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1 Microbial Transformations of Organic Chemicals in Produced Fluid From Microbial transformations of organic chemicals in produced fluid from hydraulically fractured natural-gas wells Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Morgan V. Evans Graduate Program in Environmental Science The Ohio State University 2019 Dissertation Committee Professor Paula Mouser, Advisor Professor Gil Bohrer, Co-Advisor Professor Matthew Sullivan, Member Professor Ilham El-Monier, Member Professor Natalie Hull, Member 1 Copyrighted by Morgan Volker Evans 2019 2 Abstract Hydraulic fracturing and horizontal drilling technologies have greatly improved the production of oil and natural-gas from previously inaccessible non-permeable rock formations. Fluids comprised of water, chemicals, and proppant (e.g., sand) are injected at high pressures during hydraulic fracturing, and these fluids mix with formation porewaters and return to the surface with the hydrocarbon resource. Despite the addition of biocides during operations and the brine-level salinities of the formation porewaters, microorganisms have been identified in input, flowback (days to weeks after hydraulic fracturing occurs), and produced fluids (months to years after hydraulic fracturing occurs). Microorganisms in the hydraulically fractured system may have deleterious effects on well infrastructure and hydrocarbon recovery efficiency. The reduction of oxidized sulfur compounds (e.g., sulfate, thiosulfate) to sulfide has been associated with both well corrosion and souring of natural-gas, and proliferation of microorganisms during operations may lead to biomass clogging of the newly created fractures in the shale formation culminating in reduced hydrocarbon recovery. Consequently, it is important to elucidate microbial metabolisms in the hydraulically fractured ecosystem. The numerous nitrogen and carbon sources injected in input fluid mixtures may sustain shale-associated microorganisms, prompting a need to investigate the capacity of ii microbial life to enzymatically transform organic chemicals commonplace to hydraulic fracturing operations. In Chapter 2, we investigated the putative microbial metabolisms of two bacterial genera frequently identified in the first few weeks to months after hydraulic fracturing occurs (Marinobacter and Arcobacter). Using microbial culture-dependent methods (e.g., genomics, salinity range and carbon source growth testing) and microbial culture- independent methods (e.g., metagenomics) coupled to geochemical measurements from four Appalachian Basin natural-gas wells, we determined Marinobacter and Arcobacter likely play significant roles in biogeochemical cycling weeks to months after fracturing. There is evidence that Marinobacter can utilize a wide variety of nitrogen and carbon compounds including hydrocarbons, whereas Arcobacter can use a reductive TCA cycle coupled to sulfur oxidation. In Chapter 3, we tested the ability of the dominant shale-associated bacterial genera, Halanaerobium, to transform frequently used polyglycol surfactants. We used a variety of microbial and analytical chemical methods both in situ during production of a hydraulically fractured Utica-Point Pleasant natural-gas well, and in the laboratory during batch growth of Halanaerobium congolense WG10. Our results revealed that Halanaerobium can enzymatically transform alkyl polyethoxylates, polypropylene glycols, and monomeric glycols, under anaerobic conditions. iii In Chapter 4, we investigated microbial (de)halogenation pathways during hydraulic fracturing of natural-gas wells using a metagenomic approach. We identified genes encoding for halogenation, hydrolytic dehalogenation, and reductive dehalogenation, months after fracturing occurred. The presence of these pathways indicates the potential for microbially-generated organohalides in produced fluids as well as reduction of organohalides in wastewaters. In Chapter 5, we surveyed the microbial community at six stages of treatment in a class (II) injection well facility. The microbial community was highly similar to produced fluids from the Marcellus Shale, despite the transport of wastewaters in trucks, exposure to oxygen, and addition of chemicals in the treatment process. iv Dedication This dissertation is dedicated to my husband, Derek. v Acknowledgments First, I would like to express my gratitude to my advisor, Dr. Paula J. Mouser. She has taught me how to be a quality scientist and has been dedicated to my growth as a researcher and has