Lawrence Berkeley National Laboratory

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.