Synergistic Enzymatic Hydrolysis of Cassava Starch and Anearobic Digestion of Cassava Waste

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Title:
Synergistic Enzymatic Hydrolysis of Cassava Starch and Anearobic Digestion of Cassava Waste
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1 online resource (323 p.)
Language:
english
Creator:
Aso, Samuel N
Publisher:
University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Agricultural and Biological Engineering
Committee Chair:
TEIXEIRA,ARTHUR A
Committee Co-Chair:
BATES,ROBERT P
Committee Members:
WELT,BRUCE ARI
YANG,WEIHUA WADE
PULLAMMANAPPALLIL,P C
SVORONOS,SPYROS A

Subjects

Subjects / Keywords:
anaerobic -- biomethane -- cassava -- digestion -- enzyme -- fertilizer -- flour -- hydrolysis -- organic -- peel -- pulp -- root -- simultaneous -- starch -- synergistic -- waste
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
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Agricultural and Biological Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Cassava is a major root crop and source of food and feedcarbohydrate for humans and livestock. Cassava provides economic andsubsistence value to more than 800 million to one billion people in Brazil,China, Haiti, India, Indonesia, Nigeria, Thailand and many other countries. However,current processes for conversion of cassava roots to value added products arefar from optimum. Glucose is produced by first extracting starch from theroots. The starch is then subjected to two separate operations calledliquefaction and saccharification. Each operation incurs many cost elements.Liquefaction for example demands elaborate equipment and energy inputs, whilestarch extraction is energy, labor and technology intensive. These requirementsplace glucose and other value added cassava products beyond the reach of manydeveloping nations. It would be efficient and advantageous if liquefaction andsaccharification could be carried out simultaneously as one operation and thestarch hydrolyzed directly without going through the extraction procedure.Furthermore, over 60 % of global cassava output (which was 252.2 x 109kg in 2011) is processed annually for human food, generating enormousquantities of organic waste matter with attendant environmental and disposalissues. Anaerobic digestion of cassava wastes could generate beneficialproducts and at the same time be environment friendly. The objectives of this study were to develop and demonstratea village-scale method of processing fresh cassava root into flour from solar-convectiondried cassava chips, produce glucose sweetener from combined liquefaction andsaccharification hydrolysis of native starch that was not first extracted, andgenerate biogas (methane) from anaerobic digestion of cassava waste. A solar convection dryer requiring no fuel or electricitywas used to dry thin slices of cassava root into dry chips that weresubsequently ground into cassava flour. The flour as well as freshly groundcassava root (pulp) was used as substrate for conversion into glucose by enzymehydrolysis. The rates of conversion (kinetics) for both substrates weredetermined at two different temperatures and compared with those ofcommercially available refined cassava starch. Cassava waste from peelings andtrimmings was used as feedstock for anaerobic digestion into methane fuel. Results showed thatrates of reaction for hydrolysis of all three substrates were similar to eachother at both temperatures, but resulted in different extent of reaction. Thecassava flour and pulp produced syrup with 3-4% glucose, while the commercialstarch produced 10% glucose within 4 hours at 60°C, and 72 hours at 37°C.Anaerobic digestion of cassava waste produced bio-methane yield of 0.25 liter/ gramvolatile solids with a production efficiency of 71 %. These findings wouldsuggest the possibility of a cottage industry cassava processing operation thatwould be self-sufficient in which fresh cassava roots could be solar-convectiondried to make flour. The flour and grated root pulp could be hydrolyzed in onestep to make glucose syrup sweetener, while the waste peelings and trimmingscould be processed by anaerobic digestion to produce useful energy in the formof biogas for maintaining digester temperature and/or supplementing heatrequired for hydrolysis and in the solar dryer. In addition, amylolytic (starchhydrolyzing) enzymes could be harvested from the anaerobic digestion liquor,and the post digestion effluent applied to farm land as source of organicfertilizer.
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In the series University of Florida Digital Collections.
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Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Samuel N Aso.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: TEIXEIRA,ARTHUR A.
Local:
Co-adviser: BATES,ROBERT P.

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UFRGP
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Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0046252:00001


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1 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 RE QUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Samuel NwaneL e Aso

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3 To the memory of my parents: Chief Rufus Nyeweiba Aso [Chief Ntoyi] Mrs. Elizabeth Ndem Aso [Madam Tap root] Prophetess Mrs. Caroline Ogonda Onyenma

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4 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 pe rsons 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. Correl l 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: 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 regard s go to Dr. Thomas F Burks, Dr. James D Leary, Dr. Wendell

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5 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 expe riments; 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. Than k 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 an d 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 inte rnet. 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

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6 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, vi sion 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. Bour land. 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 cru cial 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 cont inued 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 Che monics 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 med ium to sincerely apologize and beg for full forgiveness. TO GOD BE ALL THE GLORY.

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7 TABLE OF CONTENTS P age 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: Cas sava ................................ ................................ ................................ 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

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8 2.1.8.3 Cassava Starch ................................ ................................ .............. 66 2.1.8.4 Enzymatic Hydrolysis of Starch ................................ ...................... 71 2.1.8.4.1 Alpha amylase ( amylase: EC 3.2.1.1) ................................ ...... 72 2.1.8.4.2 Beta amylase ( amylase: EC 3.2.1.2) ................................ ....... 74 2.1.8.4.2 Gamma amylase ( amylase: EC 3.2.1.3 ) ................................ .. 75 2.1.8.4.3 Comparison of starch hydrolyzing enzymes ................................ 78 2.1.9 Cassava Toxicity ................................ ................................ ..................... 80 2.1.10 Cassava Detoxification ................................ ................................ .......... 86 2.1.11 Cassava Food Products ................................ ................................ ........ 96 2.1.11.1 Akpakpuru ................................ ................................ .................... 97 2.1.11.2 Attieke ................................ ................................ .......................... 97 2.1.11.3 Casabe ................................ ................................ ......................... 98 2.1.11.4 Chickwangue ................................ ................................ ............... 99 2.1.11.5 Farina ................................ ................................ ......................... 100 2.1.11.6 Fufu ................................ ................................ ............................ 100 2.1.11.7 Fuku ................................ ................................ ........................... 101 2.1.11.8 Gaplek ................................ ................................ ........................ 101 2.1.11.9 Gari ................................ ................................ ............................ 101 2.1.11.10 Konkonte ................................ ................................ .................. 102 2.1.11.11 Lafun ................................ ................................ ........................ 102 2.1.11.12 Landang (cassava rice) ................................ ............................ 103 2.1.11.13 Peujeum or Tapai ................................ ................................ ..... 103 2.1.11.14 Puttu ................................ ................................ ......................... 103 2.1.11.15 Thundam ................................ ................................ .................. 104 2.1.11.16 Other Cassava food and bev erage products ............................ 104 2.2 Part 2: Production of Amylolytic Enzymes ................................ ...................... 105 2.2.1 Background ................................ ................................ ........................... 105 2.2.2 Amylolytic Enzymes Production by Solid State Fermentation ............... 107 2.2.2.1 Substrates support structures ................................ ...................... 110 2.2.2.2 Carbon supplementation ................................ .............................. 117 2.2.2.3 Nitrogen supplementation ................................ ............................ 117 2.2.2.4 Porosity and aeration ................................ ................................ ... 118 2.2.2.5 Moisture content and water activity ................................ .............. 119 2.2.2.6 Incubation temperature ................................ ................................ 122 2.2.2.7 Inoculums concentration ................................ .............................. 123 2.2.2.8 Fermentation time and pH ................................ ............................ 125 2.2.3 Bioreactor Designs in Solid State Fermentatio n ................................ .... 126 2.2.3.1 Packed bed reactor ................................ ................................ ...... 126 2.2.3.2 Tray bioreactor ................................ ................................ ............. 127 2.2.3.3 Drum bioreactor ................................ ................................ ........... 127 2.2.3.4 Fluidized bed bioreactor ................................ ............................... 128 2.2.4 Submerged Fermentation (SmF) ................................ ........................... 12 9 2.2.4.1 Temperature and pH ................................ ................................ .... 129 2.2.4.2 Carbon and nitrogen ................................ ................................ .... 131 2.2.4.3 Salts ................................ ................................ ............................. 133 2.2.5 Summary on Enzyme Production ................................ .......................... 134

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9 3 PHYSICAL PROPERTIES OF CASSAVA FLOUR MADE FROM SOLAR CONVECTION DRIED CASSAVA CHIPS ................................ ............................ 135 3.1 Background ................................ ................................ ................................ ..... 135 3.2 Objectives ................................ ................................ ................................ ....... 136 3.3 Methods and Procedures ................................ ................................ ................ 136 3.3.1 Scope of Work ................................ ................................ ....................... 136 3.3.2 Fresh Cassava Root ................................ ................................ .............. 137 3.3.2.1 Sam ple preparation ................................ ................................ ...... 137 3.3.2.2 Moisture content and water activity for sorption isotherms ........... 138 3.3.2.3 Drying curve ................................ ................................ ................. 138 3.3.3 Cassava Flour ................................ ................................ ....................... 139 3.3.3.1 Sample preparation ................................ ................................ ...... 139 3.3.3.2 Physical prop erties ................................ ................................ ....... 140 3.4 Results ................................ ................................ ................................ ............ 141 3.4.1 Sorption Isotherm and Drying Curves for Fresh Cassava Root Pulp ..... 141 3.4.2 Physical Properties of Cassava Flour ................................ .................... 142 3.5 Summary ................................ ................................ ................................ ........ 145 4 SYNERGISTIC ENZYMATIC HYDR OLYSIS OF CASSAVA STARCH ................ 146 4.1 Background ................................ ................................ ................................ ..... 146 4.2 Objectives ................................ ................................ ................................ ....... 154 4.3 Materials and Methods ................................ ................................ .................... 155 4.3.1 Enzymes ................................ ................................ ................................ 155 4.3.2 Substrates ................................ ................................ ............................. 156 4.3.2.1 Roots ................................ ................................ ............................ 157 4.3.2.2 Flour ................................ ................................ ............................. 157 4.3.2.3 Starch ................................ ................................ ........................... 157 4 .3.3 Sample Preparation ................................ ................................ ............... 157 4.3.3.1 Root ................................ ................................ ............................. 157 4.3.3.2 Flour ................................ ................................ ............................. 159 4 .3.3.3 Starch ................................ ................................ ........................... 159 4.3.4 Reactor ................................ ................................ ................................ .. 159 4.3.5 Analytical Methods ................................ ................................ ................ 160 4.3.5.1 Proximate composition, starch, and sugar analysis ..................... 160 4.3.5.2 Glucose Analysis ................................ ................................ .......... 161 4.3.5.3 Determination of Reducing Su gar ................................ ................ 162 4.3.6 Experimental Procedure ................................ ................................ ........ 165 4.3.7 Mass Balance Stoichiometry ................................ ................................ 170 4.3.8 Reaction Kinetics ................................ ................................ ................... 171 4.3.9 Statistical Analysis ................................ ................................ ................. 173 4.4 Results and Discussion ................................ ................................ ................... 173 4.4.1 Compositional analysis of different substrates ................................ ...... 173 4.4.2 Enzyme hydrolysis at 60C ................................ ................................ .... 173 4.4.3 Enzyme hydrolysis at 37C ................................ ................................ .... 178 4.4.4 Reaction Kinetics of Enzyme Hydrolysis ................................ ............... 181

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10 4.4.4.1 First order rate constants ................................ ............................. 181 4.4.4.2 Activation energy ................................ ................................ .......... 183 5 ANAEROBIC DIGESTION OF CASSAVA WASTE FOR PRODUCTION OF BIOGAS AND AMYLOLYTIC ENZYMES ................................ .............................. 186 5.1 Background ................................ ................................ ................................ ..... 186 5.2 Objectives ................................ ................................ ................................ ....... 187 5.3 Materials and Methods ................................ ................................ .................... 188 5.3.1 Cassava Roots ................................ ................................ ...................... 188 5.3.2 Reactor ................................ ................................ ................................ .. 188 5.3.3 Inoculum ................................ ................................ ................................ 190 5.3.4 Bulking Agent ................................ ................................ ........................ 190 5.3.5 Sample Preparation ................................ ................................ ............... 190 5.3.5.1 Root com ponents proportion ................................ ........................ 190 5.3.5.2 Anaerobic digestion samples ................................ ....................... 193 5.3.6 Anaerobic Digestion Procedure ................................ ............................. 193 5.3.7 Analysis ................................ ................................ ................................ 194 5.3.7.1 Biogas composition ................................ ................................ ...... 194 5.3.7.2 Soluble chemical oxygen demand (sCOD) ................................ .. 196 5.3.7.3 pH ................................ ................................ ................................ 197 5.3.7.4 Total solids (TS), volatile solids (VS), and other properties .......... 197 5.3.8 Enzyme Extraction and Enzyme Activity ................................ ............... 199 5.3.8.1 Enzyme extraction ................................ ................................ ........ 199 5.3.8.1.1 Cell free enzyme ................................ ................................ ....... 199 5.3.8.1.2 Cell associated enzyme ................................ ............................ 200 5.3.8.1.3 Biofilm associated enzyme ................................ ........................ 200 5.3.8.2 Enzyme activity ................................ ................................ ............ 201 5.4 Results and Discussion ................................ ................................ ................... 202 5.4.1 Cassava Root Components Proportio ns ................................ ............... 202 5.4.2 Composition of Cassava Peel Waste ................................ .................... 203 5.4.3 Biogasification of Cassava Peel Waste ................................ ................. 205 5.4.4 Soluble Chemical Oxygen Demand (sCOD) ................................ .......... 206 5.4.5 Total Solids (Dry Matter) and Volatile Solids Reduction ........................ 208 5.4.6 Enzyme Activity ................................ ................................ ..................... 209 5.5 Dividends and Applications of Anaerobic Digestion of Cassava Peel Waste .. 210 5 .5.1 Biomethane Potential and Applications of Biogas ................................ 210 5.5.2 Fertilizer Value Potential of Effluent from Mesophilic Anaerobic Digestion of Cassava Peel Waste ................................ ............................... 213 5.5.2.1 Nitrogen ................................ ................................ ....................... 215 5.5.2.2 Phosphorus ................................ ................................ .................. 216 5.5.2.3 Potassium ................................ ................................ .................... 216 6 CONCLUSIONS AND RECOMMENDATIONS ................................ ..................... 219 6.1 Conclusions ................................ ................................ ................................ .... 219 6.2 R ecommendations for Future W ork ................................ ................................ 222

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11 APPENDIX A PHOTOGRAPHS OF SOME MATERIALS AND EQUIPMENT USED AND THE PRODUCTS CREATED ................................ ................................ ........................ 224 B RECOMMENDED REFERENCES FOR FU THER READING .............................. 265 LIST OF REFERENCES ................................ ................................ ............................. 272 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 322

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12 LIST OF TABLES Table P age 1 1 Agricultural Commodities in the whole World in 2011 R anked by Production Quantity ................................ ................................ ................................ .............. 25 1 2 Agricultural Com modities in the whole World in 2 011 Ranked by Monetary Value ................................ ................................ ................................ ................. 26 1 3 Agricultural Commodities in Africa in 2011 R anked by Production Quantity ...... 27 1 4 Agricultural Commodities in Africa in 2 011 Ranked by Monetary Value ............ 28 1 5 Agricultural Commodities in Asia in 2011 Ran ked by Production Quantity ........ 29 1 6 Agricultural Commodities in Asia in 2011 Ranked by Monetary Value .............. 30 1 7 Agricultural Commodities in the Am ericas in 2011 R anked by Production Quantity ................................ ................................ ................................ ............. 31 1 8 Agricultural Commodities in the Americas in 2 011 Ranked by Monetary Value ................................ ................................ ................................ ........................... 32 1 9 Global Rank of Cassava in 2011 among all Agricultural Commodities in terms of Production Quantity and Monetary Value ................................ ...................... 33 2 1 u ral Commodities: 1999 to 2011 ......... 44 2 2 Global Statistics on Cassava Production in 2011, by Country ........................... 45 2 3 Global St atistics on Cassava Production in 2011, by Region ............................ 46 2 4 Some Properties of Amyloses from Cereal, Legume and Root and Tuber Starches ................................ ................................ ................................ ............ 60 2 5 Some Properties of Amylopectins from Cereal, Legume and Root and Tuber Starches ................................ ................................ ................................ ............ 65 2 6 Some Distinguishing Properties between Amylose and Amylopectin ................. 65 2 7 Some Properties of Cassava Starch ................................ ................................ ... 68 2 8 Degree of Hydrolysis of Native Cassava Starch Achieved with Various Enzy mes and Operating Conditions ................................ ................................ ... 81 2 9 Detoxification of Cassava Components and Products by various Processing Methods ................................ ................................ ................................ .............. 87

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13 2 10 Advantages and Disadvantages of Solid State Fermentation (SSF) ................ 108 2 11 Substrates used in Microbial Fermentations, Organisms Employed and the Products Generated ................................ ................................ ......................... 111 2 12 Optimum moisture content [% W/W] for various combinations of microorganism and substrate in enzyme production ................................ ........ 121 2 13 Optimum temperature for enzyme production and activity with various combinations of microorganism and substrate ................................ ................. 124 3 1 Saturated Salt Solutions and Associated Equilibrium Relative Hum idity when placed in Desiccator Jars. ................................ ................................ ................. 139 3 2 Physical properties of cassava flour with particle size 0.5 mm. ..................... 144 4 1 Some Results of Alpha Amylase Hydrolysis of Starch from Sources other than corn ................................ ................................ ................................ .......... 152 4 2 Statistics of Enzymes used in the Research Work ................................ ........... 155 4 3 Assay Methods used by ABC Research Laboratories for Analysis of Samples 161 4 4 Experimental Design for Synergistic Enzymatic Hydrolysis of Differe nt Cassava Substrates ................................ ................................ ......................... 169 4 5 Compositional Analysis o f Different Cassava Substrates ................................ 174 4 6 Maximum Starch Conve rted to Glucose in Different Cassava Substrates after 24 hours of Enzyme Hydrolysis at 60 o C ................................ ......................... 176 4 7 Maximum Starch Converted to Glucose in Different Cassava Substrates after 96 hours of Enzyme Hydrolysis at 37 o C ................................ ......................... 179 4 8 Reaction Rate Constant (k), Correlation Coefficient (R 2 ) and Regression Equation of First Order Reaction for Enzyme Hydrolysis of Different Cassav a Substrates at 60 o C. ................................ ................................ ......................... 183 4 9 Reaction Rate Constant (k), Correlation Coefficient (R 2 ) and Regression Equation of First Order Reaction for Enzyme Hydrolysis of Different Cassava Subst rates at 37 o C. ................................ ................................ ......................... 183 4 10 Activation Energy (E A ) and Regression Equations for Estimating the Arrhenius Temperature Dependency of Rate Constant for Enzyme Hydrolysis of Different Cassava Substrates ................................ ................................ ....... 184 4 11 Reaction Kinetics Parameters for Enzyme Hydrolysis of Starch in Different Cassava Substrates ................................ ................................ ......................... 185

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14 5 1 Formulae for Methane Yield Analysis and for Determination of Properties of Cassava Peel Waste used as Feedstock/Substrate in Anaerobic Digestion for Biogas and Enzyme Production ................................ ................................ .. 198 5 2 Mass Distribution of Cassava Root Components ................................ ............. 202 5 3 Substrate Characteristics and Bio Methane Potentials of Mesophilic Anaerobic Digestion of Cassava Peel Waste ................................ ................... 204 5 4 Dry Matter and Volatile Solids Reduction during Anaerobic Digestion of Cassava Peel Waste. ................................ ................................ ....................... 208 5 5 The Activities of Enz ymes Produced by Anaerobic Digestion of Cassava Peel Waste ................................ ................................ ................................ ............. 209 5 6 The Activities of Enzymes Produced by Anaerobic Digestion of Pieces of Whole Unpeeled Cassava Roots ................................ ................................ .... 209 5 7 Specific Activities of Enzymes Produced by Anaerobic Diges tion of Cassava Root Materials ................................ ................................ ................................ 210 5 8 Methodologies used for the Analysis of Fertilizer Components of Post Anaerobic Digest ion Liquor ................................ ................................ .............. 214 5 9 Organic Fertilizer Potential of Anaerobic Digestion of Cassava Peel Waste .... 218

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15 LIST OF FIGURES Figure P age 1 1 Components of Cassava Crop: Roots; Leav es; and Seeds .............................. 23 1 2 Overview/Global Architecture of Scheme of Study ................................ ............. 37 2 1 Schematic of Unit Operations for the Manufacture of Cassava Chips, Fl our, and Starch ................................ ................................ ................................ ........ 52 2 2 An Overview of the Structure of Starch Gra nule ................................ ................. 58 2 3 Structural Formula and Linear C onfiguration of Amylose ................................ .. 60 2 4 Structural Features of Amylopectin Highlighti ................................ ................................ ............................ 61 2 5 Structural Formula and Profiles of Amylo pectin Branches and Clusters ........... 62 2 6 Basic labeling of chains in amy lopectin ................................ ............................. 63 2 7 A model of how the clusters of amylopectin build up the semi crystalline granular rings ................................ ................................ ................................ .... 64 2 8 Structure o f Alpha Amylase with calcium and chloride ions visible .................... 72 2 9 Schematics of how Amylase randomly splits Starch Molecules to liberate Glucose, Maltose and Limit Dextrins. ................................ .............................. 73 2 10 Structural Features of Be ta Amylase. ................................ ................................ 74 2 11 Limit Dextrins ...... 75 2 12 Structure of Glucoam ylase ................................ ................................ ................ 76 2 13 Glucoamylase Model and Mechanism of Starch Digestion ................................ 77 2 14 Hydrolysis of Starch by Amylase to liberate Glucose Molecules ..................... 78 2 15 Overview of the Pattern of Starch Digestion by major Starch Hydrolyzing En zymes ................................ ................................ ................................ ............ 79 2 16 Structures of the Aliphatic Cya n ogenic Glucosides of Cassava : Lina marin and Lotaustralin ................................ ................................ ................................ 82 2 17 The Biosynthesis Pathway of Linamarin showing Valine as the Precursor ........ 83

