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, such as carbon, and is tightly linked to climate-feedback processes. Key to the behavior of climate and marine ecosystems is the fate of nitrogen (N), which has been greatly altered by human activities over the past century. Nitrogen (N) is an essential element for all life on Earth. Organisms need N to form proteins and DNA and will compete vigorously for N to survive and grow. The pool sizes of different portions of the marine N cycle are often fairly well understood. What is not well known are the identities of microbes that are responsible for the inter-conversion of different forms of N and the rates at which they perform these transformations. A failure to capture the ‘who’ and the ‘how much’ dimensions of the N cycle can have large consequences for our understanding of global geochemical fluxes. Hence, this grant focused on utilizing 15N-DNA stable isotope probing (SIP) techniques in combination with 15N-uptake rate measurements to further our understanding of microbial N transformation in marine systems. The goal was to specifically link N uptake from different sources to individual microbial populations. DNA-SIP has been used as a powerful tool for the investigation of carbon metabolism in microbial communities. Substantially less work has, however, focused using 15N as a tracer, mainly because of the greater technical challenge. We applied 15N-based SIP in marine systems in order to investigate N uptake by phyto- and bacterioplankton populations. Historically, it has been perceived that inorganic forms of N are utilized by phytoplankton, while organic N is primarily a source of nutrients for bacteria. Evidence suggests that this distinction may be too simplistic and that there can be considerable plasticity within the range of a given organism’s use of N substrates. It is also unclear how phytoplankton and heterotrophic 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 and arctic, coastal communities was conducted using a range of 15N-labeled organic and inorganic substrates. In subtropical, coastal environments we observed that multiple organic and inorganic sources of N were fueling cyanobacterial and diatom primary production simultaneously. This is contrary to the traditional new-versus-recycled production paradigm, which is frequently invoked for coastal environments, and our study is 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 heterotrophic 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 with 15N-SIP and RNA-based functional microarray analysis of whole community gene expression. The aim was to gain a more comprehensive perspective of N cycling by considering whole community transcription of N cycling genes, while targeting nitrate utilization more specifically. Most importantly, the study allowed us for the first time to demonstrate nitrate uptake into several specific marine bacterioplankton taxa. We also conducted work in the Artic, which has focused on urea cycling and dark carbon fixation in coastal arctic environments during summer and winter seasons. Urea can be a large component of the dissolved organic N (DON) pool particularly in the Arctic Ocean, and may stimulate bacterial production during the long winter dark period, as urea has been implicated in marine nitrification during the long polar winter by marine archaea. Using SIP, we observed that bacterial and archaeal plankton utilize urea during the winter, but no evidence of uptake was observed for summer. An investigation into dark C fixation in Arctic winters samples provided molecular evidence for the differential utilization of bicarbonate as a C source by bacterial and archaeal taxa. This grant supported outreach to the community by creating high-school science lessons and curriculum as well as web and video content for teaching students about the importance of N in aquatic ecosystems. We hosted high school and undergraduate students from the US, Europe, and China, who actively participated in the work, and engaged in community outreach through public lectures.
