Enhancing the Environmental Sustainability of the Production and Degradation of PHA Polymers
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SUNY College of Environmental Science and Forestry Digital Commons @ ESF Dissertations and Theses Spring 5-10-2018 Enhancing the Environmental Sustainability of the Production and Degradation of PHA Polymers Joshua Harris [email protected] Follow this and additional works at: https://digitalcommons.esf.edu/etds Recommended Citation Harris, Joshua, "Enhancing the Environmental Sustainability of the Production and Degradation of PHA Polymers" (2018). Dissertations and Theses. 35. https://digitalcommons.esf.edu/etds/35 This Open Access Thesis is brought to you for free and open access by Digital Commons @ ESF. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of Digital Commons @ ESF. For more information, please contact [email protected], [email protected]. ENHANCING THE ENVIRONMENTAL SUSTAINABILITY OF THE PRODUCTION AND DEGRADATION OF PHA POLYMERS by Joshua R. Harris A thesis submitted in partial fulfillment of the requirements for Master of Science Degree State University of New York College of Environmental Science and Forestry Syracuse, NY May, 2018 Department of Chemistry Approved by: _____________________________ Christopher T. Nomura Major Professor _____________________________ Ivan Gitsov Ivanov Department Chair _____________________________ _____________________________ Gregory G. McGee S. Scott Shannon, Dean Thesis Defense Chair The Graduate School Acknowledgements First and foremost, I would like to give thanks to my advisor Christopher Nomura for allowing me the opportunity to be a part of his research group, both as an undergraduate student and for my master’s degree. I remember first meeting Chris in 2011 and asking to join his lab the very next day. Since then he has been a major influence in my life as a mentor, a role-model, and a friend. He has helped guide me in the realms of science, and sometimes life, and I give him a lot of credit for helping direct the trajectory of my career as scientist. I would also like to thank my other research mentor Benjamin Lundgren for the excellent hands on training and day-to-day guidance he has given me over the past several years. I have a lot of respect for Ben’s patience and willingness in his direction on how to work and write in a research laboratory at a professional level. In addition, I would like to thank some of the other post-docs that were part of the Nomura Research Group; Ata Pinto and Zaara Sarwar. I am very grateful to have been a part of this lab during the time that it had such phenomenal researchers so willing to help an aspiring scientist such as myself. Many thanks to my other steering committee members Gregory Boyer and Lee Newman. Dr. Newman was also my undergraduate advisor and her openness as a person and dedication as a scientist continue to be an inspiration. I would like to thank the Department of Chemistry here at SUNY-ESF as well as the university as a whole. I am proud to have had the privilege to earn my BS and MS degrees at such a wonderful and conscious institution. During my time here as a student I have formed many meaningful and lasting relationships with other students, especially my close friends Joseph Shoytush and Christopher Thomas. I am lucky to have had such rad buds that were willing to take the leap into graduate school with me at the ii same time and also willing to play frisbee in the quad when I needed a break from research (among other extracurricular activities). Thanks to some of the other members of the department Ryan Scheel and Robert Moesch and a special thanks to Joy Logan with whom I have had many long and meaningful conversations about science and the importance of this career path. Lastly, I would like to thank my parents for their continued encouragement to pursue whatever makes me happy. They don’t always understand exactly what I am doing, or why, but they have never stopped showing interest and unrelenting support for my choices in life. I consider myself to be exceedingly fortunate to have such a wonderful mother and father and I try to express that gratitude as often as possible. iii Table of Contents List of Figures ..………………………………………………………...……………………… vii List of Tables ………………………………………………………………………………...... viii Abstract ……………...…………………………….…………………………………...……..... ix Chapter 1. Introduction and Background 1.1 The ‘Age of Plastics’ 1.1.1 Global Plastic Production …………………………………………………………... 1 1.1.2 Plastics in Society ………………………………………………………………...... 2 1.1.3 Fossil Fuels and Plastics …..………………………………………………….…..... 3 1.1.4 Accumulation of Plastic in the Environment ………………………...…………...... 5 1.1.5 Chemical Structure of Plastics …………………………………………....……...… 6 1.1.6 Degradation of Synthetic Plastics ………………………………………………...... 