University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2019-04-30 Assessment of Anaerobic Bioremediation Potential in Hydrocarbon Contaminated Aquifers in Alberta, Canada Kharey, Gurpreet Singh Kharey, G. S. (2019). Assessment of Anaerobic Bioremediation Potential in Hydrocarbon Contaminated Aquifers in Alberta, Canada (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/110251 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 Assessment of Anaerobic Bioremediation Potential in Hydrocarbon Contaminated Aquifers in Alberta, Canada by Gurpreet Singh Kharey 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 APRIL, 2019 © Gurpreet Singh Kharey 2019 Abstract Anaerobic biodegradation of hydrocarbon fuels (mono-aromatic and short chain alkane hydrocarbon) was demonstrated under both field and microcosm experiments, with detection of signature metabolites, fumarate addition genes, and microbial community composition to determine the potential of hydrocarbon contaminated sites for bioremediation and assess the evidence of its past and/or present occurrence. It was determined here that the potential for bioremediation is unique to both the contaminated sites, but also to the hydrocarbon load present. Quantification of the fumarate addition genes using a newly designed mixture of primers was done. These qPCR assays for assA and bssA abundances concluded that an upper limit to gene abundance is present according to hydrocarbon concentrations, of approximately 5 ppm hydrocarbon. Monitoring hydrocarbon loss from groundwater collected at these sites, paired with fumarate addition gene abundances concluded that the contaminated sites in question have the potential for bioremediation, but is limited with higher hydrocarbon concentrations. Keywords: anaerobic hydrocarbon degradation, fumarate addition, qPCR, signature metabolite, hydrocarbon, contamination. ii Acknowledgements If I were left to achieve all that I have by myself, I’m afraid I would not have made it past Kindergarten. Heartbroken that playtime was over, and my toys would need to be put away, I refused. I continue to refuse to this day. To thank all those who have had a hand in keeping me on-track and allowing me to grow into the person I am today would be a thesis unto itself, because I am truly grateful for all those that helped me to be where I am, and where I will go from here. Firstly, I thank the Almighty for everything; all glory is yours, no one shall know my name. I thank my parents, for they had more influence over my successes than I did. For always supporting me, pushing me to be better, and giving me their experience, love, and counsel, even when they didn’t understand what I do. Absolutely none of this would have been even a dream if it wasn’t for my parents. Because of them, and for them, this dream became a reality. I thank my supervisor, Dr. Lisa Gieg, for believing and taking a chance on a skinny, awkward kid that had no idea what to do in life and telling me that I’m doing better than I think. Thank you for always supporting and teaching me that I can succeed. Also, thanks to my committee members, Dr. Raymond Turner and Dr. Casey Hubert for their support and expertise in making my work better. Thank you to Dr. Fredrick Biddle and Dr. David Hansen for giving me the opportunity to teach in my undergraduate, the true motivator for me to pursue a Graduate degree. Thank you to the United Farmers of Alberta (UFA) for providing samples and related information on the sites in this study, and to NSERC for funding the research. Huge thanks to my lab-mates/science-friends, past and present, for all the things they have taught me about science and life, and just being amazing people in general (and putting up with my insanity/aggressive humility). There’s just too many to acknowledge (in no order): to iii Gabrielle for doing the initial groundwater analyses and helping me get my project on its feet; to Corynne for teaching me all the molecular biology techniques; to Courtney and Carolina for teaching me how to be a good scientist; to Nicole and Danika for being my most trusted advisors in all things hydrocarbon and beyond; to Matija for your priceless advice in all things qPCR and beyond; to the lab Blackboard for being there to absorb all my frustrations and sparking my interest in art; to Steven for the countless hours spent in lab as my student; and to all the others for being such great friends and making my Masters the best years of my academic career. Huge thanks to my non-science friends: Sumare, Harman, Farwa, Kelsey, and all the others, for keeping me motivated to achieve, inspiring me, keeping me sane, and pushing me to be a better man every day. I thank you all for your years of support, trust, and inspiration; for celebrating my successes, and your unmatched counsel when I struggled. I cannot put into words the impact each one of you has had on my academic and personal lives. I would be half the man I am today without you all, and you all will make me a much better man than I can imagine in the future. I am eternally grateful. iv Dedication To my parents, Sardar Jagtar Singh and Sardarni Gurdeep Kaur without you, there is nothing. v Table of Contents Abstract .............................................................................................................................. ii Acknowledgements .......................................................................................................... iii Dedication ...........................................................................................................................v Table of Contents ............................................................................................................. vi List of Tables .................................................................................................................... ix List of Figures and Illustrations .................................................................................... xii List of Symbols, Abbreviations and Nomenclature .................................................... xvi Epigraphs ....................................................................................................................... xvii Chapter 1 : Literature Review ..........................................................................................1 1.1 Introduction ............................................................................................................1 1.2 Physical and Chemical Remediation Approaches ..............................................2 1.3 Bioremediation .......................................................................................................4 1.4 Anaerobic Hydrocarbon Biodegradation ............................................................6 1.5 Diagnostic Hydrocarbon Metabolites to Indicate In Situ Anaerobic Bioremediation .....................................................................................................11 1.6 Anaerobic HC Biodegradation Genes – Advantages and Limitations ............13 1.7 Use of Microcosms to Study Hydrocarbon Biodegradation ............................20 Chapter 2 : Research Objectives and Goals ..................................................................23 Chapter 3 : Analysis of Field Sites for Evidence of Anaerobic Biodegradation ........26 3.1 Introduction ..........................................................................................................26 3.2 Methods: ...............................................................................................................28 3.2.1 Site Description. .............................................................................................28 3.2.1.1 Site A. .................................................................................................28 3.2.1.2 Site B. .................................................................................................30 3.2.2 Sample collection. ...........................................................................................31 3.2.3 Hydrocarbon and Electron Acceptor Analyses ...........................................32 3.2.4 Biomass Collection. ........................................................................................33 3.2.5 DNA Extraction. .............................................................................................33 3.2.6 16S rRNA Gene Sequencing Sample Preparation and Analysis ...............33 3.2.7 assA and bssA PCR Presence/Absence Assay ..............................................35 3.3 Metabolite Extraction and Analysis ...................................................................36 3.4 Results and Discussion .........................................................................................37 3.4.1 Site A ...............................................................................................................37