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16 2 18 The Separation cum Distribution of Linamarin and Linamarase i n the Plant System ................................ ................................ ................................ .............. 84 2 19 Mechanism of Hydrolysis of Cyanogenic Glucosides and Liberatio n of Hydrogen Cyanide ................................ ................................ ............................. 85 2 20 Production and Toasting of Casabe on a Wood F ired Griddle ......................... 99 2 21 Three Dimensi onal Image of A lpha Amylase ................................ ................... 106 2 22 Schematic of a Fluidized Bed Bioreactor that uses Humidified Air for Pneumatic Agitation. ................................ ................................ ......................... 128 3 1 Prototype Solar Convection Dryer used for Drying Cassava Chips .................. 137 3 2 Air Flow Apparatus for Determination of Permeability ................................ ...... 140 3 3 Sorption Isotherm of Fresh Cassava Root Pulp Slic es for Drying into Chips .. 142 3 4 Replicate Drying Curves for Drying Cassava Slices into C hips. ....................... 143 3 5 Particle Size Distribution in Cassava Flour from Grinding Dry Cassava Chips under various Blender Operating Conditions. ................................ ......... 143 4 1 Size Reduction Devices used in Sample Preparations. ................................ ... 158 4 2 Reactor Devices and Systems for Hydro lysis Experiments ............................ 160 4 3 Stoichiometry for the Detection of Reducing Sugars as Glucose through Reactions with 3,5 dinitrosalicylic acid (DNS) Reagents ................................ .. 163 4 4 Sp ectrophotometers used for Measurement of Absorbance in Samples Reacted with DNS Reagent in Order to Determine Reduci ng Sugars as Glucose ................................ ................................ ................................ .......... 164 4 5 Calibration Curve used for Estimation of Reducing Sugars (As Glucose) in the 3, 5 dinitrosalicylic acid (DNS) Analytical Method. ................................ ...... 165 4 6 Scales used f or Mass Measurements ................................ ............................. 166 4 7 Pipettes used in Sample withdrawals and Analysi s ................................ ......... 167 4 8 Monitoring Devices for pH, and Soluble Solids ................................ ............... 168 4 9 Vacuum Evaporator Used for Glucose Syrup Conc entration .......................... 170 4 10 Mass Balance Stoichiometry for Enzymatic Hydrolysis of Different Cassava Substr ates ................................ ................................ ................................ ...... 171

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17 4 11 Profile of Glucose Content Vs. Time during Enzyme Hydrolysis of Commercial Cassava Starch at 60 o C ................................ ............................ 175 4 12 Profile of Glucose Content Vs. Time during Enzyme Hydrolysis of Flour Produced from Solar Dried Cassava Chips at 60 o C ................................ ...... 175 4 13 Profile of Glucose Content Vs. Time during En zyme Hydrolysis of Ground Fresh Cassava Root Pulp at 60 o C ................................ ................................ 176 4 14 Comparison Profile of Glucose Content Vs. Time during Enzyme Hydrolysis of Commercial Cassava Starch, Flour Produc ed from Solar Dried Cassava Chips and Ground Fresh Cassava Root Pulp at 60 o C ................................ ... 177 4.15 Conversion Profile of Enzyme Hydrolysis of Starch to Glucose in Different Cassava Substrates at 60 o C. ................................ ................................ ......... 177 4 16 Profile of Glucose Content Vs. Time during Enzyme Hydrolysis of Commercial Cassava Starch at 37 o C ................................ ............................. 178 4 17 Profile of Glucose Content Vs. Time during Enzyme Hydrolysis of Flour Produced from Solar Dried Cassava Chips at 37 o C ................................ ....... 178 4 18 Profile of Glucose Content Vs. Time dur ing Enzyme Hydrolysis of Ground Fresh Cassava Root Pulp at 37 o C ................................ ................................ .. 179 4 19 Comparison Profile of Glucose Content Vs. Time during Enzyme Hydrolysis of Commercial Cassava Starch, Flour P roduced from Solar Dried Cassava Chips and Ground Fresh Cassava Root Pulp at 37 o C ................................ .... 18 0 4 20 Conversion Profile of Enzyme Hydrolysis of Starch to Glucose in Different Cassava Substrate s at 37 o C ................................ ................................ ........... 180 4 21 Semi log Plot of Unaccomplished Glucose Conversion over Time for Estimation of First Order Reaction Rate Constants for Enzyme Hydrolysis of Starch with Different C assava Substrates at 60 o C ................................ ......... 182 4 22 Semi log Plot of Unaccomplished Glucose Conversion over Time for Estimation of First Order Reaction Rate Constants for Enzyme Hydrolysis of Starch with Different Cassava Substrates at 37 o C ................................ ......... 182 4 23 Semi log Plot of First Order Reaction Rate Constants versus Reciprocal Absolute Temperature for Estimation of Arrhenius Activation Energy for Enzyme Hydrolysis of Cassava Starch at 37 o C and 60 o C ............................ 184 5 1 Anaerobic Digestion System Design ................................ ................................ 189 5 2 My Weigh Shipping Scale used to weigh out Cassava Roots ........................... 190

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18 5 3 Implements used to Peel Cassava Roots and Prepare the Peelings for Experiment s ................................ ................................ ................................ .... 191 5 4 Components of C assava Root ................................ ................................ ......... 192 5 5 Gas Chromatograph (GC) System used to Estimate the Methane and Carbon dioxide Contents of Biogas Gene rated by Anaerobic Digestion of Cassava Peel Waste ................................ ................................ ................................ ..... 195 5 6 Laboratory Equipment used in sCOD analysis of Leachate from Anaerobic Digestio n of Cassava Peel Waste ................................ ................................ .. 196 5.7 Clinical Centrifuge used for Enzyme Extractio n ................................ ............... 199 5 8 Methane Yield of Mesophilic Anaerobic Digestion of Cassava Peel Was te ...... 205 5 9 Profiles of Soluble Chemical Oxygen Demand (sCOD) for Mesophilic Anaerobic Digestion of Cassava Peel Waste ................................ ................... 207 5 10 Mass Balance for Anaerobic Digestion of Peel Waste from one tonne (1000 kg) of fresh Cassava Root ................................ ................................ ................ 211 5 11 Mass Balance for Anaerobic Digestion of 1 tonne (1000 kg) of C assava Peel Waste ................................ ................................ ................................ .............. 213

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19 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYNERGISTIC ENZYMATIC HYDROLYSIS OF CASSAVA STARCH AND ANEAROBIC DIGESTION OF CASSAVA WASTE By Samuel NwaneLe Aso December 2013 Chair: Arthur A. Teixeira Major: Agricultural and Biological Engineering Cassava is a major root crop and source of food and feed carbo hydrate for humans and livestock. Cassava provides economic and subsistence value to more t han 800 million to one billion people in Brazil, China, Haiti, India, Indonesia, Nigeria, Thailand and many other countries However, current processe s for conversio n of cassava r oot s to value added products are far from optimum. Glucose is produced by first extracting starch from the roots. The starch is then subjected to two separate operation s called liquefaction and sa ccharification. Each operation incurs many cos t elements. Liquefaction for example demands elaborate equi pment and energy inputs, while starch extraction is energy, labor and technology intensive. These requirements place glucose and other value added cassava products beyond the reach of many developi ng nations. It would be efficient and advantageous if liquefaction and sa ccharification could be carried out simultaneously as one operation and the starch hydrolyzed directly without going through the extraction procedure. Furthermore, over 60 % of global cassava output (which was 252.2 x 10 9 kg in 2011) is processed annually for human food, generating enormous quantities of organic waste matter with

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20 attendant environmental and disposal issues. Anaerobic digestion of cassava wastes could generate beneficia l products and at the same time be environment friendly. The objectives of this study were to develop and d emonstrate a village scale method of process ing fresh cassava root into flour from solar convection dried cassava chips, produce glucose sweetener f rom combined liquefaction and sa ccharification hydrolysis of native starch that was not first extracted, and generate biogas (methane) from anaerobic digestion of cassava waste. A solar convection dryer requiring no fuel or electricity was used to dry thi n slices of cassava root into dry chips that were subsequently ground into cassava flour. The flour as well as freshly ground cassava root (pulp) was used as substrate for conversion into glucose by enzyme hydrolysis. The rates of conversion (kinetics) for both substrates were determined at two different temperatures and compared with those of commercially available refined cassava starch. Cassava waste from peelings and trimmings was used as feedstock for anaerobic digestion into methane fuel. Results show ed that rates of reaction for hydrolysis of all three substrates were similar to each other at both temperatures, but resulted in different extent of reaction. The cassava flour and pulp produced syrup with 3 4% glucose, while the commercial starch produce d 10% glucose within 4 hours at 60C, and 72 hours at 37C. Anaerobic digestion of cassava waste produced bio methane yield of 0.25 liter/ gram volatile solids with a production efficiency of 71 %. These findings would suggest the possibility of a cottage industry cassava processing operation that would be self sufficient in which fresh cassava roots could be solar convection dried to make flour. The flour and grated root pulp could be hydrolyzed in one step to make glucose syrup sweetener, while the

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21 waste peelings and trimmings could be processed by anaerobic digestion to produce useful energy in the form of biogas for maintaining digester temperature and/or supplementing heat required for hydrolysis and in the solar dryer. In addition, amylolytic (starch h ydrolyzing) enzymes could be harvested from the anaerobic digestion liquor, and the post digestion effluent applied to farm land as source of organic fertilizer.

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22 CHAPTER 1 INTRODUCTION 1.1 Background Cassava is known botanically as Manihot esculenta Cra ntz. It is variously referred to as Mandioca (in Portuguese); Manioc (in French); Yuca (in Spanish); Cassave (in Haitian Creole) and Akpakpuru (in Ikwerre, a Nigerian language). Cassava is a perennial plant widely grown in more than 90 countries in tropica l and sub tropical regions of the world (Wilson and Dufour, 2002). It is chiefly cultivated for its starchy roots (FAO, 1990; Chadha, 1961). However, young cassava leaves are consumed by humans as a vegetable (Bokanga, 1994 a; Koch et al., 1994; FAO, 1990; Hahn, 1983; Lancaster and Brooks, 1983), or used as protein supplement in livestock feeds (Borin et al., 2005; Wanapat et al., 2000; Ravindran, 1993, 1992; Hahn et al., 1992; Balagopalan et al., 1988; Moore, 1976). The seeds are used mainly in breeding pr ograms (Ceballos et al., 2012; Nassar and Ortiz, 2010; Rajendran et al., 2005; El Sharkawy, 2004; Iglesias et al. 1994; Nartey, 1978). Figure 1 1 shows some useful components of the cassava crop. 1.1.1 Importance Cassava has been reported to be the fourth most important food crop in developing nations after rice, wheat and maize/c orn (Johnson et al ., 2005; Koch et al carbohydrates for human food (Nassar and Ortiz, 2010 ; Claude and Denis, 1990; Fauquet and Fargette, 1990; Phillips, 1982). In Zaire, 60% of the daily calorie intake is provided by cassava r oot s, while 20% of protein com es from cassava leaves (Koch et al 1994).

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23 Figure 1 1 C omponents of Cassava Crop: Root s; Leaves; and Seeds (bing.com, 2013 a). Globally, cassava provides subsistence for over 800 million to one billion people (Clement et al., 2010; Nassar and Ortiz, 2010; FAO, 2008; Phillips, 1982), ser ving as food security or hunger alleviator; income provider; employment generator; market developer, including export markets; as well as input for industrial products like chips, flour, starch, sweetener, fuel alcohol and biochemicals (Hakizimana, 2010; W idowat i and Hartojo, 2010; Johnson et al ., 2009 ; Eneas, 2006; Srinorakutara et al 2006; El Sharkawy, 20 04; FAO, 2001; Lynam, 1994; Balagopalan et al 1988; Berghofer a nd Sarhaddar, 1988; Srikanta et al ., 1987; Matsumoto et al ., 1982; Ueda et al 1981; Okigbo, 1980; Lindeman and Rocchiccioli, 1979).

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24 1.1 .2 Advantages per unit land area per unit of time is said to be significantly higher than that of oth er staple food crops (Shetty et al ., 2007; Koch et al 1994; Balagopalan et al 1988; Okigbo, 1980). As a root crop, cassava has high biological ef ficiency because the most important edible carbohydrate part (the r oot) lies underground and does not require support from heavy stems and branches (Coursey and Haynes, 1970). Cassava can be planted most time of the year and is available all year long; is productive on poor soils; tolerant of adverse climatic conditions; adaptable to various farming systems and can be intercropped with beans, maize (corn), yam and several other crops. Cassava is resistant to pests and diseases; is easily propagated by stem cuttings and its production is less labor intensive than yam. Also, cassava has more than two years harvest window. This advanta ge precludes the need for expensive energy intensive preservation cum storage technique like refrigeration, affords farmers protection against famine, and guarantees flexible labor management schedules. Furthermore, cassava is endowed with good quality sta rch (Tonukari, 2004; Westby, 2002; International Starch Institu al 1 994; Hahn, 1992; Balagopalan et al 1988); that can be transformed to products with huge industrial applications. 1.2 Justifications 1.2.1 Rationale De spite huge economic and social relevance demonstrated in the enumerated importance and advantages, cassava crop is considered an orphan crop, only suitable for poor subsistence farmers of low economic status. Cassava has traditionally received relatively m inor investment and attention for resear ch and development (Ceballos et al

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25 2012; FAO, 2008; Cock, 1985; Hahn and Keyer, 1985; Rogers, 1965). The rankings of top 20 agricultural commodities produced in the world in 2011 in terms of quantity and monetary v alue are presented in Table 1 1 and Table 1 2 f or entire planet earth; Table 1 3 and Table 1 4 for Africa; Table 1 5 and Table 1 6 for Asia; and Table 1 7 and Table 1 8 for the Americas. Table 1 1 Agricultural Commodities in the whole World in 2011 Ran ked by Production Quantity (Source: Compiled from FAO online statistical data base; FAO, 2013) Rank Commodity Production Quantity (1 x 10 9 kg) 1 Sugar cane 1794.35 2 Maize 883.46 3 Rice, paddy 722.76 4 Wheat 704.08 5 Cow milk, whole, fresh 606.66 6 Potatoes 374.38 7 Sugar beet 271.64 8 Vegetables fresh 268.37 9 Soybeans 260.91 10 Cassava 252.20 11 Tomatoes 159.02 12 Barley 134.27 13 Indigenous Pig meat 108.64 14 Bananas 106.54 15 Watermelons 104.47 16 Sweet potatoes 104.25 17 Buffalo milk, whole, fresh 93.01 18 Indigenous Chicken Meat 89.55 19 Onions, dry 85.37 20 Apples 75.63

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26 Table 1 2 Agricultural Commodities in the whole World in 2011 Ranked by Monetary Value (Source: Compiled from FAO online statistical data base; FAO, 2013) R ank Commodity Monetary Value (Int $1 x 10 9 ) 1 Rice, paddy 187.88 2 Cow milk, whole, fresh 181.27 3 Indigenous Cattle Meat 168.94 4 Indigenous Pig meat 167.00 5 Indigenous Chicken Meat 127.56 6 Wheat 86.27 7 Soybeans 64.14 8 Tomatoes 58.10 9 Mai ze 57.42 10 Sugar cane 56.63 11 Hen eggs, in shell 53.86 12 Potatoes 50.27 13 Vegetables fresh 46.12 14 Grapes 39.81 15 Cotton lint 37.30 16 Buffalo milk, whole, fresh 36.55 17 Apples 31.84 18 Bananas 29.57 19 Cassava 24.59 20 Mangoes, mangoste ens, guavas 23.30

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27 Table 1 3. Agricultural Commodities in Africa in 2011 Ranked by Production Quantity (Source: Compiled from FAO online statistical data base ; FAO, 2013) Rank Commodity Production Quantity (1 x 10 9 kg) 1 Cassava 140.96 2 Sug ar cane 83.45 3 Maize 65.05 4 Yams 54.35 5 Plantains 28.81 6 Cow milk, whole, fresh 27.50 7 Rice, paddy 26.53 8 Potatoes 26.32 9 Wheat 22.08 10 Sorghum 20.78 11 Vegetables fresh 17.49 12 Sweet potatoes 17.10 13 Tomatoes 16.55 14 Bananas 15.39 15 Millet 10.78 16 Sugar beet 9.92 17 Groundnuts, with shell 9.43 18 Onions, dry 8.34 19 Oranges 7.13 20 Taro (cocoyam) 7.07

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28 Table 1 4 Agricultural Commodities in Africa in 2011 Ranked by Monetary Value (Source: Compiled from FAO online statistic al data base ; FAO, 2013) Rank Commodity Monetary Value (Int $1 x 10 9 ) 1 Cassava 14.63 2 Indigenous Cattle Meat 13.57 3 Yams 11.34 4 Cow milk, whole, fresh 8.33 5 Rice, paddy 7.06 6 Indigenous Chicken Meat 6.51 7 Maize 6.22 8 Tomatoes 6.1 1 9 Plantains 5.38 10 Bananas 4.33 11 Groundnuts, with shell 3.96 12 Potatoes 3.96 13 Indigenous Sheep Meat 3.53 14 Vegetables fresh 3.29 15 Cocoa beans 3.00 16 Sorghum 2.94 17 Wheat 2.87 18 Indigenous Goat Meat 2.75 19 Sugar cane 2.65 20 Olive s 2.59

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29 Table 1 5. Agricultural Commodities in Asia in 2011 Ranked by Production Quantity (Source: Compiled from FAO online statistical data base; FAO, 2013) Rank Commodity Production Quantity (1 x 10 9 kg) 1 Sugar cane 710.93 2 Rice, paddy 653.2 4 3 Wheat 317.86 4 Maize 270.86 5 Vegetables fresh 231.29 6 Potatoes 174.63 7 Cow milk, whole, fresh 163.34 8 Tomatoes 96.47 9 Buffalo milk, whole, fresh 90.16 10 Watermelons 87.75 11 Sweet potatoes 83.07 12 Cassava 76.68 13 Indigenous Pig meat 61.88 14 Bananas 61.64 15 Onions, dry 56.73 16 Cucumbers and gherkins 56.72 17 Cabbages and other brassicas 51.85 18 Coconuts 49.19 19 Apples 48.13 20 Eggplants (aubergines) 44.15

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30 Table 1 6. Agricultural Commodities in Asia in 2011 Ranked by Mone tary Value (Source: Compiled from FAO online statistical data base ; FAO, 2013) Rank Commodity Monetary Value (Int $1 x 10 9 ) 1 Rice, paddy 169.11 2 Indigenous Pig meat 95.13 3 Cow milk, whole, fresh 48.96 4 Wheat 43.40 5 Indigenous Chicken Me at 42.34 6 Vegetables fresh 39.42 7 Indigenous Cattle Meat 36.22 8 Buffalo milk, whole, fresh 35.44 9 Tomatoes 35.04 10 Hen eggs, in shell 31.67 11 Potatoes 26.12 12 Cotton lint 25.54 13 Sugar cane 21.90 14 Apples 20.27 15 Palm oil 18.37 16 Mangoes, mangosteens, guavas 18.10 17 Bananas 17.10 18 Other bird eggs, in shell 15.10 19 Maize 13.56 20 Grapes 12.12

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31 Table 1 7 Agricultural Commodities in the Americas in 2011 Ranked by Production Quantity (Source: Compiled from FAO online statis tical data base ; FAO, 2013) Rank Commodity Production Quantity (1 x 10 9 kg ) 1 Sugar cane 972.68 2 Maize 438.38 3 Soybeans 223.70 4 Cow milk, whole, fresh 179.29 5 Wheat 110.35 6 Potatoes 41.56 7 Indigenous Chicken Meat 39.43 8 Rice, paddy 3 7.87 9 Oranges 36.15 10 Cassava 34.36 11 Indigenous Cattle Meat 30.29 12 Sugar beet 28.85 13 Bananas 27.86 14 Tomatoes 24.19 15 Sorghum 19.96 16 Indigenous Pig meat 18.03 17 Barley 16.63 18 Rapeseed 15.12 19 Grapes 15.11 20 Hen eggs, in shell 1 3.20

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32 Table 1 8. Agricultural Commodities in the Americas in 2011 Ranked by Monetary Value (Source: Compiled from FAO online statistical data base ; FAO, 2013) Rank Commodity Monetary Value (Int $1 x 10 9 ) 1 Indigenous Cattle Meat 81.83 2 Soybea ns 56.80 3 Indigenous Chicken Meat 56.17 4 Cow milk, whole, fresh 54.01 5 Maize 33.67 6 Sugar cane 31.18 7 Indigenous Pig meat 27.72 8 Wheat 15.54 9 Hen eggs, in shell 10.95 10 Rice, paddy 10.32 11 Tomatoes 8.94 12 Grapes 8.63 13 Cotton lint 8.2 3 14 Bananas 7.70 15 Oranges 6.98 16 Potatoes 6.51 17 Coffee, green 5.10 18 Indigenous Turkey Meat 4.39 19 Rapeseed 4.19 20 Apples 3.88 A careful review of Tables 1 1 through 1 8 reveals that cassava is not receiving commensurate financial valu e for the quantity produced. Apart from Africa, other regions of the world rewarded large quantities of cassava produced with low monetary

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33 values. Why should a commodity ranked 10 th or 12 th in terms of quantity produced be ranked 19 th or not even within th e first 20 in te rms of pecuniary value (Table 1 9)? This question begs for reasonable and creative answers. Perhaps a realistic solution might be to diversify cassava into generating industrial scale value added products such as flour, starch, and chips on the one hand. On the other hand, the potential of cassava in the production of single cell protein, alcohol, enzymes, sweeteners, and biogas could commodities tables. Table 1 9 Global Rank of Cassava in 2011 among all Agricultural Commodities in terms of Production Quantity and Monetary Value ( Source: Compiled from FA O online statistical data base; FAO, 2013) Region Production Quantity & Monetary Value Quantity [1 x 10 9 kg] Rank Value [Int $1 x 10 9 ] Rank Africa 140.96 1 14.63 1 Americas 34.36 10 2.05 Below 20 Asia 76.68 12 7.88 Below 20 Oceania 0.19 Below 20 0.01 Below 20 World 252.20 10 24.59 19 1.2.2 Challenges and Opportunities Challenges that limit utilization of cassava crop include shelf life and mass; two step hydrolysis procedure; source and format of starch substrate; and enzyme availability. Cassava r oot s are bulky and heavy, and therefore expensive to transpo rt over long distances. The r oot s are also perishable, and must be either consumed or

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34 processed within a few days after harvest Transforming cassava root into value added product such as glucose sweetener could alleviate this challenge. A major limitation of conventional method of produc ing glucose sweetener is the procedure of two separate operations called liquefaction and saccharification. Liquefaction is the first stage whereby starch is gelatinized by heat treatment and thermostable alpha amylase enzyme partially hydrolyses the starc h into maltodextrins. Saccharification commences in the second stage where the partially hydrolyzed starch is converted to glucose by the action of glucoamylase. This protocol requires more process vessels, piping, and associated equipment; conditioning fo r enzyme systems; and starch gelatinization is energy intensive. An advantageous alternative approach would be to deploy alpha amylase and glucoamylase enzymes simultaneously, and carry out both liquefaction and saccharification processes inside the same r eactor vessel as a single unit operation. This option should be simpler, and more efficient. It is termed synergism and is the hydrolysis technique investigated in this study. Maize (corn) is the dominant raw materia l for global starch production (De Baere 1999; Gordon, 1999; Swinkels, 1985 ), accounting for more than 80% of the total starch processed ( Johnson et al., 2005 ; De Braganca and Fowler, 2004; De Baere, 1999) This has relegated other sources of starch such as cassava to the background. In the conv entional method of glucose sweetener production, starch is first extracted in the pure form from the native raw material. Subsequently, the extracted starch is subjected to hydrolysis to produce glucose. However, the starch extraction process is time, ener gy, technology, labor and cost intensive; and therefore not feasible in most developing nations. A more feasible alternative is to use the direct conversion method.

