中国科学院海洋研究所机构知识库

氮是海洋生态系统中重要的生源要素，也是初级生产力的限制性因素。由于人为因素的干扰，往往导致大量的活性氮在河口及邻近海域积累。过度的氮负荷改变了沿海生态系统的氮循环平衡，从而产生了一系列如富营养化、有害藻类爆发、周期性或永久性的缺氧区域扩张，以及一氧化二氮（N₂O）排放等的生态环境问题。因此，沿海海洋生态系统的氮去除途径及其环境影响因素受到了广泛关注。厌氧氨氧化和反硝化反应作为海洋生态系统中氮去除的主要过程，可以通过将水生生态系统中的活性氮转移到大气中来抑制氮的积累。边缘海虽然仅占海洋总面积的8%左右，但是在全球范围内约45%的氮去除是依靠邻近海域、河口和大陆架沉积物的厌氧氨氧化作用和反硝化作用。因此，探索陆架海域氮的来源、迁移转化、去除途径及影响机制有助于提高对沉积物中氮去除和机理因素的认识。本研究选取东海陆架海域作为研究区域，分析表层沉积物的相关微生物氮转化过程及氮去除途径，并通过测定沉积物柱状样的生物标志物梯烷脂反演了此区域上世纪的厌氧氨氧化活动，从时间和空间的尺度上解释了沉积物的氮去除途径及机理过程。此外，通过外海的研究发现人为因素是造成东海陆架海域氮过量输入的重要原因。为了阐明富营养化海水的氮去除机制，构建实验室海水模拟体系，该模拟体系接种由东海陆架海域表层沉积物富集培养的海洋厌氧氨氧化菌（MAB），并通过人为控制氮输入量，模拟研究了海水体系的氮去除机制，为富营养化海水中氮的去除提供了一种方案。总之，本研究系统的探讨了外海的氮去除途径及生态因素和人为因素的影响，并通过室内培养实验提高了富营养化海水的有效氮去除。获得了如下的主要研究结果：

（1）基于东海陆架海域不同粒度表层沉积物的物理化学特性及相关微生物氮转化过程的研究发现，有机碳是海洋生态系统微生物厌氧氨氧化和反硝化氮去除途径的关键影响因素。东海陆架海域表层沉积物氮去除以反硝化作用为主，且泥质沉积物的氮去除速率显著高于砂质沉积物。有机碳含量丰富的泥质沉积物中反硝化和厌氧氨氧化作用相较于砂质沉积物更强烈，可能是因为有机碳的分解产生亚硝酸盐（NO₂^–），而NO₂^–既是反硝化的中间产物，也是厌氧氨氧化的底物，反硝化与厌氧氨氧化产生了耦合作用。

东海陆架海域泥质沉积物中的总有机碳（TOC）含量显著高于砂质沉积物，并且TOC浓度与沉积物平均粒径之间呈现显著负相关关系（r = –0.78，p < 0.01），说明沉积物粒径对控制TOC是十分重要的。研究区域的碳氮比（C/N）（6.04至7.18）和δ¹³C值（–22.50‰至–20.88‰）均表明表层沉积物的有机碳主要来自海洋环境。最终端元混合模型结果表明，海洋有机碳在东海陆架海域表层沉积有机碳中占主导地位，平均约占78%。源于陆地的有机碳在进入海洋生态系统后，经历了广泛的微生物改变，转化为海洋有机碳进行反应。东海陆架海域的氮去除以反硝化作用为主，泥质沉积物的氮去除速率（厌氧氨氧化速率：2.73 nmol N g^–1 h^–1，反硝化速率：14.39 nmol N g^–1 h^–1）显著高于砂质沉积物的氮去除速率（厌氧氨氧化速率：1.57 nmol N g^–1 h^–1，反硝化速率：5.55 nmol N g^–1 h^–1）。这主要是因为泥质沉积物中丰富的TOC含量，增加了与厌氧氨氧化和反硝化作用相关的功能基因的拷贝数。有机碳的分解产生NO₂^–，而NO₂^–既可作反硝化的中间产物又可作厌氧氨氧化的底物，因此，加强异养反硝化作用也可以提高厌氧氨氧化的活性。此外，东海陆架海域表层沉积物总梯烷脂获得高的数值（24-354 ng g⁻¹ dw），意味着近年来人为因素导致营养物质大量由长江口输入至东海，导致东海陆架海域持续的氮流失加剧。

