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Development of Efficient Bioprocesses for the Conversion of Carbon Monoxide : Fermentative Strategy for Bioethanol and Enzymatic Conversion for Formic Acid

  • 김태환 (서강대학교 일반대학원)

초록 (요약문)

This abstract outlines the collective insights and breakthroughs of a comprehensive study focused on the development of bioprocesses for the production of value-added products using carbon monoxide (CO). Recognizing the potential of CO as a feedstock, this study presents pioneering research in converting CO into valuable chemical commodities, particularly bioethanol and formic acid, through bioprocessing. The potential of using carbon monoxide for converting into value-added chemicals is a major area of interest in current environmental and industrial research. Notably, the processes for bioethanol and formic acid production have been highlighted as exemplary, demonstrating significant potential in leveraging CO for the sustainable generation of these chemicals. Despite the promising outlook, the development of these bioprocesses encounters several limitations, including the challenges associated with the cultivation of anaerobic microbes, the inefficiency and instability of biocatalysts, and the low CO mass transfer rates which collectively impede efficacy and commercial scalability of the process. To address these challenges, this research has developed two groundbreaking bioprocesses. The first innovation encompasses an efficient CO to ethanol conversion process utilizing a novel two-stage continuous system with Clostridium autoethanogenum, achieving unprecedented bioethanol concentrations. This method ingeniously integrates high-density cell cultivation with a co-feeding strategy and a cell recycling approach, resulting in the highest bioethanol titer produced continuously over the last three decades. The second advancement pertains to the efficient conversion of CO to formic acid, explored through two distinct approaches. The initial method employs permeabilized whole-cell biocatalysts and an external electron mediator, significantly enhancing formic acid production rates by circumventing the need for enzyme purification or immobilization. The second approach leverages non-conventional media with elevated CO solubility to improve CO mass transfer rates, utilizing a combination of engineered biocatalysts and immobilized enzymes. This innovative strategy effectively overcomes the limitations posed by low CO solubility, showcasing a substantial increase in the efficiency of formic acid production. In summary, this dissertation not only identifies and addresses the key limitations hindering the bioproduction of value-added chemicals from CO but also introduces novel bioprocesses that significantly enhance the production efficiencies of bioethanol and formic acid. These contributions underscore the feasibility of using CO as a renewable feedstock for the sustainable and efficient generation of valuable chemicals, marking a significant stride towards the advancement of biotechnological applications and environmental sustainability. Keywords Carbon monoxide, Syngas fermentation, Bioprocess, Bioethanol, Formic acid

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목차

1. Introduction 1
1.1 Overview 1
1.2 Objectives of this research 2
1.3 References 7
2. Two-stage continuous CO fermentation process strategy for high-titer bioethanol production using Clostridium autoethanogenum 9
2.1 Introduction 9
2.2 Materials and methods 14
2.2.1 Microorganism and media 14
2.2.2 Investigation of growth and production enhancement factors in serum vial 15
2.2.3 Cell recycling for high-density cultivation 16
2.2.4 Reactor setup and operation 17
2.2.5 Analytical procedure 22
2.3 Results and discussions 22
2.3.1 Co-feeding effects with fructose and syngas 22
2.3.2 Product tolerance investigation 26
2.3.3 Cell-recycling for high-density cultivation 28
2.3.4 Evaluating bioethanol production potential after rapid high-density cell culture in favorable growth conditions 30
2.3.5 Sodium tungstate effects on bioethanol production 36
2.3.6 Two-stage process for continuous high-titer bioethanol production 39
2.4 Conclusion 47
2.5 References 48
3. Novel CO to formic acid bioconversion process using permeabilization-boosted inter-cellular cascade reaction between whole-cell biocatalysts 53
3.1 Introduction 53
3.2 Materials and methods 57
3.2.1 Preparation of whole cell biocatalysts 57
3.2.2 Preparation of enzymes 57
3.2.3 Intercelluar cascade reaction using whole cell biocatalysts 58
3.2.4 Optimization of intercelluar cascade reaction using whole-cell biocatalysts 59
3.2.5 Comparison of intercelluar cascade reaction system using whole-cell biocatalysts and immobilized enzyme system 60
3.2.6 Comparison of activity assays for enzymes and whole-cell biocatalysts 62
3.2.7 Changes in cascade reaction rate following treatment of M. extorquens AM1 with permeabilizing agents 63
3.2.8 Performance measurement of M. extorquens AM1 treated with 1% Triton X-100 Compared to untreated whole-cell and immobilized Enzyme 63
3.2.9 Confirmation of permeabilization by TEM imaging 65
3.2.10 Permeabilization-boosted intercellular cascade reaction in BCR 65
3.2.11 Analytical procedure 66
3.3 Results and discussion 67
3.3.1 Characterization of the intercellular cascade reaction of whole-cell biocatalysts using an extracellular electron mediator 67
3.3.2 Optimization of intercellular cascade CO to formic acid reaction using whole-cell biocatalysts 70
3.3.3 Permeabilization-boosted intercellular cascade reaction to overcome mass transfer limitation of ethyl viologen dibromide 77
3.3.4 Bioprocess operation using permeabilization-boosted intercellular cascade reaction system 86
3.4 Conclusion 90
3.5 References 91
4. Development of high-speed CO to formic acid conversion bioprocess using high CO soluble alternative solvents as reaction media 95
4.1 Introduction 95
4.2 Materials and methods 99
4.2.1 Preparation of whole-cell biocatalysts 99
4.2.2 Preparation of free enzymes and immobilized enzymes 100
4.2.3 Investigation and characterization of high-co soluble alternative solvents 101
4.2.4 Measurement of whole-cell biocatalyst stability in solvents over 3 hours 102
4.2.5 Measurement of enzyme stability in solvents over 3 hours 104
4.2.6 SEM imaging of whole-cell biocatalysts 104
4.2.7 Verification of increased volumetric productivity using high CO soluble solvent in serum vial 105
4.2.8 5 days viability of whole-cell biocatalysts 106
4.2.9 Investigation of conversion rates in different reaction media with identical gas solubility 107
4.2.10 Verification of increased maximum volumetric productivity due to enhanced CO solubility in high-solubility solvents 108
4.2.11 High-speed CO to formic acid conversion process using high-CO soluble solvents 110
4.2.12 Analytical procedure 111
4.3 Results and discussion 112
4.3.1 Investigation and feasibility assessment of high CO-soluble alternative solvents 112
4.3.2 Evaluation of biocatalyst viability in high CO soluble alternative solvents 117
4.3.3 Investigation of factors contributing to enhanced production rates in solvents 131
4.3.4 Verification of theoretical and actual CO to formic acid conversion rate improvements with high-CO soluble solvent 134
4.3.5 High-speed CO to formic acid conversion process using high-CO soluble alternative solvent 136
4.4 Conclusion 141
4.5 References 142
5. Conclusion and Perspective 147
5.1 Conclusion 147
5.1.1 Two-stage continuous CO fermentation process strategy for high-titer bioethanol production using Clostridium autoethanogenum 147
5.1.2 Novel CO to formic acid bioconversion process using permeabilization-boosted inter-cellular cascade reaction between whole-cell biocatalysts 148
5.1.3 Development of high-speed CO to formic acid conversion bioprocess using high CO-soluble alternative solvents as reaction media 149
5.1.4 Final remarks 150
5.2 Perspective of Further study 151
5.2.1 Further development of CO conversion processes using high-CO soluble alternative solvents 151
5.2.2 Development of modulated CO to formic acid conversion system 152
5.2.3 Development of a novel CO to formate ester conversion bioprocess 153
Vita 155
Achievements 156

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