LELAND STANFORD JUNIOR UNIVERSITY, THE

"INTELLECTUAL MERIT Harmful levels of nitrogen (N) in estuaries can be diminished through tightly coupled processes in the microbial nitrogen cycle, including nitrification (chemoautotrophic oxidation of ammonia to nitrite and nitrate) and denitrification (the dissimilatory reduction of nitrate to N2 gas). Despite the biogeochemical importance of both nitrification and denitrification in estuarine systems, surprisingly little is known regarding the underlying microbial communities responsible for these processes, or how they are influenced by key physical/chemical factors. San Francisco Bay --the largest estuary on the west coast of the United States --is an ideal ecosystem in which to utilize molecular, biogeochemical and cultivation approaches to explore how the distribution, diversity, abundance, and activities of key N-cycling communities are influenced by environmental gradients over temporal and spatial scales. Denitrifying communities will be studied using functional genes (nirK and nirS) encoding the key denitrification enzyme nitrite reductase, while genes encoding ammonia monooxygenase subunit A (amoA) will be used to study both ammonia-oxidizing bacteria (AOB) and the recently-discovered ammonia-oxidizing archaea (AOA) -- members of one of the most ubiquitous and abundant prokaryotic groups on the planet, the mesophilic Crenarchaeota. Analyzing sediments from sites spanning a range of physical and chemical conditions in the Bay, seasonally over the course of several years, will represent an unprecedented opportunity to examine spatial, physical/chemical, and temporal effects on both denitrifier and ammonia-oxidizer communities in this large, urban estuary. Concurrently, an intensive cultivation effort will also be undertaken, in order to compile a novel culture collection of estuarine denitrifiers and ammonia-oxidizers, for which virtually nothing is currently known. Taken together, these complimentary approaches will help reveal how complex physical/chemical gradients influence the diversity and functioning of key estuarine N-cycling communities over time and space. BROADER IMPACTS Nitrification and denitrification play pivotal roles in the cycling and removal of nitrogen in San Francisco Bay --a large, urban estuary which encompasses nearly 178,000 km2 and drains approximately 40% of the area of the entire state of California. This project will provide critical information regarding how the underlying N-cycling microbial communities are influenced by complex and fluctuating environmental gradients over time and space in this estuary. The resultant collections of novel nirK, nirS, and amoA gene sequences, cultivated estuarine denitrifiers and AOA and AOB, and biogeochemical data will provide a valuable resource to the scientific community and, ultimately, help reveal insights into the ecology and regulation of these biogeochemically-important processes in all estuarine systems. In addition, the broader impacts of this research will be communicated and investigated through several key educational and outreach activities: (1) a sophomore-level course will be developed and taught focused on San Francisco Bay as a model system for understanding the biogeochemical and societal importance of large estuaries; (2) the target audience of the intensive, 4-week Hopkins Microbiology Summer Course will be expanded to include the participation of Bay Area high school biology teachers, providing a truly unique and valuable professional development experience; (3) I will develop and contribute a `microbial community' module to the REAL (Redwood Environmental Academy of Learning) Program, focused on the hands-on ecological teaching of `continuation' students at nearby Redwood High School --located adjacent to a creek feeding directly into San Francisco Bay; and (4) I will mentor and train graduate, undergraduate, and high school students, who will be directly involved in this research project over the next 5 years."

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