PhD Thesis on Microbial Conversion of Vegetable Oils to Hydroxy Fatty Acids and Bio-based Polyurethanes by Tran Tuan Kiet, Chungbuk National University

工學博士學位論文 미생물을 이용한 식물성 오일의 하이드록시 지방산 전환 및 바이오 기반 폴리우레탄 합성 응용 Microbial Conversion of Vegetable Oils into Hydroxy Fatty Acids and Their Application in Bio-based Polyurethane Synthesis 忠北大學校大學院 化學工學科 化學工學專攻 TRAN TUAN KIET 2019 年 08 月 工學博士學位論文 미생물을 이용한 식물성 오일의 하이드록시 지방산 전환 및 바이오 기반 폴리우레탄 합성 응용 Microbial Conversion of Vegetable Oils into Hydroxy Fatty Acids and Their Application in Bio-based Polyurethane Synthesis 指導敎授 金 範 洙 化學工學科 化學工學專攻 TRAN TUAN KIET 이 論文을 工學博士學位論文으로 提出함 2019 年 08 月 A Dissertation for the Degree of Doctor of Philosophy Microbial Conversion of Vegetable Oils into Hydroxy Fatty Acids and Their Application in Bio-based Polyurethane Synthesis Department of Chemical Engineering Chungbuk National University TRAN TUAN KIET August 2019 Microbial Conversion of Vegetable Oils into Hydroxy Fatty Acids and Their Application in Bio-based Polyurethane Synthesis Advisor: Professor Kim, Beom Soo By Tran Tuan Kiet, Department of Chemical Engineering, Chungbuk National University, Cheongju, South of Korea A thesis submitted to the Chungbuk National University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the department of Chemical Engineering August 2019 本 論文을 TRAN TUAN KIET 의 工學博士學位 論文으로 認定함. 위 원 장 김대수 ㊞ 부위원장 김범수 ㊞ 위 원 신채호 ㊞ 위 원 나병기 ㊞ 위 원 문종호 ㊞ 忠 北 大 學 校 大 學 院 2019 年 08 月 Contents Contents. VII List of Figures. X List of Tables.

XIV Chapter 1: Introduction. Hydroxy fatty acids (HFAs). Synthesis of HFAs. Types of HFAs.

α-HFA and β-HFA. Applications of HFAs. Pseudomonas aeruginosa PR3 and Pseudomonas aeruginosa KNU-2B. Synthesis of Polyurethanes.

Rigid polyurethane foams. Flexible polyurethane foams. Cell-free synthetic biology. Cell-free protein engineering.

Cell-free metabolic engineering. Artificial cell engineering. Aims and objectives of this study. 22 Chapter 2: Microbial Conversion of Vegetable Oil to 7,10-dihydroxy- 8(E)-octadecenoic acid and Its Application to Bio-Based Polyurethane Synthesis.

Materials and Methods. Media and Cultivation Conditions. Production of DOD from Olive Oil. Preparation of PUs Based on DOD.

Preparation of PUs Based on DOD and PEG or PCLDO. DSC and TGA. Results and Discussion. Production of DOD from Olive Oil.

Synthesis of PUs from DOD (PU-DOD). Synthesis of PUs from DOD and PEG or PCLDO (PU‐DOD/PEG or PU-DOD/PCLDO). 49 Chapter 3: Microbial Conversion of Castor Oil to 7,10,12-trihydroxy- 8(E)-octadecenoic acid and Its Application to Bio-Based Polyurethane Synthesis. Materials and Methods.

Media and cultivation conditions. Production of TOD from castor oil. Preparation of PUs from TOD. Preparation of blended PUs: PU-TOD/PEG and PU-TOD/PCLDO.

NMR and FTIR. DSC and TGA. Results and discussion. Production of TOD from castor oil.

Synthesis of PUs from TOD (PU-TOD). Synthesis of PUs from TOD and its blend (PU-TOD/PEG or PU- TOD/PCLDO). 84 Chapter 4: Production of 7,10-dihydroxy-8(E)-octadecenoic acid from Oleic Acid by Using Cell-free Synthesis Method. Materials and methods.

Media and cultivation conditions. DOD production by cell-free method. Analysis of products. Results and Discussion.

Identify the structure of product. DOD production by cell-free method. Optimum condition for DOD production from oleic acid by cell-free method. Effect of pre-culture time.

Effect of carbon sources. Effect of nitrogen sources. Effect of oleic acid concentration. Effect of initial pH and incubation temperature.

109 Chapter 5: Conclusions and Future Prospects. 113 V Acknowledgements I would like to express my sincere thanks and deepest respect to my supervisor Prof. Kim Beom Soo. Thank you for your instruction, guidance, support and encouragement throughout the duration of this study.