（2）基于东海陆架海域沉积柱的理化特征及厌氧氨氧化特异性生物标志物梯烷脂的研究发现，总梯烷脂含量近60年来在增加，厌氧氨氧化作用逐渐增强。总梯烷脂含量在20世纪60年代之前含量较低，但自1960年之后显著增加，与中国经济快速发展时间相对应。可能是人为排入大量营养盐和有机碳进入东海，在海水富营养化现象愈发严重的同时，也导致水体中的溶解氧被大量消耗，进而造成海水缺氧严重，为厌氧氨氧化菌提供了良好的生存环境。

anammox 16S rRNA基因建立的系统发育树结果检索到的序列主要为Candidatus Scalindua，与长江口表层沉积物（KU218333.1、JX243219.1）、舟山群岛海洋沉积物（KF029964.1）以及中国南海表层沉积物（HQ665746.1）等环境来源的序列密切相关。各组分梯烷脂的浓度之间以及与总梯烷脂的含量之间的变化趋势类似，均表现出随时间增长的趋势，说明东海陆架海域厌氧氨氧化活动近年来加强的现象。悬浮颗粒物中较高的各组分梯烷脂含量主要出现在底层水中，这一现象表明东海陆架海域悬浮颗粒物中的梯烷脂是在水柱中产生的，而不是来自陆地环境或沉积物再悬浮。总梯烷脂含量在20世纪60年代之前波动于相对较低水平（16.36-47.11 ng g⁻¹ dw），然后在20世纪60年代之后开始显著增加（45.31-233.60 ng g⁻¹ dw）。厌氧氨氧化作用增强的时期与1960年后中国经济的快速发展相对应，因为大量的营养物质被排入东海陆架海域，加速了厌氧氨氧化菌的生长，进而导致了长江口附近的富营养化和缺氧现象。1960年后，沉积柱样DH5-0中TOC的含量与总梯烷脂含量有显著正相关关系（r = 0.366，p = 0.031），说明研究区域的富营养化与厌氧氨氧化反应密切相关。长江口悬浮颗粒物的搬运作用和人为因素导致的大量有机碳的输入是沉积有机碳增长的主要原因。支链GDGTs与总梯烷脂呈现出显著的正相关关系，这进一步表明了此区域因为缺氧的原因导致了厌氧氨氧化活性的增强。

（3）东海陆架海域沉积物时间和空间尺度上的研究发现人为影响是造成区域氮过量输入的重要原因。基于海水模拟系统中梯烷脂分析和¹⁵N同位素示踪技术的测定获得了富营养化海水的氮去除机制。海水模拟系统中厌氧氨氧化为主要氮去除过程，因为接种的MAB独特的生理特性使其在高盐度环境中表现出极大的耐受性，并使其能够提高富营养化海水的氮去除。模拟系统中海洋厌氧氨氧化菌属Candidatus Scalindua占据优势地位，且其丰度随着人为控制氮输入量的增大而增长（10.10%至81.71%），系统内环境适宜该菌属生长。

在实验室构建了海水模拟体系，并接种由东海陆架海域表层沉积物富集培养的MAB，通过人为控制氮输入量，研究了富营养化海水中氮的去除与细菌群落的关联性。结果表明，NH₄-N（Strength1为83.57%，Strength2为87.19%，Strength3为90.14%）、NO₂-N（Strength1为75.20%，Strength2为76.99%，Strength3为83.57%）和TN（Strength1为74.86%，Strength2为78.92%，Strength3为83.52%）的平均去除效率随着水体氮输入强度的增加而显著提高，氮输入最高的Strength3获得最佳氮去除。因为随着进水强度的增加降低了模拟系统水体中的溶氧水平，提高了相关厌氧微生物的生存空间和活性，从而促进了厌氧氨氧化菌的生长，提高了系统对氮的去除效率。氮负荷强度的增加增强了MAB接种的模拟系统的氮去除能力，厌氧氨氧化速率占总氮去除速率比值（ra）都在95%以上，表明厌氧氨氧化是该系统的主要氮去除途径。此外，高通量测序的结果显示，模拟系统以Candidatus Scalindua为主（Strength1 = 10.10%，Strength2 = 25.89%，Strength3 = 81.71%），说明系统内的环境适宜MAB的生长生存。nosZ和anammox 16S rRNA基因的丰度随着氮输入强度的增加而增加，与系统中氮去除的影响相对应。本研究通过接种MAB和调整氮输入量强度，提高了富营养化海水的氮去除效率，使其可应用于广泛的盐水原废水处理。

