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UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE CHARACTERIZATION OF SUBSURFACE MICROBIAL COMMUNITIES INVOLVED IN BIOREMEDIATION OF URANIUM AND NITRATE A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By ANNE MARIE SPAIN Norman, Oklahoma 2009 CHARACTERIZATION OF SUBSURFACE MICROBIAL COMMUNITIES INVOLVED IN BIOREMEDIATION OF URANIUM AND NITRATE A DISSERTATION APPROVED FOR THE DEPARTMENT OF BOTANY AND MICROBIOLOGY BY __________________________ Dr. Lee R. Krumholz, Chair __________________________ Dr. Joseph M. Suflita __________________________ Dr. Michael J. McInerney __________________________ Dr. Ralph S. Tanner __________________________ Dr. Elizabeth C. Butler Copyright by ANNE SPAIN 2009 All Rights Reserved. I dedicate this to my mom, Kristan Spain Acknowledgements The work presented in this dissertation thesis has been aided by the efforts of several people. First, I would like to thank each of my committee members, each of whom I have learned much from. Dr. Joe Suflita, Dr. Michael McInerny, Dr. Ralph Tanner, and Dr. Elizabeth Butler: not only have I learned much from each of you in classes and in department seminars, but also I have also gained a lot from your students who have used what they have learned from you to help me with my research efforts in various ways. The cooperation and fellowship among faculty and graduate students at the University of Oklahoma has been of extreme value to me, and is one of the things that drew me here for my graduate studies. And so again, I thank each of you for your positive attitudes, and your genuine willingness to help me as well as all graduate students here at OU. And of course, I would like to acknowledge and thank the support of my major advisor, Dr. Lee Krumholz. It has been a trying seven years, full of hard work and stress. Dr. Krumholz has always pushed me when I have needed it the most, and has never made it unclear what his expectations of me were. He has pushed me to strive for the best science I can possibly achieve, within reason, and because of his expectations, I don’t feel I could have learned how to be a better scientist at any other research institution. Dr. Krumholz has given me a lot of freedom in my academic and research pursuits, and while there has been some struggle associated with that freedom, the process has been a rewarding, gratifying, and humbling experience. He has also taught me that with science, some balance can exist along with personal happiness, as he has always stressed the importance of a balanced life. To sum it up, I iv may not have chosen to come to OU if not for Lee Krumholz; when I came for my interview visit, I knew that he was the right advisor for me. Sartre states that “to choose one’s adviser is another way to commit oneself,” and I could not be more happy or grateful for the choice that I have made and for what has been given to me. I would also like to thank my fellow graduate students with whom I have shared my graduate school experience. Namely, Dr. Kristen Savage, Dr. Kelli Palmer, and Dr. Matthew Traxler – all of you have been my partners in this graduate program, and I am so happy to have been able to form true friendships with each of you. I also would like to thank Dr. John Senko for his role in training me when I first started and for all of his helpful conversations, and Dr. Mostafa Elshahed for his role as a fill-in advisor at times and for helping me so much along the way in the lab and with writing. As well, I would like to thank John Frink, who, as a dedicated undergraduate in the lab assisted me with several tasks, the most time-consuming and appreciated of which was the set up and monitoring of pure and mixed culture groundwater experiments. And I would like to thank Neil Wofford for all of his assistance with experiment design and troubleshooting. And of course, all other past and members of the Krumholz lab and OU Micro graduate student community – working with you has been a priveledge. I would also like to thank the support of my undergraduate advisor, Dr. Elizabeth Wheeler Alm, from Central Michigan University. As with Dr. Krumholz, I feel like I have learned about the kind of scientist and human being I want to be from you; you have been a true example for me, and a good friend since I have left CMU. Lastly, I need to thank the following family and friends who have helped me along the way. My mother, Kristan Spain, and sister, Jessica Shore (and family!) for v their years of encouragement, emotional support, for understanding and reluctantly accepting my absence in times when I have not been able to be a part of family functions, and for visiting me at the times I have needed you the most. My brother, Mason Spain and my father, John Spain for their adoring attitudes and for always believing that I can be the best. And my cousin and long-time BFF, Lauren Rossi Harroun for her support and consistent presence throughout my life. My undergraduate friends, Margaret Fedder, Darlene Rosteck, and Matthew Kutch for their continuing presence in my life, and for giving me the feelings of family, love and support. And my friends and current and former roommates here in Norman, particularly Annissa and Avery Huckabee, Dusty Wheatley, Kristen Savage, Lauren Bray, Lauren Ehrsam, Katherine DeWitt, Joshua Cooper, and Genevieve Stotler, for providing me with consistency and balance during my time spent here. vi Table of Contents Acknowledgements…………………………………….