云南沼气发酵生态系统的原核生物群落时空动态研究

发布时间：2018-01-16 05:11

本文关键词：云南沼气发酵生态系统的原核生物群落时空动态研究　出处：《云南大学》2016年博士论文　论文类型：学位论文

【摘要】：沼气工程的推广应用在保护生态环境和减少温室气体排放等方面具有重要的意义。在农村地区发展户用沼气工程能够改进农村能源使用途径,治理农业面源污染和促进农业生态的良性循环。沼气发酵生态系统中大量的原核生物与系统的高效稳定运行有着密切关系。沼气发酵过程中的工艺参数和原料来源等环境因子从多方面影响着发酵系统中原核生物的数量、群落结构和群落功能,而原核生物的代谢活动反过来又会影响沼气发酵生态系统的稳定运行。因此,沼气发酵系统中原核生物的群落结构组成对产气效率起着十分重要的作用。目前,云南农村户用沼气池数量多,但沼气池在管理和使用过程中仍然存在许多问题,如沼气池产气不稳定、产气量少、冬季停用现象。针对云南农村户用沼气池的问题,目前的研究主要从工程角度出发,多集中在沼气池的工艺参数优化、保温措施和活性污泥驯化等方面研究上,但是对云南农村户用沼气发酵生态系统的重要生物因素—原核生物及其与环境因子相关性的研究几乎无人涉及。因此,对云南农村户用沼气发酵生态系统中原核生物的群落结构和功能、原核生物与环境因子相互关系的研究十分有意义。通过生态因子综合分析,揭示沼气发酵系统中原核生物群落的演替规律等基本生态学问题,能够为设计具有应用前景的高效沼气发酵系统,活性污泥驯化,发酵条件提供最优监控方案和规模化应用奠定基础。本研究是从云南4个不同气候类型与地理区域的代表性农村户用沼气发酵池中采集活性污泥,用Illumina高通量测序技术,系统地分析云南不同气候类群与地理区域的农村户用沼气池中原核生物的类群组成、群落结构和群落功能,并阐述其与环境中非生物因子间的相关性,进而探讨不同气候区域的沼气池中原核生物群落空间动态(地理格局)分布。其次,运用DGGE和Illumina高通量测序技术,研究模拟云南热带气候区域的农村户用沼气发酵过程中原核生物群落的时间动态变化。深入分析整个过程中原核生物群落结构,探讨原核生物群落在时间上的动态演替规律。研究结果显示,云南农村户用沼气发酵生态系统中存在丰富的原核生物类群。在不同气候类型与地理区域的沼气发酵生态系统中,原核生物群落结构存在较大差异。在模拟沼气发酵过程中,不同的原核生物类群的动态变化规律不一样。1.云南农村户用沼气发酵生态系统中存在丰富的原核生物类群。Illumina高通量测序结果显示,云南4个不同气候区域的农村户用沼气生态系统中存在着丰富的细菌和古菌类群。从10个沼气池中平均获得61,000多条有效序列,聚类分析发现发酵系统中有16个已知门类群：泉古菌门(Crenarchaeota)、广古菌门(Euryarchaeota)、拟杆菌门(Bacteroidetes)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、绿菌门(Chlorobi)、纤维杆菌门(Fibrobacteres)、绿弯菌门(Chloroflexi)、厚壁菌门(Firmicutes)、芽单胞菌门(Gemmatimonadetes)、变形菌门(Proteobacteria)、螺旋体门(Spirochaetes)、Synergistetes、热袍菌门(Thermotogae)、疣微菌门(Verrucomicrobia)和Caldithrix等和5个候选门类群。其中,厚壁菌门(Firmicutes)、广古菌门(Euryarchaeota)和拟杆菌门(Bacteroidetes)在该生态系统中占着绝对的优势地位。2.PCA结果显示,不同气候区域的沼气发酵系统中原核生物的群落结构存在差异,同一气候区域的原核生物群落结构相近。厚壁菌门(Firmicutes)所占的比例范围为22-55%,其中它在北温带的XGP2和XGP3比例最高,分别为54.6%和41.1%。广古菌门(Euryarchaeota)的丰度变化范围为4.1%到46%,同样差异明显。与厚壁菌门(Firmicutes)丰度相反,广古菌门(Euryarchaeota)在XGP2和XGP3中比例最低,仅为8.5%和4.1%。拟杆菌门(Bacteroidetes),在各个样点中的丰度存在差异,为10-27%。原核生物的群落结构与沼气发酵系统的产气量存在一定的相关性。3.应用DGGE指纹图谱技术研究模拟沼气发酵过程,结果表明,系统中的细菌和古菌类群具有很高的多样性,细菌类群的数量高于古菌类群。对原核生物的动态变化进行聚类分析,发现整个发酵过程中细菌和古菌的群落结构不断在变化；细菌类群的丰度变化明显大于古菌类群。整个发酵过程中细菌和古菌的群落结构变化规律可以分为3个阶段,但细菌类群的变化阶段明显早与古菌类群的变化。4. Illumina高通量测序结果表明,模拟沼气发酵过程中涉及的原核生物类群主要归属于19个门一级的系统发育类群：泉古菌门(Crenarchaeota)、广古菌门(Euryarchaeota)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、螺旋体门(Spirochaetes)、Synergistetes、绿弯菌门(Chloroflexi)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、纤维杆菌门(Fibrobacteres)、绿菌门(Chlorobi)、疣微菌门(Verrucomicrobia)、Armatimonadetes、Caldithrix、 Lentisphaerae、热袍菌门(Thermotogae)和OP9、OP11等。广古菌门(Euryarchaeota)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)和变形菌门(Proteobacteria)同样是沼气发酵系统中的主要优势类群。在模拟沼气发酵过程中,沼气发酵系统的原核生物变化规律同样分为3个阶段,不同类群的原核生物变化趋势不一致。广古菌门(Euryarchaeota)在发酵过程中的丰度变化趋势为增加,相对丰度由发酵初期的32.4%到发酵末期的60.5%。厚壁菌门(Firmicutes)相对丰度由发酵初期的19.2%到发酵末期的6.4%,与广古菌门(Euryarchaeota)的变化趋势相反。拟杆菌门(Bacteroidetes),相对丰度由发酵初期的15.7%到发酵末期的5.9%,变化趋势同样是减少。一些细菌类群的相对丰度变化与产气量变化相一致,与产气量存在一定的相关性。5.云南不同气候类型与地理区域的农村户用沼气发酵系统中,原核生物的群落分布与环境因子(发酵条件)存在着显著的相关性。RDA分析结果显示,沼气发酵系统中的总固体(TS)和化学需氧量(COD)是影响细菌类群地理空间分布的最主要非生物因子,而温度是影响古菌属群落分布的主要环境因子。而在模拟沼气发酵过程中,TS和COD同样是影响沼气发酵细菌类群时间动态变化的主要非生物因子;恒温条件下,主要产甲烷古菌类群受到发酵条件的影响不大。
[Abstract]:It has important significance to promote the application of Biogas Engineering in protecting ecological environment and reduce greenhouse gas emissions. The development of household biogas project can improve the rural energy use way in rural areas, management of agricultural non-point source pollution and promote agricultural ecological circulation. There is a close relationship between the efficient and stable operation of methane generator systems with a large number of prokaryotes and yeast in the ecological system. The process parameters and sources of raw materials and other environmental factors in the process of biogas fermentation from many aspects affect the number of fermentation system of prokaryotic, community structure and function, and metabolic activity of prokaryotic organisms will in turn affect the stable operation of Biogas fermentation ecosystem. Therefore, the community structure of the biogas fermentation system of Central Plains