Nitrite Oxidation in Oxygen Minimum Zones

这项研究的基础是氮在限制海洋产量方面的基础作用。亚硝酸盐是氮循环中的关键化合物：它可以被氧化成硝酸盐，从而保留为一种有效的营养物质，或者它可以被还原为氮气，从而从生物可利用氮库中消失。亚硝酸盐氧化细菌(NOB)氧化亚硝酸盐是产生硝酸盐的唯一生物途径，所有已知的NOB都需要氧气才能维持生命。还原途径也是由微生物进行的，在这种情况下，细菌只有在没有氧气的情况下才能茁壮成长。然而，在以前的实验中，在没有氧气的情况下，在同一海洋水域的样品中检测到亚硝酸盐的氧化和还原。在一次到秘鲁海岸低氧水域的研究巡航中，我们将调查亚硝酸盐在没有氧气的情况下明显氧化的三种解释：1)存在不需要氧气的未知种类的NOB；2)称为错位的新反应，这在自然界中是可能的，但从未被检测到；3)与NOB中的氧气压力有关的人工制品。这项研究可能导致发现新的机制和/或新的生物，决定亚硝酸盐的命运和氮的可用性，以支持长期的初级生产。这个项目将促进发现和理解，同时促进教学、培训和学习，为普林斯顿大学的学生提供机会，让他们参与实验室和潜在的海上研究并获得实践经验。本科生和研究生都将通过实习和实地经验参与研究。我们还将根据巡航期间收集的数据，通过视频和作业将我们在海上的工作融入课堂教学。亚硝酸盐氧化是唯一已知的产生硝酸盐的生物过程，它构成了海洋中最大的固定氮库。亚硝酸盐氧化是由亚硝酸氧化细菌(NOB)进行的，所有已知的物种都是专性好氧细菌。亚硝酸盐在多个微生物途径中还原为氮气，通常在缺氧条件下进行。尽管这两个过程在氧气方面明显不相容，但在氧气最小区域(OMZ)的低氧或缺氧水域中都能检测到。因此，亚硝酸盐在OMZ中的命运对全球固定N预算有影响。在OMZ区最缺氧的深度段，亚硝酸盐氧化在基本上为零的氧气水中被检测到，这表明一些亚硝酸盐氧化菌可能具有厌氧代谢能力。亚硝酸盐歧化(或歧化)，亚硝酸盐同时被氧化为硝酸盐并还原为氮气，是一个热力学上有利的反应，它将在一个有机体中连接两个过程-但在自然界中从未观察到这种现象。这里提出的研究将解决关于海洋中亚硝酸盐的两大问题：1)厌氧亚硝酸盐氧化是如何工作的？2)是什么决定了亚硝酸盐的命运？实验方法将探讨厌氧亚硝酸盐氧化的三种可能的解释：1)不同种类的NOB将亚硝酸盐氧化为硝酸盐，表现出不同的氧气耐受性/要求；2)亚硝酸盐歧化，也由NOB执行，部分解释了亚硝酸盐的氧化和还原共存；3)表面上，厌氧亚硝酸盐氧化确实是生物介导的，但并不总是代表亚硝酸盐从亚硝酸盐中净产生硝酸盐；相反，它是酶催化的亚硝酸盐和硝酸盐相互转化过程中同位素平衡的结果。这些问题将通过在位于海洋中一个主要的海洋保护区--东热带南太平洋--内外的站点进行一套15N示踪剂孵化来解决。将确定速率过程对氧浓度的依赖关系，并将评估微生物组合的组成，以确定不同环境条件下是否涉及不同的微生物成分。将跨氧梯度和氧操作测量参与低氧浓度下氧化/还原/呼吸代谢的基因的表达，以确定它们在支持“厌氧”亚硝酸盐氧化方面的潜在作用。显然的厌氧亚硝酸盐氧化是由于亚硝酸盐和硝酸盐之间的酶水平的相互转化，这不会导致净硝酸盐产生，也与亚硝酸盐氧化细菌的生长无关，这一可能性也将被调查。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This research is grounded in the fundamental role of nitrogen in limiting production in the ocean. Nitrite is a pivotal compound in the nitrogen cycle: it can be oxidized to nitrate, and thus retained as an available nutrient, or it can be reduced to dinitrogen gas, and thus lost from the bioavailable nitrogen pool. Oxidation of nitrite by nitrite oxidizing bacteria (NOB) is the only biological pathway by which nitrate is produced, and all known NOB require oxygen for life. The reduction pathway is also carried out by microbes, in this case, bacteria that thrive only in the absence of oxygen. In previous experiments, however, both oxidation and reduction of nitrite were detected in the same samples from ocean waters in the absence of oxygen. We will investigate three explanations for the apparent oxidation of nitrite in the absence of oxygen on a research cruise to the low oxygen waters off the coast of Peru: 1) The presence of unknown kinds of NOB that do not require oxygen; 2) a new reaction called dismutation, which is possible but never detected in nature; 3) an artifact associated with oxygen stress in NOB. This research could lead to discovery of novel mechanisms and or novel organisms that determine the fate of nitrite and the availability of nitrogen to support primary production in the long run. This project will advance discovery and understanding while promoting teaching, training and learning by providing opportunities for Princeton students to get involved in and have hands on experience in research in the lab and potentially at sea. Both undergraduate and graduate students will participate in the research through