高效纤维素分解菌的筛选及其包埋固定化

发布时间：2018-03-07 06:00

本文选题：纤维素分解菌　切入点：酶活测定　出处：《东北农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来,随着资源短缺和环境污染问题不断加剧,秸秆纤维素作为可再生的生物质资源,其处理和利用不仅关乎新能源的开发,缓解全球能源短缺的现状,同时也关系到农村的生态保护以及新农村建设。因此秸秆还田作为最环保、利用率最高的处理方式受到国内外研究者的广泛关注。随着分子生物学的不断发展,秸秆还田的方式也已经发生改变,从传统的焚烧、机械粉碎等方式逐渐倾向于向秸秆中投加纤维素类微生物菌剂,来促进秸秆纤维素的腐解,此方法既不会造成环境污染,又能提高土壤的肥力,改良土壤结构。目前已经从土壤中筛选出的纤维素类微生物已经有上千株,但总体来讲,它们对秸秆的降解能力比较单一,降解效果一般。因此筛选高效的纤维素分解菌是降解秸秆纤维素的重中之重。本试验基于获得高效纤维素分解菌的目的,通过分离纯化初步得到30株菌株,利用刚果红染色法进行初筛,共得到14株纤维素分解菌,采用滤纸条崩解试验进一步筛选,共得到5株效果较好的纤维素分解菌,利用秸秆和麸皮作为碳源,对筛选出来的5株菌株进行连续5d的发酵产酶培养,并每隔24h取样,利用DNS显色法测定其CMCase活力和FPA活力,最终确定4株纤维素优势分解菌,并分别命名为X-1,X-6,X-7和X-11,同时测定4株菌株的CMCase活力、FPA活力以及β-Gase活力,4株菌株的CMCase活力均较高,分别为71.17 U/m L,57.48 U/m L,65.96 U/m L和69.2 U/m L;FPA活力最低和最高的菌株分别为X-6和X-11,酶活分别可达到34.17 U/m L和53.41 U/m L;菌株X-11的β-Gase活力最高,为74.43 U/m L,而菌株X-7的β-Gase活力最低也可达到35.79 U/m L。综合考虑,筛出的4株纤维素分解菌均具有较高的产酶能力。将该4株纤维素优势分解菌应用于秸秆的固液态发酵,在为期30d发酵中,同自然状态下进行固态发酵相比,其对秸秆的降解率分别提高了28.92%,30.40%,30.33%和37.99%;同自然状态下进行液态发酵相比,其对秸秆的降解率分别提高了31.92%,40.15%,35.29%和39.98%,表明4株菌均具有相对高效的降解秸秆纤维素的能力。此外,以菌株X-11为例,用海藻酸钠包埋法对其进行固定化,通过正交试验确定包埋剂的浓度为5%、固定剂的浓度为5%、包埋剂与菌液比为2:1以及交联时间为24 h时,制备的固定化菌体小球质量最佳。并根据Logistic方程和Luedeking-Piret方程,利用Origin8.0软件,构建了固定化菌体X-11的动力学模型,为探索固定化微生物在降解纤维素领域的应用提供了理论依据。经过对4株高效纤维素分解菌进行分子鉴定,根据16Sr DNA序列比对结果发现,菌株X-1,X-7和X-11均为粪产碱杆菌;菌株X-6属于解糖假苍白杆菌。由于该菌属应用于降解纤维素领域的研究较少,本研究的结论为该菌属在此类应用上的后续研究提供了一定的理论依据和实际意义。
[Abstract]:In recent years, with the shortage of resources and the increasing problems of environmental pollution, straw cellulose as a renewable biomass resources, its processing and utilization is not only related to the development of the new energy, to alleviate the current situation of global energy shortage, but also related to the ecological protection in rural areas and new rural construction. So the straw as the most environmentally friendly, the utilization rate of treatment the highest attention of researchers at home and abroad. With the development of molecular biology, the straw has changed from the traditional way of burning, mechanical crushing gradually tend to straw cellulose adding microbial agents, to promote the straw cellulose decomposition, this method not only will not cause environmental pollution. It can improve the soil fertility, improve soil microbial structure. The cellulose has been screened from soil has thousands of lines, but generally speaking They, on the degradation of straw degradation ability is relatively simple, general effect. So the screening of high efficient cellulose decomposing bacteria is the priority among priorities. Straw cellulose degrading the efficient cellulose decomposing bacteria on 30 strains were obtained by the separation and purification, were screened using Congo red staining method, 14 strains of cellulose decomposing bacteria are common, with the collapse of filter test further screening, 5 strains of cellulose decomposing bacteria better effect can be obtained, using straw and wheat bran as carbon source for screening out of 5 strains of 5D continuous fermentation culture, and every 24h sampling, to determine the CMCase activity and FPA activity by DNS colorimetric method finally, we selected 4 strains of cellulose decomposing bacteria advantage, and were named as X-1, X-6, X-7 and X-11, the simultaneous determination of 4 strains of CMCase activity, FPA activity and beta -Gase activity, 4 strains of CMCase activity were higher, U/m were 71.17 L, 57.48 U/m L, 65.96 U/m L and 69.2 U/m L; the activity of FPA in the lowest and highest strains were X-6 and X-11 respectively, the enzyme activity can reach 34.17 U/m L and 53.41 U/m L; strain X-11 beta -Gase activity is the highest, 74.43 U/m L, and strain X-7 beta -Gase activity the minimum can be reached 35.79 U/m L. considering the enzyme production capacity of the screened 4 strains of cellulose decomposing bacteria were high. The liquid and solid fermentation of 4 strains of cellulose decomposing bacteria used in the advantages of straw, in a 30d fermentation, with the natural state of solid state fermentation, the degradation rate of straw respectively. Increased by 28.92%, 30.40%, 30.33% and 37.99%; with the natural state of liquid fermentation, the degradation rate of straw were increased by 31.92%, 40.15%, 35.29% and 39.98%, showed that 4 strains with degradation of straw cellulose with relatively high efficiency ability. In addition, the strain X-11 as an example, It was immobilized on sodium alginate embedding method, through orthogonal test to determine the concentration of embedding agent is 5%, the concentration of fixing agent 5%, embedding agent and bacteria liquid ratio 2:1 and reaction time is 24 h, the preparation of immobilized cell beads of the best quality. And according to the Logistic equation and Luedeking-Piret equation using Origin8.0 software, constructs dynamic model of immobilized bacteria X-11, and provides a theoretical basis for the application of immobilized microorganisms in cellulose degradation field. After 4 strains of cellulose decomposing bacteria, molecular identification, according to the 16Sr DNA sequence alignment results showed that strains X-1, X-7 and X-11 were Alcaligenes faecalis strain X-6; belong to the sugar solution. Because of the false Ochrobactrum sp. used in cellulose degradation area is less, the conclusion of this study provides certain theory basis for further research and the bacteria in such application. Practical significance.

【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X712;X172

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