9 1.1.7 Toxicity of Petroleum-Based Plastics ……………………..…………………….... 11 1.1.8 Moving Forward in the Plastic Age ………………………………….…………..... 13 1.2 The Biodegradable Plastic Revolution 1.2.1 Birth of an Industry …………………..…………………………………………… 14 1.2.2 Biosynthesis of Polyhydroxyalkanoates ………..………………………………… 16 1.2.3 Mechanical Properties of PHAs …..…………………….………………………… 19 1.2.4 PHA Biodegradation ………………………...……………………………….…… 22 1.2.5 Advancements in the PHA Industry …………………………...………….……… 24 1.2.6 Modern Plastic Society …………………………………...………….…………… 26 1.3 References ………………….…………………………………...………….…………… 28 Chapter 2. A rapid and efficient electroporation method for transformation of Halomonas sp. O-1 Abstract …………………………………………………………….……………………….. 37 2.1 Introduction 2.1.1 Halomonas; Halophilic PHA Producers …………………………………………... 38 2.1.2 Molecular Methods for Engineering Halomonas Bacteria ………………………... 39 2.1.3 Objectives of this Study ………………...…………………………….…………... 42 2.2 Materials and Methods 2.2.1 Bacterial Strains, Plasmids, and Plasmid Preparations ……….…………………… 43 2.2.2 Optimization of Halomonas Growth Conditions for Electroporation ….………… 44 2.2.3 Antibiotic Screening …..………………………………..………………………… 44 2.2.4 Preparation of Electrocompetent Cells ………………………........………….…… 45 2.2.5 Electroporation of Halomonas sp. O-1 ………………………...………….……… 45 2.2.6 Cell Viability Assays ……………………………………...………….…………… 46 iv 2.3 Results 2.3.1 Development of a Low Ionic Strength Growth Medium ……….………………… 46 2.3.2 Antibiotic Screening …..………………………………..………………………… 48 2.3.3 Preparation and Viability of Electrocompetent Cells ………………….…….…… 49 2.3.4 Electroporation of Halomonas sp. O-1 ………………………...………….……… 50 2.4 Discussion ………………….…………………………………...………….…………… 54 2.5 Conclusion ……………………………………………………...………….…………… 58 2.6 References ………………….…………………………………...………….…………… 59 Chapter 3. The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 (HbrC) and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1 Abstract …………………………………………………………….……………………….. 64 3.1 Introduction 3.1.1 Natural Occurrence of (R)-3-Hydroxybutyrate …………………………………... 65 3.1.2 Metabolism of (R)-3-HB in Pseudomonas aeruginosa …………...…….………... 66 3.1.3 Objectives of this Study ………………...…………………………….…………... 68 3.2 Materials and Methods 3.2.1 Bacteria, Plasmids and Media ………………………….…….…………………… 69 3.2.2 Standard DNA Procedures ….………………………………………………….… 70 3.2.3 Cloning of the bdhA (PA2003) and PA2004 Genes …………………..…..……… 70 3.2.4 Cloning of the PA2005 ORF ……………………...…………........………….…… 70 3.2.5 Construction of the PA2004-lacZ Reporter ………………………....…….……… 71 3.2.6 Growth studies of P. aeruginosa on (R,S)-3-HB ………………………......……… 71 3.2.7 b-Galactosidase (LacZ) Assays …………………………………......…….……… 72 3.2.8 Purification and Electrophoretic Mobility Shift Assays of His6-PA2005…………. 73 3.3 Results 3.3.1 Transposon Insertions into the bdhA (PA2003), PA2004 or PA2005 Gene Hindered the Growth of P. aeruginosa PAO1 on (R,S)-3-HB ……………..…… 74 3.3.2 Expression of a PA2004-lacZ Construct was Induced by (R)-3-HB ……………… 76 3.3.3 RpoN was Required for the Induction of PA2004- lacZ by (R)-3-HB ……………. 78 3.3.4 Induction of the PA2004-lacZ Reporter was Dependent on PA2005 ………...…… 79 3.3.5 (R)-3-HB Induced Expression of PA2004-lacZ in E. coli Top10 that Heterologously Expressed PA2005 …………………….……………….……… 80 3.3.6 Promoter Region of PA2004 was Bound by Purified His6-PA2005 ……...……… 82 3.3.7 Assimilation of (±)-1,3-butanediol Requires the bdhA Gene ................………….. 83 3.4 Discussion ………………….………………………………......………….…….……… 85 3.5 Conclusion ………………….……………………………….....………….…….……… 88 3.6 References ………………….………………………………...….………..……..……… 90 v Chapter 4. Conclusions and Outlook ……………………………………...………………….. 94 References ………………………………………………………………….....………. 100 Appendix A. Supplementary Material for Chapter 3 ……………….…………………...…. 102 Appendix B. Peer-review Publication Covers ……………….………….……………..……. 104 Appendix C. Additional Publication Contributed to by this Author ………………...……. 106 Vita ……………………………………...………………………..…..…….…………………. 115 vi List of Figures Chapter 1 Figure 1.1 Global Plastic Production Since 1950 .......................................................................... 2 Figure 1.2 Chemical Structure of Ethylene and Poly(ethylene) .................................................... 6 Figure 1.3 U.S. Resin Identification Classes .................................................................................. 8 Figure 1.4 Structures of Bisphenol A Compared to Estrogen products ........................................ 12 Figure 1.5 General Structure of Polyhydroxyalkanoates …………….......................................... 16 Figure 1.6 Structure of Selected PHA Monomers ……………..…………………...................... 17 Figure 1.7 Common Pathway