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个投入和损失可能不平衡，因此低估N个投入可能解释这种不平衡。气态氮气转化为生物有效氮(氮气固定)是海洋中新氮的最大来源。有可能通过鉴定新的氮固定来源来解释“缺失的”N。与其他海洋微生物不同，氮气的固定依赖于一组微生物，称为重氮菌，它们利用氮气进行生长。重氮菌分为两类，一类是能够通过光合作用获取能量的蓝藻重氮细菌，另一类是非蓝藻重氮细菌(NCDs)，它们需要非光能源。对非传染性疾病的生态学和生物学几乎一无所知，除了它们普遍存在于海洋中并含有固氮基因，但没有直接测量它们的氮气固定活性。在单细胞水平上研究生物体的最新分子进展使测量NCDs固定氮气成为可能。这项研究的重点是确定海洋非传染性疾病是否确实在环境中固定氮气，并了解它们的氮气固定是如何调节的。确定非传染性疾病活动是否是全球海洋中一个重要的缺失氮源，有可能填补我们对海洋氮循环的一个关键空白。该项目通过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，包括加州大学洛杉矶分校的加州少数民族参与联盟(CAMP)。氮素固定，即将氮气转化为生物可用氨的微生物过程，是海洋中氮的重要来源。历史上，研究一直集中在最明显的重氮菌，如毛霉菌，但在公开海洋中发现的单细胞蓝藻和非蓝藻重氮菌(NCDs)揭示了比之前认为的更广泛的多样性。关于非传染性疾病的大部分知识仅限于基因调查中的存在、丰度估计和转录活动。非传染性疾病分布在全球沿海和贫营养环境中，然而，目前尚不清楚非传染性疾病是否提供N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正在积极修复氮气。这项研究的重点是测量北太平洋和北冰洋光照充足、富氧的沿海和低营养表层水域中各种类群的单细胞NCD-N_2固定率。研究人员正在使用一种名为GeneFISH的独立于培养的技术来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仪测量单个细胞中15N2的掺入情况。除了现场测量NCD氮气固定率外，还在进行实验，以确定单细胞NCD氮气固定的环境控制因素(光、温度、溶解有机物、溶解无机N和铁)。从一系列分类群、不同实验条件下以及沿海和低营养环境中获得单细胞NCD氮固定率将提供信息，将它们的存在与氮固定活动联系起来，确定海洋环境中NCDs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NSF的法定使命，并通过使用基金会的智力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.