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ABSTRACT Title of Dissertation ABSTRACT Title of Dissertation: ANTIBIOTIC CONCENTRATIONS AND THE COMPOSITION OF BACTERIAL COMMUNITIES IN MUNICIPAL WASTEWATER AND RECLAIMED WATER Prachi Kulkarni, Doctor of Philosophy, 2016 Dissertation directed by: Dr. Amy R. Sapkota, Maryland Institute for Applied Environmental Health Before reclaimed water is used more widely within the current United States (U.S.) wastewater treatment infrastructure, it is important to examine the potential public health impacts of this emerging, alternative freshwater resource. My dissertation evaluated antibiotic concentrations and the composition of bacterial communities in conventionally treated municipal wastewater and resulting reclaimed water. I also evaluated the efficacy of a point-of-use filtration system in reducing antimicrobials present in reclaimed water. My objectives were to: 1) Assess the fate of antibiotics and; 2) Characterize the total bacterial community structure of differentially treated wastewater, and reclaimed water that has undergone on-site treatment and storage; and 3) Evaluate zero-valent iron (ZVI)-biosand filtration as a potential point-of use treatment technology for the reduction of antimicrobials from conventionally treated reclaimed water. I extracted nine antibiotics and total genomic deoxyribonucleic acid (DNA) from differentially treated wastewater and reclaimed water samples from two Mid-Atlantic and two Midwest WWTPs, and one associated Mid-Atlantic spray irrigation site. I quantified the presence of antibiotics using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and characterized total bacterial community structure using 16S rRNA gene sequencing. I also used HPLC-MS/MS to quantify the reduction of thirteen antimicrobials from conventionally treated reclaimed water after ZVI-biosand filtration. Statistical analyses included the Kruskal Wallis test, paired Wilcoxon signed-rank test, and differential abundance using normalization achieved by cumulative sum scaling. Activated sludge treatment used at all four WWTPs resulted in the reduction of some antibiotics and the increase of genera containing potentially pathogenic bacteria (Mycobacterium and Legionella). Treatment plant chlorination and spray irrigation site ultraviolet radiation (UV) treatment and open-air storage reduced the concentration of azithromycin and increased the relative abundance of Mycobacterium. ZVI-biosand filtration achieved significant reductions in azithromycin, ciprofloxacin, erythromycin, linezolid, oxolinic acid, pipemidic acid, penicillin and vancomycin. This research provided additional scientific evidence that activated sludge treatment and chlorination alone may not be sufficient for the removal of antimicrobials and potentially pathogenic bacteria from municipal wastewater and resulting reclaimed water. However, ZVI-biosand filtration may be an efficient reuse site technology for the reduction of antimicrobials from conventionally treated reclaimed water. ANTIBIOTIC CONCENTRATIONS AND THE COMPOSITION OF BACTERIAL COMMUNITIES IN MUNICIPAL WASTEWATER AND RECLAIMED WATER by Prachi Kulkarni Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2016 Advisory Committee: Dr. Amy R. Sapkota, Chair Dr. Hector Corrada Bravo Dr. Shirley A. Micallef Dr. Emmanuel F. Mongodin Dr. Mihai Pop © Copyright by Prachi Kulkarni 2016 Dedication For my husband Rob and mother Shaku. ii Acknowledgements Thank you, Dr. Amy Sapkota, for your mentorship and support, and for giving me the opportunity to do incredible and exciting research, for encouraging me to do my best, and for being an amazing role model. Thank you to my committee members Dr. Hector Corrada Bravo, Dr. Shirley Micallef, Dr. Emmanuel Mongodin and Dr. Mihai Pop for your encouragement and guidance. Anthony Bui, Rhodel Bradshaw, Cary Coppock, Cheryl East, Meghan Fisher Holbert, Eric Handy, Nate Olson, Dr. Joe Paulson, Dr. Greg Raspanti, Russell Reynells, Maurice Rocque and Dr. Manan Sharma - thank you for your kindness, support, hard work, advice and optimism. Kelsey Babik and Rianna Murray, thanks for all of our chats. Dr. Hugh Bruck, Dr. Anne Lucas and Dr. Paul Turner thank you for your advice, support and encouragement and for the lab and life skills I learned from you. Chitra Phadke, without your help, encouragement and wisdom, none of this would have been possible. Adam Buxton, Joe Cornish, Elis James, John Robins and the paw team, thank you for helping me get through the past year. Finally, I would like to thank the two people, without whom I wouldn’t have been able to accomplish this task – my mother and my husband. I would like to thank my mother for showing me that women are capable of great things, and for dedicating her life to making sure that I never had to face any of the hurdles she did. Rob, my rock, and my puncture repair, thank you for believing in me. Thank you for your love, humor, patience, support and encouragement. I couldn’t have done this without you. iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv List of Tables .............................................................................................................. vii List of Figures ............................................................................................................ viii Chapter 1: Introduction ..................................................................................................1 Chapter 2: Background ..................................................................................................7 Municipal Wastewater Treatment in the United States ............................................ 7 Reclaimed Water Use in the United States ............................................................... 9 Regulations Governing Reclaimed Water Use in the United States ....................... 10 Antibiotics in Wastewater and Reclaimed Water ................................................... 12 Bacteria in Wastewater and Reclaimed Water ....................................................... 15 Exposure to Wastewater and Reclaimed Water ...................................................... 16 Agricultural and Landscape Irrigation with Reclaimed Water in the United States ................................................................................................................................ 18 Impact of Irrigation with Reclaimed Water ............................................................ 19 Chapter 3: The Application of Zero-valent Iron Technology for the Reduction of Antibiotic Residuals .....................................................................................................24 Zero-valent Iron Technology .................................................................................. 24 Laboratory Studies of Antibiotic Removal by Zero-valent Iron Technology ........ 25 Chapter 4: Characterization of the Total Bacterial Community Structure of Environmental Samples ...............................................................................................28 Introduction ............................................................................................................. 28 Structure and Function of the 16S rRNA gene ....................................................... 29 Bacterial Identification Using the 16S rRNA gene ................................................ 31 Bacterial Community Analysis using 16S rRNA gene Sequencing ....................... 32 Experimental Design and 16S rRNA gene Sequencing Data Analysis .................. 34 Experimental Design ........................................................................................... 34 Sequence Filtering .............................................................................................. 35 Data Transformation ........................................................................................... 35 Taxonomic Summaries and Heatmaps ................................................................ 37 Diversity Analysis ............................................................................................... 37 Multivariate Exploratory Analyses ..................................................................... 38 Multivariate Environmental Interpretation Analyses .......................................... 38 Differences Between Groups .............................................................................. 39 Longitudial Analysis ........................................................................................... 39 Bacterial Community Analysis Using 16S rRNA gene Sequencing-Advantages .. 40 Bacterial Community Analysis Using 16S rRNA gene Sequencing-Limitations .. 40 16S rRNA gene Sequence ................................................................................... 40 Multiple 16S rRNA Operon Copy Numbers....................................................... 41 Genes and Species Definition ............................................................................. 42 Assignment and Clustering Approches ..............................................................
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