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Genome-Scale Metabolic Reconstruction and Analysis of a Pure Culture Anaerobic Digester

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Genome-Scale Metabolic Reconstruction and Analysis of a Pure Culture Anaerobic Digester ! AN ABSTRACT OF THE THESIS OF Tyler M. Kirkendall for the degree of Master of Science in Environmental Engineering presented on August, 24, 2015. Title: Genome-Scale Metabolic Reconstruction and Analysis of a Pure Culture Anaerobic Digester. Abstract approved: ______________________________________________________ Tyler S. Radniecki Anaerobic digestion is a biological process in which organic matter is decomposed by a community of microbes in the absence of oxygen. The end product of anaerobic digestion is biogas, composed of methane and carbon dioxide, which is often recovered and used to generate energy. Commonly, biogas is not produced in sufficient quantities suitable for economic recovery, resulting in the flaring of biogas to the atmosphere. To optimize anaerobic digestion performance, in silico models have been created. Current models examine the critical components of anaerobic digestion, approaching the system at a macroscopic level. To address the limits of current macroscopic in silico models, a genome-scale model (GEM) of a pure culture anaerobic digester was constructed to evaluate both the individual organism’s metabolic activity and the community level fitness. An in silico pure culture anaerobic digester GEM was defined to include the acidogenic bacteria Clostridium acetobutylicum (iCac802), the syntrophic short chain fatty acid oxidizer Syntrophomonas wolfei (iTK530), and the methanogenic archaea Methanosarcina barkeri (iMG746). While GEMs for both C. acetobutylicum and M. barkeri have been previously published, a novel GEM for S. wolfei ( iTK530) was curated. Flux balance analysis was performed on the S. wolfei GEM to determine reaction fluxes ! ! through the system for various experimental conditions. The pure culture anaerobic digester was analyzed through the application of OptCom and descriptive-OptCom, which utilized multi-level and multi-objective optimization techniques. The construction of a pure culture anaerobic digester GEM presents a high resolution platform for in silico analysis and continued investigation of anaerobic digestion at the genome level. ! ! ©Copyright by Tyler M. Kirkendall August 24, 2015 All Rights Reserved ! ! Genome-Scale Metabolic Reconstruction and Analysis of a Pure Culture Anaerobic Digester by Tyler M. Kirkendall A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented August 24, 2015 Commencement June 2016 ! ! Master of Science thesis of Tyler Kirkendall presented on August 24, 2015. APPROVED: Major Professor, representing Environmental Engineering Head of the School of Chemical, Biological, and Environmental Engineering 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. Tyler M. Kirkendall, Author ! ! ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Tyler Radniecki for serving as my advisor and mentor throughout my entire engineering education. From working as an undergraduate in his lab at San Diego State University to working with him as a graduate student at Oregon State University, Dr. Radniecki has continued to foster my academic, personal, and professional growth. I greatly appreciate the unrelenting support, dedication, and enthusiasm Dr. Radniecki has provided me with and for always looking towards the bright side, even when our research isn’t going as smoothly as expected. I would also like to express my sincerest thanks to Dr. Frank Chaplen, who not only served as a spark of inspiration for this project, but who has continued to serve as an advisor and as a source of invaluable expertise on the subject. I would like to thank Dr. Chaplen for providing the patience, guidance, support, and modeling knowledge that directly enabled the success of this project. I would also like to Inara Scott for serving as my Minor Professor and for providing me with the opportunity to connect my engineering knowledge to real world and business situations. The guidance and expertise that Inara provided has played a critical role in my growth as an engineer. I would like to thank Dr. Mark Dolan for serving as a committee member and I would also like to thank Dr. Ingrid Arocho for serving as my Graduate Council Representative. I would like to express my sincerest gratitude to all the CBEE graduate students, faculty, and graduate students in Owen 406 who have continued to provide support, advice, and a much needed break from research these past two years. Most importantly, I would like to thank my parents, Mike and Pam, and my brother, David, for continuing to provide me the support and stability to allow me to accomplish my goals. ! ! TABLE OF CONTENTS Page 1 INTRODUCTION .............................................................................................................. 1 2 LITERATURE REVIEW ................................................................................................... 3 2.1 History of Anaeorbic Digestion ........................................................................ 3 2.2 Introduction to Microbiology of Anaerobic Digestion ..................................... 5 2.3 Primary Processes of Anaerobic Digestion ....................................................... 5 2.3.1 Hydrolyrsis .............................................................................................. 6 2.3.2 Acidogenesis ........................................................................................... 9 2.3.3 Acetogenesis ......................................................................................... 11 2.3.3.1 Homoacetogens ............................................................................ 12 2.3.3.2 Syntrophic Acetogens .................................................................. 13 2.3.4 Methanogenesis ....................................................................................... 15 2.3.4.1 Acetoclastic Methanogens ........................................................... 15 2.3.4.2 Hydrogenotrophic Methanogens .................................................. 17 2.4 Anaerobic Digestion Microbial Community ................................................... 19 2.5 Review of Anaerobic Digestion Modeling ..................................................... 22 2.6 Genome-Scale Modeling ................................................................................. 24 2.7 Genome-Scale Construction ........................................................................... 25 2.7.1 Automated GEM Draft Reconstruction ................................................ 26 2.7.2 Submitting a Genome to RAST for Genome Annotation ..................... 26 2.7.3 RAST Annotation Review .................................................................... 27 2.7.4 Converting RAST Annotation to Draft GEM ....................................... 29 ! ! TABLE OF CONTENTS (Continued) Page 2.7.5 Developing GEM Draft Biomass Compostion Reaction ...................... 29 2.7.6 Auto-completion of a Draft GEM ......................................................... 31 2.7.6.1 Errors Introduced During an Automated Reconstruction ............ 33 2.7.7 Completed GEM Review ...................................................................... 34 2.8 Flux Balance Analysis .................................................................................... 36 2.8.1 Mathematics of FBA .................................................................................... 36 2.9 Modeling Microbial Communities With GEMs ............................................. 38 2.9.1 Development of OptCom to Model Microbial Communities ............... 40 2.9.2 Descriptive-OptCom ............................................................................. 40 3 MATERIALS AND METHODS ..................................................................................... 44 3.1 Definition of a Pure Culture Anaeorbic Digester ............................................ 44 3.2 Acidogenic Organism ..................................................................................... 45 3.3 Acetogenic Organism ...................................................................................... 45 3.4 Methanogenic Organism ................................................................................. 46 3.5 Construction of a Genome Scale Model for S. wolfei ..................................... 46 3.6 Draft Reconstruction ....................................................................................... 46 3.7 Manual Curation ............................................................................................. 48 3.7.1 Growth Media ......................................................................................... 48 3.7.2 Biomass Composition Reaction .............................................................. 49 3.7.3 Model SEED Gap Filled Reactions ......................................................... 51 3.8 GEM Evaluation ............................................................................................. 51 ! ! TABLE OF CONTENTS (Continued) Page 3.8.1 Manual Gap Filling ................................................................................. 51 3.8.1.1 Biomass Composition Reaction ................................................... 52 3.8.2 Futile Cycles and Unbalanced Reactions ................................................ 53 3.8.3 Substrate Uptake
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