Nitrogen is often considered to be an important biogenic element of marine ecosystems and a limiting factor in primary productivity. The intensification of anthropogenic disturbances has led to the accumulation of large amounts of reactive nitrogen in the estuary and adjacent waters. Excessive nitrogen loading alters the balance of the nitrogen cycle in coastal ecosystems, resulting in a range of ecological problems such as eutrophication, harmful algal outbreaks, periodic or permanent expansion of hypoxic zones, and emissions of nitrous oxide (N₂O). Nitrogen removal pathways in coastal marine ecosystems and their environmental influences have therefore received much attention. Anaerobic ammonia oxidation (anammox) and denitrification reactions, the primary pathways for nitrogen removal in marine ecosystems, can inhibit nitrogen accumulation by transferring reactive nitrogen from the aquatic ecosystem to the atmosphere. Although marginal seas cover only about 8% of the total ocean area, about 45% of global nitrogen removal is accomplished through denitrification and anammox of sediments in estuaries, adjacent seas and continental shelves. Therefore, exploring the sources, transport transformations, removal pathways and influencing mechanisms of nitrogen in shelf seas can help improve the understanding of nitrogen removal from sediments and the mechanistic factors. In this study, the East China Sea shelf waters were selected as the study area to analyze the relevant microbial nitrogen transformation processes and nitrogen removal pathways in the surface sediments. Moreover, the anammox activities in this area in the last century were inferred by measuring the biomarkers of sedimentary column sample, explaining the nitrogen removal pathways and mechanistic processes in the sediments from the temporal and spatial scales. In addition, anthropogenic factors were found to be an important cause of excessive nitrogen input to the East China Sea shelf waters through studies in the open sea. In order to elucidate the nitrogen removal mechanism of eutrophic seawater, a laboratory seawater simulation system was constructed, which was inoculated with marine anammox bacteria (MAB) cultivated by surface sediment enrichment in the East China Sea shelf, and the nitrogen removal mechanism of the seawater system was simulated and studied by artificially controlling the nitrogen input, providing a scheme for nitrogen removal in eutrophic seawater. This study systematically investigated the nitrogen removal pathways in the open sea and the influence of ecological and anthropogenic factors, and improved the effective nitrogen removal from eutrophic seawater through laboratory-scale incubation experiments. The following main findings were obtained:

(1) Based on the physicochemical properties of surface sediments with different grain sizes and related microbial nitrogen transformation processes in the East China Sea shelf, it was found that organic carbon is a key factor in the microbial anammox and denitrification nitrogen removal pathways in marine ecosystems. The nitrogen removal from surface sediments in the East China Sea shelf was dominated by denitrification, and the nitrogen removal rate of muddy sediments was significantly higher than that of sandy sediments. Denitrification and anammox were more intense in muddy sediments rich in organic carbon compared with sandy sediments, probably because the decomposition of organic carbon produced nitrite (NO₂^–), which was both an intermediate product of denitrification and a substrate of anammox, and denitrification and anammox produced coupling effects.

The total organic carbon (TOC) content of muddy sediments in the East China Sea shelf was significantly higher than that of sandy sediments, and the mean sediment grain size was negatively correlated with TOC concentration (r = –0.78, p < 0.01), suggesting that sediment grain size is important in controlling TOC. The carbon to nitrogen ratio (C/N) (6.04 to 7.18) and δ¹³C values (–22.50‰ to –20.88‰) for the study area both indicate that the organic carbon in the surface sediments is predominantly from the marine environment. The results of the most terminal meta-mixing model indicate that marine organic carbon dominates the surface sedimentary organic carbon of the East China Sea shelf, averaging about 78%. Organic carbon of terrestrial origin undergoes extensive microbial alteration after entering the marine ecosystem and is converted to marine organic carbon for reaction. Nitrogen removal in the East China Sea shelf was dominated by denitrification, with rates of nitrogen removal from muddy sediments (denitrification rate: 14.39 nmol N g⁻¹ h⁻¹, anammox rate: 2.73 nmol nmol N g⁻¹ h⁻¹) significantly higher than the nitrogen removal rates from sandy sediments (denitrification rate: 5.55 nmol N g⁻¹ h⁻¹, anammox rate: 1.57 nmol N g⁻¹ h⁻¹). This is mainly due to the abundant TOC content of the muddy sediment, which increases the copy number of functional genes associated with anammox and denitrification. The decomposition of organic carbon produces NO₂⁻, which can be used as both an intermediate product of denitrification and a substrate for anammox; therefore, enhanced heterotrophic denitrification can also increase the activity of anammox. In addition, high values (24-354 ng g⁻¹ dw) were obtained for summed ladderane lipids in surface sediments of the East China Sea shelf, implying that anthropogenic factors have led to a large input of nutrients from the Yangtze River estuary to the East China Sea in recent years, resulting in an increase in the ongoing nitrogen loss from the shelf of East China Sea.

