Use of carbon stable isotopes to monitor biostimulation and electron donor fate in chromium-contaminated groundwater.

Hexavalent chromium Cr(VI) is a common inorganic contaminant in industrial areas and represents a serious threat to human health due its toxicity. Here we report experimental results from a field-scale investigation of Cr(VI) bio-immobilization at Hanford 100H reservation, a U.S Department of Energy facility (Washington State, USA). Microbial Cr(VI) reduction was stimulated via injection of a13C-labeled sodium lactate solution into the high-permeability aquifer consisting of gravel and coarse sand sediments. Concentrations and carbon isotope ratios of metabolites, including dissolved inorganic carbon and total organic carbon, and compound-specific analysis of acetate and propionate, together with phospholipid fatty acids (biomass) have been analyzed to help provide an understanding of the predominant redox processes accompanying Cr(VI) reduction. Results of our study indicate that the injection of an electron donor caused a sharp decrease of Cr(VI) concentration from ∼32 to ∼10 nM. Cr(VI) reduction was associated with a decrease in the concentration of carboxylic acids, such as lactate (∼6 mM to undetectable), propionate (∼9 mM to undetectable), and acetate (∼6 mM to undetectable), as well as dissolved inorganic carbon (30-10 mM C). Carbon isotope data indicate carbon transfers from the original substrate to organic byproducts and mineralized carbon. Concentrations of metabolites and stable isotope data as well as carbon isotope mass balance calculations were used to monitor biologically mediated reduction of Cr(VI).

Reoxidation of Chromium(III) Products Formed under Different Biogeochemical Regimes.

Hexavalent chromium, Cr(VI), is a widespread and toxic groundwater contaminant. Reductive immobilization to Cr(III) is a treatment option, but its success depends on the long-term potential for reduced chromium precipitates to remain immobilized under oxidizing conditions. In this unique long-term study, aquifer sediments subjected to reductive Cr(VI) immobilization under different biogeochemical regimes were tested for their susceptibility to reoxidation. After reductive treatment for 1 year, sediments were exposed to oxygenated conditions for another 2 years in flow-through, laboratory columns. Under oxidizing conditions, immobilized chromium reduced under predominantly denitrifying conditions was mobilized at low concentrations (≪1 µM Cr(VI); ∼ 3% of Cr(III) deposited) that declined over time. A conceptual model of a limited pool of more soluble Cr(III), and a larger pool of relatively insoluble Cr(III), is proposed. In contrast, almost no chromium was mobilized from columns reduced under predominantly fermentative conditions, and where reducing conditions persisted for several months after introduction of oxidizing conditions, presumably due to the presence of a reservoir of reduced species generated during reductive treatment. The results from this 3-year study demonstrate that biogeochemical conditions present during reductive treatment, and the potential for buildup of reducing species, will impact the long-term sustainability of the remediation effort.

Physiological and transcriptional studies of Cr(VI) reduction under aerobic and denitrifying conditions by an aquifer-derived pseudomonad.

Cr(VI) is a widespread groundwater contaminant that is a potent toxin, mutagen, and carcinogen. In situ reductive immobilization is a favored approach for Cr(VI) bioremediation, and Cr(VI) reduction has been reported in a variety of aerobic, facultative, and anaerobic bacteria, including a number of pseudomonads. However, studies comparing Cr(VI) reduction under aerobic and denitrifying conditions in the same organism are not available. We have conducted studies with strain RCH2, a bacterium similar to Pseudomonas stutzeri that we isolated from a Cr-contaminated aquifer. Cell suspension studies with lactate demonstrated that Cr(VI) reduction could occur under either denitrifying or aerobic conditions (at comparable specific rates) and that reduction was at least 20-fold more rapid when the terminal electron acceptor (i.e., nitrate or O(2)) was present. Our results suggest that Cr(VI) reduction by strain RCH2 under either aerobic or denitrifying conditions is primarily cometabolic in the sense that the physiological electron acceptor (oxygen or nitrate) appears to be required. Under both aerobic and denitrifying conditions, the gene(s) associated with chromate reduction are not inducible by Cr. Continuous culture (chemostat) studies showed strong correlations (r(2) values >0.93) between nitrate reduction rate and the transcript copy number of either nirS (cytochrome cd(1) nitrite reductase) or narG (nitrate reductase α subunit). As our studies indicate that anaerobic Cr(VI) reduction by this pseudomonad requires active denitrification and that denitrification and chromate reduction rates are highly correlated (r(2) > 0.99), monitoring expression of such denitrification genes in biostimulated aquifers could provide valuable proxy information for in situ chromate reduction by similar bacteria even if the specific genes involved in chromate reduction have not been identified. We also report incomplete removal of reduced Cr from solution and on artifacts in the widely used diphenylcarbazide assay for Cr(VI), most notably, its complete inactivation in the presence of millimolar nitrite.

