Collaborative Research: Cryptic nitrogen cycling in the anoxic subterranean estuary

氮是维持沿海生态系统高生产力的重要营养物。然而，过量的氮输送会导致严重的水质恶化，包括有害的藻类繁殖、鱼类死亡和无氧死区。在沿海环境中，大量的氮转化调节着氮输送和氮去除之间的平衡，其中大多数反应发生在沉积物中，海水穿过地下并与来自高地的地下水混合。这个混合区被称为地下河口，它的地球化学特征与上面的海水或下面的地下水非常不同。因此，它有可能承载各种独特的反应，影响氮对上覆水的可用性。来自威廉玛丽学院、弗吉尼亚海洋科学研究所（VIMS）和康涅狄格大学（UConn）的科学家们提议研究一个隐氮循环的重要性，这是一个在地下河口中新颖且可能广泛存在的氮循环过程。隐氮循环包括缺氧氨氧化生成亚硝酸盐（缺氧硝化）与厌氧氨氧化（厌氧氨氧化）或反硝化反应，产生无害的二氮气体。拟议的项目代表了高度变革性的科学，因为它有可能改变目前在缺氧环境中详细描述生物地球化学氮循环操作的范例。隐氮循环的发生将对陆地和地下水系统以及沿海海洋的氮收支产生广泛的影响。对隐氮循环的表征将使我们更好地理解氮、金属和硫循环之间的相互作用，以及人类对沿海环境的持续改变的潜在影响。该项目的教育贡献主要集中在研究生和本科生的培养上。VIMS和康涅狄格大学的两名研究生将接受微生物学、分子生态学和生物地球化学的跨学科培训，而通过VIMS REU（本科生研究经验）计划和康涅狄格大学海洋科学项目招募的几名本科生也将参与该项目。此外，将从汉普顿大学（Hampton University）招募三名暑期本科生实习生，这是一所历史悠久的黑人学院，他们将接受培训，以加强少数族裔在海洋科学方面的教育和研究。公众外展活动将会在一些受欢迎的地点举行，例如VIMS海洋科学日，以及VIMS的非工作时间公众讲座系列。康涅狄格大学的Tobias还通过康涅狄格大学海洋学者和早期大学体验项目以及神秘水族馆的展览提供教育贡献和推广工作。隐氮循环是一种将缺氧硝化与厌氧氨氧化、反硝化等微生物脱氮途径相结合的新工艺。与厌氧氨氧化不同，厌氧氨氧化指的是亚硝酸盐氧化铵形成二氮（N2）气体，缺氧硝化是在没有氧气的情况下，使用其他常见的化学氧化剂，如金属氧化物（即铁和锰）或硫酸盐，在许多海洋和沿海系统中丰富。这些反应的热力学有利性依赖于通过这些氧化剂与厌氧氨氧化或反硝化反应形成的亚硝酸盐的耦合。由于这种耦合作用，亚硝酸盐在缺氧海洋系统中不会积累或无法测量。因此，在缺氧环境中，负责亚硝酸盐生产的隐态N循环可能作为一种新的N转化过程发生，是N2生产的重要环节，并减弱从地下河口（STE）排放的N负荷。位于弗吉尼亚州约克河河口的STE的初步结果显示，在亚硝酸盐和硝酸盐都检测不到的含铁和硫化物带，大量的N2产量显示了50-75%的固定地下水氮的去除。利用15N示踪剂的稳定同位素培养实验和微生物群落的分子分析表明，在STE中，缺氧硝化和厌氧氨氧化的耦合过程是主要的N2生产途径，而不是典型的反硝化过程。因此，沿海地下水中耦合的厌氧硝化-厌氧氨氧化可能是陆海界面固定氮未被识别的主要汇。除了沿海地下水外，在缺氧带和海洋盆地中，隐氮循环也具有潜在的重要性。这一建议的重点是STE，因为地球化学条件似乎最适合所提出的反应发生，我们的初步数据显示了一个隐N循环的有力证据。拟议的工作使用地球化学、15N同位素示踪剂和微生物学相结合的方法来评估环境对隐氮循环的控制，并估计其对减少沿海海洋固定氮通量的贡献。提出了四种方法：(1)对地下河口缺氧硝化反应及相关微生物群落进行现场表征；(2)实验室培养实验，确定隐氮循环热点；(3)控制微观实验，确定地球化学对缺氧硝化的控制作用；(4)缺氧硝化的原位评价，以估计沿海含水层中隐氮循环的重要性。

Nitrogen is an important nutrient that maintains high coastal ecosystem productivity. Yet excess nitrogen delivery can cause serious water quality deterioration including harmful algal blooms, fish kills, and oxygen free dead zones. Numerous nitrogen transformations regulate the balance between nitrogen delivery and nitrogen removal in coastal environments and the majority of these reactions occur in sediments where seawater passes through the subsurface and mixes with groundwater transported from uplands. This mixing zone, referred to as the subterranean estuary, is characterized by very different geochemistry than either the seawater above it or the groundwater below it. Thus, it has the potential to host a variety of unique reactions that affect nitrogen availability to the overlying water. Scientists from the College of William and Mary, Virginia Institute of Marine Science (VIMS), and the University of Connecticut (UConn) propose to examine the importance of a cryptic nitrogen cycle, a novel and potentially widespread nitrogen cycling process in the subterranean estuary. The cryptic nitrogen cycle comprises anoxic ammonium oxidation to nitrite (anoxic nitrification) coupled with anaerobic ammonium oxidation (anammox) or denitrification producing harmless dinitrogen gas. The proposed project represents highly transformative science because it has the potential to change the current paradigm detailing operation of the biogeochemical nitrogen cycle in anoxic environments. Occurrence of the cryptic nitrogen cycle would have broad implications for the nitrogen budget of terrestrial and groundwater systems and the coastal ocean. Characterization of the cryptic nitrogen cycle will allow us to better understand interactions among the nitrogen, metals, and sulfur cycles, and potential impacts of ongoing human modification of coastal environments. Educational contribution of this