nuclear biological composition plays an important role in gas production efficiency. At present, the number of household biogas digesters in rural areas of Yunnan, but the tube There are still many problems and the use of the process, such as methane stable gas production, gas consumption, winter stop phenomenon. For biogas plants in rural areas of Yunnan, the current study mainly from the engineering point of view, optimization of process parameters and more concentrated in the digester, research on thermal insulation measures and activated sludge etc. However, research on important biological factors in Yunnan Prokaryotae rural household biogas fermentation ecosystem and its correlation with environmental factors almost nobody involved. Therefore, the community structure and function of biogas fermentation ecosystem of prokaryotic households in rural areas of Yunnan, the relationship between prokaryotes and environmental factors is of great significance. Through comprehensive analysis ecological factors, reveal the biogas fermentation system of prokaryotic community succession and other basic ecological problems, efficient biogas to design with application prospect Gas fermentation system, activated sludge, fermentation conditions to provide optimal solutions and lay the foundation for monitoring large-scale applications. This study is the active sludge collected from Yunnan biogas 4 different types of climate and geographical areas representative of rural household fermentation tank, using Illumina high-throughput sequencing technology, system analysis of Yunnan climate and different groups geographical area of rural household biogas digesters of prokaryotic species, community structure and function, and expounds the relationship between the non biological factors and the environment, and to explore the different climatic regions of biogas pool of prokaryotic community spatial dynamic (geographical pattern) distribution. Secondly, using DGGE and Illumina high-throughput sequencing technology dynamic research on Simulation of Yunnan tropical climate region of the rural household biogas fermentation process of prokaryotic organisms. In-depth analysis of the whole process of prokaryotic biological community To investigate the structure, dynamic succession at the time of the prokaryotic community. The results showed that the presence of prokaryote taxa rich Yunnan rural household biogas fermentation ecosystem. Biogas fermentation ecosystem in different types of climate and geographical area, there is a big difference between the prokaryotic community structure. In the simulation process of biogas fermentation, high.Illumina groups of prokaryotes the sequencing results showed the presence of abundant amount of dynamic changes of different groups are not the same as the prokaryote.1. Yunnan rural household biogas fermentation ecosystem, there are bacteria and archaea rich groups of Yunnan in 4 different climate regions of the rural household biogas system. The average obtained more than 61000 effective sequences from 10 digesters in the cluster analysis showed that the fermentation system there are 16 known categories: Crenarchaeota group (Crenarchaeota), Euryarchaeota (Euryarcha Eota), Bacteroidetes (Bacteroidetes), acidobacteria (Acidobacteria), actinobacteria (Actinobacteria), green (Chlorobi), bacterial phylum fibrobacteres (Fibrobacteres), Chloroflexi (Chloroflexi), Firmicutes (Firmicutes), gemmatimonadetes (Gemmatimonadetes). Proteobacteria (Proteobacteria), spiral gate (Spirochaetes), Synergistetes, thermotogae (Thermotogae), verrucomicrobia (Verrucomicrobia and Caldithrix) and 5 candidate door groups. Among them, the Firmicutes (Firmicutes), Euryarchaeota (Euryarchaeota) and Bacteroidetes (Bacteroidetes in the ecological system) dominating the.2.PCA absolute that community structure in different climate regions of the biogas fermentation system of prokaryotic differences, the same group of prokaryotes in climatic regions