【作者】 董梁；

【导师】 宋长春；

【作者基本信息】 大连理工大学 ， 水文学及水资源， 2025， 博士

【摘要】 自工业化以来,人类活动导致自然环境中氮含量的持续增加,而河口近海区域成为氮的主要汇流富集区之一,过量的氮负荷不断汇入,引发该区域产生一系列的水环境与生态问题。河口作为陆海交汇的重要系统,在活性氮的转化与去除中发挥着关键作用。近几十年来,人类活动与气候变化叠加作用改变了河口环境格局,例如径流量减少、海水入侵、人为氮输入、海冰过程变化以及水体缺氧频发等。这些自然与人为驱动因素通过调节盐度、溶解氧及无机氮等关键环境因子,进而影响沉积物脱氮过程。反硝化与厌氧氨氧化是河口区域主要的微生物脱氮途径,可依据反应底物来源划分为非耦合脱氮与耦合脱氮两种形式,前者所需的硝酸盐和亚硝酸盐来自上覆水,而后者的底物则由硝化作用生成。此外,脱氮过程中还伴随生物途径引起的氧化亚氮（N₂O）排放。然而,寒区河口及近海沉积物脱氮过程与N₂O生成对变化环境的响应机制仍不明确,相关人工干预的氮去除技术也有待提升。基于此,本文重点选取我国东北寒区的辽河口为典型研究区,综合运用野外监测、室内模拟实验、氮同位素示踪与分子生物学技术,基于“过程响应—环境效应—应对方法”的研究思路,系统探讨径流变化、海冰过程以及氮-氧-盐协同作用下的河口脱氮与N₂O生成机制,评估其潜在环境效应,并探索适用于低温环境的人工氮去除技术。取得的主要研究成果如下:（1）基于野外观测与实验研究发现,在正常径流与低径流时期,反硝化作用是辽河口沉积物中主要的脱氮途径,平均贡献率分别为86.5%和94.0%。正常径流期以非耦合脱氮为主,而低径流期则表现为耦合与非耦合过程并存,其中非耦合过程占比约为51%,耦合过程占比约为49%。低径流时期的海水入侵显著提升了辽河口区盐度,促使氨氧化古菌（AOA）向氨氧化细菌（AOB）转变,并促进硝化细菌Nitrospina、Nitrosomonas和Nitrosomonadaceae的丰度提升,从而增强了硝化–反硝化耦合脱氮过程,同时无机氮浓度被稀释,导致反硝化速率下降。结果揭示了径流变化通过改变环境因子与微生物群落调控河口沉积物的脱氮速率与途径。（2）通过海水结冰和融化模拟实验研究,发现温度通过调控硝化微生物丰度与结构,从而成为影响河口沉积物硝化速率的关键因子。冻结初期硝酸盐的积累增强了微生物反硝化作用,但随后高盐和低温抑制了反硝化细菌丰度,导致反硝化速率下降。厌氧氨氧化速率在冻结期同样受到盐度升高的抑制而降低,其在融化期逐步恢复。估算表明,辽河口沉积物氮转化过程每年可去除约28%的径流输入无机氮通量,其中约88%的氮素通过沉积物反硝化作用去除。而在海冰期,去除通量约占年沉积物无机氮去除通量的7%。结果揭示了海冰过程通过改变水体环境来调控河口沉积物中微生物氮去除过程。（3）采用野外监测与¹⁵N–¹⁸O双同位素标记实验方法,发现弱氧环境下低盐条件能够通过促进nir S和nor B基因丰度显著提高N₂O生成速率,同时硝酸盐浓度升高通过增强了反硝化作用,进一步促进N₂O生成速率提升约35%;相反,高盐环境则通过抑制反硝化细菌Pseudomonas丰度,降低了N₂O生成速率。异养反硝化是河口N₂O生成的主要途径,其中在低盐度环境中主要由反硝化速率增强驱动,而在高盐度环境中则主要受不完全反硝化过程促进。同时,高盐度条件下优势硝化细菌Nitrosomonas丰度升高,可能导致环境中亚硝酸盐的积累,从而增强硝化细菌反硝化途径的N₂O生成;同时Nitrospina和Nitrospira的丰度增加,促进硝酸盐的生成,提升了硝化耦合反硝化途径对N₂O生成的相对贡献。常氧环境中硝化过程更趋完整,氮氧化物被进一步转化为硝酸盐,导致硝化细菌的N₂O贡献降低。通过促进硝化过程的完整进行,从而降低了硝化细菌硝化途径的贡献。研究结果揭示了氮氧盐多因子协同作用下沉积物N₂O生成的微生物机制。进一步分析发现,辽河口区域N₂O排放水平整体高于近海水域,并在弱氧时期更易形成排放热点。当弱氧条件下无机氮输入增加一倍时,沉积物N₂O排放通量增加约78%,凸显了缺氧与氮负荷双重胁迫下河口N₂O排放的加剧效应。（4）利用低温环境下构建的沉积物微生物燃料电池（SMFC）实现了10°C条件下有效氨氮去除,同时未引发过量N₂O排放。比选发现不锈钢电极通过其优良导电性表现出更快的去除速率,而木炭复合电极则通过提供丰富微生物附着位点,在溶解氧波动环境中显示出较高稳定性。阳极SM1A02细菌在底层厌氧环境以电极为电子受体中将氨氮氧化为亚硝酸盐,产物扩散至上层有氧水体后进一步氧化为硝酸盐,同时氧气在阴极作为电子受体被还原。部分生成的硝酸盐再扩散回阳极厌氧区,被阳极反硝化细菌Marinicella转化为氮气。此外,Methylotenera与Methylobacter的共生作用促进了硝酸盐与亚硝酸盐的还原效率,从而增强整体氮去除率。研究结果系统评估了SMFC在低温条件下的稳定脱氮能力,厘清了SMFC系统内电极参与驱动的微生物氮去除途径。总体而言,本研究揭示了寒区河口径流变化、海冰过程和氮–氧–盐协同作用对多途径微生物脱氮及N₂O生成的调控机制。同时,建立并优化了适用于低温环境的SMFC氮去除技术,阐明了电极参与的氮转化途径,为寒区河口氮污染治理提供了新的技术方向。更多还原

【Abstract】 Since the beginning of industrialization,human activities have led to a continuous increase in nitrogen levels in the natural environment.Estuarine and coastal regions have become major convergence and accumulation zones of nitrogen,where excessive nitrogen inputs have caused a series of water quality and ecological problems.As critical interfaces between land and sea,estuaries play a key role in the transformation and attenuation of reactive nitrogen.Over the past decades,the combined impacts of human activities and climate change have significantly altered estuarine and coastal environments,including reduced runoff,seawater intrusion,increased anthropogenic nitrogen inputs,sea-ice dynamics,and frequent hypoxia events.These natural and anthropogenic drivers