【作者】 芦光新；

【导师】 陈秀蓉；

【作者基本信息】 甘肃农业大学 ， 草地生物多样性， 2012， 博士

【摘要】 在我国北方尤其是高寒地区,油菜秸秆通常被农牧民用作燃料,造成了资源浪费和环境污染。探索一种构建高效降解油菜秸秆细胞壁物质多酶系菌群的方法,采用微生物发酵技术降解油菜秸秆,对促进油菜秸秆资源循环和再生利用具有十分重要的意义。论文以项目组前期分离获得的来自东祁连山高寒草地土壤中的真菌菌株作为研究材料,采用底物显色的初步筛选法和酶活力复选法,筛选出产羧甲基纤维素酶、木聚糖酶和漆酶菌株,通过rDNA-ITS基因序列分析、培养特性、液体发酵粗酶液的酶学性质及不同菌株液体发酵粗酶液之间的协同效应分析,构建多酶系菌群,并研究多酶系菌群对油菜秸秆的降解活性,取得了以下主要结果：(1)从125株真菌菌株中筛选得到56个纤维素分解菌株,其中：有49个菌株能在滤纸平板培养基上生长,且不同菌株对滤纸的分解程度不同；筛选出34个既能产羧甲基纤维素酶又能产木聚糖酶的菌株,稳定性测定后,最终获得4株高产羧甲基纤维素酶和木聚糖酶的菌株,经rDNA-ITS基因序列分析,4个菌株初步鉴定为：阿太菌属(Athelia sp.)(菌株23a)、Saccharicola sp(菌株24d)、埃里砖格孢属(Embellisia sp.)(菌株26B)、梭孢壳属(Thielavia sp.)(菌株H)。在56个菌株中,有9个菌株在-萘酚-PDA培养基和愈创木酚-PDA培养基上都能产生褐色氧化带,其中有6个菌株能在分别以愈创木酚、邻苯二酚、邻苯甲苯胺为底物的选择性培养基上生长,产生褐色氧化带；以愈创木酚为底物测定漆酶活力,结果发现只有菌株310b产生漆酶。经rDNA-ITS基因序列分析,初步鉴定为：小皮伞属(Marasmiussp.)。(2)对菌株310b产漆酶条件的研究结果表明：菌株310b在25℃、pH=4.0时产漆酶的酶活力最大；9种碳源抑制菌株产漆酶,蛋白胨诱导菌株产漆酶,且酶活力最高；邻甲苯胺、-萘酚和吐温-80等3种非营养有机物促进菌株产漆酶,愈创木酚对菌株产漆酶没有明显的作用；邻苯二酚、单宁酸、吲哚乙酸抑制菌株产漆酶,其中吲哚乙酸抑制作用最强烈；随Cu2-增加,产漆酶活力增加,0.025g/L时产酶活力最大；随着接种量的增加,诱导菌株产漆酶；转速在0～180r min-1范围内,随着转速的增加,漆酶的酶活力增加,且转速达到180r min-1时,酶活力达到最大。(3)5个菌株生长的最适温度范围在25-35℃之间,25℃条件下菌株两两混合培养7d后,发现5个菌株间可以共存。5个菌株产酶的种类及酶活力大小不同,菌株26B产生的羧甲基纤维素酶活力最高,24d产生的木聚糖酶活最高；将不同菌株的发酵粗酶液两两等体积混合后,与对应的单个菌株的酶活力相比较,反应体系中羧甲基纤维素酶、木聚糖酶和漆酶的酶活力下降,5个菌株间随机组合进行液体发酵后,单个菌株和组合菌株反应体系中的酶活力t测验结果表明,漆酶的酶活力变化差异显著(p<0.05),但在菌株26B、310b和24d的不同组合中,羧甲基纤维素酶和木聚糖酶的酶活力变化差异不显著(p>0.05)。(4)产羧甲基纤维素酶菌株26B在55℃、pH=6时酶的催化活性达到最大值,菌株23a在30℃、pH=5时酶的催化活性达到最大值,菌株H在50℃、pH=5.5时酶的催化活性达到最大值；菌株26B产生羧甲基纤维素酶对热不稳定,菌株23a和H的羧甲基纤维素酶在0-40℃相对稳定。产木聚糖酶菌株24d在55℃、pH=5时酶的催化活性达到最大值。菌株23a在35℃、pH=5.5时酶的催化活性达到最大值,菌株H在55℃、pH=5.0时酶的催化活性达到最大值。菌株24d产生的木聚糖酶对热不稳定,菌株23a和H的木聚糖酶在0-35℃相对稳定。产漆酶菌株310b在30℃、pH=4.5时酶的催化活性达到最大值,且在0-45℃酶活力相对稳定。(5)将菌株26B、310b和24d等3个菌株组合后进行固体发酵,结果表明,相比于其他组合,固体发酵60d后,反应体系中的相对还原糖含量高,相对纤维素含量低,油菜秸秆颜色变浅、菌丝附着多,扫描电镜结果显示,与对照相比,秸秆表面、断裂处、秸秆髓部等都发生了明显的变化。菌株310b、24d和26B组合的多酶系菌群对油菜秸秆的降解效果明显。更多还原

