Table Of Content!
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:
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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
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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.
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©Copyright by Tyler M. Kirkendall
August 24, 2015
All Rights Reserved
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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
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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
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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.
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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
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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
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TABLE OF CONTENTS (Continued)
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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 and Product Secretions ................................................ 54
3.8.4 Continued Evaluation .............................................................................. 54
3.9 Construction of a Pure Culture Anaerobic Digestion GEM ........................... 55
4 iTK530 RESULTS AND DISCUSSION .......................................................................... 56
4.1 iTK530 Summary ............................................................................................ 56
4.2 iTK530 Validation .......................................................................................... 57
4.3 Maximum Acetate Production ........................................................................ 58
4.4 Maximum Formate Production ....................................................................... 60
4.5 Maximum PHB Production ............................................................................. 62
4.6 Maximum H Production ................................................................................ 65
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4.7 Uptake of Select Growth Media Compounds ................................................. 68
4.8 S. wolfei GEM (iTK530) Conclusions ............................................................ 70
5 PURE CULTURE ANAEROBIC DIGESTER GEM
RESULTS AND DISCUSSION ...................................................................................... .72
5.1 Pure Culture Anaerobic Digester GEM .......................................................... 72
5.2 Pure Culture Anaerobic Digester GEM Validation with OptCom ................. 72
5.3 Varying Methanogenic Population with OptCom .......................................... 75
5.4 Varying Methanogenic Population with Descriptive-OptCom ....................... 77
5.5 Growth Dynamics with Descriptive-OptCom ................................................. 78
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TABLE OF CONTENTS (Continued)
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5.5.1 Rate Limiting Step Identification with Descriptive-OptCom ................. 80
5.5.2 Methane Production Validation with Descriptive-OptCom .................... 81
5.6 Model Improvement Through Constraints of S. wolfei ................................... 82
5.6.1 Growth Dynamics of Modified Model Through Descriptive-OptCom .. 83
5.6.2 Methane Validation of Modified Model Through Descriptive-OptCom 85
5.7 Effects of Acetate Uptake Rate Through Descriptive-OptCom ...................... 87
5.7.1 Population Dynamics With Acetate Unconstrained ................................ 89
5.7.2 Methane Validation of the Modified GEM ............................................. 90
5.8 Comparison of the Pure Culture Anaerobic Digester GEM to a Real World
Anaerobic Digester ............................................................................................... 91
5.9 Pure Culture Anaerobic Digestion Conclusions ............................................. 95
6 CONCLUSIONS ............................................................................................................... 97
6.1 iTK530 and Pure Culture Anaerobic Digester GEM Conclusions ................. 97
6.2 Future Work .................................................................................................... 98
BIBLIOGRAPHY .............................................................................................................. 100
APPENDIX ......................................................................................................................... 110
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Description:not only served as a spark of inspiration for this project, but who has continued to serve as Upon submittal to the RAST server for annotation, .. solventogenic phase and transforms acetate and butyrate to acetone and butanol. In.
