Microbial and Geochemical Characterization of Wellington Oil Field, Southcentral Kansas, and Potential Applications to Microbial Enhanced Oil Recovery By Breanna L. Huff Submitted to the graduate degree program in Geology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Master of Arts. ________________________________ Jennifer A. Roberts, Chair ________________________________ David A. Fowle, Co-Chair ________________________________ Gwendolyn L. Macpherson ________________________________ Leigh A. Stearns Date Defended: May 12, 2014 The Thesis Committee for Breanna L. Huff certifies that this is the approved version of the following thesis: Microbial and Geochemical Characterization of Wellington Oil Field, Southcentral Kansas, and Potential Applications to Microbial Enhanced Oil Recovery ________________________________ Jennifer A. Roberts, Chairperson Date approved: May 12, 2014 ii Abstract The aqueous geochemistry and microbiology of subsurface environments are intimately linked and in oil reservoir fluids. This interdependence may result in a number of processes including biodegradation of oil, corrosion of pipes, bioclogging of porous media, and biosurfactant production. During production of oil and reinjection of production water, surface exposed fluids are introduced to oxygen and exogenous microbes, both of which may alter reservoir biogeochemistry. In this study, production waters from six wells within the Wellington Field in SE Kansas, which has been water flooded continuously for 60 years, were sampled and analyzed for geochemistry, microbial ecology, microbial biomass, and biosurfactant production to better understand the relationship between the microbiology and oil production in the field. Minor differences in aqueous geochemistry were detected among the five production wells and single injection well, and data analysis and modeling indicate that depth-specific water-rock reactions play a major role in controlling the major ion geochemistry in the field. Microbial diversity in fluids produced from the wells indicated a system that is in steady state, with microbial community composition linked to the stratigraphic location of pumping rather than injection of recycled fluids. Further, analyses of surface tension, a proxy for biosurfactant production, and its relationship to microbial biomass and oil production, suggests that while biosurfactants may be produced, biomass is likely clogging porosity and inhibiting oil recovery. Biociding practices, the injection of chemicals toxic to microorganisms, are implemented in the Wellington field to keep microbial biomass low. This study suggests their effectiveness iii may need to be addressed further. Known biosurfactant-producing microbes isolated in this study may be targeted for in-situ stimulation to increase biosurfactant production through the introduction of nutrient and energy sources into the reservoir that can increase oil production. iv Acknowledgements I would like to thank my advisors Jennifer Roberts and Dave Fowle for their direction, helpful conversations, and their excitement for this project. I would also like to acknowledge Lynn Watney for supporting my research and always being prompt to answer any question I had. I need to express gratitude to Belinda Sturm for her tremendous input on the DNA side of this project and her willingness to be available to train and put up with me as I learned completely new methods. I need to thank Christa Jackson, whom when I was away, struggled with the trials and tribulations of DNA work in all its frustration. Christa dedicated an entire summer to advancing my project and I am forever in her debt. I would like to acknowledge Masato Ueshima for being my field partner and analyzing samples I collected over two years. I want to show my gratitude to Karla Leslie for analyzing my samples, helping me with lab procedures and instruments and simply being always excited and available to talk science when I felt I had gone of course. I would like to thank Karen Peltier and Stephen Johnson from the Tertiary Oil Recovery Program. I want to acknowledge funding from the Kansas Geological Survey, the University of Kansas Department of Geology, the Geological Society of America, the Association of Women Geoscientists and the American Association of Petroleum Geology. Lastly, I need to thank Jessica Clatterbuck for all her love and unyielding support over the years and all of my friends and family who have always stood by me. v Table of Contents ABSTRACT ................................................................................................................................. III ACKNOWLEDGEMENTS .......................................................................................................... V LIST OF TABLES .................................................................................................................... VIII LIST OF FIGURES ..................................................................................................................... IX CHAPTER 1: INTRODUCTION ................................................................................................. 1 MICROBIAL ECOLOGY OF OIL FIELD BRINES ............................................................................... 1 REDOX CONDITIONS AND NUTRIENT AVAILABILITY OF OIL FIELD BRINES ............................... 3 INFLUENCE OF INJECTION WATERS ON THE MICROBIOLOGY OF PRODUCTION WELLS .............. 4 BIOCLOGGING AND BIOSURFACTANT PRODUCTION BY MICROORGANISMS ............................... 6 OVERVIEW OF THESIS CHAPTERS ................................................................................................ 9 CHAPTER 2: FIELD SITE DESCRIPTION ............................................................................ 11 SITE DESCRIPTION: WELLINGTON OIL FIELD ............................................................................ 11 CHEMICAL TREATMENT OF WELLINGTON OIL FIELD ................................................................ 12 DESCRIPTION OF SAMPLED WELLS ............................................................................................ 13 STRUCTURAL BACKGROUND OF KANSAS; PRE TO POST-MISSISSIPPIAN .................................. 15 DEPOSITIONAL SETTING OF MISSISSIPPIAN-AGED ROCKS IN KANSAS ...................................... 16 MINERALOGY OF THE MISSISSIPPIAN PAY ZONE ....................................................................... 18 CHAPTER 3: MATERIALS AND METHODS ........................................................................ 20 RESEARCH APPROACH ............................................................................................................... 20 SAMPLE COLLECTION AND GEOCHEMICAL ANALYSIS .............................................................. 21 PHREEQC: AQUEOUS GEOCHEMICAL MODELING ................................................................... 22 MOST PROBABLE NUMBER ANALYSIS (MPN) ........................................................................... 22 LIPID PHOSPHATE BIOMASS ANALYSIS ..................................................................................... 23 MICROBIAL BIOSURFACTANT EXPERIMENTS-SURFACE TENSION MEASUREMENTS ................. 24 DNA EXTRACTION ..................................................................................................................... 24 POLYMERASE CHAIN REACTION (PCR) AND GEL ELECTROPHORESIS ...................................... 26 DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) ANALYSIS .................................... 27 DGGE QUANTITATIVE AND QUALITATIVE ANALYSIS .............................................................. 28 DNA CLONING METHODS .......................................................................................................... 28 DNA SEQUENCING AND PHYLOGENETIC TREE CONSTRUCTION ............................................... 29 CHAPTER 4: RESULTS ............................................................................................................. 30 AQUEOUS GEOCHEMISTRY OF PRODUCTION FLUIDS, INJECTION WELL AND DRILL STEM TEST .................................................................................................................................................... 30 Major element geochemistry .................................................................................................. 30 Saturation indices of oilfield minerals in aqueous solution .................................................. 32 Biogeochemistry of production fluids .................................................................................... 33 MICROBIOLOGY OF WELLINGTON OIL RESERVOIR ................................................................... 34 Most Probable Number Analysis-Cultured Reservoir Microbes ........................................... 34 Microbial Biomass and Oil Production ................................................................................. 35 Surface Tension Measurements on Raw Production Fluid ................................................... 37 COMMUNITY DIVERSITY OF OILFIELD MICROORGANISMS ........................................................ 39 MICROBIAL COMMUNITY DIVERSITY AS A FUNCTION OF DISTANCE FROM THE INJECTION WELL .......................................................................................................................................... 42 MICROBIAL COMMUNITY DIVERSITY AND SPECIES RICHNESS ................................................