Molecular analysis of phosphate limitation in Geobacteraceae during the bioremediation of a uranium-contaminated aquifer.

Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified by microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high-affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU upregulated the most. Quantitative PCR analysis of pstB and phoU transcript levels in G. sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium-bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve because of the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.

Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation.

Reduction of soluble uranium U(VI) to less-soluble uranium U(IV) is a promising approach to minimize migration from contaminated aquifers. It is generally assumed that, under constant reducing conditions, U(IV) is stable and immobile; however, in a previous study, we documented reoxidation of U(IV) under continuous reducing conditions (Wan et al., Environ. Sci. Technol. 2005, 39:6162-6169). To determine if changes in microbial community composition were a factor in U(IV) reoxidation, we employed a high-density phylogenetic DNA microarray (16S microarray) containing 500,000 probes to monitor changes in bacterial populations during this remediation process. Comparison of the 16S microarray with clone libraries demonstrated successful detection and classification of most clone groups. Analysis of the most dynamic groups of 16S rRNA gene amplicons detected by the 16S microarray identified five clusters of bacterial subfamilies responding in a similar manner. This approach demonstrated that amplicons of known metal-reducing bacteria such as Geothrix fermentans (confirmed by quantitative PCR) and those within the Geobacteraceae were abundant during U(VI) reduction and did not decline during the U(IV) reoxidation phase. Significantly, it appears that the observed reoxidation of uranium under reducing conditions occurred despite elevated microbial activity and the consistent presence of metal-reducing bacteria. High-density phylogenetic microarrays constitute a powerful tool, enabling the detection and monitoring of a substantial portion of the microbial population in a routine, accurate, and reproducible manner.

Colloid formation at waste plume fronts.

Highly saline and caustic tank waste solutions containing radionuclides and toxic metals have leaked into sediments at U.S. Department of Energy (DOE) facilities such as the Hanford Site (Washington state). Colloid transport is frequently invoked to explain migration of radionuclides and metals in the subsurface. To understand colloid formation during interactions between highly reactive fluids and sediments and its impact on contaminant transport, we simulated tank waste solution (TWS) leakage processes in laboratory columns at ambient and elevated (70 degrees C) temperatures. We found that maximum formation of mobile colloids occurred at the plume fronts (hundreds to thousands times higher than within the plume bodies or during later leaching). Concentrations of suspended solids were as high as 3 mass %, and their particle sizes ranged from tens of nanometers to a few micrometers. Calcium carbonate is always one of the dominant phases of the plume front colloids, while the other phases varied with solution pH and temperature. During infiltration of the leaked high-Na+ waste solution, rapid and completed Na+ replacement of exchangeable Ca2+ and Mg2+ from the sediment caused accumulation of these divalent cations at the moving plume front. Precipitation of supersaturated Ca2+/Mg2+-bearing minerals caused dramatic pH reduction atthe plume front. In turn, the reduced pH caused precipitation of other minerals. This understanding can help predict the behavior of contaminant trace elements carried by the tank waste solutions and could not have been obtained through conventional batch studies.

pH neutralization and zonation in alkaline-saline tank waste plumes.

At the Hanford Site in Washington State, the pH values of contaminant plumes resulting from leaking of initially highly alkaline-saline radioactive waste solutions into the subsurface are now found to be substantially neutralized. However, the nature of plume pH neutralization has not previously been understood. As a master geochemical variable, pH needs to be understood in order to predict the fate and transport of contaminants carried by the waste plumes. Through this laboratory study, we found that the plume pH values spanned a broad range from 14 (within the near-source region) down to the value of 7 (lower than the pH value of the initial soil solution) while the plume was still connected to an actively leaking source. We defined two zones within a plume: the silicate dissolution zone (SDZ, pH 14-10) and the neutralized zone (NZ, pH 10-7). Quartz dissolution at elevated temperature and precipitation of secondary silicates (including sodium metasilicate, cancrinite, and zeolites) are the key reactions responsible for the pH neutralization within the SDZ. The rapid and thorough cation exchange of Na+ replacing Ca2+/Mg2+, combined with transport, resulted in a dynamic Ca2+/Mg2+-enriched plume front. Subsequent precipitation of calcite, sodium silicate, and possibly talc led to dramatically reduced pH within the plume front and the neutralized zone. During aging (after the plume source became inactive), continued quartz dissolution and the secondary silicate precipitation drove the pH value lower, toward pH 11 at equilibrium within the SDZ, whereas the pH values in the NZ remained relatively unchanged with time. A pH profile of 11 from the plume source to pH 7 at the plume front is expected for a historical plume. This laboratory-based study provided realistic plume pH profiles (consistent with that measured from borehole samples) and identified underlying mechanisms responsible for pH evolution.