RCEES OpenIR  > 中国科学院环境生物技术重点实验室
人工湿地中植物对氮功能微生物的调控机制研究
Alternative TitleRegulation mechanisms of the plant on the microbial community responsible for nitrogen removal in constructed wetland
孙海曙
Subtype博士
Thesis Advisor庄绪亮
2020-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword人工湿地 植物根系泌氧 模式分泌物 植物 -氮 功能微生物互作 硝 化 -反硝化作用 constructed WetlAnds, Radial Oxygen Loss, Artificial Root Exudates, Interactions Of Plant And Microbes Responsible For n Removal, Nitrification-denitrification
Abstract

     氮(N )循环是由植物和微生物驱动的一系列氧化还原反应,尽管 N 循环主要由微生物驱动, 植物可以改变微生物种群及其驱动的 N 循环过程 。 人工湿地作为 主要由植物和微生物组成的水生生态系统,是一种 具有较大潜力的污水修复技术,最大的优点 是 成本低、能耗少、管理运营简单且环境友好 。由于 长期或定期水淹条件, 人工湿地 是水化学与微生物 N 循环反应发生的热区 ,其次 存在复杂互作界面,尤其根际界面 ;同时 具有时间和空间异质性,异质性由于根际效应而增加 。目前人工湿地中 植物和 N 功能微生物的互作机制仍不清楚 。揭示植物与 N 功能微生物的互作机制对于 湿地优化设计和强化生物脱氮过程有重要意义 。
     本研究通过建立可以高效稳定处理高浓度畜禽养殖废水的人工湿地,研究植物对N 功能微生物种群丰度、结构和互作关系的影响机制,解析植物根系泌氧和模式分泌物对 N 功能种群的调控机制,并利用 D NA 稳定同位素探针技术探究了驱动植物 微生物联合脱 N 过程中的活性关键功能微生物。主要研究结果如下:
     (1)经过 600天的运行,种植绿狐尾藻( M. aquaticum)的人工湿地实现了对 COD, 氨氮和总氮高效而稳定的去除,去除率分别为 88.2%, 98.3%和 95.9%。植物存在下对 N功能微生物的 主要作用热区 位于 沉积物中 。定量 PCR结果表明硝化基因 AOA amoA AOB amoA 和反硝化基因 nirK, nirS, nosZ在 沉积物中被 M. aquaticum显著增加 。 高通量测序揭示了 M. aquaticum显著 改变了微生物群落的结构,硝化细菌 、 厌氧氨氧化菌和反硝化细菌 被显著富集。 分子生态网络分析表明, N功能微生物 之间的正相关 互作 关系被 M. aquaticum促进。
     (2) 溶氧 微电极技术在空间上 测 得 M. aquaticum根系 周围 存在 一定的泌氧区域 (<400 μm)),柠檬酸钛比色法进一步 证明 M. aquaticum的泌氧能力为 0.019 mg/h/plant。 植物这种根系泌氧特征 可能 构建了沉积物 水体 根表面反硝化作用逐渐增强的有序生态位 ,与兼性厌氧相关的基因和编码反硝化酶的基因显著上调 反硝化功能基因 nirS nirK, nosZ显著增加 尤其是根表面 nosZ基因的丰度与沉积物相比增加 21倍 兼性厌氧的反硝化菌如 Aeromonas(气单胞菌属Pseudomonas(假单胞菌属 21.4%)和 Acinetobacter 不动杆菌属 等在根表面被 显著富集 。 通过对湿地系统 N去除过程中氧气来源和消耗进行理论衡算显示 ,大气复氧 2.8 g/m2/d)可能是水体好氧层 0-5 cm 发生 硝化作用 所需氧气( 2.5 g/m2/d 的 主要来源,植物根系泌氧( 0.3 g/m2/d)则促进兼性厌氧的反硝化菌在中层水体( 5-10 cm)和根表面的生长,从而 实现 湿地 的反硝化作用。
     (3 )通过建立根系泌氧和初级模式分泌物的微宇宙实验模型,揭示了 根系泌氧和分泌物对反硝化过程 的 复合调控作用,发现 模式 分泌物显著提高反硝化活性 。 qPCR结果表明相比模式分泌物,根系泌氧显著影响 narG, nirS, nirK和 nosZ基因的丰度 。 基于 16S rRNA基因的测序结果表明,相较于根系泌氧,模式分泌物更 显著 改变了种群结构, 富集了属于厚壁菌门的 Clostridium sensu stricto 和Bacillus等微生物 。 结构方程模型( SEM 进一步解析了这种调控作用的因果关系,揭示了 根系分泌物 和种群结构 是反硝化过程 中 的重要驱动力; 即 根系分泌物显著改变种群结构,种群结构进一步显著提高反硝化活性。
     (4) DNA-SIP技术 在 根系泌氧 和 初级模式分泌物的模型中 成功实现了对关键的活性反硝化微生物的标记 。 相较于 根系 泌氧, 模式 分泌物对种群结构影响更为显著 。 Candidatus Koribacter Azospirillum(固氮螺菌属 Burkholderia(伯克氏菌属), Arthrobacter(节杆菌属 Anaeromyxobacter(厌氧粘细菌 Nocardia(诺卡氏属 Magnetospirillum 磁螺菌属 和 Ralstonia(青枯菌属 等 是添加模式分泌物处理中 关键的 活性 反硝化 微生物,尤其是 Magnetospirillum是对照处理中( 0.1%)的 15倍,被模式分泌物激活并富集。
      本研究从新的角度揭示了人工湿地高 N去除率下, 湿地 植物 特性 对 N功能微生物的 调控 机制 建立 了 植物特性与 N功能微生物种群之间的联系 。 对于寻找关键 的植物和 功能微生物并推进湿地优化设计提供了理论基础和技术 应用的新途径 。

