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为同时和有效的金属还原和脱氯。这项研究提出了三个具体目标：第一，生物产生的氧化还原活性剂，包括分泌因子，腐殖酸和硫化铁矿物，将用于构建电子传输链，允许有效的电子转移，以减少金属和脱氯;在第二个项目中，将对一种模式细菌进行鉴定和改造，以改善脱氯和金属还原的电子流;在第三个目标中，将在含水层沉积物微观世界中评价前两个目标中开发的生物体和电子转移过程，并通过分离能够同时进行脱氯和金属还原的细菌，以及培养能够产生硫化亚铁矿物质的生物体。使用这些程序产生的因素和FeS将表征其对动力学和转化产物的影响。以腐败希瓦氏菌MR-1为模型菌，可满足第二个具体目标。将监测电子传递所需基因的表达，并将其与氯化溶剂、铬酸盐、分泌因子和人体物质的不同浓度和组合的脱氯和金属还原速率进行比较。将使用含有菌株MR-1的每个开放阅读框的DNA微阵列评价基因表达。在表征野生型MY-1中的电子流之后，将产生突变体。四氯化碳转化为氯仿所需的基因将被敲除，并将添加通过不产生氯仿的途径转化四氯化碳降解的基因。 将监测电子传递所需基因的表达以及脱氯和金属还原速率，并与野生型进行比较。在最后一个具体目标中，将对使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的含水层进行脱氯和金属还原试验。群落结构的变化将通过定量PCR、群落基因组阵列和16 S rDNA片段分析进行监测。

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.