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

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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.