The objective of this study is to examine the bacterial community structure in wells of varying heavy metal and acid contamination to determine which contaminants have the greatest effect. Five monitoring wells and an uncontaminated background well from the Field Research Center (FRC) site of the U.S. DOE ERSP (Environmental Remediation Science Program) at Oak Ridge, Tennessee, were sampled to provide a gradient of groundwater nitrate, pH and uranium concentrations. DNA from these samples was analyzed with a comprehensive functional gene array containing 24,243 probes for >10,000 genes involved in carbon, sulfur, nitrogen, and metal cycling. Genes with the highest signal intensities from each sample were correlated with the groundwater geochemistry of that well. Wells with similar geochemical profiles had greater gene overlap than dissimilar wells. A higher percentage of nitrogen fixation genes were detected in groundwater with lower nitrate concentrations, while the percentage of nitrate reduction genes generally decreased with decreasing nitrate. Wells with elevated sulfate concentrations had a greater percentage of genes dedicated to sulfate reduction, and higher signal intensities for dsrAB genes than the background, indicating a greater abundance of those genes. Contaminated wells did not have a higher percentage of metal reduction and resistance genes than the background, but the total signal intensity of those genes was 1.4- to 2.3-fold greater than the background, indicating that metal-related genes were more prevalent in the contaminated wells. Uranium, nitrate and sulfate were identified by CCA as important factors in determining community structure. This study provides an overall view of the functional genes present in a highly contaminated environment, and shows the differences in functional populations between wells with varying contamination. As indicated by this work, contaminant level has significant effects on bacterial community structure, the knowledge of which may be important in planning and implementing successful bioremediation strategies in the future.

A pilot-scale system was established for biostimulation of subsurface U(VI) reduction by injection of ethanol at the U.S. DOE's Field Research Center (FRC) in Oak Ridge, TN. After U(VI) reduction was achieved, the stability of the bioreduced area was examined by suspension of ethanol injections (resting state) and reoxidation of the area by introducing dissolved oxygen (DO) for two months. After the reoxidation, ethanol injections were resumed. Geochip 2.0, a comprehensive 50mer microarray containing probes for genes involved in the geochemical cycling of N, S, and C, metal resistance and contaminant degradation, was used to monitor the dynamics of the groundwater microbial community structure and function. The immobilized U was stable during the resting state. After DO was introduced to the reduced area, the monitoring well (FW101-2) located closest to the injection well, had a greater increase in DO (2 mg L-1) than the well located further away (FW102-3; <0.4-0.5 mg L-1). Based on canonical correspondence analysis and Mantel test results, ethanol showed the greatest correlation to community structure, although sulfide did correlate with changes in the functional community. Detrended correspondence analysis showed a shift towards a different community structure after ethanol injections resumed compared to the periods of starvation and exposure to DO. Changes in the functional community structure were similar in the two wells; however, the community in FW101-2 was more affected by DO than in FW102-3. Hierarchical clustering showed that cytochrome c genes grouped based on DO exposure, resting state, or ethanol addition, while dissimilatory sulfite reductase (dsr) genes grouped only by resting state or ethanol addition. However, the relative abundance of dsr genes did decrease when DO levels increased while the relative abundance of cytochrome genes seemed unaffected by changes in DO. Overall, results indicated that ethanol was the main factor affecting community structure, although some changes could be attributed to DO.