Light-Dependent Growth Kinetics and Mathematical Modeling of Synechocystis sp. PCC 6803 by Levi Straka A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved March 2017 by the Graduate Supervisory Committee: Bruce Rittmann, Chair Peter Fox César Torres ARIZONA STATE UNIVERSITY May 2017 ABSTRACT One solution to mitigating global climate change is using cyanobacteria or single- celled algae (collectively microalgae) to replace petroleum-based fuels and products, thereby reducing the net release of carbon dioxide. This work develops and evaluates a mechanistic kinetic model for light-dependent microalgal growth. Light interacts with microalgae in a variety of positive and negative ways that are captured by the model: light intensity (LI) attenuates through a microalgal culture, light absorption provides the energy and electron flows that drive photosynthesis, microalgae pool absorbed light energy, microalgae acclimate to different LI conditions, too-high LI causes damage to the cells’ photosystems, and sharp increases in light cause severe photoinhibition that inhibits growth. The model accounts for all these phenomena by using a set of state variables that represent the pooled light energy, photoacclimation, PSII photo-damage, PSII repair inhibition and PSI photodamage. Sets of experiments were conducted with the cyanobacterium Synechocystis sp. PCC 6803 during step-changes in light intensity and flashing light. The model was able to represent and explain all phenomena observed in the experiments. This included the spike and depression in growth rate following an increasing light step, the temporary depression in growth rate following a decreasing light step, the shape of the steady-state growth-irradiance curve, and the “blending” of light and dark periods under rapid flashes of light. The LI model is a marked improvement over previous light-dependent growth models, and can be used to design and interpret future experiments and practical systems for generating renewable feedstock to replace petroleum. i ACKNOWLEDGMENTS There are many people whose support, intellectual and moral, has allowed me to prepare this dissertation. I would like to thank everybody who has been part of my life for the past six years. Specifically, and this is not exhaustive: I would like to thank my peers in the Photobioreactor team, Biodesign Swette Center for Environmental Biotechnology, and IGERT SUN fellowship program, for their mentorship, collaboration, feedback and tolerance. This work would not be possible from myself alone. A few members include: Chao Zhou, Hyun Woo Kim, Binh Nguyen, Alex Zevin, Matt Thompson, Brendan Cahill, Everett Eustance, Joseph Laureanti, Anna Beiler, Lisa Dirks, and Leah Holton. I would like to thank my friends and family who kept me going throughout this process. The path to a PhD can be tedious, lonely, and often feel unrewarding. These people vitalize me, and remind me that my life is about more than work. In addition to the peers listed above I would like to specifically recognize the following people: Haley Lowrance, Dave Hanigan, Allan Greenfield, Matt Miles, Chase Holton, Steve Goodman, Sara Carey, Onur Apul, and Frank, Donna, Luke, and Umnia Straka. Finally, I would like to thank Arizona State University and all the faculty and staff who have worked with me, taught a class that I took, or coordinated what I need to complete my work. This includes my graduate supervisory committee, Bruce Rittmann, Peter Fox, and César Torres, and our lab managers Carole Flores and Diane Hagner. I underscore that none of this work would have been possible without Bruce, who provided me with the opportunity to come back to school, guidance throughout my degree, and funding for the work. This work was made possible by Dean’s funding from ASU, the NSF IGERT SUN fellowship, and Brian Swette for his support to the Swette Center. ii TABLE OF CONTENTS Page LIST OF TABLES .............................................................................................................. vii LIST OF FIGURES ........................................................................................................... viii CHAPTER 1. INTRODUCTION ..................................................................................................... 1 1.1. Global Warming, The Big Problem .................................................................. 1 1.2. Microalgae as Part of the Solution.................................................................. 5 1.3. Molecular Mechanisms of Photosynthesis ..................................................... 8 1.4. Structure of the Dissertation ......................................................................... 13 1.5. Carbon kinetics .............................................................................................. 16 1.6. Nitrogen kinetics ........................................................................................... 19 1.7. Community Considerations ........................................................................... 21 2. THE ROLE OF HETEROTROPHIC BACTERIA IN ASSESSING PHOSPHORUS STRESS TO SYNECHOCYSTIS SP. PCC 6803 ..................................................... 26 2.1. Abstract ......................................................................................................... 26 2.2. Introduction ................................................................................................. 27 2.3. Materials and Methods ................................................................................ 29 2.3.1. Synechocystis Growth Conditions ....................................................... 29 2.3.2. Bench-top PBR ..................................................................................... 29 2.3.3. Sampling and Analytical Methods ........................................................ 31 2.4. Theory / Calculations ................................................................................... 33 2.5. Results and Discussion ................................................................................. 36 2.5.1. Synechocystis Batch-Growth Experiments .......................................... 36 2.5.2. Measuring Heterotrophic Biovolume .................................................. 39 iii CHAPTER Page 2.6. Conclusions ................................................................................................... 41 2.7. Supplementary Material ............................................................................... 42 3. LIGHT ATTENUATION CHANGES WITH PHOTOACCLIMATION IN A CULTURE OF SYNECHOCYSTIS SP. PCC 6803 ................................................. 43 3.1. Abstract ......................................................................................................... 43 3.2. Introduction ................................................................................................. 44 3.3. Materials and Methods ................................................................................ 47 3.4. Results and Discussion ................................................................................ 50 3.5. Conclusion .................................................................................................... 54 3.6. Supplementary Material .............................................................................. 55 4. LIGHT-DEPENDENT KINETIC MODEL FOR MICROALGAE EXPERIENCING PHOTOACCLIMATION, PHOTODAMAGE, AND PHOTODAMAGE REPAIR .. 58 4.1. Abstract ......................................................................................................... 58 4.2. Introduction ................................................................................................. 59 4.3. Modeling Growth with Photoinhibition Phenomena .................................. 62 4.4. Modeled Results and Discussion ................................................................. 68 4.4.1. Steady-state Photoacclimated Growth-Irradiance-Curve .................... 68 4.4.2. Growth Response - Increasing Light Steps from 75 µmol m-2 s-1 ........ 70 4.4.3. Growth Response - Increasing Light Steps from 25 µmol m-2 s-1 ........ 74 4.4.4. Growth Response - Decreasing Light-Steps from 600 µmol m-2 s-1 ..... 75 4.4.5. Implications .......................................................................................... 77 4.5. Conclusions .................................................................................................. 79 5. DYNAMIC RESPONSE OF SYNECHOCYSTIS SP. PCC 6803 TO CHANGES IN LIGHT INTENSITY .............................................................................................. 80 iv CHAPTER Page 5.1. Abstract ......................................................................................................... 80 5.2. Introduction .................................................................................................. 81 5.3. Materials and Methods ................................................................................ 83 5.3.1. Synechocystis Growth-rate Experiments ............................................. 83 5.3.2. Parameterizing the Model to the Growth Data .................................... 85 5.4. Results and Discussion .................................................................................. 91 5.5. Conclusions ................................................................................................. 105 5.6. Supplementary Material ............................................................................