Carbonyl inhibition and detoxification in microbial fermentation of biomass hydrolysates by Rui Xie A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama May 4, 2014 Keywords: Lignocellulosic biomass, fermentation, detoxification, carbonyl inhibitors Copyright 2014 by Rui Xie Approved by Maobing Tu, Chair, Associate Professor of Forestry Yoon.Y. Lee, Uthlaut Family Professor of Chemical Engineering Maria L. Auad, Associate Professor of Polymer and Fiber Engineering Eduardus (Evert) Duin, Associate Professor of Chemistry and Biochemistry Abstract Bioconversion of lignocellulosic biomass to biofuels has great potential to supplement petroleum-derived fuels. One of the major barriers to bioconversion is the release of considerable amount of carbonyl degradation compounds in the pretreatment of biomass, which because of their high reactivity toward biological nucleophiles inhibit the subsequent microbial fermentation. The development of a cost-effective detoxification approach and identification of the reaction mechanisms would alleviate the issue. Overliming has been widely used to detoxify biomass hydrolysates. However, the chemical mechanisms were not very well understood. My initial work therefore was to explore possible detoxification mechanisms by using a carbonyl model compound o-phthalaldehyde. At 1 mM, o-phthalaldehyde completely inhibited ethanol production by Saccharomyces cerevisiae, but interestingly, the inhibition disappeared under alkaline conditions (pH~10) at 60°C for 2 h in the presence of a reducing sugar. Non-reducing sugar had no effect. LC/MS analysis of the detoxification mixture revealed an aldol condensation reaction between o-phthalaldehyde and a reducing sugar. The reducing sugar converted to its enolate ion under alkaline conditions, which then reacted with one of the aldehyde groups through nucleophilic addition. Loss of one aldehyde group could be the key for the detoxification. In following work amino acids were used to detoxify the biomass hydrolysates for ethanol production by S. cerevisiae. I found cysteine was one of the amino acids that effectively detoxified loblolly pine hydrolysates. Ethanol production rate at 6 h increased from 0.18 in the ii untreated hydrolysate to 1.77 g/L/h and the final yield from 0.02 to 0.42 g/g, significant increases in both production rate and yield. The extraordinary detoxification by cysteine was probably due to its reactive thiol group that, in addition to its amine group, reacted with aldehydes to form thiazolidine derivatives. Meanwhile, the amine group could attack the carbon of aldehydes/ketones via electrophilic substitution to form imines. To understand the mechanism of aromatic aldehyde inhibition on yeast fermentation, I further investigated the structure-inhibition relationships using thirteen benzaldehydes. The results indicated that fermentation inhibition of benzaldehydes appeared related to their ortho- group (CHO > OH > OCH ) and position of the OH group in the benzene ring (ο-OH > m-OH > 3 p-OH). Correlating the molecular descriptors to inhibition efficiency revealed a strong association between Log P and inhibitory activity. iii Acknowledgments I would have never been able to finish my dissertation without the guidance of my committee members, friends, and support from my family, especially my mom and fiancé. I would like to express my deep gratitude to my advisor, Dr. Maobing Tu, for his guidance and patience, he has provided me with an excellent atmosphere for doing research, and inspired me by example of being persistent and hard-working. I also would like to thank Dr. Yoon Y. Lee, Dr. Maria Auad and Dr. Evert Duin for their continuous advice and support inthe past four years. They helped me to develop my background in Chemical Engineering, Polymer and Fiber Engineering, and Biochemistry. My special thanks to Dr. Xinyu Zhang at department of Polymer and Fiber Engineering, who participated in my final defense requested at the last minute. I would also like to extend my appreciation to Dr. Yonnie Wu at the mass spec lab in the Chemistry Department for his numerous discussion, guidance of my research project, and editing my papers. I would like to thank Dr. Thomas Elder at the USDA-Forest service Alexandria Forestry Center for his help with some of the data analysis. I would like to thank my fellow student Chenfeng Zhou and Pixiang Wang, who always provided their helping hands and be my companion, often late in the lab, it would have been a lonely lab without them. Many thanks to others in the lab including Anshu Shrestha, Jamarius Carvin, Mi Li, Jing Li, Chenhuan Lai, Daihui Zhang, Zhiqiang Shi, Hao Shi, Xiaofei Wang and iv those in our neighboring laboratory of Dr. Brian Via’s. My research would not be complete without their help. I would like to thank my parents and my younger sister. They always support me and encourage me with their best wishes. Last but not the least, I would like to thank my fiancé, Kyle Malinowski. He always loves me, supports me and cheers me up in every possible every way he could. v Table of Contents Carbonyl inhibition and detoxification in microbial fermentation of biomass hydrolysates . i Abstract .......................................................................................................................................... ii Acknowledgments ........................................................................................................................ iv List of Tables ................................................................................................................................. x List of Figures ............................................................................................................................... xi List of Abbreviations ................................................................................................................. xiv Chapter 1: Introduction ............................................................................................................... 1 1.1 Background - the significance of lignocellulosic biofuel production and current issues ...... 1 1.2 Chemical composition of lignocellulosic biomass ................................................................ 4 1.3 Biochemical conversion of lignocellulosic biomass ............................................................. 