South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange Electronic Theses and Dissertations 2020 Developing Microbial Based Process to Produce High Value Natural Antimicrobial (Glyceollin) in Soybeans and Pullulan from Sucrose Andrea Zavadil South Dakota State University Follow this and additional works at: https://openprairie.sdstate.edu/etd Part of the Agronomy and Crop Sciences Commons, Environmental Microbiology and Microbial Ecology Commons, and the Plant Biology Commons Recommended Citation Zavadil, Andrea, "Developing Microbial Based Process to Produce High Value Natural Antimicrobial (Glyceollin) in Soybeans and Pullulan from Sucrose" (2020). Electronic Theses and Dissertations. 5002. https://openprairie.sdstate.edu/etd/5002 This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. DEVELOPING MICROBIAL BASED PROCESS TO PRODUCE HIGH VALUE NATURAL ANTIMICROBIAL (GLYCEOLLIN) IN SOYBEANS AND PULLULAN FROM SUCROSE BY ANDREA ZAVADIL A thesis submitted in partial fulfillment of the requirements for the Master of Science Major in Biological Sciences Specialization in Microbiology South Dakota State University 2020 ii THESIS ACCEPTANCE PAGE Andrea Zavadil This thesis is approved as a creditable and independent investigation by a candidate for the master’s degree and is acceptable for meeting the thesis requirements for this degree. Acceptance of this does not imply that the conclusions reached by the candidate are necessarily the conclusions of the major department. Bishnu Karki Advisor Date Heike Bucking Department Head Date Nicole Lounsbery, PhD Director, Graduate School Date iii This thesis is dedicated to my family. To my mom and dad, for pushing me to work hard and believe in myself from the start, while always remembering to take the time to laugh and remain humble. To Nicole and Jason for being the best friends I can laugh with along the way. This work would not have been possible without their constant love and encouragement. iv ACKNOWLEDGEMENTS I would like to thank the following people for their support in obtaining my Masters degree: Dr Bishnu Karki for encouraging my interest in industrial microbiology from my undergraduate to graduate career, for her patience and forever willingness to extend guidance for my research, conferences, and other goals. To Dr. William Gibbons, for allowing me to continue on with learning in my graduate career, and always giving guidance on any project I undertook. To my graduate committee members Dr. Tofuko Woyengo and Dr. Zhong Hu for their input and support throughout my graduate career. To Stephanie Wootton and Burgandy Zschedtzsche for teaching me the ropes when I first joined the lab, staying patient, and making my graduate career so fun. To my campus co- workers Dr. Tom Kasiga, Dr. Jaimie Gibbons, Ahmad Alhomodi, Camille Massmann, and Allison Braun for never saying no to calls for an extra hand, helping count yeast cells, and making my time in lab so enjoyable. To Dr. Mark Berhow of the USDA for his expertise for HPLC analysis and giving guidance throughout my projects. To Nathan Morris, Alethea Caraway, and all team members at Prairie Aquatech for their patience and teaching me how to use the LECO 528 for protein analysis. And finally, to the South Dakota Oilseed Initiative, the South Dakota Oilseed Commission, and the SD Agricultural Experiment Station at SDSU for their funding support that made my research possible. v TABLE OF CONTENTS ABBREVIATIONS ............................................................................................... vii LIST OF FIGURES .............................................................................................. viii LIST OF TABLES .................................................................................................. x ABSTRACT .......................................................................................................... xii Chapter I. Literature Review .................................................................................. 1 Chapter II. Introduction ......................................................................................... 5253 Chapter III. Fungal Inoculation by Trichoderma reesei and Aspergillus sojae on Glycine max Seeds at Varying Maturity Level to Enhance Glyceollin Production ............ 59 Introduction ........................................................................................................... 60 Materials and Methods .......................................................................................... 62 Results. .................................................................................................................. 66 Discussion .............................................................................................................. 78 Conclusion ............................................................................................................. 82 Chapter IV. UV Light Cotreatment of A. sojae Inoculated Soybeans to Elicit Glyceollin Expression ............................................................................................................. 84 Introduction ........................................................................................................... 85 Materials and Methods .......................................................................................... 87 Results. .................................................................................................................. 90 Discussion .............................................................................................................. 97 Conclusion ........................................................................................................... 101 vi Chapter V. Determination of Aureobasidium pullulans Growth on Various Concentrations of Sucrose Based Media in Batch Fermentation for Pullulan Production…. ....................................................................................................... 103 Introduction ......................................................................................................... 104 Materials and Methods ........................................................................................ 106 Results. ................................................................................................................ 111 Discussion ............................................................................................................ 127 Conclusion ........................................................................................................... 135 Chapter VI. Summary and Conclusions .............................................................. 137 Chapter VII. Literature Cited .............................................................................. 144 vii ABBREVIATIONS CYP93A1 – 3, 9 – di hydroxy pterocarpan 6a monooxygenase DDGS – distiller dried grains with solubles HPLC – high performance liquid chromatography GES – glyceollin enriched soybeans GYE – glucose yeast extract G2DT - glycinol 2 – dimethylallytransferase G4DT – glycinol 4 – dimethylallytransferase IFS – isoflavone synthase IFS1 – 2 hydroxy isoflavone synthase 1 IFS2 - 2 hydroxy isoflavone synthase 2 MG – maturity group PDA – potato dextrose agar PAL – phenylalanine ammonium – lyase SSF – solid state fermentation TFA – trifluoracetic acid UGT – uridine diphosphate glycotransferase UV – ultraviolet light 2HID – 2hydroxyisoflavone dehydratase viii LIST OF FIGURES Figure 1.1: Soybean maturity levels in the United States. ................................................ 18 Figure 1.2: Germination process of soybean seed ............................................................. 20 Figure 1.3: Phenylpropanoid pathway for the production of Flavonoids and Isoflavonoids ........................................................................................................................................... 24 Figure 1.4: Biosynthesis of three glyceollin isomers I, II, III, starting with precursor molecule daidzein .............................................................................................................. 30 Figure 1.5: Glyceollin I, II, and III biosynthesis from daidzein as part of the phenylpropanoid pathway ................................................................................................ 32 Figure 1.6: Genes that are upregulated or downregulated in cell suspension (green) and cotyledon tissues (red) after stress from 2% oligosaccharides. ......................................... 33 Figure 1.7: Basic structure of pullulan .............................................................................. 43 Figure 3.1: Comparison of T. reesei spore’s vs mycelium inoculums on Total Glyceollin (mg/g) production at 96 h of incubation by different soybean varieties. .......................... 67 Figure 3.2: Growth of AG69X6 soybeans at (a) 0 h uninoculated (b) 96 h control (c) 96 h T. reesei inoculated and (d) 96 h A. sojae inoculated. ....................................................... 68 Figure 3.3: Total Glyceollin (mg/g) produced