渤海及北黄海表层沉积物中脱氮功能微生物及其活性的时空分布

	渤海及北黄海表层沉积物中脱氮功能微生物及其活性的时空分布
	杨安静
学位类型	硕士
导师	龚骏 ; 杨建敏
	2014-05-20
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	环境工程
关键词	渤海北黄海沉积物反硝化厌氧氨氧化丰度群落结构脱氮速率及贡献率环境因子
摘要	氮是所有生物体必需的营养元素之一，在生命活动中占有极重要的地位，被称之为生命元素。然而，氮也是水体富营养化发生和演变的关键要素之一，其负荷量与近海赤潮爆发、低/缺氧区形成、生态系统退化等具有密切的相关性。近几十年来，由于高强度的人类活动，导致陆地河流氮素输入量逐年上升，河流携带陆源物质(主要为泥沙和溶解质)输入海洋，使海洋水体富营养化，对海洋生态环境造成严重威胁，成为当今世界面临的一个重大环境问题。微生物主导的反硝化与厌氧氨氧化过程是海洋生态系统氮自净功能的主要途径。因此，研究富营养化海区的氮输出过程对生态建模、环境容量评估等具有重要意义。本研究以渤海、北黄海为研究区域，利用15N 稳定同位素示踪技术测定表层0-5cm沉积物中反硝化与厌氧氨氧化脱氮速率，通过夏、冬季节釆样获得研究区域反硝化和厌氧氨氧化作用的季节和空间变化特征及其与环境因子的关系；以相关基因（反硝化细菌nosZ基因和厌氧氨氧化菌16S rRNA基因）为分子标记，利用群落指纹图谱分析及定量 PCR技术，揭示反硝化与厌氧氨氧化菌丰度和群落结构的时空动态，探讨相关基因与脱氮速率之间的耦合关系。研究结果显示：不同海域和季节之间环境因子存在显著差异，夏季样品拥有更高的盐度、溶氧、叶绿素a、pH、硝态氮和亚硝态氮含量，氮磷比以及总金属Pb含量；其他重金属（如Cd、Fe、Co、Ni、Cu、Zn、Cd）的含量则为冬季含量显著高于夏季（P<0.05）；与渤海相比，北黄海海域具有更高的水深、盐度及沉积物粒径，更低的氮磷比和重金属Mn含量（P<0.05）。而且，除中心位点夏季氮磷比小于16，不论冬、夏，渤海和北黄海海域底层海水的氮磷比基本都在30以上，富营养化现象非常严重。反硝化和厌氧氨氧化菌的群落结构和多样性表现了显著的季节差异（P<0.05），但地域差异不显著（P>0.05）。典范对应分析（CCA）结果显示，亚硝氮浓度（P=0.001）、重金属Pb（P=0.001）浓度、N/P（P=0.045）和水深（P=0.005）对沉积物反硝化细菌种群分布影响最为显著；而冗余分析（RDA）结果显示，氨氮浓度（P=0.001）、Cr（P=0.001）、Cd（P=0.001）浓度以及上覆水叶绿素a含量（P=0.017）是影响沉积物厌氧氨氧化菌种群分布最显著得因素。反硝化菌nosZ基因丰度远高于厌氧氨氧化菌16S rRNA基因的丰度，且夏季功能基因丰度高于冬季；反硝化菌和厌氧氨氧化菌丰度的高峰值均出现在渤海湾口、黄河口和莱州湾口一带。冗余分析结果表明，水深（P=0.014）、pH（P=0.001）、氨氮浓度（P=0.048）、N/P（P=0.025）、沉积物粒径（P=0.029）、重金属Zn（P=0.004）、Cu（P=0.021）浓度、以及沉积物叶绿素a（P=0.047）是影响沉积物nosZ和厌氧氨氧化菌-16S rRNA基因丰度分布最显著的因素。沉积物厌氧脱氮过程以反硝化为主；厌氧氨氧化过程在渤海和北黄海脱氮过程中占有重要地位（<50%），在渤海中的贡献高于北黄海，黄河口附近区域最高，且冬季贡献率高于夏季；莱州湾口、渤海湾口和黄河口附近是渤海脱氮过程的重要区域。功能基因丰度与脱氮速率没有显著相关性（P>0.05），但是厌氧氨氧化菌16S rRNA基因丰度与该过程的贡献率显著相关（ρ=0.34，P=0.038）。冗余分析表明，沉积物粒径（P=0.011）、氨氮（P=0.045）和重金属Co（P=0.015）是沉积物反硝化和anammox脱氮速率分布最显著的影响因素。本研究揭示了渤海、北黄海表层沉积物反硝化与厌氧氨氧化菌群落结构和丰度的时空动态，首次利用稳定同位素示踪技术得出了研究区域沉积物反硝化与厌氧氨氧化脱氮速率及贡献率；明确了影响脱氮速率及相关功能微生物类群的主控因子。有关功能基因表达与其生态功能的耦合关系还需要进一步实验证实。
其他摘要	Nitrogen (N) is one of the necessary elements for organisms, and overloading of N causes eutrophication in coastal ecosystems. There are close correlations between nitrogen overloading and the outbreak of red tide, the formation of anoxia and ecosystem degradation. In recent decades, the nitrogen inputs from terrestrial sources increased dramaticly, which becomes a serious threat to the marine environment. Denitrification and anaerobic ammonia oxidation (anammox) driven by microbes are two main ways of nitrogen removal from marine ecosystems. Understanding the diversity, distribution and controlling mechanisms of N-removal microbes and related processes in eutrophied seas is of vital significance in ecological modeling and environmental capacity assessment. In this study, the denitrification and anammox rates in surface sediments (0-5 cm), which were collected from Bohai and the North Yellow Sea in summer and winter of 2012, were determined using slurry incubation and 15N isotope tracer method. The seasonal and spatial variations of community structure and abundance of denitrifiers and anammox bacteria were assessed using nosZ gene and anammox bacterial 16S rRNA gene as molecular markers, and in combination of a range of molecular tools, such as Terminal Restriction Fragment Length Polymorphism（T-RFLP）and Real-time Quantitative PCR (Q-PCR). Main results: There were significant differences of environmental factors between Bohai and the North Yellow Sea and between summer and winter. The summertime samples has higher salinity, dissolved oxygen, Chl-a, pH, NO2--N, NO3--N, N/P and heavy metal Pb, and lower other metals such as Cd、Fe、Co、Ni、Cu、Zn、Cd (P<0.05). Compared with Bohai Sea, the North Yellow Sea has higher salinity, sediment grain size and lower concentration of metal Mn (P<0.05). The diversities and community structure of denitrifying and anammox bacteria exhibited significant seasonal (P<0.05) but no regional differences (P>0.05). CCA results showed that NO2--N (P=0.001), Pb (P=0.001), N/P (P=0.045) and depth (P=0.005) significantly influenced the distributions of denitrifying bacterial community. However, RDA results showed that NH4+-N (P=0.001), Cr (P=0.001), Cd (P=0.001) and Chl-a (P=0.017) significantly affected the distribution of anammox bacterial community. The nosZ gene of denitrifying bacteria were muchmore abundant than 16S rDNA of anammox bacteria. For abundance of each gene, the summertime samples were much higher than the wintertime. The peak values of the two genes occurred near the mouth of Laizhou Bay and Bohai Bay and the Yellow River estuary. RDA results show that water depth (P=0.014), pH (P=0.001), NH4+-N (P=0.048), N/P (P=0.025), sediment grain size (P=0.029), metals Zn (P=0.004), Cu (P=0.021), and Chl-a (P=0.047) contributed most to the abundance variations of denitrifying and anammox bacteria. In surface sediments of Bohai and the North Yellow Sea, denitrification predominated in nitrogen removal, but anammox also played an important role in nitrogen removal with a contribution up to 50% in some sites. The rates and contributions of anammox were higher in Bohai than that in the North Yellow Sea, and higher in winter than that in summer. The mouth of Laizhou Bay and Bohai Bay and the Yellow River estuary were the most hot-spot regions. Thers were no significant correlations between nitrogen removal rates and revelant functional gene abundances, but a significant relationship between anammox-16S rRNA gene abundance and anammox contribution was found (ρ=0.34，P=0.038). RDA results indicate that sediment grain size (P=0.011), NH4+-N (P=0.045) and metal Co (P=0.015) contributed most to the variations of microbial nitrogen removal activities. Tish study reveals the temporal and spatial dynamics of community structure and abundance of denitrification and anammox bacteria, and the denitrifying rate and anaerobic ammonium oxidation rate and their contribution rate in the study area has been determined by using 15N isotope tracer method firstly; the main environmental factors which impact the nitrogen removal rate and the spatial and temporal characteristics about related functional microbial have been figured out. However, the relationship between gene expression and its ecological functions need to be further confirmed.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/6801
专题	中国科学院烟台海岸带研究所知识产出_学位论文
推荐引用方式 GB/T 7714	杨安静. 渤海及北黄海表层沉积物中脱氮功能微生物及其活性的时空分布[D]. 北京. 中国科学院研究生院,2014.

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