University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2016 Aerobic Hydrocarbon-degrading Microbial Communities in Oilsands Tailings Ponds Rochman, Fauziah Rochman, F. (2016). Aerobic Hydrocarbon-degrading Microbial Communities in Oilsands Tailings Ponds (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/24733 http://hdl.handle.net/11023/3508 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 Aerobic Hydrocarbon-degrading Microbial Communities in Oilsands Tailings Ponds by Fauziah Fakhrunnisa Rochman 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 DECEMBER, 2016 © Fauziah Fakhrunnisa Rochman 2016 Abstract Oilsands process-affected water (OSPW), produced by the surface-mining oilsands industry in Alberta, Canada, is alkaline and contains salts, various metals, and hydrocarbon compounds. In this thesis, aerobic communities involved in several key biogeochemical processes in OSPW were studied. Degradation of several key hydrocarbons was analyzed in depth. Benzene and naphthalene were used as models for aromatic hydrocarbons, in which their oxidation rates, degrading communities, and degradation pathways in OSPW were researched. The potential oxidation rates were 36.7 μmol L-1 day-1 for benzene and 85.4 μmol L-1 day-1 for naphthalene. Via stable isotope probing (SIP), and high-throughput sequencing of 16S rRNA gene amplicons, it was discovered that strains of the genera Methyloversatilis and Zavarzinia were the main benzene degraders, while Thiococcus and Pseudomonas were the main naphthalene degraders. Cultivated strains of Zavarzinia and Pseudomonas were shown to be growing on benzene and naphthalene. Metagenomics analysis revealed genes encoding oxygenases active against aromatic compounds, as well as catechol oxidases. Although these belonged to many phylogenetically diverse bacteria, only few bacteria were predominant in the SIP experiments. A highly divergent pmoA-like gene was also detected in the metagenome data. Here, the possibility of this gene allowing growth on short alkanes (C1 to C3) was examined. This gene was investigated via SIP and quantitative PCR. Results showed that the monooxygenase encoded by the gene has high affinity toward ethane and mostly propane. For the study of lighter hydrocarbons, methane, ethane, and propane were chosen as model compounds. OSPW was capable of supporting methane oxidation with a rate of 108.2 −1 −1 −1 −1 μmol of CH4 L OSPW d , ethane oxidation with a rate of 83.2 μmol of C2H6 L OSPW d , −1 −1 and propane oxidation with a rate of 58.6 μmol of C3H8 L OSPW d . SIP analysis uncovered Methyloparacoccus to be predominant in methane-incubated samples, whereas Methyloversatilis was predominant in ethane and propane-incubated samples. SIP technique was also employed to study photosynthetic bacterial communities and indigenous aerobic bacterial communities that assimilate methanol, acetate, and protein extracts. All OSPW photosynthetic ‘heavy-DNA’ samples were dominated by unidentified Planctomycetes. Predominant groups in methanol, acetate, and protein extract-SIPs were Betaproteobacteria, Alphaproteobacteria, and Bacteroidetes. Finally, via a modified cultivation technique, a novel Verrucomicrobia was isolated from OSPW. The aerobic bacterium was named Oleiharenicola alkalitolerans gen. nov., sp. nov., and it was studied in depth via phylogenetic, chemotaxonomic and whole-genome sequencing techniques. iii Acknowledgements One of the lessons that I learned while earning this degree is that none of my achievements could have been attained without the help of others. It is, therefore, a pleasure to thank the many people who made this dissertation possible. First and foremost, I would like to thank my supervisor, Dr. Peter Dunfield, for providing me the opportunity to explore the microbial world. I barely knew how to use a micropipette when I first came, and now I have learned so many scientific methods and laboratory techniques due to your encouragement and dedication. Thank you for your patience, thoughtful guidance, critical comments, and insights in my research. My sincerest thanks to my committee, Dr. Gerritt Voordouw, Dr. Lisa Gieg, and Dr. Patrick Hettiaratchi, for your insightful comments and valuable time. I’d also like to thank my examiners Dr. Thomas Oldenburg and Dr. Tariq Siddique for giving your time and insights to improve this dissertation. My dissertation would not be possible without the following funding providers: Institute for Sustainable Energy, Environment and Economy (ISEEE), Genome Alberta, Genome Canada, Hydrocarbon Metagenomics group, and Syncrude Canada, Ltd. Sincere thanks to Dr. Lisa Gieg for allowing me to complete some experiments in your lab. Also, to Dr. Carolina Berdugo and Courtney Toth from the Gieg lab who helped me operate their lab GC for aromatics detection. I really appreciate the work dedicated by the departmental staff: Karen Barron, Christine Goodwin, Sophia George, David Bininda, Rebecca Lee, Carol Sprague, Laureen Clement, Hayley Harris, Bill Huddleston, Cate McRae, and Paulette Harrison, who have all made my time as a graduate student and teaching assistant at the U of C a lot easier. I am indebted to Dr. Allyson Brady, who was very helpful and accommodating in my first two years in the lab. Thank you for patiently teaching me how to operate all lab equipment. Thank you to Dr. Joongjae Kim, my classic-microbiology mentor, for generously sharing your vast knowledge and thoughts in the field. I will be forever grateful! I am also very thankful to Dr. Ivica Tamas, for introducing me to the basics of bioinformatics in a way that made it seemed so easy, and to Dr. Christine Sharp for all your help with the GC, SIP, QIIME, and countless other things. To Gareth Jones, thank you for taking care of the gas tanks business, and for having your arms wide open to help with every little problem in and out of the lab that I had to deal with. ii Thank you to Dr. Angela Smirnova, for helping me with so many orders, Roshan Khadka, for sharing the valuable pmoA trees and ARB databases, and Ilona Ruhl for the very helpful and useful Illumina guidance. Thanks to Alireza Saidi-Mehrabad, for the times we shared working together on the tailings, bugging Joongjae for help, and talking about microbes and politics. To Emily Wang, Evan Haupt, Gul Zeb, Emad Albakistani, and Andriy Sheremet: thanks for always being wonderful friends! And to all past and present Dunfield lab members: thank you for the great times and the valuable friendship. I wish you all the best in life! The journey for this degree was not always smooth, especially that it happened at the same time as I embarked the new adventure of motherhood. But I was lucky to have endless family support. This doctorate wouldn’t be possible if my father didn't let my mom travel halfway around the world and stay with me in Calgary for my first two years of motherhood. Thank you so much umi and buya for your prayers and sacrifices! I’d also like to thank my sisters, Rya and Ifa, for coming over to help keep me sane in numerous ways. Especially to my children, Ibrahim and Mariam, thank you for being the most wonderful and understanding kids! Finally, to my husband, Mochamad, thanks for the fantastic support – I couldn't have asked for more. iii To my family− whose encouragement, inspiration, prayers, support and love sustained me throughout my years pursuing education. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................. ii Table of Contents .................................................................................................................v List of Tables ................................................................................................................... viii List of Figures and Illustrations ........................................................................................ xii List of Symbols, Abbreviations and Nomenclature ....................................................... xviii CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Research objectives....................................................................................................1 1.2 Dissertation structure .................................................................................................4 CHAPTER TWO: LITERATURE REVIEW ......................................................................7 2.1 Oilsands in Alberta ....................................................................................................7 2.1.1 Background........................................................................................................7 2.1.2 Oilsands recovery ..............................................................................................7