Collaborative Research: Quantifying N2 fixation rates of noncyanobacterial diazotrophs and environmental controls on their activity

氮（N）是海洋中的一种重要元素，它限制了微型海洋植物浮游植物的生长。估计表明，在现代海洋中，N输入和损失可能不平衡，因此N输入的低估可能解释这种不平衡。气态N2转化为生物可利用氮（N2固定）是海洋新氮的最大来源。这是可能的，“失踪”的N可以通过确定新的N2固定源来解释。N2固定依赖于一组称为“固氮生物”的微生物，它们利用N2生长，不像其他海洋微生物。固氮生物分为两类，蓝藻固氮生物，能够通过光合作用获得能量，和非蓝藻固氮生物（NCD），需要非光基能源。对非传染性疾病的生态学和生物学几乎一无所知，除了它们在海洋中无处不在并含有固氮基因，但没有直接测量其固氮活性。最近在单细胞水平上研究生物体的分子进展现在使得测量NCD的N2固定成为可能。这项研究的重点是确定海洋非传染性疾病是否真的在环境中固定N2，并了解它们的N2固定是如何调节的。确定NCD活动是否是全球海洋中重要的缺失氮源，有可能填补我们对海洋氮循环理解的关键空白。该项目支持早期职业STEM研究人员，包括一名研究生和一名博士后科学家，以及通过包括UCSC的加州少数民族参与联盟（CAMP）在内的多个项目支持本科生。固氮是将N2转化为生物有效氨的微生物过程，是海洋中氮的重要来源。从历史上看，研究一直集中在最明显的固氮生物，如束毛藻，但在开放的海洋中发现的单细胞蓝藻和非蓝藻固氮生物（NCD）揭示了比以前认为的更广泛的多样性。关于非传染性疾病的大部分了解仅限于存在情况、丰度估计和基因调查的转录活性。非传染性疾病在全球范围内分布于沿海和贫营养环境中，但目前尚不清楚非传染性疾病是否提供氮来支持初级生产力。需要对海洋非传染性疾病进行测量，以确定非传染性疾病是否积极固定N2。本研究的重点是测量单细胞NCD N2固定率从各种类群生活在光照充足，富氧的沿海和贫营养表面沃茨在北太平洋和北冰洋。研究人员正在使用一种名为geneFISH的独立于培养的技术，在显微镜下观察和定位NCD，并使用纳米级二次离子质谱法测量15N2掺入单细胞的情况。除了测量原位NCD N2固定率之外，正在进行实验以确定对单细胞NCD N2固定的环境控制（光、温度、溶解的有机物、溶解的无机N和铁）。在不同的实验条件下以及在沿海和贫营养环境中，从一系列分类群中获得单细胞NCD N2固定率将提供信息，将其存在与N2固定活性联系起来，确定NCD在海洋环境中的定量意义，该奖项反映了NSF的法定使命，并被认为是值得支持的，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。

Nitrogen (N) is an important element in the ocean that limits the growth of the microscopic marine plants, phytoplankton. Estimates suggest N inputs and losses may not be balanced in the modern ocean, and thus an underestimation of N inputs may explain this imbalance. The conversion of gaseous N2 to biologically available N (N2 fixation) is the largest source of new N to the ocean. It is possible that the “missing” N can be explained by identifying new sources of N2 fixation. N2 fixation relies on a group of microorganisms, termed “diazotrophs,” that utilize N2 for growth, unlike other marine microorganisms. Diazotrophs fall into two groups, cyanobacterial diazotrophs, which are able to derive energy through photosynthesis, and non-cyanobacterial diazotrophs (NCDs), which require a non-light-based energy source. Next to nothing is known about the ecology and biology of NCDs, except that they are ubiquitous in the ocean and contain the nitrogen fixing gene, but no direct measurements of their N2 fixation activity exist. Recent molecular advances for studying organisms at the single cell level now makes the measurement of N2 fixation by NCDs possible. This study is focused on determining whether marine NCDs are actually fixing N2 in the environment and understanding how their N2 fixation is modulated. Determining if NCD activity is an important missing N source in the global oceans has the potential to fill a critical gap in our understanding of the marine N cycle. This project supports early career STEM researchers including a graduate student and a postdoctoral scientist, as well as undergraduate students through several programs including UCSC’s California Alliance for Minority Participation (CAMP).Nitrogen fixation, the microbial process of converting N2 into biologically available ammonia, is an important source of N in the oceans. Historically, research has focused on the most conspicuous diazotrophs, such as Trichodesmium, but the discovery of unicellular cyanobacterial and non-cyanobacterial diazotrophs (NCDs) in the open ocean revealed a broader diversity than previously thought. Much of what is known about NCDs is restricted to presence, abundance estimates and transcriptional activity from gene surveys. NCDs are globally distributed throughout coastal and oligotrophic environments, however, it is not known whether NCDs supply N to support primary productivity. Measurements of marine NCDs are needed to determine if NCDs are actively fixing N2. This study is focused on measuring single cell NCD N2 fixation rates from a variety of taxa living in well-lit, oxygen-rich coastal and oligotrophic surface waters in the North Pacific and Arctic Oceans. The investigators are using a cultivation-independent technique called geneFISH to microscopically visualize and localize NCDs and measuring the incorporation of 15N2 into single cells using nanoscale secondary ion mass spectrometry. Beyond measuring in situ NCD N2 fixation rates, experiments are being conducted to determine environmental controls on single cell NCD N2 fixation (light, temperature, dissolved organic matter, dissolved inorganic N, and iron). Obtaining single cell NCD N2 fixation rates from a range of taxa, under different experimental conditions and in coastal and oligotrophic environments will provide information to link their presence to N2 fixation activity, determine the quantitative significance of NCDs in the marine environment, and set the stage for their inclusion in biogeochemical models.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.