Field Deployment for In-situ Metal and Radionuclide Stabilization by Microbial Metabolites

A novel biotechnology is reported here that was demonstrated at SRS that facilitates metal and actinide immobilization by incorporating the physiology and ecology of indigenous bacteria. This technology is based on our previous work with pyomelanin-producing bacteria isolated from SRS soils. Through tyrosine supplementation, overproduction of pyomelanin was achieved, which lead ultimately to metal and actinide immobilization, both in-vitro and in-situ. Pyomelanin is a recalcitrant microbial pigment and a humic type compound in the class of melanin pigments. Pyomelanin has electron shuttling and metal chelation capabilities and thus accelerates the bacterial reduction and/or immobilization of metals. Pyomelanin is produced outside the cell and either diffuses away or attaches to the cell surface. In either case, the reduced pyomelanin is capable of transferring electrons to metals as well as chelating metals. Because of its recalcitrance and redox cycling properties, pyomelanin molecules can be used over and over again for metal transformation. When produced in excess, pyomelanin produced by one bacterial species can be used by other species for metal reduction, thereby extending the utility of pyomelanin and further accelerating metal immobilization rates. Soils contaminated with Ni and U were the focus of this study in order to develop in-situ, metal bioimmobilization technologies. We have demonstrated pyomelanin production in soil from the Tims Branch area of SRS as a result of tyrosine amendments. These results were documented in laboratory soil column studies and field deployment studies. The amended soils demonstrated increased redox behavior and sequestration capacity of U and transition metals following pyomelanin production. Treatments incorporating tyrosine and lactate demonstrated the highest levels of pyomelanin production. In order to determine the potential use of this technology at other areas of SRS, pyomelanin producing bacteria were also quantified from metal contaminated soils at TNX and D areas of SRS. A bacterial culture collection from subsurface studies near P Area of SRS were also evaluated for pyomelanin production. Bacterial densities of pyomelanin producers were determined to be >10{sup 6} cells/g soil at TNX and D areas. In addition, approximately 25% of isolates from P area demonstrated pyomelanin production in the presence of tyrosine. Biogeochemical activity is an ongoing and dynamic process due, in part, to bacterial activity in the subsurface. Bacteria contribute significantly to biotransformation of metals and radionuclides. An understanding and application of the mechanisms of metal and radionuclide reduction offers tremendous potential for development into bioremedial processes and technologies. This report demonstrates the application of recent advances in bacterial physiology and soil ecology for future bioremediation activities involving metal and actinide immobilization

Evaluation of Natural and in-Situ Remediation Technologies for a Coal-Related Metals Plume

Metals contamination exceeding drinking water standards (MCLs) is associated with acidic leachate generated from a coal pile runoff basin at the Savannah River Site (SRS) in Aiken, South Carolina. The metals plume extends over 100 acres with its' distal boundary about onehalf mile from the Savannah River. Based on the large plume extent and high dissolved iron and aluminum concentrations, conventional treatment technologies are likely to be ineffective and cost prohibitive. In-situ bioremediation using existing groundwater microbes is being evaluated as a promising alternative technology for effective treatment, along with consideration of natural attenuation of the lower concentration portions of the plume to meet remedial goals. Treatment of the high concentration portion of the groundwater plume by sulfate-reducing bacteria (SRB) is being evaluated through laboratory microcosm testing and a field-scale demonstration. Organic substrates are added to promote SRB growth. These bacteria use dissolved sulfate as an electron acceptor and ultimately precipitate dissolved metals as metal sulfides. Laboratory microcosm testing indicate SRB are present in groundwater despite low pH conditions, and that their growth can be stimulated by soybean oil and sodium lactate. The field demonstration consists of substrate injection into a 30-foot deep by 240-foot long permeable trench. Microbial activity is demonstrated by an increase in pH from 3 to 6 within the trench. Downgradient monitoring will be used to evaluate the effectiveness of SRB in reducing metal concentrations. Natural attenuation (NA) is being evaluated for the low concentration portion of the plume. A decrease in metal mobility can occur through a variety of abiotically and/or biotically mediated mechanisms. Quantification of these mechanisms is necessary to more accurately predict contaminant attenuation using groundwater transport models that have historically relied on simplified conservative assumptions. Result s from matched soil/porewater samples indicate higher soil/water partition coefficients (Kds) with increasing distance from the source. In addition, site-specific metals availability is being assessed using sequential extraction techniques, which more accurately represent environmental conditions as compared to default EPA extraction methods. Due to elevated sulfate levels in the plume, SRB are most likely to be the dominant biotic contributor to NA processes

Chromate Reduction by a Pseudomonad Isolated from a Site Contaminated with Chromated Copper Arsenate

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter(−1) and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K(m) of 23 mg liter(−1) (437 μM) and a V(max) of 0.98 mg of Cr h(−1) mg of protein(−1) (317 nmol min(−1) mg of protein(−1)). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments