University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2021-01-08 Biodegradation of Aromatic Hydrocarbons by Methanogenic Consortia and Groundwater-Associated Microbial Communities Taylor, Nicole Taylor, N. (2021). Biodegradation of Aromatic Hydrocarbons by Methanogenic Consortia and Groundwater-Associated Microbial Communities (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/112987 master 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 Biodegradation of Aromatic Hydrocarbons by Methanogenic Consortia and Groundwater- Associated Microbial Communities by Nicole Taylor A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA JANUARY, 2021 © Nicole Taylor 2021 Abstract The biodegradation of hydrocarbons is an important environmental process responsible for in situ remediation of crude oil and gas components. Microorganisms of many lineages and redox conditions have been characterized to degrade numerous types of petroleum hydrocarbons, including those with aromatic structures. Alkyl-substituted mono- and polycyclic aromatic hydrocarbons are more chemically reactive than their unsubstituted counterparts, and as such their anaerobic degradation pathways have been studied to varying degrees. Aromatic hydrocarbons require enzymatic functionalization before biodegradation can occur; these activation enzymes and products are often unique to anaerobic reactions, therefore identifying the metabolites produced or the enzymes carrying out these reactions lends evidence to identifying in situ bioremediation of aromatic hydrocarbon contamination. Hydrocarbon biodegradation in the deep subsurface is often associated with methanogenesis. Anaerobic toluene degradation has been extensively studied and has been shown in multiple studies to involve an activation process known as fumarate addition, however methanogenic biodegradation of other alkylbenzenes and polycyclic aromatic hydrocarbons is comparatively poorly understood. In this work, the biodegradation of ethylbenzene and p-xylene was examined in the presence of toluene; p-toluic acid was found as a metabolite of p-xylene biotransformation, but no evidence of fumarate addition to either p-xylene or ethylbenzene were observed. A second methanogenic biodegradation study of naphthalene, 2-methylnaphthalene, and phenanthrene revealed 2-naphthoic acid as the primary metabolite produced by microbial cultures. A third study involved evaluating the use of a trapping device for passively sampling microorganisms from groundwater contaminated with aromatic hydrocarbons; this study showed that the chosen sorptive material did not influence the biodiversity of microbial communities, did ii not influence the rate of hydrocarbon biodegradation, and the presence of hydrocarbons was correlated to higher biomass recovery. iii Acknowledgements My time in this program has been some of the best years of my life. The support of my family, friends and colleagues have been instrumental to my success and I am incredibly grateful. In particular I would like to thank my sister, parents, and grandparents, who despite the physical distance have always been there for me when I needed them; and to all the members of the University of Calgary community who have made my time here a success. To my supervisor Dr. Lisa Gieg for her encouragement, guidance, advice, and faith in me; and to my supervisory committee Drs. Hubert and Dunfield for their valuable insight and feedback. To Gabrielle, Gurpreet, Danika, Julie, Ciara, Natalie, Yin, Rita, Gloria, Nuno, Mohita, and all past or present members of the Gieg lab family for being friends more than colleagues. Your constant advice, suggestions, problem solving, and sometimes just venting about frustrations made a molehill out of what would otherwise have been a mountain. Lunchtime laughs, coffee walks, and ice cream trips were indispensable mental health breaks. Special thanks to my mentor Dr. Courtney Toth for teaching me the essentials for working with hydrocarbons and TOLDC, to Gurpreet Kharey for teaching me the fundamentals of molecular biology, and to Dr. Victoria Collins for collaborating on experiments described in Chapter Six. Funding for this project was provided by NSERC Discovery, Western Canadian Innovation Offices, and the Genome Canada LSARP grants all awarded to Dr. Lisa Gieg. Isaac Newton once wrote, “if I have seen further it is by standing on the shoulders of giants.” By lifting me up, you are all giants to me and for that I will always be grateful. iv Dedication To Bradley: my partner, my love. I owe this all to you. v Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Dedication ............................................................................................................................v Table of Contents ............................................................................................................... vi List of Tables .......................................................................................................................x List of Figures and Images ................................................................................................ xii List of Symbols, Abbreviations and Nomenclature ....................................................... xviii Chapter One: Introduction ...................................................................................................1 1.1 Research objectives ..................................................................................................1 1.2 Thesis structure and summary .................................................................................2 Chapter Two: Literature Review .........................................................................................4 2.1 Hydrocarbons in the environment ............................................................................4 2.2 Origins of crude oil ..................................................................................................5 2.3 Chemical and physical properties of petroleum hydrocarbons ................................5 2.3.1 Features of aromatic hydrocarbons ....................................................................6 2.4 Hydrocarbon biodegradation ...................................................................................8 2.5 Aerobic biodegradation of aromatic hydrocarbons ................................................11 2.6 Anaerobic biodegradation of toluene .....................................................................14 2.6.1 Hydrocarbon addition to fumarate (fumarate addition) ...................................16 2.6.2 Benzylsuccinate synthase .................................................................................17 2.7 Anaerobic activation of other alkylbenzenes .........................................................19 2.7.1 Ethylbenzene ....................................................................................................20 2.7.2 Xylenes ............................................................................................................22 2.7.3 Alkylated PAHs ...............................................................................................23 2.8 Activation of unsubstituted aromatic hydrocarbons ..............................................24 2.9 Substrate range of hydrocarbon-biodegrading microorganisms ............................26 2.10 Remediation technologies ......................................................................................27 2.11 Research needs .......................................................................................................30 Chapter Three: General Materials and Methods ................................................................32 3.1 Microbial cultures and strains ................................................................................32 3.2 General cultivation of anaerobic microorganisms .................................................32 3.2.1 Media recipes and procedures ..........................................................................32 3.2.2 Addition of BTEX substrates ...........................................................................33 3.2.3 Addition of PAH substrates .............................................................................33 3.2.4 Adsorptive materials (Amberlite and Tenax) ..................................................33 3.3 Gas chromatography-flame ionization detection (GC-FID) ..................................34 3.3.1 Hydrocarbon analysis.......................................................................................34 3.3.2 Methane analysis ..............................................................................................35