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MICROBIOLOGY of HYDRAULIC FRACTURING WASTEWATER By MICROBIOLOGY OF HYDRAULIC FRACTURING WASTEWATER by Daniel Lipus Bachelor of Science in Environmental Science, Iona College, 2012 Master of Science in Civil Engineering, University of Pittsburgh, 2014 Submitted to the Graduate Faculty of Swanson School of Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2017 UNIVERSITY OF PITTSBURGH SWANSON SCHOOL OF ENGINEERING This dissertation was presented by Daniel Lipus It was defended on June 15, 2017 and approved by Kyle J. Bibby, Ph.D., Assistant Professor, Civil and Environmental Engineering Department Radisav D. Vidic, Ph.D., Professor, Civil and Environmental Engineering Department Leanne Gilbertston, Ph.D., Assistant Professor, Civil and Environmental Engineering Department Kelvin Gregory, Professor, Department of Civil and Environmental Engineering (Carnegie Mellon University) Dissertation Director: Kyle J. Bibby, Ph.D., Assistant Professor, Civil and Environmental Engineering Department ii Copyright © by Daniel Lipus 2017 iii MICROBIOLOGY OF HYDRAULIC FRACTURING WASTEWATER Daniel Lipus, Ph.D. University of Pittsburgh, 2017 The extraction of natural gas and oil from shale formations using hydraulic fracturing generates large volumes of wastewater, often termed produced water. One of the biggest challenges associated with produced water management is microbial activity. Microorganisms growing in produced water may have the ability to form biofilms and produce acids and sulfides, which can contribute to biocorrosion and gas souring. This dissertation investigates the microbial ecology of microorganisms living in produced water by studying their community structure and metabolic potential as well as the active, genetic response of Pseudomonas biofilms to the biocide sodium hypochlorite to inform microbial control. First, storage guidelines for hydraulic fracturing produced waters intended for microbiological analysis were developed. Results suggested microbial communities in produced water samples to remain stable when stored at 4oC for three days or less. Next, the microbial ecology of 42 Marcellus Shale produced water samples was analyzed. Samples were dominated by the taxa Halanaerobiales, specifically the genus Halanaerobium. Subsequently, metagenomic sequencing and binning allowed the recovery and annotation of a Halanaerobium draft genome. Annotation results suggested Halanaerobium to have the metabolic potential for acid production and sulfide production through thiosulfate reduction. Microbiological assessment of produced waters from 18 Bakken Shale wells, sampled across a six-month time frame, confirmed the presence of Halanaerobium in produced water. iv However, the microbial community structure was found to change temporally, and the majority of the samples were dominated by the order Bacillales. Finally, the active, genetic response to the broad-spectrum biocide sodium hypochlorite, which is also used for microbial control in hydraulic fracturing operations, was assessed. Pseudomonas fluorescens biofilms were exposed to sublethal concentrations of sodium hypochlorite and differential genes expression was analyzed. Results suggested genes involved in oxidative stress response pathways and multidrug efflux mechanisms to be upregulated, demonstrating genetic components to be involved in sodium hypochlorite resistance. Ultimately, findings from this dissertation enhance the current understanding of microbial community dynamics in produced water and may help to limit corrosion, control fouling and souring issues, protect well infrastructure, and minimize unnecessary biocide application. v TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... XII LIST OF FIGURES ................................................................................................................... XIII ACKNOWLEDGEMENTS ......................................................................................................... XV 1.0 INTRODUCTION ................................................................................................................ 1 1.1 MOTIVATION AND OBJECTIVE ...................................................................... 1 1.1.1 Advances in hydraulic fracturing and importance of produced water management ....................................................................................................... 1 1.1.2 Microbial activity in hydraulic fracturing systems and its role in produced water management ............................................................................................. 2 1.1.3 Economic impact of microbial activity on the oil and gas industry .................. 3 1.1.4 The importance of biocide efficacy during produced water management ........ 5 1.2 DISSERTATION ORGANIZATION ................................................................... 6 2.0 A REVIEW OF THE MICROBIOLOGY ASSOCIATED WITH HYDRAULIC FRACTURING PRODUCED WATER ........................................................................... 11 2.1 INTRODUCTION ............................................................................................... 11 2.2 MICROBIAL COMMUNITY STRUCTURE AND BIOLOGICAL ACTIVITY IN PRODUCED WATER FROM THE MARCELLUS SHALE REGION ............ ............................................................................................................................. 12 2.3 MICROORGANISMS IN PRODUCED WATERS FROM BARNETT SHALE WELLS ................................................................................................................. 20 vi 2.4 MICROBIAL ECOLOGY IN PRODUCED WATERS FROM THE ANTRIM SHALE GAS REGION......................................................................................... 23 2.5 MICROBIAL ECOLOGY IN PRODUCED WATERS FROM THE BAKKEN SHALE REGION.................................................................................................. 25 2.6 USE OF BIOCIDES TO CONTROL MICROBIAL ACTIVITY IN PRODUCED WATERS .............................................................................................................. 26 2.7 CONCLUSIONS ................................................................................................. 32 3.0 THE INFLUENCE OF SAMPLE STORAGE CONDITIONS ON MICROBIAL COMMUNITY COMPOSITION OF HYDRAULIC FRACTURING PRODUCED WATER ............................................................................................................................... 34 3.1 INTRODUCTION ............................................................................................... 35 3.2 MATERIALS AND METHODS ........................................................................ 36 3.2.1 Sampling .......................................................................................................... 36 3.2.2 Sample Processing ........................................................................................... 37 3.2.3 Chemical analysis ............................................................................................ 37 3.2.4 DNA Extraction ............................................................................................... 38 3.2.5 DNA processing and sequencing ..................................................................... 38 3.2.6 Computational analysis.................................................................................... 39 3.3 RESULTS AND DISCUSSION .......................................................................... 40 3.3.1 Sampling and geochemical characterization ................................................... 40 3.3.2 Changes in microbial community structure ..................................................... 40 3.3.3 Changes in microbial diversity within and between samples .......................... 42 3.3.4 Implications and produced water storage recommendations ........................... 46 3.4 SUMMARY AND CONCLUSIONS .................................................................. 48 4.0 PREDOMINANCE AND METABOLIC POTENTIAL OF HALANAEROBIUM IN PRODUCED WATER FROM HYDRAULICALLY FRACTURED MARCELLUS SHALE WELLS ................................................................................................................. 49 vii 4.1 INTRODUCTION ............................................................................................... 50 4.2 MATERIALS AND METHODS ........................................................................ 53 4.2.1 Samples ............................................................................................................ 53 4.2.2 Chemical analysis ............................................................................................ 53 4.2.3 DNA extraction, PCR, and sequencing ........................................................... 54 4.2.4 qPCR ................................................................................................................ 55 4.2.5 16S rRNA gene data processing ...................................................................... 55 4.2.6 Correlation analysis ......................................................................................... 56 4.2.7 Metagenome library preparation ..................................................................... 56 4.2.8 Quality control and assembly .......................................................................... 57 4.2.9 Reference genome mappings ..........................................................................
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