UNIVERSITY OF CALIFORNIA RIVERSIDE Factors Contributing to Recalcitrance of Poplar to Deconstruction A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemical and Environmental Engineering by Samarthya Bhagia June 2016 Dissertation Committee: Dr. Charles E. Wyman, Chairperson Dr. Eugene A. Nothnagel Dr. Ian Wheeldon Copyright by Samarthya Bhagia 2016 The Dissertation of Samarthya Bhagia is approved: Committee Chairperson University of California, Riverside ACKNOWLEDGEMENTS I express my gratitude to the Department of Chemical and Environmental Engineering in Bourns College of Engineering at the University of California Riverside for giving me the opportunity to carry out graduate study towards a Doctor of Philosophy in Chemical and Environmental Engineering. I am grateful for funding by the Office of Biological and Environmental Research in the Department of Energy (DOE) Office of Science through the BioEnergy Science Center (BESC) at Oak Ridge National Laboratory (Contract DE-PS02- 06ER64304). I also acknowledge the Center for Environmental Research and Technology (CE-CERT) of the Bourns College of Engineering for providing the facilities and the Ford Motor Company for funding the Chair in Environmental Engineering that facilitates projects such as this one. I’m indebted to my adviser Prof. Charles E. Wyman for making me part of his biofuel research team, support and caregiving over the past four and a half years. His teachings and valuable guidance have helped me to perform research in the field of biofuels. I thank Prof. Eugene A. Nothnagel and Prof. Ian Wheeldon for their guidance on this dissertation. I also thank Dr. Rajeev Kumar for teaching me the necessary skills for carrying out this research as well as suggestions, guidance and support over the years. I would like to acknowledge Prof. Charles E. Wyman’s research team Abhishek Patri, Charles Cai, Christian Alcaraz, Glen Svenningsen, Hongjia Li, Jerry Tam, May- Ling Lu, Nikhil Nagane, Ninad Kothari, Rachna Dhir, Taiying Zhang, Vanessa Thomas, Yen Nguyen, and Xiadi Gao, as well as members of BESC for their support. iv DEDICATIONS This dissertation is dedicated to my mummy, Dr. Minoo Bhagia. v ABSTRACT OF THE DISSERTATION Factors Contributing to Recalcitrance of Poplar to Deconstruction by Samarthya Bhagia Doctor of Philosophy, Graduate Program in Chemical and Environmental Engineering University of California, Riverside, June 2016 Dr. Charles E. Wyman, Chairperson Cellulosic ethanol as a transportation fuel can cut greenhouse gas emissions for a sustainable earth and lower dependence on fossil fuels. However, successful penetration into an oil dominated market can only come through cost-competitiveness with gasoline, but biological conversion of cellulosic biomass to fuels is currently stymied by the need for high enzyme doses to realize commercially relevant yields for the widely pursued biological route of pretreatment followed by enzymatic hydrolysis to produce fermentable sugars. Achieving high sugar yields at low enzyme loadings can benefit from a better understanding of factors that contribute to biomass recalcitrance to deconstruction. When poplar varieties with lower lignin content due to a rare natural mutation associated with lignin biosynthesis were subjected to a high throughput pretreatment and co-hydrolysis platform, they gave higher sugar yields than standard poplar, but temperature pretreatment severity affected their rankings. In another approach to understand factors responsible for recalcitrance, application of flowthrough and batch pretreatment with dilute acid and just liquid hot water to standard poplar indicated that vi flowthrough pretreatment solubilized and removed 65 to 70% of the lignin before it could react further to low solubility lignin rich fragments that otherwise deposit on biomass in batch operations and hinder enzyme action. In subsequent work, combinations of enzymes and bovine serum albumin applied to solids produced by aqueous flowthrough and batch pretreatments revealed how polysaccharides in poplar were successively deconstructed layer by layer through pretreatment and subsequent hydrolysis by cellulase and xylanase combinations and through BSA addition. The standard strong acid hydrolysis procedure for measuring carbohydrate and lignin content in biomass was shown to be robust over a range of particle sizes, reaction times, and filtration strategies. Finally, elimination of shaking resulted in enzyme loadings of only 5 mg protein/g glucan able to realize nearly complete cellulose conversion, similar yields to those achieved with shaking if surfactants were added. This surprising result suggest that surfactants protect enzymes from deactivation that only occurs in shaken flasks at low enzyme to substrate ratios. vii Table of Contents List of figures……………………………………………………………………………xii List of Tables……………………………………………..