(2) Based on the physicochemical characteristics of sediment columns and anammox-specific biomarkers in the East China Sea shelf, it was found that the summed ladderane lipids content was increasing in the last 60 years and the anammox was gradually enhanced. Summed ladderane lipid content was low until the 1960s, but increased significantly since 1960, corresponding to the time of rapid economic development in China. It may be that anthropogenic discharge of large amounts of nutrient and organic carbon into the East China Sea has led to a large depletion of dissolved oxygen in the water column while the eutrophication of seawater has become more serious, which in turn has caused serious hypoxia in seawater and provided a good living environment for anammox bacteria.

The results of the phylogenetic tree established by the anammox 16S rRNA gene retrieved sequences mainly from Candidatus Scalindua, closely related to sequences from environmental sources such as the surface sediments of the Yangtze River estuary (KU218333.1, JX243219.1), marine sediments of the Zhoushan Islands (KF029964.1) and surface sediments of the South China Sea (HQ665746.1). Similar trends were observed between the concentrations of the component ladderanes and with the summed ladderanes content, all showing an increasing trend with time, indicating that the anammox activity in the East China Sea shelf has intensified in recent years. The higher levels of ladderanes in suspended particulate matter were mainly found in bottom water layer, a phenomenon that suggests that ladderanes in suspended particulate matters from the East China Sea shelf is generated in the water column rather than from the terrestrial environment or sediment resuspension. Summed ladderane lipids content fluctuated at relatively low levels (16.36-47.11 ng g⁻¹ dw) until the 1960s and then began to increase significantly (45.31-233.60 ng g⁻¹ dw) after the 1960s. The period of enhanced anammox corresponds to the rapid economic development of China after 1960, which accelerated the growth of anammox bacteria due to the discharge of large amounts of nutrients into the coastal shelf of the East China Sea, which in turn led to eutrophication and anoxia near the mouth of the Yangtze River. After 1960, there was a significant positive correlation between TOC content and summed ladderane lipids content in DH5-0 sedimentary column samples (r = 0.366, p = 0.031), indicating that eutrophication in the study area is closely related to anammox reactions. The transport of suspended particulate matter in the Yangtze estuary and the input of large amounts of organic carbon due to anthropogenic factors are the main reasons for the increase in sedimentary organic carbon. The branched GDGTs showed a significant positive correlation with the summed ladderane lipids, which further suggests that this region has enhanced anammox activity due to hypoxia.

(3) Studies on the temporal and spatial scales of sediments in the East China Sea shelf have identified anthropogenic influences as an important cause of excessive regional nitrogen inputs. The nitrogen removal mechanism of eutrophic seawater was obtained based on the determination of ladderane lipids analysis and ¹⁵N isotope tracing technique in a seawater simulation system. Anammox is the main nitrogen removal process in the seawater simulation system because the unique physiological properties of the inoculated MAB make it extremely tolerant in high salinity environments and enable it to enhance nitrogen removal from eutrophic seawater. The genus Candidatus Scalindua, a marine anammox bacterium, was dominant in the simulated system, and its abundance increased with increasing anthropogenic control of nitrogen input (10.10% to 81.71%), and the environment in the system was suitable for the growth of this genus.

A seawater simulation system was constructed in the laboratory and inoculated with MAB enriched and cultured from surface sediments of the East China Sea shelf, and the correlation between nitrogen removal from eutrophic seawater and bacterial communities was investigated by artificially controlling the nitrogen input. The results show that the average effluent removal efficiencies of NH₄-N (83.57% for Strength1, 87.19% for Strength2, and 90.14% for Strength3), NO₂-N (75.20% for Strength1, 76.99% for Strength2, and 83.57% for Strength3) and TN (74.86% for Strength1, 78.92% for Strength2, and 83.52% for Strength3) were significantly higher as the intensity of the influent nitrogen input increased. The optimal nitrogen removal was obtained by the system Strength3 with the highest influent nitrogen input. This is because increasing influent strength reduces the dissolved oxygen level in the water of the seawater system, increasing the space and activity of the associated anaerobic microorganisms, thus promoting the growth of anammox bacteria and improving the removal efficiency of nitrogen. The increase in nitrogen loading strength enhanced the nitrogen removal capacity of the MAB-inoculated seawater system, and the ratio of anammox rate to total nitrogen removal rate (ra) was all above 95%, indicating that anammox was the main nitrogen removal pathway of the system. In addition, the results of high-throughput sequencing showed that Candidatus Scalindua was dominant within the constructed seawater system (10.10% in Strength1, 25.89% in Strength2, and 81.71% in Strength3), indicating that the environment within the system was suitable for the growth and survival of MAB. nosZ and anammox 16S rRNA genes abundance increased with increasing nitrogen input strength, corresponding to the effect of nitrogen removal in the system. This study improved the nitrogen removal efficiency of eutrophic seawater by inoculating MAB and adjusting the strength of nitrogen input, allowing it to be applied to a wide range of saline raw wastewater treatments.

