University of Tennessee, Knoxville Trace: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2016 Bioremediation of Chlorinated Ethenes: pH Effects, Novel Dechlorinators and Decision- Making Tools Yi Yang University of Tennessee, Knoxville, [email protected] Recommended Citation Yang, Yi, "Bioremediation of Chlorinated Ethenes: pH Effects, Novel Dechlorinators and Decision-Making Tools. " PhD diss., University of Tennessee, 2016. http://trace.tennessee.edu/utk_graddiss/4117 This Dissertation is brought to you for free and open access by the Graduate School at Trace: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of Trace: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Yi Yang entitled "Bioremediation of Chlorinated Ethenes: pH Effects, Novel Dechlorinators and Decision-Making Tools." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Civil Engineering. Frank Löffler, Major Professor We have read this dissertation and recommend its acceptance: Chris Cox, Terry Hazen, Qiang He, Gary Sayler Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Bioremediation of Chlorinated Ethenes: pH Effects, Novel Dechlorinators and Decision-Making Tools A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Yi Yang December 2016 Copyright © 2016 by Yi Yang All rights reserved. ii DEDICATION To my parents and my beloved wife Jia Xue. iii iv ACKNOWLEDGEMENTS “Masters open the door. You enter by yourself.” And it is my advisor Dr. Frank Löffler who opened the microbial world to me. Without his guidance, I would have never entered such an amazing world. I am so lucky to get a chance to learn from him. His enthusiasm for science, comprehensive knowledge about microbiology, and incisive mind towards scientific questions make him a role model for me. I would like to thank Dr. Löffler for his teaching, discussion, encouragement and help during this process. I am also grateful for the advice from my committee members: Dr. Chris Cox, Dr. Terry Hazen, Dr. Qiang He and Dr. Gary Sayler. Thank you all for your time, helpful suggestions and valuable comments on my research. It has been an enriching experience to discuss science and learn from you. I am also thankful to all labmates who I worked with in the Löffler lab for their help and tolerance towards me. Especially, I want to thank Dr. Jun Yan, who gave me enormous help and valuable advice. I also want to thank soon to be Dr. Burcu Simsir, Dr. Jeongdae Im, Dr. Silke Nissen, Dr. Jarrod Pollock, Nannan Jiang and soon to be Dr. Steve Higgins for their friendship and help. Many thanks to soon to be Dr. Jenny Onley and Cindy Swift for keeping the lab organized. I am also honored for the opportunities working with Dr. Kurt Pennell and Dr. Natalie Cápiro at Tufts University. I greatly appreciate Dr. Cápiro’s patience and help editing my manuscript drafts. I am also greatly indebted to all other v UTK faculty, staff and graduate students I have met and who have made my experiences so much more colorful. I want to thank my parents and my wife. Your unconditional love and support make me the luckiest person in the world. vi ABSTRACT Chlorinated solvents have been widely used in different areas of modern society. Usage of these chlorinated solvents was not necessarily accompanied with proper handling and disposal of these hazardous compounds, which caused a variety of environmental problems and continues to affect human health. Remediation of chlorinated ethenes contaminated sites has high priority for state regulators and site owners. Among the available treatment technologies, bioremediation shows great promise as a cost-effective corrective strategy for a variety of environmental pollutants. Prerequisites are that the microbiology involved in contaminant degradation and geochemical factors, such as pH, are understood, so that bioremediation technologies can be confidently implemented. The aims of this dissertation work were 1) to enrich and isolate PCE dechlorinators under low pH conditions, 2) to investigate how pH fluctuations affect the microbial community of a PCE-to-ethene consortium, 3) to determine the pH tolerance of Dehalococcoides mccartyi (Dhc), 4) to identify a non-Dehalococcoides type microorganism responsible for reductive dechlorination of vinyl chloride, 5) to identify and characterize a novel vinyl chloride reductase gene and 6) to develop an Excel-based tool to guide remedial practitioners to select suitable remediation strategies. Only one enrichment culture out of total sixteen sites samples showed PCE dechlorination activity at pH 5.5 and stoichiometric conversion to cDCE occurred after repeated transfers. The analysis of 16S rRNA gene sequencing data revealed the genera Desulfovibrio, Sulfurospirillum, and Megasphaera were most abundant in pH 5.5 enrichment. Two PCE-dechlorinating vii isolates (strains PLC-TCE and PLC-DCE) were obtained from a pH 5.5 enrichment, and identified as members of the genus Sulfurospirillum. Experiments with a Dhc-containing consortium demonstrated that exposure time affected Dhc ability to recover reductive dechlorination activity following low pH exposure. Low pH conditions affected Dhc strains differently, and Dhc strains carrying the vcrA gene responsible for reductive dechlorination of the human carcinogen vinyl chloride (VC) were least tolerant to low pH. Enrichment and isolation efforts led to the discovery of a Dehalogenimonas (Dhgm) species capable of respiring chlorinated ethenes, including VC. These research findings advance understanding of the microbial reductive dechlorination process and will improve the implementation of in situ bioremediation. viii TABLE OF CONTENTS CHAPTER I Introduction ................................................................................................................ 1 Physical requirement for bioremediation: pH ............................................................................. 6 Microbial requirement for bioremediation: dechlorinators ......................................................... 8 Summary ................................................................................................................................... 11 References ................................................................................................................................. 13 CHAPTER II Reductive dechlorination of chlorinated ethenes under low pH conditions ........... 19 Abstract ..................................................................................................................................... 20 Introduction ............................................................................................................................... 21 Materials and Methods .............................................................................................................. 24 Results ....................................................................................................................................... 30 Discussion ................................................................................................................................. 36 References ................................................................................................................................. 42 Appendix ................................................................................................................................... 47 CHAPTER III Recovery of Dehalococcoides mccartyi exposed to low ph and distribution of Dehalococcoides mccartyi in groundwater with two pH ranges ................................................... 53 Abstract ..................................................................................................................................... 54 Introduction ............................................................................................................................... 55 Materials and Methods .............................................................................................................. 57 Results ....................................................................................................................................... 61 Discussion ................................................................................................................................. 66 References ................................................................................................................................. 72 Appendix ................................................................................................................................... 74 CHAPTER Iv Grape pomace compost as a habitat for strictly organohalide-respiring Dehalogenimonas species harboring novel reductive dehalogenase genes ................................... 76 Abstract ..................................................................................................................................... 77 Significance ..............................................................................................................................