Assessing Outdoor Algal Cultivation in Panel and Raceway Photobioreactors for Biomass and Lipid Productivity by Everett Eustance A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved July 2015 by the Graduate Supervisory Committee: Milton Sommerfeld, Co-Chair Peter Fox, Co-chair Paul Westerhoff ARIZONA STATE UNIVERSITY August 2015 ABSTRACT Over the past decade, there has been a revival in applied algal research and attempts at commercialization. However, the main limitation in algal commercialization is the process of cultivation, which is one of the main cost and energy burdens in producing biomass that is economically feasible for different products. There are several parameters that must be considered when growing algae, including the type of growth system and operating mode, preferred organism(s), and many other criteria that affect the process of algal cultivation. The purpose of this dissertation was to assess key variables that affect algal productivity and to improve outdoor algal cultivation procedures. The effect of reducing or eliminating aeration of algal cultures at night, in flat panel photobioreactors (panels), was investigated to assess the reduction of energy consumption at night. The lack of aeration at night resulted in anoxic conditions, which significantly reduced lipid accumulation and productivity, but did not affect log phase biomass productivity. In addition, the reduction in aeration resulted in lower pH values, which prevented ammonia volatility and toxicity. Raceways are operated at deeper cultivation depths, which limit culture density and light exposure. Experimentation was accomplished to determine the effects of decreasing cultivation depth, which resulted in increased lipid accumulation and lipid productivity, but did not significantly affect biomass productivity. A comparison of semi-continuous cultivation of algae in raceways and panels in side-by-side experiments showed that panels provided better temperature control and higher levels of mixing, which resulted in higher biomass productivity. In addition, sub-optimal morning temperatures in raceways compared to panels were a significant factor in reducing algae biomass productivity. The results from this research i indicate that increasing lipid productivity and biomass productivity cannot be completed simultaneously. Therefore, the desired product will determine if lipid or biomass productivity is more crucial, which also dictates whether the system should be operated in batch mode to either allow lipid accumulation or in semi-continuous mode to allow high biomass productivity. This work is a critical step in improving algal cultivation by understanding key variables that limit biomass and lipid productivity. ii DEDICATION This dissertation is dedicated to my family who I love dearly. To my parents Camie and Randy Eustance and my sister Chanee Eustance who all showed me how to work hard and made sacrifices so that I could pursue my dream. To my wife Jessica Eustance, who has always supported me and has been amazing, as I have worked towards getting my Ph.D. To my baby girl Brianne Eustance, who has been my inspiration during these last few months. iii ACKNOWLEDGMENTS I would like to thank my advisor Dr. Milton Sommerfeld. Without his guidance and support, I would not have had the freedom to pursue the research that will help shape the future of algal cultivation. I would also like to thank my committee members, Dr. Peter Fox and Dr. Paul Westerhoff, for their support and questioning during this process. Additionally, I would like to thank Dr. Robert Gardner who initially mentored me during my Master’s degree, as he taught me a strong foundation for conducting excellent research. I would also like to thank Joshua T. Wray, Shahrzad Badvipour, and Emil Puruhito, who are excellent co-authors and colleagues, which provided a great community for collaboration during this process. Lastly, I would like to thank the technicians of the Arizona Center for Algae Technology and Innovation for their help and support during this process. Specifically, I would like to thank Sarah Arrowsmith for her help with the biochemical analysis that was completed during this process. iv TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ............................................................................................................ x CHAPTER 1. INTRODUCTION ..................................................................................................... 1 1.1. Commercialization of Algae ......................................................................... 1 1.2. Limitations to Commercialization of Algae .................................................. 4 1.3. Project goal: Improving the Algal Cultivation Process ................................. 5 1.4. Overview of Dissertation .............................................................................. 6 2. BACKGROUND ..................................................................................................... 10 2.1. What is Required for Algal Growth ............................................................ 10 2.2. How are Algae Grown? ............................................................................... 23 2.3. What Affects Algal Growth? ....................................................................... 30 2.4. What are the Benefits of using Algae over Terrestrial Plants? ................... 31 3. MANUSCRIPT 1: THE EFFECTS OF LIMITING NIGHT TIME AERATION ON PRODUCTIVITY AND LIPID ACCUMULATION OF SCENEDESMUS DIMORPHOUS TO MINIMIZE AMMONIA VOLATILIZATION ..................... 34 3.1. Introduction: ................................................................................................ 36 3.2. Materials and Methods ................................................................................ 