…………………………..iv-vi List of Tables…………………………………………………………...…………...x-xi List of Figures………………………………………………………...…….........xii-xiii Preface……………………………………………………………………...…...xiv-xvii Abstract………………………………………………………………...………xviii-xix CHAPTER 1: Introduction and Literature Review: The Importance and Composition of Nitrate-Reducing Bacteria at the Oak Ridge Integrated Field Research Challenge Site Site Description and the Effects of Nitrate on Uranium Bioremediation……………1-3 Nitrate-Reducing Bacteria at the OR-IFRC………………....……….……………..4-11 References…………………………………………………………………………12-16 Tables and Figures…………………………………………………...……………17-22 CHAPTER 2: Preliminary Microcosm Studies: Uranium Reduction by Microbial Subsurface Communities Stimulated Under Iron-Reducing, Sulfate-Reducing, and Methanogenic Conditions Abstract………………...………………………………………………………….23-24 Introduction………………………………………………………………………..25-27 Materials and Methods…………………………...……………………………….28-32 Results and Discussion……………………………………………...…………….33-40 vii References…………………………………………………………………………41-46 Tables and Figures…………………………………………………...……………47-56 CHAPTER 3: Identification and Isolation of a Castellaniella Species Important During Biostimulation of an Acidic Nitrate- and Uranium-Contaminated Aquifer Abstract…...……………………………………………………………………….57-58 Introduction………………………………………………………………………..59-60 Materials and Methods…………………………...……………………………….61-67 Results……………………………………………………………………………..68-76 Discussion……………………………….………...………………………………77-83 References…………………………………………………………………………84-91 Tables and Figures…………………………………………………….....………92-101 CHAPTER 4: Interactions Among Denitrifying Bacteria Growing in Acidic High Nitrate Groundwater Abstract…………………………………………………………………………102-103 Introduction……………………………………………………………………..104-106 Materials and Methods…………………………………………...…………….107-119 Results…………………………………………………………………………..120-131 Discussion…………………………………………………………...……….…132-136 References………………………………………………………………………137-143 Tables and Figures……………………………………………………...………144-153 viii APPENDIX I: Composition, Abundance, Diversity, and Novelty of Soil Proteobacteria Abstract………………………………………………………………………....154-155 Introduction………………………………………………………………...…...156-157 Materials and Methods………………………………………………...…….…158-160 Results and Discussion……..………………………………………..…..……..161-168 References………………………………………………………………………169-171 Tables and Figures……………………………………………………...………172-179 APPENDIX II: Phylogeny and Environmental Distribution of the Phylum Fibrobacteres Phylogeny of the Phylum Fibrobacteres …………………….………...……….180-181 Environmental Distribution of the Phylum Fibrobacteres ……………..……...…...182 References…………………………………………………….………………….….183 Tables and Figures…………………...…………………………………………184-186 ix List of Tables Chapter 1 Table 1. Geochemical GW data from Area 1 at the OR-IFRC……….………17 Table 2. 16S rRNA libraries generated from the OR-IFRC…….…..…….18-20 Table 3. nirK and nirS clone libraries from the OR-IFRC………….………..21 Chapter 2 Table 1. Summary of treatment group amendments………………………….47 Table 2. PLFA composition of different treatment groups…………………..48 Table 3. Correlation coefficients between PLFA groups and U(IV)…………49 Chapter 3. Table 1. Groundwater and sediment chemistry data…………..………...…....92 Table 2. Descriptive diversity statistics of sediment cores……………….…..93 Table 3. Phylogenetic distribution of 16S rRNA clones……………...….…..94 Table 4. PLFAs from sediment cores……………………………….….…….95 Table 5. nirK and nirS OTUs from sediment cores………………………….96 Chapter 4 Table 1. Summary of cultivation approach………..…….….……………….144 Table 2. pH and temp ranges of OR-IFRC isolates…………………………145 Table 3. pH-dependent nitrite accumulation…………………………….….146 Table 4. Generation times of OR-IFRC isolates………………...………….147 Table 5. Summary of growth in OR-IFRC groundwater………………..….147 Table 6. pH-dependent nitrite MICs………………………..…….…………148 x Table 7. Michaelis Menten kinetic parameters, K m and V max ……………….148 Appendix I Table 1. Composition and abundance of Proteobacteria in KFS and other soils among published studies………………………………..…………….….….172