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.
The ocean’s microbial realm is the largest and arguably most complex ecosystem on Earth. It plays a central role in controlling global element cycles and is tightly linked to climate-feedback processes. Nitrogen (N) is an essential element for all life on Earth. Organisms need N to form proteins and deoxyribonucleic acids (DNA) and will compete vigorously for N to survive and grow. The total concentrations of the inorganic and organic N pools in the ocean are fairly well understood. What is not well known are the identities of the microbes that take up the different forms of N and the rates with which they do it. Not capturing the ‘who’ and the ‘how much’ dimensions of the N cycle can have large consequences for our understanding of global geochemical fluxes. This grant used a stable isotope of N (15N) to trace the incorporation of N into microbial (phytoplankton, bacteria, and archaea) DNA and to determine how quickly different N compounds (for example, nitrate or urea) were taken up. Our goal was to specifically link N uptake from different sources to individual microbial populations. Isotopes of N can be distinguished between one another by their molecular weight. For example, 15N is heavier than the more common 14N isotope that accounts for 99.6% of all global N. Since 15N is heavier, we can determine whose DNA has incorporated the 15N from the added substrate and whose DNA still contains the naturally abundant 14N and thus did not use the added N. This work is done through stable isotope probing (SIP) techniques. SIP has been used as a powerful tool for the investigation of carbon metabolism in microbial communities but technical challenges limited its use in N studies. We used 15N-SIP in a study to investigate N uptake by phytoplankton and bacterial populations in the ocean. Historically, it has been perceived that inorganic forms of N (for example, nitrate) are utilized by phytoplankton, while organic N (for example, urea) is primarily a source of N for bacteria. Evidence suggests that this distinction may be too simplistic and that there can be considerable flexibility in the N substrates used by a given organism. It is also unclear how phytoplankton and bacteria interact in the environment when forced to compete for limited N resources, and what the N substrate ranges of bacterial and phytoplankton populations are under natural conditions. A series of experiments on subtropical (Chesapeake Bay) and polar (Arctic) coastal communities was conducted using a range of 15N-labeled organic (urea and amino acids) and inorganic (ammonium, nitrate and nitrite) substrates. In subtropical coastal environments we observed that multiple organic and inorganic sources of N were fuelling cyanobacterial and diatom primary production simultaneously. This result is particularly interesting because it challenges the traditional hypothesis that nitrate utilization is a good proxy for primary production. Our work shows that this may not always be the case in coastal environments. Our study is also among only a few in the literature that directly demonstrate the uptake of a specific form of N into individual phytoplankton species in the environment. In a subsequent related study we focused on bacterial nitrate utilization. Molecular evidence had suggested that nitrate-utilizing bacteria are abundant, wide-spread, and potentially active in marine systems, but direct evidence of incorporation of N from nitrate into individual bacterial species had not been reported. We combined uptake rate measurements and15N-SIP with ribosomal nucleic acid (RNA)-based functional microarray analysis which is a technique that provides information of genes expressed by the whole community. Through this combination of techniques, we were able to gain a more comprehensive perspective of N cycling by considering all of the active N cycling genes of the whole community, while targeting nitrate utilization more specifically. Most importantly, the study allowed us to demonstrate nitrate uptake into several specific marine bacterioplankton taxa for the first time. Application of SIP in the Arctic showed that ammonium but not nitrate was universally taken up by all bacterial and archaeal taxa, which aligns with current paradigms of preferred N sources. Urea, which can be a large component of the dissolved organic N pool in the Arctic Ocean, may stimulate bacterial growth even during the dark winter period. Using SIP, we observed that bacterial and archaeal plankton utilize urea during the winter, but no evidence of uptake was observed for summer, when larger phytoplankton likely outcompete bacteria and archaea. In marine systems, it is assumed that in the absence of light as an energy source, only microorganisms that thrive in low oxygen conditions fix carbon. An investigation into dark carbon fixation in Arctic winter samples provided molecular evidence for utilization of carbon by certain bacterial and archaeal taxa during the long Arctic winter, potentially giving those groups a competitive advantage.
