Reductive processes for bioremediation of chlorinated solvent/metal mixtures

这项研究的总体假设是，电子传输可以被设计成同时有效地进行金属还原和脱氯。这项拟议的研究从三个具体目标解决了这一假设：第一，生物产生的氧化还原活性物质，包括分泌因子、腐植酸和铁硫化矿物，将被用来构建电子传输链，允许电子有效地转移用于金属还原和脱氯；第二，将表征和设计一种模式细菌，以改善用于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，以及通过培养能够产生FeS矿物的生物，在含水层沉积微宇宙和生长在不溶性电子受体或营养物质上的生物体和富集物中评估在前两个目标中形成的生物体和电子转移过程。使用这些程序产生的因子和FeS将因其对转化动力学和产物的影响而被表征。第二个具体目标将以腐烂希瓦氏杆菌MR-1为模式菌来实现。电子传递所需基因的表达将被监测，并与不同浓度和组合的氯化溶剂、铬酸盐、分泌因子和人类物质的脱氯和金属还原速率进行比较。基因表达将使用包含菌株MR-1的每个开放阅读框架的DNA微阵列进行评估。在鉴定了野生型My-1的电子流之后，将产生突变体。四氯化碳转化为氯仿所需的基因将被敲除，四氯化碳降解转化为氯仿的途径将被添加。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检测，以确定利用前两个具体目标中开发的工艺处理的含水层沉积物微观世界。群落结构的变化将通过定量聚合酶链式反应、群落基因组阵列和分析16S rDNA片段进行监测。

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.