The overall hypothesis of this research is that electron transport can be engineered for simultaneous and efficient metal reduction and dechlorination. The proposed research addresses this hypothesis in three specific aims: in the first, biologically generated redox active agents, including secreted factors, humic acids, and iron sulfide minerals, will be used to construct electron transport chains that permit efficient transfer of electrons for reduction of metals and dechlorination; in the second, a model bacterium will be characterized and engineered to improve electron flow for dechlorination and metal reduction; and, in the third, the organisms and electron transfer processes developed in first two aims will be evaluated in aquifer sediment microcosms and the organisms and enrichments grown on insoluble electron acceptors or nutrients, by isolating bacteria capable of simultaneous dechlorination and metal reduction, and by cultivating organisms capable of generating FeS minerals. Factors and FeS generated using these procedures will be characterized for their effects on the kinetics and products of transformation. The second specific aim will be satisfied using Shewanella putrefaciens MR-1 as the model bacterium. The expression of genes required for electron transport will be monitored and compared with rates of dechlorination and metal reduction for varied concentrations and combinations of chlorinated solvents, chromate, secreted factors, and human substances. Gene expression will be evaluated using a DNA microarray containing each of the open reading framers of strain MR-1. After characterizing electron flow in wild type MY-1, mutants will be created. Genes required for carbon tetrachloride transformation to chloroform will be knocked out and genes for conversion of carbon tetrachloride degradation by a pathways that does not produce chloroform will be added. The expression of genes required for electron transport as well as rates for dechlorination and metal reduction will be monitored and compared with the wild type. In the final specific aim, dechlorination and metal reduction will be assayed for aquifer for aquifer sediment microcosms treated using processes developed in the first 2 specific aims. Changes in community structure will be monitored by quantitative PCR, by community genome arrays, and by analysis of 16S rDNA fragments.
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