黄河入海口沉积物反硝化细菌分布特征及其影响因素研究

发布时间：2018-06-20 01:48

  本文选题：黄河入海口 + nirK　； 参考：《山东农业大学》2017年硕士论文

【摘要】：河口位于海陆交汇的地带,其各种生物地球化学过程之间的相互作用十分活跃。氮是组成生物有机体的主要元素,也是影响河口富营养化发生和演变的关键生源要素之一。黄河入海口位于黄河、渤海湾和莱州湾的交汇处,是胜利油田的所在地,黄河携带的大量泥沙、陆源污染物以及周边人类活动的影响使得该地区生态环境极为复杂多变。目前大量人为氮素的输入使黄河入海口环境日益恶化,使其承受着巨大的生态压力。反硝化被认为是去除活性氮的关键过程。本研究以黄河入海口5个典型采样点(SA-SE)的沉积物样品为研究对象,利用克隆文库和定量PCR的方法研究两种基因型反硝化细菌的多样性、群落结构及丰度,结合理化因子进行相关性分析,揭示环境因子与反硝化细菌群落的关系。主要研究结果如下:(1)以nirK、nirS基因为分子标记,建立克隆文库,得到阳性克隆子并测序,获得nirK基因有效序列370条,nirS基因有效序列356条。将序列按照95%的相似性进行OTU(操作分类单元)划分,nirK基因共获得120个OTUs,nirS基因共获得126个OTUs。对于nirK基因,每个采样点的OTUs个数在19-55之间,nirS基因各个采样点OTUs的个数在30-39之间。(2)基于OTU水平的α-多样性分析表明,入海口nirS型反硝化细菌多样性整体略高于nirK型反硝化细菌多样性,且无论nirK或nirS基因,均表现为采样点SE反硝化细菌多样性较高。基于OTU水平的β-多样性分析结果表明,两种基因型的反硝化细菌其物种组成的空间分布方式不同,对nirK型反硝化细菌,SB和SE点的物种组成相似,而SA和SE点的nirS型反硝化细菌物种组成较相似。(3)NirK基因系统发育分析表明,与绝大部分OTUs亲缘关系较近的克隆序列主要来自黄河入海口水体、旧金山湾河口沉积物、重金属污染的土壤、活性污泥、沿海微生物垫和各种农田土等环境。NirK基因系统进化树可划分为7个Clusters(Cluster I-VII),其中,Cluster I、III、IV、V的绝大部分序列主要分布在溶解氧含量较低的采样点(SB、SC、SE),其他Clusters则分布在溶解氧含量较高的采样点(SA、SD)。NirS基因系统发育分析表明,与绝大部分OTUs亲缘关系较近的克隆子主要来自长江入海口、东湖、海河、北运河、珠江口和切萨皮克湾等河口沉积物环境,nirS基因系统进化树可划分为5个Clusters(Cluster I-V),与nirK型反硝化细菌群落结构不同,nirS型反硝化细菌群落结构不存在类似的空间分布模式。(4)利用定量PCR估算nirK和nirS型反硝化细菌丰度,nirK型反硝化细菌丰度为4.17×103-9.72×105 copies/g(干重),nirS型反硝化细菌丰度为4.34×104-2.62×105copies/g(干重)。单因素方差分析显示,样品SA、SC和SD中的nirS型反硝化细菌丰度显著地高于其他采样点(P0.05)。对于nirK,样本间的基因丰度显著不同(P0.001)。对两种基因型反硝化细菌丰度比较分析,相比nirS,nirK型反硝化细菌在潮汐区域(SA、SC和SD)显示较高的丰度,在河流汇合点(SB和SE)丰度较低。(5)环境因子与两种基因型反硝化细菌的多样性、丰度及优势OTUs的斯皮尔曼(Spearman)相关性分析表明,相比nirK型反硝化细菌,黄河入海口沉积物nirS型反硝化细菌的多样性及丰度对环境因子的响应更为敏感。基于优势OTUs与全部OTUs的典型对应分析(Canonical Correspondence Analysis,CCA)表明,nirK型反硝化细菌群落结构对环境因子的响应较为敏感,即nirK型反硝化细菌群落结构受到溶解氧的显著影响(P0.05),而nirS型反硝化细菌群落结构受环境因子影响较小,可能主要受其他未被检测到的环境因子所影响。
[Abstract]:The estuary is located in the area where the sea and land converge. The interaction between various biogeochemical processes is very active. Nitrogen is one of the main elements to form a biological organism. It is also one of the key factors that affect the occurrence and evolution of eutrophication in the estuary. The Yellow River entrance to the sea is located in the Yellow River, Bohai Bay and Laizhou Bay, which is a Shengli Oilfield. The impact of a large amount of sediment, land source and surrounding human activities in the Yellow River makes the ecological environment in the region very complex and changeable. A large amount of artificial nitrogen input makes the environment of the Yellow River entrance to the sea increasingly worsening, making it bear great ecological pressure. Denitrification is considered as a key process to remove active nitrogen. Based on the sediment samples of 5 typical sample points (SA-SE) in the Yellow River entrance to the sea, the diversity, community structure and abundance of two genotypic denitrifying bacteria were studied by cloning library and quantitative PCR, and the relationship between the environmental factors and denitrifying bacteria community was revealed. The results are as follows: (1) take nirK and nirS gene as molecular marker, establish clone library, obtain positive clones and sequence, obtain 370 effective sequence of nirK gene and 356 effective sequence of nirS gene. The sequence according to the similarity of 95% is divided into OTU (operation classification unit), nirK gene obtains 120 OTUs, nirS gene obtains 126 OTUs. for nirK. The number of OTUs at each sampling point is 19-55 and the number of OTUs at each sampling point of the nirS gene is 30-39. (2) the