Identifying and Quantifying Active Denitrifiers in Complex Environments Using Functional Gene Expression Analysis

确定负责特定生物转化过程的微生物的身份仍然是环境微生物学和环境工程的主要挑战之一。在研究反硝化细菌时，使用16S rRNA基因序列检测和分析环境样品中细菌群落而不进行分离和培养的经典方法是不可能的，因为它们具有系统发育多样性。考虑到反硝化在水和废水污染、温室气体排放和臭氧层破坏中的重要性，这是一个重大问题。本提案旨在开发并优化一种新的分子技术，以确定活性污泥和土壤中功能活性硝酸盐，亚硝酸盐，一氧化氮和一氧化二氮还原剂的系统发育特性。具体目标是：(1)优化序列mRNA FISH流式细胞术（SmRFF）方法（之前在我们的实验室开发），并研究其在其他微生物过程中的潜力；(2)确定絮体结构、DO和碳氮水平对活性反硝化群落的影响；(3)鉴定反硝化氨氧化菌（AOB）；(4)考察反硝化AOB处理高氨、低COD废水的生物反应器的反硝化生态。序列mRNA FISH-Flow Cytometry （SmRFF）方法具有巨大的前景，并可能导致显著的结果，以及开辟新的研究领域。这个提议的项目将是第一批进行以下研究的项目之一：(1)分离细胞，积极表达硝酸盐-，亚硝酸盐-，一氧化氮-和氧化亚氮？减少基因;(2)捕获活性反硝化菌的近全长16S rRNA基因；(3)应用组合流式细胞术鉴定表达nirS和nirK基因的AOB。确定mRNA FISH所需的最低mRNA拷贝量将允许SmRFF更广泛地应用于其他微生物生态领域，如生物修复。拟议的研究如果成功，将是一个潜在的重大方法进步，将影响一般微生物生态学以及环境工程中的生物过程。该项目将促进微生物学、分子生物学和工程学领域的多学科教育。在过去的7年里，PI已经指导了24名REU学生，并将继续让本科生接触研究和发现的兴奋。该项目还将潜在地有利于市政和工业废水处理厂进行氮去除。此外，该项目的成果将纳入PI定期组织的高中生暑期讲习班和污水处理专业人员微生物讲习班。

0853864de los Reyes IIIDetermining the identity of microorganisms responsible for specific biotransformation processes remains one of the major challenges in environmental microbiology and environmental engineering. The classic approach of using 16S rRNA gene sequences to detect and analyze bacterial communities in environmental samples without isolation and cultivation is not possible when studying denitrifying bacteria, because they are phylogenetically diverse. This is a major issue, given the importance of denitrification in water and wastewater pollution, greenhouse gas emission, and ozone layer destruction. This proposal is to develop and to optimize a novel molecular technique to determine the phylogenetic identity of functionally active nitrate-, nitrite-, nitric oxide-, and nitrous oxide-reducers in activated sludge and soil. Specific objectives are: (1) to optimize the Sequential mRNA FISH Flow Cytometry (SmRFF) method (developed previously in our lab) and investigate its potential for other microbial processes; (2) to determine the effect of floc structure, DO and carbon and nitrogen levels on the active denitrifying community; (3) to identify ammonia oxidizing bacteria (AOB) that denitrify; and (4) to examine the denitrifying ecology of a bioreactor that uses denitrifying AOB for treating high ammonia, low COD wastewater. The Sequential mRNA FISH-Flow Cytometry (SmRFF) method holds tremendous promise and may lead to significant results, as well as open new areas of research. This proposed project will be among the first studies to perform the following: (1) separate cells that are actively expressing the nitrate-, nitrite-, nitric oxide-, and nitrous oxide ?reducing genes; (2) capture nearly full-length 16S rRNA genes of active denitrifiers; and (3) apply combinatorial flow cytometry to identify AOB that are expressing the nirS and nirK genes. Determining the minimum level of mRNA copies needed for mRNA FISH will allow the more general application of SmRFF to other microbial ecology areas, such as bioremediation. The proposed research if successful would be a potentially significant methodological advance that would impact general microbial ecology as well as biological processes in environmental engineering. This project will contribute to the multidisciplinary education in the areas of microbiology, molecular biology and engineering. The PI has mentored 24 REU students in the last 7 years, and will continue to expose undergraduates to research and the excitement of discovery. This project will also potentially benefit municipal and industrial wastewater treatment plants that perform nitrogen removal. In addition, results from this project will be incorporated in summer workshops for high school students, and microbiology workshops for wastewater treatment professionals that are regularly organized by the PI.