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(SIRE) Followed by Back-Extraction (BE) Process for Efficient Fermentation of Ketose Sugars to Products

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(SIRE) Followed by Back-Extraction (BE) Process for Efficient Fermentation of Ketose Sugars to Products A Dissertation entitled Optimizing Simultaneous-Isomerization-and-Reactive-Extraction (SIRE) Followed by Back-Extraction (BE) Process for Efficient Fermentation of Ketose Sugars to Products by Peng Zhang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Engineering ________________________________________ Dr. Patricia Relue, Co-Committee Chair ________________________________________ Dr. Sasidhar Varanasi, Co-Committee Chair ________________________________________ Dr. Sridhar Viamajala, Committee Member ________________________________________ Dr. Stephen Callaway, Committee Member ________________________________________ Dr. Randall Ruch, Committee Member ________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo May 2018 Copyright 2018, Peng Zhang This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Optimizing Simultaneous-Isomerization-and-Reactive-Extraction (SIRE) Followed by Back-Extraction (BE) Process for Efficient Fermentation of Ketose Sugars to Products by Peng Zhang Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Engineering The University of Toledo May 2018 Lignocellulosic biomass is an abundant, inexpensive feedstock. It is mostly composed of cellulose (38-50% of the dry mass) and hemicellulose (23-32% of the dry mass). Cellulose and hemicellulose are polysaccharides that can be hydrolyzed to monosaccharides, mostly glucose and xylose. These sugars can eventually be fermented to many different products, such as ethanol and 2,3-butanediol. Fuel ethanol, which is currently produced from food-based sugars, can also be produced via fermentation of sugars derived from lignocellulosic biomass. The fermentation of xylose is essential for the cost-effective bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose because the metabolic pathways convert xylose to xylitol via an NADPH-linked xylose reductase. Fermentation of xylose to ethanol through xylulose does occur in organisms which possess an NADH-linked aldose reductase, indicating that a balanced supply of NADH and NADPH must be maintained to avoid xylitol production. Although S. cerevisiae does not convert xylose to ethanol, it does have the metabolic pathway for the conversion of xylulose, the ketose isomer of xylose, to ethanol. Conversion of xylose to iii xylulose in high yield and at low cost from biomass hydrolysate has the potential to bypass the barrier to ethanol production from C5 and C6 sugars with native yeast. 2,3-Butanediol (2,3-BD) is a key building block and a promising bulk chemical due to its extensive industrial applications in making polymers, plastics, and hydrocarbon fuels. For example, 2,3-BD can be readily converted to butenes, butadiene, and methyl ethyl ketone that are used in the production of hydrocarbon fuels. Enterobacter cloacae NRRL B-23289, isolated from decaying wood/corn soil samples by the USDA Agricultural Research Service (Peoria, IL), is a natural producer of 2,3-BD. Previous work at the USDA ARS has shown that this strain is more efficient in converting ketose than aldose sugars to 2,3-BD. Particularly interesting is that fermentation of fructose showed higher 2,3-BD yield within a much shorter period of time as compared to glucose. Converting aldoses to ketoses involves isomerization, typically conducted enzymatically. However, the isomerization does not have a favorable equilibrium with respect to ketose formation. We have previously developed a method of simultaneous isomerization and reactive extraction (SIRE) to produce the ketose isomer of xylose (xylulose) in high yield and purity. SIRE-followed by back-extraction (BE) allows recovery of xylulose in nearly pure form. Although successfully implemented with low concentration xylose, SIRE has not been tested for high concentration sugars (C5 and C6 mixtures), which would be relevant for biomass hydrolysates. Optimization of SIRE-BE with high concentrations of both C5 (xylose/xylulose) and C6 (glucose/fructose) is one of the objectives of this dissertation. Using this innovative and optimized method to pretreat the aldose sugars (glucose and xylose) and produce large quantities of nearly pure, concentrated ketose sugars iv (fructose and xylulose), the production of 2,3-butanediol was investigated. Ketose sugar fermentation yielded more 2,3-butanediol and in a shorter time than the aldose fermentations. Using this method to produce large quantities of nearly pure, concentrated xylulose, the production of ethanol was also investigated. The native yeast produced 0.44- 0.45 g ethanol/ g xylulose in xylulose fermentation. v This dissertation is dedicated to my father (1954-1999), my grandmother (1929-2003) and my grandfather (1929-1995). Acknowledgements I would like to express the deepest appreciation to my advisors: Dr. Patricia Relue and Dr. Sasidhar Varanasi. Without their guidance and persistent help, my research and this dissertation would not have been possible. I would like to thank my committee members: Dr. Sridhar Viamajala, Dr. Stephen Callaway, and Dr. Randall Ruch, for giving me advice on my research. In addition, a thank you to Tammy Phares, our lab coordinator, who helped me with my experimental set-ups. I also thank my lab mates: Bin Li, Heng Shao, Kelly Marbaugh, and Jeremy Schreur, for their assistance and support. I would also like to thank to the College of Engineering, especially the Department of Bioengineering, for giving me the opportunity and supporting my Ph.D. study here. Last but not least, a special thank you to my family, especially my wife and my mother, for their endless love, encouragement and support during this journey. A sincere thank you to my best friends, I feel fortunate to have them at my back. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables ......................................................................................................................x List of Figures .................................................................................................................... xi 1 Overview ..................................................................................................................1 1.1 Background ........................................................................................................1 1.1.1 Simultaneous-isomerization-and-reactive-extraction (SIRE), followed by back-extraction (BE) .......................................................4 1.1.2 Ethanol Production..............................................................................8 1.1.3 2,3-Butanediol Production ................................................................10 1.2 Objectives and Significance .............................................................................12 1.2.1 Optimize the SIRE-BE process with high concentrations of both C5 and C6 sugars ....................................................................................13 1.2.2 Evaluate the ethanol yield of native S. cerevisiae on fermentation of xylulose and xylulose/glucose mixed sugars from the SIRE-BE process ..............................................................................................13 1.2.3 Investigate 2,3-butandiol production from single and mixed sugars by Enterobacter cloacae NRRL B-23289 ........................................13 vi 1.3 Organization of the Dissertation ......................................................................13 2 Kinetics of Isomerization and SIRE-BE Process with High Concentration of Single Sugar… .......................................................................................................15 2.1 Introduction ......................................................................................................15 2.2 Method and Material ........................................................................................16 2.2.1 Chemicals and materials ....................................................................16 2.2.2 Sugar isomerization ...........................................................................16 2.2.3 SIRE-BE with high concentration of single sugar ............................17 2.2.4 Analytical techniques and data analysis ............................................18 2.3 Results and Discussion .....................................................................................18 2.3.1 Kinetic of glucose isomerization .......................................................18 2.3.2 Kinetic of xylose isomerization .........................................................21 2.3.3 Kinetic of mixed sugars isomerization ..............................................21 2.3.4 SIRE with 165 mM and 490 mM N2B in the organic phase for C6 sugars ................................................................................................26 2.3.5 SIRE with
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