Direct Identification and Characterization of Marine Heterotrophic Nitrogen Fixers by Stable Isotope Probing

海洋中的氮循环极其复杂，它不断产生惊喜。在这个项目中获得的知识将直接有助于解决一个悬而未决的重要问题，即海洋中氮的来源，因为氮预算的不平衡已经提出了我们对所有氮输入和去除的量化程度的问题。虽然固氮蓝藻一直被认为是海洋固氮的重要贡献者，但异养固氮细菌（HNFB）是海洋固氮群落中一个特性不佳但潜在重要的组成部分。该项目将采用先进的方法，开发对海洋中新颖、活跃的高通量生物的新认识。这些生物的鉴定及其系统发育多样性和生物地球化学意义的评估将为了解它们在海洋氮循环中的作用提供重要的见解。该项目将开发一种新的稳定同位素探测（SIP）方法，结合高通量测序。SIP利用了同位素富集底物进入核酸（例如DNA）的吸收，而高通量测序应该允许一般固氮细菌的鉴定，特别是HNFB，这样首席研究员就可以直接将氮的生物地球化学与这些固氮细菌的系统发育联系起来。该项目的目标是提供概念的证明，并使用这种新方法确定重要的HNFB谱系，并开始对HNFB种群对海洋固氮和氮循环的贡献进行定量估计。这个项目将促进研究生的发展，他将在他的博士学位项目的各个方面工作，工作将在科学会议和公共宣传场所进行展示。本科生也将在研究生和首席研究员的指导下从事该项目。参与者还将参与南加州大学青年研究人员计划（YRP），该计划招募来自不同和贫困的当地学校的高中生进行暑期研究体验。学生们将在这个项目的实验室方面得到指导，并在YRP研讨会上展示他们的工作。首席研究员还将继续参加海洋科学教育卓越中心-西部项目的科学课程开发研讨会。

The nitrogen cycle in the ocean is extremely complex and it continues to yield surprises. Knowledge gained in this project will directly contribute to resolving an unsettled and important question regarding the sources of nitrogen in the oceans because imbalances in nitrogen budgets have raised questions on how well we have quantified all nitrogen inputs and removal. While nitrogen-fixing cyanobacteria have long been recognized as important contributors to nitrogen fixation in the oceans heterotrophic nitrogen-fixing bacteria (HNFB) are a poorly characterized, but potentially important, component of the marine nitrogen-fixing community. Using advanced methodologies, this project will develop the means to obtain a new understanding of novel, active, HNFB in the ocean. The identification of these organisms and the assessment of their phylogenetic diversity and biogeochemical significance will provide important insights about their role in the marine nitrogen cycle. The project will develop a novel stable isotope probing (SIP) methodology coupled with high-throughput sequencing. SIP exploits the uptake of isotopically enriched substrates into nucleic acids (e.g., DNA) while high-throughput sequencing should allow the identification of nitrogen-fixing bacteria generally, and HNFB specifically, so that the principal investigator can directly link the biogeochemistry of nitrogen with the phylogeny of those nitrogen-fixers. The objective of this project is to provide a proof of concept effort and identify important HNFB lineages using this novel approach and begin to develop quantitative estimates for the contribution of HNFB populations to marine nitrogen-fixation and the nitrogen cycle. This project will foster the development of a graduate student who will work on all aspects of the project for his PhD and the work will be presented at science conferences and in public outreach venues. Undergraduates will also work on the project under the guidance of the graduate student and principal investigator. The participants will also engage with the USC Young Researchers Program (YRP), which recruits high school students from diverse and underprivileged local schools for summer research experiences. Students will be guided in the laboratory-based aspects of this project, and present their work at an YRP symposium. The principal investigator will also continue to participate in science curriculum development workshops of the Center for Ocean Sciences Education Excellence-West program.