Without your help, I would not be able to complete my research. I would like to thank Dr. Prasun Kumar and all the members of the Biochemical Engineering Lab for helping me a lot in my research and making my time in the lab more interesting. I would also like to thank my friends at Chungbuk National University for sharing unforgettable memories during my time in Korea.

Finally, I would like to express my sincere thanks to my family. Thanks for always loving, caring, supporting and cheering for me. The love of family is a great motivation to help me overcome all difficulties in life. Thank you very much.

VI Microbial Conversion of Vegetable Oils into Hydroxy Fatty Acids and Their Application in Bio-based Polyurethane Synthesis Tran Tuan Kiet Department of Chemical Engineering Chungbuk National University Cheongju, South of Korea Supervised by: Professor Kim, Beom Soo Abstract New polyurethanes were synthesized based on dihydroxy fatty acid obtained by the microbial conversion of olive oil. Monounsaturated 7,10‐ dihydroxy‐8(E)‐octadecenoic acid (DOD) was produced from olive oil by Pseudomonas aeruginosa KNU-2B and reacted with hexamethylene diisocyanate (HMDI) at different ratios to form polyurethanes. Fourier transform infrared spectroscopy and gas chromatography/mass spectrometry confirmed the synthesis of DOD. The thermal and tensile properties of the polyurethanes were investigated by differential scanning calorimetry, thermogravimetric analysis, and a universal testing machine.

At an isocyanate/hydroxyl ratio of 1.4, the polyurethane exhibited an elongation at break of 59.2% and a high tensile strength of 37. DOD was also mixed VII with polycaprolactone diol or polyethylene glycol at different weight ratios and then reacted with HMDI to produce new polyurethanes of various properties. These polyurethanes displayed higher elongation at break and good thermal stability. This is the first report on the synthesis of polyurethanes based on DOD produced by the microbial conversion of vegetable oil.

Castor oil is a vegetable oil containing 85-95% ricinoleic acid, a substrate for tri-hydroxy fatty acids production. In this study, castor oil was used as a raw material to synthesize 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) by the strain Pseudomonas aeruginosa KNU-2B. Subsequently, HMDI was used in different ratios and reacted with TOD to produce new polyurethanes. Fourier transform-infrared spectroscopy, nuclear magnetic resonance spectroscopy, and gas chromatography/mass spectrometry were used to confirm the identity of TOD.

Differential scanning calorimetry, thermo-gravimetric analysis, and tensile property testing were used to investigate the thermal and mechanical properties of polyurethanes. Polyurethanes synthesized based on TOD had a tensile strength of 45.4 MPa with low elongation at break of 8.16% at an isocyanate/hydroxyl ratio of 2. To modify properties of polyurethane, TOD was blended separately with polyethylene glycol or polycaprolactone diol at different weight ratios before reacting with HMDI. The modified polymer showed improved thermal stability and comparatively higher elongation at break.

This is the first study VIII demonstrating the conversion of castor oil into TOD that can be used stand alone or as a blend for polyurethane synthesis. Pseudomonas aeruginosa KNU-2B is a new strain isolated from Pseudomonas aeruginosa PR3. This new strain was also capable of producing 7,10‐dihydroxy‐8(E)‐octadecenoic acid (DOD) from oleic acid but the production yield was lower than by PR3. Cell-free synthesis method is known for many advantages such as fast production rate, high product content.

In this study, for the first time, cell-free synthesis method was applied to DOD production from oleic acid by strain KNU-2B to improve this process. The results showed faster production time and higher product concentration than conventional method. The production of DOD from oleic acid reached the highest of 6.41 g/L of DOD in 36 hours. Various factors were changed to find the optimal conditions for production.

Among the carbon sources tested, galactose was most effective for DOD production. Sodium glutamate and diammonium phosphate were most effective for DOD production as using for sole nitrogen source. The optimal pH and incubation temperature were 8 and 27oC, respectively. IX List of Figures Figure 1.

Biosynthetic pathway of oleic acid to 7,10-dihydroxy-8(E)- octadecenoic acid. Application of Polyurethanes. The chemical reaction of Polyurethanes (A) and their classification (B). Types of polyol.

Engineering protein, metabolism and artificial cell in the open cell-free system. FTIR spectra of the product produced from olive oil by P. EI mass spectra of the major product produced from olive oil by P. FTIR spectra of polyurethane‐7,10‐dihydroxy‐8(E)‐octadecenoic acid (PU-DOD) with different NCO/OH ratios.

