Microbial Aspects of Shale Flowback Fluids and Response to Hydraulic Fracturing Fluids THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Maryam Ansari Cluff Graduate Program in Environmental Science The Ohio State University 2013 Master's Examination Committee: Paula J. Mouser, Advisor, Assistant Professor of Civil, Environmental and Geodetic Engineering, The Ohio State University John J. Lenhart, Associate Professor of Civil, Environmental and Geodetic Engineering, The Ohio State University Charles J. Daniels, Professor of Microbiology, The Ohio State University Copyrighted by Maryam Ansari Cluff 2013 Abstract Recent technological advancements in hydraulic fracturing and horizontal drilling as applied to shale formations have revived interest in Ohio’s oil and natural gas reserves. In many cases, short and long-term impacts to the environment from this exploration are not well understood as production in the field outstrips conducted research. The following two studies explore microbial community dynamics in shale well flowback fluids and their response to synthetic fracturing fluid exposure, respectively, and may yield insight into ecological impacts to the surface and subsurface as a result of shale gas development. Microbial diversity in the shale well fluids studied decreased significantly. The microbial ecology of these fluids shifted from one dominated by microbes present in source waters to one consistent with a brine system. In addition, significant enrichment of various hydrocarbon-degrading biomarkers was observed in an aquifer response to frack fluid exposure. Overall, significant dissolved organic carbon attenuation, largely attributed to biodegradation, was observed in both studies. Characterizing microbial community content and dynamics of fluids through hydraulic fracturing, flowback and production periods of shale gas stimulation may aid well operators in maximizing natural gas recovery and practitioners in making informed decisions on wastewater management strategies. In addition, examining how the biogeochemistry of a typical aquifer system responds to fracking fluid exposure can be used as a timely indicator of surface and groundwater pollution by these shale gas-associated fluids. ii Dedication I lovingly dedicate this thesis to my best friend and husband Taylor, whose limitless support never fails to build me up and confirm in me my ability to accomplish so much more than I ever thought possible. iii Acknowledgments I would like to express my greatest gratitude to Dr. Paula J. Mouser, my thesis advisor, for countless moments of help and support throughout my graduate education at The Ohio State University. Her scientific knowledge and aptitude in this field are praiseworthy and have played an invaluable part of my academic and professional success here at OSU. Thank you to Dr. John J. Lenhart and Dr. Charles J. Daniels for agreeing to serve on my committee and offering knowledge and advice along the way. Special thanks to Dr. Angela Hartsock, post-doctoral research associate at the Department of Energy’s National Energy Technology Laboratory, for facilitating access to samples and information crucial to shale well flowback fluid study. An enormous thank you to Dr. Jean D. MacRae, Associate Professor of Civil and Environmental Engineering at the University of Maine, for her invaluable contribution of raw data reduction for the majority of the data presented herein. I wish to thank my graduate colleague Shuai Liu (MS in Civil, Environmental and Geodetic Engineering) for his help in coordinating and carrying out the microcosm portion of this research and his subsequent contribution of geochemical data. A sincere thanks to Mike Brooker (MS in Environmental Science) for offering up his wealth of research knowledge and advice whenever I was in need of help. He is always willing to help out his labmates even in inconvenient times and for that I am very grateful. Thanks to Mike Zianni and Anthony McCoy, Senior Research Associate and Research Associate Laboratory Technician, respectively, at the Plant-Microbe Genomics iv Facility at The Ohio State University for their technical expertise in sequencing technologies. Lastly, I wish to thank the entities which are responsible for the funding of this research, namely the Ohio Water Development Authority, Environmental Science Graduate Program at The Ohio State University through a GAA appointment, and an Ohio State University Fellowship. v Vita 2011 ............................................................... B.S. Biology, The University of Maine 2013 ............................................................... M.S. Environmental Science, The Ohio State University Fields of Study Major Field: Environmental Science vi Table of Contents Abstract ............................................................................................................................... ii Acknowledgments .............................................................................................................. iv Vita ..................................................................................................................................... vi Fields of Study ................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi Chapter 1: Introduction ...................................................................................................... 1 Brief History of Oil and Natural Gas Development in Ohio ........................................... 1 Early Exploration Techniques ......................................................................................... 2 Recent Development in Ohio’s Marcellus and Utica Shale ............................................ 3 The Formation of Hydrocarbons in the Marcellus and Utica Shale ................................ 8 Physical Characteristics of Shale .................................................................................. 11 Origin of Methane in Marcellus and Utica Shale .......................................................... 12 Horizontal Hydraulic Fracturing Technique ................................................................. 13 Hydraulic Fracturing Fluids .......................................................................................... 16 Flowback and Produced Water ..................................................................................... 20 vii Management of Flowback Fluids .................................................................................. 22 Microbial Sources ......................................................................................................... 24 Microorganisms Associated with Shale ........................................................................ 25 Microorganisms Associated with Oil and Gas Operations ........................................... 26 Conclusion ..................................................................................................................... 28 Chapter 2: Microbial Community Shifts in Shale Well Flowback Fluids ........................ 30 Introduction ................................................................................................................... 30 Methods ......................................................................................................................... 31 Results ........................................................................................................................... 32 Discussion ..................................................................................................................... 43 Conclusions ................................................................................................................... 51 Introduction ................................................................................................................... 52 Methods ......................................................................................................................... 53 Results ........................................................................................................................... 54 Discussion ..................................................................................................................... 75 Conclusions ................................................................................................................... 80 Chapter 4: Research Implications ..................................................................................... 82 Bibliography ..................................................................................................................... 84 Appendix A: Microbial Community Shifts in Shale Well Flowback Fluids Methods ... 102 viii Sample Collection and Preparation ............................................................................. 102 DNA Extractions, PCR Amplification, Cloning and Sequencing ............................... 104 16s rRNA Gene Sequence Analysis ...........................................................................