ASSESSING AND ENHANCING METHANE PRODUCTIVITY FROM ANAEROBIC DIGESTION USING CYANOTHECE BG0011 AS FEEDSTOCK By NGUYET T M DOAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Nguyet T M Doan To my beloved parents Luc Huu Doan and Tuyet Thi Bach Tu, my mother in-law Du Thi Tran, husband Tanh Tran Nhan Nguyen and my son Phuc Huu Doan Nguyen ACKNOWLEDGMENTS I would like to express the great gratitude to my academic supervisor Dr Pratap C Pullammanappallil for advising, supporting, and encouraging me during my PhD study at University of Florida I admired him by his knowledge, gentleness, and kindness The next person I would like to appreciate is my co-advisor, Dr Spyros A Svoronos for insightful ideas Both of them gave the best supports and led me to have the right way to accomplish my study I would like to give special thanks to my PhD committee members for helping me to enhance my knowledge and have a good vision for my PhD project Thanks to Dr Ray A Bucklin who supported me all the time for my PhD program Dr Edward J Phlips promoted me to study for Cyanobacteria BG0011 and Dr Ben Koopman’s flexibility advices were useful for my research I would like to thank Dr Melanie J Correll, Dr Bruce A Welt, Dr Eric McLamore, Dr Bin Gao from Department of Agricultural and Biological Engineering, UF for supporting me in using centrifuge, lab space, gas detector, microscope, and nano and distilled water; Dr Mark Brenner, Department of Geological Science at University of Florida, who showed me how to collect soil samples and gave me sediment samples; Dr Keelnatham T Shanmugam, Microbiology and Cell science at University of Florida, for helping me for using High Performance Liquid Chromatography (HPLC) to test volatile fatty acid; Dr Kien Pham, Biomedical Science at University of Florida, for helping me in using centrifuge and microscope I would like to send my thanks to my colleagues and my friends who spent time with me during my study and research at University of Florida: Yingxiu Zhang, Tung Chen, Caesar Moreira, Ziynet Boz, Samriddhi Sinha, Sunchang Yang, Wen Ji, Na Wu, Wei Wu, Patrick Dube, Bailey Slagle, Samuel Sammy, Nusheng Chen, Lidwine Hyppolite, Arnoldo Poveda, Steve Miller, Saman Souri, Rubi Raymundo, Daniel Preston, Lane Paul, and Robin Snyder I will also never forget the support and comradery of undergraduate students as we worked together in a team: Kavir Maharaj, Kevin Gilroy, Bianca Gouvea, Nicole Giatti, Quan Nguyen, Jennifer Jackson, Logan McCoy, Christian Svetics, Calvin Heimburg, Raquel Bradley, James Clover, Dylan Wald, and Alexson Joseph I would like to thank my Gainesville families for supports and treating me as their family member: Dr Khe Chau’ family, Dr Dolores Krausche and grandma, Mr Lac’s family, Ms Lien Duong’s family, Mr Phuoc’s family, Mr Roger Sedlacek’s family, and Mr Donny Dillon’s family I would like send my beloved thanks to Vietnamese students in University of Florida I would like to thank Dr Dorota Z Haman who gave me the best environment in the ABE department for study I am proud to be part of the family of Department of Agricultural and Biological Engineering, University of Florida, USA Many thanks are given to An Giang University, Vietnam; Department of Agricultural and Biological Engineering, University of Florida, USA for PhD research assistantship; Office of Energy, Florida Department of Agricultural and Consumer Services, USA; PhD Fellowship Program of Vietnam’s Ministry of Agriculture and Rural Development; Vietnam’s Ministry of Education and Training Finally, a personal record I would like to express all my gratitude to my mom, my dad, my son, and my beloved husband who are always with me all the time and all their life to support and encourage me on all my ways I would like to appreciate my friends and my relatives in my hometown (An Giang) who supported my parents and my son TABLE OF CONTENTS page ACKNOWLEDGMENTS TABLE OF CONTENTS LIST OF TABLES 10 LIST OF FIGURES 11 ABSTRACT 15 CHAPTER INTRODUCTION 18 Motivation 