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Diversity of Methane and Short Chain Hydrocarbon Degrading Bacteria with an Emphasis on Methane Biofilter Systems

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University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2018-10-03 Diversity of methane and short chain hydrocarbon degrading bacteria with an emphasis on methane biofilter systems Khadka, Roshan Khadka, R. (2018). Diversity of methane and short chain hydrocarbon degrading bacteria with an emphasis on methane biofilter systems (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/33152 http://hdl.handle.net/1880/108810 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Diversity of methane and short chain hydrocarbon degrading bacteria with an emphasis on methane biofilter systems by Roshan Khadka A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA SEPTEMBER, 2018 © Roshan Khadka 2018 Abstract Methanotrophs house enzymes capable of methane oxidation, act as a sink for atmospheric methane and play a key role in the global carbon cycle. This study conducted multiple studies on methanotrophs, including: examination of the evolutionary history of copper membrane monooxygenases (CuMMOs), application of methanotrophic communities in protocol design for monitoring methane biofilter systems, and the analyses of single cell genomes containing new CuMMO-encoding genes. CuMMOs are encoded by three genes, usually in an operon of xmoCAB, and oxidize ammonia, methane, and short chain alkanes and alkenes. To examine the evolutionary history of CuMMOs, phylogenetic inferences and compositional genome analyses were applied to a set of 66 genomes. Individual phylogeny of all genes xmoA, xmoB, and xmoC closely matched in almost all genomes, indicating this operon evolved as a unit. However in Verrucomicrobia pmoB has a distinct phylogeny from pmoA and pmoC. The gammaproteobacteria AMO (Nitrosococcus spp.), the gammaproteobacterial Pxm, the thaumarcheotal AMO and the NC10 pMMO showed little or no compositional bias in the xmo operon indicating similar compositional biases to its genome. Based on the analysis, possible lateral gene transfer events of xmoCAB genes were predicted. The nitrifying bacterium Nitrosococcus postulated as the donor of pmoCAB to both the alpha- and gammaproteobacterial methanotrophs. To design a monitoring protocol that would allow a simple, cost effective and accurate estimation of whether a methane biofilter is operating efficiently, microcosms using compost as a biofilter material were tested via growth and starvation experiments for long periods. Analysis of 16S rRNA gene sequences suggested that non-methanotrophic methylotrophic bacteria belonging the family Methylophilaceae showed a rapid response to biofilter methane oxidation ii activity and may be a good monitoring target. A monitoring system based on these “methanotroph-associated methylotrophs” is proposed and a ratio of Methylophilaceae to Methylococcaceae of 0.35 was related to high methane activity and 0.1 to low activity. Novel copper membrane monooxygenase encoding operons (xmoCAB) were detected while screening metagenomes obtained from oil sands environments. Quantitative PCR assays were developed for detection of xmoCAB genes in methane, ethane and propane enrichment cultures from environmental samples. Single cell genomes were sequenced from the xmoCAB positive sorted cells of a propane enrichment culture. Screening the genomes identified Polaromonas and Rhodoferax as containing multiple xmoCAB operons. Potential propane oxidation pathways were predicted based on enzymes present in single cell genomes of these two genera. iii Preface This research results section of this dissertation consists of one research paper under review and two additional thesis chapters. Chapter 3 is in review (Frontiers in Microbiology). Chapter 3: Roshan Khadka, Lindsay Clothier, Lin Wang, Chee Kent Lim, Martin G. Klotz and Peter F. Dunfield (2018). Evolutionary history of copper membrane monooxygenases. Frontiers in Microbiology. (accepted with minor revisions) Supplementary information from the above paper is included in Appendix A. An additional co-author research paper that is equally contributed as first author is included as appendix B and is based in part on the results presented in Chapter 5. Appendix B: Rochman FF, Khadka R, Tamas I, Lopez-Jauregui AA, Malmstrom RR, Dunfield PF, and Verbeke TJ (2018). New copper containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing betaproteobacteria in oilsands tailings. The results presented in Chapter 4 are not yet submitted for publication. Additional co-author research papers that I contributed to during my thesis research time that are not included in this dissertation are: Sharp, C.E., Smirnova, A.V., Graham, J.M., Stott, M.B., Khadka, R., Moore, T.R., Grasby, S.E., Strack, M., and Dunfield, P.F. (2014). Distribution and diversity of Verrucomicrobia methanotrophs in geothermal and acidic environments. Environ. Microbiol. doi:10.1111/1462- 2920.12454 Alireza Saidi-Mehrabad; K Dimitri Kits; Joong-Jae Kim; Ivica Tamas; Peter Schumann; Roshan Khadka; W Irene C Rijpstra; Jaap Sinninghe Damsté; Peter F Dunfield (2017). Methylomicrobium oleiharenae sp. nov., an aerobic methanotroph isolated from an oil sands tailings pond. International Journal of Systematic and Evolutionary Microbiology (submitted) In Chapter 3, Lidsay Clothier provided guidance and involved to perform compositional analysis methods to detect possible lateral gene trasfer. In Chapter 5, Fauziah Rochman provided 13 13 13 13 C-alkane ( CH4, C2H6, and C3H8) SIP fraction of MLSB and BML oilsands samples. Single cell sorting, single cell genome sequencing and their assembly and annotation were performed by iv DOE Joint Genome Institute-Integrated Microbial Genomes and Microbiomes (JGI-IMG). Screening of single sorted cell samples to confirm copper monooxygenase-encoding genes was done by former summer student Abraham Lopez-Jauregui. v Acknowledgements First and foremost, I would like to express my sincere gratitude to my supervisor Dr. Peter Dunfield. I have had so much fun and gained tons of experience during my time at University of Calgary due to your support and guidance. You are an amazing mentor. I would like to express my deepest thanks to my committee members Dr. Gerrit Voordouw and Dr. Casey Hubert for their feedback and guidance during my thesis. Special thanks to the Department of Biological Sciences, University of Calgary, Mitacs, and Natural Sciences and Engineering Research Council of Canada (NSERC) for providing financial support. Thanks to all past and present Dunfield Lab members. To Dr. Angela Smyrnova, thank you for giving your valuable time and providing guidance especially to verrucomicrobia project. Thanks to JoongJae for your valuable thoughts in Biofilter project, Fauziah for sharing your SIP fractions, especial Evan Haupt for those amazing days, you are a great friend and roommate, Emad, who was always excited to learn about methanotrophs and the stories you share, Ilona, Gareth, Andrey, Gul for always being supportive. My special thanks to my father Rudra Dhoj Khadka and mother Binda Khadka for your love, support and encouragement. This work would not be possible without my wife Susanna Kc who was with me all the time and provide me strength, confidence, support, encouragement, and guidance. My sincere thanks and appreciation to all my family members for your encouragement. vi To my family…. vii Table of Contents Abstract .............................................................................................................................. ii Preface ............................................................................................................................... iv Table of Contents ........................................................................................................... viii List of Tables .................................................................................................................... xi List of Figures and Illustrations ................................................................................... xiii List of Symbols, Abbreviations and Nomenclature .................................................... xix CHAPTER ONE: INTRODUCTION ..............................................................................1 1.1 Research objectives ..................................................................................................1 1.2 Dissertation structure ..............................................................................................2 CHAPTER TWO: LITERATURE REVIEW .................................................................4 2.1 Methane as a greenhouse gas ..................................................................................4 2.2 Global methane cycle ...............................................................................................5 2.3 Aerobic methanotrophic bacteria ...........................................................................7 2.3.1 Methanotrophic proteobacteria .....................................................................7
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