Sustainable Cultivation of Microalgae Using Diluted Anaerobic Digestate for Biofuels Production A dissertation presented to the faculty of the Russ College of Engineering and Technology of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Husam A. Abu Hajar August 2016 © 2016 Husam A. Abu Hajar. All Rights Reserved. 2 This dissertation titled Sustainable Cultivation of Microalgae Using Diluted Anaerobic Digestate for Biofuels Production by HUSAM A. ABU HAJAR has been approved for the Department of Civil Engineering and the Russ College of Engineering and Technology by R. Guy Riefler Associate Professor of Civil Engineering Dennis Irwin Dean, Russ College of Engineering and Technology 3 ABSTRACT ABU HAJAR, HUSAM A., Ph.D., August 2016, Civil Engineering Sustainable Cultivation of Microalgae Using Diluted Anaerobic Digestate for Biofuels Production Director of Dissertation: R. Guy Riefler Microalgae cultivation has gained considerable interest recently as a potential source for the production of biofuels. Nevertheless, many obstacles still face the industrial application of microalgal biofuels such as the high production costs due to nutrient requirements and the high energy input for cultivating and harvesting the microalgal biomass. In this study, the utilization of the anaerobic digestate as a nutrient medium for the cultivation of two microalgal species was investigated. The anaerobic digestate was initially characterized and several pretreatment methods such as hydrogen peroxide treatment, filtration using polyester filter bags, and supernatant extraction were applied to the digestate. It was found that the supernatant extraction was the simplest and most effective method in decreasing the turbidity and COD of the diluted anaerobic digestate while maintaining sufficient nutrients (particularly nitrogen) for microalgae cultivation. The microalga Neochloris oleoabundans was cultivated using the diluted anaerobic digestate supernatant on a bench-scale. It was found that 100 mg N/L dilution was sufficient to maximize the biomass concentration of this microalga. It was attempted to scale up the cultivation of the microalga N. oleoabundans to 100 L raceway ponds; however, the culture was contaminated with other algal species. 4 The microalga Scenedesmus dimorphus was then cultivated using the anaerobic digestate supernatant on a bench-scale. The highest biomass concentration recorded was 654 mg/L. Furthermore, it was found that 50 – 100 mg N/L dilutions were sufficient to maximize the specific growth rate of this microalga while still producing relatively high biomass concentrations. As a result, the microalgae cultivation was scaled up to 100 L raceway ponds using 100 mg N/L dilution at 454 and 317 µmol/m2/s light intensities and 50 mg N/L dilution at 384 and 234 µmol/m2/s light intensities. The highest biomass concentration achieved was 432 mg/L in the 100 mg N/L – 454 µmol/m2/s culture. Nitrogen removal efficiencies were in the 65 – 72% range with complete ammonia removal. Phosphorus removal efficiencies were in the 63 – 100% range while COD removal efficiencies as a result of the bacteria presence in the unsterilized nutrient media were in the 78 – 82% range. The effect of mixing on the growth of the microalga S. dimorphus was evaluated by cultivating this microalga in a raceway pond at 0.1, 0.2, and 0.3 m/s water surface velocities. It was concluded that the biomass concentration and growth rate increased with an increase in the mixing velocity. However, by balancing the power required to operate the pond at different velocities with the potential energy yield from biodiesel synthesis, it was found that operating the pond at 0.1 m/s surface velocity was the only case with a positive net energy. Finally, two biomass growth models were developed and tested on the growth of the microalga S. dimorphus. It was found that the logistic model, which assumes a maximum bearing capacity of the culture, represented the biomass growth better than the 5 exponential growth model, which assumes that the microalgae grow exponentially at specific growth rates equal to or less than the maximum specific growth rate depending on the culture conditions. 6 PREFACE This dissertation is divided into two parts. Part I (chapters 1 through 7) titled “Sustainable Cultivation of Microalgae Using Diluted Anaerobic Digestate for Biofuels Production” addresses my research work over the past 3 years of my Ph.D. program, while Part II (Appendix A) titled “Selective Precipitation of Aluminum and Iron in Acid Mine Drainage” covers my earlier Ph.D. research which focused on the treatment of acid mine drainage in eastern and southeastern Ohio. 7 DEDICATION Dedicated to my beloved family in Jordan and to all my friends in Athens, Ohio. My success is because of your love, support, and encouragement 8 ACKNOWLEDGMENTS I would like to express my sincere gratitude to my adviser Dr. R. Guy Riefler for his full support, guidance, and encouragement throughout my Ph.D. program. I am also very thankful to Dr. Ben J. Stuart for his endless support and for giving me the chance to work on the “Sustainable Housing through Holistic Waste Stream Management and Algal Cultivation” project. I would like also to thank my committee members, Dr. David Bayless, Dr. Morgan Vis, and Dr. Kurt Rhoads for agreeing to serve on my Ph.D. committee and for their valuable feedback and guidance. I would like also to thank all Civil Engineering faculty, staff, graduate and undergraduate fellows who helped and supported me throughout my graduate career at Ohio University. Also, I would like to extend my gratitude to the faculty, staff, and students of the Institute for Sustainable Energy and the Environment. To all my friends in Athens, thank you for being there for me, I have been lucky enough to have met wonderful people like you, and you will always be my family. Finally, I would like to acknowledge the National Science Foundation (NSF) for funding the “Sustainable Housing through Holistic Waste Stream Management and Algal Cultivation” project through the Sustainable Energy Pathways (SEP) program (Award # 1230961) and the Wayne National Forest, U.S. Forest Service and the Ohio Department of Natural Resources, Division of Mineral Resources Management for funding the “Selective Precipitation of Aluminum and Iron in Acid Mine Drainage” project. 9 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Preface................................................................................................................................. 6 Dedication ........................................................................................................................... 7 Acknowledgments............................................................................................................... 8 List of Tables .................................................................................................................... 13 List of Figures ................................................................................................................... 15 Chapter 1. Introduction ..................................................................................................... 18 1.1 Energy Sources and the Environment ........................................................ 18 1.2 Microalgal Biofuels .................................................................................... 19 1.3 Research Objectives ................................................................................... 20 1.4 Dissertation Outline .................................................................................... 20 Chapter 2. A Review of the Cultivation of the Microalga Neochloris oleoabundans for Biofuels Production and other Industrial Applications ..................................................... 24 2.1 Abstract ..................................................................................................... 24 2.2 Introduction ................................................................................................ 25 2.3 Microalgae Cultivation ............................................................................... 27 2.3.1 Microalgae definition and growth conditions ................................... 27 2.3.2 Applications and products from microalgae ..................................... 28 2.3.3 Lipids accumulation in microalgae ................................................... 30 2.3.4 Challenges facing microalgae cultivation and potential solutions .... 31 2.4 Neochloris oleoabundans ........................................................................... 33 2.4.1 Introduction to the microalga Neochloris oleoabundans .................. 33 2.4.2 N. oleoabundans biomass composition............................................. 35 2.4.3 Stress conditions in N. oleoabundans and lipids accumulation ........ 39 2.4.4 Harvesting ......................................................................................... 40 2.4.5 Applications ...................................................................................... 42 10 2.5 Factors Affecting the Growth of N. oleoabundans .................................... 43 2.5.1 Nutrients ............................................................................................ 43 2.5.2 Light .................................................................................................