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Hexavalent Chromium is a widespread contaminant found in soil, sediment, and groundwater. In order to stimulate microbially-mediated reduction of Cr(VI), a poly-lactate compound (HRC) was injected into the Chromium-contaminated aquifer at the Hanford (WA) 100H site in 2004. Cr(VI) concentrations rapidly declined to below the detection limit and remained so for more than three years after injection. Based on the results of the bacterial community composition using high-density DNA 16S rRNA gene microarrays, we observed the community to transition through denitrifying, ironreducing and sulfate-reducing populations. As a result, we specifically focused isolation efforts on three bacterial species that were significant components of the community. Positive enrichments in defined anaerobic media resulted in the isolation of an iron-reducing Geobacter metallireducens-like isolate, a sulfate-reducing Desulfovibrio vukgaris-like strain and a nitrate-reducing Pseudomonas stutzeri-like isolate among several others. All of these isolates were capable of reducing Cr(VI) anoxically and have been submitted for genome sequencing to JGI. To further characterize the microbial, and geochemical mechanisms associated with in situ Cr(VI) reduction at the site, additional HRC was injected in 2008. The goal was to restimulate the indigenous microbial community and to regenerate the reducing conditions necessary for continued Cr(VI) bio-immobilization in the groundwater. Analysismore » of the microbial populations post-injection revealed that they recovered to a similar density as after the first injection in 2004. In this study, we present the results from our investigation into microbially-mediated Cr(VI) reduction at Hanford, and a comparison of the microbial community development following two HRC injections four years apart.« less