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35 Direct conversion procedure involves hydrolyzing native starch in raw materials with minima l value added history. Two approaches to the direct conversion technique were investigated in this work. The first was to hydrolyze cassava flour. The flour was produced from cassava chips that were dried with a solar convection dryer developed at the Univ ersity of Florida (Schiavone et al., 2013). However, the cassava flour accumulated some cost and value adding processes; but not to the extent of extracted starch. The second approach investigated wet ground cassava root as substrate for enzyme hydrolysis. This latter approach would appear to be the best and simplest method over flour and starch because it accumulated the least cost and value adding operations. Enzymes are expensive. In 2011 a gram of alpha amylase could cost as much as US $252. Worldwide e nzyme sales were over $1.5 billion in 1998 (Beilen and Li, 2002). This value is projected to rise to $7 billion in 2013 ( Rep ortLinker, 2009) Because culture medium and substrate are important factors in the synthesis of industrial enzymes, the use of low cost substrates such as cassa va peels and other biomaterials or agricultural byproducts can help reduce the cost of sweetener production Cassava wastes such as peels are generated during the processing and production of numerous cassava based food product s such as chikwangue, farinha de mandioca, and gari. With manual peeling, the peel can constitute 20 35 % of the total weight of the root (Ekundayo, 1980). Therefore, enormous organic waste is generated from cassava processing. This waste should be good feed stock for anaerobic digestion and fermentation processes that could generate biogas and hydrolytic enzymes. (Adeniran and Abiose, 2011 ; Silva et al., 2009; Cuzin et al., 1992). It will appear to be a win and

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36 win again opportunity if cassava peel is di gested to produce biogas that could be used as a source of energy while extracting hydrolytic enzymes from the digestate at the same time. This study explored both opportunities. 1.3 Objectives The objectives of the study were to: 1. Demonstrate a cottage in dustry level process fo r the production of cassava flour from solar convection dried cassava chips. The flour could be used for the manufacture of bread and other bakery products. 2. Demonstrate a cottage industry level process for the production of glucose sweetener from synergistic hydrolysis with commercial enzymes on the following substrates: a) Commercially available refined cassava starch. b) Flour produced from solar convection dried cassava chips. c) Freshly ground cassava root pulp The sweeteners would s erve as sugar substitutes to consumers; and as industrial ingredient for use in the beverage, confectionery, pharmaceuti cal and the allied industries. 3. Estimate first order kinetic parameters (rate constant along with the Arrhenius activation energy) from the enzyme hydrolysis of each substrate at two different temperatures 4. Demonstrate anaerobic digestion of cassava waste by performing experiments and reporting performance indicators such as: methane yield, s COD, pH, etc. 5. Isolate and determine the activity of enzymes from post anaerobic digestion broth. The architecture of the entire scheme of study is presented in Figure 1 2.

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37 Figure 1 2. Overview/Global Architecture of Scheme of Study Peels Package: Product Clean Peeled Root s Enzyme Biogas Glucose Sweetener Wet Slurry Flour Chips Chipping & Dicing, Drying; Grinding/Milling, Sieving Package: Product Synergistic Enzymatic Hydrolysis Package: Product Anaerobic Digestion Grating/Rasping Package: Product Package: Product Washing and Peelin g of Cassava Roots

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38 1.4 Organization of Dissertation The work report ed in this dissertation wa s presented in a series of self contained chapters as follows Chapter 1: i ntroduction ; Chapter 2: literature r eview which consisted of two parts. Part 1 dealt with c assava, i ts products and potential for enzyme hydrolysis Part 2 discussed the p roduction of enzymes by submerged and solid state fermentations Chapter 3 dealt with physical properties of cassava flour made from solar convection dried cassava c hips Chapter 4 reported on synergistic enzymatic hydrolysis of cassava s t arch Chapter 5 reported on experiments done on a naerobic digestion of cassava waste for production of biogas and amylolytic e nzymes Chapters 3, 4, and 5 each bega n with a concise background that contained a n over v iew of the literature pertinent to that ch apter, fol lowed by methodology and results of the work reported in that chapter. Each chapter serves as the basis for preparing a manuscript suitable for submission to be published in a scientific journal.

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39 CHAPTER 2 REVIEW OF LITERATURE The subject of thi s dissertation project concerns the enzyme hydrolysis of cassava starch for conversion to glucose. Therefore a review of the literature was undertaken in two parts PART 1: Cassava I ts origin, classification, agro climatic conditions, propa gation, product ion, utilization, packaging, etc. PART 2: Production of enzymes by submerged and solid state fermentations. 2.1 Part 1: Cassava 2.1.1 Origin and Distribution Numerous reports have supported the view that c assava originated in South America (Clement et al ., 2010; Nassar and Ortiz, 2010; Lotard et al., 2009; Allem, 2002; Olsen and Schaal, 2001, 1999; FAO, 1990; Fauquet and Fargette, 1990; Balagopalan et al., 1988; Lancaster et al., 1982; Okigbo, 1980; Coursey and Haynes, 1970, Rogers, 1965, Chadha, 1961). Cassava is said to have been domesticated as far back as 4000 BC. By 1500 AD, Portuguese explorers cultivated cassava plantations in South America. The Portuguese introduced cassava into Congo river basins of Africa around 1558 in the 16 th century; into Ja va in 1810; India in 1840; and Singapore and Malaysia in 1850 (FAO, 1990; Fauquet and Fargette, 1990; Balagopalan et al., 1988; Cock, 1985; Lancaster et al., 1982; Hohnholz, 1980; Okigbo, 1980). Today, Cassava is widely distributed and cultivated in most t ropical and sub tropical regions of planet earth. In the year 2011, one hundred countries in the world cultivate d cassava (FAO, 2013).

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40 2.1.2 Classification There has been ongoing scientific debate about the botanic classification and domestication of cassa va. On the one hand, there is the view that cassava emanated as a hybridization of several species (Ugent et al., 1986, Rogers and Appan, 1973; Rogers, 1965). On the other hand, cassava is postulated to have a sole progenitor (Lotard et al., 2009; Allem, 2002; Olsen and Schaal, 2001; Roa et al., 2000; Olsen and Schaal, 1999; Allem, 1994). Allem (1994) argued the recognition of three subspecies viz: Manihot esculenta esculenta ; Manihot esculenta peruviana ; and Manihot esculenta flabellifolia Manihot escule nta esculenta comprises the domesticate and includes all cultivars in cultivation. Manihot esculenta Crantz belongs to the family Euphorbiaceae and is the flagship of cultivated cassava crop. This family is generally divided into two main groups; the bitte r or toxic variety and the sweet or non toxic variety (Elias et al., 2004; Nye, 1991). The bases for classification have ranged from subjective opinions about organoleptic tastes to objective quantifiable cyanide content. Scientific reports have correlated bitter cassava with high cyanogenic potential ( Pereira et al., 1981; Sinha and Nair, 1968). I t was reported that the ratio of total cyanide in the peel to that in the parenchyma is low for bitter cassava varieties and high for the sweet varieties. In othe the linamarin concentration of the peel is considerably higher than the linamarin concentration of the parenchyma whe linamarin concentration is more evenly distributed throughout the entire root (Dufou r 2007, 1988 ; Gomez et al. 1985). Consequently sweet cassava with high cyanogenic potential and bitter cassava with low cyanogenic potential have been found (Bokanga, 1994 b; Sinha and Nair, 1968). There is little correlation between the cyanogen ic conte nt of the peeled root and the leaves. The latter

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41 tend to be high in cyanogens in both low (sweet) and high (bitter) cyanogenic cultivars (Gomez et al. 1985) Furthermore, it w as also reported that there is no strong morphological relationship establishe d between cyanogenic potential and other traits (Mahungu, 1994; Nye, 1991; Rogers, 1965). Therefore, using sensory taste to determine cynogenic potential may be misleading as there is considerable overlap between the cyanide content of bitter and sweet cla sses. This was demonstrated by Coursey (1973). By graphically representing the results of Sinha and Nair (1968), Coursey (1973), clearly illustrated the overlapping of cyanide content of Sweet, Non bitter, Bitter, and Very bitter cassava varieties; which w ere respectively (in ppm HCN [or mg HCN/kg]): 40 to 130, 30 to 180, 80 to 412.5, and 280 to 490 (Okigbo, 1980). Nevertheless, some criteria have been used to distinguish between bitter and sweet cassava varieties. In one characterization arising from a su rvey within the framework of the Collaborative Study of Cassava in Africa (COSCA), the sweet non toxic cassava varieties were defined as those which can be eaten raw or after simple boiling; while the bitter toxic varieties were those that must be processe d adequately before consumption (Nweke and Bokanga, 1994). This classification has been echoed by others (Clement et al., 2010; Wilson and Dufour, 2002 ; Mckey and Beckerman, 1993). Bolhuis (1954) noted that Koch (1933) was the author who tried to classify cassava roots according to their toxicity (cyanide content). In that work, three levels of classification were proposed (Tewe, 2004; Balagopalan et al., 1988; Okigbo, 1980; Coursey, 1973; Bolhuis, 1954; Koch, 1933): Innocuous: Less than 50 mg HCN per kg of fresh peeled root. Moderately poisonous: 50 to 100 mg HCN per kg fresh peeled root.

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42 Dangerously poisonous: More than 100 mg HCN per kg fresh peeled root. It should be noted that this classification wa s based on the premise that 50 to 60 mg HCN is the the oretical lethal dose for an adult weighing 50 kg (Boorsma, 1905). However, citing Rosling (1988), Lynam (1994) noted that up to 100 mg HCN equivalent per day can be detoxified by the body under normal dietary circumstances. Based on the foregoing, some wor kers now classify sweet cassava varieties as those with low cyanogenic potential defined as having less than 50 mg HCN equivalent per kilogram of fresh peeled root, and bitter cassava varieties as those with more than 50 mg HCN equivalent per kilogram of f resh peeled root (Clement et al., 2010; Elias et al., 2004; Wilson and Dufour, 2002). Other workers have used 100 mg HCN equivalent per kilogram of fresh peeled root to define the limit of low cyanogenic potential ( Dufour, 2007; Dixon et al., 1994). On the other hand, World Health Organization (WHO) standard (Codex Alimentarius Standard) for the total hydrocyanic acid content of edible cassava flour is 10 mg/kg maximum (FAO/WHO, 1995). 2.1.3 Agro Climatic C onditions There is a range of agro climatic conditi ons conducive for the production of cassava. The crop is suitably cultivated in the areas between 30 o North and 30 o South of the equator; at elevations from sea level to 2,500 meters and from areas with as little as 500 mm of rainfall, to tropical rain forest zones with more than 6,000 mm of rain per year. Cassava can adapt to soils pH of 3 to 9.5; temperatures between 8 and 33 o C; and relative humidity of 15 % to near saturation at 90 % (Wilson and Dufour, 2002; Consultative Group on International Agri cultural Research [CGIAR], 1997; Cock, 1985; Hohnholz, 1980; Lozano et al., 1980; Okigbo, 1980; Rogers, 1965) Cassava is noted to perform poorly under flooded soil conditions and at temperatures below 17 o C or above

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43 37 o C (El Sharkawy, 2004; Balagopalan et al., 1988; Hohnholz, 1980). Conditions reported for optimum performance of cassava include 1000 to 1500 mm of annual rainfall, pH of 5 to 8, and temperatures between 25 o C and 30 o C (El Sharkawy, 2004; Cock, 1985). 2.1.4 Propagation Cassava may be pr opagated by stem cuttings or seeds. Although seeds have been used for propagation operations (Rajendran et al., 2005; Sambatti et al., 2001; Rajendran et al 2000; Iglesias et al., 1994), most farmers use stem cuttings for multiplication and planting purp oses (Ceballos et al 2012; Iglesias et al., 1994; Kakes, 1990; Cock, 1985; Nartey, 1978; Chadha, 1961). Stem cuttings or stakes used for propagation may vary between 15 a nd 30 cm in length (Ceballos et al 2012; El Sharkawy, 2004; Hahn and Keyser, 1985) Each plating stake can have 5 to 11 nodes and may be planted horizontally, inclined or vertically on ridges or mounds formed over shallow pits and troughs. Using different stake lengths of 6.25 cm, 16 cm and 25 cm, it was shown that c assava can be culti vated at densities of 1000 to 400 0 stakes per hectare (Keating et al 1988). However, the number of plants per hectare ranges between 10000 and 15000 (Shetty et al., 2007). 2.1.5 Production Cass ava has emerged as one of the top twenty agricultural commodi ties in the World. In terms of quantity produced, cassava has consistently ranked among the first ten for the past co nsecutive twelve years (Table 2 1). Between 1999 and 2011, cassava output worldwide increased by more than 48%, from 169781525 to 252203769 metric tonnes (169.78 to 252.20 x 10 9 kg) (Table 2 1). The increase in global output may be attributed to improved varieties, better farming systems and increased cultivated land

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44 Table 2 1. r anking among World Agricult ural Commodities: 1999 to 2 011 ( Values were achieved, compiled, deduced and/or computed from FA O online statistical data base; FAO, 2013) Year Production Quantity [1 x 10 9 kg] Production Quantity Rank Monetary Value [Int $1 x 10 9 ] Monetary Value Rank 2011 252 .20 10 24 59 19 2010 236 70 9 23 00 19 2009 235 45 8 22 89 19 2008 232 00 8 22 45 20 2007 226 42 9 21 64 20 2006 223 56 9 21 31 20 2005 205 64 10 19 51 20 2004 201 11 10 19 08 20 2003 190.8 2 9 18 22 20 2002 184 28 9 17 50 20 2001 181 92 9 17 35 20 2000 176 27 9 16 82 20 1999 169 78 9 16 18 20 areas (Phillips et al., 2004; Tewe, 2004; Cock, 1985). Under traditional farming practices, cassava crop yield has been reported at 0.53 to 0.78 kg per plant (Wilson and Dufour, 2002); and 5 to 20 tonnes per hectare (Tewe, 20 04; Balagopalan et al., 1988; Keating et al., 1988; Cock, 1985; Hahn and Keyser, 1985; Hahn, 1983; Amsterdam, 1980; FAO, 1977; Chadha, 1961). In 2011, the average yield in India was 3 6.4 tonnes per hectare (Table 2 2). With modern farming techniques and in puts, yields over 60 tonnes per hectare may be normal (FAO, 1977). Cassava can produce 250 x 10 3 calories per hectare per day compared to 200 x 10 3 for maize (corn); 176 x 10 3 for rice; 114 x 10 3 for sorghum; and 110 x 10 3 for wheat (Coursey and Haynes, 19 70). The top countries that produce cassava in the world are shown in Table 2 2. Nigeria is the world leader in cassava production. In 2011, Nigeria produced 52403500 metric tonnes (52.40 x 10 9 kg) of cassava worth about Int $5.47 x 10 9 This production

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45 T able 2 2. Global Statistics on Cassava Production in 2011, by Country ( Values were achieved, compiled, deduced and/or computed from FAO online statistical data base; FAO, 2013) Country Rank of Country in Cassava production Rank of Cassava Amon g All the Crops Produced in the Country Production Quantity and Proportion Production Value (Int $1 x 10 9 ) Area Harvested [1 x 10 6 Ha] Yield [1 x 10 3 kg/Ha] Quantity [1 x 10 9 kg] Proportion [%] Nigeria 1 1 52.40 20.7 5.47 3.73 14.0 Brazil 2 5 25.44 10.0 1.32 1.74 14.6 Indonesia 3 2 24.00 9.5 2.45 1.18 20.3 Thailand 4 3 21.91 8.6 2.28 1.13 19.2 Democratic Republic of the Congo 5 1 15.56 6.1 1.61 2.17 7.1 Angola 6 1 14.33 5.6 1.49 1.07 13.3 Ghana 7 1 14.24 5.6 1.48 0.8 8 16.0 Viet Nam 8 3 9.87 3.9 1.03 0.56 17.6 India 9 20 8.07 3.2 0.84 0.22 36.4 Mozambique 10 1 6.26 2.4 0.65 0.97 6.4 Uganda 11 2 4.75 1.8 0.49 0.42 11.1 United Republic of Tanzania 12 1 4.64 1.8 0.46 0.73 6.2 China 13 Below 20 4.51 1.7 0.4 2 0.27 16.3 Cambodia 14 2 4.36 1.7 0.45 0.20 21.2 Malawi 15 1 4.25 1.6 0.44 0.19 21.5 Cameroon 16 1 3.90 1.5 0.36 0.28 13.9 Benin 17 1 3.60 1.4 0.37 0.25 13.9 Madagascar 18 2 3.30 1.3 .34 0.36 8.9 Rwanda 19 2 2.57 1.0 0.26 0.20 12.3 Paraguay 20 4 2. 45 0.9 0.13 0.18 13.5 Haiti 32 2 0.65 0.2 0.06 0.15 4.1 World 10 252.20 100.0 24.59 19.64 12.8 quantity accounted for 20.7% of the global output. The production quantity was harvested from 3737090 hectares of land at the yield rate of 14.0 tonnes per hectare. Nigeria was followed by Brazil, second producer in the world; Indonesia, third; Thailand, fourth; Democratic Republic of the Congo, fifth; Angola, sixth; and Ghana, seventh. T hese seven nations accounted for more than 60% of global cassava output in 2011( Table 2 2). Haiti was ranked 32 nd ; having produced about 0.2% of world output

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46 However, Haiti is a major cassava producer in the Sout h America and Caribbean region ( Henry and Hershey, 2 002) In 2011, Haiti was the largest cassava producer in the C aribbean, seconded by Cuba; and cassava was produced in the s econd largest quantity in Haiti, exceeded only by sugar cane (FAO, 2013). The 2011 statistics of global cassava output from the different regions of th e world is presented in Table 2 3. Africa d ominated world production with 55.8% share and 39 countries in active production. Asia followed with 30.4% and 14 countries. The Americas was third with 35 countries producing 13.6% of world cassava output. Oceania produced less than 0.1% of global cassava output in 2011 (Table 2 3). Table 2 3. Global Statistics on Cassava Production in 2011, by Region ( Values were achieved, compiled, deduced and/or computed from FA O online statistical data base; FAO, 2013). Region Global Rank of Region in Cassava P roduction Rank of Cassava Among All the Crops Produced in the Region Production Quantity and Proportion Monetary Value (Int $1 x 10 9 ) Area Harvested [1 x 10 6 Ha] Total No. of Countries Quantity [1 x10 9 kg ] Proportion [%] Africa 1 1 140.96 55.8 14.63 13.04 39 Western 1 76.06 30.1 7.94 5.70 15 Middle 1 36.25 14.3 3.72 4.02 9 Eastern 2 28.64 11.3 2.97 3.30 15 Asia 2 12 76.68 30.4 7.88 3.91 14 South Eastern 3 63.79 25.2 6.59 3.39 10 Eastern Below 20 4.51 1.7 0.42 0.27 1 Southern Below 20 8.36 3.3 0.86 0.24 3 Americas 3 10 34.36 13.6 2.05 2.66 35 South 5 32.30 12.8 1.86 2.34 11 Caribbean 5 1.35 0.5 0.12 0.26 16 Central Below 20 0.70 0.2 0.06 0.05 8 Oceania 4 Below 20 0.19 < 0.1 0.01 0.01 12 Melanesia 13 0.1696 < 0.1 0.0171 0.01 4 Polynesia 6 0.0145 < 0.1 0.0013 0.000929 7 Micronesia 4 0.0089 < 0.1 0.0009 0.000738 1 World 10 252.20 100.0 24.59 19.64 100

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47 2.1.6 Cassava U tilization Constraints that limit the utilization of cassava crop include sh elf life and mass. Cassava root s are bulky and heavy commodities and therefore expensive to transp ort over long distances. The r oot s are also extremely perishable, thus must be either consumed or processed within a few days after har vest (Eneas, 2006; Balagopalan et al., 1988; Kuppuswamy, 1961). To overcome these limitations, it may be p rudent to process cassava r oot s near the source of production; and into products that are less bulky and with longer shelf life. Such products include chips, flour, and starch. Other constraints on the utilization of cassava crop are associated with cost and technology as highlighted below. 2.1.6.1 Starch C assava r oot is richly endowed with good qua lity starch to the extent of 30 32 wt% (Westby, 2002; al., 1988). In addition, Cass ava starch exhibit s vital benefits and advantages over other sources of starch such as wheat, corn, and waxy maize. The reported advantages and benefits includ e the following (Central Tuber Crops Research Institute (CTCRI), 2010; Juszczak et al., 2003; Larotonda, et al., 2003; Balagopalan, 2002; Lopez Ulibarri and Hall, 1997; Balagopalan et al., 1988; Franco et al., 1988; Vijayagopal et al., 1988; Cereda and Wos iacki, 1985; Moorthy, 1985; Swinkels, 1985): Unique functional and structural qualities. Most bland flavor starch in the world. Does not have any mealy or undesirable starch flavors. Gelatinizes and swells rapidly in a narrow temperature range.