Differential scanning calorimetry (DSC) analysis of PU‐DOD with different NCO/OH ratios. (a) TGA and (b) derivative thermogravimetry (DTG) thermograms of PU-DOD with different NCO/OH ratios. Stress-strain curves of PU‐DOD with different NCO/OH ratios. Reaction schemes for the synthesis of PU‐DOD/PCLDO and PU‐ DOD/PEG.

FTIR spectra of (a) PU-DOD/PEG200, (b) PU-DOD/PEG20K, and (c) PU-DOD/PCLDO. TGA and derivative thermogravimetry (DTG) thermograms of (a) PU-DOD/PEG200, (b) PU-DOD/PEG20K, and (c) PU-DOD/PCLDO. Tensile properties of (a) PU-DOD/PEG200, (b) PU- DOD/PEG20K, and (c) PU-DOD/PCLDO. Bio-conversion of castor oil to TOD by P.

FTIR spectra of the major product produced from castor oil by P. GC/MS of the major product produced from castor oil by P. 1H-NMR spectra of product produced from castor oil by P. FTIR spectra of PU-TOD with different NCO/OH ratios and PU- CO.

DSC analysis of PU-TOD with different NCO/OH ratios. (a) TGA and (b) derivative thermogravimetry (DTG) thermograms of PU-TOD with different NCO/OH ratios. Tensile properties of PU-TOD with different NCO/OH ratios and PU-CO. Reaction scheme for the synthesis of PU-TOD, PU-TOD/PCLDO and PU-TOD/PEG.

FTIR spectra of PU-TOD/PEG200, PU-TOD/PEG20K, and PU- TOD/PCLDO. TGA and derivative thermogravimetry (DTG) thermograms of (a) PU-TOD/PEG200, (b) PU-TOD/PEG20K, and (c) PU-TOD/PCLDO. Tensile properties of (a) PU-TOD/PEG200, (b) PU- TOD/PEG20K, and (c) PU-TOD/PCLDO. EI mass spectra of the major product produced from oleic acid by cell-free method.

1H-NMR spectra of product produced from oleic acid by cell-free method. Comparison of DOD production from oleic acid between conventional method and cell-free method. Effect of pre-culture time on DOD production from oleic acid. Effect of carbon source on DOD production from oleic acid.

Effect of organic nitrogen source on DOD production from oleic acid. Effect of inorganic nitrogen source on DOD production from oleic acid. Effect of substrate concentration on DOD production from oleic acid. Effect of initial pH on DOD production from oleic acid.

Effect of incubation temperature on DOD production from oleic acid. 107 XIII List of Tables Table 1. Production of DOD and TOD from fatty acid and vegetable oil by P. Thermal and mechanical properties of PU‐DOD.

Thermal and mechanical properties of PUs from DOD with PEG or PCLDO. Gel content of PU-TOD, PU-TOD/PEG and PU-TOD/PCLDO. Thermal and mechanical properties of PU-TOD. Thermal and mechanical properties of PU-TOD/PEG and PU- TOD/PCLDO.

86 XIV Chapter 1: Introduction 1. Hydroxy fatty acids (HFAs) HFAs are saturated or unsaturated fatty acids with a branchless long carbon chain containing one or more hydroxyl (-OH) groups and a carbonyl (- COOH) group at the end of the circuit. In nature, HFAs are found in waxes, cerebellum, and lipids of animals, plants and microorganisms. Compared with normal fatty acids, HFAs have higher stability, viscosity, reactivity and solubility with solvents.

Synthesis of HFAs Although possessing outstanding properties, HFAs are not commercially available because of their high cost and their synthetic pathways are not environmentally friendly. The chemical synthesis pathway of HFAs has many limitations: multiple steps, poor selectivity, harsh reaction conditions, toxic chemicals and low product diversity [1]. These factors have created barriers to the potential of commercialization of HFAs. However, the emergence of microorganisms capable of synthesizing HFAs has created a new step in improving the value of these fatty acids.

These strains are reported to synthesize HFAs through the conversion of fatty acids or vegetable oils with a variety of different enzymes. In general, there are four enzymes involved in the conversion of fatty acids into HFAs: hydratase, cytochrome P450 1 monooxygenase, lipoxygenase, and hydroxylase [2]. Many studies have shown that different enzymes metabolize different HFAs such as 13- hydroxyoctadecadienoic acid (HODE), 10-hydroxystearic acid and ω-hydroxy fatty acid produced by lipoxygenase [3], hydratase [4] and cytochrome P450 monooxygenase (P450) [5], respectively. Compared to chemical synthesis, the production of HFAs through microbial processes is easier, does not affect the environment, and reduces production costs.