fall. Similar structures of Firmicutes (Firmicutes) the proportion of the range of 22-55%, in which it North temperate XGP2 and XGP3 highest proportion, respectively 54.6% and 41.1%. Euryarchaeota (Euryarchaeota) abundance ranged from 4.1% to 46%, the same difference. And Firmicutes (Firmicutes) abundance in Euryarchaeota (Euryarchaeota) in XGP2 and XGP3 in the lowest rate, only 8.5% and 4.1%. (Bacteroidetes), Bacteroidetes abundance in each sample point of difference for community structure and gas production of biogas fermentation system 10-27%. in prokaryotes have certain correlation.3. DGGE fingerprint technique of simulated biogas fermentation process, the results show that the system of bacteria and archaea, bacteria with high diversity the number of groups is higher than that of bacteria, archaea. The dynamic changes of the prokaryotic community structure found by clustering analysis of bacteria and archaea in the whole fermentation process of the continuous changes in the abundance of bacterial groups significantly; More than archaea. Community structure changes of bacteria and archaea in the whole fermentation process can be divided into 3 stages, but the change was earlier stage groups of bacteria and archaea. The changes of.4. Illumina high-throughput sequencing results showed that prokaryotic organisms belonging mainly involved in the process of biogas fermentation system simulation in 19 a development group: Crenarchaeota (Crenarchaeota), Euryarchaeota (Euryarchaeota), (Firmicutes) the Firmicutes, Bacteroidetes and Proteobacteria (Bacteroidetes) (Proteobacteria), spiral gate (Spirochaetes), Synergistetes, Chloroflexi (Chloroflexi), acid bacillus the door (Acidobacteria), actinobacteria (Actinobacteria), fibrobacteres (Fibrobacteres), green (Chlorobi), bacterial phylum verrucomicrobia (Verrucomicrobia), Armatimonadetes, Caldithrix, Lentisphaerae, thermotogae (Thermotogae) and OP9, OP11 and so on. The Archaea The door (Euryarchaeota), (Firmicutes) the Firmicutes, Bacteroidetes and Proteobacteria (Bacteroidetes) (Proteobacteria) is also the dominant group biogas fermentation system. In the simulation process of biogas fermentation, prokaryotes variation of biogas fermentation system of the same is divided into 3 stages, the change trend of different groups of prokaryotes inconsistent. Euryarchaeota (Euryarchaeota) abundance change trend in the process of fermentation was increased by 60.5%., the relative abundance of Firmicutes to 32.4% at the end of fermentation at the initial stage of fermentation of the relative abundance (Firmicutes) from 19.2% to 6.4% at the end of fermentation fermentation stage, and Euryarchaeota (Euryarchaeota) changes on the contrary. The Bacteroidetes (Bacteroidetes), the relative abundance of from 15.7% to 5.9% at the end of fermentation fermentation stage, the same trend is reduced. Some bacterial taxa relative abundance changes and the changes of the gas production. The correlation between.5. and Yunnan in different climate types with a certain geographic region and gas production of rural household biogas fermentation system, community distribution and environmental factors in prokaryotes (fermentation) there is a significant correlation between.RDA analysis results showed that the total solids in the biogas fermentation system (TS) and chemical oxygen demand (COD) is the influence of geographical space distribution of bacterial taxa most major abiotic factors, while temperature is the main environmental factors affecting the community distribution of ancient bacteria. And in the simulation of methane fermentation process, TS and COD are also major abiotic factors affecting the change of population dynamic time fine biogas fermentation fungus; under the condition of constant temperature, mainly produced little effect methane Archaea by fermentation conditions.

【学位授予单位】：云南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S216.4

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