18 Problem Statement 23 Objectives 25 Research Approach 25 METHODOLOGY 28 Measurement of Optical Density of Microalgal Cell Culture Suspensions 28 Salinity Adjustment 28 Cell and Supernatant Dry Weight 29 Volatile Solid Measurement 33 Cell Specific Growth Rate Assessment 35 Extraction of Exopolysaccharide 35 Determination of Reducing Sugar 38 Prepare Dinitrosalicylic Acid Reagent 38 Calibration Curve for Reducing Sugar in Microalgae 38 Total Volatile Fatty Acid Measurement 42 Biochemical Methane Potential (BMP) Measurement 44 LONG-TERM SEMI-CONTINUOUS CULTIVATION OF CYANOTHECE BG0011 47 Rationale 47 Methods 49 Experimental Apparatus 49 Assessment 51 Results and Discussions 52 Cell Mass, EPS, and pH Variation 52 BG0011 Growth 57 Summary 63 EFFECT OF NITROGEN SOURCES ON GROWTH OF CYANOTHECE BG0011 64 Rationale 64 Methods 67 Microalgal Strain and Medium Conditions 67 Experimental Design and Materials 67 Measurement and Analysis 69 Cell Growth Assessment 69 Results and Discussions 70 Cell Dry Weight and Specific Growth Rate 70 Monod Kinetics 74 Summary 80 EFFECT OF LIGHT:DARK DURATION ON CYANOTHECE BG0011 GROWTH AND EPS PRODUCTION 82 Rationale 82 Methods 83 Experimental Apparatus 83 Measurement 84 Data Analysis 85 Results and Discussions 85 Cell Growth 85 EPS 91 Summary 96 ANAEROBIC DIGESTION IN HIGH SALINITY CONDITIONS 97 Rationale 97 Methods 99 Anaerobic Digesters 99 Inoculum 99 Operation 102 Measurement 104 Results and Discussions 104 Salt-acclimation and Methane Production 104 Salt-acclimation Mechanism 113 Volatile Fatty Acids (VFAs) 114 Summary 119 BIOCHEMICAL METHANE POTENTIAL OF CYANOTHECE BG0011 CULTURES 120 Rationale 120 Methods 120 Results and Discussion 123 BMP Assays Using Inoculum from Salt Acclimated Mesophilic Digester 123 BMP of Assays Using Inoculum from Mesophilic Dairy Manure Digester 126 Summary 131 ASSESSING THE EFFICIENCY OF HYDROLYSIS AND BIOGASIFICATION OF CYANOTHECE BG0011 CULTURES 132 Rationale 132 Methods 134 Experimental Apparatus and Materials 134 Measurements 137 Results and Discussions 137 Temperature and pH 137 Enzymes 139 TiO2 and UV 143 BMP after Hydrolysis with TiO2 and UV 144 Summary 149 CONCLUSIONS 150 APPENDIX 153 A VFA CALCULATION 153 B LIGHT DARK DURATION 155 C MEDIUM FORMULA FOR CYANOTHECE BG0011 164 D HYDROLYSIS 166 E EQUIPMENTS 167 F MEETING 185 LIST OF REFERENCES 186 BIOGRAPHICAL SKETCH 198 LIST OF TABLES Table 2-1 2-2 2-3 2-4 4-1 5-1 5-2 6-1 6-2 7-1 7-2 7-3 8-1 8-2 8-3 8-4 8-5 page Absorbance substrates for the level of 1.5 mg/L 40 Absorbance substrates for the level of 0.75 mg/L 40 Absorbance substrates for the level of 0.45 mg/L 41 Absorbance substrates for the level of 0.15 mg/L 41 Effect of concentration of Nitrate (NO3-N) and Ammonium (NH4-N) on BG0011 cell growth 77 Experimental design 84 Specific growth rate and EPS 95 Sample site description 100 Salt acclimation for mesophilic and thermophilic digesters 105 Various concentrations of molasses and sugar as substrate for BMP 122 BMP assays using mesophilic dairy-manure digester inoculum 123 Methane yield in experiments with mesophilic dairy-manure digester inoculum 128 Photocatalytic experiments with TiO2 136 BMP assays for pretreatments with TiO2 and UV 136 Enzymatic hydrolysis with samples (5 mL EPS), phosphoric acid 2N (0.1 mL or 0.08 mL), heating for 30 142 Effects of UV and TiO2 exposure for 30 on hydrolysis 143 VFA results from treatments 149 10 ... Requirements for the Degree of Doctor of Philosophy ASSESSING AND ENHANCING METHANE PRODUCTIVITY FROM ANAEROBIC DIGESTION USING CYANOTHECE BG0011 AS FEEDSTOCK By Nguyet T M Doan ABSTRACT April 2017... microbial biomass was conducted as a pretreatment step for anaerobic digestion, it enhanced methane yields in several cases For methane yield from anaerobic digestion of Nannochloropsis was increased... conditions was 0.33 day-1 From biochemical methane potential studies, using entire BG0011 culture as feedstock, an average methane yield of 217.16 ± 37.3 mL at STP (g VS-1) was obtained from assays