RCEES OpenIR  > 环境水质学国家重点实验室
湿地岸边带亚区尺度微生物群落生态与氮循环模式 宏基因组分析
Alternative TitleMetagenomic Analysis of Microbia l Communities and Nitrogen Cycle at Subregional Scale in Riparian Zone
周立光
Subtype博士后
Thesis Advisor祝贵兵
2019-05
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学
Degree Discipline环境工程
Keyword氮循环 Metagenome Soils Sediments Riparian Zone. 宏基因组 土壤 沉积物 岸边带 keywords: Nitrogen Cycle
Abstract

        水生生态系统和陆地生态系统之间的 缓冲区域形成了湿地,其面积广泛,覆盖了大约 6% 的地球表面积。湿地 最重要的功能之一就是进行氮素的转化。人类工业化引起了土地的变化,已经显著地减少了湿地的面积。现在湿地的重要性越来越受到重视,但一些成功构建的人工湿地,往往侧重于恢复洪泛平原和宏观生态,而不是生物地球化学循环所需的微生物生态学 。 然而 一旦湿地生态系统被打破, 通过湿地修复 或者构建人工湿地 的方式 似乎很难在短时间内达到自然湿地生态系统的生态学功能。因而 对自然湿地生态系统中微生物参与的生物地球化学循环的了解显得尤为重要。
        目前对湿地生态系统的微生物地球化学循环的研究主要集中在水饱和的生境,并多为对单一生物学过程的研究,例如氮循环。这些研究不能同时、全面了解湿地岸边带生态系统微生物地球化学循环的全貌,无法同时分析不同 生物学过程对环境因子的响应,更无法分析不同生物学过程的相互作用。本研究 依水文情势,选择 远岸 旱地土壤(表层和亚表层)、水陆交界面土壤(表层和亚表层)和沉积物、开阔水体沉积物为研究对象。 依 托宏基因组测序,分析白洋淀湿地岸边带微生物群落组成及潜在的生物学功能,重点解析微生物氮循环模式的异质性。
        本研究第一部分工作旨在对湿地生态系统微生物组成及其所发挥的生物学功能的潜势进行分析。 6 个样品(每个样 品三个生物学重复)的宏基因组测序,质控后获得总 clean reads 为 845,931,439 条,平均每个样本为140,988,573.2±6,869,231.688 条。每个样本质控后的 clean reads 均占 raw reads 的99% 以上。 6 个样品共预测获得 20,711,413 个开放阅读框( Op en Reading Frame,ORF ORFs 总长度为 8,721,548,820 bp ,平均长度为 421.1bp 。分析结果表明:① 湿地岸边带微生物驱动的生物地球化学循环仍主要以细菌为主。在门水平上,Proteobacteria 和Actinobacteria 两类微生物的相对丰度分别在沉积物和土壤中占主导。古菌中 Euryarchaeota 和 Thaumarchaeota 是相对丰度最高的两个门,丰度和占总古菌丰度的 88.29% 以上。 ② 湿地岸边带微生物多样性进化稳定:共有物种分析发现,共有的属 超过总数的 86.7%86.7%,共有的种超过总数的 82.5% 。③不同样点间微生物群落组成结构存在极大差异:最明显的差异体现在土壤和沉积物之间,其次是水陆交界面沉积物和开阔水体沉积物之间。微生物群落组成结构上的极大差异暗示微生物驱动的生物学过程可能也存在明显的不同。 ④ 基于 eggNOG 数据库和 KEGG 数据库的微生物功能分析发现,样点间微生物功能的差异也主要体现在功能组成结构上。土壤与沉积物之间微生物群落组成,功能组成的极大差异表明,在同一个生态系统中微生物群体发挥的生物学功能在不同区域存在极大的差异,即使是同一水 陆交界面的土壤和沉积物(样点间距离仅有6 m )之间。这些结果暗示如果单纯的从微生物多样性的角度去恢复湿地功能或构建人工湿地,似乎很难恢复或发挥湿地真正的生态学功能。
        氮循环可能是微生物活动影响最大的生物地球化学循环,而湿地在氮的生物地球化学循环中发挥重要 作用。本研究的第二部分基于宏基因组数据分析,结合理化参数和实时荧光定量 PCR qPCR )结果,分析白洋淀湿地岸边带 的 氮循环模式。目前对湿地生态系统氮循环微生物群落结构及其对湿地氮循环贡献的研究主要集中在水饱和的生境,本部分的研究涉及了远水岸旱地土壤。研究结果表明:① narGH/nxrAB 基因在氮循环基因家族中最为丰富,推测硝酸盐和亚硝酸 盐相互转化过程可能是岸边带总氮循环的驱动力,而且受环境因素影响弱 。 ② 土壤和沉积物的亚硝酸盐还原过程 为 两种不同的模式:土壤中以 nirK 型反硝化和ANRA 为主;沉积物中以 nirS 型反硝化和 nrfA 型 DNRA 为主,并存在 anammox过程。 ③ 反硝化过程仍然是白洋淀湿地岸边带主导的氮 循环过程,而固氮作用、硝化作用和 N damo 都是相对较弱的氮循环过程。 ④ 氨氧化过程与 nirK 型反硝化作用密切相关,并且对 土壤氮循环 的贡献大于对沉积物氮循环的贡献 。 ⑤ 尽管固氮作用很弱,但在沉积物氮循环中的占比高于土壤中,这可能与沉积物中的厌氧环境有关。 ⑥ 值得关注的是 anammox 在土壤和沉积物中的表现明显不同,基于宏基因组数据的分析发现土壤中的 anammox 途径不完整,暗示其可能不易在土壤环境中发生,这解释了为什么 anammox 在水生环境中广泛发生的现象。⑦ 本部分内容还揭示 了 生境对岸边带氮循 环微生物及氮循环功能基因相对丰度的影响。 ⑧ 确定白洋淀湿地岸边带水陆交面是微生物生物量高的区域,也分析了与氮循环过程紧密相互作用的微生物及微生物对氮循环过程的贡献。
 

Other Abstract

        The interaction between aquatic and terrestrial ecosystems forms wetlands, which has widespread areas and cover approximately 6% of the Earth’s surface. Wetlands known as the kidneys of the earth, one of the most important functions is perform transformation of nitrogen.
        Human industrialization has led to land changes, which have dramatically reduced the area of wetlands. Now the importance of wetlands has been paid more and more attention. Successful constructed wetlands often focus on restoring flood plains and macro eco systems rather than microbial ecology required for biogeochemical cycling.
Once natural wetland ecosystem is broken, it is difficult to recovery the ecological function by constructed ecosystem in a short time. Therefore, it is particularly important to un derstand the biogeochemical cycle in natural wetland ecosystem in which microorganisms are involved.
       At present, the study of microbial geochemical cycle in wetland ecosystem mainly focuses on water saturated habitats, most of researches are for single bi ological processes, such as one nitrogen cycling process. These studies can not simultaneously and comprehensively understand the whole microbial biogeochemical cycle of wetland ecosystem, nor can they analyze the interaction of different biological proces ses and the response to environmental factors. Based on metagenome, we analyzed the composition and potential biological functions of microbial communities in the riparian zone of Baiyangdian Lake, and emphasis on the heterogeneity of microbial nitrogen cycle patterns. Hydric soils, land water ecotone and “far shore” dryland soil" in riparian zone were selected as the research sites.
