Diatom Photorespiration and Microbial Carbon and Nitrogen Cycling in the Upper Ocean: An Integrated Molecular/Biochemical Approach.

E. Virginia Armbrust and Richard Keil, University of Washington and

Jennifer Cherrier, Florida A & M University

Marine diatoms are estimated to be responsible for >25% of the 45-50 billion tonnes of organic carbon fixed annually in the sea. Diatoms are predicted to play an even larger role in global carbon cycling than that of all terrestrial rainforests combined. Recent analyses suggest that oscillations in diatom abundance over geological time may have influenced global climate by changing the oceans' capacity to take up atmospheric CO₂. Thus, it is critical to understand how diatom primary productivity in today's oceans influences carbon and nitrogen fluxes out of the upper ocean and into deeper waters, a process that helps mitigate rising atmospheric CO₂ levels. The goal of the research project is to understand how environmental factors such as changes in light conditions and nutrient status influence the fate of diatom-produced organic matter. Organic matter that remains in the surface waters is almost immediately consumed by bacteria and will ultimately be returned to the atmosphere as CO₂. A combination of physiological, molecular biological, and biogeochemical studies are employed to understand carbon and nitrogen cycling in these critical microbial food-webs. Gene expression patterns are monitored in diatoms and bacteria as sensitive measures of the response of these organisms to environmental changes. A suite of organic compounds released by diatoms will be measured, and their use by bacteria will be determined both in laboratory and field settings. Ultimately, these studies will help to better understand how the global carbon cycle is influenced by changing environmental conditions.

Molecular Approaches for In Situ Studies of Nitrate Utilization by Marine Bacteria

Marc E. Frischer, Peter G. Verity, and Stuart Whipple, Skidaway Institute of Oceanography; Matthew Gilligan, Savannah State University; Deborah A. Bronk, Virginia Institute of Marine Sciences; Melissa G. Booth, Roanoke College

Although the importance of inorganic nitrogen (N) for the growth of marine phytoplankton has been well-recognized, inorganic N utilization by bacteria has received less attention. Although it is difficult to differentiate between bacterial and phytoplankton N uptake using biogeochemical techniques, the application of molecular approaches to this problem is yielding important new insights. Understanding the controls and rates of NO₃^- assimilation by ocean bacteria is critical for understanding ocean carbon cycling. Assimilation of N by bacteria may impact mechanisms that increase atmospheric CO₂ levels and enhance global warming.

This project will develop molecular tools for studying the role of bacterial utilization of NO₃^-. Project components include the development of flow cytometer based methodologies to sort individual cells with the identification of their ability to incorporate NO₃^-, molecular methods to study urea uptake, and implementation of an existing Ocean Carbon Model to explore the global significance of NO₃^- uptake by marine bacteria.

In addition, this project will expand the partnership between the Skidaway Institute of Oceanography (SkIO) and a historically African-
American institute, Savannah State University (SSU), and build a new research/education partnership between the SkIO and a second teaching institution, Roanoke College. The project also depends on partnership with the Virginia Institute of Marine Sciences and collaboration with the University of Georgia and the University of Maryland. This project will support the undergraduate and graduate marine science programs at SSU. The project will provide support for up to 10 undergraduate and 4 graduate student research assistantships.

Microbial Ecology of Denitrifying Bacteria in the Coastal Ocean

and the Geochemical RNA Integration Study (GRIST)

Lee Kerkhof, Sybil Seitzinger, and Gary Taghon, Rutgers University; Jorge Corredor, University of Puerto Rico-Mayaguez

The project focuses primarily on using the genes for nitrous oxide reductase (nosZ) to measure activity and abundance of denitrifying bacteria in coastal sediments and on the GRIST experiment which was designed to link molecular measurements of activity with specific biogeochemical processes. Recent research efforts included re-design of nosZ primers and T-RFLP screening protocols to identify and isolate important denitrifying strains from coastal sediments. Additionally, experiments were conducted using bromo-deoxyuridine (BrDU--a thymidine analog) to capture newly synthesized DNA in water column and sediment samples. Furthermore, intact ribosomes have been characterized to determine the fraction of the population actively growing by amplifying both the 16S and 18S rRNA genes. Preliminary results by T-RFLP analysis in the water column during GRIST indicate that BrDU incorporation and ribosome fingerprinting are targeting the same bacterial populations. Active members were spread throughout the Proteobacteria and Cytophaga/Flavobacter groups. Few active 16S rRNA gene clones matched sequences current in Genbank. The 18S rRNA data corroborates studies on RUBISCO mRNA and primary production measurements indicating diatoms and prymnesiophytes are the major contributors to the carbon fixed during the diel studies off the New Jersey Coast. Finally, genetic tools to monitor nitrogenase activity (nifH) and nitrification (amoA) are being applied to a Caribbean Time Series site (CaTS) to better understand the biogeochemistry of nitrogen species in the tropical environment.

Cycling of Dissolved Organic Carbon and Dissolved Organic Nitrogen by Novel Heterotrophic and Photoheterotrophic Bacteria in the Ocean

David Kirchman, University of Delaware and David Royer, Lincoln University

Current models of C and N cycles in the oceans are very simplistic in depicting the interactions between microbes and the largest pools of organic C and N in aquatic ecosystems, dissolved organic carbon (DOC) and dissolved organic nitrogen (DON), which are components of dissolved organic material (DOM).  These models have a single component for heterotrophic bacteria, because little is known about DOM processing by specific bacterial groups.  This project is using molecular approaches for unraveling the roles of specific bacterial groups in DOM uptake in Delaware coastal waters and the Sargasso Sea.  The organic compounds (e.g. free amino acids and protein) used by an abundant bacterial group, SAR11, and by newly-discovered photoheterotrophic bacteria and cyanobacteria (Synechococcus and Prochlorococcus) are being examined.  The hypothesis being tested is that SAR11 is an oligotroph and uses low molecular weight (LMW) compounds, whereas biopolymers and other high molecular weight (HMW) material are used mainly by Cytophaga-like bacteria.  The biogeochemical significance of photoheterotrophic bacteria remains to be established, and although cyanobacteria are well known to be important in primary production, their contribution to DOM uptake is not clear.  The main approach for examining these questions combines identification of bacteria with fluorescence in situ hybridization (FISH) and detection of DOM uptake by microautoradiography (Micro-FISH).  This project will begin to determine the ecological role of specific bacterial groups and to unravel the coupling between C and N cycles as linked by DOM processes.   

