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Methylotrophic Methanogenesis in Hydraulically Fractured Shales

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Methylotrophic Methanogenesis in Hydraulically Fractured Shales Methylotrophic Methanogenesis in Hydraulically Fractured Shales Master’s Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By: Daniel Nimmer Marcus, B.S. Graduate Program in Microbiology The Ohio State University 2016 Thesis Committee: Kelly Wrighton PhD, Advisor Joseph Krzycki PhD Charles Daniels PhD Matthew Sullivan PhD Copyright by Daniel Nimmer Marcus 2016 ABSTRACT Over the last decade shale gas obtained from hydraulic fracturing of deep shale formations has become a sizeable component of the US energy portfolio. There is a growing body of evidence indicating that methanogenic archaea are both present and active in hydraulically fractured shales. However, little is known about the genomic architecture of shale derived methanogens. Here we leveraged natural gas extraction activities in the Appalachian region to gain access to fluid samples from two geographically and geologically distinct shale formations. Samples were collected over a time series from both shales for a period of greater than eleven months. Using assembly based metagenomics, two methanogen genomes from the genus Methanohalophilus were recovered and estimated to be near complete (97.1 and 100%) by 104 archaeal single copy genes. Additionally, a Methanohalophilus isolate was obtained which yielded a genome estimated to be 100% complete by the same metric. Based on metabolic reconstruction, it is inferred that these organisms utilize C-1 methyl substrates for methanogenesis. The ability to utilize monomethylamine, dimethylamine and methanol was experimentally confirmed with the Methanohalophilus isolate. In situ concentrations of C-1 methyl substrates, osmoprotectants, and Cl- were measured in parallel with estimates of community membership. The appearance of the methanogenic genus Methanohalophilus followed different geochemical trends in the two shale environments. To adapt to the high osmolarities measured in the two shale systems, all recovered ii genomes encoded three distinct strategies for osmoprotection. This includes the synthesis of the compatible organic solute glycine betaine from glycine, which has been shown to be a capability in some methanogenic archaea. The overall scheme of carbon cycling for the three analyzed Methanohalophilus genomes is described. Collectively, this investigation constitutes the first in depth genomic and complementary physiological analysis of methanogens obtained from the economically important shale environment. iii Dedication I would like to dedicate this document to my family & close friends for their support, and for constantly reminding me of the person I strive to be. I would also like to recognize the positive influence 李琪 (Amelia) has had on my life; I look forward to what will come next… iv Acknowledgements I would like to acknowledge individuals who assisted with my work: • Dr. Wrighton for serving as my advisor • Dr. Krzycki, Dr. Daniels, and Dr. Sullivan for serving as members of my thesis committee • Rebecca Daly for training and handling logistics in the laboratory • David Hoyt from Pacific Northwest National Lab for help in performing and analyzing Nuclear Magnetic Resonance (NMR) measurements • Pearlly Yan from the OSU Comprehensive Cancer Center for assistance in sequencing my Methanohalophilus isolate I would also like to acknowledge my funding sources: • National Science Foundation DEB Grant #1342701 • Department of Energy BER EMSL/JGI Grant #48483 • The Marcellus Illumina sequencing was made possible by the Deep Carbon Observatory’s Census of Deep Life supported by the Alfred P. Sloan Foundation • The Ohio State Comprehensive Cancer Center core support grant #P30CA016058 provided sequencing of Methanohalophilus fracturphilus v Vita The Ohio State University – Columbus, OH 2014–present M.S. with Thesis - Department of Microbiology, GPA = 3.9 University of Michigan – Ann Arbor, MI 2006–2010 B.S. - Program in the Environment, School of Natural Resources, GPA = 3.5 Research Laboratory Specialist 2013–2014 Raskin Laboratory, Department of Environmental and Water Resources Engineering, University of Michigan Research Laboratory Specialist 2013–2014 Dick Geomicrobiology Laboratory, Department of Earth and Environmental Sciences, University of Michigan Lab Manager/Research Technician 2010–2014 Dick Geomicrobiology Laboratory, Department of Earth and Environmental Sciences, University of Michigan Network Administrator/Computer Support Staff 2006–2008 Department of Earth and Environmental Sciences, University of Michigan PUBLICATIONS • A.J. Pinto, D.N. Marcus, U.Z. Ijaz, Q.M.B. -de lose Santos, G.J. Dick, and L. Raskin. (2015). “Metagenomic Evidence for the