Advancing Chain Elongation Technology for Medium Chain Carboxylic Acids Production from Waste Streams by Shilva Shrestha A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Environmental Engineering) in The University of Michigan 2021 Doctoral Committee: Professor Lutgarde Raskin, Chair Assistant Professor Matthew T. Agler, Friedrich Schiller University Jena Professor Glen Daigger Associate Professor Xiaoxia Lin Professor Steve Skerlos Shilva Shrestha [email protected] ORCID iD: 0000-0001-5062-3634 DEDICATION To Baba and Mama for their innumerable sacrifices and unparalleled love that paved the way to where I am today, and to my late grandmother, Pushpa Shrestha for seeding confidence and self- belief in a little girl. ii ACKNOWLEDGEMENTS This dissertation research was financially supported by the National Science Foundation Sustainability Research Networks (grant no 1444745) and the Department of Energy Small Business Innovation Research grant. I was fortunate to receive several fellowships during my graduate studies that gave me the flexibility to pursue my research without any financial constraints. I would like to acknowledge the Water Environment Federation Canham Graduate Scholarship, the Burroughs Wellcome Fund Integrated Training in Microbial Systems (ITiMS) Fellowship, and the University of Michigan Rackham Predoctoral Fellowship. I would like to express my sincere thanks to my PhD advisor, Lutgarde Raskin, for her mentorship and wisdom, for helping me grow as an independent researcher, for believing in me and giving me the freedom to shape my dissertation research according to my interest, and for her endless support as my career goals evolved. I would like to express my gratitude to my committee members Steven Skerlos, Glen Daigger, Nina Lin, and Matthew Agler for lending me their expertise and providing constructive feedback throughout my PhD. I am thankful to Steve for his insightful suggestions and guidance with the life cycle assessment work. Several PhD, Master’s, and undergraduate students were instrumental in making this research possible. I would like to sincerely thank Dianna Kitt, Hang (Alex) Song, Caro Tryuers, Siqi Xue, Maxim Muermans, Brittany Colcord, Lucy Aley, and Liem Ellen Setiawan for their hard work and significant contributions at various stages of this research. Working with such a dedicated team of individuals and learning from each of them was one of the most gratifying experiences of my graduate life at the University of Michigan. Samir Khanal, my master’s thesis advisor, has supported me from the day I arrived from Nepal until today. I got the opportunity to interact and collaborate with wonderful researchers like Xavier Fonoll, Ilse Smets, Matt Scarborough, Josh Neufeld, Ryan Ziels, and Pranav Sampara and would iii like to thank them for all the discussions and helpful input on my research. The bioreactor work was made possible through the technical assistance of CEE Technical Staff, especially Steve Donajkowski, Ethan Kennedy, and Jan Pantolin. I would like to extend my thanks to Thomas Yavaraski for helping me troubleshoot the analytical methods. Thank you to the CEE administrative team for their help with purchases and reimbursements. A heartfelt thanks to my Environmental Biotechnology family, particularly all the current and former members of the Raskin Research Group for their friendship, doses of encouragement and fun over coffee and happy hours, and invaluable feedback on research and conference presentations. I have thoroughly enjoyed being a part of such a collaborative and stimulating research environment and have learned a tremendous amount from this exceptionally talented group of researchers. Many thanks to my friends Molly, Binita, Ramila, Indu, Shikha, Prashamsha, Abhishek, and Swechchha for having my back and lifting my spirits during some of the challenging times of my PhD. Finally, my deepest love and appreciation to my family for their unconditional love and encouragement. I am indebted to my mom, dad, and brother, my biggest supporters through every step of this journey, for providing me with resources, often beyond their capacity, that made it possible for me to come this far. My sincere gratitude also goes to my grandparents for their love and my in-laws for their unfailing support. Last but not the least, I am grateful to my husband, Bibhushan, who has been by my side throughout, for his unwavering love, companionship, and relentless support. His perseverance, optimism, and encouragement have helped me keep life challenges in perspective. iv TABLE OF CONTENTS DEDICATION ................................................................................................................................ ii ACKNOWLEDGEMENTS ........................................................................................................... iii LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ....................................................................................................................... xi LIST OF APPENDICES ............................................................................................................. xvii ABSTRACT ............................................................................................................................... xviii Chapter 1 Introduction .................................................................................................................... 1 1.1 Resource recovery from organic waste streams ................................................................... 1 1.2 Chain elongation for MCCA production .............................................................................. 2 1.3 Problem statement and research motivation ......................................................................... 4 1.4 Dissertation outline ............................................................................................................... 6 1.5 References ............................................................................................................................. 7 Chapter 2 Resource Recovery of Medium Chain Carboxylic Acid from Brewery and Pre- fermented Food Waste Streams: Effect of Microbial Immigration on Chain Elongation ............ 12 2.1 Abstract ............................................................................................................................... 12 2.2 Introduction ......................................................................................................................... 13 2.3 Materials & Methods .......................................................................................................... 16 2.3.1 Inoculum and Substrate ................................................................................................ 16 2.3.2 Experimental setup ....................................................................................................... 16 2.3.3 Chemical analyses ........................................................................................................ 18 2.3.4 Microbial analyses ....................................................................................................... 18 2.3.5 Statistical analyses ....................................................................................................... 20 2.4 Results and Discussion ....................................................................................................... 21 2.4.1 MCCAs recovery from brewery and pre-fermented food waste streams .................... 21 2.4.2 MCCAs production correlated with Clostridiales and Pseudoramibacter activity ..... 24 2.4.3 Effect of pH and SRT on methanogens ....................................................................... 26 v 2.4.4 Influent plays a greater role in shaping the bioreactor microbiome than the inoculum28 2.4.5 Microbial immigration from influent to downstream, bioreactor microbial community ............................................................................................................................................... 30 2.5 Conclusions ......................................................................................................................... 36 2.6 References ........................................................................................................................... 36 Chapter 3 Effect of Methanogenic Inhibitor on Competitive Reactions During Ethanol Chain Elongation ..................................................................................................................................... 44 3.1 Abstract ............................................................................................................................... 44 3.2 Introduction ......................................................................................................................... 45 3.3 Materials & Methods .......................................................................................................... 47 3.3.1 Experimental setup and operating conditions .............................................................. 47 3.3.2 Chemical analyses ........................................................................................................ 48 3.3.3 Microbial community analyses ...................................................................................