Nitrogen-dependent Carbon Fixation by Picoplankton in Culture and in the Mississippi River Plume

John H. Paul and Boris Wawrik, University of South Florida, F. Robert Tabita, Ohio State University, and Thomas E. Smith, Howard University

Understanding the fate of anthropogenic CO₂ in the oceans is important to the U.S. Department of Energy mission in climate change. The Mississippi River Plume may play a vital role in carbon fixation in the Gulf of Mexico and the North American carbon budget. Nitrate and phosphate-rich freshwater (derived from anthropogenic activities) floats on the saline oligotrophic Gulf of Mexico water; this freshwater lens is in intimate contact with atmospheric CO₂. The overall goal of the proposed research is to understand the control of CO₂ fixation by nitrogen in phytoplankton both in culture and in a “natural laboratory” for macronutrient enrichment, the Mississippi River Plume. Although the Mississippi River Plume only accounted for 2.75% of the surface area of the oligotrophic Gulf of Mexico, carbon fixation by this plume was estimated to be 41% of the total surface carbon fixation in the Gulf of Mexico in July, 2001. A bacterial artificial chromosome (BAC) library containing 3200 clones made from DNA obtained from the Prochlorococcus-dominated depth from the Gulf of Mexico in July, 2001 yielded 10 clones hybridizing to the Form IA rbcL. The influence of nutrient stress, namely nitrogen and iron levels, on rbcL expression is being studied in marine picocyanobacteria. This has led to the finding that the transcriptional regulators NtcA and GlnB have varying abilities to influence rbcL transcription, which is dependent on the nitrogen status of the cells. Moreover, research in this project has led to the discovery of a small antisense RNA that may play an important role in the regulation of nitrogen metabolism in marine picoplankton.

Molecular Approaches to Understanding Carbon and Nitrogen Dynamics and Their Role in the Global Carbon Cycle.

James M. Tiedje, Michigan State University, Jizhong Zhou, Oak Ridge National Laboratory, Allan H. Devol, University of Washington, and Arturo A. Massol-Deya, University of Puerto Rico.

The world’s global oceans are a major site of carbon cycling and sequestration, the latter accomplished by the permanent burial in sediments of sinking fixed carbon. By far, the largest site of carbon burial is in continental margin sediments (>90%). This is due to the abundant productivity in waters overlying margin sediments and the short travel distance from the sea surface to the sediment surface, along with relatively high burial efficiencies. An important control on carbon sequestration is the cycling of important nutrients back into elemental cycles during carbon degradation. Nitrogen thought to be limiting in many oceanic ecosystems and continental margins accounts for half of the nitrogen cycling taking place in the oceans. Several questions remain to be answered to understand the microbial and/or chemical regulation of carbon burial efficiency and nitrogen cycling in margin marine sediments. Does oxygen and nitrogen exposure time control the organic carbon:surface area relationship in marine sediments and hence affect carbon sequestration? Do oxygen consumers and denitrifiers coexist and actively grow in the same zone and hence influence the nitrogen economy of the sediments? Are the novel denitrifiers being found in marine sediments functionally important members of the denitrifier community? This project addresses these questions using molecular and cultivation-based approaches along with DNA microarray-based detection methods for analyzing denitrifier, nitrifier, and sulfate reducer communities. The study will allow a link to be established between the microbial catalysts of the key sediment carbon and nitrogen processes, and hence a better understanding of the mechanisms controlling sediment carbon sequestration.

The Coupling between Carbon and Nitrogen Cycles in Coastal Upwelling Ecosystems: Biogeochemical Cycling and its Molecular Basis

Frances Wilkerson and Richard Dugdale, San Francisco State University; Jonathon Zehr , University of California at Santa Cruz; and Bess Ward, Princeton University

This project concerns the connections at the molecular level between carbon fixation and sequestration and nitrogen cycling by ocean margin microbial communities, and their links to biogeochemical and global scale feedbacks. Ocean margins and coastal upwelling regions are ecosystems where nutrient concentrations are high (especially nitrate), the biological pump is active, and the atmospheric to ocean flux of carbon dioxide due to new production by phytoplankton may be significant. The composition of the phytoplankton communities and their physiological states has implications for the quantity of vertical carbon fluxes as well as trophic transfer through the food chain. One of the limitations of traditional biogeochemical approaches for studying phytoplankton is the inability to determine how the different components of the microbial community are responding. Molecular approaches offer ways to evaluate the contribution by individual taxa and groups. This project has characterized eukaryotic and cyanobacterial genes that best describe the coupling/decoupling of carbon and nitrogen cycles (i.e. RuBP carboxylase, nitrogen transporters, nitrate reductase and glutamine synthetase). Assays for these genes will be applied to field samples from a coastal upwelling site in northern California and the LEO-15 site off New Jersey to evaluate the differential gene expression of different microbial components at different stages of productivity. The molecular and biogeochemical data provide a mechanistic view of the assimilatory processes being carried out by different microbial groups, and will aid future researchers in developing molecular approaches to understanding water column biogeochemical transformations.