Nitrite Oxidation in Oxygen Minimum Zones

这项研究是基于氮在限制海洋生产中的基本作用。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，从而作为一种有效的营养物质保留下来，或者它可以被还原成二氮气体，从而从生物有效氮库中消失。亚硝酸盐氧化菌（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都需要氧气来维持生命。还原途径也由微生物进行，在这种情况下，细菌只能在缺氧的情况下茁壮成长。然而，在先前的实验中，在没有氧气的海水中，在相同的样品中检测到亚硝酸盐的氧化和还原。我们将在秘鲁海岸低氧水域进行研究巡航，研究在缺氧情况下亚硝酸盐明显氧化的三种解释：1)不需要氧气的未知NOB的存在；2)一种被称为突变的新反应，这是可能的，但在自然界中从未被发现；3) NOB中与氧应激相关的伪影。这项研究可能会导致发现新的机制和/或新的生物，这些机制和/或新的生物决定了亚硝酸盐的命运和长期支持初级生产的氮的可用性。该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参与和实践实验室和海上研究经验的机会。本科生和研究生都将通过实习和实地体验参与研究。我们还将根据航行期间收集的数据，通过视频和作业，将我们在海上的工作整合到课堂教学中。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盐氧化细菌（NOB）进行的，所有已知的物种都是专性需氧菌。亚硝酸盐还原成N2发生在多种微生物途径中，通常在缺氧条件下。尽管它们在氧气方面明显不相容，但在氧气最低带（OMZs）的低氧或缺氧水中都可以检测到这两个过程。因此，亚硝酸根的命运对全球固定氮收支具有影响。在OMZ区域缺氧深度层段的零氧水中，亚硝酸盐氧化率很高，这表明一些亚硝酸盐氧化剂可能具有厌氧代谢能力。亚硝酸盐歧化（或歧化），即亚硝酸盐同时被氧化为硝酸盐和还原为N2，是一个热力学上有利的反应，它将两个过程联系在一起，但在自然界中从未观察到过。这里提出的研究将解决海洋中亚硝酸盐的两个大问题：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解释：1)亚硝酸盐被不同分支的NOB氧化为硝酸盐，这些分支对氧的耐受性/要求不同；2) NOB也进行了亚硝酸盐的失配，部分解释了亚硝酸盐氧化还原的共同发生；3)显然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的净产量；相反，它是由酶催化亚硝酸盐和硝酸盐相互转化过程中的同位素平衡引起的。这些问题将通过在位于海洋中一个主要omz（东热带南太平洋）内外的站点进行一套15n示踪剂孵育来解决。将确定速率过程对氧浓度的依赖性，并评估微生物组合的组成，以确定在不同的环境条件下是否涉及不同的微生物成分。在低氧浓度下，参与氧化/还原/呼吸代谢的基因的表达将在氧气梯度和氧气操作中进行测量，以确定它们在支持“厌氧”亚硝酸盐氧化中的潜在作用。亚硝酸盐的厌氧氧化可能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的产生，也与亚硝酸盐氧化细菌的生长无关，也将进行研究。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.