Reductive processes for bioremediation of chlorinated solvent/metal mixtures

本研究的总体假设是，电子传递可以被设计为同时有效的金属还原和脱氯。提出的研究在三个具体目标中解决了这一假设：首先，生物生成的氧化还原活性剂，包括分泌因子，腐植酸和硫化铁矿物，将用于构建电子传递链，允许有效转移电子以还原金属和脱氯；其次，将对模型细菌进行表征和设计，以改善脱氯和金属还原的电子流；在第三个目标中，将通过分离能够同时脱氯和金属还原的细菌和培养能够产生FeS矿物的生物，在含水层沉积物微观环境和在不溶性电子受体或营养物上生长的生物和富集物中评价前两个目标中发展的生物和电子转移过程。使用这些程序产生的因子和FeS将被描述为它们对动力学和转化产物的影响。第二个特定目标将被满足使用腐烂希瓦氏菌MR-1作为模式细菌。将监测电子传递所需基因的表达，并与不同浓度和组合的氯化溶剂、铬酸盐、分泌因子和人体物质的脱氯和金属还原率进行比较。基因表达将使用包含菌株MR-1的每个开放阅读框的DNA微阵列进行评估。在确定了野生型MY-1的电子流特征后，将产生突变体。四氯化碳转化为氯仿所需的基因将被敲除，而通过不产生氯仿的途径转化四氯化碳降解的基因将被添加。电子传递所需基因的表达以及脱氯和金属还原的速率将被监测并与野生型进行比较。在最后的具体目标中，将对含水层的脱氯和金属还原进行分析，使用前两个具体目标中开发的工艺处理含水层沉积物微观环境。群落结构的变化将通过定量PCR、群落基因组阵列和16S rDNA片段分析来监测。

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.