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

氮(N)是海洋中的重要元素，它限制了微观海洋植物浮游植物的生长。估计表明，在现代海洋中，氮的输入和损失可能不平衡，因此，对氮输入的低估可能解释了这种不平衡。气态氮转化为生物可利用氮（氮固定）是海洋新氮的最大来源。有可能通过确定新的氮固定源来解释“缺失”的氮。固氮依赖于一组被称为“重氮营养体”的微生物，它们利用氮气生长，这与其他海洋微生物不同。重氮营养体分为两类，蓝藻重氮营养体能够通过光合作用获得能量，而非蓝藻重氮营养体（ncd）需要非光能来源。我们对非传染性疾病的生态学和生物学几乎一无所知，只知道它们在海洋中无处不在，并且含有固氮基因，但没有对它们固氮活性的直接测量。在单细胞水平上研究生物体的最新分子进展使得测量非传染性疾病对N2的固定成为可能。本研究的重点是确定海洋非传染性疾病是否实际上在环境中固定N2，并了解它们的N2固定是如何被调节的。确定非传染性疾病活动是否是全球海洋中缺失的一个重要氮源，有可能填补我们对海洋氮循环的理解中的一个关键空白。该项目通过UCSC的加州少数民族参与联盟（CAMP）等几个项目支持早期职业STEM研究人员，包括一名研究生和一名博士后科学家，以及本科生。固氮是将N2转化为生物可利用氨的微生物过程，是海洋中氮的重要来源。从历史上看，研究主要集中在最明显的重氮营养体上，如Trichodesmium，但在开阔海洋中发现的单细胞蓝藻和非蓝藻重氮营养体（ncd）揭示了比以前认为的更广泛的多样性。对非传染性疾病的了解大多局限于基因调查的存在、丰度估计和转录活性。非传染性疾病在全球沿海和少营养环境中分布，然而，非传染性疾病是否提供氮来支持初级生产力尚不清楚。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主动固定N2。本研究的重点是测量生活在北太平洋和北冰洋光照充足、富氧的沿海和低营养地表水中的各种分类群的单细胞NCD N2固定率。研究人员正在使用一种称为geneFISH的非培养技术，在显微镜下观察和定位非传染性疾病，并使用纳米级二次离子质谱法测量15N2进入单个细胞的情况。除了测量原位NCD - N2固定率外，正在进行实验以确定单细胞NCD - N2固定的环境控制（光、温度、溶解的有机物、溶解的无机氮和铁）。在不同的实验条件下，在沿海和少营养环境中，从一系列分类群中获得单细胞NCD的N2固定率，将提供信息，将它们的存在与N2固定活性联系起来，确定海洋环境中NCD的定量意义，并为将其纳入生物地球化学模型奠定基础。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。

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.