SYSTEMS ANALYSIS OF ENGINEERED AND NATURAL MICROBIAL CONSORTIA by Hans Christopher Bernstein A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Engineering MONTANA STATE UNIVERSITY Bozeman, Montana May 2013 ©COPYRIGHT by Hans Christopher Bernstein 2013 All Rights Reserved ii APPROVAL of a dissertation submitted by Hans Christopher Bernstein This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citation, bibliographic style, and consistency and is ready for submission to The Graduate School. Dr. Ross P. Carlson Approved for the Department of Chemical and Biological Engineering Dr. Jeffrey Heys Approved for The Graduate School Dr. Ronald W. Larsen iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non- exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Hans Christopher Bernstein May 2013 iv DEDICATION This thesis is dedicated to my loving wife, Katharina Frank Bernstein. It’s been a long road and Katha has supported me from my undergraduate degree through this Ph.D. work. This accomplishment is as much hers as my own. v ACKNOWLEDGEMENTS I would like to acknowledge my main Ph.D. advisor, Dr. Ross Carlson, for giving me a shot at graduate school when others would not. However, I am near certain he is satisfied about his decision. For if he had not picked me up as a student; then he may have perished by accidently locking himself in a bathroom at the Goldschmidt conference, in the Czech Republic. You never know when you may need a graduate student to kick a door down. I have also received support and encouragement from my secondary thesis advisors, Drs. Jeff Heys, Brent Peyton and Robin Gerlach. Finally, I would also like to thank Dr. Bill Inskeep for additional support through the NSF-IGERT fellowship program and by acting as my field-research mentor. vi TABLE OF CONTENTS 1. GENERAL INTRODUCTION ....................................................................................... 1 2. MICROBIAL CONSORTIA ENGINEERING FOR CELLULAR FACTORIES: IN VITRO TO IN SILICO SYSTEMS ................................................... 6 Contribution of Authors and Co-Authors ....................................................................... 6 Manuscript Information Page ......................................................................................... 7 2.1. Abstract ................................................................................................................... 8 2.2. Introduction ............................................................................................................. 8 2.3. Ecology as the Foundation for Engineered Consortia ............................................. 9 2.4. Consortial Interaction Motifs ................................................................................ 11 2.5. Consortia Types and Case Studies ........................................................................ 13 2.5.1. Artificial Consortia ..................................................................................... 15 2.5.2. Synthetic and Semi-Synthetic Consortia .................................................... 17 2.5.3. Natural Consortia ........................................................................................ 21 2.6. Microbial Consortia in Industry ............................................................................ 21 2.7 In silico Analysis of Microbial Consortia .............................................................. 22 2.8 Broader Impact and Future Directions ................................................................... 26 2.9. Acknowledgments ................................................................................................. 26 2.10. Citation ................................................................................................................ 27 3. DESIGN, CONSTRUCTION AND CHARACTERIZATION METHODOLOGIES FOR SYNTHETIC MICROBIAL CONSORTIA ..................... 28 Contribution of Authors and Co-Authors ..................................................................... 28 Manuscript Information Page ....................................................................................... 29 3.1. Abstract ................................................................................................................. 30 3.2. Introduction ........................................................................................................... 30 3.3. Materials for In Silico, Stoichiometry-Based Metabolic Pathway Analysis. .............................................. 32 3.3.1. Stoichiometric Modeling Software ............................................................. 32 3.3.2. Matrix Manipulation Software .................................................................... 32 3.3.3. Stoichiometric Model ................................................................................. 33 3.4 P1 Phage Transduction and Antibiotic Resistance Cassette Curing Materials ................................................................ 33 3.4.1. Antibiotic Stock Solutions (1000x running concentration) ........................ 33 3.4.2. Luria-Bertani (Low Salt-LB) Medium Variants ......................................... 33 3.4.2. Citrate Buffer .............................................................................................. 33 3.4.3. P1 Phage Lysate .......................................................................................... 33 vii TABLE OF CONTENTS – CONTINUED 3.4.4. E. coli Keio Knock-Out Collection (Thermo Scientific) ............................ 34 3.5. Culturing Materials and Reactors .......................................................................... 34 3.5.1. Culturing Medium ....................................................................................... 34 3.5.2. Reactors For Batch and Continuous Culturing ........................................... 35 3.5.3. Colony Biofilms .......................................................................................... 35 3.6. HPLC Materials and Standards ............................................................................. 35 3.6.1. HPLC .......................................................................................................... 35 3.6.2. Column and Mobile Phase .......................................................................... 35 3.6.3. Standards and Dilution Matrix .................................................................... 36 3.7. Microscopy Preparation Materials ........................................................................ 36 3.7.1. Microscope and Image Analysis Software ................................................. 36 3.7.2. Cryosectioning ............................................................................................ 36 3.7.3. Fluorescent Reporter Protein Plasmids ....................................................... 37 3.8. Oxygen Microsensor Materials and Standards ...................................................... 37 3.8.1. Oxygen Microsensors (Microelectrodes) .................................................... 37 3.8.2. Amplification Hardware and Data Acquisition Software .......................... 37 3.8.3. Positioning Equipment ................................................................................ 37 3.8.4. Calibration Standards .................................................................................. 38 3.9. In Silico Design and Testing Methodology ........................................................... 38 3.10. Deletion Mutant Construction Methodology ...................................................... 41 3.11. Batch and Continuous Culturing Methodology ................................................... 45 3.12. Extracellular Metabolite Analysis (HPLC) ......................................................... 49 3.13. Colony Biofilm Culturing ................................................................................... 51 3.14. Microscopy Preparation Methods ....................................................................... 53 3.15. Oxygen Microsensor Analysis ............................................................................ 55 3.16. Notes .................................................................................................................... 57 3.17. Acknowledgements ............................................................................................. 61 4. SYNTHETIC ESCHERICHIA COLI CONSORTIA ENGINEERED FOR SYNTROPHY DEMONSTRATE ENHANCED BIOMASS PRODUCTIVITY ............................................................... 63 Contribution of Authors and Co-Authors ....................................................................