internships and field experiences. We will also integrate our work at sea into teaching in the classroom via videos and assignments based on data collected during the cruise.Nitrite oxidation is the only known biological process that produces nitrate, which comprises the largest fixed nitrogen reservoir in the ocean. Nitrite oxidation is carried out by nitrite oxidizing bacteria (NOB), and all known species are obligate aerobes. Nitrite reduction to N2 occurs in multiple microbial pathways, generally under anoxic conditions. Despite their apparent incompatibility regarding oxygen, both processes are detected in the low oxygen or anoxic waters of oxygen minimum zones (OMZs). Thus, the fate of nitrite in OMZs has implications for the global fixed N budget. Nitrite oxidation is detected at high rates in essentially zero oxygen water in the most oxygen depleted depth intervals in OMZ regions, which suggests that some nitrite oxidizers might possess anaerobic metabolic capabilities. Nitrite disproportionation (or dismutation), in which nitrite is simultaneously oxidized to nitrate and reduced to N2, is a thermodynamically favorable reaction, which would link the two processes in one organism – but it has never been observed in nature. The research proposed here will address two big questions about nitrite in the ocean: 1) How does anaerobic nitrite oxidation work? 2) What determines the fate of nitrite? The experimental approach will investigate three possible explanations for anaerobic nitrite oxidation: 1) Nitrite is oxidized to nitrate by different clades of NOB, which exhibit different tolerances/requirements for oxygen; 2) Nitrite dismutation, also performed by NOB, partially explains the cooccurrence of oxidation and reduction of nitrite; 3) Apparently anaerobic nitrite oxidation is indeed biologically mediated but does not always represent net production of nitrate from nitrite; rather it results from isotopic equilibration during enzyme-catalyzed interconversion of nitrite and nitrate. These questions will be addressed by performing a suite of 15N-tracer incubations at stations located within and outside of one of the major OMZs in the ocean, the Eastern Tropical South Pacific. The dependence of the rate processes on oxygen concentrations will be determined, and the composition of the microbial assemblages will be assessed in order to determine whether different microbial components are involved under different environmental conditions. The expression of genes involved in oxidation/reduction/ respiratory metabolisms at low oxygen concentrations will be measured across oxygen gradients and in oxygen manipulations to identify their potential role in supporting “anaerobic” nitrite oxidation. The possibility that the apparently anaerobic nitrite oxidation is due to an enzyme level interconversion between nitrite and nitrate, which does not lead to net nitrate production and is not linked to growth of nitrite oxidizing bacteria, will also be investigated.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.