S10: Denitrifying strains isolated from contaminated groundwater at Oakridge FRC

At the U.S. Department of Energy’s Field Research Center in Oak Ridge the groundwater contains high plumes of nitrate up to 10000 mg/L, along with uranium, technetium, volatile organic compounds and a pH gradient from 3-10. Nitrate levels above 10mg/L is considered toxic, as it impairs the oxygen-carrying capacity of red blood cells. We investigated the diversity of anaerobic microbes present across these geochemical gradients capable of reducing nitrate to non-toxic end products. Groundwater samples collected from several wells across a nitrate gradient were incubated under anaerobic conditions with several compounds including lactate, acetate, glycerol, simple sugars or simple fatty acids as carbon and electron donors. Close to two hundred isolates were obtained and identified by 16S-rDNA sequencing. These isolates mostly belonged to Pseudomonadales, Caulobacterales, Rhodocyclales, Burkholderiales, Actinomycetales, Clostridiales, Bacillales, Neisseriales. Phenotypic studies with these isolates show a differential response in their growth rates and in their ability to reduce nitrate. For example, Intrasporangium calvum strain GW247B1 grew robustly at nitrate concentration of upto 300mM with lactate as sole carbon and electron source, while Chromobacterium strain FW507F1did not grow at nitrate concentrations beyond 180mM. Some denitrifiers preferentially utilized nitrite as the electron acceptor. Phenotypic and Genotypic characterization of these denitrifying isolates and their involvement in the cycling of metal and humic substances will be discussed.