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48 The film ha s high: clarity, gloss, transparency, smoothness, continuity, flexibility, plasticity, folding endurance, internal strength, tensile strength, solubility, and rewetting capacity. High paste viscosity, clarity, and stability. High binding force and thickeni ng power. Good solubility and dry starch mobility. Fair resistance to retrogradation and syneresis (weeping). Less corrosion of granules from enzyme attacks. Smoother granules structure than potato starch granules. No foam building. Easy extractability. Co mplete and easier hydrolysis. The imperfections that have been noted for cassava starch are instability of hot gels and stickiness in cooking, fall in viscosity at elevated temperatures, and poor shear resistance (CTCRI, 2010; Balagopalan et al., 1988; Sw inkels, 1985). results with syrups from tapioca, wheat or potato starch which are as good as those advantages and quality of cassava starch, there is no significant application of its use in the manufacture of sweeteners such as glucose and high fructose syrup; unlike the case for corn starch. The limited application appears to be due to the extensive cost and technological requirements associated with the industrial production of glucose and high fructose syrup from corn (Johnson et al 2009; Hoehn et al., 1983). The devices, equipment, and techniques exploited for cooking/heating; clarification/filtr ation; concentration/evaporation; refining; enrichment/fractionation; crystallization, dehydration

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49 and other duties include the use of steam, extrusion, centrifugation, vacuum evaporation, carbon/ion exchange chromatographic separation regimes, membrane se paration (ultra filtration) reactors, encapsulation, spray dehydration, and various proprietary polishing, decolorizing and other unit operations methodologies (Cavette, 2010; Wikipedia, 2010; PUROLITE, 2007; Akdogan 1999; Lopez Ulibarri and Hall, 1997; P razeres and Cabral, 1994; Hanover and White, 1993; Darnoko et al., 1989; Hoehn et al., 1983). These levels of input can surpass the financial and technological capabilities of developing nations. Alternative, less cost and technology intensive approaches t o the manufacture of sweeteners from cassava starch are therefore necessary; and will appear to be desired options for farmers in developing countries. 2.1.6.2 Flour Cassa va flour is more difficult to preserve in storage (Balagopalan et al., 1988). This ma y be attributed to the hygroscopic nature of cassava flour (Kuppswamy, 1961), which is more than those of cassava chips and starch (CTCRI, 1986). Cassava flour at 6.7% moisture content that was packed in cloth or gunny bags picked up additional 5% moisture within 6 months of storage (Kuppswamy, 1961). Also, cassava flour is most commonly infested by Tribolium castaneum insect species (CTCRI, 1986; McFarlane, 1982; Ingram and Humphries, 1972); Bacillus and Enterococci species of bacteria, and Aspergillus f ungi species (Ingram and Humphries, 1972). In one comparative evaluation of packaging materials, polyethylene lined jute bags were found to be most effective for storage of cassava flour for up to 90 days (Etorma, 1936). Proper initial drying and the maint enance of l ow moisture content with the use of water vapor transmission resistant packaging material have been suggested for the packaging of cassava flour in order to prevent fungal or bacterial infection (Balagopalan et al., 1988; McFarlane, 1982). On th e

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50 other hand, cassava starch is thought to be neither attacked nor relished by the insects (Nanda and Potty, 1985; MacFarlane, 1982). However, high density polyethylene (HDPE) bags or polyethylene impregnated jute bags were recommended for the storage of c assava starch for up to 180 days in order to determine moisture, viscosity, total microbial count, and viable bacterial and fungal populations (Nanda and Potty, 1985). 2.1.6.2.1 Gluten free flour Celiac disease is a condition in which the mucous membrane of the small intestine of gluten intolerant people is damaged by gluten, resulting in inflammation and poor absorption of nutrients and consequently, weight loss, diarrhea, anemia, fatigue, flatulence, deficiency of folate and osteopenia (Blades, 1997; Tho mpson, 1997). The o eliac disor Gee (St. Bartholomew's Hospital Reports of 1888 ). C o eliac disorder was defined as a chronic indigestion occurring in people of all ages, particularly in o ne to five years old children. Cassava is free from gluten ( Rudrappa, 2013). Cassava flour and starch can be used to formulate gluten free breads ( Ahlborn et al., 2005; Lpez et al., 2004; Sanchez et al., 2002; Eggleston et al., 1992 ). Eating gluten free diet helps people with celiac disease control the symptoms and prevent complications ( Mayo Clinic, 2013). 2.1.6.3 Chips The requirement to transform cassava tubers to dried chips may be justified on account of the fact that the tubers are very perishable, bulky, and heavy. By transforming into chips, it will be possible to transport cassava economically (by mass and volume) to far distances on the one hand. On the other hand, cassava can then be preserved for a prolonged period because the chips store bett er and longer than the physiologically active tubers. Consequently, it will be possible to produce flour or starch

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51 from the chips at any time; and from facilities/plants that are distant or remote from the cassava crop production centers. 2.1.6.4 Packagi ng A food package is expected to provide protection against humidity, light, oxygen, moisture/condensation, heat/temperature, compression, shock, and vibration exposure that are associated with not only the physical environment, but also the storage, distr ibution and transportation system (Robertson, 2006 ; Soroka, 2002 ). Cassava starch, flour and chips must be protected from unwanted interaction with the environment. Water vapor transmission rate (WVTR) resistance is frequently the prime consideration in pr otective packaging of cassava products (Balagopalan et al., 1988). This is because to various degrees starch, flour and chips but especially flour is hygroscopic (CTCRI, 1986 ; Kuppuswamy, 1961 ). These products absorb moisture freely and hence suffer from biochemical deterioration, and biological spoilage from insect infe station, microbial growth, etc (Nanda and Potty, 1985; McFarlane, 1982; Parker and Booth, 1979 ; Balagopal and Nair, 1976 ) The relevant unit operations involved, as well as the equipment re quirement for the production of cassava prod ucts are identified in Figure 2 1 (Aso, 2004; Olomo and Ajibola, 2003; Lopez Ulibarri and Hall, 1997; Onabolu et al., 1998; Osunsami et al., 1989). 2.1.7 Sweetener Production Sweeteners are in great industrial demand for applications in the food, confectionary, beverage, pharmaceutical, and allied industries ( Kroger et al., 2006 ; BeMiller, 2002; Coulston and Johnson, 2002 ; Vuilleumier, 1993; Balagopalan et al., 1988 ) In these industries, sweeteners may be used to achieve, control, modify, or

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52 Drying Dewatering & Drying Grinding/ Milling Screening/Sieving Drying Centrifugation & Decantation Washing & Peeling Reception Of Cassava Roots Grating/Rasping Slicing/Dicing Solubilization & Sieving Pulverization & Screening/Sieving Solubilization & Sieving Centrifugation & Decantation Drying Pulverization & Screening/Sieving Pulverization & Screening/Sieving Figure 2 1. Schematic of Unit Operations for the Manufacture of Cassava Chips, Flour, and Starch. Equipment/Machinery Required for Cassava Chips, Flour & Starch Production include: Washers; Peelers; Chipping/Dicing Machines; Wet Grinding/Rasping Machines; Pressing Machines (to dewater); Dryers; Dry Grinding/Milling Machines; Dry Sieving Machines; Wet Mixing Machines (to Solubilize); Wet Sieving Machines; Centrifuges; Packaging Systems. Fresh Roots Peeled Roots Wet Chips Cassava Flour Dry Chips Product Ground Meal Cassava Starch Starch Milk Wet Starch Cake Dry Starch Cake Cassava Flour Cassava Starch Starch Milk Wet Starch Cake Dry Starch Cake Wet Pulp Dry Pulp

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53 influence one or more of the following properties: viscosity, hygroscopicity, flavor, size, adhesiveness, appearance, emulsification, etc ( Coulston and Johnson, 2002 ; International Pectin Producers Association (IPPA) 2001; McWilliams, 2001; Nabors, 2001; Hanover and White, 1993; Grenby, 1991 ; White and Lauer, 1990; Hoehn et al., 1983; Palmer, 1975) 2.1.7.1 Historical ove r view The genesis of nutritive sweeteners can be traced to 1811 when Kirchhoff, a Russian chemist discovered that heating a starch water mixture with dilute acid produced a sugar like sweet substance (Roberts et al., 1995; Balagopalan et al., 1988; MacAllister, 1980 ). At th e time it was thought that the substance was common sugar (sucrose). However, in 1814 Saussure proved that the substance was indeed glucose (Radley, 1940 ). In 1866, Dextrose was produced from corn starch ; and in 1957 enzymatic conversion of d glucose to d fructose was developed (Marshall and Kooi, 1957) Today, a gargantuan quantity of sweeteners is used to produce an astonishing array of products in the bakery, beverage, confectionery, food, pharmaceutical, and other industries. However, the growth in the sweetener arena benefited from and was boosted by advances in other sectors such as starch crop agronomy, enzyme engineering, and process technologies. Following is a brief sketch of the historical background on the production of sweeteners and H igh F ruct ose S yrup or HFS (Tomasik, and Horton 2012; Cavette, 2010; Wikipedia 2010; BeMiller, 2009; C orn Refiners Association, 2007; En symm Consulting on Biotechnology 2005; Hein et al., 2005; Me dical Ecology, 2004; Olson, 1995; Roberts et al., 1995; Bagopalan et al., 1988; MacAllister, 1980; Marshall and Kooi, 1957; Dale and Langlios, 1940). Early 1700s. Device to shell corn (remove kernels from cob) was patented.

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54 1811. GSC Kirchoff (a Russian chemist) produced dextrose and other starch derived sweeteners by heat ing potato starch in a weak solution of sulfuric acid. [Genesis of acid hydrolysis of starch?]!! 1841. The wet milling process (separation of starch from kernels) was patented by Orlando Jones. 1842. The first commercial wet milling plant in the USA was es tablished by Thomas Kingsford. 1844. The first dedicated corn starch plant in the world operated in Jersey City, New Jersey, USA. 1866. Dextrose was produced from corn starch. This was the first corn sweetener produced by adapting the acid conversion of st arch method. 1882. First manufacture of refined corn sugar (Anhydrous sugar). 1900. Introduction of thin boiling starches. 1915. Introduction of chlorinated starches. 1921. Introduction of crystalline dextrose hydrate. 1940. Dale and Langlois introduced en zyme conversion systems for syrups that were preliminarily made by acid hydrolysis. [Genesis of enzyme catalyzed hydrolysis of starch?]!! 1940s. Introduction of waxy maize starch. Mid 1950s. Introduction of high amylose corn starch. Development of various corn sweeteners; Introduction of Maltodextrins and low DE (Dextrose Equivalent) syrups. 1957. Richard O Marshall and Earl P Kooi developed the enzymatic method of converting d Glucose to d Fructose. 1967. First commercialization of batch process enzyme cat alyzed High Fructose Syrup ( HFS ) production; at 15% Fructose content. 1968. First commercial shipment of enzyme catalyzed HFS production; at 42% Fructose content. 1972. A continuous method of enzyme catalyzed starch conversion process was introduced. 1975 1985. HFS became the sweetener of choice; and was used extensively by the processed foods and soft drinks industries.

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55 1996. About 11.4 billion kilograms of corn were converted to corn syrup and other sweeteners; accounting for over 55% of the nutritive swe etener market in the USA. 2.1.7.2 Acid or enzyme h ydrolysis Depending on the type of sweetener desired (glucose or fructose for example), production may require two or three process steps; excluding downstream processing. Basically starch is hydrolyzed to glucose in two steps (liquefaction and saccharification); and then the glucose is transformed by isomerization into fructose (Aschengreen, 1975; Johnson et al., 2009). Starch hydrolysis may be achieved with the use of acids and or enzymes. Acid hydrolysis is often avoided because of several disadvantages such as low yield, extensive product purification and equipment demands, destruction of starch macromolecules, discoloration and product contamination, charring and dehydration reactions, and the toxic, er osive and hazardous nature of all acids (Aschengreen, 1975; Heitmann and Mersmann, 1997; Johnson et al., 2005; Srinorakutara et al., 2006). For these reasons, the work reported here was conducted with enzyme hydrolysis. 2.1.7.3 Amylolytic e nzymes E nzymes employed in industrial hydrolysis of starch have been noted to be the second largest consume amylase, glucoamylase), account for up to 33% of the ma rket share (Nguyen et al., 2002) decades. The market value rose from $180 million i n 1960 to $817 million in 1985 ( Enzy mes used in food industry, 2011) In 1998, worldwide enzyme sales were over $1.5 billion (Beilen and Li, 2002). This value is projected to rise to $7 billion in 2013 ( Rep ortLinker, 2009) B ecause culture medium and substrate are important factors in

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56 the synthesis of industrial enzymes, the use of low cost substrates such as cassava peels and other biomaterials/agricultural byproducts can help reduce the cost of sweetener production (Kar et a l., 2010; Roses and Guerra, 2009 ; Silva et al., 2009; Rajagopalan and Krishnan 2008; Spier et al., 2006; Ayerno et al., 2002; Pandey et al., 2000 a ) In addition, the onsite production of such enzymes will obviate transportation and associated logistical costs. 2.1.7.4 S ynergistic and direct methods of glucose sweetener p roduction Perhaps the two major constraints to conventional method of glucose production are the two step liquefaction cum saccharification procedure, and starch extraction process. The t wo step procedure requires that liquefaction be carried out first, followed by saccharification. This protocol is inefficient in use of resources: process vessels, piping, and associated equipment, enzyme conditioning etc. Similarly, the starch extraction is time energy and labor intensive. It would be advantageous if liquefaction and saccharification processes can be combined as a single unit operation, inside one reactor vessel; and if minimally processed starch source such as fresh cassava root is used as the substrate The former is called synergism and the latter, direct conversion. Some authors have tried to study these phenomena. Gauss et al., (1976) patented a process for alcohol production by simultaneous saccharification and fermentation of cellu losic materials Arasaratnam and Balasubramaniam (1993), observed that synergistic hydrolysis of dry milled starch was more efficient than wet milled starch Linko and Javanainen (1996) claimed 98% yield of lactic acid when 130 g/L starch was simultaneousl y liquefied, saccharified and fermented. Kearsley and Nketsia Tabiri (1979) performed direct hydrolysis of nati ve starches in grains, roots and tubers and

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57 reported that unprocessed native starches compared favorably with commercial starches, especially in total starch conversion. However, little work has been reported in the literature on direct conversion techniques coupled with the synergistic procedure, particularly where glucose sweetener is the desired end product. Most of the reported work was done for the production of alcohol or lactic acid where a reducing sugar, such as glucose, is a transition i t em to be fermented to the desired end product. The work undertaken in this study was an attempt to combine both direct conversion and synergistic techni ques in the production of glucos e sweetener, using cassava substrates as the raw materials source. Further objectives included determination of Arr henius type reaction kinetics: rate constant and activation energy of each substrate at two different tempera tures. 2.1.8 Starch Chemistry Starch, with the chemical or molecular formula [(C 6 H 10 O 5 ) n ] is a polysaccharide carbohydrate composed of glucose monomer units that are joined by glycosidic bonds. Starch occurs widely in plant tissues in the form of storage granules in fruits (banana, breadfruit), roots (cassava, arrowroot), seeds (corn/maize, rice and wheat), tubers (yam, potato), stem pith (sago) and other parts (Buleon and Colonna, 2007; Eliasson, 2004; Jane et al., 1994; Jenner, 1982). Starch contributes 50 70% of the energy in the human diet (Copeland et al., 2009), and is the principal components of such staple foods like beans, bread, and pasta products (Wang et al., 1998). Pure starch has an auto ignition temperature of 410 o C; gelatinization tempera ture of 60 o C 80 o C; density of about 1.5 g/cm 3 ; is white, odorless, tasteless, insoluble in cold water or alcohol; and is composed of two major molecules known as amylose and amylopectin (Encyclopedia Britannica: http://www.britannica.com/EBchecked/to pic/563582/starch [Accessed

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58 10/27/2013]; Water Structure and Science: http://www.lsbu.ac.uk/water/hysta.html#r1758 [Accessed 10/27/2013]; Wikipedia: http://en.wikipedia.org/wiki/Starch [Accessed 10/27/2013] ; Copeland et al, 2009; Yuryev et al., 2007) Figure 2 2 illustrates an overview of the structure of starch granule. Figure 2 2. An Overview of the Structure of Starch Gra nule. Source: Purdue University, http://www.cfs.purdue.edu/class/f&n630/pdfs/Starch_yao.pdf [Accessed 10/30/2013] Depending on botanical source, starch generally may have crystallinity of 15 % to mposed of 17 % to 25% amylose and 75 % to 83% amylopectin by weight (Perez and Bertoft, 2010; Charles et al., 2004; Lindeboom et al., 2004; Bahnassey and Breene, 1994). However,

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59 waxy starches could have amylopectin contents of over 99% (Bahnassey and Breen e, 1994). The structure of starch (Figure 2 2) and the methods of studying it have been reported by many authors (Blazek and Gilbert, 2011; Lopez Rubio and Gilbert, 2009; Belton, 2007; Limbach and Kremer, 2006; Sanguansri and Augustin, 2006; Ubbink and Mez zenga, 2006; Lindeboom et al., 2004; Molinero et al., 2004; Donald et al., 2001; You et al., 1999; Jenkins and Donald, 1996; Aberle et al., 1994; Jenkins et al., 1993; Hizukuri, 1985; Blanshard et al., 1984; Rosenthal et al., 1974). St arch has varied utili ties in the paper, textile, pharmaceutical, bioplastics, and oil drilling and other industries. It is the source of energy and carbon in food and feed; used f o r texture and functionalities in processed fo ods; and serves as feed stock for sweetener and etha nol product ion. 2.1.8.1 Amylose A mylose is a 4) linked D glucopyranosyl units. T he 1 carbon on one glucose molecule is linked to the 4 carbon on the next glucose molecule giving rise to 4 ) ) bonds or linkages. However recent studies are revealin g that amylose contain s slight degree of branching with 6) linkage (Hoover, 2001; Takeda et al. 1992 ; Hizukuri et al. 1981 ) Amylose has a packed structure with propensity to retrograde. With its l ow degree of branching, amylo se easily form s insoluble se mi crystalline aggregates of tough gels and strong films that are resistant to d igestion. Amylose influence s stickiness, texture, water absorption capacity and the digestibility of processed foods (Copela nd et al., 2009). It is reported that amylose is pos itively correlated to hardness and gumminess of starch gels (Sandhu and Singh, 2007). Figure 2 3 represents the structural formula and linear profile of amylose. Some amylose properties of the starches from cereal, legume and root and tuber crops are prese nted in Table 2 4.

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60 Figure 2 3. Structural Formula and Linear Configuration of Amylose (Sources: Water Structure and Science, http://www.lsbu.ac.uk/water/hysta.html [Accessed 11/06/2013]; Wikipedia, http://en.wikipedia.org/wiki/Amylose [Acces sed 11/06/2013]) Table 2 4. Some Properties of Amyloses from Cereal, Legume and Root and Tuber Starche s (Source: Hoover, 2001) S/N Property Starch Cereals Legumes Roots and Tubers 1 Blue Value (BV) 1.39 1.45 1.38 1.56 2 Iodine Affinity (I 2 : g /100g) 9.0 20.9 16.0 22.0 18.3 20.5 3 Organic Phosphorus (ppm) 1.0 14.0 2.0 10.0 4 Amylosis Limit (%) 61.0 95.0 79.0 86.9 57.0 97.5 5 Number of Branch Linkages (BL) 0.90 5.5 2.2 12.0 6 Number Degree of Polymerization (DPn ) 690 1690 1000 1900 1273 8025 7 Weight Degree of Polymerization (DPw) 1810 5450 3320 8040 8 Apparent Degree of Polymerization Distribution (DP) 180 25200 480 40000

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61 Specifically f or cassava starch, reported amylose values include the following. Blue value: 1.47; Iodine affinity: 16.2 % amylosis limit: 75 %; Number of chains per molecule: 6 8; Chain length: 340; Number degree of polymerization (DPn): 2600 3642; Weight degree of polymerization (DPw): 6680; Apparent degree of polymerization distribution (DP) : 580 22400; and Limiting viscosity number: 384 mL/g (Hoover, 2001; Hizukuri and Takeda, 1984; Takeda et al., 1984; Ketiku and Oyenuga, 1972). 2.1.8.2 Amylopectin Amylopectin is the larger of the two components of starch. It consists of 4) linked D g lucopyranosyl units, but with more than 5 % 6) linkages ; Figure 2 4. Figure 2 4. Structural Features of Amylopectin Highlighting 4) Linkages (in red) and 6) branch points (in blue) (Source: Water Structure and Sc ience, http://www.lsbu.ac.uk/water/hysta.html [Accessed 11/06/2013]) This composition gives amylopectin a highly branched tree like structure, an architecture that led to formation of the cluster model generally used to describe the three dimensional con figuration of amylopectin (Manners, 1989; Hizukuri, 1986, 1985).

