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© 2018 Suo Xiao All Rights Reserved © 2018 SUO XIAO ALL RIGHTS RESERVED PHAGOTROPHIC ALGAE BASED APPROACHES FOR ADVANCED WASTEWATER TREATMENT A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Suo Xiao December, 2018 PHAGOTROPHIC ALGAE BASED APPROACHES FOR ADVANCED WASTEWATER TREATMENT Suo Xiao Dissertation Approved: Accepted: Advisor Department Chair Dr. Lu-Kwang Ju Dr. Michael Cheung Committee Member Interim Dean of the College Dr. Nic D. Leipzig Dr. Craig Menzemer Committee Member Dean of the Graduate School Dr. Christopher M. Miller Dr. Chand Midha Committee Member Date Dr. John M. Senko Committee Member Dr. Zhenmeng Peng ii ABSTRACT Municipal wastewater treatment plants (WWTPs) generate huge quantities of organic solids such as waste activated sludge (WAS) and waste grease (WG). Large volumes of organics-rich wastewater are also generated by the food industry. The increasing wastewater production and more stringent environmental regulations have created an urgent need in sustainable management of wastewater and organic solids. The microalga Ochromonas danica has unique phagotrophic capability, grows faster than common photosynthetic algae, and produces polyunsaturated fatty acids (PUFA). These properties make O. danica an excellent candidate as the new microbial agent for resource recovery from wastewater by direct ingestion of particulates. In this research, three new phagotrophic algae-based processes were studied for carbon recovery from different wastewater organics including waste activated sludge (WAS), waste grease (WG) and high-strength food wastewaters. WAS contains concentrated bacteria and particulate organics. Currently, its disposal requires costly treatment. Ultrasonication was studied to release WAS particulates and bacteria for direct ingestion by O. danica. Destruction/lysis of strong bacterial cell wall was unnecessary, thus minimizing energy-requirement. Effects of sonication power, duration, and WAS volume were studied with a 3 × 6 × 3 factorial design. Quantitative correlations describing the extent of particulate organics (as iii Volatile Solids VS) release were established. By proper increase of initial WAS pH, the VS release by sonication could be further improved with lower energy consumption. O. danica growth on the released WAS VS was found to follow the Monod-type kinetics but, unlike the typical Monod dependency for soluble substrates, the specific cell growth rate, , correlated with the prey-to-predator ratio, i.e., the ratio of (fed VS concentration)-to-(initial O. danica concentration), significantly better than -1 with the VS alone. The best-fit kinetics had the following parameters: μmax = 0.198 h and KM = 1.056 (g-VS/g-algae). Batch cultivations in fermentors at pH 5 confirmed algae production under nonsterile conditions using this new technology, giving a high volumetric algae productivity of 2.8 g/L-day with 38% VS reduction and 44.5% O. danica VS yield. Aerobic digestion of the remaining WAS from the sonication step was also compared with digestion of the non-sonicated WAS. The total oxygen uptake required (in mg O2/L) for the remaining WAS to reach the Class B Biosolids requirement in specific oxygen uptake rate was approximately 80% lower, indicating substantially reduced aeration cost. Compared to the conventional aerobic digestion, the new ultrasonication-phagotrophic algae process could offer enhanced overall WAS digestion with lower energy consumption, while producing algae biomass and products. O. danica cultivation was also studied with waste grease (WG, collected from a municipal WWTP) and two types of industrial food wastewaters (from cheese- and apple juice-making plants). The O. danica growing on WG synthesized and iv accumulated PUFAs, mainly C18:2n6, C18:3n3, C18:3n6, C20:4n6 and C22:5n6, to up to 67% of intracellular FAs, from the WG with only 15% PUFA. The study showed feasibility of converting WG to PUFA-rich O. danica algae culture, possibly as aquaculture/animal feed. The two industrial food wastewaters were tested for toxicity to O. danica, analyzed for compositions, and identified for major issues to address for the algal production. New process designs were proposed and evaluated. The growth kinetics and yield of O. danica in these food wastewaters were determined. Overall, the research provided fundamental insights of phagotrophic algal growth on different particulate organics including bacteria-sized particles from WAS, WG, and the bacteria grown from food wastewaters. The processes developed will find applications in bioconversion of organics solids for microalgae production and sustainable wastewater treatment. v ACKNOWLEDGEMENTS I want to thank my advisor Dr. Lu-Kwang Ju for his passion