regulate environmental factors such as salinity,dissolved oxygen,and inorganic nitrogen,thereby influencing nitrogen removal processes.Denitrification and anammox are the primary microbial pathways for nitrogen removal in estuarine sediments.Depending on the source of the substrates,nitrate and nitrite required for uncoupled nitrogen removal originate from the overlying water,whereas the substrates for coupled nitrogen removal are generated through nitrification.In addition,the nitrogen removal processes are accompanied by emissions of nitrous oxide（N₂O）via microbial pathways.However,the mechanisms of nitrogen removal and N₂O production in cold-region estuarine and coastal sediments in response to environmental changes remain unclear,and artificial nitrogen removal technologies require further improvement.Thus,the Liao River Estuary in cold northeastern China was selected as typical research areas.Using field monitoring,laboratory simulations,nitrogen isotope tracing,and molecular biological analyses within a“process response–environmental effect–mitigation approach”framework,the study investigated nitrogen removal and N₂O production in estuarine sediments under the effects of runoff,sea ice,and the combined influence of nitrogen,oxygen,and salinity,and further assessed their potential environmental impacts while exploring low-temperature nitrogen removal technologies.The main research findings are as follows:（1）Field observations and experimental studies indicate that denitrification is the dominant nitrogen removal pathway in Liao River Estuary sediments,accounting for an average of 86.5%and 94.0%of total nitrogen removal during normal and low-runoff periods,respectively.Uncoupled nitrogen removal dominated during normal runoff,whereas coupled and uncoupled processes coexisted during low runoff,accounting for approximately 51%and 49%of total nitrogen removal,respectively.Seawater intrusion during low runoff periods significantly increased salinity in Liao River Estuary,promoting a shift from ammonia-oxidizing archaea（AOA）to ammonia-oxidizing bacteria（AOB）and enhancing the abundance of nitrifiers such as Nitrospina,Nitrosomonas,and Nitrosomonadaceae,thereby strengthening the coupled nitrification–denitrification pathway.These results highlight how river runoff alters nitrogen removal by reshaping environmental conditions and microbial communities.（2）Through simulated experiments of seawater freezing and melting,it was found that temperature is a key factor influencing nitrification rates in estuarine sediments by regulating the abundance and structure of nitrifying microorganisms.Nitrate accumulation during early freezing enhanced denitrification,but subsequent low temperatures and high salinity suppressed denitrifier abundance and activity,leading to a decrease in denitrification rates.The rate of anammox was also inhibited during freezing due to increased salinity,but recovered during melting.It was estimated that nitrogen transformation processes in Liao River Estuary sediments can annually remove approximately 28%of inorganic nitrogen flux input from runoff,of which around 88%is eliminated through sedimentary denitrification.These findings indicate that sea-ice processes regulate microbial nitrogen removal in estuarine sediments by altering water environment conditions.