菌是在自然界广泛存在的一种好氧的产芽孢杆菌。它可以产生蛋白酶、淀粉酶、纤维素酶和半纤维素酶等酶,是农作物秸秆、畜禽粪便等有机物料腐熟菌剂的主要菌种之一。芽孢数和有效活菌数是腐熟菌剂质量的重要指标。生产成本也是微生物菌剂生产企业所关心的重要问题。本课题研究了枯草芽孢杆菌DK36的产芽孢条件,优化了发酵工艺。在此基础上,研究了菌剂的制备工艺。为了降低生产成本,进行了以脱酚棉籽蛋白为氮源培养枯草芽孢杆菌DK36的研究。其主要研究结果如下:1)枯草芽孢杆菌DK36的产芽孢条件研究。当葡萄糖、蔗糖、果糖、可溶性淀粉、玉米粉、麸皮为碳源时,菌体生长良好。葡萄糖和蔗糖作碳源时均不产生芽孢。当麸皮作为碳源时,形成的芽孢最多。其次为玉米粉、可溶性淀粉。蛋白胨、酵母浸粉、大豆蛋白胨、豆饼粉、硝酸钾、硝酸钠、硝酸铵均可使菌体大量形成芽孢,其中大豆蛋白胨的效果最好。尿素抑制菌体生长和芽孢形成。添加Ca2+、Mg2+、Na+、Fe2+、Mn2+均可促进芽孢的形成,其中效果最明显的是硫酸锰。Cu2+对生长和形成芽孢均有很大抑制作用。产芽孢最佳培养基:麸皮15.0 g/L、大豆蛋白胨5.0g/L、硫酸锰0.1 g/L、碳酸钙3.0 g/L。最佳产芽孢发酵条件:培养温度30℃、转速200 r/min、接种为2%、培养基初始pH为7.0,芽孢数达3.6×109 cfu/mL。2)脱酚棉籽蛋白培养枯草芽孢杆菌DK36的研究。棉籽蛋白作为氮源时,菌体生长良好。碳源的种类对产芽孢有显著影响。葡萄糖、蔗糖、玉米粉、麸皮均使菌体生长良好。但葡萄糖和蔗糖为碳源,菌体不形成芽孢,而玉米粉和麸皮可使菌体产生大量芽孢。添加Ca2+、Mn2+、Mg2+和K+对芽孢形成有明显的促进作用,并且发现钾盐与镁盐、锰盐、钙盐复合效果更佳。研究还发现,脱酚棉籽蛋白作为氮源会造成芽孢形成缓慢。脱酚棉籽蛋白培养枯草芽孢杆菌的最佳培养基:玉米粉30.0g/L、棉籽蛋白30.0 g/L、硫酸镁2.0 g/L、磷酸二氢钾4.5 g/L。其最佳发酵条件:培养温度30℃、摇床转速200 r/min、接种量为8%,芽孢数和芽孢率分别达到3.5×109cfu/mL和98.7%。3)枯草芽孢杆菌DK36菌剂的制备。尽管芽孢能耐高温,但随着干燥温度升高,菌剂有效活菌数显著下降。发酵液pH7.0时,菌剂活菌数最高。载体种类对菌剂质量影响十分显著。麸皮、硅藻土和活性炭作载体时,菌剂有效活菌数相近。次面粉,玉米粉作菌剂载体效果较差。当添加葡萄糖、淀粉、糊精时,对菌剂活菌数影响不显著。枯草芽孢杆菌菌剂最佳制备工艺:发酵液与载体比例为1.5:1、干燥温度70℃、菌液pH为7.0、载体为麸皮,水分含量为3.3%,菌剂有效活菌数为26亿/g。
[Abstract]:Rotting bacteria mainly refers to a class of microorganism agents which are studied and produced for the treatment of difficult decomposable organic matter such as animal manure, plant residues and some domestic wastes, and are widely used in crop stalks. Bacillus subtilis is a aerobic bacillus widely found in nature. It can produce enzymes such as protease, amylase, cellulase and hemicellulase. The number of spores and the number of viable bacteria are the important indexes of the quality of the rotting agent. The production cost is also an important issue concerned by the microbial agent production enterprises. The spore production conditions of Bacillus subtilis DK36 were studied. The fermentation process was optimized. On the basis of this, the preparation process of the bacterial agent was studied. In order to reduce the production cost, The DK36 of Bacillus subtilis cultured with desphenol cottonseed protein as nitrogen source was studied. The main results were as follows: 1) the spore production conditions of Bacillus subtilis DK36 were studied. When glucose, sucrose, fructose, soluble starch, corn meal, glucose, sucrose, fructose, soluble starch, corn flour, When wheat bran was carbon source, bacteria grew well. When glucose and sucrose were used as carbon source, no spores were produced. When wheat bran was used as carbon source, spore formation was the most, followed by corn flour, soluble starch, peptone, yeast extract, soybean peptone. Soybean cake powder, potassium nitrate, sodium nitrate and ammonium nitrate can make the bacteria form spores in large quantities, among which soybean peptone is the best. Urea inhibits the growth of bacteria and spore formation. Addition of Ca2 to mg _ 2, Na, Fe _ 2 and mn _ 2 can promote the formation of spores. The best medium for spores production was: bran 15.0 g / L, soybean peptone 5.0 g / L, manganese sulfate 0.1 g / L, calcium carbonate 3.0 g / L. A study on the culture of Bacillus subtilis DK36 with denatured cottonseed protein at 200rmin, inoculation at 2um, initial pH 7.0, spore number 3.6 脳 109cfumL. 2). When cottonseed protein was used as nitrogen source, the bacteria grew well, and the species of carbon source had a significant effect on spores production. Sucrose, corn flour and bran all made the bacteria grow well, but glucose and sucrose were the carbon source, the bacteria did not form spores, but corn flour and bran could make the bacteria produce a large number of spores. The addition of Ca2, mn 2, mg 2 and K could obviously promote the formation of spores. It was also found that the combination of potassium salt and magnesium salt, manganese salt, calcium salt is better. Dephenol cottonseed protein as a nitrogen source causes slow spores formation. The best medium for dephenol cottonseed protein culture of Bacillus subtilis is corn flour 30.0g / L, cottonseed protein 30.0g / L, magnesium sulfate 2.0 g / L, potassium dihydrogen phosphate 4.5 g / L. its optimum fermentation conditions are as follows:. The culture temperature is 30 鈩，

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