University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2020-01-23 Biostimulation for Enhanced Bioremediation of Crude Oil and Diesel Fuel by Marine Sediment Communities of Canada’s Subarctic: A Microcosm-Simulated Oil Spill Study Murphy, Sean Michael Colin Murphy, S. M. C. (2020). Biostimulation for Enhanced Bioremediation of Crude Oil and Diesel Fuel by Marine Sediment Communities of Canada’s Subarctic: A Microcosm-Simulated Oil Spill Study (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/111552 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 Biostimulation for Enhanced Bioremediation of Crude Oil and Diesel Fuel by Marine Sediment Communities of Canada’s Subarctic: A Microcosm-Simulated Oil Spill Study by Sean Michael Colin Murphy 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, 2020 © Sean Michael Colin Murphy 2020 Abstract Increases in shipping traffic, future mining, and oil and gas developments represent significant oil spill risks in Canada’s subarctic marine environment. The impact of oil on marine ecosystems and the traditional activities of local indigenous peoples are of major concern. To understand the response of local microbial communities to oil contamination and nutrient biostimulation, microcosm-simulated oil spills contaminated with diesel fuel or crude oil and incubated at 4°C were constructed using marine sediments from Hudson Bay and the Labrador Sea. Changes in microbial community structure, diversity, and composition were monitored by DNA extraction, the amplification of 16S rRNA genes, followed by sequencing and taxonomic classifications. Additionally, hydrocarbon degradation in response to bioremediation was monitored by changes in gas compositions with GC, and through hydrocarbon extractions and GC-MS analysis. Results suggested that petroleum hydrocarbons decreased observed microbial diversity and led to dominance by Gammaproteobacteria in both sediments, where many hydrocarbonoclastic bacteria (HCB) increased heavily in abundance at both sites, including Cycloclasticus, Marinobacter, Oleispira, Paraperlucidibaca, Pseudomonas, Thalassolituus, and Zhongshania. The same OTUs were found to increase in abundance in both high and low nutrient treatments, but biostimulation was found to increase initial rates of biodegradation by accelerating the succession and dominance of these HCB. Increase in the relative abundance of Cycloclasticus was noted as signifying succession in response to hydrocarbon degradation and biostimulation. The Labrador Sea sediment community was found to be more responsive to oil spills and biostimulation mitigation strategies, which could be tied to historical exposures of the community to natural oil seepages in the region. Porticoccus and Oleispira are suggested as robust bioindicators for cold seawater environments contaminated by diesel or crude oil, respectively. A comparison of three PCR primer pairs for HCB detection found 341F/806R was the preferred choice for detecting HCB taxa and assessing environmental baselines in areas at- risk of oil spills. Microbial biodiversity baselines and in situ rates of microbial degradation should be included in future environment assessments by industry. Overall, this study provided a first account of key crude oil- and diesel-degrading bacteria among marine sediments in this subarctic region. ii Preface This thesis is the original, unpublished, independent work by the author Sean Murphy. iii Acknowledgments I would like to start by thanking my supervisor Dr. Casey Hubert for taking a risk on a student with no microbiology background and for supporting such a diverse, inclusive, and exciting research group. Carmen Li for showing me how to do my first DNA extraction, and for all the continued support and sequencing thereafter. Amy Noël for taking me under her wing when I first joined EBG and showing me the ropes in lab and in the field. Margaret Cramm, not only for collecting the sediment samples from which this project grew, but for being an amazing co-worker, slo-pitch teammate, and friend. To the GENICE team, thank you all for your help, support, and love of Arctic research! María Bautista for inspiring others to be better microbiologists and for being an amazing field work companion on that first expedition. Srijak Bhatnagar for being the best chief scientist, always having answers for everything, and for an endless supply of spicy mangos. Alastair Smith for always giving great suggestions and for helping to setup my first field experiment aboard the Amundsen. And to Rhonda Clark, the best project manager there is, thank you for always taking care of us in the field and in the office. A big thank you to the crew members and scientists of the 2017 Amundsen expeditions and the 2018 William Kennedy expedition for all your help. To my committee members, Dr. Lisa Gieg and Dr. Steven Vamosi, thank you both for your feedback and expertise offered during our meetings and after graduate seminar presentations, your encouragement and suggestions have been greatly appreciated. Thank you to all members of EBG for your inputs during weekly meetings, for providing a welcoming and enjoyable workplace, and for sharing your incredible science. Specifically, I would like to thank those that have been instrumental in providing help with methods and analysis of the present study, including Anirban Chakraborty, Emil Ruff, Jayne Rattray, Jianwei Chen, Jackie Zorz, Xiaoli Dong, and from PRG, Kim Nightingale and Priyanthi Weerawardhena. Finally I wish to thank MEOPAR and Genome Canada for financial support in addition to the scholarship agencies that recognized the potential in my research, including the Government of Alberta (Queen Elizabeth Scholarships), Alberta Innovates (Technology Futures Graduate Student Scholarship), the Association of Canadian Universities for Northern Studies and The W. Garfield Weston Foundation (W. Garfield Weston Award for Northern Research), iv the Natural Sciences and Engineering Research Council (Canada Graduate Scholarship), and to the ArcticNet Training Fund for funding my Amundsen HUET training, without which I could never have taken this picture of the Amundsen in Frobisher Bay (2017), my favourite photo. v Dedication I would like to dedicate this work to my parents Margaret Hackett and Thomas Murphy for their love and continued support of my education and scientific curiosity, and to my fiancé Carson Harris for always celebrating every achievement, no matter how small. vi Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Acknowledgments ........................................................................................................................ iv Dedication ..................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Supplementary Tables and Figures .............................................................................. xiv List of Abbreviations .................................................................................................................. xv Chapter 1: Introduction ............................................................................................................... 1 1.1 Arctic and subarctic environments ................................................................................................ 1 1.2 Oil spill risks: future marine shipping and oil production ............................................................ 3 1.3 Insights from past oil spills ........................................................................................................... 5 1.4 Hydrocarbon degrading microorganisms in the marine environment ........................................... 7 1.5 Bioremediation as an oil spill response ......................................................................................... 9 1.6 Oil accumulation and biodegradation among marine sediments ................................................ 13 1.7 Rare oil degraders in the Canadian subarctic .............................................................................. 14 1.8 Microbial biodiversity for environmental baselines ..................................................................