in research that motivated me, for his critical analysis that influenced my view of different challenging scientific and engineering questions, for his patience in revising my manuscripts that encouraged me to complete my research work, and for his efforts to fund my PhD study. Learning his diligence and his attitude towards science can help me face whatever challenges in my future life and career. I would also like to thank Dr. Nic D. Leipzig, Dr. Christopher M. Miller, Dr. John M. Senko and Dr. Zhenmeng Peng as my committee members for giving valuable comments on my research for the past years. I would like to thank those anonymous reviewers for giving me suggestions on my submitted manuscripts to help me finally publish my work. I would also thank Chemosphere, Water Research and Applied Microbiology and Biotechnology for publishing my research work. I would like to thank all my lab mates: Dr. Nicholas Callow, Dr. Yajie Chen, Dr. S. M. Mahfuzul Islam, Dr. Qian Li, Dr. Abdullah Al Loman, Dr. Shida Miao, Dr. Soroosh Soltani Dashtbozorg, Dr. Cong Li, Ms. Krutika Invally, Mr. Ashwin Sancheti, Ms. Napaporn Vongpanish and Mr. Jacob Kohl. vi I would like to thank Mr. Gilbert Stadler for assistance in waste sludge and waste grease sampling at Akron WRF. I also want to thank Ovivo USA LLC for funding research in sludge digestion and food wastewater projects for two years. I would like to thank my parents Yu Xiao and Xiangyuan Li for their constant support of me while encouraging me with their best wishes always. Finally, I would like to thank my wife Weixiu Zeng who has always been there cheering me up and onward while standing by me in unwavering support. vii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... xv LIST OF FIGURES .................................................................................................... xvi CHAPTER I. INTRODUCTION .................................................................................................... 1 II. BACKGROUND ....................................................................................................... 6 2.1. Sustainable Wastewater Treatment .................................................................... 6 2.2. Waste Activated Sludge (WAS) ........................................................................ 8 2.3. Waste Grease (WG) ......................................................................................... 13 2.4. Phagotrophic Algae .......................................................................................... 15 2.5. Ultrasonication and its application in WAS management ............................... 19 2.6. Cheese wastewater and fruit juice wastewater ................................................. 21 2.6.1. Cheese wastewater ............................................................................. 22 2.6.2. Fruit juice wastewater ........................................................................ 23 III. PRELIMINARY OBSERVATIONS ..................................................................... 25 Summary ...................................................................................................................... 25 3.1. Introduction ...................................................................................................... 25 3.2. Materials and Methods ..................................................................................... 27 3.2.1. Materials ............................................................................................. 27 viii 3.2.2. Sludge as substrate for O. danica cultivation ..................................... 28 3.2.3. Aerobic digestion of Akron sludge .................................................... 29 3.2.4. Effect of O. danica on aerobic digestion of Akron sludge................. 30 3.2.5. Analytical methods ............................................................................. 30 3.3. Results and Discussion .................................................................................... 30 3.3.1. Feasibility of using sludge to grow phagotrophic alga Ochromonas danica 30 3.3.2. Aerobic digestion of WAS ................................................................. 32 3.3.3. Inoculating O. danica in WAS aerobic digestion at pH 5 .................. 34 3.4. Conclusion ....................................................................................................... 36 IV. ENERGY-EFFICIENT ULTRASONIC RELEASE OF BACTERIA AND PARTICULATES TO FACILITATE INGESTION BY PHAGOTROPHIC ALGAE FOR WASTE SLUDGE TREATMENT AND ALGAL BIOMASS AND LIPID PRODUCTION ...........................................................................................................38
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