SO, MARK JASON S. Application of Sequential mRNA Fluorescence in situ Hybridization and Fluorescence-Assisted Cell Sorting (SmRFF) in Separating Active Nitrite-Reducers from Mix Cultures and Environmental Samples. (Under the direction of Dr. Francis de los Reyes III.) Denitrification, considered the

SO, MARK JASON S. Application of Sequential mRNA Fluorescence in situ Hybridization and Fluorescence-Assisted Cell Sorting (SmRFF) in Separating Active Nitrite-Reducers from Mix Cultures and Environmental Samples. (Under the direction of Dr. Francis de los Reyes III.) Denitrification, considered the primary method of nitrogen-removal during wastewater treatment, is a process of sequentially converting nitrate to nitrogen gas through a series of reactions catalyzed by several genes found in a broad variety of unrelated microorganisms. To optimize denitrification, it is important to determine which specific microorganism is responsible for a specific biological process/step. However, current methods of identifying active microorganisms in complex environments using standard molecular methods for linking microbial community structure to function are extremely difficult since PCR amplification of denitrification genes are often limited to assessing gene diversity, and not microbial function. In this study, a novel “sequential mRNA fluorescence in situ hybridization and fluorescence-assisted cell sorting” (SmRFF) is described to potentially separate and phylogenetically identify in situ nitrite reducers in mixed culture preparations. The novel method was demonstrated using mixed cultures consisting of: (1) actively denitrifying bacterium (2) non-active denitrifier and (3) non-denitrifier. The process involves specific tagging of the active denitrifiers by hybridization of expressed nirS, with a HRP-labeled oligonucleotide probe or a DIG-labeled LNA probe followed by single tyramide signal or two-pass amplification. Under fluorescence microcopy, it was observed that both hybridization techniques yielded appreciable fluorescent signal that differentiate active denitrifiers from other cells. Once the target cells are fluorescently tagged, the actively denitrifying bacteria, with a noticeably higher fluorescence intensity than non-denitrifying bacteria can then be physically sorted using gated flow cytometry, based on relative signal intensities. However in using two different types of probes, LNA probes seem to provide the best signal during flow cytometry having effectively separate 40% active denitrifier from the original mix culture and with a 88% purity. In spiked activated sludge, the use of LNA probes also provided a high concentration of the target denitrifier (90.56%), however, in terms of recovery, the procedure only acquired 10.57% of the original denitrifying population. This research focuses on addressing the fundamental question of which microorganism is responsible for a specific bioprocess in a given system by developing and optimizing a novel molecular method for separating, identifying, and quantifying bacteria that actively express specific functional genes in mixed culture samples. This method could also be applied to other complex environments (e.g., soil; groundwater) and may also allow the identification of active microorganisms responsible for different biological processes (e.g., bioremediation). Application of Sequential mRNA Fluorescence in situ Hybridization and Fluorescence-Assisted Cell Sorting (SmRFF) in Separating Active Nitrite-Reducers from Mix Cultures and Environmental Samples