……………………......….xviii Chapter 1 Introduction………….……………….......…………………………...……..1 1.1 The Need for Sustainable Fuels………………………………………………...….2 1.2 Biological Conversion of Cellulosic Biomass to Ethanol………………………….5 1.3 Thesis Organization………………………………………………………………..7 1.4 References………………………………………………………………………….9 Chapter 2 Pretreatment Options for Reducing Recalcitrance of Lignocellulosic Biomass……………………………………………………………………………….…12 2.1 Abstract………………………………………………………...…………………13 2.2 Introduction……………………………………………………………………….13 2.3 Pretreatment Options……………………………………………………………...16 2.3.1 Mechanical Pretreatment…………………………………………………….16 2.3.2 Leading Thermochemical Pretreatments…………………………………….17 2.3.2.1 Liquid Hot Water (LHW) Pretreatment………………………………..17 2.3.2.2 Dilute Acid (DA) Pretreatment………………………………………...18 2.3.2.3 Flowthrough Pretreatment……………………………………………...19 2.3.2.4 Steam Explosion Pretreatment…………………………………………20 2.3.2.5 Lime Pretreatment……………………………………………………...20 2.3.2.6 Ammonia Fiber Expansion (AFEX)……………………………………21 2.3.2.7 Ammonia Recycle Percolation (ARP)…………………………………22 2.3.2.8 Soaking in Aqueous Ammonia (SAA)…………………………………22 2.3.2.9 Alkaline Hydrogen Peroxide (AHP)…………………………………...23 2.3.2.10 Pretreatments in Organic Solvents……………………………………24 2.3.2.11 Pretreatment with Ionic Liquids………………………………………25 2.4 Biological Pretreatment…………………………………………………………...26 2.4.1 Overview…………………………………………………………………….26 2.4.2. Classes of Rot-fungi………………………………………………………...28 2.4.2.1 White-rot fungi…………………………………………………………28 2.4.2.2 Brown-rot fungi………………………………………………………...29 2.4.2.3 Soft-rot fungi…………………………………………………………...29 2.4.3 Wood Degradation by Bacteria……………………………………………...30 2.4.4 Characteristics and Mechanism of Wood Depolymerization by Fungi……..30 2.4.5 Future of Biological Pretreatment……………………………………...……35 2.5 Conclusions……………………………………………………………………….35 2.6 References………………………………………………………………………...37 viii Chapter 3 Natural Genetic Variability Reduces Recalcitrance in Poplar…………..47 3.1 Abstract…………………………………………………………………………...48 3.2 Introduction……………………………………………………………………….49 3.3 Materials and Methods……………………………………………………………52 3.3.1 Plant Materials……………………………………………………………….52 3.3.2 Compositional Analysis…………………………...………………………...53 3.3.3 Processing Conditions………………………………………………..….…..54 3.3.4 Yield Calculations…………………………...………………………………57 3.3.5 Statistical Analysis…………………………………………..……………....58 3.4 Results and Discussion……………………………………………………………60 3.5 Conclusions……………………………………………………………………….80 3.6 Abbreviations……………………………………………………………………..81 3.7 Acknowledgements……………………………………………………………….81 3.8 References………………………………………………………………………...83 Chapter 4 Flowthrough Pretreatment with Very Dilute Acid Provides Insights into High Lignin Contribution to Biomass Recalcitrance………………………….….….87 4.1 Abstract………………………………………………………………………...…88 4.2 Introduction……………………………………………………………………….89 4.3 Materials and Methods……………………………………………………………94 4.3.1 Materials……………………………………………………………………..94 4.3.2 Pretreatments………………………………………………………………...94 4.3.3 Biomass Composition and Pretreatment Liquor Analysis…………………..96 4.3.4 Enzymatic Hydrolysis……………………………………………………….97 4.3.5 Calculations………………………………………………………………….98 4.4 Results and discussion……………………………………………………....…..100 4.4.1 Solids compositions and yields…………………………………………….100 4.4.2 Xylan Removal from the Solids and Yields in Solution…………………...105 4.4.3 Glucan Removal from the Solids and Yields in Solution………………….114 4.4.4 Lignin Removal…………………………………………………………….115 4.4.5 Enzymatic Hydrolysis……………………………………………………...117 4.5 Conclusions……………………………………………………………………...121 4.6 Abbreviations……………………………………………………………………121 4.7 Acknowledgements……………………………………………………………...121 4.8 References……………………………………………………………………….123 4.9 Additional Information………………………………………………...………..128 4.9.1 Material balances…………………………………………………………...128 ix Chapter 5 Effects of Dilute Acid and Flowthrough Pretreatments and BSA Supplementation on Enzymatic Deconstruction of Poplar by Cellulase and Xylanase…………………………………………………………………………..…...135 5.1 Abstract………………………………………………………………………….136 5.2 Introduction……………………………………………………………………...137 5.3 Materials and Methods…………………………………………………………..140 5.3.1 Materials……………………………………………………………………140 5.3.2 Pretreatments……………………………………………………………….141 5.3.3 Solids Composition………………………………………………………...142 5.3.4 Enzymatic Hydrolysis………………………………………………...........144 5.3.5 Sugars Determination………………………………………....……………146 5.3.6 Calculations………………………………………………………………...147 5.3.7 Sugar Yields from Enzymatic Hydrolysis without Pretreatment…………..148 5.4 Results and Discussion…………………………………………………………..149 5.4.1 Sugar yields