Western University Scholarship@Western Electronic Thesis and Dissertation Repository 9-9-2016 12:00 AM ABE Fermentation From Low Cost Substrates Kai Gao The University of Western Ontario Supervisor Lars Rehmann The University of Western Ontario Graduate Program in Chemical and Biochemical Engineering A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Kai Gao 2016 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Biochemical and Biomolecular Engineering Commons, Bioresource and Agricultural Engineering Commons, and the Biotechnology Commons Recommended Citation Gao, Kai, "ABE Fermentation From Low Cost Substrates" (2016). Electronic Thesis and Dissertation Repository. 4087. https://ir.lib.uwo.ca/etd/4087 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract The high cost of substrate and product inhibition in the fermentation broth remain two major problems associated with bio-butanol production. This thesis aims to solve these problems by examining abundant lignocellulosic biomass as potential feedstocks and exploring novel substrates such as carbohydrates derived from microalgae for ABE (Acetone-butanol-ethanol) fermentation. The commonly observed toxic effect after pretreatment of lignocelluslosic biomass was removed by resin adsorption, where the resin could also serve as an in-situ butanol recovery device. Corn cobs (an agricultural waste), switchgrass (an energy crop) and phragmites (an invasive plant in North America) were investigated as substrates for ABE fermentation by Clostridium saccharobutylicum DSM 13864. NaOH pretreatment followed by a washing step was used to reduce the biomass recalcitrance and facilitate the subsequent enzymatic hydrolysis. Total sugar yields for corn cobs, switchgrass and phragmites were 475, 365, and 385 g/kg of raw biomass, respectively. After the subsequent fermentation, an ABE yield of 166, 146, and 150 g/kg raw biomass was obtained. Although biofuel production from lignocellulosic biomass is considered more sustainable than biofuel from food crops, it still faces many challenges. In order to demonstrate a possible biofuel production strategy using microalgal biomass, lipid extracted microalgae (LEA) was also used as substrates for ABE fermentation. To convert the carbohydrate fraction into solvents (ABE), LEA was either acid hydrolysed into glucose or directly fermented. The highest butanol titers (8.05 g/L) was obtained with the fermentation of acid hydrolysates. However fermenting the hydrolysate required detoxification via a resin, while direct fermentation did not, significantly simplifying the LEA to butanol process. The resin that was used to detoxify acid hydrolysates of LEA was further investigated for detoxification of lignocellulosic hydrolysates and in-situ butanol recovery. Detoxifcation of acid hydrolyzed phragmites by resin L-493, improved the fermentability signficantly. Resin L-493 was efficient in removing phenolic comopunds present in the phragmites hydrolysates, as well as butanol produced during fermentation. Keywords lignocellulosic biomass, NaOH pretreatment, ABE fermentation, resin, pervaporation, microalgae ii Co-Authorship Statement Contents of Section 2 were published to refereed journals and were co-authored by Dr. Lars Rehmann, who provided editorial and technical advice. Simone Boiano provided experimental assistance to portions of the work performed in section 2.4 and is listed as an co-author; Dr. Antonio Marzocchela provided advice on the draft of manuscript and is listed as a co-author. In section 2.5, Valerie Orr contributed to cultivation and lipid extractions of microalgae, and revision of the first draft of manuscript, and is listed as a co-author; Kai Gao designed and conducted all fermentation experiments, composition analysis and collected data; Kai Gao drafted the manuscript. iii Acknowledgments First and foremost I would like to thank Prof. Lars Rehmann for taking me as a PhD student four years ago. It was a great adventure for me to come over to Canada and I did not expect to be where I am today. For the last several years, Prof. Lars has inspired me both academically and mentally. Without his contribution, I would not have such a great journey and fruitful results during my PhD. I would like to thank him for providing many opportunities to go to conferences to present my research; these great experiences substanially improved my presentation and networking skills. Thanks to Dr. Erin Johnson, Dr. Luis Luque, Valerie Orr, Sascha Kießlich, Dr. Ethan Su, and Dr. Jeff Wood. It was a great pleasure to work with them and they have made the lab such a great place. I have to thank the Department of Chemical and Biochemical Engineering, the Natural Science and Engineering Research Council of Canada, and China Scholarship Council for funding. I would like to thank Biqiong Wang for sharing all the moments with me for the last several years. Her helps in all aspects of my life is greatly appreciated. Finally, many thanks to my mom (Ying Lin) and dad (Yinxiang Gao) in China, for their unconditional love and constant support. iv Table of Contents Abstract ................................................................................................................................ i Co-Authorship Statement ................................................................................................... iii Acknowledgments .............................................................................................................. iv Table of Contents ................................................................................................................ v List of Tables ..................................................................................................................... vi List of Figures .................................................................................................................. viii Section 1 - Introduction and Literature Review .................................................................. 1 1.1. Introduction ............................................................................................................. 1 1.2. Literature review ..................................................................................................... 4 Section 2 - Experimental data and interpretation .............................................................. 40 2.1 Structure of the thesis ............................................................................................ 40 2.2 General Objective ................................................................................................. 41 2.3 Specific Objectives ............................................................................................... 41 2.4 ABE fermentation from enzymatic hydrolysate of NaOH-pretreated corncobs ... 43 2.5 Cellulosic butanol production from alkali-pretreated switchgrass (Panicum virgatum) and phragmites (Phragmites australis) ................................................. 57 2.6 Butanol fermentation from microalgae-derived carbohydrates after ionic liquid extraction ............................................................................................................... 72 2.7 Combined Detoxification and In-situ Product Removal by a Single Resin During Lignocellulosic Butanol Production ......................................................... 94 Section 3 - Summary and Conclusions ........................................................................... 114 3.1 Summary .............................................................................................................. 114 3.2 Conclusions .......................................................................................................... 116 3.3 Future work and recommendation ....................................................................... 116 References ....................................................................................................................... 118 v List of Tables Table 1.1 Butanol fermentation from starch-based substrates ................................................ 16 Table 1.2 Percentage of major components of common lignocellulosic biomass .................. 20 Table 1.3 Effect of various pretreatment methods .................................................................. 26 Table 1.4 Comparison of major inhibitor concentrations obtained after different pretreatment methods .............................................................................................................. 27 Table 1.5 Sugar yields from switchgrass pretreated by various pretreatment methods .......... 32 Table 1.6 Comparisons of ABE fermentation from lignocellulosic materials ........................ 34 Table 2.1 Comparison of enzymatic hydrolysis of corncobs and corn stover pretreated with alkali and wet disk milling .............................................................................................. 50 Table 2.2 Comparison of ABE production from alkali-pretreated corncobs and reported ABE production from lignocellulosic materials pretreated with other method ...................... 55 Table 2.3 Chemical compositions of raw and pretreated biomass .........................................