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Design and Implementation of Degenerate Qpcr/Qrt-PCR Primers to Detect Microbial Nitrogen Metabolism in Wastewater and Wastewater-Related Samples

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Design and Implementation of Degenerate Qpcr/Qrt-PCR Primers to Detect Microbial Nitrogen Metabolism in Wastewater and Wastewater-Related Samples University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School August 2019 Design and Implementation of Degenerate qPCR/qRT-PCR Primers to Detect Microbial Nitrogen Metabolism in Wastewater and Wastewater-Related Samples Ryan F. Keeley University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Ecology and Evolutionary Biology Commons, Environmental Sciences Commons, and the Microbiology Commons Scholar Commons Citation Keeley, Ryan F., "Design and Implementation of Degenerate qPCR/qRT-PCR Primers to Detect Microbial Nitrogen Metabolism in Wastewater and Wastewater-Related Samples" (2019). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/7826 This Thesis is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Design and Implementation of Degenerate qPCR/qRT-PCR Primers to Detect Microbial Nitrogen Metabolism in Wastewater and Wastewater-Related Samples by Ryan F. Keeley A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology with a concentration in Environmental and Ecological Microbiology Department of Integrative Biology College of Arts and Sciences University of South Florida Major Professor: Kathleen Scott, Ph. D. Sarina Ergas, Ph. D. Valerie Harwood, Ph. D. Date of Approval: June 24, 2019 Keywords: Degenerate Primer, amoA, nxrB, nosZ, nitrification, denitrification, Photo- sequencing batch reactor, qPCR, qRT-PCR Copyright © 2019, Ryan F. Keeley TABLE OF CONTENTS List of Tables ................................................................................................................................. iv List of Figures ................................................................................................................................. v Abstract ......................................................................................................................................... vi Chapter 1 Introduction .................................................................................................................... 1 Wastewater Treatment ........................................................................................................ 1 Nitrogen Transformation Pathways in Prokaryotes and Algae........................................... 1 Shortcut Nitrogen Removal Facilitated by Algae ............................................................... 4 Molecular Methods Used to Analyze N Transformations .................................................. 6 Objectives of this Study .................................................................................................... 10 Figure 1.1 Canonical Nitrogen Metabolism .......................................................... 11 Figure 1.2 Shortcut Nitrogen Removal ................................................................. 12 Chapter 2 Degenerate PCR Primers for Assays to Track Steps of Nitrogen Metabolism by Taxonomically Diverse Microorganisms in a Variety of Environments ..................... 13 Abstract ............................................................................................................................. 13 Introduction ....................................................................................................................... 14 Methods............................................................................................................................. 18 Database mining and sequence collection ............................................................ 18 Clustering genes to facilitate primer design.......................................................... 19 Environmental sampling ....................................................................................... 21 qPCR standards ................................................................................................... 211 qPCR ................................................................................................................... 23 Environmental amplicon sequences and phylogeny ............................................. 23 Results ............................................................................................................................... 24 Primer matches to aligned sequences ................................................................... 24 Limit of quantification of target sequences with new primers ............................. 24 Primer performance in engineered or environmental system ............................... 24 Discussion ......................................................................................................................... 25 Figures............................................................................................................................... 28 Figure 2.1 Nitrogen Cycle ..................................................................................... 28 Figure 2.2 Phylogenetic Composition of Sequence Alignments used for Primer Design. .......................................................................................... 29 Figure 2.3 Clusters of amoA, nxrB, and nosZ Sequences Drawn from Integrated Microbial Genomes Database .................................................. 30 Figure 2.4 Schematics of Wastewater Treatment Plant Tracks ............................ 31 i Figure 2.5 Primer Mismatches Against Sequence Alignment for Respective Biomarkers from IMG ............................................................ 32 Figure 2.6 Biomarker Detection and Quantification in Environmental Samples. .................................................................................................... 33 Figure 2.7 Comparison of Environmental Amplicon Sequence Identity to Top Environmental and Cultured Hits from GenBank. ............................ 34 Figure 2.8 Phylogenetic Analysis of amoA Amplicon Sequences ........................ 35 Figure 2.9 Phylogenetic Analysis of nxrB and nosZ Amplicon Sequences .......... 36 Tables ............................................................................................................................... 37 Table 2.1 Primers Previously Used to Study Nitrogen Metabolism. .................... 37 Table 2.2 Primers Developed in this Study .......................................................... 38 Table 2.3 qPCR Reaction Conditions. .................................................................. 39 Table 2.4 Primer Degeneracy and Threshold of Detection for qPCR Assay ........ 40 Chapter 3 Shortcut Nitrogen Removal in a Photo-Sequencing Batch Reactor with Variable Solids Retention Time ........................................................................................ 41 Introduction ....................................................................................................................... 41 Methods............................................................................................................................. 43 Molecular analysis ................................................................................................ 43 Results and Discussion ..................................................................................................... 45 Nitritation .............................................................................................................. 46 Nitratation ..............................................................................................................47 Denitrification ........................................................................................................48 Figures............................................................................................................................... 50 Figure 3.1 DNA Copy Numbers for Biomarkers in SRT 15 Bioreactor .............. 50 Figure 3.2 RNA Copy Numbers for Biomarkers in SRT 15 Bioreactor. .............. 51 Tables .................................................................................................................................52 Table 3.1 qPCR Primers for Nitrogen Metabolism .............................................. 52 Chapter 4 Conclusion .................................................................................................................... 53 Objective 1: Designing and validating degenerate primers for qPCR and qRT- PCR assays to track the presence and activity of microorganisms catalyzing steps of the nitrogen cycle relevant to evaluating PSBR performance .......................................................................................................... 53 Objective 2: Verifying that ammonium oxidation is stimulated during the illuminated portion of the PSBR diel cycle .......................................................... 55 Objective 3: Verifying that minimal nitrite oxidation is occurring in the PSBR. ............. 55 Objective 4: Verifying that denitrification is stimulated during the dark portion of the PSBR diel cycle .............................................................................................. 55 Future Directions for Nitrogen Metabolism Evaluation using Molecular Tools .............. 56 Bibliography ................................................................................................................................
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