MONITORING THE METABOLIC STATE OF GEOBACTERACEAE DURING IN SITU U(VI) BIOREMEDIATION

Stimulation of metal reducing microorganisms such as Geobacter species is a promising strategy for the reductive immobilization of U(VI) in contaminated aquifers. An understanding of the rates of microbial metal reduction will help develop strategies for optimization of the bioremediation process. However, it is difficult to monitor in situ metabolic rates using geochemical techniques. Therefore, a strategy for monitoring expression of genes that are indicative of metal reduction rates and the associated metabolic states of microorganisms participating in U(VI) reduction was developed. Relevant Geobacteraceae genes were identified from microarray experiments conducted under a variety of environmentally relevant conditions. Field experiments were conducted at a uranium-contaminated site in Rifle, CO. Acetate was injected into the subsurface to stimulate the growth of metal-reducing microorganisms. During the active phase of U(VI) reduction, Geobacteraceae accounted for 90% and 57% of the bacterial sequences in the groundwater and sediments, respectively. The in situ metabolic state of Geobacteraceae was determined by tracking the expression of stress response, nutrient limitation, and chemotaxis and motility genes. Results show that the oxidative stress genes, cydA and sodA, as well as the heavy metal efflux gene, cusA, were expressed during bioremediation. Moreover, genes indicative of nitrogen (nifD), phosphorous (phoU), and Fe(II) (feoB and ideR) limitations were also expressed. Interestingly, expression of the chemotaxis and motility gene, pilT, was inversely correlated with Fe(II) concentrations in the groundwater. As was expected, the number of mRNA transcripts from genes involved in central metabolism (gltA, mdh, and korA) and electron transport (mcpA and ompB) was correlated with acetate concentrations in the groundwater. These results demonstrate that monitoring the in situ transcript levels of key genes can provide insight into the rates of metabolism and nutrient requirements of Geobacteraceae during in situ bioremediation of uranium.