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Denitrification by Zero-Valent Iron-Supported Mixed DENITRIFICATION BY ZERO-VALENT IRON-SUPPORTED MIXED CULTURES by Inyoung Kim A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Civil Engineering Fall 2018 © 2018 Inyoung Kim All Rights Reserved DENITRIFICATION BY ZERO-VALENT IRON-SUPPORTED MIXED CULTURES by Inyoung Kim Approved: __________________________________________________________ Sue McNeil, Ph.D. Chair of the Department of Civil and Environmental Engineering Approved: __________________________________________________________ Levi T. Thompson, Ph.D. Dean of the College of Engineering Approved: __________________________________________________________ Douglas J. Doren, Ph.D. Interim Vice Provost for Graduate and Professional Education I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Daniel K. Cha, Ph.D. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Julia Anne Maresca, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Pei C. Chiu, Ph.D. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Jeffry Fuhrmann, Ph.D. Member of dissertation committee ACKNOWLEDGMENTS I would like to express my sincere gratitude to my advisor, Dr. Daniel K. Cha for his continuous guidance, assistant, and expertise that I needed during my degree. I gratefully acknowledge my Ph.D. committee members, Dr. Julia A. Maresca, Dr. Pei C. Chiu, and Dr. Jeffry Fuhrmann for their time and insightful feedback for my research and thesis. My sincere thanks also goes to Mr. Michael Davidson for his tremendous technical and substantial support for research and safety in the laboratory. I am thankful to Mrs. Christine Reoli, a graduate academic advisor of the department, for her guidance and support for better graduate school life. I also thank lab manager, Dr. Yu-han Yu, and staffs of the Department of Civil and Environmental Engineering, Mrs. Karen Greco, Sarah Palmer, and Christine Murray, for their tremendous help and support. The members of the Cha group have contributed immensely to my personal and professional time at UD. The members have been good friends as well as collaborators. All past and present group members that I have had the pleasure to work with are undergraduate and graduate students Beom-seok Kim, Taylor Smith, Philip McGuire, Yaseen Al-Qaraghuli, Xiangmin Liang, Larissa Gaul, Mingjun Shao, Aidan Meese, and Rachel Aukamp; and the numerous visiting scholars who have come through the lab. In regard to the technical support, I thank the staffs Deborah Powell at UD Bioimaging Center and Drs. Choaying Ni and Yong Zhao at Keck Center for iv Advanced Microscopy and Microanalysis for advising to acquire confocal and scanning electron microscopic images. Also, I thank Brewster F. Kingham at UD Sequencing & Genotyping Center and Dr. John Hanson, Rocio Navarro, and Lars Koenig at RTL genomics to help me to better understand microbial sequencing area. I am deeply thankful to my family: my parents and to my brother for supporting me spiritually throughout my life in general. Most of all for my loving, supportive, and encouraging husband Minho whose faithful support during the Ph.D. is appreciated. v TABLE OF CONTENTS LIST OF TABLES .................................................................................................... ix LIST OF FIGURES .................................................................................................... x ABSTRACT............................................................................................................. xii Chapter 1 INTRODUCTION .......................................................................................... 1 1.1 Nitrate .................................................................................................... 1 1.2 Nitrate Treatment Techniques ................................................................ 2 1.3 Objectives .............................................................................................. 3 2 LITERATURE REVIEW ............................................................................... 6 2.1 Hydrogenophillic Denitrifiers................................................................. 9 2.2 Ferrous-oxidizing Denitrifiers .............................................................. 10 2.3 Anaerobic Ammonium Oxidation (Anammox) ..................................... 11 2.4 Microbial-meditated Corrosion and Surface Colonization .................... 12 3 MICROBIAL COMMUNITY ANALYSIS OF DENITRIFYING CULTURES GROWN ON ZERO-VALENT IRON ..................................... 14 3.1 Introduction ......................................................................................... 15 3.2 Materials and Methods ......................................................................... 19 3.2.1 Microorganisms and Chemicals ................................................ 19 3.2.2 Denitrification Test with Various Electron Donors ................... 19 3.2.3 Chemical Analysis .................................................................... 20 3.2.4 DNA Extraction and Sequencing for Microbial Identification ... 21 3.2.5 Bacterial 16S rRNA Gene Sequence Analysis .......................... 21 3.2.6 Statistical Analysis with Sequence Results ............................... 22 3.3 Results and Discussion ......................................................................... 22 3.3.1 Nitrate Reduction in Batch Reactors ......................................... 22 3.3.2 Microbial Diversity under Different Conditions ........................ 24 3.3.3 Principal Component Analysis .................................................. 27 vi 4 EFFECT OF LOW TEMPERATURE ON ABIOTIC AND BIOTIC NITRATE REDUCTION BY ZERO-VALENT IRON ................................. 38 4.1 Introduction ......................................................................................... 39 4.2 Materials and Methods ......................................................................... 42 4.2.1 Chemicals and Microorganisms ................................................ 42 4.2.2 Batch Reduction Experiments ................................................... 43 4.2.3 Analytical Procedures ............................................................... 43 4.3 Results and Discussion ......................................................................... 44 4.3.1 Effects of Temperature on Nitrate Reduction by ZVI ................ 44 4.3.2 Effects of Temperature on Nitrate Reduction by ZVI and Microorganisms ........................................................................ 45 4.3.3 Enhanced Denitrification by Microbial Induced Corrosion ....... 46 4.4 Conclusion ........................................................................................... 48 5 NITROGEN REMOVAL BY MICROBIALLY-COLONIZED IRON GRANULES: ANAMMOX-LIKE PROCESS .............................................. 57 5.1 Introduction ......................................................................................... 58 5.2 Materials and Methods ......................................................................... 61 5.2.1 Batch Reduction Tests with Anammox Mixed Cultures ............ 61 5.2.2 Denitrification in Anaerobic Fluidized Bioreactor ..................... 63 5.2.3 Surface Study of Zero-valent Iron ............................................. 64 5.2.3.1 Confocal Microscopy ................................................. 64 5.2.3.2 Scanning Electron Microscopy ................................... 65 5.2.4 Identification of Bacterial Community Colonizing Zero-valent Iron .......................................................................................... 65 5.3 Results and Discussion ......................................................................... 66 5.3.1 Anammox Activities in the Presence of Zero-valent Iron: Preliminary Tests ...................................................................... 66 5.3.2 Enhanced Nitrate Reduction in Anaerobic Fluidized Bioreactor 67 5.3.2.1 Characterization of Biofilms on Zero-valent Iron ....... 68 5.3.2.2 Diversity of Microbial Communities in the Biofilm .... 69 6 CONCLUSIONS .......................................................................................... 85 vii 6.1 Summary of Results ............................................................................. 85 6.1.1 Denitrification Performances in the Mixed-culture System with Various Electron Donors and Zero-valent Iron .......................... 85 6.1.2 Enhanced Nitrate Removal by Microorganisms and Zero- valent Iron under Low Temperature .........................................
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