40 3.3. Results: ........................................................................................................ 45 3.4. Discussion: .................................................................................................. 54 3.5. Conclusions: ................................................................................................ 60 v CHAPTER Page 3.6. Acknowledgements ..................................................................................... 61 3.7. References ................................................................................................... 61 4. MANUSCRIPT 2: THE EFFECTS OF CULTIVATION DEPTH, AREAL DENSITY AND NUTRIENT LEVEL ON LIPID ACCUMULATION OF SCENEDESMUS ACUTUS IN OUTDOOR RACEWAY PONDS ........................ 66 4.1. Introduction: ................................................................................................ 68 4.2. Materials and Methods ................................................................................ 72 4.3. Results ......................................................................................................... 75 4.4. Discussion ................................................................................................... 84 4.5. Conclusions ................................................................................................. 87 4.6. Acknowledgments ....................................................................................... 88 4.7. References ................................................................................................... 88 5. MANUSCRIPT 3: BIOMASS PRODUCTIVITY OF TWO SCENEDESMUS STRAINS OPERATED SEMI-CONTINUOUSLY IN OUTDOOR RACEWAY PONDS AND FLAT-PANEL PHOTOBIOREACTORS ....................................... 91 5.1. Introduction: ................................................................................................ 93 5.2. Materials and Methods ................................................................................ 96 5.3. Results ......................................................................................................... 99 5.4. Discussion ................................................................................................. 109 5.5. Conclusions ............................................................................................... 116 5.6. Acknowledgments ..................................................................................... 117 5.7. References ................................................................................................. 118 vi CHAPTER Page 6. SYNTHESIS CHAPTER: IMPROVING ALGAL CULTIVATION ................... 121 6.1. Lipid Accumulation Improves Lipid Productivity .................................... 122 6.2. Key Variables that Influence Biomass Productivity ................................. 124 6.3. Implications of the Research in the Field of Algal Cultivation ................. 129 7. SUMMARY CONCLUSION AND FUTURE RESEARCH ............................... 132 7.1. Key Findings from the Research Chapters ................................................ 132 7.2. General Conclusions and the Implications from Synthesis Chapter ......... 135 7.3. Future Research Recommendations .......................................................... 136 8. WORK CITED ...................................................................................................... 140 vii LIST OF TABLES Table Page 2.1 Modified from Williams and Laurens (2010) Highlighting the High Annual Biomass Production Compared to Current Terrestrial Crops. .................................32 3.1 Timeline and Description of Experiments with Different Nighttime Aeration Conditions and Ammonium Sources ........................................................................44 3.2 Biochemical Composition of Strain LB 0414 with and without Aeration at Night During Different Growth Phases.. ............................................................................49 3.3 Biochemical Composition of Strain LB 0414 with Different Sparging Frequencies at Night During Different Growth Phases. ..........................................51 3.4 Biochemical Composition of Strain LB 0414 with Low Aeration (Approximately 60% or 80% DO Saturation) at Night During Different Growth Phases .................54 4.1 Biochemical Composition of Scenedesmus acutus Strain 0414 during Different Stages of Growth Under Different Nutrient Levels and the Same Depth ................78 4.2 Biochemical Composition of Scenedesmus acutus Strain 0414 during Different Stages of Growth Under Different Depths and the Same Areal Nutrient Concentration (mg-N/m2) .........................................................................................80 4.3 Average Culture Productivity During Log Phase, Stress Phase and Overall Cultivation Time ......................................................................................................82 5.1 Description of Changes that Occurred During Experimentation from February 17th to March 27th .................................................................................................100 5.2 Average ± S.D. for Solar Irradiance, Biomass Productivity, and Photosynthetic Efficiency for Experiments Completed in February and March ............................102 viii Table Page 5.3 Description of changes that occurred during experimentation from April 2nd to May 7th ..................................................................................................................105 5.4 Average ± S.D. for Solar Irradiance, Biomass Productivity, and Photosynthetic Efficiency for Experiments Completed in April and May .....................................107 ix