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

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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.