SYNERGISTIC ENZYMATIC HYDROLYSIS OF CASSAVA STARCH AND ANEAROBIC DIGESTION OF CASSAVA WASTE By SAMUEL NWANELE ASO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013 1 © 2013 Samuel NwaneLe Aso 2 To the memory of my parents: Chief Rufus Nyeweiba Aso [Chief Ntoyi] Mrs. Elizabeth Ndem Aso [Madam Taproot] Prophetess Mrs. Caroline Ogonda Onyenma 3 ACKNOWLEDGMENTS I want to thank the President of the University of Florida, Bernie Machen, and his team of administrators for enabling environment conducive for academic work. I express great gratitude to members of the supervisory graduate committee: Dr. Arthur A. Teixeira; Dr. Bruce A. Welt; Dr. Pratap C. Pullammanappallil; Dr. Robert P. Bates; Dr. Spyros A. Svoronos; and Dr. Weihua Yang; for advice, encouragement, guidance and support. I am grateful to Dr. Ray A. Bucklin for providing laboratory space for this project; and to various persons that allowed me access to their labs and facilities. Dr. Bin Gao for use of spectrophotometer, vortex machine, pipettes and distilled water; Dr. Melanie J. Correll for use of microscope, convection oven, spectrophotometer and distilled water; Dr. Bruce A. Welt for use of vacuum oven, dessicator jars and chemicals; Dr. Pratap C. Pullammanappallil for use of centrifuges, gas partitioner, anaerobic digester, incubator, Hach reactor and colorimeter; Dr. Weihua Yang for portable refractometer; Dr. Keith R. Schneider for water bath; Dr. Jesse F. Gregory for the two books: Fennema’s Food Chemistry and Principles of Enzymology for the food Sciences; Dr. Robert P. Bates, Dr. Charles A. Sims, Rob Pelick and Bridget Stokes of the department of food science and human nutrition for portable and table top refractometers, vacuum evaporator, freeze dryer, cooking range and accessories. I also thank DSM, Genencor, Novozymes, BioCat, and BioSun corporations for donating enzymes used in this project. I thank all the persons I have met and worked with: The librarians, folks at Agricultural and Biological Engineering Department, as well as colleagues; for friendship and support. My regards go to Dr. Thomas F. Burks, Dr. James D. Leary, Dr. Wendell 4 A. Porter, Robin, Veronica, Paul, Billy, Orlando, Steve, Patrick, James, Cesar, Donna, Amy, Christan, Shannon, etc. I am thankful to my student assistants and mentees: Brandon A. Doan, Jeremy D. Foxx, Alex Hyyti, Garrett Gant, Kelly Calderon, and Frances de Asis. Whether it was peeling cassava roots, slicing and drying peeled roots to produce cassava chips; milling chips into flour; conducting physical properties tests; conducting hydrolysis experiments; or cataloging equipment used, I appreciate all the help rendered. I would like to thank doctors, nurses and staff at the student health care center. Phillip A. Barkley, M.D. and Guy W. Nicolette, M.D. thanks for the care and support provided. Thank you also to the Disability Resource Center [DRC]. When I sustained incapacitating wrist injury while conducting research experiments, doctors did their job and DRC supported my application for work place accommodation. Thank you to everyone. Furthermore, I dish out special regards to members of my family and relations for relentless support, encouragement and prayers. To Nnenne, Apu, Uzoma, Chibuzor, Ozioma, and Papa Edwin Onyenma, I say meka weh. My nephews and nieces: Chimezie, Junior, Joy, Stephanie and Caro; my cousins: Nnanna (Chief of staff), Chibuenyi (Boyoyo), Bright (Britoe) and Obi (Obilolo); and Mr. Iroha Iroha (Alias Agadi) and Mrs. Iroha (Madam Agadi), I say bravo to you all. I want to use this opportunity to thank those that created the internet. The information cyber space is making it easier to access knowledge and do research. I thank the scientists, thinkers and builders that worked assiduously to develop the theories, equations and numerous other facilities we take for granted today. The likes of 5 Thomas Edison, Isaac Newton, Nikola Tesla, Albert Einstein, Alexander Graham Bell, Galileo Galilei, Johannes Kepler and many others. I also remember Nicolas Appert, Louis Pasteur, Carl Sagan and Karl Jansky. Thanks to you all for your service, vision and creativity. You all are indeed the giants on whose shoulders present-day workers are seeing from. I send a shout out to my space friends and inspirers: NASA, ESA, RKA (Roscosmos), Robert Farquhar, Robert Zubrin, Peggy A Whitson and Charles T. Bourland. Thanks for keeping space enthusiasm alive. Funding: I am hugely appreciative of all funding assistances that supported the PhD endeavor. Government of Rivers State of Nigeria, Mr. Stephen Majebi and Mrs. Silverline Majebi assisted me to make the crucial down payment that started the PhD program. Without the down payment, seed of PhD program most probably may not have been planted in the first place, and we would not be at this juncture today. I thank them immensely for the seed funding and their continued support over the years. After a seed is planted and germinated, it needs nourishment to grow and blossom. Dr. Tex worked hard to arrange funding support from Chemonics. For this I greatly thank Dr. Tex and Chemonics. When the funding support from Chemonics expired, Dr. Dorota Z. Haman was benevolent and assisted the project. Thank you Dr. Haman. At some points in the course of this project, I may have been anxious to get the work done and thus could have offended people without knowing. I use this medium to sincerely apologize and beg for full forgiveness. TO GOD BE ALL THE GLORY. 6 TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 12 LIST OF FIGURES ........................................................................................................ 15 ABSTRACT ................................................................................................................... 19 CHAPTER 1 INTRODUCTION .................................................................................................... 22 1.1 Background ....................................................................................................... 22 1.1.1 Importance .............................................................................................. 22 1.1.2 Advantages ............................................................................................. 24 1.2 Justifications ..................................................................................................... 24 1.2.1 Rationale ................................................................................................. 24 1.2.2 Challenges and Opportunities ................................................................. 33 1.3 Objectives ......................................................................................................... 36 1.4 Organization of Dissertation .............................................................................. 38 2 REVIEW OF LITERATURE .................................................................................... 39 2.1 Part 1: Cassava ................................................................................................ 39 2.1.1 Origin and Distribution ............................................................................. 39 2.1.2 Classification ........................................................................................... 40 2.1.3 Agro Climatic Conditions ......................................................................... 42 2.1.4 Propagation ............................................................................................. 43 2.1.5 Production ............................................................................................... 43 2.1.6 Cassava Utilization .................................................................................. 47 2.1.6.1 Starch ............................................................................................. 47 2.1.6.2 Flour ............................................................................................... 49 2.1.6.2.1 Gluten free flour .......................................................................... 50 2.1.6.3 Chips .............................................................................................. 50 2.1.6.4 Packaging ...................................................................................... 51 2.1.7 Sweetener Production ............................................................................. 51 2.1.7.1 Historical overview ......................................................................... 53 2.1.7.2 Acid or enzyme hydrolysis ............................................................. 55 2.1.7.3 Amylolytic enzymes........................................................................ 55 2.1.7.4 Synergistic and direct methods of glucose sweetener production .. 56 2.1.8 Starch Chemistry ..................................................................................... 57 2.1.8.1 Amylose ......................................................................................... 59 2.1.8.2 Amylopectin ................................................................................... 61 7 2.1.8.3 Cassava Starch .............................................................................. 66 2.1.8.4