A Dissertation entitled Microalgae Fractionation and Production of High Value Nylon Precursors by Godwin Ameh Abel Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Engineering ________________________________________ Dr. Sasidhar Varanasi, Committee Chair ________________________________________ Dr. Sridhar Viamajala, Committee Member ________________________________________ Dr. Kana Yamamoto, Committee Member ________________________________________ Dr. Maria Coleman, Committee Member ________________________________________ Dr. Patricia Relue, Committee Member ________________________________________ Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2017 Copyright 2017, Godwin Ameh Abel 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 Microalgae Fractionation and Production of High Value Nylon Precursors by Godwin Ameh Abel Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Engineering The University of Toledo August 2017 Liquid fuels from microalgal biomass have become less attractive recently due to the fall in prices of petroleum and natural gas. However, interest in microalgae as a renewable feedstock for value-added bioproducts such as oleo-chemicals and sugar- derived platform molecules has been on the rise. This is due to the economic and environmental benefits associated with the processing of these higher value products from microalgae biomass. Microalgae biomass consists mainly of carbohydrate, protein and lipids. When efficiently fractionated, the lipid and carbohydrate portions could be used for synthesizing oleochemicals and sugar-based platform molecules, respectively. The protein rich residue could serve as feed for animals. This study is a part of the microalgae processing methodologies being explored in our group, with the purpose of developing an integrated approach for producing renewable high value chemicals from microalgae via environmentally sustainable pathways. For this purpose, a specific pathway for processing microalgae after it is harvested following cultivation is proposed. The proposed pathway entails subjecting harvested microalgae to enzymatic digestion to fractionate and affect depolymerization of iii the individual fractions: Simple sugars resulting from the hydrolysis of the carbohydrate fraction can be converted to value-added products via fermentation using product-specific microorganisms. Similarly, oleic acid, the major component of the lipid fraction, can form the feed-stock for the production of value-added compounds such as industrial nylon precursors. As conventional (chemical/physical) methods of isolating carbohydrates from microalgae are deemed unsustainable, in our first project we explored an enzymatic hydrolysis method for isolation of simple sugars directly from microalgae biomass. Lipids are isolated from the enzymatically digested algal slurry through immiscible solvent extraction. Following phase separation, the aqueous phase retains the oligomeric/monomeric sugars from carbohydrates and the protein fragments, and serves as a tailor-made broth for converting the sugars to value-added platform molecules via fermentation methods. Alternately, the monomeric sugars can be selectively extracted from this medium and can separately be converted into specific chemicals/fuels through appropriate chemical conversion strategies. This new fractionation method lowers cost and energy required over the traditional approaches due to the mild processing conditions and allows for the isolation of the simple sugars in high yield. Our second project provides the synthesis of precursors for nylon 12 and 13 from methyl oleate/microalgal lipids. These nylons, developed in the 1980s for use in the automotive industries, are high-strength polymers that find applications in many industrial sectors (medical, electronic and sport industries). The production of these nylons currently uses petroleum feedstock or exotic fatty acids and involves 4 – 6 steps. We have developed a new simple 2-step procedure featuring use of cross metathesis iv (CM) reaction as the key step. This approach starts with cross-metathesis (CM) of methyl oleate (methyl ester of oleic acid) with alkenyl nitriles (allyl cyanide or homoallyl cyanide), to form cyano esters. This step is followed by hydrogenation of the unsaturated alkene and nitrile groups of the cyano-esters to produce the desired amino esters (nylon precursors). We have also developed a ring-closing metathesis approach as an alternative pathway to cross-metathesis for producing nylon 12 precursors. This alternative pathway had been extended for the synthesis of precursors for nylons 11, 12, and 13 from oleic acid, the most abundant natural fatty acid in microalgae. The alternative pathway is also simple, but 3-step procedure featuring use of ring-closing metathesis (RCM) reaction as the key step. The first step (amide formation) and third step of this approach (hydrogenation) can be performed with no major issues. Therefore, only the key ring- closing metathesis step was optimized. This second strategy (ring closing metathesis approach) involves first converting the oleic acid to alkenyl amides. Cross-coupling of free amines is not possible because of catalyst poisoning of amines. The amides were then subjected to ring closing metathesis generating the unsaturated lactams, which are then hydrogenated to lactams. This approach avoids the use of high-pressure hydrogenation and produces fewer undesired by-products than CM approach. v This dissertation is dedicated to my beloved parents (Abel Ameh and Anthonia Ameh), as a tribute to their sacrifices, prayers, support, encouragement and unceasing love. Acknowledgements This dissertation could not have been possible without the grace and blessings of God, acceptance and guidance of my advisors and committee members, assistance from lab mates and friends, and prayers and support from my parents, brothers and sisters. To my advisors, Drs. Sasidhar Varanasi and Sridhar Viamajala. I would like to express my deepest gratitude for accepting me into their group and giving me the opportunity to perform research. I would like to thank them for their guidance, genuine care, patience, and providing the necessary atmosphere for research. To my committee members, Drs. Kana Yamamoto, Maria Coleman and Patricia Relue. Thank you so much for your guidance, understanding and willingness to participate in my defense committee. Your time is very much appreciated. To all of my friends, lab mates and postdocs, Christopher Anukwu, Engr. Patrick Nwokolo, Dr. Ajith Yapa, Yaser Shirazi, Xiaofei Zhao, Matin Hanifzadeh, Ravi Gogar, Arsalan Sepehri, Alessandra Krusciel, Jayachandra Kopalli, Drs. Brahmaiah Pendyala, Jehad Almaleety, and Pramod Poudel. Thank you so much for your friendship, support and guidance. It was my pleasure to work with you guys. To my beloved parents (Abel Ameh and Anthonia Ameh), brothers and sisters, thank you so much. They were always supporting me with their prayers and best wishes. Finally, I would like to thank God almighty for making everything beautiful in His time. To Him be the glory. vi Table of Contents Abstract…………………………… .................................................................................. iii Acknowledgements………….. .......................................................................................... vi Table of Contents………… .............................................................................................. vii List of Tables……….. .......................................................................................................x List of Figures………….. .................................................................................................. xi List of Abbreviations…………….. ................................................................................. xiii 1 Downstream Processing of Microalgae Biomass ....................................................1 1.1 Background .......................................................................................................1 1.1.1 Microalgae .......................................................................................1 1.1.2 Advantages of using microalgae for the production of bioproducts……………… ...................................................................................................2 1.1.3 Microalgae harvesting and biomass concentration………… ..........2 1.1.4 Microalgae cell disruption ...............................................................3 1.2 Use of renewable feedstock in the production of bioproducts ..........................5 1.2.1 Nylons ..............................................................................................7 1.2.2 Olefin metathesis ...........................................................................11 1.3 Dissertation overview .....................................................................................12 References for chapter 1………………….. ......................................................................15 vii 2 Microalgae Fractionation and Recovery of Native Components through Application of Low-Cost Enzymes………….. ..................................................................23 2.1 Introduction ......................................................................................................23