【Abstract】 Rape straw is usually used as fuel and becomes waste and pollution sources in northern China especially in the alpine region. Methods by using fungi system of multi-enzyme to degradation rape straw cell wall material were carried out by traditional microbiological methods. Fungal strains, produce high enzyme activity, was screened, including cellulose, xylanase and laccase. Gene sequences of rDNA-ITS, cultural characteristics, enzymatic properties of liquid crude enzyme, and the synergies between different enzyme was analyzed as well. Construction of fungi system of multi-enzymes for degradation of rape straw cell wall material and its degradation characteristics was studied. The results obtained as follows:(1) Based on preliminary work of isolating125fungal strains from the east Qilian Mountain alpine grassland soil.Fifty six fungal strains were screened by using traditional chromogenic substrate screening method, and forty nine fungal strains can grow on filter paper plate medium,thirty four fungal strains that could produce high enzyme activity of both cellulose and xylanase were screened by enzyme activity screening method in flask liquid fermentation while rape straw used as sole carbon source. Four fungal strains with high yield of cellulose and xylan enzyme activity, Embellisia sp.(strain26B), Saccharicola sp.(strains24d), Athelia sp.(strain23a), Thielavia sp.(strain H) were obtaied. One fungal strain, code310b, producing laccase was obtained, and identified as Marasmius sp., Based on rDNA-ITS gene sequence analysis.(2) To Marasmius sp., the optimum temperature of laccase activity producing is25℃with pH value4.0; Add sugar source will inhibit laccase activity of the strains; peptone induced the highest laccase activity among the nine nitrogen sources tested; O-toluidine, a-naphthol and Tween-80can promote the strains laccase activity while catechol, tannic acid, indole acetic acid inhibit the strains laccase activity, and indole acetic acid inhibit it strongest among the test acid; Guaiacol had no siginificant effect on laccase activity. Laccase activity increased with the content of Cu2+increasing, and it reached up to maximum when Cu2+was added to0.025g/L, Laccase activity arosen with the increase of rotar speed.(3) Culture characteristics of five tested strains showed that the optimum growth temperature range between25-35℃. It was conducive that the five strains can coexist under the culture conditions of25℃. The type of enzyme and enzyme activity of five fungal strains were different determined in flask liquid fermentation while rape straw used as sole carbon sources. Strain26B,23a, H,24d can produce both cellulase and xylanase, Carboxymethyl cellulose activity (CMCase) of strain26B was the highest, xylanase activity of strain24d was the highest. Three kinds of enzyme activity was measured after crude enzyme solution of single strain was mixed by equal volume in order to further understand synergies between different type of enzyme of crude enzyme of five strains. Multi-enzyme activity decreased to a certain extent. And combination strains were inoculated and mixed culture in flask liquid fermentation was measured, As opposed to single strain fermentation, enzyme activity of mixed strain fermentation was decline, the reason was probably the environmental conditions of mixture reaction system change, and was related to changes of pH value and reducing sugar content in closely.(4) To strain26B, the optimum temperature of CMCase activity producing is55℃with pH value6.0, it is unstable on the temperature.to strain23a, the optimum temperature of CMCase activity producing is30℃with pH value5.0; and to strain H, the optimum temperature of CMCase activity producing is50℃with pH value5.5; Strains23a and H are relatively stable under the condition of temperature range between0-40℃. To strain24d, the optimum temperature of xylanase activity producing is55℃with pH value5.0, it is unstable on the temperature. to strain23a, the optimum temperature of xylanase activity producing is35℃with pH value5.5, to strain H, the optimum temperature of xylanase activity producing is55℃with pH value5.0, Strain24d is instability on the temperature, the xylanase of strain23a and H is relatively stable under the condition of temperature range between0～35℃. To strain310B, the optimum temperature of laccase activity producing is30℃with pH value4.5, the laccase activity reaches its maximum, and the enzyme activity is relative stability under the condition of temperature range between0～45℃(5) In the solid fermentation system which made up of strains310b,24d and26B, the CMCase, xylanase and laccase activity could detect in the crude enzyme of fermentation. A conclusion that there was high correlation between three kinds of enzymes activity in the number of combinations of the reaction system and the relative cellulose content, the same as relative reducing sugar content did were obtained by analysis the mutual relations of the relative content of reducing sugar and enzyme activity and the relative cellulose content in the different reaction system of combinations strain. It indicated that the decomposition effect of cell wall material of rape straw was better in multi-enzymes fungi system.更多还原