EVALUATION OF FACTORS THAT INFLUENCE MICROBIAL COMMUNITIES AND METHANE PRODUCTION IN COAL MICROCOSMS by Lisa K. Gallagher A thesis submitted to the Faculty and Board of Trustees of the Colorado School of Mines in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Environmental Science and Engineering) Golden, Colorado Date _____________ Signed: ___________________________________ Lisa K. Gallagher Signed: ___________________________________ Dr. Junko Munakata Marr Thesis Advisor Golden, Colorado Date _____________ Signed: ___________________________________ Dr. John E. McCray Department Head Civil and Environmental Engineering ii ABSTRACT Vast reserves of coal represent a largely untapped resource that can be used to produce methane gas, a cleaner energy alternative compared to burning oil or coal. The methane produced in subsurface coal seams, referred to as coalbed methane, represents an increasingly important source of domestic energy in the United States comprising approximately 10% of natural gas production. Biogenic coalbed methane is formed via the activity of microorganisms and contributes about 40% to the total amount of CBM produced in the world. Enhancement of biogenic coalbed methane has become an area of active research due to its potential impact on energy reserves as well as the positive environmental implications associated with its use. Enrichment cultures from coal were incubated and evaluated by DNA sequencing, qPCR analysis and gas chromatography to determine whether the presence of specific organisms was correlated to methane production and whether microbial community structure differed between productive and unproductive coal microcosms. Additionally, microcosm experiments were designed to assess how prior exposure of coal to oxygen might influence methane production and microbial community structure and dynamics. Microcosms with oxidized coal consistently produced between 50 and 100 micromoles less methane per gram of coal than the un-oxidized coal microcosms. Additionally, un-inoculated microcosms produced levels of methane comparable to their inoculated counterparts, demonstrating the importance of native, coal-associated microbial assemblages in biogenic methane production. Pelobacter was strongly correlated to methane production, suggesting its relevance for methane production. Productive microcosms in this study also had methanogenic populations that were dominated by Methanosarcina, a group of metabolically versatile methanogens. Copy number thresholds for methanogens (mcrA gene; 107 copies/mL), sulfate reducing bacteria (dsrA; 106 copies/mL) and acetogens (fhs; 105 copies/mL) were identified, iii below which methane production was limited. The results of this research add to the existing body of knowledge and provide findings that may help with increasing natural gas yields by stimulating existing seam activity or by reviving depleted wells. Further work is needed to better understand the intricacies of this process and ways to ensure coalbed methane remains a feasible energy resource. iv TABLE OF CONTENTS ABSTRACT ...................................................................................................................... iii TABLE OF CONTENTS ................................................................................................... v LIST OF FIGURES .......................................................................................................... ix LIST OF TABLES ............................................................................................................ xii ACKNOWLEDGEMENTS .............................................................................................. xiii CHAPTER ONE: INTRODUCTION ................................................................................. 1 1.1: Background ................................................................................................... 1 1.1.1: Microbial Capabilities ........................................................................ 2 1.1.2: Microbial Ecology of Coal Systems ................................................. 4 1.1.3: Characterization of Coalbed Microbial Communities ....................... 6 1.1.4: Basins of Interest ............................................................................. 8 1.2: Scope of Research ...................................................................................... 10 1.3: Research Objectives ................................................................................... 10 1.3.1: Task 1: Methanogenic Potential of Coal-Associated Microbial Populations ............................................................................................... 11 1.3.2: Task 2: Characterization of Microbial Communities Associated with Productive and Unproductive Coals ......................................................... 12 1.3.3: Task 3: Microorganisms as Indicators of Methane Production ..... 12 1.3.4: Task 4: Impact of Coal Oxidation ................................................. 13 1.4: Organization of Dissertation ........................................................................ 14 CHAPTER TWO: CHARACTERIZATION OF MICROBIAL COMMUNITIES OF PRODUCTIVE AND UNPRODUCTIVE COALS ............................................................ 17 2.1: Abstract ....................................................................................................... 17 2.2: Introduction .................................................................................................. 18 2.3: Materials and Methods ................................................................................ 19 2.3.1: Gas Chromatography .................................................................... 22 2.3.2: DNA Extraction .............................................................................. 22 v 2.3.3: DNA Sequencing and Analysis ...................................................... 23 2.3.4: Quantitative PCR Analysis ............................................................ 25 2.3.5: Statistical Analysis ......................................................................... 29 2.4: Results......................................................................................................... 29 2.4.1: Methane Production ...................................................................... 30 2.4.2: Phylogeny ...................................................................................... 31 2.4.4: Diversity ......................................................................................... 39 2.5: Discussion ................................................................................................... 43 2.5.1: Methane Production ...................................................................... 43 2.5.2: Phylogeny ...................................................................................... 45 2.5.3: Organism Quantification ................................................................. 50 2.5.4: Diversity ......................................................................................... 54 2.6: Summary and Conclusions ......................................................................... 55 CHAPTER THREE: THE EFFECT OF COAL OXIDATION ON METHANE PRODUCTION AND MICROBIAL COMMUNITY STRUCTURE IN POWDER RIVER BASIN COAL .................................................................................................................. 57 3.1: Abstract ....................................................................................................... 57 3.2: Introduction .................................................................................................. 58 3.3: Materials and Methods ................................................................................ 59 3.3.1: Coal Microcosm Preparation ......................................................... 60 3.3.2: Gas Chromatography .................................................................... 61 3.3.3: DNA Extraction and Sequencing ................................................... 62 3.4: Results......................................................................................................... 63 3.4.1: Effect of Coal Oxidation on Methane Production ........................... 63 3.4.2: Microbial Community Structure ..................................................... 64 3.5: Discussion ................................................................................................... 69 3.5.1: Methane Production ...................................................................... 70 3.5.2: Microbial Community Structure ..................................................... 73 3.6: Summary and Conclusions ......................................................................... 75 vi CHAPTER FOUR: TEMPORAL DYNAMICS OF THREE FUNCTIONAL MICROBIAL GROUPS ASSOCIATED WITH METHANE PRODUCTION FROM POWDER RIVER BASIN COAL INCUBATIONS ........................................................................................ 76 4.1: Abstract ....................................................................................................... 76 4.2: Introduction .................................................................................................. 77 4.3: Materials and Methods ................................................................................ 79 4.3.1: Gas Chromatography and High Performance Liquid Chromatography ......................................................................................