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6 2 Figure 2 5 highlights r e presentative structural formula and branching profile s of amylopectin Figure 2 5 Structural Fo rmula and Profiles of Amylopectin Branches and Clusters (Sources: P urdue Uni versity, http://www.cfs.purdue.edu/class/f&n630/pdfs/Starch_yao.pdf [Accessed 11/02/2013]; Water Structure and Science, http://www.lsbu.ac.uk/water/hysta.html [Accessed 11/06 /2013]; Wikipedia, http://en.wikipedia.org/wiki/Amylopectin [Accessed 11/06/2013]

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63 The structural design enables amylopectin to impart unique characteristics and functional properties to food systems; influencing crystallinity, gelatinization, retrogradat ion, cooking and pasting properties (Copeland et al., 2009; Tran et al., 2001; Jane e t al., 1999). Amylopectin is reporte d to play a role in the palatability of rice (Tran et al., 2001), staling of bread and cakes (Copeland et al., 2009), as well as suppor t the crystalline domains and enthalpy of gelatinization of starch granules (Rolland Sabate et al., 2012; Perez and Bertoft, 2010). Figure 2 6. Basic labeling of chains in amylopectin. Schemati c shows A, B and C chains with the glucose reducing end att ached to the C chain ; Amorphous and crystalline lamellae; the Hilum or origin of starch formation ; as well as (1 6 ) linkage point s (Source: Water Structure and Science, http://www.lsbu.ac.uk/water/hysta.html [Accessed 11/06/2013])

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64 Amylopectin is compose d of three classes of glucose chains or branches designated as A, B and C chains (Copeland et al., 2009; Wang et al., 1998; Manners, 1989). As represented in Figure 2 6, t he A chains are defined as unsubstituted; they only bind to the B chains. On the othe r hand, B chains are said to be substituted in that they can bind to other B chains or to the C chains The C chains feature as the backbone of amylopectin molecules. There is only one C chain per amylopectin molecule, and it is the C chain s that carry the reducing glucose ( Figure 2 6 A ). Studies have shown that A chains are the shortest, B chains the longest, while C chains are intermediate in length (Hizukuri, 1986, 1985). Figure 2 7 depicts the building block construction model for amylopectin. Figure 2 7. A model of how the clusters of amylopectin build up the semi crystalline granular rings. (a) 3D illustration of the building block structure of the cluster of amaranth amylopectin. The blocks are attached to a backbone forming a network of chains in the amorphous lamella. Numbers indicate types of building blocks with a corres ponding number of chains. IB CL = inter block chain length. (b) The same structure with the external chains added forming five double helices symbolized as dark cylinders in the crystalline lamella. (c) Several hundred double helices from a large number of clusters interact to build up the crystalline lamella (Source: Perez and Bertoft, 2010). Some a mylopectin properties of the starches from cereal, legume and root and tuber crop s are presented in Table 2 5 Sp e cific values for cassava amylopectin include Blue value: 0.104; Iodine affinity: 0 % amylosis limit: 57 %; and Average

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65 Table 2 5. Some Properties of Amylop e ctin s from Cereal, L egume and Root and Tuber Starche s (Source: Hoover, 2001) S/N Property Starch Cereals Legumes Roots and Tubers 1 Blue Value (BV) 0.049 0.441 0.104 0.245 2 Iodine Affinity (I 2 : g/100g) 0.39 4.63 1,0 5.3 0.06 1.1 3 Organic Phosphorus (ppm) 19 119 21 900 4 A mylosis Limit (%) 56 61 56 66.5 43.8 66.7 5 Average Chain length (CL) 19 32 20 34 19 44 chain length (CL) : 21 27.6 (Hoover, 2001; Jane et al., 1999; Hizukuri, 1986, 1985; Ketiku and Oyenuga, 1972). In Table 2 6 are presented some di stinguishing properties between amylose and a mylopectin Table 2 6. Some Distinguishing Properties between Amylose and Amylopectin S/N Property Unit Amylose Amylopectin Reference(s) 1 Chemical Interaction with Iodine Blue Color Purple; Reddish Br own Color Wang et al., 1998; Whistler and Daniel, 1984 2 Iodine Affinity % 19 % 20 % < 0.2 % Wang et al., 1998; Rickard et al., 1991 3 General Structure Linear Branched Perez and Bertoft, 2010; Jane et al., 1999; Aberle et al., 1994; Rickard et al ., 1991 4 Linkages Mainly 4]; Less than 0.5 % 6] 4] with more than 5 % 6] Copeland et al., 2009; Eliasson, 2004

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66 Table 2 6 Continued. Some Distinguishing Properties between Amylose and Amylopectin S/N Property Unit Amylose Amylopectin Reference(s) 5 M olecular Weight g/mole 1 x 10 5 1 x 10 6 1 x 10 8 Copeland et al., 2009; Wang et al., 1998 6 Degree of Polymerization (DP) Glucose Units 1000 10000 Up to 1 x 10 6 or more Copeland et al., 2009; Manners, 1989 7 Retrogradation Time Minutes Days Minut es Hours Hours Days Copeland et al., 2009 8 Amylosis Limit % 73 95 55 60 Hoover, 2001; Takeda et al., 1986, 1984 9 Average Chain Length (Glucose Residues) CL 100 10000 20 30 Rickard et al., 1991 10 Solubility in Water % Variable Soluble Rickard et al., 1991 11 Stability in Aqueo us Solution Retrogrades Stable, may retrograde at high conc. Rickard et al. 1991 12 Conversion to amylase % Rickard et al. 1991 2.1.8.3 Cassava Starch Starch is the principal component of cassava crop especially the root Cassava starch is of great value as an important source of nutritional calories in both human food and animal feed. Cassava starch is also finding utilities in the industrial sector. It is used in the production of glucos e and high fructose syrup, in the s izing of paper and textiles,

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67 and in the manufacture of adhesives and alcohol (Tonukari, 2004; Rickard et al., 1991 ; Chadha, 1961 ). Furthe rmore, cassava starch has be en recommended for the manufacture of extruded snacks, processed baby foods and as filler and bonding agent in the bi s cuit and confectionery industries. These applications are possible because cassava starch is reported to impart to the products a fine structure, light color, transparent look, smooth surface, and bland flavor (Seibel and Hu, 1 994). Cassava starch is increasingly being extracted and used in many countries. It is a major source of starch in Brazil, China, India, Indonesia, Philippines and Thailand (Moorthy, 2002; Rickard et al., 1991). Studies have demonstrated that there are v ariabilities in the physicochemical properti es of starch not only between different botanical sources, but also within the same species (Rolland Sabate et al., 2012; Martinez Bustos et al., 2007; Charles et al., 2004; Moorthy, 2002; Hoover, 2001; Santisopa sri et al. 2001; Defloor et al., 1998; As a o ka et al., 1991; Rickard et al., 1991 ). However, Perez and Bertoft ( 2010) stated that the following facts have been established for starch: Occurrence of segments of amylopectin with a left handed, parallel st rand double helical structure. Two types of stable arrangement of double helices can occur, as found in the A and B polymorphs. Up to 200 double helical segments are densely packed in platelet nanocrystals, which are polar and chiral. The 1 6 branching do es not preclude the formation of double helical arrangements. components.

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68 Some of the i mportant properties that have been reported for cassava starch are presented in Table 2 7. Table 2 7 S ome Properties of Cassava Starch S/N Property Unit Value Reference(s) 1 Amylose Content (Normal crop) % 13.6 27 Sanchez et al., 2009; Ceballos et al., 2008; Charles et al., 2005 a 2004; Hoover, 2001; Jane et al., 1999; Defloor et al., 1998; Asoaka et al., 1991; Olorunda et al., 1981; Ketiku and Oyenuga, 1972 Amylose Content (Mutated crop) % 28 36 Rolland Sabate et al., 2012; Ceballos et al., 2008 2 Ash % 0.02 0.49 Nwokocha et al., 2009; Asoaka et al., 1991; Rickard et al., 1991; Rosenthal et al., 1974 ; Rasper, 1969 3 Crude Fat % 0.08 1.54 Asoaka et al., 1991; Rickard et al., 1991; Soni et al., 1985; Rosenthal et al., 1974 4 Crude Protein % 0.03 0.61 Asoaka et al., 1991; Rickard et al., 1991; Rosenthal et al., 1974 Gelatinization 5 Start Tempt. (T o ) o C 54 64 Charles et al., 2004; Santisopasri et al. 2001; Bahnassey and Breene, 1994; Defloor et al., 1988 6 Peak Tempt. (T p ) o C 63 76 Charles et al., 2004; Santisopasri et al. 2001; Bahnassey and Breene 1994 ; Defloor et al., 1988 7 Complete Tempt. (T c ) o C 72 83 Charles et al., 2004; Bahnassey and Breene, 1994 ; Defloor et al., 1988 8 Enthalpy J/g 9 16.9 Rolland Sabate et al., 2012; Charles et al., 2005 a 2004; Santisopasri et al. 2001; Defloor et al., 1988

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69 Table 2 7 Continued S ome Properties of Cassava Starch S/N Property Unit Value Reference(s) 9 Granule Shape Oval, Round, Spherical, Truncated Ceballos et al., 2008; Soni et al., 1985; Ketiku and Oyenuga, 1970 10 Granule Size 2.4 32 Nwokocha et al., 2009; Ceballos et al., 2008; Santisopasri et al. 2001; Defloor et al., 1998; Jane et al., 1994; Asoaka et al., 1991; Ketiku and Oyenuga, 1970 1 1 Size of Amylopectin Cluster (Repeat Distance) nm 9.1 Jenkins et al., 1993 12 Nitrogen % 0.008 0.013 Nwokocha et al., 2009; Defloor et al., 1998 13 Phosphorus % 0.0075 0.021 Asoaka et al., 1991; Soni et a l., 1985; Rosenthal et al., 1974 14 Starch % 73.7 98.7 Valetudie et al., 1993; Rickard et al., 1991; Rickard and Behn, 1987 ; This Diss ertation (Chapter 4; Table 4 5 ) 15 Total Pectin mg GA/kg 314 This Dissertation (Chapter 4; Table 4 5 ) 16 Crystalli nity % 15.3 40 Rolland Sabate et al., 2012; Hoover, 2001; Asoaka et al., 1991 Rheological (Pasting) Characteristics 17 Pasting Temperature (PT) o C 58.8 74.6 Sanchez et al., 2009; Ceballos et al., 2008; Charles et al., 2005 a ; Santisopasri et al. 2001 ; Rasper, 1969 18 Peak Viscosity (PV) % RVA 24.2.0 142.6 Charles et al., 2005 a ; Bahnassey and Breene, 1994; Def fenbaugh and Walker, 1989 a, 1989 b

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70 Table 2 7 Continued S ome Properties of Cassava Starch S/N Property Unit Value Reference(s ) 18 Cont. Peak Viscosity (PV) mPa.s 22.0 1505.0 Sanchez et al., 2009; Ceballos et al., 2008 19 Minimum or Hot Paste Viscosity at 95 o C (HPV) % RVA 11.0 30.5 Charles et al., 2005 a ; Bahnassey and Breene, 1994; Def fenbaugh and Walker, 1989 a 20 Final or Cold Paste Viscosity at 50 o C (CPV) % RVA 12.5 127.0 Charles et al., 2005 a ; Bahnassey and Breene, 1994; Def fenbaugh and Walker, 1989 a 21 Breakdown (PV HPV) % RVA 12.2 118.0 Charles et al., 2005 a ; Bahnassey and Breene, 1994; Def fenbaugh and Walker, 1989 a mPa.s 28.1 859.0 Sanchez et al., 2009 22 Setback (CPV PV) % RVA ( 18.8) to ( 9) Charles et al., 2005 a ; Bahnassey and Breene, 1994; Def fenbaugh and Walker, 1989 a mPa.s ( 702.0) to (273.0) Sanchez et al., 2009 23 Co nsistency (CPV HPV) % RVA 0.5 102.4 Charles et al., 2005 a ; Bahnassey and Breene, 1994; Def fenbaugh and Walker, 1989 a mPa.s 0.0 620.0 Sanchez et al., 2009 24 Cooking Ability (Time to PV Time to PT) Min. 1.0 5.6 Sanchez et al., 2009; Bahn assey and Breene, 1994 25 Solubility at 35 o C 95 o C % 0.2 48 Sanchez et al., 2009; Ceballos et al., 2008; Charles et al., 2005 a ; Moorthy, 2002; Hoover, 2001; Rickard et al, 1991

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71 Table 2 7 Continued S ome Properties of Cassava Starch S/N Proper ty Unit Value Reference(s) 26 Swelling Power at 35 o C 95 o C g/g 0.8 71.0 Sanchez et al., 2009; Ceballos et al., 2008; Charles et al., 2005 a ; Moorthy, 2002; Hoover, 2001; Rickard et al, 1991; Rosenthal et al., 1974 27 Paste Clarity % 12.5 96.6 Sanchez et al., 2009; Ceballos et al., 2008 28 Storage Modulus o C and [90 o C] Pa 4970 19370 [471.7 1937] Charles et al. 2004 29 Loss Shear 25 o C and [90 o C] Pa 453 3060 [51.3 219] Charles et al. 2004 3 0 Loss Tangent at 25 o C and [90 o C] Tan 0.12 0.19 [0.10 0.13] Charles et al. 2004 3 1 Iodine Affinity % 3.07 4.7 Jane et al., 1999; Rosenthal et al., 1974 3 2 Water Holding Capacity % 71.8 Soni et al 1991 2.1.8.4 Enzymatic Hydrolysis of Starch Enzyme technologies are increasi ngly being used in numerous processes in the pharmaceutical, fine chemi cal, food, brewing, fermentation, distilling, paper and textile industries, as well as in clinical, medicinal and analytical chemistries (Pandey et al., 2000 b ; Solomon, 1978 ). One of t he areas of high application is starch conversion (Satyanarayana et al., 2004; Nguyen et al., 2002). It has been reported that enzyme technology applied to the processing of starch provides higher yields, significant

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72 improvements in product quality, as wel l as energy savings (Reichelt, 1983; Ueda, 1981). A variety of enzymes may be used for starch conversion. These include alpha amylase, beta amylase, amyloglucosidase, isoamylase, pullulanase, etc. (Tomasik and Horton, 2012; Uhlig, 1998; James and Lee, 19 97; Roberts et al., 1995; Whitaker, 1994; Fogarty, 1983; Lyons, 1983 ; Solomon, 1978; Fogarty and Griffin, 1973 ). 2.1.8.4.1 Alpha amylase ( amylase: EC 3.2.1.1) amylase is alternatively known as 1 ,4 D glucan glucanohydrolase or dextrogenic amylase. It is widely distributed in microorganisms such as bacteria and fungi and has an optimum pH range of 5 7. The structure of amylase is shown in Figure 2 8. Figure 2 8. Structure of Alpha Am ylase with calcium ion (khaki) and chloride ion (green) visible. (Source: Wikipedia, http://en.wikipedia.org/wiki/Fil e:Salivary_alpha amylase_1SMD.png [Accessed 11/09/2013])

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73 The amylase is referred to as endo acting enzyme in that it cleaves the substrate links in the interior of the molecule acting from any side of the chain If starch is treated with amylase enzyme the enz ym e will randomly hydrolyze 4 ) glucosidic bo nds in amylose and amylopectin, whereas 6) linkages are not attacked. This reaction gives rise to the formation of maltose, maltotriose, glucose, and limit dextrins. The dextrins contain all the 6) linkages (Fogarty, 1983 ; Solomon, 1978 ) an d polysaccharide chain segments. Figure 2 9 illustrates the operatin g mechanism of alpha amylase during the hydrolysis of starch = Figure 2 9. Schematics of how A mylase randomly splits Starch Molecules t o liberate G l ucose, M altose and Limit D extrins. links 6 ) link Glucose units Maltose units Limit Dextrins Amylose The Reducing Ends Amylopectin

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74 2.1.8.4 .2 Bet a amylase ( amylase: EC 3.2.1.2) amylase enzyme is also known as 1 ,4 D glucan maltohydrolase or saccharogenic amylase. This enzym e is widely distributed in plants like sweet potatoe s and soybeans but can also occur as extracellular enzyme in microorganisms. It has the optimum pH of about 4 5. The structure of amylase is re presented in Figure 2 10. Figure 2 10. Structural Features of Beta Amylase. (Source: Wikipedia, http://en.wikipedia.org/wiki/File:2xfr_b_amylase.png [Accessed 11/09/2013]) amylase is an exo acting enzyme that hydrolyses every second 4 ) linkage from the non reducing end splitting off two glucose units (maltose ) at a time. The amylase does not bypass or cleave 6) linkages (James and Lee, 1997) and therefore produces incomplete starch hydrolysis ; resulting in 50 % 60 % limit dextr ins (Perez and Bertoft, 2010; Uhlig, limit dextrins contain all unhydrolyzed 6)

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75 branches and other polysaccharide chain segments. Consequently, if starch is treated amylase, the alternate 4 ) linkage s o f the starch molecules (amylase and amylopectin) are quickly hydrolyzed to maltose (and perhaps some amounts of maltotriose). The 6) branches are not hydrolyze, but rather constitute part of the limit dextrins; Figure 2 11. = Figure 2 11. Hydrolysis of Starch by A mylase to lib e Limit Dextrins 2.1.8.4.2 Gamma amylase ( amylase: EC 3.2.1.3 ) The gamma amylase is popularly known as g lucoamylase. However, it is also alterna tively called 1,4 D glucan glucohydrolase Exo 1,4 glucosidase and amyloglucosidase. The structure of glucoamylase is shown in Figure 2 12. The glucoamylase enzyme is exo acting and lib e rates glucose molecules from the non reducing chain ends of pol ysaccharides (Whitaker, 1994; Ueda, 1981). It has an optimum pH range of about 3.5 5 and is found mostly in fungi. links 6 ) link splits split splits Amylose The Reducing Ends Amylopectin Limit Dextrins Maltose units

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76 Figure 2 12. Structure of Glucoamylas e (Source: bing.com/images, htt p://www.public.iastate.edu/~cfford/GAstructure.jpg [Accessed 11/09/2013]) 6) linkages are hydrolyzed more slowly. When starch is exposed to glucoamylase, the ; Solomon, 1978 ) to release glucose molecules. Therefore, if incubated for sufficient amount of time, the glucoamylase is able to completely hydrolyze starch (James and Lee, 1997). A model for gluco amylase and its mechanism of hydrolysis of starch has been proposed b y the institute of food research in the United Kingdom; Figure 2 13. In this paradigm, glucoamylase is considered a multi domain enzyme, consisting of

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77 A B C Figure 2 13. Glucoamylase Model and Mechanism of Starch Digestion. A). Proposed model of glucoamylase; B). Coupling of glucoamylase model with starch molecule; C). Locking/binding of glucoamylase model with starch chain ends and subsequent cleavage. (Source: Institute of Food Research, http://www.ifr.ac.uk/SPM/glucoamylase.html [Accessed 11/09/2013]) smaller starch binding domain (SBD) which is attached to the larger catalytic domain (LCD); Figure 2 13A. Complex formation wi th starch molecule is dominated by the SBD with, the catalytic domain decorating the outside of the starch SBD ring as illustrated in Figure 2 13B. The nature of starch (amylose) SBD complex suggests a role for the SBD. The SBD is considered to recognize and dock onto the ends of amylose double helices. This locks the otherwise mobile chain ends at the end of the helix in the vicinity of the LCD (Figure 2 13C), facilitating binding and subsequent cleavage. When this happen s bond digestion of starch to glucose as represented in Figure 2 14. SBD LCD

PAGE 78

78 = Figure 2 14. Hydrolysis of Starch by Amylase to lib e rate Glucose Molecules 2.1.8.4.3 Comp a rison of starch hydrolyzing enzymes In Figure 2 15 is presented a global view of the major end products achieved by various starch hydrolyzing enzymes. It can be seen how the capabilities of the enzymes vary in amylase (glucoamylase) that could achieve complete digestion of starch to glucose, the other enzymes exhibited incomplete digestion with varying degrees of glucose, maltose, dextrins, etc (Figure 2 15). However, the d igestibility or hydrolysis of starch is a function of numerous factors. These include starch related matters such as botanic source, amylose/amylopectin ratio, extent of molecular association between starch components, degree of crystallinity, gelatinizati on, retrogradation, germination/sprouting, granule size, available specif ic surface area, amylose chain length, amylose lipid complexes, links 6 ) link splits split splits Amylose The Reducing Ends Amylopectin Glucose units

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79 Figure 2 15. Overview of the Pattern of Starch Digestion by major Starch H ydrolyzing E nzymes (Tomasik and Horto n, 2012) porosity, presence of activators/ inhibitors, moisture content during heat treatment, structural inhomogeneities and degree of integrity ; a s well as many other enzyme associated issues ( Chen et al., 2011; Copeland et al., 2009; Shariffa et al., 2009; J ayakody and Hoover, 2008 ; Hoover and Vasanthan, 1994 ; Jood et al., 1988; Ring et al., 1988; Hoover and Sosulski, 1985; Dreher et al., 1984; Fogarty, 1983; Snow and O'Dea, 1981 Wiseman, 1973 )

