【作者】 谢久凤；

【导师】 喻子牛；

【作者基本信息】 华中农业大学 ， 微生物学， 2010， 硕士

【摘要】 猪粪,牛粪中的粗纤维经亮斑扁角水虻幼虫生物转化之后有明显的降低,水虻肠道微生物在其中也起着比较重要的作用。从环境保护和发现利用新的菌种资源角度来讲,有必要对亮斑扁角水虻幼虫肠道内具有纤维素酶活的菌株进行鉴定和分析。本课题以猪粪饲养的亮斑扁角水虻幼虫为研究对象,在对猪粪消化能力最强的4龄幼虫肠道内筛选并鉴定了七株具有纤维素酶活性的细菌；通过进一步比较分析,获得一株纤维素酶活性较高的菌株X4；分别提取猪粪和水虻处理后的猪粪残渣中的粗纤维,使用X4菌株对其进行降解；设计正交试验,对使用X4菌株生产纤维素酶的发酵条件进行优化,为其应用提供参考。试验结果：从水虻幼虫肠道内筛选出七株具有纤维素酶活性的菌株X1,X2,X3,X4, X5, X6, X7并做系统进化树分析；抑菌试验表明：X1,X2和X3菌对核盘菌无抑制作用,X4, X5, X6, X7菌对核盘菌有明显的抑制作用,其中X4菌对大肠杆菌有抑制作用,X5对金黄色葡萄球菌有抑制作用。酶活测定结果表明,在相同条件下X4菌株纤维素酶活为7个国际单位。测定X4菌株的生长曲线时发现,5-7小时可达到对数生长期。提取新鲜猪粪和水虻幼虫转化后的猪粪残渣中粗纤维,使用X4菌株进行降解,培养48小时后计算降解率,发现猪粪中12.64%的干物质被降解,残渣中10%的干物质被降解。用玉米粉和豆粕粉配制产酶发酵培养基,对X4发酵培养条件进行优化。结果显示,培养基初始pH为9,发酵温度45℃,发酵时间36小时,产酶活性最高,可达117.13 IU,比优化前提高了近17倍。这为工业化生产纤维素酶提供了参考。更多还原

【Abstract】 The coarse fiber of swine manure and shard was significantly low after digesting by Hermetia illucens L. maggots and during the process microbe in intestines of maggots may play an important role. Considering the environmental protection and utilization new strains, it was necessary to screen and identify strains with enzyme activity of cellulose. Seven strains with the activity of cellulose had been screened and identified from the 4th larvae when they had the best digestion to swine manure. Through the determination of CMCase activity and FPA activity, obtain a strain named X4 with high cellulose activity. Extract the crude fiber from swine manure and residue that had been digested by Hermetia illucens L. maggots and degrade with X4. Design orthogonal test to optimize the conditions of fermentation. The results were as follow.Seven strains had been screened. There were named X1, X2, X3, X4, X5, X6 and X7. Determine the seven strains by the methods of morphological characteristics and 16s rDNA. The antibiotic spectra of the seven showed X4 could inhibit Escherichia coli and Sclerotinia sclerotiorum, X5 could inhibit Staphylococcus aureus and Sclerotinia sclerotiorum, X6 and X7 could inhibit Sclerotinia sclerotiorum. Determine enzyme activity of the seven strains with the methods of DNS and FPA. X4 had the highest enzyme activity (7 IU).Determine the X4-growth curve with beef cream-peptone culture medium. The result showed that the logarithmic growth phase of X4 was 5-7h. Culture them for 48h and calculate the degradation rate. The results were that the degradation rate of swine manure was 12.64% and the degradation rate of residue was 10%.Make the fermentation medium with corn and soybean. The optimal condition of producing cellulose was studied by three factors and three levels orthogonal experiment and the results were the best initial pH 9.0, incubating temperature 45℃and incubating time 36h. The enzyme activity was 117.131U and increased nearly 17 times than before. Introduced reliable technical parameters are suitable for commercial production更多还原