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Methanogenic Biodegradation of Crude Oil and Polycyclic Aromatic Hydrocarbons

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Methanogenic Biodegradation of Crude Oil and Polycyclic Aromatic Hydrocarbons University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2015-01-29 Methanogenic biodegradation of crude oil and polycyclic aromatic hydrocarbons Berdugo-Clavijo, Carolina Berdugo-Clavijo, C. (2015). Methanogenic biodegradation of crude oil and polycyclic aromatic hydrocarbons (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26891 http://hdl.handle.net/11023/2043 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 Methanogenic biodegradation of crude oil and polycyclic aromatic hydrocarbons by Carolina Berdugo-Clavijo A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA JANUARY, 2015 © Carolina Berdugo-Clavijo 2015 Abstract The methanogenic biodegradation of crude oil is an important process occurring in many subsurface hydrocarbon-associated environments, but little is known about this metabolism in such environments. In this thesis work, the methanogenic biodegradation of crude oil and polycyclic aromatic hydrocarbons (PAH) was investigated. Methanogenic cultures able to metabolize light and heavy crude oil components were enriched from oilfield produced waters. Metabolites (e.g., alkylsuccinates) and genes (e.g. assA and bssA) associated with a fumarate addition mechanism were detected in the light oil-amended culture. A Smithella sp. dominated the community, suggesting this organism was involved in the degradation of the hydrocarbon components. In experiments conducted in sandstone-packed column systems simulating marginal oil fields, the light oil-amended culture was shown to bioconvert alkanes and aromatic hydrocarbons to CH4. Other oil-associated microbial inocula also enhanced CH4 production from oil in the column systems. Shifts in the microbial communities were observed after the inocula were incubated in the columns. Methanogenic hydrocarbon metabolism was also investigated using new enrichment cultures that biodegraded 2-ringed PAHs under methanogenic conditions. Metabolite and marker gene analyses were conducted on these cultures to investigate the mechanism(s) involved in PAH metabolism. The PAH-utilizing enrichments were dominated by methanogens closely affiliating with Methanosaeta and Methanoculleus, and bacterial members most closely related to the Clostridiaceae family. Further qPCR analysis with a 2- methylnaphthalene-amended culture suggested that Clostridium was the main hydrocarbon degrader in the enrichment. The results of these studies have added new knowledge to the field of methanogenic hydrocarbon biodegradation that may find application in bioremediation or microbial enhanced energy recovery. ii Acknowledgements These years of graduate school have been for me an incredible opportunity filled with challenges, but also full of fun and satisfactory experiences. I would like to express my sincere gratitude to my supervisor Dr. Lisa Gieg from whom I have learned a lot during these years of my PhD. I appreciate your unconditional help, and your patience for guiding me in the writing of this thesis. Your encouragement and positive attitude at all times helped me to be persistent and enjoy my work in the lab. I also want to thank the members of my committee. Dr. Gerrit Voordouw for his guidance and reinforcement to do conscientious research, and Dr. Peter Dunfield for his advice. I want to thank my external examiners Dr. Angus Chu and Dr. Joel Kostka for their feedback to improve this dissertation. I also would like to thank Dr. Xiaoli Dong and Dr. Jung Soh for their support in sequencing and phylogenetic analyses. I want to express my gratitude to Dr. Jane Fowler, Dr. Esther Ramos and Dr. Sandra Wilson for their help on genetics and molecular work. Also, my sincere thanks to Courtney Toth for her help setting up columns and enrichments, and Kathy Semple for her guidance on the oil fractionation analysis. My special thanks to Dr. Rhonda Clark who facilitated many things for me during this Ph.D. Thanks to the past and present members of the Gieg and Voordouw lab who were with me during this journey, especially my friends Yetty, Ginny, Jane, Esther and Shawna. I would like to thank all my family for being always supportive, especially my mother for her unconditional love and encouragement to seek success in what I do. Finally, I want to thank my fiancé Tim for joining me in this “Canadian adventure”, and for giving me the strength to finish this thesis. “It is good to have an end to journey toward; but it is the journey that matters, in the end” -Ernest Hemingway iii For my grandparents, an inspiration of hard work, endurance, and love iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii Table of Contents .................................................................................................................v List of Tables ..................................................................................................................... ix List of Figures and Illustrations ........................................................................................ xii List of Symbols, Abbreviations and Nomenclature ......................................................... xvi CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Rationale and significance of the project ...................................................................1 1.2 Research objectives ....................................................................................................2 1.3 Organization of thesis ................................................................................................3 CHAPTER TWO: LITERATURE REVIEW ......................................................................6 2.1 Microorganisms in hydrocarbon-laden environments ...............................................6 2.2 Anaerobic biodegradation of hydrocarbons ...............................................................7 2.2.1 Metabolic mechanisms involved in anaerobic crude oil biodegradation ..........9 2.2.1.1 Fumarate addition ....................................................................................9 2.2.1.2 Carboxylation .........................................................................................14 2.2.1.3 Methylation ............................................................................................16 2.2.1.4 Hydroxylation ........................................................................................17 2.3 Syntrophy and methanogenesis ...............................................................................18 2.3.1 Principles of syntrophic metabolism ...............................................................18 2.3.2 Syntrophy in methanogenic hydrocarbon degradation ....................................20 2.4 Tools for assessing anaerobic hydrocarbon biodegradation pathways ....................23 2.4.1 Hydrocarbon metabolomics .............................................................................23 2.4.2 PCR -based approaches ...................................................................................25 2.4.2.1 Functional gene-based analysis .............................................................25 2.4.2.2 Pyrotag sequencing ................................................................................27 2.4.2.3 qPCR ......................................................................................................28 2.5 Biotechnology applications of petroleum microbiology .........................................29 2.5.1 Crude oil bioremediation .................................................................................29 2.5.2 Improved energy production ...........................................................................31 2.6 Conclusions ..............................................................................................................33 CHAPTER THREE: METHANOGENIC BIODEGRADATION OF HYDROCARBON COMPOUNDS FROM LIGHT AND HEAVY CRUDE OIL .................................36 3.1 Introduction ..............................................................................................................36 3.2 Materials and Methods .............................................................................................38 3.2.1 Development of a crude oil-degrading enrichment culture .............................38 3.2.2 Methane measurements ...................................................................................38 3.2.3 Metabolite analysis ..........................................................................................39 3.2.4 DNA isolation from light oil-degrading culture ..............................................40 3.2.5 Identification of putative bssA and assA sequences ........................................40
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