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80 In general, it has been reported that ungelatinized roo t and tuber starches are more resistant to enzyme hydrolysis than their cereal counterparts ( Valetudie et al., 1993; Rickard et al., 1991; Dreher et al., 1984; Ueda, 1981; Rasper et al., 1974; Leach and Schoch, 1961 ). On the other hand, cassava starch was found to be the least resistant to enzymatic digestion among the non cereal starches (Valetudie et al., 1993; Gorinstein and Lii, 1992; Rickard et al., 1991; Rasper et al., 1974), with digestibilities reported to be comparable to t hose of corn starch ( Ueda 1981 ; Leach and Schoch, 1961 ). The activity of an enzyme on the digestibility of native (raw, ungelatinized) starch is often expressed as the percentage of the activity of the enzyme on the native starch as compared to the activity of the enzyme on the gelatinized starch. However, i t is difficult to make direct comparisons among the digestibilities of various starches reported in the literature due to a number of reasons. The reasons among others include differences in the type and sources of enzyme used enzyme purity, enzyme concentration, botanic sources of the starch, varie tal differences of the starch; as well as experimental conditions such as time of hydrolysis, pH, temperature, etc. Nevertheless an att e mpt has been made here to present some of th e reported data on the digestibility of native cassava starch in Table 2 8. 2.1.9 Cassava Toxicity A major concern for the handling, consumption and use of cassava products is their potential to engender cyanide poisoning. It was reported that diets high in cassava led to increased levels of cyanide in the blood, and elevated concentrations of thiocyanate in the urine (Delange et al., 1973; Osuntokun, 1973). The consumption of cassava has also been associated with such diseases as TAN: tropical ataxic

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81 T able 2 8. Degree of Hydrolysis of Native Cassava Starch Achieved with Various Enzymes and Operating Conditions S/N Starch Conc. [%] Type/Name of Enzyme Enzyme Activity Enzyme Conc. Hydrolysis Tempt. [ o C] Hydrolysis pH Hydrolysis Time [Hours] Degree of H ydrolysis Achieved Reference(s) 1 25 STARGEN 001 TM 3736 units/g 1 % 35 5 6 24 36 % Shariffa et al., 2009 2 4 Bacillus subtilis amylase 2.8 starch 37 6.7 24 44.0 % Valetudie et al., 1993 3 4 Porcine Pancreatic amylase 2.8 starch 37 6.7 24 52.9 % Valetudie et al., 1993 4 17 Bacterial amylase 3490 SKB units 37 5.2 36 57.5 % Franco et al., 1987 5 80 Glucoamylase from Rhizopus spp 10 mg/mL 55 4.8 2 14 16 Rickard and Behn, 1987 6 Glucoamylase from Endomycopsis fibuligera 1.71 units/mL 40 4.5 24 34.9 % Ueda and Saha, 1983 7 10 Bacterial amylase 10 EU/mg 30 6.4 48 4.7 % Maltose Rasper et al., 1974 8 10 Bacterial amylase 10 EU/mg 30 6.4 24 2.5 % Maltose Rasper et al., 1974 9 10 Fungal Glucoamylase Grade II 30 5.0 48 4 .1 % Maltose Rasper et al., 1974 10 10 Fungal Glucoamylase Grade II 30 5.0 24 2 % Maltose Rasper et al., 1974 11 20 Bacterial amylase 16000 SKB 0.5 % 50 6.5 24 55.7 % Leach and Schoch, 1961 12 10 (Root Pulp) Bacterial amylase (25%) Plus Fungal Gluc oamylase (75 %) 5 x 10 6 BAAU/g Plus 1000 AG/g 0.015 % 37 4.3 6.6 96 55 % This Dissertation; Chapter 4

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82 neuropathy (Osuntokun, 197 3; Osuntokun et al., 1970), endemic goiter (Bourdoux et al., 1978; Ekpechi, 1973, 1967) and perhaps konzo (Tylleskar et a l. 1992 ; Mlingi et al. 1991 ) The source of these disorders is believed to be the cyanogenic potential of cassava. The cassava crop is reported to accumulate two aliphatic cyanogenic glucosides known as linamarin D glucopyranosyloxy) isobutyronitrile which is the major glucoside in the plant and to a lesser extent, a higher homologue of linamarin called lotaustralin D glucopyranosyloxy) 2 methylbutyronitrile (Cooke et al., 1978 ; Zitnak et al., 1977; Na rtey, 1968) The structures of these cyanogens are shown in Figure 2 16 T he biosynthesis pathway of linamarin with valine as the precursor is presented in Figure 2 17. A B Figure 2 16. Structures of the A liphatic Cyanogenic Glucosides of Cassava A: Linamarin and B: Lotaustralin. (Sources: http://en.wikipedia.org/wiki/File:Linamarin_3D_sticks.png [Accessed 11/14/2013 ]; http://en.wikipedia.org/wiki/File:Lotaustralin_3D_sticks.png [Accessed 1 1/14/2013]; Nart ey, 196 8)

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83 Figure 2 17 The Biosynthesis Pathway of Linamarin showing Valine as the Precursor (Kakes, 1990). glucosidase called linamarase EC 3.2.1.21 also known as linamarin D glucoside glucohydrolase (I k ediobi et al. 1980; Cooke et al., 1978) and another catabolic enzyme Hydroxynitrile lyase EC 4.1.2.37 (HNL, acetone cyanohydrin lyase, or oxynitrilase ) (Conn, 1994; Hughes et al., 1994; Conn, 1969) Linamarase is an enzyme that catalyzes the hydrolysis of the glucosides to ultimately lib erate hydro gen cyanide. The cyanogenic glucoside, linamarin, is thought to

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84 be synthesized in the leaves and petioles and accumulated in the vacuoles (Selmar, 1994; Kakes, 1990 ) where it is separated from the enzymes ; Figure 2 18. Figure 2 18 The Separation cum Distribution of Linamarin and Linamarase in the Plant System (Kakes, 1990) The linamarin is thereafter translocated to o the r parts of the plan t via the phloem. The peel of the root and the leaves are the main sink s for the cyanogenic glu cosides ( Koch et al., 1994; Nambisan and Sundaresan, 1994; Pereira et al., 1981; de Bruijn, 1973 ; Nartey, 1968). Bokanga ( 1994 a ) noted that the leaves including the petioles

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85 possess the highest cyanogenic potential in a plant. Howeve r, it has been argued that synthesis of linamarin does occur in the root also, and at rates comparable to those in the leaves (White et al., 1994) In intact plants, the cyanogenic compounds and the enzymes capable of hydrolyzing them are separated in diff erent compartments and shielded by the cell walls and other cell components; Figure 2 18. However, if the plant is physically injured or the cell wall becomes ruptured, the enzyme s will make contact with the glu coside s thereby facilitating hydrolysis of t he later and the subsequent production of hydrogen cyanide. The mechanism of hydrolysis is demonstrated in Figure 2 19 and briefly explained here A1 + Linamarase pH = 5.5 A2 + A3 B1 Hydroxynitrile Lyase B2 + B3 C Figure 2 19. Mechanism of Hydrolysis of Cyanogenic Glucosides and Lib e ration of Hydrogen Cyanide ( Conn, 1994; Ikediobi et al., 1980; Meuser and Smolnik, 1980; Cooke, 1978) pH > 5.0 pH < 5 .0

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86 The process is a two step reaction. First, l inamarin sho wn in Figure 2 19 A1 is initially glucosidase, linamarase, to form free glucose; Figure 2 19 A2 and the acetone cyanohydrin or 2 hydroxyisobutyronitrile identified by Figure 2 19 A3. This step is favored by pH of about 5.5 (Meuser and Smolnik, 1980) In the second step if the pH is alkaline or greater than 5, or the temperature is greater than 35 o C acetone cyanohydrins will dissociate spontaneously into hydrogen cyanide (HCN); Figure 2 19 B2 ( Dufour, 2007 ; Cooke, 1978) and acetone ; Figure 2 19 B3 (Conn, 1994). Other wise, the reaction can be catalyzed by the second catabolic enzyme, hydroxynitrile lyase to achieve the dissociation (White et al., 1998) Figure 2 19 C illustrates the discussed phenomenon as applied to the hydrolysis of lotaustralin (Ikediobi et al., 198 0). Lotaustralin is the higher homologue of linamarin (Nartey, 1968). H ydrogen cyanide (HCN) has a density of about 0.69 g/cm 3 ; and the boiling and freezing points of about 26 o C and 1 4 o C respectively. These properties enable HC N to rapidly vaporize an d dissipate at near room temperature. However, HCN is highly toxic and poison ous. I t inhibits cellular oxidative processes and therefore hazardous to human and animal health if consumed. Because cassava roots and leaves are food and feed items, they must b e processed to reduce the content of poisonous cyanogens [the cyanogenic glucosides and the ir hydrolysis products ( cyanohydrins and free cyanide ions ) ] to safe levels in the final products before consumption. The Codex Alimentarius Commission requires that the total hydrocyanic content of edible cassava flour not exceed 10 mg/kg (FAO/WHO, 1995). 2.1.10 Cassava Detoxification The prime goal of detoxifying cassava is to render it safe for consumption, whether as human food or animal feed. A number of proce ssing techniques have been

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87 used to achieve the detoxification of cassava products. Some of the methods used are dehydration, boiling, baking, steaming, frying, soaking, fermentation, storage, maceration, etc. (Nambisan, 1994; Oke, 1994; Balagopalan et al., 1988). These p rocessing techniques can and have been used in combinations. In Table 2 9 are presented typical detoxification levels achieved with som e of the methods. Among the more efficient processes reported are combined grating/pounding, fermentation /dewatering, followed by drying/roasting. These combinations have been reported to detoxify cyanogens in cassava by 80 % to 100 % (Nambisan, 2011 ; Mahungu et al., 1987 ) Table 2 9. Detoxification of Cassava Components and Products by various Processing Me thods S/N Cassava Component Process Applied Detoxification of Total Cyanogens Achieved [%] Reference(s) Leaf and Leaf Products 1 Pounded; then boiled for 30 minutes 96.3 99.6 Ngudi et al., 2003 2 Pounded for 15 minutes 63.0 73.0 Bokanga, 1994 a 3 Pounded for 15 minutes, then Boiled for 15 minutes 99.1 99.5 Bokanga, 1994 a 4 Chopped and then boiled for 15 minutes 85.5 Nambisan, 1994 5 Crushed or pounded and then boiled 97.0 Nambisan, 1994 6 Wilting at 30 o C for 5 hours 63 Nam bisan, 1994 7 Wilting at 30 o C for 12 hours 76 Nambisan, 1994 8 Chopped and then dried at 70 o C 30 Nambisan, 1994

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88 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Processing Methods S/N Cassava Component Process Applied Detoxification of Total Cyanogens Achieved [%] Reference(s) Leaf and Leaf Products 9 Kisamvu preparation: Pound fresh young leaves into small pieces 86.0 Gidamis et al., 1993 10 Kisamvu: Pound fresh young leaves into small pieces, boil for 30 to 45 minutes 95.7 Gidamis et al., 1993 11 Apical leaves are grated to a pulp 76.9 83.8 Dufour, 1989 12 Apical leaves are grated to a pulp, then cooked for 10 minutes 98.6 99.1 Dufour, 1989 13 Apical leaves are grated to a pulp, then cooked for 20 minutes 98.9 99.5 Dufour, 1989 14 Apical leaves are grated to a pulp, then cooked for 30 minutes 99.0 99.6 Dufour, 1989 15 Apical leaves are grated to a pulp, cooked for 30 minutes, then cooled to ready to eat 99.3 99.6 Dufour, 1989 16 Dried at 45 o C for 24 hours 74.0 80.1 Padmaja, 1989 17 Dried at 60 o C for 24 hours 78.7 85.9 Padmaja, 1989 18 Wilted at 30 o C for 16 hours then Dried at 60 o C for 24 hours 87.3 91.7 Padmaja, 1989 19 Wilted at 30 o C for 16 ho urs, Chopped to 1 cm length and then Dried at 60 o C for 24 hours 77.3 87.3 Padmaja, 1989 20 Dried at 75 o C for 24 hours 64.2 84.4 Padmaja, 1989 21 Mpondu: Blanch, pound, cook 95.7 Mahungu et al., 1987 22 Dried leaves 3.7 Bourdoux et al., 19 82 23 Mpondu preparation: Wash fresh leaves 6.9 Bourdoux et al., 1982 24 Wash fresh leaves, grind and boil for 15 minutes 94.6 Bourdoux et al., 1982

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89 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Processing Metho ds S/N Cassava Component Process Applied Detoxification of Total Cyanogens Achieved [%] Reference(s) Leaf and Leaf Products 25 Mpondu: Wash fresh leaves, grind and boil for 30 minutes 98.3 Bourdoux et al., 1982 26 Mpondu : Blanch for 10 minutes mash/pound into a pulp 62.0 Mad uagwu and Umoh, 1982 27 Mpondu: Blanch for 10 minutes, mash/pound into a pulp, boil for 80 minutes 92.3 Mad uagwu and Umoh, 1982 Root and Root Products 28 Boiling 50 g pieces 25 Nambisan, 1994 29 Boiling 25 g pieces 50 Nambisan, 1994 30 Boiling 5 g pieces 75 Nambisan, 1994 31 Pieces are blanched ( boiled for 5 to 10 minutes) 50.0 Nambisan, 1994 32 Peeled and boiled 69.8 Mahungu et al., 1987 33 Crushed and sun dried for 8 hours 96.8 98.5 Nambisan and Sundaresan, 1985 34 Sliced into 3 cm x 2 cm x 1 cm pieces and then boiled for 30 minutes 44.5 47.2 Nambisan and Sundaresan, 1985 35 Sliced into 3 cm x 2 cm x 1 cm pieces and then oven dried at 70 o C for 24 hours 25.2 29.2 Nambisan and Sunda resan, 1985 36 Sliced into 3 cm x 2 cm x 1 cm pieces and then baked at 110 o C for 20 minutes 12.9 15.0 Nambisan and Sundaresan, 1985 37 Sliced into 3 cm x 2 cm x 1 cm pieces and then steamed in a pressure cooker for 20 minutes 13.5 18.3 Nambisan and Sundaresan, 1985

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90 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Processing Methods S/N Cassava Component Process Applied Detoxification of Total Cyanogens Achieved [%] Reference(s) Root and Root Products 38 Sliced into 3 cm x 2 cm x 1 cm pieces and then fried in oil for 5 minutes 8.8 14.4 Nambisan and Sundaresan, 1985 39 Sun drying 10 mm thick chips 67.4 72.2 Nambisan and Sundaresan, 1985 40 Oven drying 10 mm thick chips at 50 o C 49.5 53.6 N ambisan and Sundaresan, 1985 41 Oven drying 10 mm thick chips at 70 o C 40.0 46.2 Nambisan and Sundaresan, 1985 42 Sun drying 3 mm thick chips 41.6 48.0 Nambisan and Sundaresan, 1985 43 Oven drying 3 mm thick chips at 50 o C 35.8 40.0 Nambis an and Sundaresan, 1985 44 Oven drying 3 mm thick chips at 70 o C 20.0 25.8 Nambisan and Sundaresan, 1985 45 Oven drying : Peeled chips (5 to 10 x 2 to 4 x 0.3 to 0.6) cm dimensions at 20 kg/m 2 loading rate, at 60 o C for 20 to 22 hours 72.0 Gomez et al., 1984 46 Oven drying : 1 cm thick peeled slices at 20 kg/m 2 loading rate, at 60 o C for 20 to 22 hours 49.0 Gomez et al., 1984 47 Oven drying : Irregular root peel pieces at 14 to 15 kg/m 2 loading rate, at 60 o C for 20 to 22 hours 64.0 Gomez et al., 1984 48 Sweet variety boiled for 20 minutes 87.9 Bourdoux et al., 1982

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91 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Processing Methods S/N Cassava Component Process Applied Detoxification of Total Cyanog ens Achieved [%] Reference(s) Root and Root Products 49 Pulp: Freeze dried 51.2 Meuser and Smolnik, 1980 50 Pulp: Air dried at 40 o C 98.5 Meuser and Smolnik, 1980 51 Pulp: Drum dried 99.1 Meuser and Smolnik, 1980 52 Fermented pulp: Drum dried 86.5 Meuser and Smolnik, 1980 53 Fermented pulp: Dried with heated air at 180 o C 91.4 Meuser and Smolnik, 1980 54 Chips: Dried with heated air at 180 o C 98.4 Meuser and Smolnik, 1980 55 Chips: Air dried at 40 o C 98.5 Meuser and Smolnik, 1 980 56 Slices: Flash dried 52.0 Meuser and Smolnik, 1980 57 Chips (40 mm x 8.2 mm x 6.8 mm) oven dried at 46.5 o C for 18 hours 29 % of bound cyanide detoxified; 82.5 % of free cyanide detoxified Cooke and Maduagwu, 1978 58 Chips (40 mm x 8.2 mm x 6.8 mm) oven dried at 60 o C for 18 hours 26 % of bound cyanide detoxified; 83 % of free cyanide detoxified Cooke and Maduagwu, 1978 59 Chips (40 mm x 8.2 mm x 6.8 mm) boiled for 25 minutes 55 % of bound cyanide and > 90 % of free cyanide detoxified C ooke and Maduagwu, 1978 60 Chips (40 mm x 8.2 mm x 6.8 mm) soaked and stirred in cold (24 o C) water for 18 hours 50 % of bound cyanide detoxified; 90 % of free cyanide detoxified after 4 hours Cooke and Maduagwu, 1978 61 Storage: R oots packed in plas tic bags, each bag stapled shut, bags then stored in the shade at ambient conditions for 72 hours. 9.93 Dufour, 1988

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92 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Processing Methods S/N Cassava Component Process App lied Detoxification of Total Cyanogens Achieved [%] Reference(s) Root and Root Products 62 Whole roots of sweet cassava stored in field clamps for two weeks 50.0 Booth et al., 1976 63 Whole roots of bitter cassava stored in field clamps for two weeks Toxicity increased by 19.7 % Booth et al., 1976 64 Attieke Peel, grate, ferment, add oil and salt, dewater, sun dry 95.9 Mahungu et al., 1987 65 Casabe Scrape, grate, wash to separate starch, ferment for 48 hours, dewater, cook/toast 92.4 98. 5 Dufour, 200 7; 1989 66 Scrape, grate, wash to separate starch, ferment for 24 hours, dewater, cook/toast 86.8 96.8 Dufour, 1989 67 Scrape, grate cook/toast ; obtain fresh casabe 85.4 95.2 Dufour, 1989 68 Baton de Manioc Peel, cut into cubes 20 mm to 40 mm size, remove fibers, soak/ferment for 2 days, dewater for 1 hour, pound/grind to fine paste, wrap in leaves and boil for 20 to 30 minutes 99.3 100 (Mean = 99.6) O'Brien et al., 1992 69 Chikwangue Peel, ferment (3 days), sieve, steam (45 minutes), knead, boil (1 to 2 hours) 100 Mahungu et al., 1987 70 Chinyanya Peel, slice and pound to small pieces, sun dry for 2 to 3 hours, re pound, sun dry for 2 to 3 hours, sieve, obtain final flour 82.2 86.6 Mlingi et al., 1995; Mlingi and Ba inbridge, 1994 71 Eba: Derived from gari Gari (stored for 4 months), soak in boiling water (2 to 5 minutes), knead into soft paste 99.8 Mahungu et al., 1987 72 Farina Soak whole roots for 2 to 4 days, peel, sift, toast 99.2 Dufour, 1989 73 Farine de Manioc Peel, cut into 60 mm to 90 mm thick chunks, soak/ferment for 2 days, crush and dewater for 24 hours, sun dry, pound into flour, boil flour to form thick porridge 91.3 99.9 (Mean = 94.4) O'Brien et al., 19 92

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93 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Processing Methods S/N Cassava Component Process Applied Detoxification of Total Cyanogens Achieved [%] Reference(s) Root and Root Products 74 Farinha P eel, soak, grate, ferment, dewater, toast 99.0 Dufour, 200 7 75 Flour preparation Soak flour 1 : 1.25 ratio (flour : water ) spread the mixture out in a thin layer, heat in the oven at 30 o C for 5 hours 83.3 Cumbana et al., 2007 76 Soak/Wet flour a t 30 o C for 5 hours 55 Bradbury, 2006 77 Flour production Peel, cut into 4 cm x 4 cm dimensions, sun dry for 4 days, mill into flour 27.8 Muzondo and Zvauya, 1995 78 Reconstitute flour at 30 % to 35 % moisture content, steam for 20 minutes, cool to r oom temperature, oven dry for 2 days at 60 o 35.6 Muzondo and Zvauya, 1995 79 Ferment gelatinized cassava flour at 40 o C with formulate d media for 50 hours, 64.6 Muzondo and Zvauya 1995 80 Peel; heap, cover and incubate for 3 days; remove mold; crush; sun dry; pound; obtain flour 82.7 98.5 Essers, 1994 81 Fufu Ghana style: Peel, boil, pound 80.4 Mahungu et al., 1987 82 Nigeria style: Peel, soak (1 day), grate, dehydrate, steam, pound 95.4 Mahungu et al., 1987 83 Zaire style: Peel, ferment (3 days), sun dry, mill, cook 99.8 Mahungu et al., 1987 84 Preparation: Soak whole roots for 3 days 82.6 Bourdoux et al. 1982 85 Peel Soaked roots, break into small fragments a nd sundry for 3 days 85.9 Bourdoux et al. 1982 86 Grind sun dried fragments into Flour (uncooked fufu) 97.8 Bourdoux et al. 1982