（3）Field monitoring and isotope labelling experiments with ¹⁵N–¹⁸O revealed that low-salinity,weak hypoxia enhanced N₂O production by increasing nir S and nor B gene abundances and elevated nitrate,boosting rates by 35%.In contrast,high salinity suppressed denitrifying Pseudomonas,reducing N₂O production.Heterotrophic denitrification is the main pathway for N₂O production in estuarine sediments.Under low-salinity conditions,N₂O production is primarily driven by elevated denitrification rates,whereas in high-salinity environments it is mainly attributed to incomplete denitrification.Increased Nitrosomonas,Nitrospina,and Nitrospira under high salinity promoted nitrite and nitrate accumulation,increasing coupled nitrification–denitrification contributions.Normoxia promotes the complete progression of the nitrification,reducing the contributions from nitrifier nitrification pathways.N₂O emissions were higher in the Liao River Estuary than offshore,with emission hotspots forming during weak hypoxia.Doubling nitrate inputs under weak hypoxia increased sediment N₂O emissions by 78%,emphasizing the compounded effects of hypoxia and nitrogen loading.（4）The Sediment Microbial Fuel Cell（SMFC）constructed under low-temperature conditions achieved effective ammonium removal at 10°C without excessive N₂O emissions.Comparative analysis revealed that stainless steel electrodes achieved faster removal due to superior conductivity,while charcoal composite electrodes provided higher stability under fluctuating oxygen by offering abundant microbial attachment sites.Anodic SM1A02 bacteria oxidized ammonium to nitrite under anaerobic conditions using the electrode as an electron acceptor;the nitrite diffused to overlying oxic water for further oxidation to nitrate,some of which returned to the anode and was reduced to nitrogen gas by denitrifying Marinicella.Additionally,synergistic interactions between Methylotenera and Methylobacter enhanced nitrate and nitrite reduction efficiency,improving overall nitrogen removal.The findings systematically evaluated the stable denitrification capacity of SMFC under low-temperature conditions and clarified electrode-driven microbial nitrogen removal pathways.Overall,this study elucidates how variations in cold-region estuarine runoff,sea-ice processes,and nitrogen–oxygen–salinity interactions regulate multi-pathway microbial nitrogen removal and N₂O production.In parallel,SMFC technology for nitrogen removal under low-temperature conditions was developed and optimized,revealing the electrode-mediated nitrogen transformation pathways.These findings offer a novel approach for mitigating nitrogen pollution in cold estuarine environments.更多还原