OTU level based alpha diversity analysis shows that the diversity of nirS denitrifying bacteria at the entrance to the sea is slightly higher than that of the nirK denitrifying bacteria, and the nirK or nirS genes are not discussed, all of which represent the SE denitrification finer at the sampling point. The beta diversity analysis based on OTU level showed that the spatial distribution of two genotypes of denitrifying bacteria was different, the species composition of nirK type denitrifying bacteria, SB and SE points was similar, while the species composition of nirS type denitrifying bacteria at SA and SE points was similar. (3) the phylogenetic analysis of NirK gene showed that the phylogenetic analysis of NirK gene showed that the species composition of denitrifying bacteria was similar. The most closely related clones of OTUs were mainly from the Yellow River entrance water, the sediments of San Francisco Bay Estuary, heavy metal contaminated soil, activated sludge, coastal microbial pads and various farmland soils could be divided into 7 Clusters (Cluster I-VII), including Cluster I, III, IV, V. Some sequences were mainly distributed in SB, SC, SE of low dissolved oxygen content, and other Clusters were distributed at the sampling point (SA, SD).NirS gene phylogenetic analysis of high dissolved oxygen content (SA, SD) showed that the clones closest to most OTUs were mainly from Changjiang River to Haikou, East Lake, Haihe River, North Canal, Pearl River mouth and Chesapeake The phylogenetic tree of the nirS gene could be divided into 5 Clusters (Cluster I-V), and the structure of the denitrifying bacteria community was different from that of nirK type denitrifying bacteria. (4) the abundance of nirK and nirS type denitrifying bacteria was estimated by quantitative PCR, and the abundance of nirK type denitrifying bacteria was 4.. 17 x 103-9.72 x 105 copies/g (dry weight), nirS type denitrifying bacteria abundance was 4.34 x 104-2.62 x 105copies/g (dry weight). Single factor variance analysis showed that the abundance of nirS type denitrifying bacteria in SA, SC and SD was significantly higher than that of other sampling points (P0.05). For nirK, the abundance of genes between samples was significantly different (P0.001). Against two types of denitrification. Compared with nirS, nirK type denitrifying bacteria showed higher abundance in Tidal Regions (SA, SC and SD) and lower abundance in river junction (SB and SE). (5) the diversity, abundance and Spielman (Spearman) correlation analysis of environmental factors and two genotypic denitrifying bacteria showed that compared to nirK type denitrification. The diversity and abundance of nirS type denitrifying bacteria in the sediments of the sea entrance of the Yellow River were more sensitive to environmental factors. Based on the typical corresponding analysis of the dominant OTUs and all OTUs (Canonical Correspondence Analysis, CCA), the response of the nirK type denitrifying bacteria community structure to environmental factors was more sensitive, that is, nirK type denitrification. The bacterial community structure was significantly affected by dissolved oxygen (P0.05), while the nirS type denitrifying bacteria community structure was less affected by environmental factors, and may be affected mainly by other environmental factors that were not detected.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q938

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