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94 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Processing Methods S/N Cassava Comp onent Process Applied Detoxification of Total Cyanogens Achieved [%] Reference(s) Root and Root Products 87 Fufu Soak whole roots for 3 days, Peel soaked roots, break into small fragments and sundry for 3 days, Grind sun dried fragments into f lou r Cook the flour in boiling water to form firm paste, (obtain fufu) 98.7 Bourdoux et al. 1982 88 Fuku Peel, cut into small pieces, dry (2 days), grind into flour (uncooked fuku) 83.6 Bourdoux et al. 1982 89 Peel, cut into small pieces, dry (2 day s), grind into flour, cook flour into a gruel (cooked fuku) 95.3 Bourdoux et al. 1982 90 Gari/gari production Peel, grate, add water at 75 % (v/w); heat at 50 o C for 6 hours > 99 % Sokari, 1992 91 Peel, grate, dehydrate, ferment, fry 83.2 Mahungu et al., 1987 92 Peel, grate, dehydrate, ferment, fry, store (4 months) 98.1 Mahungu et al., 1987 93 Peel, grate, dewater with screw press for 24 hours, garify (roast) 99.0 99.6 Estimated from Maduagwu & Oben, 1981 94 Peel, grate, dewater with t raditional press (weights of heavy stones) for 72 hours, garify (roast) 93.5 97.9 Estimated from Maduagwu & Oben, 1981 95 Peel, grate, ferment (48 hours), press (48 hours), roast 71.3 Ketiku et al., 1978 96 Ijapu Peel, boil for 25 minutes, slice, s oak slices in cold water for 24 hours, rinse 84.5 95.3 Sokari, 1992 97 Konkonte (unfermented), mill, cook 95.4 Mahungu et al., 1987 98 Lafun Peeling, soaking, fermenting, mashing, drying 88.1 Ketiku et al., 1978 99 M akopa; normal size Peel, split longitudinally, sun dry on rock/platform for 8 days, pound, obtain flour 45.7 70.7 Mlingi et al., 1995; Mlingi and Bainbridge, 1994

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95 Table 2 9 Continued. Detoxification of Cassava Components and Products by various Proce ssing Methods S/N Cassava Component Process Applied Detoxification of Total Cyanogens Achieved [%] Reference(s) Root and Root Products 100 Makopa; normal size Peel, split longitudinally, sun dry on rock/platform for 17 days, pound, obtain flour 63.2 73.1 Mlingi and Bainbridge, 1994 101 Manicuera Boiled cassava starch juice beverage 98.6 99.6 Dufour, 1989 10 2 Ntuka Zaire style: Peel, ferment (3 days), steam (2 hours) 99.8 Mahungu et al., 1987 10 3 Oyoko Zaire style: Peel, grate, mix with fermenting pulp (3:1), steam 90.8 Mahungu et al., 1987 10 4 Plakali fermenting pulp (3:1), sun dry, pound, cook 96.6 Mahungu et al., 1987 10 5 Ugali wa mhogo (from peeled root) Air Drying Method: Slice peeled roots into 30 mm to 50 mm thick pieces, oven dry at 40 o C for 3 days 93.7 Gidamis et al., 1993 10 6 Pound dried pieces and sieve into flour 95.1 Gidamis et al., 1993 10 7 Prepare Ugali wa mhogo by boiling 1 : 2 (flour : water) ratio and stirring 95.3 Gidamis et al., 1993 10 8 Ugali wa mhogo (from unpeeled root) Soaking Method: Soak unpeeled root in water at 30 o C for 8 days, 62.7 Gidamis et al., 1993 1 09 Slice soaked roots and oven dry at 40 o C for 3 days 96.4 Gidamis et al., 1993 11 0 Pou nd dried pieces and sieve into flour 96.8 Gidamis et al., 1993 11 1 Prepare Ugali wa mhogo by boiling 1 : 2 (flour : water) ratio and stirring 98.3 Gidamis et al., 1993 11 2 Animal Feed Silage: Unpeeled roots were washed and cut into chips. Chips were p acked into wooden silos lined with sheet metal and plastic. Silos were covered with thick plastic sheet and made airtight wit h wood shavings. Silos were maintained in shaded area and ensiled for 26 weeks 64 74 8 Gomez and Valdivieso, 1988

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96 Cardoso et al., (2005), proposed equations that may be used to relate the initial cyanide content of cassava to the percent cyanide retention after processing. Based on their analyses, it may be possible to estimate the initial maximum total cyanide content for a spe cific processing method to achieve the 10 ppm cyanide safety standard of WHO. Consequently, the more efficient processing methods such as grating/pounding, fermentation/dewatering, that are used to produce farinha and gari should start with cassava having initial maximum total cyanide content of about 222 ppm. On the other hand, to achieve the same safety margin (10 ppm cyanide in the final product), the less efficient process such as frying or the sun drying used for flour production should start with cass ava having initial maximum total cyanide content of about 33 ppm (Cardoso et al., 2005). Below is a brief discussion on cassava foo d systems from around the world. 2 .1.11 Cassava Food Products A wide range of cassava foo d products have been developed in d ifferent parts of the world. Procedures for these food systems may vary from country to country; and even between different regions of the same country. Cassava food systems may be fermented; grated; dried; fried; boiled; or subjected to various other unit operations or combinations thereof. Among food products of cassava prepared and consumed around the world are: akpakpuru, attieke, casabe, chickwangue, farina, fou fou, fuku, gaplek, gari, ijapu, konkonte kpokpo gari, l afun, landa ng (cassava rice), mpond u, ntuka, peujeum or tapai, puttu, t apioca thundam, etc In many products, grating, fermenti n g drying etc. minimize hydrocyanic acid concentration (Mahungu et al., 1987; Ketiku et al., 1978). Some of the mentioned cassava food systems are briefly highligh ted.

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97 2.1.11 .1 Akpakpuru In the Rivers State of Nigeria and various states in Eastern and Southern Nigeria, a dumpling type fermented cassava product is produced and consumed both in the rural areas and in the cities. The dumplings have acquired personality of sorts. They are sold as 10 12 cm balls wrapped with polyethylene sheets in city streets and touted by locality to locality. The Ikwerre people of Rivers State of Ni geria call both cassava crop and this product the same name: Akpakpuru. The traditional preparation method is as follows. Whole cassava roots are soaked in 90 100 cm diameter 10 25 cm deep dug out pits or fence protected areas at the edges of tidal str eams for five or more days. Fermentation occurs during this soaking period, softening the roots parenchyma considerably. Root peels and central fib er s are removed; the soft parenchymatous pulp is crumbled and sieved into jute sacks with raffia basket mesh. Mass of sieved pulp is dewatered, wrung dried and stored in the sacks until required for consumption. Akpakpuru is prepared for consumption by molding the pulp into 10 12 cm diameter balls, boiling for 10 15 minutes and pounding the balls into dough with pestle and mortar. The boiling and pounding is repeated a couple of times or until the dough is well cooked. Akpakpuru is then eaten as dumpling when served with delicately prepared stews and soups rich in vegetables, game meat, and sea foods such as clams, crabs, oysters, periwinkles and shrimps. 2.1.11 .2 Attieke Balagopalan et al., 1988; Mahungu et al., 1987; Lancaster et al., 1982). It is produced through laborious and time cons uming manual unit operations which include peeling,

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98 washing, grating, fermenting, dewatering, sieving, granulating, drying and steaming. Cassava roots are peeled, washed and grated or mashed. A starter culture of 5 10 % of the mass of mash is added to f acilitate fermentation. About 1 % of palm oil and some salt are also added after which fermentation is allowed to proceed for fifteen hours. The fermented mass is dewatered, sieved and granulated. The granules are sun dried briefly for about an hour and th en gelatinized by steaming over a boiling pot of water for thirty minutes. While steaming, the granules are stirred with a spatula to prevent them from sticking together. The fermentation process is reported to impart a slightly sour taste to the finished product (Ross, 2012; Balagopalan et al., 1988; Lancaster et al., 1982). Attieke can be prepared for eating like couscous. Mix equal parts of attieke and water (or broth of choice) in a saucepan and stand for ten minutes. Attieke will absorb the fluid and s well. After the ten minutes wait add salt, butter and desired spices and herbs. Stir and steam cook for ten minutes or until desired tenderness. Like couscous, Attieke may be served and eaten with stewed meats such as beef, fish, pork and poultry; and with vegetables and milk. Attieke can also be used as replacement of grains in various dishes (Ross, 2012). 2.1.11 .3 Casabe Casabe is a flat, cracker like bread that is made from cassava root. It is a traditional food of the Amazonians, and its preparation ca n range from simple to elaborate. In the simplest form, fresh cassava roots are peeled and grated into a mash. The mash is subsequently dewatered and placed on hot clay or stone griddle, pressed down into a thin layer and toasted on each side (Figure 2 2 0 ) The resulting flat circular cake is the cassava bread known as casabe (Lancaster et al., 1982 ; Seigler and

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99 Pereira, 1981 ). A more elab orate method of preparing casabe was described in the literature (Dufour, 2007; 1994 ; 1989 ). Figure 2 20. Production a nd Toasting of Casabe on a Wood Fired Griddle (Wikipedia, 2013 B). 2.1.11.4 Chickwangue Chickwangue is a fermented cassava food product. Variations of it are prepared and consumed in many parts of Africa, including Zaire, Sudan, Gabon, Congo, Central Afric an Republic, Cameroon, and Angola. It has been reported to be the most popular processed food from cassava in Zaire (Hahn, 1992). Basically, the cassava root is ferme nted in water for 3 5 days. Peeling of the root may be effected before or after the fe rmentation process. Following fermentation, the soft pulp is recovered, washed clean and pounded into a thick paste. The paste is subsequently securely wrapped and tied in l eaves of Megaphrynium macrostachyum banana, plantain, or leaves of other plant of Marantaceae or Zingiberaceae family ( Congocookbook.com 2013 ; Hahn, 1992). The wrapped paste is then steamed for several hours to near consistency of modeling clay. Viola, chickwangue is done. Sizes of the product vary. In Central Africa,

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100 two sizes of chic kwangue wraps are common: 2.54 5.08 cm diameter by 30.48 cm long and 10.16 cm diameter by 30.48 cm long (Congocookbook.com, 2013). In group. Myondo is 1.5 2.0 cm in diamete r by 15.0 20.0 cm long; whil e Bobolo is 2.0 4.0 cm in diameter by 30.0 40.0 cm long (Hahn, 1992). Chickwangue and its group of product s may be served with soup, stew, or any sauce dish (Congocookbook.com, 2013). 2.1.11 .5 Farina Farina is a popular me thod of preparing cassava in tropical America. It is prepared in the form of a meal or granules and also called farinha de mandioca in Brazil (Lages and Tannenbaum, 1978). In the preparation of farina, fresh roots are peeled, grated, dewatered and sifted t o obtain an even textured mash. The mash is roasted on a griddle with continuous stirring until cooked to less than 10 % moisture content (Oke, 1994). In traditional Amazonia, the grater board, tipiti (a basket work sleeve press), and clay griddle are used respectively for grating/rasping the root, dewatering and roasting the mash (Dufour, 1994). In some preparation styles, the cassava root is soaked or fermented in water for some days before processing (Balagopalan et al., 1988). 2.1.11 .6 Fufu Cassava fuf u may be processed in a variety of ways. In one production style, cassava roots are peeled, washed and grated. The grated mash is packed into sacks, weighted down with heavy objects to squeeze out contained juice, and allowed to ferment for a few days. The resulting dough is used to produce fufu (Lancaster et al., 1992). In Ghana, the fufu is said to be prepared by adding quantities of the dough into boiling water and stirring until desired consistency is achieved (Doku, 1969). In another

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101 production method, the peeled cassava roots are first soaked in water and allowed to ferment for several days. The soft root pulp is mashed, sieved in a basin of water, and allowed to sediment or settle. The sediment paste is thinned down in a pot and cooked on low fire wit h constant stirring until desired consistency is ac hieved (Ayankunbi et al., 1991) 2.1.11 .7 Fuku Fuku is a cassava root product consumed in Zaire. Cassava roots are peeled, cut into pieces and dried in the sun. Dried chips are pounded into flour and partia lly fermented with corn. Fermentation is stopped by grilling, and the fuku flour is consumed as a meal gruel prepared in boiling water (Bourdoux et al., 1983). 2.1.11 .8 Gaplek Gaplek is a popular traditional famine protector cassava food product in Java an d other parts of Indonesia. Fresh cassava roots are peeled and sliced into 15.24 20.32 cm pieces. The pieces are dried in the sun for 1 3 or more days depending on weather conditions. The dried pieces are stored in a cool and dry place as vanguard agai nst hunger. When other sources of food become scarce or expensive, gaplek is retrieved from storage, pounded into smaller pieces and cooked and consumed like rice. 2.1.11 .9 Gari Gari is a popular cassava food product consumed extensively in many parts of W est Africa including Benin, Ghana, Guinea, Nigeria, and Togo. In these countries, gari may be consumed as a snack in various forms or eaten as the main dish. It is eaten plain just as it is; eaten with groundnuts, coconuts, or palm kernel; eaten soaked in cold water with or without the addition of milk and sugar; or eaten as eba in combination of

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102 soups of fish, beef, game meat, poultry, vegetables, etc. Eba is derived when gari is soaked in hot water and kneaded. The consistency of eba ranges from that of a soft paste to jell to modeling clay. The preparation of gari is similar to that of farina, but requires more elaborate equipment, processes and time. The basic unit operations for gari production are washing, peeling, grating, dewatering/fermenting, sifti ng, and then garification ; which consists of roasting/frying (Aso, 2004; Collard and Levi, 1959). Fresh cassava roots are grated or ground into a mash after washing and peeling operations. The mash is packed into a jute or polypropylene sack and wrung drie d/fermented for a few days. The clumped cake is crumbled and sifted through sieves. The sifted mass is then garified (roasted/fried) in shallow iron pots to produce gari. Palm oil may be added during fermentation, and or during garification stage to improv e nutritional (Vitamin A) content and minimize sticking to the iron pots. After cooling, the gari may be sieved to obtain homogenous granules. 2.1.11 .10 Konkonte the Caribbean It is often regarded as poverty food for low income earners. Konkonte is fairly easy to prepare. Fresh cassava roots are peeled, sliced into chips and dried in the sun. Dried chips are milled into flour and consumed as a gru el or runny porridge, being en joyed with a specially prepared soup 2.1.11 .11 Lafun For "lafun" production in the Western States of Nigeria, peeled or unpeeled cassava roots are immersed in a stream, in stationary water (near a stream) or in an earthenware vessel, and fermented until t he roots become soft. The peel and central fibers of the fermented roots are manually removed and the recovered pulp is hand

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103 mashed or pounded and sun dried. The dried mash is then milled into lafun flour (Oke, 1994; Hahn, 1992). To prepare the lafun, an e stimated amount of lafun flour is mixed with an estimated amount of boiling water. The mixture is allowed to cook while stirring continuously to prevent clumps and burning, until the desired consistency is achieved (Ayankunbi et al., 1991). A similar produ ct is reported to be processed in Angola where the dried pulp is called bombo or makessu, and the ground flour is referr ed to as fuba (Alberto, 1958). 2.1.11 .12 Landang (cassava r ice) In the Philippines, a popular dish known as landing or cassava rice is p roduced by first peeling and grating cassava roots. The grated mass is loaded into jute sacks and the juice squeezed out with applied weight. The mass is then winnowed into small pellets with a basket, dried, steamed, and dried again (Lancaster et al., 198 2). Landang can be eaten as is or it may be cooked and eaten like rice or maize. 2.1.11 .13 Peujeum or Tapai Peujeum is a traditional cassava food product in Java. However, it is wide spread in Asia where it is known by various names including tapai ketela in Indonesia; tapai ubi kayu in Malaysia; and binuburang ube or tapay panggi in the Philippines. Peeled fresh cassava roots are steamed to tenderness, cooled to 30 o C and dusted with a special mixture of flour and spices known as ragi. The dusted root is layered in a basket or wrapped in banana leaves in an earthenware pot and allowed to ferment for a couple of days. The peujeum may be eaten as is or baked before consumption. 2.1.11 .14 Puttu In Sothern India, boiled cassava roots are grated and mixed wit h shredded coconut. The product is called puttu and is often served with cardamom seed. Grated

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104 roots may be minced, mixed with fried onions, cashew nuts, black gram and coriander leaves. The mixture is then molded into balls, dusted with Bengal gram flour and fried. This product is then called pakkoda (Lancaster et al., 1982). Also, parboiled cassava pieces could be mixed with fat, ginger, coriander and curry leaves, mustard seeds, Bengal dhal and black gram dhal to produce uppuma (Balagopalan et al., 1988) 2.1.11 .15 Thundam In Southern India, thundam is prepared by removing the whole peel from cassava roots, slicing and drying the peeled roots. Dried chips may be boiled or fried for consumption. Alternativel y, chips are converted to flour for a variety o f uses. 2.1.11 .16 Other Cassava f ood and beverage p roducts A variety of nonconventional food systems have been produced from cassava. These include cassava rava, porridge, pappads, and wafers (Nanda et al, 2002). Preparations from cassava flour include dum plings, cakes, and fritters. Similarly several beverages have been produced from cassava. Alcoholic drinks include beers, banu or uala, kasili, yaraqui, amoiuare, paya, yakari, tapana, malicha, paiwa r ri (Balagopalan et al., 1988; Lancaster et al., 1982). L ancaster et al., ( 1982 ) reported that t he Karinya people produce a non alcoholic beverage called karato from cassava. Dufour (1989, 1988), noted that Tukanoan Indians in Northwest Amazonia also process bitter cassava Mowat (1989), describ ed Chicha as the generic name applied to native beer and other beverages in South America. Chicha may be derived from sweet or bitter cassava, maize, sugar cane, and various fruits. Amon g the beverages made from cassava by the Amazonian Indians are the non alcoholic drinks like boiled juice that was expressed from grated cassava root with the tipiti; and boiled cassava starch. Beers and other

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105 fermented alcoholic beverages from cassava inc lude bernia, kachiri, kurai, masato, paiwarri and puchokwa (Mowat, 1989). 2.2 Part 2: Production of Amylolytic Enzymes 2.2.1 Background The development of enzymatic processes and the ma rket for enzymes is growing because the use of enzymes has numerous advantages The s e include cheaper production ; great specificity and enhanced rate reactions; application to various current and frontier fields like agriculture, chemicals, clean energy, food, pharmaceuticals, clinical, medicinal and analytical chemistries materials, textile, brewing and distilling industries ; the potential to address issues of health, raw materials, sustainability and environment; as well as the possibility to benefit from use of genetics knowledge to produce enzymes specifically tail ored for special applications (Beilen and Li, 2002 ; Pandey et al., 2000 b ). The study of amylolytic enzymes is of great importance for the improvement of industrial processes (Gupta et al., 2003), treatment of waste waters and animal nutrition applications (Bu end ia et al., 2 003), among others. There are man y sources of amylolytic enzymes. Perhaps the most important source for industrial processes is microbial; especially from some strains of bacteria and fungi ( Tomasik and Horton, 2012; Sous a and Magalhes 2 01 0 ; Uhlig, 1998; Srivastava and Baruah, 1986; Tani et al., 1986; Pestana and Castillo, 1985; Michelena and Castillo, 1984; Forgarty, 1983 ). amylase are starch converting enzymes used for the production of a wide range of products such as dextrins, modified starches, glucose syrups organic acids, flavor and aroma compounds, mushrooms and ethanol from

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106 starch degradation ( Couto and Sanroman, 2006; Gupta et al., 2003 ; Stamford et al., 2002; Pandey et al., 2000 a; Labeille et al., 1997 ). Gluco amylase or amyloglucosidase (EC 3.2.1.3), is an exo amylase that produce s glucose as final product from starch, hydrolyzing glucose units from the non reducing ends ( 1,6 linkages) of am ylose and amylopectin ( Tomasik and Horton, 2012; Anto et al., 2006; Ellaiah et al., 2002; Uhlig, 1998; James and Lee, 1997; Roberts et al., 1995). On t he other hand, amylase (EC 3.2.1.1) catalyzes the hydrolysis of interna l 1,4 O glycosidic bonds in polysaccharides. Indeed amylase is the most reported endo amylase that produces oligodextrins as main product (Castro et al., 2011). Figure 2 21 is a photo image of Alpha Amylase. Figure 2 21. Three Dimensional Image of Al pha Amylase (bing.com, 2013 b; Mobini Dehkordi and Javan, 2012) Some of the enzymes that are available for industrial applications do not withstand the extreme processes and conditions in the industries. This has led to the search for extremophile microor ganisms that survive non standard conditions It is presumed that the enzymes from these microbes c a n be adapted and optimized for

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107 extreme e nvironmental factors (Atomi, 2005; Van Den Burg, 2003; Demirjian et al., 2001; Hough and Danson, 1999; Niehaus et a l., 1999). The production of exogenous enzymes has been studied using : solid state fermentation ( Hashemi, et al., 2011; Murado et al., 1997); submerged fermentation (Aguilar et al., 2008; Amoozegar et al., 2003; Aguilar et al., 2001; Soccol et al., 1994); encapsulated strains (Konsoula and Liakopoulou Kyriakides, 2006 a; 2006 b); and with the advances in genetics, using recombinant microorganisms ( Rasiah and Rehm, 2009; Murakami et al., 2007; Hashim et al., 2005; Lin et al., 2003; Bruins et al., 2001; Lo e t al., 2001). This review highlights fermentat ion processes and microorganisms used for the production of am ylolytic enzymes as well as the process variables. 2.2.2 Amylolytic Enzymes Production by Solid State Fermentation Solid State Fermentation (SSF) may be defined as a fermentation process that occurs on solid matrix in virtual absence of free water. The s olid matrix or substrate provides physical support and may contain sufficient moisture in absorbed form to supply nutrients (Couto and Sanroman, 20 06; Pandey, 1992). SSF technology is used for many applications in Asia E xample s include the production of soy sauce, miso and koji. Koji is made f rom rice with A spergillus oryzae and is used as a starter culture for processing several food products (Hlk er and Lenz, 2005). Durand (2003) described suitable for growth of fungi which are the microorganism s commonly used (Aguilar et al., 2008; Viniegra Gonzlez et al., 2003 ). SSF cha racteristics are similar to natural environment, thus increasi ng the fungi activity ( Barrios Gonzalez, 2012 ; Couto and Sanroman, 2006; Hlker and Lenz, 2005 ; Pandey, 2003; Castil ho et al., 2 000 ). Fungi