Presence of Comammox Nitrospira-Like Bacteria in a Drinking Water System.” MSphere 1: e00054-15. • A.A. Voorhies, S.D. Eisenlord, D.N. Marcus, M.B. Duhaime, B.A. Biddanda, J.D. Cavalcoli, and G.J. Dick. (2016). “Ecological and genetic interactions between cyanobacteria and viruses in a low-oxygen mat community inferred through metagenomics and metatranscriptomics.” Environmental Microbiology 18(2): 358-371. • A.A. Voorhies, B.A. Biddanda, S.T. Kendall, S. Jain, D.N. Marcus, S.C. Nold, N.D. Sheldon, and G.J. Dick. (2012). "Cyanobacterial life at low O2: community genomics and function reveal metabolic versatility and extremely low diversity in a Great Lakes sinkhole mat." Geobiology 10: 250-267. Fields of Study Major Field: Microbiology vi Table of Contents Abstract…………………………..…………………………………….…..…….ii Dedication……………………….………………………………………...…….iv Acknowledgements……………..………………………………………….…….v Vita…………………………………..……………………………………….….vi Table of Contents……………….…………….…………………………..…….vii List of Tables…………………………………..…………………………...….viii List of Figures…………..……………………………………………………….ix Chapter 1: Introduction……….………………..………….……...…….……….1 Chapter 2: Genome Announcement……...……………………....….…………10 Chapter 3: Main Thesis Text………………………………….....….………….13 Introduction………………….……….…………………............13 Results and Discussion……….……….………………...............13 Geochemistry….…………………….…………...............13 16S rRNA Gene Analysis….………...…….…….............21 Methanohalophilus Isolate Recovered…….…….............24 Methanohalophilus Genomes Recovered…….….……....30 Genomic Analysis…….….…………….………………...31 Substrate Catabolism……………………….….32 Oxidative Branch Methanogenesis…….………36 Reductive Branch Methanogenesis……………36 Chemiosmotic Energy Conservation…………..37 Inputs Carbon Assimilation……………………38 Citric Acid Cycle………………………………39 Gluconeogenesis……………………………….42 Pentose Sugar Synthesis….……………………43 Acetyl-CoA Synthesis….………………………46 Mechanisms of Osmo-Tolerance………………48 Gene Synteny for Glycine Betaine Synthesis….49 Global Genome Comparison…………………..53 Conclusions….…………………………………………………55 Materials and Methods….……………………………………...56 Chapter 4: Future Directions…………………………..….…………………..64 References…………….………………………………………………………...67 Appendix A….…...……...……………………………………………………...75 vii List of Tables Table 1.1 Energetics of Methanogenesis………………………………..……….4 ------------------------------------------------------------------------------------------ Table 3.1 Media Revision 1 Recipe………….…………………………………25 Table 3.2 Media Revision 2 Recipe…………………………………………….26 Table 3.3 Primers used for PCR…………………………………………….…..27 Table 3.4 Comparison of 16S rRNA Gene Sequences...……………………......27 Table 3.5 Genome Statistics……………………...……………………….…….31 Table 3.6 Description of Chemical Reactions Carried out by Bifunctional Methyltransferases Involved in Glycine Betaine Synthesis.…………….……….49 Table 3.7 Alignment Statistics of Bifunctional Methyltransferases Involved in Glycine Betaine Synthesis..…………………………..………………………….49 Table 3.8 Average Nucleotide Identity (ANI) Comparison of Methanohalophilus genomes………………………………………….…………54 viii List of Figures Figure 1.1 The Distribution of and Estimated Reserves in U.S. Shales…………2 Figure 1.2 Prior Microbiological Investigations of U.S. Shales………....………5 Figure 1.3 16S rRNA Gene Tree of Methanogens and Optimum NaCl Concentrations for Growth...…………………………………………...….8 ------------------------------------------------------------------------------------------ Figure 3.1 Time Series Profile of Marcellus Production Fluids…….…….…….14 Figure 3.2 Time Series Profile of Utica Production Fluids……….….…...……..19 Figure 3.3 Utica Sample Read Mappings to Methanohalophilus Genomes...…..22 Figure 3.4 Plot of Halanaerobium Dominance in Utica Production Water...…..24 Figure 3.5 Brightfield Micrograph of Methanohalophilus fracturphilus……….28 Figure 3.6 Growth Curve of Methanohalophilus fracturphilus …………..........28 Figure 3.7 16S rRNA Gene Tree of Methanohalophilus with Substrate Range..29 Figure 3.8 Amino Acid Alignments of Methylamine Methyltransferases……..32 Figure 3.9 Genome Cartoon (Energy Metabolism) of Methanohalophilus Genome………………………………………………..…..34 Figure 3.10 Central Carbon Metabolism from Methanohalophilus Genomes....40 Figure 3.11 Citric Acid Cycle from Methanohalophilus Genomes………...….41 Figure 3.12 Gluconeogenesis from Methanohalophilus Genomes…………….43 Figure 3.13 Partial Pentose Phosphate Pathway from Methanohalophilus Genomes…………………………………………………...45 Figure 3.14 Proposed Alternate Entrance into the Shikimic Acid Pathway.…...45 ix Figure 3.15 Acetyl-CoA Biosynthetic Pathway from Methanohalophilus Genomes...……………………………………………...….47 Figure 3.16 Phylogenetic Tree of Glycine Sarcosine Methyltransferases.….….51 Figure 3.17 Proposed Supportive Metabolic Framework for Glycine Betaine Synthesis..………………………………………………….….53 x Chapter
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