URANIUM IMMOBILIZATION BY THE ACTIVITIES OF MICROBIAL PHOSPHATASES

The U.S. Department of Energy is tasked with the remediation of over 120 sites with subsurface metal and radionuclide contaminates resulting from nuclear weapons research activities. We previously demonstrated phosphatase activities of subsurface microbes, isolated from contaminated Oak Ridge, TN FRC (ORFRC) subsurface soils, released phosphate extracellularly during growth in the pH range 5-7. Phosphate liberated from glycerol-3-phosphate, provided as the sole carbon and phosphorus (P) source, was sufficient to precipitate >95% of uranium [U(VI)] as low solubility uranium-phosphate minerals. Presently, the substrate and pH range of phosphatase enzymes harbored by subsurface microbial communities exposed to long-term metal and radionuclide contamination is unknown, and their potential role in immobilizing metals and radionuclides is poorly characterized. This project continues pure culture studies, using ORFRC Rahnella sp. Y9602, to investigate the use of phytic acid as a potential substrate for subsurface remediation. Preliminary results obtained after 25-day incubations indicate the Rahnella sp. is capable of liberating 1.8 mM and 21 mM P when grown at pH 7 and pH 5.5, respectively. We have also begun mixed microbial community analyses of ORFRC soil slurries supplemented with glycerol-2-phosphate (G2P) to determine if microbial phosphatase activity present in subsurface communities can liberate sufficient phosphate to promote the precipitation of U(VI) under both oxic and anoxic growth conditions at pH 5.5 or pH 6.8. Oxic slurry incubations demonstrate that organophosphate hydrolysis rates are greater at low pH. Anoxic soil slurry incubations initiated with 10 mM G2P and 15 mM nitrate amendments under acidic anoxic conditions reveal denitrifying and organophosphate hydrolyzing activities. Total DNA extractions from soil slurry incubations are being analyzed via high-density oligonucleotide microarray. Preliminary data suggests that under oxic conditions, the microbial community structure is enriched in proteobacterial taxa at low pH when compared to the diversity of unamended soils. By using culture-dependent and -independent approaches, we aim to identify molecular mechanisms and microbial community members optimized for metal and uranium remediation of subsurface environments.