Acetate Availability and its Influence on Sustainable Bioremediation of Uranium-Contaminated Groundwater

Field biostimulation experiments at the U. S. Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, Colorado, have demonstrated that uranium concentrations in groundwater can be decreased to levels below the U.S. Environmental Protection Agency's (EPA) drinking water standard (0.126 mu M). During successive summer experiments - referred to as "Winchester" (2007) and "Big Rusty" (2008) - acetate was added to the aquifer to stimulate the activity of indigenous dissimilatory metal-reducing bacteria capable of reductively immobilizing uranium. The two experiments differed in the length of injection (31 vs. 110 days), the maximum concentration of acetate (5 vs. 30 mM), and the extent to which iron reduction ("Winchester") or sulfate reduction ("Big Rusty") was the predominant metabolic process. In both cases, rapid removal of U(VI) from groundwater occurred at calcium concentrations (6 mM) and carbonate alkalinities (8 meq/L) where Ca-UO2-CO3 ternary complexes constitute > 90% of uranyl species in groundwater. Complete consumption of acetate and increased alkalinity (> 30 meq/L) accompanying the onset of sulfate reduction corresponded to temporary increases in U(VI); however, by increasing acetate concentrations in excess of available sulfate (10 mM), low U(VI) concentrations (0.1-0.05 mu M) were achieved for extended periods of time (> 140 days). Uniform delivery of acetate during "Big Rusty" was impeded due to decreases in injection well permeability, likely resulting from biomass accumulation and carbonate and sulfide mineral precipitation. Such decreases were not observed during the short-duration "Winchester" experiment. Terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes demonstrated that Geobacter sp. and Geobacter-like strains dominated the groundwater community profile during iron reduction, with C-13 stable isotope probing (SIP) results confirming these strains were actively utilizing acetate to replicate their genome during the period of optimal U(VI) removal. Gene transcript levels during "Big Rusty" were quantified for Geobacter-specific citrate synthase (gltA), with ongoing transcription during sulfate reduction indicating that members of the Geobacteraceae were still active and likely contributing to U(VI) removal. The persistence of reducible Fe(III) in sediments recovered from an area of prolonged (110-day) sulfate reduction is consistent with this conclusion. These results indicate that acetate availability and its ability to sustain the activity of iron-and uranyl-respiring Geobacter strains during sulfate reduction exerts a primary control on optimized U(VI) removal from groundwater at the Rifle IFRC site over extended time scales (>50 days).