DISSERTATION ANAEROBIC DIGESTION OF SUBMERGED MACROPHYTES —BIOCHEMICAL APPROACH FOR ENHANCING THE METHANE PRODUCTION— 2016 SOKA UNIVERSITY GRADUATE SCHOOL OF ENGINEERING MITSUHIKO KOYAMA ANAEROBIC DIGESTION OF SUBMERGED MACROPHYTES —BIOCHEMICAL APPROACH FOR ENHANCING THE METHANE PRODUCTION— March 2016 MITSUHIKO KOYAMA SOKA UNIVERSITY Author: Mitsuhiko Koyama Title: Anaerobic digestion of submerged macrophytes —Biochemical approach for enhancing the methane production— Department: Environmental Engineering for Symbiosis Faculty: Engineering Degree: Ph.D. Convocation: March 2016 Permission is herewith granted to Soka University to circulate and copy for non- commercial purposes, at its discretion, the above title request of individual or institutions. We certify that we have read this dissertation and that, in our opinion, it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Engineering March 2016 DISSERTATION COMMITTEE Prof. Dr. Tatsuki Toda Prof. Dr. Shuichi Yamamoto Prof. Dr. Kiyohiko Nakasaki Contents page ACKNOWLEDGMENTS i ABSTRACT iii Chapter I GENERAL INTRODUCTION 1.1. Emerging issues of aquatic macrophytes 1 1.2. Harvesting of submerged macrophytes and treatment of the harvested phytomass 2 1.3. Anaerobic digestion of lignocellulosic biomass and enhancement of CH4 recovery 5 1.4. Objectives 7 Tables 9 Figures 10 ChapterⅡ CHEMICAL COMPOSITION AND ANAEROBIC DIGESTIBILITY OF FIVE SUBMERGED MACROPHYTE SPECIES DOMINANT IN LAKE BIWA 2.1. Introduction 16 2.2. Materials and methods 17 2.2.1. Substrates and inoculum 17 2.2.2. Batch anaerobic digestion of submerged macrophytes 18 2.2.3. Analytical parameters 19 2.2.4. Calculation 20 2.3. Results and discussion 21 2.3.1. Chemical composition of submerged macrophytes 21 2.3.2. CH4 production rate and CH4 yield 23 2.3.3. Relationship between CH4 recovery and lignocellulose content 25 2.4. Conclusions 27 Tables 29 Figures 33 ChapterⅢ EFFECT OF ALKALINE PRE-TREATMENT ON ANAEROBIC DIGESTIBILITY OF LIGNIN-RICH SUBMERGED MACROPHYTE 3.1. Introduction 39 3.2. Materials and methods 43 3.2.1. Substrates and inoculum 43 3.2.2. Hydrolysis test of submerged macrophytes using alkaline pre-treatment 43 3.2.3. Batch anaerobic digestion test of pre-treated macrophytes 44 3.2.4. Methanogenic and hydrolysis toxicity test of dissolved lignin 45 3.2.4.1. Lignin extraction and purification 45 3.2.4.2. Methanogenic and hydrolysis toxicity test 45 3.2.5. Analytical parameters 46 3.2.6. Calculations 48 3.2.7. Kinetic models 50 3.2.8. Statistical analysis 51 3.3. Results and discussion 51 3.3.1. Chemical component change of submerged macrophytes by alkaline pre-treatment 51 3.3.1.1. Hydrolysis efficiency 51 3.3.1.2. Lignocellulose composition 52 3.3.1.3. Lignin composition 55 3.3.2. Anaerobic digestion of thermochemically pre-treated submerged macrophytes and possible inhibitor 56 3.3.3. Influence of dissolved lignin on anaerobic digestion process 59 3.3.3.1. Chemical characteristics of purified dissolved lignin 59 3.3.3.2. Influence on methanogenesis 59 3.3.3.3. Influence on acidogenesis and hydrolysis 61 3.4. Conclusions 66 Tables 67 Figures 76 ChapterⅣ EFFECT OF ALKALINE PRE-TREATMENT ON SEMI-CONTINUOUS ANAEROBIC DIGESTION OF SUBMERGED MACROPHYTE 4.1. Introduction 92 4.2. Materials and methods 94 4.2.1. Substrates and inoculum 94 4.2.2. Semi-continuous anaerobic digestion of alkali pre-treated or un-treated P. maackianus 94 4.2.3. Analytical parameters 95 4.2.4. Fractionation of soluble organics and estimation of dissolved lignin/ humic substances in the digestate 96 4.2.5. Calculations 96 4.3. Results and discussion 97 4.3.1. pH and salinity variation in the digestate 97 4.3.2. Accumulation of dissolved lignin in the digestate 98 4.3.3. Effect of dissolved lignin on methane recovery in semi-continuous operation 99 4.3.3.1. Performance of semi-continuous anaerobic digestion of alkali pre-treated and un-treated P. maackianus 99 4.3.3.2. Inhibition and acclimatization against dissolved lignin 100 4.4. Conclusions 103 Table 105 Figures 106 ChapterⅤ GENERAL DISCUSSION 5.1. Economic and thermal balance assessment of alkaline pre-treatment 112 5.1.1. Scenario design 113 5.1.2. Recovery of CH4, electricity and thermal energy 115 5.1.3. Economic and thermal balance 116 5.1.3. Effect of electricity tariff of economic balance 116 5.1.3. Effect of species composition on economic and thermal balance 117 5.2. Further studies for more efficient anaerobic digestion of submerged macrophytes 118 5.2.1. Lignin recovery from alkali pre-treatment liquor prior to anaerobic digestion 118 5.2.2. Effect of seasons on the digestibility of submerged macrophytes 119 Tables 121 Figures 123 REFERENCES 127 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor Professor Tatsuki Toda for all the guidance throughout my Ph.D. study and for his strict but warm hearted encouragement rendered in the completion of this dissertation. I sincerely appreciate for his tremendous academic support, as well as for giving me a lot of wonderful opportunities throughout this six years in his laboratory, especially Lake Biwa Project. I am also deeply thankful to my co-supervisor Professor Shuichi Yamamoto and Professor Kiyohiko Nakasaki for taking so much time for my dissertation and for their insightful comments and suggestions regarding the present study. I am grateful to Professor Syuhei Ban for his advice and guidance, especially during the application of research grant and implementation of research project. I appreciate to Dr. Kanako Ishikawa for the knowledge about submerged macrophytes and for heling harvest of macrophytes in Lake Biwa. I deeply appreciate to Mr. Chiaki Niwa for his professional advices and helpful suggestions, especially during experimental set-up and data analysis. I am thankful to Professor Satoru Taguchi, Professor Norio Kurosawa, Professor Tatsushi Matsuyama, Professor Junichi Ida, Associate Professor Shinji Sato, Dr. Norio Nagao, Dr. Keiko Watanabe, Dr. Minako Kawai, Dr. Kenji Tsuchiya and Dr. Shinichi Akizuki for their invaluable advice and support. I am sincerely grateful to Mr. Yuichi Ezawa for his guidance, support and invaluable discussion throughout the first three years of the laboratory life, especially during my experiments, writings and presentations. I also appreciate to Mr. Koichi Izumi and Mr. Nobuyuki Nakatomi for advices during writings and presentations. I deeply thank to all my seniors and friends belong to Laboratory of Restoration Ecology for their support, encouragement and friendship. Special thanks goes to Ms. Hiroyo Takee, Ms. Keiko Mizubayashi, Mr. Kiyoshi Nakamura, Mr. Masatoshi Kishi and Ms. Yukiko Sasakawa, for their encouragement and sharing the invaluable time throughout three to six years. I i am also grateful to Ms. Hiromi Joo, Mr. Nobuo Nakahashi, Mr. Kazuhiro Kaneda, Mr. Hideyuki Taira, Mr. Masaaki Fujiwara, Ms. Midori Goto, Mr. Naoki Mizuno and Mr. Tang Jau Jiun for assistance of my experiments. Many thanks to “University-Industry Joint Research” Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology) 2009- 2013, “Environment Research and Technology Development Fund” from the Ministry of the Environment, Japan (4-1406), and Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows (15J12677) for funding this research. I also appreciate to Hokubu Sludge Treatment Center, Lake Biwa Policy Division and Ohmi Environment Conservation Foundation for the donation of materials for the present study. I would love to thank my parents and sisters and friends for their support, trust and understanding. I could never have done this work without their support, understanding and love. Finally, I am sincerely grateful to the founder of Soka University, Dr. Daisaku Ikeda for his sincere trust, great expectation, heartwarming guidance and tremendous encouragement. ii ABSTRACT In recent decades, aquatic macrophytes including floating, emergent and submerged macrophytes have been excessively propagated and causing various environmental problems in lakes, dams and reservoirs worldwide. In the case of Japan, submerged macrophytes have been propagated and causing water stagnation, foul odor, fishing interference and landscape fouling. The treatment of harvested macrophytes is one of the largest concern, but the effective and low-cost treatment has not been established yet. Anaerobic digestion enables the recovery of bioenergy as methane gas from biomass with high moisture content. From this point of view, the application of anaerobic digestion has been receiving its attention for the effective alternative treatment. Anaerobic digestion has been employed to the treatment of wide variety of bio-wastes such as wastewater, food waste, animal manure, agriculture waste, and sewage sludge in commercial scale. In recent years, lignocellulosic biomass has been expected for the prospective feedstocks for biomethane recovery. Substrates with slow degradation rate and/or low methane recovery are generally regarded as unsuitable for anaerobic digestion. Hence, the improvement of biomass biodegradability is required for lignocellulosic biomass such as submerged macrophytes, in order to successfully apply anaerobic digestion treatment. The overall research aim of this Ph.D. thesis is to examine anaerobic digestion process of submerged macrophytes and to enhance the anaerobic digestibility by chemical pre-treatment. First, anaerobic digestibility of five dominant