【作者】 汪婷；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2005， 硕士

【摘要】 沼气发酵过程是一个由多种微生物联合，交替作用的复杂生化过程。在发酵过程中，不产甲烷细菌和产甲烷菌之间，相互依赖，又互相制约，在发酵过程中处于平衡状态。而在发酵过程中微生物的代谢活动与相互作用也体现在发酵反应中物料的理化性质的改变上。 在沼气发酵中，产甲烷菌是沼气发酵微生物的核心，作为自然界碳素循环中厌氧生物链的最后一个成员，分析沼气发酵过程中产甲烷菌的变化，对于了解发酵程度，优化发酵条件，提高沼气产量，具有十分重要的意义。目前已知，自然环境中的微生物有90-99％是还未可培养的，加之产甲烷菌的严格厌氧性、培养周期长等特点，使得通过传统的培养分离技术对产甲烷菌群落结构的分析十分困难，随着现代分子生物学的发展，特别是16S rDNA分析的方法引入厌氧环境微生物群落的研究中，使得快速、全面、准确地研究厌氧系统内的产甲烷菌的群落结构的多样性成为可能。 本文研究了以奶牛粪便为原料的沼气发酵过程中的物料理化性质的改变、参与发酵过程的六大微生物生理群的数量变化。同时采用不依赖于培养的16SrDNA分析的方法研究了沼气发酵的不同阶段沼液中产甲烷菌的多样性的变化。并通过传统的Hungate厌氧技术从发酵液中分离出产甲烷菌并进行了初步的鉴定。 一、沼气发酵过程中主要微生物生理群的演替与物料的变化 研究了以奶牛粪便为原料的沼气发酵过程中六大微生物生理群(好氧产酸菌、厌氧产酸菌、好氧氨化菌、厌氧氨化菌、厌氧纤维素降解菌、产甲烷菌)数量的变化，以及在此过程中物料理化性质的变化。实验表明：发酵前期，以好氧微生物活动为主，随着发酵的进行，厌氧微生物，特别是产甲烷菌的作用越来越显著。 在发酵过程中，挥发性有机酸，全氮、pH、氧化还原电位都发生变化，其变化与沼液中微生物类群变化相关。 二、沼液总DNA的提取和纯化方法 通过直接提取法和间接提取法的比较，确立了从沼液中提取总DNA的方法。比较结果表明，尽管直接提取法的得率要高于间接法，但由于沼液样品中的腐殖质等杂质的含量较高，经过纯化回收后的直接法提取的总DNA，仍无法通过PCR扩增的方法得到所需的片断，无法进行后续试验。而间接法的得率虽然较低，但所得总DNA纯度高，无需纯化，即可进行后续操作。 三、沼气发酵过程中产甲烷多样性的变化 采用不依赖于培养的16SrDNA分析的方法研究了沼气发酵的不同阶段产甲烷菌的多样性的改变。首先提取了发酵前、发酵启动期、盛产期前期、盛产期、发酵末期五个阶段的沼液总DNA，应用产甲烷菌特异性的16SrDNA引物通过PCR扩增了五个阶段的沼液样品的总DNA，建立了五个沼液样品16SrDNA文库，应用HaeⅢ-HpaⅡ两种四碱基限制性核酸内切酶对五个文库中的16SrDNA片段进行了限制性片段长度多态性分析。共获得127种酶切类型。应用各种多样性和均度参数对五个文库HaeⅢ-HpaⅡ酶切类型的丰富度和分布进行了比较。对五个文库的16SrDNA主要HaeⅢ-HpaⅡ酶更多还原

【Abstract】 The methane fermentation, is a process that unites by the many kinds of microorganisms, with replacement complex biochemistry process in it. In the fermentation, The interdependence and rectriction of no-produce -methane bacterium and methanogens makes a equilibrium in the fermentation. But microorganism’s metabolic activity and affection also manifests in the physical and chemical changes of material in fermentation.Methanogen is the core of the methane fermentation. And methanogen is the last member of the anaerobe chain in nature carbon cycle. The study on changes of methanogen in the methane fermentation can make us realize fermentation degree, optimize the fermentation condition and enhance the methane output. It is well known that more than 90% of the microorganisms existing in nature are refractory to selective enrichment cultures. Being anaerobic microorganism, the raise cycle of methanogen is long. These characteristics cause it extremely difficult to study community structure of methanogen through the traditional cultivation techniques.To overcome these drawbacks, molecular biological techniques especially 16SrDNA analysis are increasingly employed in the analysis of microorganisms in anaerobic natural habitats.This article has been studied the changes of chemical property of material and six major microorganisms in the methane fermentation with cow’s feces as the raw material .At the same time the culture-independent 16SrDNA analysis method was used to investigate the genetic diversity of methanogen in the methane fermentation.As the same time three methanogen strains have been isolated and characterized from the fermentative fluid through Hungate techniques.1. Changes of major microorganism and chemical property of material in methane fermentationThe quantitative changes of six major microorganisms and the transformation of material’s chemical property were investigated in the present study. The result indicate that the aerobic microorganisms are active and play an important role in early period of fermentation, while the anaerobic microorganisms especially methanogen are more and more remarkable later.In the fermentation the material’s chemical properties such as the volatile organic acid, the entire nitrogen, PH, the redox potential have great changes. The changes are correlated with microorganisms ’ changes.2. Extraction and purification of moors fluid microbial total DNA.A method of extracting moors fluid total DNA was established through the comparison of the direct and indirect extraction method. Although the extract rate of direct method is higher, the target gene is unable to be amplified by the PCR amplification from the sample even after the purification because of the high content of impurity. It is unable to carry on更多还原