gone to great lengths to ensure my success. My appreciation and respect for her are unquantifiable. I would like to thank my committee members, Dr. Gil Bohrer, Dr. Matthew Sullivan, Dr. Ilham El-Monier, and Dr. Natalie Hull, for their valuable time and service on my committee. Thank you to Dr. Kelly Wrighton, Dr. Mike Wilkins, Rebecca Daly, Mikayla Borton, and Dr. Anne Booker, for their assistance on this dissertation. I would like to express my appreciation to Dr. Andrea Hanson and Dr. Jenna Luek, whose advice, editing, and moral support helped me endure even the toughest times in my graduate career. Thanks to Jenny Panescu, our lab manager and fellow student, who helped me transition into graduate school and taught me proper microbiology procedures in the lab. Thanks to my fellow students, July Laszakovits, Billy Fagan, Sharon Scott Grove, Anton Rosi, Nick Nastasi, Dr. Michael Brooker, Kate Villars, Katie Heyob, Ryan Trexler. Their assistance in my research and thought- provoking scientific discussions helped on even the most difficult of days. I would like to express my appreciation for my family, friends, and husband, who were patient with me during this time, and helped keep me afloat these last 4 years. Funding for this research was provided by the Fay Graduate Fellowship (ESGP), the National Science Foundation vi (CBET award no. 1342701), and DOE National Energy Technology Laboratory through the Marcellus Shale Energy and Environmental Laboratory (project #DE-FE0024297). vii Vita 2015………………………………………... B.S. Chemistry, The Ohio State University Fay Fellow, Environmental Science 2015-2016………………………………….. Graduate Program, The Ohio State University 2016-2017………………………………….. Graduate Administrative Assistant, Ohio Water Resources Center, The Ohio State University 2017-2019………………………………….. Graduate Research Associate, Department of Civil, Environmental, and Geodetic Engineering, The Ohio State University viii Publications Evans, M. V., Panescu, J., Hanson, A. J., Welch, S. A., Sheets, J. M., Nastasi, N., et al. (2018). Members of Marinobacter and Arcobacter influence system biogeochemistry during early production of hydraulically fractured natural gas wells in the appalachian basin. Frontiers in Microbiology 9, 2646. doi:10.3389/fmicb.2018.02646. Evans M. V., Getzinger G., Luek J. L., Hanson A. J., McLaughlin M. C., Blotevogel J., et al. In situ transformation of ethoxylate and glycol surfactants by shale-colonizing microorganisms during hydraulic fracturing. (In Review at the ISME Journal). Evans M. V., Daly R. A., Luek J. L., Wrighton K. C., Mouser P. J.. Microbial (de)halogenation pathways in hydraulically fractured shale. (In Preparation) Evans M. V. and Mouser P. J. Microbial communities in a class (II) injection well disposal facility receiving produced fluids from the Marcellus Shale. (In Preparation) Rogers, J. D., Thurman, E. M., Ferrer, I., Rosenblum, J. S., Evans, M. V., Mouser, P. J., et al. (2018). Degradation of polyethylene glycols and polypropylene glycols in microcosms simulating a spill of produced water in shallow groundwater. Environ. Sci.: Processes Impacts. doi:10.1039/C8EM00291F. Heyob, K. M., Blotevogel, J., Brooker, M., Evans, M. V., Lenhart, J. J., Wright, J., et al. (2017). Natural Attenuation of Nonionic Surfactants Used in Hydraulic Fracturing Fluids: Degradation Rates, Pathways, and Mechanisms. Environ. Sci. Technol. 51, 13985–13994. doi:10.1021/acs.est.7b01539. ix Fields of Study Major Field: Environmental Science x Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii Table of Contents ............................................................................................................... xi List of Tables ................................................................................................................... xvi List of Figures ................................................................................................................. xvii Chapter 1. Introduction ....................................................................................................... 1 1.1 Problem Description ................................................................................................. 1 1.1.1 Microbial biogeochemical cycling in hydraulically fractured natural-gas wells ..................................................................................................................................... 4 1.1.2 Microbial xenobiotic pathways in hydraulically fractured shale ......................
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