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Identifying and Characterizing Novel Bacilli Capable of Degrading Recalcitrant Polymers

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Identifying and Characterizing Novel Bacilli Capable of Degrading Recalcitrant Polymers University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2019 Breaking the Chain: Identifying and Characterizing Novel Bacilli Capable of Degrading Recalcitrant Polymers Kyle Bonifer University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Bonifer, Kyle, "Breaking the Chain: Identifying and Characterizing Novel Bacilli Capable of Degrading Recalcitrant Polymers. " PhD diss., University of Tennessee, 2019. https://trace.tennessee.edu/utk_graddiss/5738 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Kyle Bonifer entitled "Breaking the Chain: Identifying and Characterizing Novel Bacilli Capable of Degrading Recalcitrant Polymers." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Microbiology. Todd Reynolds, Major Professor We have read this dissertation and recommend its acceptance: Elizabeth Fozo, Gladys Alexandre, Jennifer Debruyn Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Breaking the Chain: Identifying and Characterizing Novel Bacilli Capable of Degrading Recalcitrant Polymers A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Kyle Sabastian Bonifer December 2019 Copyright © 2019 by Kyle S. Bonifer All rights reserved. ii ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. Todd Reynolds, for all his help during my six years in graduate school. I have struggled tremendously throughout this process, having had many failed projects and ideas that did not come to successful fruition. Dr. Reynolds has been the most patient and understanding person that I could have hoped for, helping me find my way and allowing me to grow as a scientist. I will always hold him in the highest regard and strive to be as great a scientist and a person as he is. In graduate school, I have had committee members and collaborators who have always been there to shed new light and provide perspectives on the problems I was trying to solve. My former and current committee members, Drs. Jeffrey Becker and Robert Wheeler, Elizabeth Fozo, Jennifer Debruyn and Gladys Alexandre have always been willing to take my questions and critique my work. I have learned a lot from them and would not have been able to complete this work without their help. The people who I have worked with in the Reynolds lab have been a driving force for new ideas, collaboration and comedic relief. I would like to thank Bob Tams, Chelsi Cassilly, Tian Chen, Sahar Hasim, Sarah Davis, Andy Wagner, Billy Carver, Elise Phillips and Aeric Zhou for making the thousands of hours we’ve spend together memorable and enjoyable. I would also like to thank all the people in the Microbiology department for contributing to both my scientific and social development. I will miss the camaraderie and council I have had with all my peers, many of whom have always been willing to talk about science and life over beers. I also want to extend a huge thank you to the love of my life, Tamara Demchenko, who has been with me since the beginning of my journey in graduate school. Whether it be her words of wisdom, love and affection or cleaning up my graphs and posters with her designer magic, she has contributed more to my success than anything else. Without her support and confidence in me, I don’t think I could have finished this process. Finally, I’d like to send out a thank you to all my friends and family for their support, patience and understanding these past six years. No process like this can be completed by one person and having this group of people in my life has been key to my success. iii ABSTRACT The development of plastic polymers has revolutionized modern society. These polymers are found ubiquitously in commodities and the production of these plastics is one of the most profitable industries in the world. However, the very characteristics that make plastics so universal and useful is also leading to detrimental ecological impacts. Due to their prevalence, finding novel microorganisms capable of breaking down these plastics is an important task. Another way these problems have are being addressed is by creating plastics that are more amenable to degradation. These plastics contain moieties more easily recognized by enzymes created by microbial life. Due to the increased adoption of these plastics, its important to understand their effect on the biology of microbial life. In this dissertation we developed a biochemical assay to detect lactic acid that was being released as monomer from the biodegradable polymer polylactic acid due to bacterial degradation. We found a novel isolate of Bacillus pumilus (B12) that is capable of breaking down polylactic acid, with this degradation being able to robustly within 48 hours of incubation. Next we used our assay to observe how changing the nutrient composition allotted to B. pumilus B12 will alter its rate of PLA degradation. We saw increases in degradation when B. pumilus B12 was given maltose and mannitol as a sole carbon source, while observing decreases with adenine, potassium and phosphate supplementations. Next we used proteomic and transcriptomic tools to identify genes differentially expressed in conditions with altered PLA degradation. We found that in conditions with a lowered level of PLA degradation, genes associated with motility, chemotaxis and protein export are all downregulated. Next, we investigated contamination found in a surfactant rich environment for novel bacteria capable of breaking down polyethylene and other recalcitrant carbon sources. We found six strains of novel Bacilli that were capable of growing on polysorbates, polyethylene glycol and mineral oil-rich in alkenes. Upon incubating one isolate of Bacillus megaterium with polyethylene films, we observed microbial growth, a lowered pH and a loss of tensile strength of the plastic. These two projects contribute new assays, bacterial isolates and insight to the field of polymer degradation. iv TABLE OF CONTENTS Chapter I: Introduction .................................................................................................... 1 The Costs and Benefits of Man-Made Polymers ............................................................... 2 Microbial-Derived Enzymatic Degradation of Natural Polymers ...................................... 4 Plastic Polymer Degradation............................................................................................... 7 Summary ........................................................................................................................... 14 References ......................................................................................................................... 16 Chapter II: Bacillus pumilus B12 Degrades Polylactic Acid and Degradation is Altered by Changing Nutrient Conditions....................................................................................... 33 Abstract ............................................................................................................................. 35 Introduction ....................................................................................................................... 36 Methods............................................................................................................................. 37 Results ............................................................................................................................... 40 Discussion ......................................................................................................................... 71 References ......................................................................................................................... 81 Appendix ........................................................................................................................... 84 Chapter III: Bacillus pumilus B12 Alters its Gene Expression in Conditions that Reduce PLA Degradation ..................................................................................................................... 88 Abstract ............................................................................................................................. 90 Introduction ....................................................................................................................... 91 Methods............................................................................................................................. 92 Results ............................................................................................................................... 97 Discussion ....................................................................................................................... 115 References ......................................................................................................................
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