project focuses on graduate and undergraduate student training. Two graduate students at VIMS and UConn will receive interdisciplinary training in microbiology, molecular ecology, and biogeochemistry while several undergraduates recruited through the VIMS REU (Research Experience for Undergraduates) Program and the UConn marine science programs will also participate in the project. In addition, three summer undergraduate interns will be recruited from Hampton University, a historically Black college, and trained to enhance minority education and research in marine science. Public outreach will be achieved through popular venues such as VIMS Marine Science Day, and the VIMS After Hours Public Lecture Series at VIMS. Tobias at UConn also provides educational contributions and outreach efforts through the UConn Marine Scholars and Early College Experience programs and an exhibit at Mystic Aquarium.A cryptic nitrogen cycle is proposed as a new process coupling anoxic nitrification to microbial nitrogen removal pathways such as anammox and denitrification. Unlike anammox, which refers to the oxidation of ammonium by nitrite to form dinitrogen (N2) gas, anoxic nitrification occurs by oxidation of ammonium in the absence of oxygen using other common chemical oxidants such as metal oxides (namely, Fe and Mn) or sulfate, abundant in many marine and coastal systems. The thermodynamic favorability of these reactions relies on coupling nitrite formed via these oxidants with anammox or denitrification. Due to the coupling, nitrite will not accumulate or be measurable in anoxic marine systems. Thus, a cryptic N cycle responsible for nitrite production can occur as a novel N transforming process in anoxic environments, serve as a vital link to N2 production, and attenuate N loads discharging from a subterranean estuary (STE). Preliminary results from a STE in the York River Estuary located in Virginia showed substantial N2 production, representing removal of 50-75% of the fixed groundwater N, in ferruginous and sulfidic zones where neither nitrite nor nitrate were detectable. Stable isotope incubation experiments using the 15N tracer and molecular analysis of microbial communities suggest that coupled anoxic nitrification and anammox processes are the dominant N2 production pathways rather than canonical denitrification in the STE. Therefore, coupled anoxic nitrification-anammox in coastal groundwater may be a major unrecognized sink for fixed nitrogen at the land-sea interface. In addition to coastal groundwater, the cryptic N cycle has potential importance in anoxic zones and ocean basins. This proposal focuses on the STE because geochemical conditions there appear optimal for the proposed reactions to occur, and our preliminary data show strong evidence for a cryptic N cycle. The proposed work uses a combined geochemical, 15N isotope tracer and microbiological approach to evaluate environmental controls on the cryptic N cycle as well as to estimate its contribution to reduction of fixed N fluxes to the coastal ocean. Four approaches are proposed: (1) Field characterization of anoxic nitrification reactions and associated microbial communities in a subterranean estuary; (2) Laboratory incubation experiments to identify hotspots of the cryptic N cycle; (3) Controlled microcosm experiments to determine geochemical controls on anoxic nitrification; and (4) in situ assessment of anoxic nitrification to estimate the importance of the cryptic N cycle in a coastal aquifer.