8 1.3.1 Pretreatment .................................................................................................................... 9 1.3.1.1 Chemical pretreatment ................................................................................................... 10 1.3.1.2 Biological pretreatment .................................................................................................. 14 1.3.2 Enzymatic hydrolysis and microbial fermentation ....................................................... 15 1.4 Strategies in improving lignocellulosic fermentability ....................................................... 17 1.4.1 Identification of inhibitors and their inhibitory mechanism ......................................... 17 1.4.1.1 Carboxylic acids and their inhibition ........................................................................... 17 1.4.1.2 Aldehydes/ketones and their inhibition ........................................................................ 20 1.4.1.3 Phenols and their inhibition .......................................................................................... 25 1.4.2 Development of analytical methods for identifying inhibitors ..................................... 31 1.4.3 Detoxification strategies ............................................................................................... 33 vi 1.4.3.1 Physical detoxification ................................................................................................... 33 1.4.3.2 Chemical detoxification .................................................................................................. 35 1.4.3.3 Biological detoxification ................................................................................................ 39 1.5 Research objectives ............................................................................................................. 42 Chapter 2: Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate................................................................................................ 46 2.1 Background ......................................................................................................................... 46 2.2 Materials and methods ........................................................................................................ 47 2.2.1 Reagents and biomass hydrolysate samples ................................................................. 47 2.2.2 High performance liquid chromatography ................................................................... 48 2.2.3 Linearity and recovery .................................................................................................. 49 2.3 Results and discussion ......................................................................................................... 49 2.3.1 Dropping column temperature leads to significant improvement in separation ........... 49 2.3.2 Effect of flow rate and sulfuric acid concentration in mobile phase ............................ 54 2.3.3 The separation of acetic acid, levulinic acid, HMF and furfural from biomass hydrolysate............................................................................................................................. 57 2.3.4 Linearity and recovery .................................................................................................. 59 2.4 Conclusion ........................................................................................................................... 60 Chapter 3: Reducing sugars facilitated carbonyl condensation in detoxification of aromatic aldehyde model compounds for bioethanol fermentation ....................................................... 61 3.1 Background ......................................................................................................................... 61 3.2 Materials and methods ........................................................................................................ 62 3.2.1 Microbial strain and medium ........................................................................................ 62 3.2.2 Fermentation and growth curve .................................................................................... 63 vii 3.2.3 Alkaline detoxification of OPA .................................................................................... 63 3.2.4 HPLC and LC/MS analysis ......................................................................................... 64 3.3 Results and discussion ......................................................................................................... 65 3.3.1 Effects of OPA, vanillin, furfural and HMF on fermentation and growth of S. cerevisiae ............................................................................................................................... 65 3.3.2 Effects of OPA concentration on fermentation inhibition and growth of S. cerevisiae 68 3.3.3 Effect of inoculation size of S. cerevisiae on OPA fermentation inhibition ................ 72 3.3.4 Effects of reducing sugars on alkaline detoxification of OPA ..................................... 74 3.3.5 Identification of potential mechanism for alkaline detoxification of OPA .................. 78 3.4 Conclusion ........................................................................................................................... 83 Chapter 4: Effect of Amino acids on detoxification of biomass hydrolysate and carbonyl inhibitor cinnamaldehyde for bioethanol fermentation .......................................................... 85 4.1 Background ......................................................................................................................... 85 4.2 Material and methods .......................................................................................................... 87 4.2.1 Chemical reagents and stock preparation ..................................................................... 87 4.2.2 Preparation of biomass hydrolysate .............................................................................. 88 4.2.3 Detoxification with amino acids ................................................................................... 89 4.2.4 Microbial strain and fermentation ................................................................................ 