         The first part of this study aims to reveal the microbial composition of wetland ecosystem and the potential biological funct ions. Six samples three biological repeats per sample were sequenced and the total clean reads were 845,931,439, with an average of 140,988,573.2 ±6,869,231.688 per sample. The clean reads of each sample after quality control accounted for more than 99%of raw reads. A total of 20,711,413 open reading frames ORF were predicted for six samples. The total length of ORFs was 8,721,548,820 bp, and the average length of that was 421.1 bp. The analysis of metagenomic data discovery that: the biogeochemical cy cle was mainly driven by bacteria in the riparian zone. At the phylum level, the sum of relative abundance of Proteobacteria and Actinonobacteria were the highest in sediments and soils, respectively. In archaea, the relative abundance of Euryarchaeota and Thaumarchaeota were the highest, and the sum of their proportion accounted for more than 88.29% in total abundance of archaea. The microbial diversity was similar in evolution in different areas of the riparian zone, becauxe over 86.7% of the genera and 8 2.5% of the species were common in six sample. In the microbial community composition, there were great differences among different samples. The most obvious difference was between soils and sediments, and the next was between the two sediment samples. Thi s difference suggested that there might be significant differences in the biological processes driven by microorganisms in different sample sites. The comparative analysis based on eggNOG and KEGG databases, it was found that the microbial functions compos ition among samples were differences rather than diversity. The functions were performed by microbial communities existed greatly difference in different regions of this same ecosystem, which indicated that it seemed difficult to restore or develop the real ecological functions of wetlands, if we only restore wetland functions or construct artificial wetlands from the perspective of microbial diversity.
        Nitrogen cycle may be the biggest cycle is affected by microbial activities, and wetlands play an importa nt role in the biogeochemical cycle of nitrogen. The second part of this study was based on the analysis of metagenomic data, combined with physical and chemical parameters and quantitative PCR qPCR results, to comprehensively analyze the composition pat terns of nitrogen cycle in the riparian zone of Baiyangdian Lake, and revealed the similarities and differences of nitrogen cycle patterns under different hydrological conditions of this wetland ecosystem. At present,the research on microbial community st ructure involved nitrogen cycle, and its contribution to nitrogen cycle in wetland ecosystem mainly focuses on water−saturated habitats. In this part, the far shore dry soils were also included in the analysis. We investigated the difference regions microo rganism community, abundance and
composition of nitrogen cycling genes and revealed the difference patterns total nitrogen cycle in riparian zone of Baiyangdian Lake. Microorganisms inhabited in soils and sediments in two classifications, which were embodi ed in the biological functions of microorganisms. We found that the genomic potential for inter−conversion of nitrate and nitrite would be the dynamics for total nitrogen cycle in riparian zone, because genes narGH nxrAB were the most abundant in the nitro gen cycling gene families. The predominant of narGH nxrAB genes were less affected by environmental factors, and the nitrate reduction process might be the main, because the nitrification was weak in all samples, which presumably due to the rich exotic nit rate always existed, such as fertilization. However, the subsequent reduction of nitrite was divided into two main modes: nirK type denitrification and ANRA were dominant in soils; while nirS type denitrification and nrfA type DNRA were dominant, and exist ed Anammox in sediments. The denitrification was still the dominant nitrogen cycling process in all sites, while nitrification, N damo and nitrogen fixation were relatively weak nitrogen cycling processes. Inside, the process of ammonia oxidation was close ly interacted with nirK type denitrification. We speculated that nitrite produced in the process of ammonia oxidation might be the main substrate of nirK type denitrification. Although nitrogen fixation was very weak, it still accounted for a certain propo rtion of the nitrogen cycle in sediments, which might be related to the anaerobic environment in sediments. On the other hand, it was worth noting that the process of anammox was different in soils and sediments, the process of soil was incomplete in metag enomic data, which explained why anammox occurs widely in aquatic environment. Ultimately, this research demonstrated the impact of habitat environment on microbial nitrogen cycling pattern in riparian zone of wetlands. It was determined that the water lan d interface of riparian zone was the area with high microbial biomass. The microbial and microbial contributions to nitrogen cycle were also analyzed in text.

Pages153
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42364
Collection环境水质学国家重点实验室
Recommended Citation
GB/T 7714
周立光. 湿地岸边带亚区尺度微生物群落生态与氮循环模式 宏基因组分析[D]. 北京. 中国科学院生态环境研究中心,2019.
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