UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE THE ROLE OF GAMMAPROTEOBACTERIA IN AEROBIC ALKANE DEGRADATION IN OILFIELD PRODUCTION WATER FROM THE BARNETT SHALE A THESIS SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE BY MEREDITH MICHELLE THORNTON Norman, Oklahoma 2017 THE ROLE OF GAMMAPROTEOBACTERIA IN AEROBIC ALKANE DEGRADATION IN OILFIELD PRODUCTION WATER FROM THE BARNETT SHALE A THESIS APPROVED FOR THE DEPARTMENT OF MICROBIOLOGY AND PLANT BIOLOGY BY ______________________________________ Dr. Joseph Suflita, Chair ______________________________________ Dr. Kathleen Duncan ______________________________________ Dr. Amy Callaghan © Copyright by MEREDITH MICHELLE THORNTON 2017 All Rights Reserved. I would like to dedicate this work to my family. To my loving parents, Tim and Donna, who have sacrificed so much for me and continue to provide for and support my wildest aspirations. To my sister, Mackenzie, who inspires me to set an example worthy of following. And to my future husband, Clifford Dillon DeGarmo, who motivates me to work harder, dream bigger, and pursue the best version of myself. Acknowledgements This work would not be possible without the guidance and unwavering support of my advisor, Dr. Kathleen Duncan. Throughout this process, she has been a role model for me in more than just the academic setting; she has inspired me through her kind nature, diligent work ethic, and optimistic outlook on life. She invested so much time and effort into shaping me into a scientist and I can never express how thankful and lucky I am to have been part of her legacy. I would also like to acknowledge Drs. Joseph Suflita and Amy Callaghan for their input in my thesis and time as a graduate student. Neither of them allowed me to settle for anything less than the best and I am grateful for the high standard to which they held me. Additionally, I would like to acknowledge Brian Harriman for his patience and help in teaching me many different laboratory methods. iv Table of Contents Acknowledgments.................................................................................................. iv Table of Contents.................................................................................................... v List of Tables.......................................................................................................... vii List of Figures......................................................................................................... ix Abstract................................................................................................................... xi Chapter 1: A review of selected topics in petroleum microbiology: biocorrosion, aerobic and anaerobic hydrocarbon degradation genetic systems, and aerobic hydrocarbonoclastic microorganisms Preface.................................................................................................................... 1 Abstract................................................................................................................... 5 Microbially influenced corrosion........................................................................... 6 Biocorrosion mitigation strategies.......................................................................... 12 Hydrocarbon degradation & petroleum biodegradation......................................... 13 Aerobic hydrocarbonoclastic microorganisms....................................................... 22 Chapter 2: The role of Gammaproteobacteria in aerobic alkane degradation in oilfield production water from the Barnett Shale Abstract................................................................................................................... 28 Introduction............................................................................................................ 29 Materials & Methods.............................................................................................. 36 Results.................................................................................................................... 52 Discussion............................................................................................................... 64 References.............................................................................................................. 80 Tables..................................................................................................................... 110 v Figures.................................................................................................................... 120 Appendix 1: Supplemental Tables and Figures for Chapter 2: The role of Gammaproteobacteria in aerobic alkane degradation in oilfield production water from the Barnett Shale Tables...................................................................................................................... 139 Figures.................................................................................................................... 144 Appendix 2: Isolation of the dominant cultivable aerobic heterotrophic bacteria from oil field production water produced from the Barnett Shale Abstract................................................................................................................... 153 Introduction............................................................................................................ 155 Materials & Methods.............................................................................................. 156 Results.................................................................................................................... 160 Discussion............................................................................................................... 162 Tables...................................................................................................................... 168 Figures.................................................................................................................... 171 vi List of Tables Chapter 2: The role of Gammaproteobacteria in aerobic alkane degradation in oilfield production water from the Barnett Shale Table 1 Hydrocarbon and heterotrophic substrates amendments used for production water enrichment.............................................................. 105 Table 2 Balanced stoichiometry for complete oxidation of various hydrocarbons...................................................................................... 106 Table 3 Environmental parameters of Barnett Shale production water................................................................................................... 107 Table 4 Organic extractions of Barnett Shale production water identifying putative polar metabolites, intermediates, and other compounds....... 108 Table 5 Summary of the dominant classes and corresponding genera of bacteria detected via 16S rRNA sequencing of original Barnett Shale production water....................................................................... 109 Table 6 Aerobic enrichment of Barnett Shale production water amended with hydrocarbon and heterotrophic substrates in Widdel’s medium 110 Table 7 Summary of 16S rRNA sequence libraries from the original Barnett Shale production water and after the third round of the initial and progressive enrichments amended with n-alkanes and n-fatty acid...... 111 Table 8 Evaluation of Halomonas A11A genome for genes involved in the aerobic degradation of alkanes and aromatics.................................... 112 Table 9 Range of hydrocarbons oxidized by Halomonas A11A..................... 113 Table 10 Comparison of oxygen consumed for n-pentane versus n-decane grown cells.......................................................................................... 114 Appendix 1: Supplemental Tables for Chapter 2: The role of Gammaproteobacteria in aerobic alkane degradation in oilfield production water from the Barnett Shale Table A1 Additional progressive aerobic enrichment amended with n- alkanes (C5-C10) and n-fatty acids (C5-C10) in Widdel’s medium.... 134 Table A2 Select metabolic pathways and genes of interest in the Halomonas A11A genome................................................................................... 136 Table A3 Molar calculations of hydrocarbon oxidation................................... 138 vii Appendix 2: Isolation of the dominant cultivable aerobic heterotrophic bacteria from oil field production water produced from the Barnett Shale Table A4 Chemical parameters of Barnett Shale production water collected in December 2014.......................................................................... 163 Table A5 Isolate identity based on partial 16S rRNA gene sequences.......... 164 Table A6 Hydrocarbon degradation screening by isolates obtained from Barnett Shale production water...................................................... 165 viii List of Figures Figure 1 Diagram of enrichment design..................................................... 115 Figure 2 16S sequence libraries representing the relative abundance (%) of different classes of bacteria in the initial and progressive rounds (“transfer”) of enrichment amended with n-alkanes (C5- C10) compared to the corresponding unamended transfer ........... 116 Figure 3 16S sequence libraries representing the relative abundance (%) of different classes of bacteria in the initial and progressive rounds (“transfer”) of enrichment amended with n-fatty