Collaborative Research: Determining rates of group-specific phytoplankton and bacterial uptake of inorganic and organic nitrogen by means of stable isotope techniques

智力优势：海洋氮循环涉及不同无机和有机氮库之间复杂的生物转化网络。近年来，在确定海洋环境中的N循环过程方面取得了相当大的进展，但由于方法的局限性，一些重要问题仍未得到解决。例如，研究氮循环的传统工具无法准确评估海洋生态系统中浮游植物或细菌特定的氮使用。因此，需要开发新的技术和方法。本项目的PI最近在这方面取得了两个重要进展：（1）应用流式细胞术（FCM）分离浮游植物和细菌，分别测量这两个群体的氮吸收。先前的方法依赖于不同尺寸部分的测量，这些尺寸部分总是包含一定程度的浮游植物和细菌吸收。FCM允许细菌与浮游植物N掺入的明显分离。(2)基于氮的DNA稳定同位素探测（SIP）方法已被改编为特定的浮游植物种群的氮吸收询问。DNA SIP可以为浮游植物或细菌的特定种群吸收氮源提供证据。这种方法与传统的测量方法不同，传统的测量方法不能对单个种群或物种进行推断。该项目旨在应用这两种方法的进步，以获得下一代的氮吸收测量。浮游植物和细菌的具体吸收率将通过FCM技术进行测量，并通过DNA SIP询问浮游植物或细菌的个体群体或物种的氮吸收。这些工具将在一次夏季巡航和一次冬季巡航期间应用于切萨皮克湾发现的特征良好的营养梯度。浮游植物、细菌和古菌种群将通过多重焦磷酸测序技术沿取样断面沿着进行表征。将测量无机（NH 4+、NO3-和NO2-）和有机氮源（15 N和14 C尿素双标记和氨基酸）作为底物的氮吸收。研究人员假设，浮游植物将获得更大比例的氮营养从有机形式沿着样带（即北到南），作为竞争与细菌的铵增加。DNA SIP将应用于特定的优势浮游植物和细菌种群，以调查其N营养。通过应用这种独特的组合方法，该项目将提供前所未有的社区，群体和物种水平的分辨率N吸收在切萨皮克湾，并将为我们提供一个更好的了解N循环在海湾和海洋系统作为一个整体。更广泛的影响：该项目将被纳入高中，本科和研究生水平的学生的教育。几个研究生将直接参与进行拟议的研究，和PI将提供本科生指导的研究机会，为有才华和有动力的学生寻求研究经验。学生将在几个研究领域接受培训，包括：分子生物学，微生物生态学，生态系统生物学以及环境和分析化学。此外，PI将通过让六名俄克拉荷马州高中教师参与夏季研究项目，将K-12教育推广到社区，然后通过移动的平台进行互动视频会议，为俄克拉荷马州学校的K-12教师提供虚拟互动实地考察。这将通过与俄克拉荷马州大学的K-20教育和社区更新中心合作来实现。K-20是一个跨学科的全州中心，通过学校、大学、工业、社区和政府机构之间的互动、面向行动的伙伴关系，专注于俄克拉荷马州的教育和社区重建。该中心拥有遍布全州的500多所学校和行业合作伙伴的广泛网络。

Intellectual Merit: The marine nitrogen (N) cycle involves a complex network of biological transformations among different inorganic and organic N reservoirs. Considerable progress has been made in defining N cycling processes in marine environments in recent years, but significant questions remain unanswered in part due to methodological limitations. Traditional tools for studying N cycling, for example, cannot accurately assess phytoplankton or bacteria specific N use in marine ecosystems. Therefore there is a need to develop new techniques and methodologies. The PIs of this project have recently made two important advances in this context: (1) a flowcytometric methodology (FCM) to separate phytoplankton from bacteria was applied to separately measure N uptake by these two groups. Prior methodologies relied on measurements of different size fractions, which always contain some degree of both phytoplankton and bacterial uptake. FCM allows for the distinct separation of bacterial versus phytoplankton N incorporation. (2) N-based DNA stable isotope probing (SIP) methodology has been adapted to interrogate N uptake in specific phytoplankton populations. DNA SIP can provide evidence for the uptake of an N source into a specific population of phytoplankton or bacteria. This methodology is in contrast to traditional measurements, which cannot make inferences about individual populations or species. This project aims to apply these two methodological advances in order to obtain the next generation of N uptake measurements. Phytoplankton and bacteria specific uptake rates will be measured via the FCM technique, and the individual groups or species of phytoplankton or bacteria will be interrogated for N uptake via DNA SIP. These tools will be applied across the well-characterized nutrient gradient found in Chesapeake Bay during one summer cruise and one winter cruise. Phytoplankton, bacterial, and archaeal populations will be characterized along the sampling transect via multiplexed pyrosequencing technology. N uptake will be measured for inorganic (NH4+, NO3-, and NO2-) and organic N sources (15N and 14C urea dual-labeled and amino acids) as substrates. The investigators hypothesize that phytoplankton will derive a larger percentage of their N nutrition from organic forms along the transect (i.e. North to South), as competition with bacteria for ammonium increases. DNA SIP will be applied to specific dominant phytoplankton and bacterial populations in order to investigate their N nutrition. By applying this unique combination of methodologies, the project will provide unprecedented community, group and species level resolution of N uptake in Chesapeake Bay and will furnish us with an improved understanding of N cycling in the Bay and marine systems as a whole.Broader Impacts: The project will be integrated into the education of students at the high-school, undergraduate, and graduate levels. Several graduate students will be directly involved in conducting the proposed research, and the PIs will provide undergraduate directed research opportunities for talented and motivated students seeking research experience. Students will be trained in several research areas including: molecular biology, microbial ecology, ecosystems biology, as well as environmental and analytical chemistry. Additionally, the PIs will extend K-12 educational outreach to the community through engaging six Oklahoma high school teachers in summer research projects, followed by interactive videoconferencing via a mobile platform to provide virtual interactive field trips to K-12 teachers in Oklahoma schools. This will be achieved through collaboration with the K-20 Center for Education and Community Renewal at the University of Oklahoma. K-20 is an interdisciplinary, statewide center focusing on educational and community renewal in Oklahoma through interactive, action-oriented partnerships among schools, universities, industry, community and governmental agencies. The Center has an extensive network of over 500 schools and industry partners throughout the state.