Abstract: Denitrification is the main process by which fixed nitrogen is lost from ecosystems and the regulation of this process may directly affect primary production and carbon cycling over short and long time scales. Previous investigations of the role of bioactive metals in regulating denitrification in bacteria from permanently ice-covered Lake Bonney in the Taylor Valley of East Antarctica indicated that denitrifying bacteria can be negatively affected by metals such as copper, iron, cadmium, lead, chromium, nickel, silver and zinc; and that there is a distinct difference in denitrifying activity between the east and west lobes of the lake. Low iron concentrations were found to exacerbate the potential toxicity of the other metals, while silver has the potential to specifically inhibit denitrification because of its ability to interfere with copper binding in redox proteins, such as nitrite reductase and nitrous oxide reductase. High silver concentrations might prevent the functioning of nitrous oxide reductase in the same way that simple copper limitation does, thereby causing the buildup of nitrous oxide and resulting in a nonfunctional nitrogen cycle. Other factors, such as oxygen concentration, are likely also to affect bacterial activity in Lake Bonney. This project will investigate silver toxicity, general metal toxicity and oxygen concentration to determine their effect on denitrification in the lake by using a suite of 'sentinel' strains of denitrifying bacteria (isolated from the lake) incubated in Lake Bonney water and subjected to various treatments. The physiological responses of these strains to changes in metal and oxygen concentration will be quantified by flow cytometric detection of single cell molecular probes whose sensitivity and interpretation have been optimized for the sentinel strains. Understanding the relationships between metals and denitrification is expected to enhance our understanding of not only Lake Bonney's unusual nitrogen cycle, but more generally, of the potential role of metals in the regulation of microbial nitrogen transformations. The broader impacts of this work include not only a better understanding of regional biogeochemistry and global perspectives on these processes; but also the training of graduate students and a substantial outreach effort for school children.