University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2014-04-30 Syntrophic hydrocarbon metabolism under methanogenic conditions Fowler, Susan Jane Fowler, S. J. (2014). Syntrophic hydrocarbon metabolism under methanogenic conditions (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27962 http://hdl.handle.net/11023/1450 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 Syntrophic hydrocarbon metabolism under methanogenic conditions by Susan Jane Fowler 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 APRIL, 2014 © SUSAN JANE FOWLER 2014 Abstract Methanogenic metabolism of organic matter is a key process in both natural and engineered systems. Methanogenic hydrocarbon degradation is an important biogeochemical process in the deep subsurface, and subsurface hydrocarbon contamination is frequently remediated by methanogenic processes. Despite the importance of methanogenic processes in hydrocarbon-impacted systems, we currently have an incomplete understanding of the hydrocarbon activation and degradation pathways used by the syntrophic bacteria, the roles of the non-hydrocarbon degrading syntrophs, which are often present in high abundance, and the ways in which syntrophic bacteria and methanogenic archaea establish and maintain relationships that allow them to coordinate their metabolism. By studying methanogenic hydrocarbon degrading enrichment cultures, we remove many complicating features of natural systems and can gain a basic understanding of the primary factors governing hydrocarbon metabolism under methanogenic conditions. In this dissertation, we describe several methanogenic hydrocarbon degrading enrichment cultures with a major focus on a toluene degrading methanogenic enrichment culture. Methanogenic hydrocarbon degrading communities consist of a diverse assemblage of Archaea and Bacteria dominated by members of the Methanomicrobia, Firmicutes, Deltaproteobacteria, Chloroflexi, Spirochaetes and other bacterial phyla in smaller proportions. Using stable isotope probing, key organisms involved in the degradation of toluene were identified, including Desulfosporosinus sp., which is associated with toluene activation as well as Syntrophus- like organisms and Desulfovibrio sp. Metabolite and metagenomic analysis indicate that fumarate addition is involved in toluene activation in this culture and results from this and other cultures suggest that fumarate addition is a key mechanism involved in the activation of alkanes, monoaromatic and polyaromatic hydrocarbons ii under methanogenic conditions. Comparative metagenomic analysis suggests that key functional features that distinguish methanogenic hydrocarbon degrading cultures include enrichment of archaeal and bacterial hydrogenases, as well as functions related to the regulation of redox conditions, energy conservation and methanogenesis. Hydrogen and/or formate transfer appears to play a major role in metabolite and electron transfer in these cultures. A better understanding of the processes involved in methanogenic hydrocarbon metabolism may provide us with the knowledge to develop new tools to monitor, control and harness these technologies to the benefit of ourselves and our environment. iii Acknowledgements One of the joys of writing this dissertation is to look back to where I started and remember those who have helped me over the course of this journey. First and foremost, I would like to express my deepest gratitude to Dr. Lisa Gieg who has been an incredible mentor. You have given me the space and flexibility to grow, while all the while being patient, supportive and encouraging. Never lose your enduring sense of wonder for what we still don’t understand as this inspires many of us. I’d also like to thank my co-supervisor Dr. Gerrit Voordouw for his encouragement and support. His vast knowledge of seemingly everything always makes trips to his office enjoyable and enlightening. My sincerest thanks to my committee, Dr. Ray Turner and Dr. Peter Dunfield for your support, suggestions and your valuable time. I’d also like to thank my examiners Dr. Marc Strous and Dr. Tariq Siddique for giving their time and insight to improve this dissertation. My time as a graduate student was funded by NSERC and AI and as such I’d like to thank them for keeping a roof over my head and food in my cupboards. Best wishes to members of the Gieg lab and the rest of the PMRG, both past and present. To my friends Esther, Carolina, Sylvain, Shawna and Marcy, thank you for all the fun times and revealing discussions, both science-related and not. Also, a special thanks to Dr. Rhonda Clark, the PMRG would be lost without you. Thanks also to my collaborators and fellow scientists from the University of Calgary, University of Alberta, University of British Columbia and University of New South Wales, without whom this work wouldn’t have been possible. Brandon & Erin, thanks for the continued support and assistance in navigating the vast maze that is graduate school. Your friendship, humpdays and homebrew have made the past five years fly by. Finally, to Will, thanks for the unwavering love and support and especially for bringing me coffee on those dark, cold, winter mornings. If it weren’t for that I might still be in bed. iv Dedication For my mother, without whom I would not be here in more ways than one. v Table of Contents Abstract ............................................................................................................................... ii Dedication ............................................................................................................................v Table of Contents ............................................................................................................... vi List of Tables .......................................................................................................................x List of Figures ................................................................................................................... xii List of Symbols, Abbreviations and Nomenclature ....................................................... xviii CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Historical context and rationale .................................................................................1 1.2 Research objectives ....................................................................................................2 1.3 Organization of the thesis and contributions of co-authors .......................................3 CHAPTER TWO: LITERATURE REVIEW ......................................................................6 2.1 Syntrophy and methanogenesis .................................................................................6 2.1.1 Interspecies metabolite and electron transfer ....................................................7 2.1.1.1 Hydrogen and formate .............................................................................8 2.1.1.2 Acetate ...................................................................................................10 2.1.1.3 Direct interspecies electron transfer (DIET) ..........................................11 2.1.1.4 Other compounds ...................................................................................12 2.1.1.5 Summary ................................................................................................13 2.2 Methanogenic hydrocarbon metabolism ..................................................................14 2.2.1 Anaerobic hydrocarbon activation ..................................................................16 2.2.1.1 Fumarate addition ..................................................................................16 2.2.1.2 Carboxylation .........................................................................................19 2.2.1.3 Methylation ............................................................................................20 2.2.1.4 Hydroxylation ........................................................................................21 2.2.2 Detailed pathway of anaerobic toluene metabolism by fumarate addition .....22 2.3 Syntrophic hydrocarbon degrading microorganisms ...............................................26 2.4 Current research focus .............................................................................................29 CHAPTER THREE: METHANOGENIC TOLUENE METABOLISM: COMMUNITY STRUCTURE AND INTERMEDIATES.................................................................31 3.1 Abstract ....................................................................................................................32 3.2 Introduction ..............................................................................................................32 3.3 Experimental procedures .........................................................................................35