Elizabeth B. Kujawinski - Woods Hole Oceanographic Institution The carbon cycle in aquifer systems, and particularly the role of prokaryotic and eukaryotic microbes, is poorly understood. A large body of work in marine and freshwater systems has indicated that eukaryotes such as protozoa play fundamental roles in the carbon and nutrient cycles in all aquatic environments - ranging from nutrient-rich coastal systems to nutrient-poor mid-ocean gyres. Nevertheless, the role of protozoa in aquifer systems has been examined by a relatively small number of studies. Given the pivotal role these organisms play in other aquatic environments, it is reasonable to assume that they play fundamental roles in the carbon and nutrient cycles of the subsurface. It is time to investigate which protozoan species are present, which bacteria they graze upon and the impact of protozoan grazing on the carbon and nutrient cycles in groundwater. The goal of this work is to utilize stable isotopes in combination with geochemical and microbial methods to study the effect of protozoa on nutrient regeneration rates and carbon remineralization in a subterranean estuary on Cape Cod MA. This work will use 13C-labeled substrates such as acetate to track the active bacterial and protozoan community. Sediment will be collected from the freshwater and saline end-members of the subterranean estuary and will be used to establish flow-through columns. These columns will be incubated with groundwater pumped from the appropriate site in the estuary to maintain the in situ salinity and ion composition. Groundwater will be amended with 13C-substrates. The movement of the isotopic label from substrate to organic matter will be monitored by isotope-ratio mass spectrometry of inorganic carbon (aqueous CO2 species), bulk organic carbon (TOC / DOC) and small organic molecules (volatile organic acids and amino acids). Incorporation of the label into microbial biomass will be assessed by examination of isotopically-labeled nucleic acids and phospholipid fatty acids (PLFAs). Active bacterial and protozoan species will be identified by their isotopically-labeled rDNA or rRNA sequence. The relative roles of protozoa and bacteria will be assessed by parallel column incubations with 1.0-um filtered and non-filtered groundwater. The proposed work is the first study, to our knowledge, that directly links protozoan community composition to nutrient regeneration and carbon remineralization in fresh and saline aquifers. This group of microbes is vastly under-studied and the results of this study have the potential to elucidate recycling processes outside the current paradigm of microbial activity in aquifer sediments. Furthermore, the methods developed here can be generally applied to ecological and biogeochemical studies in numerous environments, ranging from freshwater systems to marine sediments.