Testing environmental controls on methane generation during microbial degradation of coal and oil from the Cherokee basin, Kansas by Michael A. Tummons B.S., Kansas State University, 2014 A THESIS submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Geology College of Arts and Sciences KANSAS STATE UNIVERSITY Manhattan, Kansas 2017 Approved by: Major Professor Dr. Matthew Kirk Abstract Biodegradation of crude oil to methane has long been known to exist in shallow petroleum reservoirs. It is only in the past decade, however, in which the concept of in-reservoir petroleum biodegradation has changed from a model emphasizing aerobic crude-oil degradation (with oxygen delivered down from meteoric waters) to a more recent model in which crude-oil degradation is driven by anaerobic processes (methanogenic microorganisms). In this study, we examine controls on microbial conversion of crude oil and coal into methane in middle-Pennsylvanian strata in the Cherokee Basin, Kansas, USA and how access to oil or coal influence microbial communities. Specifically, we considered the following hypotheses: 1) microorganisms in the basin are capable of generating methane by degrading crude oil or coal and 2) potential controls on the rate of methane formation include microbial diversity, formation water chemistry, nutrient abundance, and carbon dioxide abundance. To test these hypotheses, we used three sets of laboratory experiments constructed of materials from the Cherokee basin, Kansas. One set tested environmental controls on methane generation from oil, another from coal, and a third was a control experiment that utilized methanogenic substrates rather than oil or coal. In the experiments with oil and coal, environmental factors tested ammonium/phosphate availability, feedlot wastewater injection, and carbon dioxide abundance. Our experiments also tested the influence of salinity, by including materials from a well producing water with relatively low salinity and a well producing water with relatively high salinity. The cultures were allowed to incubate from approximately 75 to 170 days, during which headspace of oil and coal bioreactors were sampled periodically and analyzed for methane concentrations. Post incubation analyses included microbial DNA sequencing. We determined that a higher concertation of methanogens existed in the lower salinity well, which has higher potential for practical stimulatory injection. Of methane produced, the only significant (Mann Whitney) treatment had access to oil in lower salinity formation water. Access to coal resulted in no significant results. Microbial diversity, in the form of methanogenic archaea abundance, formation water chemistry (salinity), and wastewater nutrient often correlated with increased, yet insignificant, rates of methane production, while carbon dioxide abundance showed no benefit. Of methanogenic substrates consumed, we determined that most Cherokee basin methanogens preferred methanol over hydrogen and acetate. Table of Contents List of Figures ................................................................................................................................. v List of Tables ................................................................................................................................. vi Acknowledgements ....................................................................................................................... vii Chapter 1 - Introduction .................................................................................................................. 1 Environmental controls on methanogenesis ............................................................................... 3 The Cherokee basin .................................................................................................................... 6 Chapter 2 - Methods...................................................................................................................... 12 Field sampling ........................................................................................................................... 12 Laboratory experiments ............................................................................................................ 13 Experiment 1: Setup and treatment design (oil availability)................................................ 13 Experiment 2: Setup and treatment design (coal availability) .............................................. 15 Experiment 3: Setup and treatment design (substrate consumption) .................................... 16 Chemical analyses ..................................................................................................................... 18 Rate calculation ......................................................................................................................... 20 Extraction and sequencing of DNA .......................................................................................... 20 Microbial community analyses ................................................................................................. 23 Chapter 3 - Results ........................................................................................................................ 24 Formation water chemistry (Bailey 2-2 and Stich 1-2) ............................................................ 24 Rate variation from raw formation water ................................................................................. 24 Rate variation from coal and oil ............................................................................................... 25 Rate variation from waste ......................................................................................................... 26 Rate variation from non-significant majority ........................................................................... 26 Microbial Community Composition ......................................................................................... 29 Relative abundance of archaea from two natural gas wells .................................................. 29 Relative abundance of bacteria from two natural gas wells.................................................. 30 Relative abundance of archaea between treatments .............................................................. 32 iii Relative abundance of bacteria between treatments ............................................................. 34 Rate variation of substrate consumption reactors ..................................................................... 35 Relative archaeal abundance of substrate consumption reactors .......................................... 36 Relative bacterial abundance of substrate consumption reactors.......................................... 36 Chapter 4 - Discussion .................................................................................................................. 38 A comparison with previous Cherokee basin data ................................................................ 38 The dominant groups of microbes: Firmicutes, Proteobacteria, and Methanogens ............ 38 Methanogens as picky eaters................................................................................................. 39 Geo-environmental factors as a control on microbial habitability ........................................ 40 Impact of oil and coal availability on microbial community composition ........................... 41 Impact of waste availability on microbial community composition ..................................... 42 Visualization of beta diversity clustering via principle coordinate analysis (PCoA) ........... 44 Oppositions to the overarching methanogenic conceptual model ........................................ 47 Significance summarized ...................................................................................................... 47 Potential sources of error ...................................................................................................... 49 Conclusion and future work .................................................................................................. 50 Bibliography ................................................................................................................................. 52 Appendix 1 – Raw GC data (Experiment 1) ................................................................................. 55 Appendix 2 – Raw GC data (Experiment 2) ................................................................................. 59 Appendix 3 – Raw GC data (Experiment 3) ................................................................................. 63 Appendix 4 – Microbial Beta Diversity (phylum - family) .......................................................... 67 Appendix 5 – Cherokee basin well data ....................................................................................... 78 Appendix 6 – Cherokee basin water chemistry ............................................................................ 80 Appendix 7 – DNA concentrations (NanoDrop) .......................................................................... 82 Appendix 8 – Additional resources............................................................................................... 84 iv List of Figures Figure 1 – Well longitude, depth, and TDS .................................................................................... 8 Figure 2 – Biostimulation