AN ABSTRACT OF THE THESIS OF Avram M. Shayevitz for the degree of Master of Science in Food Science and Technology presented on September 17, 2018 Title: Do Oak Barrels Contribute To The Variability of The Microbiome of Barrel-Aged Beers? Abstract approved: ______________________________________________________ Christopher D. Curtin Lambic and other barrel-aged beer styles are gaining popularity in the United States and Europe and are often treated as a premium product that can command a premium price. However, these styles can be prone to spoilage during the barrel-aging process, which represents a significant time and product commitment by a brewery, and thus it is important to understand what exactly is happening within these barrels from a microbiological point of view. Previous studies have used microbiome analyses to establish the similarity in microbial succession between traditional Belgian Lambic beer and America Coolship Ales, but to date no studies have been performed on a large number of barrels. The focus of this study was on the influence of oak barrels on the microbiome of three distinct beers produced and matured within the state of Oregon, USA and aged in 102 barrels. It was evident that traditionally fermented beer produced outside of Belgium exhibited a similar microbial profile to traditional Lambic beers during the first 36 weeks of fermentation, with eventual dominance of Dekkera (syn. Brettanomyces) bruxellensis and Lactobacillus. During this time, previously unreported instances of Gluconoacetobacter were observed, a genera more often associated with vinegar and kombucha production than with beer. Analysis of beer that had aged up to five years in barrels showed that yeast and bacterial communities follow a conserved trend, with the eventual dominance of Dekkera (syn. Brettanomyces) bruxellensis and acetic acid bacteria. At any given point in time, however, there was substantial variation between individual barrels, meaning the beer’s rate of progress towards terminal microbiome composition was impacted by the barrel. These temporal aspects played a larger role in microbiome variability than barrel origin, which could not be linked to specific changes in the microbiome. ©Copyright by Avram M. Shayevitz September 17, 2018 All Rights Reserved Do Oak Barrels Contribute To The Variability of The Microbiome of Barrel-Aged Beers? by Avram M. Shayevitz A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented September 17, 2018 Commencement June 2019 Master of Science thesis of Avram M. Shayevitz presented on September 17th, 2018 APPROVED: _________________________________________________ Major Professor, representing Food Science and Technology _________________________________________________ Head of the Department of Food Science and Technology _________________________________________________ Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. _________________________________________________ Avram M. Shayevitz, Author ACKNOWLEDGEMENTS I would like to take the opportunity to express my deepest thanks to everyone that has helped make this work possible. There is no concise way to express the gratitude and fortune for having these people in my life, so I may as well just speak from the heart. First, to my parents and family – Dr. Jay and Susan Shayevitz, my siblings Deena and Givon, who have always been an encouraging force in my life and only questioned my decisions a little bit. Just enough to make sure I think things through. Next, to my mentor, Dr. Chris Curtin, who took the chance in taking me on as his first graduate student at Oregon State University and for having the patience to oversee my development as a scientist. And to Dr. Joy Waite-Cusic, for her unending advice, patience, and help that can be quantitatively measured in lab supplies. I would also like to express my deepest gratitude to Dr. Alan Bakalinsky for providing us the extra lab space we needed right from the beginning. Among my cohorts, you have pretty much become my extended family at this point. So thank you, Derrick Risner, my closest friend and fellow graduate student, who made sure I never gave up, no matter how stubborn I became. And to Keisha Harrison, who has provided a wealth of knowledge and comradery during those long nights in the lab, as my friend and colleague. To Maria Alessandri, Danton Batty, Dean Hauser Alex Emch, Sebastian Ramirez, Quintin Ferraris, Jared Johnson, John Jorgenson, and Eileen Chow for always being available to have a cookout, grab a beer, or just goof off when we had the time. I want to extend a special thanks to, Kamila and Elysse Bennett and Jake Heath, for the homecooked meals and amazing memories. And last, but certainly not least, deepest gratitude to my best friend, Kelsey Snyder, who kept me grounded in reality with almost indisputable words of wisdom. I would also like to extend a special thanks to my committee members, Dr. Thomas H. Shellhammer, Dr. Si Hong Park, and Dr. Christopher Beaudry for taking the time to make the last leg of his journey possible. And to all the faculty and staff of the Food Science department, who have made navigating graduate school so much easier. Thank you, all, from the bottom of my heart! TABLE OF CONTENTS Page 1 Introduction .............................................................................................................................. 1 2 Review of Literature ................................................................................................................ 5 2.1 A Brief History of Beer .................................................................................................... 5 2.1.1 Definition of Beer .................................................................................................... 5 2.1.2 The Traditional Fermentation .................................................................................. 6 2.1.3 Oak Barrel Aged Beers ............................................................................................ 9 2.2 Traditional Fermentation: Culture-Dependent Analysis ................................................ 10 2.2.1 Culture-Dependent Techniques .............................................................................. 11 2.2.2 Microbial Diversity as Revealed by Culture-Dependent Analysis ........................ 14 2.2.3 Major Limitations of Culture-Dependent Analysis................................................ 15 2.3 Traditional Fermentation: Culture-Independent Analysis ............................................. 16 2.3.1 Culture-independent analytical techniques (non-HTS based) ................................ 18 Real-Time Quantitative PCR (qPCR) ................................................................ 18 Denaturing gradient gel electrophoresis (DGGE) .............................................. 19 Flow Cytometry (FC) ......................................................................................... 20 Fluorescence in situ Hybridization (FISH) ........................................................ 21 2.3.2 Culture-independent analytical techniques (HTS-based) ....................................... 22 Technology ........................................................................................................ 23 Metabarcode Analysis ........................................................................................ 23 Metagenomics .................................................................................................... 25 Microbial Diversity as Revealed by Culture-Independent Analysis .................. 26 2.4 Microbial Ecology of Traditionally Fermented Beer ..................................................... 27 2.4.1 Microbial Phases of Traditionally Fermented Lambic Beer .................................. 30 2.5 Aims and Approach ....................................................................................................... 33 3 Materials and methods ........................................................................................................... 36 3.1 Sample collection and processing .................................................................................. 36 3.1.1 Beer ........................................................................................................................ 37 3.2 Culture-Dependent Analysis: ......................................................................................... 38 3.2.1 Media Selection ..................................................................................................... 38 3.2.2 Yeast and Bacteria Enumeration ............................................................................ 40 3.2.3 Isolate Selection ..................................................................................................... 41 3.2.4 Sanger Sequencing Identification .......................................................................... 41 3.3 Culture-Independent Analysis: ...................................................................................... 43 3.3.1 DNA Extraction ..................................................................................................... 43 3.3.2 Sample Indexing ...................................................................................................