89 4.2.5 HPLC and LC/MS analysis .......................................................................................... 90 4.2.6 SEM of yeast cell .......................................................................................................... 92 4.3 Results and discussion ......................................................................................................... 92 4.3.1 Detoxification of loblolly pine hydrolysate by amino acids ......................................... 92 4.3.2 Effect of temperature and pH on cysteine and glycine detoxification ....................... 100 viii 4.3.3 SEM image of yeast cells ........................................................................................... 105 4.3.4 Identification of potential detoxification products using a lignocellulose-derived carbonyl inhibitor cinnamaldehyde ..................................................................................... 108 4.4 Conclusion ......................................................................................................................... 111 Chapter 5: Substituents-related inhibition of benzaldehydes on fermentation and their quantitative structure-activity relationships .......................................................................... 113 5.1 Background ....................................................................................................................... 113 5.2 Materials and methods ...................................................................................................... 114 5.2.1 Chemical reagents and stock preparation ................................................................... 114 5.2.2 Microbial fermentation ............................................................................................... 115 5.2.3 Inhibitory activity ....................................................................................................... 116 5.2.4 HPLC analysis ............................................................................................................ 116 5.2.5 Calculation of physicochemical descriptors ............................................................... 117 5.3 Results and discussion ....................................................................................................... 117 5.3.1 Effects of benzaldehydes on the fermentation by S. cerevisiae ................................. 117 5.3.1.1 Fermentation inhibition by benzaldehydes with different ortho substituents ........ 122 5.3.1.2 Fermentation inhibition by benzaldehydes substituted with hydroxyl group at different positions ....................................................................................................................... 123 5.3.1.3 Fermentation inhibition by benzaldehydes substituted with different amount of hydroxyl groups .......................................................................................................................... 125 5.3.2 QSARs analysis .......................................................................................................... 129 5.4 Conclusion ......................................................................................................................... 132 Chapter 6: Future work ........................................................................................................... 133 References ........................................................................................................................................ ix List of Tables Table 1: The effect of chemical pretreatments on biomass fractionation and downstream steps ............... 15 Table 2: Carboxylic acids identified in lignocellulosic biomass after pretreatment ................................... 26 Table 3: Aldehydes and ketones identified in lignocellulosic biomass after pretreatment ......................... 28 Table 4: Phenols identified in lignocellulosic biomass after pretreatment ................................................. 30 Table 5: The effect of different detoxification on sugars, inhibitors and improvement of fermentability for biofuel production .......................................................................................................................... 41 Table 6: Effect of flow rate on retention times and capacity factors of acetic acid, levulinic acid, HMF and furfural ........................................................................................................................................... 56 Table 7: Effect of sulfuric acid in mobile phase on resolution of acetic acid and levulinic acid ................ 57 Table 8: Linearity and recovery tests of acetic acid, levulinic acid, HMF and furfural in biomass hydrolysate ..................................................................................................................................... 60 Table 9: Effects of OPA concentration on ethanol productivity and yields ................................................ 70 Table 10: Colony Forming Units (CFU) of S. cerevisiae on YDP agar plates in the presence of 0 mM (M9 control), 0.02 mM, 0.1 mM, 0.5mM and 1.0 mM OPA at 30°C ................................................... 72 Table 11: Effects of reducing sugars and non-reducing sugar on alkaline detoxification of OPA ............. 76 Table 12: The change of pH during the alkaline treatment of 1.0 mM OPA with different sugars (2h, 60°C) ....................................................................................................................................................... 78 Table 13: Detoxification by 20 amino acids (pH6, 60°C and 2 h) on improving the fermentability of concentrated loblolly pine hydrolysate by S. cerevisiae ................................................................ 97 Table 14: Effect of different types of benzaldehydes on fermentation by S. cerevisiae ........................... 120 Table 15: Molecular descriptors and inhibition efficiency of 13 benzaldehydes ..................................... 131 Table 16: Linear regression analysis between benzaldehydes’ physicochemical descriptors and their inhibition efficiency ..................................................................................................................... 131 x