Other Abstract

     The nitrogen (N) cycle can be described as a network of oxidation–reduction reactions catalyzed by plants, fungi, bacteria, and archaea. Although N cycle is mainly drived by microorganisms, plants can exert control over N transformations catalyzed by the microbes. Constructed wetlands (CWs), as aquatic ecosystems mainly composed of plants and microorganisms, have been developed as a sustainable technology for N pollution of water sources because of their technical feasibility, ecological benefits, and economic advantages. CWs is the hotspots of N cycle because of periodically or constantly flooded, second complex interaction interfaces, especially rhizosphere, are existed in CWs. Third, temporal and spatial heterogeneity increases due to rhizosphere effects. However, the relative importance of the interactions between the plants and microorganisms responsible for N removal are rarely studied. Unravelling the characteristics of plants that affect microbial communities involved in N cycle holds great potential for optimization of CWs design and bioenhancement of N removal. In this study, steady and efficient CW for N removal was constructed, and effect of plants on abundance, structure, and interactions of microbial community was studied. Regulation mechanism of radial oxygen loss (ROL) and artificial root exudates (ARE) on the functional populations for N removal was explored. Meanwhile, the key and active functional microorganisms driving the N removal by combined plant-microbe were illuminated using DNA stable-isotope probing (SIP). The main results are as follows:
    (1) Steady-state conditions were achieved throughout the 600-day operating period with high average removal efficiencies of COD (88.2%), ammonium (98.3%) and TN (95.9%) in the CWs planted with M. aquaticum treating high-strength swine wastewater. The effect hotspots of plant on N cycle microorganisms is located in the sediment. qPCR showed nitrifying and denitrifying genes were enriched significantly by M. aquaticum. M. aquaticum markedly changed the structure of the microbial community, and the nitrifiers, anammox bacteria and denitrifiers were significantly enriched in the sediment. Positive relationships among nitrifiers, denitrifiers, and other bacteria were stimulated by M. aquaticum in the sediment.
     (2) Oxygen microsensor showed the 0-400 μm from the rootlet of the M.aquaticum was the oxygen-producing zone. Ti3+-citrate colorimetric method demonstrated that ROL of M. aquaticum was 0.019 mg/h/plant. This ROL characteristic may build niches with increasing denitrification from the sediment across the water to the rhizoplane. The relative abundance of genes associated with facultative anaerobic and involved in encoding denitrifying enzymes was over-presented. qPCR showed that the abundance of nirS, nirK and nosZ were significantly increased, especially, the nosZ gene with a 21-fold increase in the rhizoplane compared with that in the sediment. These genes originated from facultative anaerobic denitrifiers, such as Aeromonas (21.8%), Pseudomonas (21.4%) and Acinetobacter (10.3%), which were significantly enriched in the rhizoplane. The theoretical calculation of oxygen sources and consumptions showed that water reaeration (2.8 g/m2/d) should provide the oxygen requirement for nitrification (2.5 g/m2/d) in the aerobic layer (0–5 cm). Moreover, the ROL (0.3 g/m2/d) could enrich the facultative anaerobic denitrifiers in the rhizoplane and water (5–10 cm) to achieve denitrification in the CW.
    (3) We set up soil microcosm models to illuminate the cross-effect of ROL and ARE on the denitrification. ARE significantly promotes the denitrification enzyme activity (DEA). qPCR showed ROL significantly changed the abundances of narG, nirK, nirS and nosZ gene compared with ARE. High-throughput sequencing of microbial 16S rRNA gene amplicons showed that ARE greatly changed the structure of microbial communities compared to ROL, and Clostridium sensu stricto and Bacillus were significantly enriched by ARE. Structural equation models (SEMs) demonstrated microbial community composition and ARE were the two most important drivers of DEA, that is, ARE significantly changed the microbial community composition, and the microbial community composition further significantly increased DEA.
     (4) DNA-SIP was successfully applied in labelling the key active denitrifiers in our microcosm models, and further validated ARE greatly changed the microbial community compositions compared with ROL. Candidatus Koribacter Azospirillum, Burkholderia Arthrobacter Anaeromyxobacter Nocardia Magnetospirillum and Ralstonia were the active and key denitrifiers in the soils amended with ARE, especially, the Magnetospirillum in the ARE treatment (1.4%) was 15-fold higher than that in the control treatment (0.1%), which was stimulated and enriched by the ARE amendment.
      This study provides a new way to investigate the molecular regulation mechanisms of wetland plants on microorganisms responsible for N removal under high N removal efficiency. Unraveling this regulation mechanism provides new theoretical basis and technical supports for the search for key plants and functional microbial communities and can advance the optimization of CWs.

Pages139
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/43648
Collection中国科学院环境生物技术重点实验室
Recommended Citation
GB/T 7714
孙海曙. 人工湿地中植物对氮功能微生物的调控机制研究[D]. 北京. 中国科学院生态环境研究中心,2020.
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