Performance evaluation of microbe and plant-mediated processes in phytoremediation of toluene in fractured bedrock using hybrid poplars by Michael Ben-Israel A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Environmental Sciences Guelph, Ontario, Canada © Michael Ben-Israel, March, 2020 ABSTRACT PERFORMANCE EVALUATION OF MICROBE AND PLANT-MEDIATED PROCESSES IN PHYTOREMEDIATION OF TOLUENE IN FRACTURED BEDROCK USING HYBRID POPLARS Michael Ben-Israel Advisor: University of Guelph, 2020 Dr. Kari Dunfield Efficacy of hybrid poplar trees for phytoremediation of toluene in fractured bedrock aquifers is unclear and active mechanisms require validation. This multi-year field study was conducted on a pilot phytoremediation system at an urban site, implemented to address aged toluene impacts to a shallow fractured dolostone aquifer. The study aimed to establish performance by quantifying phytoremedial activities at the site. Contaminant concentrations in groundwater, soil, and soil vapour, measured in high spatial resolution, showed the main residual toluene mass is coincident with the water table and located favourably for phyto-remedial uptake and biodegradation, with shallow groundwater concentrations approaching aqueous solubility in high-impact areas. Biodegradation occurring in the vadose zone was shown through metagenomic analyses that enumerated toluene degradation genes and gene transcripts in roots and root-associated soil, and compound-specific stable isotope analysis that showed enrichment of toluene stable carbon isotopes in soil vapour. Transpiration measurement, in planta contaminant quantification, and high-throughput sequencing of microbial taxonomic genes in roots and stem tissue were employed to measure toluene uptake through phytoextraction and resolve biodegradation influences upon uptake patterns. Though most phyto-available toluene was being degraded/attenuated prior to uptake, phytoextraction rates were quantified in a subset of trees over a two-week peak-season period. Phytoextraction was greatest in the site’s high-impact region. Trees there had distinct, more uniform root- colonizing bacterial communities, also surmised to have a greater toluene-degrading capacity compared to other locations. Stem phyllosphere microbiomes were shaped by in planta toluene presence as well, showing enrichment in predictive degradation capacity with increasing toluene exposure. Microbial diversity and richness in the phyllosphere were seasonally dynamic, increasing in the late growing season. Finally, a lab-scale DNA stable isotope probing study identified putatively novel toluene-degrading bacteria and fungi taxa in rhizosphere soil and their taxonomic gene sequences were made available for future studies. This study validated ways in which phytoremediation of toluene using hybrid poplars actively occurs in fractured bedrock systems and resolved contributing chemical and biological mechanisms of action on quantitative and qualitative scales. Techniques employed in this study broaden available field-validated methods to monitor and assess aromatic hydrocarbon attenuation in poplar phytoremediation systems. iv DEDICATION To Judy. v ACKNOWLEDGEMENTS First, thank-you to my advisor, Dr. Kari Dunfield, for providing me with this unique opportunity, for your mentorship, and for your steadfast support and guidance throughout this journey. I would also like to acknowledge and thank the other members of my advisory committee, Dr. Beth Parker, Dr. Elizabeth Haack, and Dr. David Tsao, for your time, guidance, and indispensable advice. I am grateful to my entire committee for this immersive, practical experience across many fields and disciplines. I would like to acknowledge the other members of the collaborative research and development program supported by BP, the University Consortium for Field-Focused Groundwater Contamination Research, and the Natural Sciences and Engineering Research Council of Canada (NSERC). Thank-you to Dr. Ramon Aravena for your guidance to help keep me on track. Thank-you to Jeremy Fernandes, for initiating and onboarding me into this project and for the solid foundation your work provided for my research. I am grateful also to Dr. Philipp Wanner, for your exceptional collaboration, helpful discussions, and technical support. Thank-you to Dr. Joel Burken, who’s expertise advanced the breadth of this work. Thank-you for technical support, training, and field supervision provided by partners at AECOM and EcoMetrix Inc., including Jeff McBride, Sean Todd, Matt Smith, Dr. Fei Luo, and many others. A special thank-you to Alan Scheibner (and colleagues at BP Canada) for your unwavering support to this project and your commitment to scientific development and education. I am grateful for invaluable technical support, training, and helpful discussion from members of the Dunfield research group, G360 research group, and the University community at large. A special thank-you to Kamini Khosla, for your time and contributions. Thank-you to Jonathan Gaiero, Dr. Jemaneh Habtewold, Dr. Micaela Tosi, Tolulope Mafa-Attoye, Anibal Castillo, Dr. Dasiel Obregon Alvarez, John Drummelsmith, Andrea Roebuck, Dr. Nicola Linton, Travis Mazurek, Dr. Elizabeth Bent, Dr. Eduardo Kovalski Mitter, Sara Low, Adrianna Wiley, James Hommersen, Maria Gorecka, Isaac Noyes, Steve Chapman, Juliana Camillo, Dr. Patryk Quinn, Rashmi Jadeja, Amanda Pierce, Sean Jordan, Ian Renaud, Steve Wilson, Dr. Dyanne Brewer, Dr. Armen Charchoglyan, Dr. James Longstaffe, Brent Coleman, and Kevin Ecott among many others. I am forever grateful for the constant support I have received from my family and friends, without whom I would not be where I am today. Finally, my utmost gratitude goes to Pooja Arora – thanks for inspiring me every day and pushing me to achieve my goals. vi TABLE OF CONTENTS Abstract .......................................................................................................................... ii Dedication ..................................................................................................................... iv Acknowledgements ....................................................................................................... v Table of Contents ......................................................................................................... vi List of Tables ................................................................................................................ ix List of Figures ............................................................................................................... x List of Abbreviations .................................................................................................. xiii 1 Introduction ............................................................................................................ 1 1.1 Motivation, study site, and research program .............................................. 1 1.2 Thesis format and objectives ......................................................................... 8 2 Toluene biodegradation in the vadose zone of a poplar phytoremediation system identified using metagenomics and toluene-specific stable carbon isotope analysis .................................................................................................... 10 2.1 Abstract ......................................................................................................... 10 2.2 Introduction ................................................................................................... 11 2.3 Materials and Methods ................................................................................. 13 2.3.1 Site description ............................................................................................ 13 2.3.2 Soil vapor and groundwater sampling ......................................................... 16 2.3.3 Toluene concentration analysis and compound-specific carbon isotope analysis (CSIA) .......................................................................................... 16 2.3.4 Rhizosphere soil, rhizoplane soil, and root samples .................................... 17 2.3.5 Extraction of nucleic acids and cDNA synthesis .......................................... 18 2.3.6 Quantitative PCR ......................................................................................... 19 2.3.7 Statistical analyses ...................................................................................... 20 2.4 Results and discussion ................................................................................ 20 2.4.1 Toluene concentrations in soil, groundwater, and soil vapor ....................... 20 2.4.2 Stable carbon isotope ratios of toluene ....................................................... 23 2.4.3 Abundance of toluene degradation genes in poplar-associated rhizosphere soil .............................................................................................................. 25 vii 2.4.4 Toluene degradation gene expression in poplar-associated rhizosphere soil ................................................................................................................... 27 2.4.5 Abundance of toluene degradation genes in poplar roots and root-associated rhizoplane soil ............................................................................................ 29 2.5 Conclusions .................................................................................................