해양 플라스틱 오염 저감을 위한 플라스틱 분해 미세조류 개발 = Development of plastic-degrading microalgae for remediation of marine plastic pollution

목차 (Table of Contents)

• CHAPTER 1. Introduction 1
• 1. Overview 1
• 1.1. Marine plastic pollution 2
• 1.2. Microplastic in food system 10
• 1.3. Current strategies in plastic pollution management 13
• 2. Plastic-degrading enzyme 16
• 2.1. Polypropylene and polyethylene 16
• 2.2. Polyvinyl chloride and polystyrene 17
• 2.3. Polyethylene terephthalate 18
• 3. Engineered microbial chassis for PET biodegradation 19
• 3.1. Prokaryotes 19
• 3.2. Eukaryotes 22
• 4. Aims of this study 24
• CHAPTER 2. Functional expression of PET-degrading enzyme in
• model microalgae 26
• 1. Introduction 26
• 2. Materials and methods 30
• 2.1. Microalgal strain and cultivation condition 30
• 2.2. Design of microalgal expression vector 31
• 2.3. Transformation process 32
• 2.4. Protein preparation and Western blot analysis 35
• 2.5. Enzymatic activity assay and HPLC analysis 36
• 2.6. Scanning electron microscopy analysis 37
• 3. Results 38
• 3.1. Construction of the PETase expression vector and transformation
• of C. reinhardtii 38
• 3.2. Confirmation of the enzyme expression in C. reinhardtii 40
• 3.3. Demonstration of the enzyme activity against PET 42
• 4. Discussion 47
• 5. Conclusions 49
• CHAPTER 3. Development of high-performance PET-degrading
• marine microalgae 50
• 1. Introduction 50
• 2. Materials and methods 53
• 2.1. Microalgal strain and cultivation condition 53
• 2.2. Design and construction of expression vector and sgRNA 54
• 2.3. CRISPR/Cas9-mediated integration of the gene into microalga
• 55
• 2.4. Protein preparation and Western blot analysis 56
• 2.5. Enzyme activity assay and HPLC analysis 57
• 2.6. Identification of intrinsic plastic-degrading enzymes 58
• 3. Results 64
• 3.1. Generation of the enhanced PETase-expressing N. salina 64
• 3.2. Characterization of PETase expressed in N. salina 67
• 3.3. Characterization of endogenous PETase-like proteins in N. salina
• 75
• 4. Discussion 84
• 5. Conclusions 88
• CHAPTER 4. Stability assessment of the microalgae in plastic-polluted
• environments 90
• 1. Introduction 90
• 2. Materials and methods 92
• 2.1. Thermal, pH, salinity stability assessment 92
• 2.2. Cell cultivation in plastic-polluted marine environment 93
• 2.3. Measurement of ROS indicators (SOD, total antioxidants, MDA
• and DCFDA) 93
• 2.4. Catalytic activity and protein expression level analysis 96
• 2.5. Simulation of plastic-polluted marine environments 97
• 3. Results 98
• 3.1. Protein stability across marine conditions 98
• 3.2. Physiological and biochemical stability under microplastic
• exposure 104
• 3.3. Performance in large-scale simulated plastic-polluted marine
• environments 114
• 4. Discussion 118
• 5. Conclusions 120
• CHAPTER 5. Application of the microalgae for reducing plastic
• bioaccumulation in marine food chain 122
• 1. Introduction 122
• 2. Materials and methods 126
• 2.1. Preparation of marine zooplankton as food chain model 126
• 2.2. Experimental setup for microplastic bioaccumulation test · 126
• 2.3. Calculation of survival population of zooplankton 127
• 3. Results 128
• 3.1. Validation of the marine food chain model: zooplankton viability
• under microplastic-polluted environment 128
• 3.2. Microcosm approach to study effects of plastic-degrading
• microalga on bioaccumulation in the marine food chain· 134
• 4. Discussion 138
• 5. Conclusions 140
• CHAPTER 6. Risk assessment of the microalgae for in situ
• environmental bioremediation 142
• 1. Introduction 142
• 2. Materials and methods 147
• 2.1. Genome-based hazard screening 147
• 2.2. Cell preparation for oral administration 148
• 2.3. Experimental animals and bioethics 148
• 2.4. In vivo risk assessment of N. salina 149
• 2.5. Molecular analysis 150
• 3. Results 151
• 3.1. Taxonomic characterization and ecological distribution 151
• 3.2. Genetic safety assessment: pathogenicity, allergenicity, and
• toxicity analyses 155
• 3.3. In vivo gastrointestinal viability and toxicity evaluation in mouse
• model 170
• 4. Discussion 174
• 5. Conclusions 176
• CHAPTER 7. Conclusions 178
• BIBLIOGRAPHY 182