第 1 章 绪论...............................................................................................1

1.1 研究背景.............................................................................................................1

1.2 海洋生态系统氮循环过程.................................................................................2

1.3 氮去除过程的主要影响因素.............................................................................6

1.3.1 反硝化过程影响因素分析..........................................................................7

1.3.2 厌氧氨氧化影响因素分析..........................................................................8

1.4 氮去除测定方法研究进展...............................................................................10

1.4.1 基因拷贝法................................................................................................10

1.4.2 生物标志物法............................................................................................11

1.4.3 15N 同位素示踪法......................................................................................13

1.5 时空分布特征研究进展...................................................................................13

1.6 拟解决的科学问题...........................................................................................14

1.7 论文研究内容...................................................................................................15

第 2 章 研究区域概况与实验方法 ........................................................17

2.1 外海研究区域概况...........................................................................................17

2.2 外海调查站位与样品采集...............................................................................17

2.3 室内培养实验...................................................................................................18

2.3.1 海水体系的配置和操作............................................................................18

2.3.2 培养实验水样采集与分析........................................................................19

2.4 样品分析方法...................................................................................................21

2.4.1 理化性质测定............................................................................................21

2.4.2 有机碳、氮及稳定碳同位素....................................................................22

2.4.3 沉积物定年................................................................................................22

2.4.4 梯烷核心脂测定........................................................................................23

2.4.5 DNA 提取与高通量测序...........................................................................24

2.4.6 定量 PCR 测定..........................................................................................24

2.4.7 沉积物培养实验........................................................................................25

2.5 数据处理...........................................................................................................26

2.5.1 沉积物氮去除速率来源............................................................................26

2.5.2 核苷酸序列登录号....................................................................................26

2.5.3 培养实验废水氮去除率............................................................................26

2.6 统计分析...........................................................................................................26

第 3 章 有机碳对东海陆架海域沉积物氮去除的影响........................27

3.1 沉积物中有机碳的来源...................................................................................27

3.2 沉积物中梯烷核心脂的分布...........................................................................32

3.3 有机碳对氮去除的反应...................................................................................35

3.4 东海陆架海域的氮去除机制...........................................................................37

3.5 环境因素与微生物丰度之间的联系...............................................................41

3.6 本章小结...........................................................................................................43

第 4 章 140 年来东海陆架海域沉积物氮去除过程的反演.................45

4.1 研究区域的理化特征.......................................................................................45

4.2 anammox 16S rRNA 基因的群落组成及系统发育分析.................................48

4.3 梯烷脂在东海中的出现和起源.......................................................................50

4.4 梯烷脂反演过去厌氧氨氧化活动的变化.......................................................53

4.5 厌氧氨氧化与富营养化和缺氧之间的联系...................................................54

4.6 本章小结...........................................................................................................57

第 5 章 富营养化海水体系氮去除机制的模拟研究............................59

5.1 海水体系的整体氮去除性能...........................................................................59

5.2 海水体系中梯烷脂的变化...............................................................................63

5.3 微生物群落结构...............................................................................................64

5.4 海水体系的氮去除机制...................................................................................66

5.5 海水体系中厌氧氨氧化和反硝化细菌的潜在活性.......................................69

5.6 本章小结...........................................................................................................70

第 6 章 结论与展望 ................................................................................73

6.1 主要结论...........................................................................................................73

6.2 论文创新点.......................................................................................................75

6.3 研究展望...........................................................................................................75

参考文献...................................................................................................77

附录 缩略语.............................................................................................93

致谢...........................................................................................................95

作者简历及攻读学位期间发表的学术论文与其他相关学术成果......97