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108 are also useful in soil bioremediation, enzyme pr oduction and as an alternative s o u r ce of animal feed. The advantages and limitations of solid s tate fermen tation are presented in Table 2 10 ( Aguilar et al., 2008; Couto and Sanroman, 2006; Viniegra Gonzlez et al., 2003 ) Table 2 10. Advantages and D isadvantages of Solid State Fermentation (SSF) S/N Advantages S/N Disadvantages 1 Simple Technology: Substrate can serve directly as medium or enriched with nutrients. 1 Process control constraints: humidity, pH, heat, CO 2 etc. are difficult to manage. 2 Concentrated product; requiring less downstream processing. 2 Reduced variety of inoculums due to low water activity environments 3 Generated enzymes have low sensitivity to catabolic repression or induction 3 Process Scale up difficulties 4 Medium is similar to natural habitat of inoculums 5 Low risk of microbial contamination 6 Low waste generation 7 Reduced operational problems 8 Reduced energy requirements 9 Reduced water requirements 10 Low cost media 11 Low cost operations 12 Higher productivity (Biomass, Protein retention, Enzyme yield, etc.) 13 Good circulation

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109 The process is relatively simple ; utilizes marginal inputs; and has low energy requirements and production cost s. SSF process es can proceed at water activities of 0.935 0.980 ( Ruijter et al. 2004; Ishida et al. 2000; Grajek & Gervais 1987 a 1987 b ) and therefore, produces less waste ( Anto et al., 2 006 ; Hlker and Lenz, 2005 ) In some cases the fermented product is also used as an enzyme source for other hydrolytic processes (Hlker and Lenz, 2005). M icroorganisms used for SSF and the sources from which they are isolated are very diverse. B acteria may be isolated from hot springs (Sodh i et al., 2 005) ; Cow dung (Swain and Ray, 2009; 2007) ; or from soils (Kar et al., 2010; Oy eleke and Oduwole, 2009 ; Kumar and Satyanarayana, 2004; Srivastava and Baruah, 1986 ). Fungi isolated from soil s and seeds are also used ( Saxena and Singh, 2011 ; Varalakshm i et al., 2 009; Alva et al., 2007; Ant o et al., 2 006 ) I n the case of amylolytic enzymes production the diversity is useful for a variety of applications such as the creation of detergents bakery products, sugar syrups, brewery products, etc. In SSF, the solid material used can be the source of nutri ents for the microorganism and or provide physical support to the system for immobilization When an inert material such as polyurethane foam, acreolite or a resin is used as SSF support structure, the extraction and purification of fermentation products b ecome less costly and cumbersome than when agro industrial wastes serve as fermentation matrix (Pandey, 2003 ; Murad o et al., 19 97 ). Important variables that r e quire control during SSF include physicochemical and biochemical parameters. O ne variable at a ti me approach has often been used to study fermentation processes for enzyme production However, t hi s kind of approach tends to overlook or cloud interactions between factors and responses that are revealed when

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110 adequate experimental designs and response su rface methodologies are adopted (Deep ak et al., 2 008 ; Kumar and Satyanarayana, 2004) Some of the SSF control variables are discussed below and include: Substrate Support Structure Carbon Supplementation Nitrogen Supplementation Porosity and Aeration Mo isture Content and Water Activity Incubation Temperature Inoculums Concentration Fermentation Time and pH 2.2.2.1 Substrates support s tructures A gricultural, biological and industrial materials, products and wastes have been used as physical support st ruc ture s in SSF processes with various microorganisms including species of bacteria: Bacillus Clostridium Streptomyces ; yeast: Kluyveromyces Saccharomycopsis Z ygosaccharomyces ; and many fungi: Aspergillus Ceratocystis Neurospora Penicillium Rhizopu s and Thermomyces P roducts generated include amylases, pectinases, proteases, flavor compounds, etc. The gamut of substrate covers seeds, grains, tubers, leaves, as well as industrial by products that are used in different formats. For example, cassava h as been used and studied in the form of starch, flour, bagasse, peel gelatinized, and fibrous residue ; maize and corn in the format of steep liquor, flour, crushed, and as corn cobs; mustard as seed cake; oat as flour; plantain as peel; potato as flour, s tarch and solid waste; c offee as husk; rice as bran, flake, flour/ powder, grains, hull, and gelatinized; soybean as meal and seeds ; sugar beet as pulp ; sugarcane as bagasse ; sun flour as seeds; while wh eat has been utilized in the form of bran, crushed see ds, flakes, flour ; and yam as peel Tab le 2 11 highlights these su bstrates, the organisms used as well as the products generated.

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111 Table 2 11 Substrates used in Microbial F ermentations Organisms Employed and the Products Generated S/N Substrate Organism Employed Product Generated Reference(s) 1 Amaranth Grain Rhizopus oryzae Flavor (Aroma Compound) Christen et al., 2000 Rhizopus oryzae ATCC 34612 Flavor (Aroma Compound) Bramorski et al., 1998 2 Apple Pomace Rhizopus oryzae Flavor (Aroma Compound) C hristen et al., 2000 Rhizopus oryzae ATCC 34612 Flavor (Aroma Compound) Bramorski et al., 1998 3 Barley Bran Thermomyces lanuginosus Amylase Enzyme Kunamneni et al., 2005 4 Cassava Bagasse Aspergillus oryzae TISTR 3605 Alpha Amylase Pengthamkeerati et al., 2012; Kluyveromyces marxianus Flavor (Aroma Compound) Medeiros et al., 2001 Rhizopus oryzae Flavor (Aroma Compound) Christen et al., 2000 A. niger LPB 21 ; A. niger NRRL 2001 ; A. niger CFTRI 30 ; Ceratocystis fimbriata; K. marxianu s; L. edodes; P. sajor caju; Rhizopus sp.; R. oryzae Aroma Compounds; Mushrooms; Organic Acids; Glucose Hydrolysates Pandey et al., 2000 B Rhizopus oryzae ATCC 34612 Flavor (Aroma Compound) Bramorski et al., 1998 NA Citric Acid Soccol, 1996 5 Cas sava (Cooked) Rhizopus delamer ATCC 34612; Rhizopus oryzae MUCL 28168; Rhizopus oryzae MUCL 28627 Protein enrichment; Alpha Amylase; Glucoamylase Soccol et al., 1994 A 6 Cassava Fibrous Residue Streptomyces erumpens MTCC 7317 Alpha Amylase Kar et al., 2010 Bacillus subtilis CM3 Alpha Amylase Swain and Ray, 2007

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112 Table 2 11 Continue d. Substrates used in Microbial Fermentations, Organisms Employed and the Products Generated S/N Substrate Organism Employed Product Generated Reference(s) 7 Cassava Fl our (Gelatinized) Aspergillus niger; Amylases Soccol, 1996 8 Cassava Flour (Steam cooked) Rhizopus oryzae Protein Enrichment Daubresse et al., 1987 9 Cassava Peel Aspergillus flavus Amylase Sani et al., 1992 Aspergillus niger Amylase Sani et al., 19 92 10 Cassava (Raw) Rhizopus delamer ATCC 34612; Rhizopus oryzae MUCL 28168; Rhizopus oryzae MUCL 28627 Protein enrichment; Alpha Amylase; Glucoamylase Soccol et al., 1994 A 11 Cassava Root Rhizopus sp. Amyloglucosidase; Single cell protein Sukara and Doelle, 1989 12 Cassava Starch Saccharomycopsis fibuligera Alpha Amylase; Glucoamylase Gonzalez et al., 2008 Rhizopus sp. Protein Enrichment Soccol, 1996 B Aspergillus sp. N 2 Glucoamylase Tani et al., 1986 13 Cassava Waste Rhizopus stolonifer Aspe rgillus niger Aspergillus terreus Cellulasses; Beta Glucosidase; Protein Enrichment Pothiraj et al., 2006 14 Chestnut Flour Bacillus subtilis amylase; galactosidase Konsoula & Liakopoulou Kiriakides, 2007 15 Coconut Oil Cake Aspergillus sp. JGI 12 Amylase Alva et al., 2007 16 Coffee Husk Ceratocystis fimbriata Flavor (Aroma Compound) Soares et al., 2000 17 Corncobs Thermomyces lanuginosus Amylase Kunamneni et al., 2005 18 Corn Flour Bacillus subtilis amylase; galactosidase Konsoula & Liakopoulou Kiriakides, 2007 19 Corn Starch Bacillus subtilis Alpha Amylase Konsoula & Liakopoulou Kiriakides, 2006 a; 2006 b

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113 Table 2 11 Continue d. S ubstrates used in Microbial Fermentations, Organisms Employed and the Products Generated S/N Substrate Organism Employed Product Generated Reference(s) 20 Corn Steep Liquor Bacillus subtilis amylase; galactosidase Konsoula & Liakopoulou Kiriakides, 2 007 Clostridium thermosulfurogenes SV2 Beta Amylase Rama Mohan Reddy et al., 2003 21 Cowpea Waste Aspergillus oryzae BS41 Gluco Amylase Kareem et al., 2009 22 Giant Palm Bran Kluyveromyces marxianus Flavor (Aroma Compound) Medeiros et al., 2001 23 G ram Husk Bacillus sp. Amylase Saxena & Singh, 2011 24 Ground Nut Oil Cake Aspergillus sp. JGI 12 Amylase Alva et al., 2007 25 Kumara; a starchy root crop Aspergillus niger Citric Acid Lu et al., 1998 26 Maize (Crushed) Thermomyces lanuginosus Amylase K unamneni et al., 2005 27 Maize Meal Thermomyces lanuginosus Amylase Kunamneni et al., 2005 28 Millet Cereal Thermomyces lanuginosus Amylase Kunamneni et al., 2005 29 Millet (Pearl Millet) Flour Clostridium thermosulfurogenes SV2 Beta Amylase Rama Mohan Reddy et al., 2003 30 Miso Zygosaccharomyces rouxii Flavor (Aroma Compound) Sugawara et al., 1994 31 Molasses Bran Thermomyces lanuginosus Amylase Kunamneni et al., 2005 32 Mustard Oil Seed Cake Bacillus sp. Amylase Saxena & Singh, 2011 33 Oat Flour Ba cillus subtilis amylase; galactosidase Konsoula & Liakopoulou Kiriakides, 2007 34 Plantain Peel Aspergillus niger Beta Amylase Adeniran et al., 2010 35 Potato Flour Bacillus subtilis amylase; galactosidase Konsoula & Liakopoulou Kiriakides, 20 07

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114 Tabl e 2 11 Continue d. Substrates used in Microbial Fermentations, Organisms Employed and the Products Generated S/N Substrate Organism Employed Product Generated Reference(s) 36 Potato Starch Bacillus subtilis Alpha Amylase Konsoula & Liakopoulou K iriakides, 2006 a; 2006 b Clostridium thermosulfurogenes SV2 Beta Amylase Rama Mohan Reddy et al., 2003 37 Potato Waste (Solid) Anaerobic sludge and waste water innocula A mylase, Carboxymethyl Cellulase, Filter paper Cellulase, Xylanase, Pectinase and Protease Parawira et al., 2005. 38 Rice Bran Bacillus sp. Amylase Saxena & Singh, 2011 Aspergillus sp. JGI 12 Amylase Alva et al., 2007 Thermomyces lanuginosus Amylase Kunamneni et al., 2005 Aspergillus terreus GTC 826 Glucoamylase Ali et al., 1 989 Aspergillus sp. N 2 Glucoamylase Tani et al., 1986 39 Rice Flake Manufacturing Waste Aspergillus sp HA 2. Glucoamylase Anto et al., 2006 40 Rice Flour Bacillus subtilis amylase; galactosidase Konsoula & Liakopoulou Kiriakides, 2007 Aspergillus awamori Glucoamylase Pestana and Castillo, 1985 Aspergillus foetidus Alpha Amylase Michelena and Castillo, 1984. 41 Rice (Gelatinized) Neurospora sp Flavor (Aroma Compoun d) Pastore et al., 1994 42 Rice Hull Aspergillus sp. N 2 Glucoamylase Tani et al., 1986 43 Rice Husks Penicillium citrinum Cellulasses Kuhad and Singh, 1993

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115 Table 2 11 Continue d. Substrates used in Microbial Fermentations, Organisms Employed and th e Products Generated S/N Substrate Organism Employed Product Generated Reference(s) 44 Rice Powder Aspergillus sp HA 2. Glucoamylase Anto et al., 2006 Aspergillus fumigatus Glucoamylase Cherry et al., 2004 45 Rice Grains Aspergillus oryzae RIB 128 Volatile Compounds: Alcohols, Aldehydes, Esthers and Ketones Ito et al., 1990 46 Rice Starch Bacillus subtilis Alpha Amylase Konsoula & Liakopoulou Kiriakides, 2006 a; 2006 b 47 Soybeans Rhizopus oryzae Flavor (Aroma Compound) Christen et al., 2000 Bacillus subtilis IFO 3013 Flavor (Aroma Compound) Besson et al., 1997 48 Soybean (Ground) Bacillus subtilis IFO 3013 Flavor (Aroma Compound) Larroche et al., 1999 49 Soybean Meal Rhizopus oryzae ATCC 34612 Flavor (Aroma Compound) Bramorski et al., 199 8 50 Sugar Beet Pulp Aspergillus niger I 1472 Feruloyl esterase Asther et al., 2002 51 Sugar Cane Bagasse Aspergillus niger UO 01 Amylase Roses and Guerra, 2009 52 Sugarcane Bagasse Hydrolysate Bacillus subtilis strain KCC103 Alpha Amylase Rajagopalan a nd Krishnan, 2008 53 Sunflower Seeds Penicillium fellutanum Protease Muller dos Santos et al., 2004 54 Tea Waste Aspergillus niger Glucoamylase Selvakumar et al., 1998

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116 Table 2 11 Continue d. Substrates used in Microbial Fermentations, Organisms Employ ed and the Products Generated S/N Substrate Organism Employed Product Generated Reference(S) 55 Wheat Bran Aspergillus oryzae IFO 30103 Alpha Amylase Dey & Banerjee, 2011 Bacillus sp KR 8104 Alpha Amylase Hashemi et al., 2011 Bacillus sp. Amylase Saxena & Singh, 2011 Aspergillus niger JGI 24 Alpha Amylase Varalakshmi et al., 2009 Aspergillus sp. JGI 12 Amylase Alva et al., 2007 Aspergillus sp HA 2. Glucoamylase Anto et al., 2006 Thermomyces lanuginosus Amylase Kunamneni et al., 2005 Bacillus sp. PS 7 Alpha Amylase Sodhi et al. 2005 Thermomucor indicae seudaticae Glucoamylase Kumar & Satyanarayana, 2004 Ceratocystis fimbriata Aroma Compound Christen et al., 1997 56 Wheat (Crushed) Thermomyces lanuginosus Amylase Kunamneni et al., 2005 57 Wheat Flakes Thermomyces lanuginosus Amylase Kunamneni et al., 2005 58 Wheat Flour Bacillus subtilis amylase; galactosidase Konsoula & Liakopoulou Kiriakides, 2007 Aspergillus oryzae Alpha Amylase Rahardjo et al., 2005 59 Wheat Powder Aspergillus fumigates Glucoamylase Cherry et al., 2004 60 Yam Peel Aspergillus niger Amyloglucosidase Adeniran et al., 2010 Aspergillus niger Amylase Uguru et al., 1997

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117 2.2.2.2 Carbon s upplementation For effective performance of microorganisms and optimal secretion of products, substrates are often supplemented with carbon sources. Various sources of carbon such as glucose, lactose, sucrose, and soluble starch, have been studied. These studies revealed that the production rate of enzyme with added sugars could vary widely depending on type of sugar, strain or type of microorganism and composition of substrate The highest production rates were obtained when sucrose was added to the culture media during fermentation with Aspergillus sp. (Ellaia h et al., 2002; Anto et al., 2 006) and Aspergillus niger (Selvakum ar et al., 19 98). However, the latter was shown to ha ve a negative impact on enzyme production when fructose and lactose were added (instead of sucrose). For Aspergillus. niger JG 24, the addition of glucose and sucrose repressed the enzyme production rate when wheat bran was used as the solid material (Vara laksh mi et al., 20 09). On the other hand, when cassava bagasse was used for enzyme production, the addition of glucose had no significant impact on enzyme yields, but amylase productio n with Aspergillus oryzae (Pengthamkee rati et al., 20 12). amylase with Clostridium thermosulfurogenes wheat bran w as used as support enriched with potato starch and corn steep liquor A gradual increase in the yield was observed whe n concentrations of potato starch and corn steep liquor were increased from 5 % to 20% and 0.5% to 2% respectively (Rama Mohan Red dy et al., 2 003). 2.2.2.3 Nitrogen s upplementation The effect of organic and inorganic nitrogen has also been studied, revealin g an improvement in production when yeast extract (YE) (An to et al., 20 06), malt extract

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118 (Selvakuma r et al., 1 998), casein, meat extract (Varalakshmi et al., 2 009), peptone, tr yptone (Kunamneni et al., 2005), and urea (Ellaiah et al., 2002; Kunamneni et al ., 2 amylase production was observed when a mixed substrate of biosludge and cassava bagasse was used with the nitrogen sources (Penghamkeer ati et al., 20 12). The addition of organic source amylase when wheat bran and Aspergillus niger were used for enzyme production; but, the opposite occurred with urea supplementation (Varalaksh mi et al., 20 09). In the case of glucoamylase production, supplementation of wheat bran, corn steep liquor and peptone with urea showed varying degrees of improvement in enzyme yields (Ellaiah et al., 2 002). 2.2.2.4 Porosity and aerat ion Porosity of the bulk solid materials used in SSF plays an important role in the efficacy of solid sta te fermentations. Porosity can be quantified by the ratio of empty air spaces (pores) to the total bulk volume of the porous material (Durand, 2003). Greater porosity will promote improved aeration enhancing oxygenation and regulation of humidity and temp erature because the combination of air and solid matrix has lower thermal diffusivity compared to water (Pandey, 2003). Porosity also contributes to the distribution of volatile compounds and amount of CO 2 produced during the process (Graminh a et al., 2 008 amylase, in which the use of different rates of air flow produce different enzymatic activities during fermentation with Aspergillus oryzae IFO 30103 as the biocatalyst and wheat bran as the subs trate. One study obtained highest enzyme activity at a flow rate of 0.1 L min 1 g 1 of wheat bran B amylase activity dropped gradually (Dey & Banerjee, 2012 ). Ant o et al. ( 2006) used wastes from rice production

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119 of flakes as carb on source for the substrate (coarse waste, medium waste, rice powder), as well as wheat bran. Maximum enzyme production was obtained with wheat bran according to Sodh i et al. (20 05) and Ellaiah et al. ( 2002), followed by coarse and medium waste. These resu lts correlated well with starch content in the samples and characteristics of the substrate used. Coarse wastes contain rice bran particles as substrate and husk as support, increasing the porosity in the media and access to the nutrients, thus allowing ge rmination of spores and growth of mycelia. Fermentation of wheat dough with A. oryzae Rahardj o et al. (2 005) resulted in amylase and biomass production rates, along with production of aerial mycelia by taking advantage of porosity to increase mac roscopic respiration rate. The effect of porosity on microorganism growth was reported by Murad o et al. ( 1997 ) in which negative signs in terms involving density of the solid support in a mathematical model showed the restriction in intra particular diffus ion when working with high densities using amylase production with A. oryzae In SSF, porosity is a key characteristic because the open pore structure of the solid used as substrate or anchorage benefits the enz yme diffusion and the substrate degradation happens inside. The fragments of substrate that are soluble in water have to diffuse out of the solid matrix where the enzymes action produces metabolizable compounds (Raghavara o et al., 20 03). 2.2.2.5 Moi sture c ontent and water a ctivity Moisture content also affects the efficacy of SSF, and should neither be too high nor too low. High moisture content decreases the porosity of the medium and impedes oxygen penetration, while too low a moisture content can impede microbial growth because of poor accessibility to nutrients (Pandey, 2003). Water activity is a measure of

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120 the amount of water for any given moisture content that is freely available to support microbial and biochemical activity. It can determine the type of microorganism that can grow in SSF systems. For example, yeasts and molds can grow at water activities much lower than the higher levels needed for bacteria. The relatively low water activities (a w ) used in SSF influence the physiological aspects of mi croorganisms, metabolite production and enzyme activity (Graminha et al., 2008). Kareem et al. ( 2009) tried different levels of initial moisture content for the production of glucoamylase by the cultures A. oryzae BS41. Highest enzymatic activity was obta ined with 60% initial mois ture content. The same moisture content (60%) was also found to be optimum in amylase by a strain of A. oryzae with cassava bagasse and amylase (Pengt hamkeer ati et al., 20 12 ). Furthermore, initial moisture content of 60% was found to be the optimum for production of glucoamylase from fermentation of tea waste with Aspergillus niger NCIM 1248, achieving maximum enzyme production o f 198.4 IU /gds (glucoamy lase units/gram dry substrate) at 96 hours of fermentation (Selvakumar et al., 19 98). The same 60%moisture content was also optimum for fermentation with Streptomyces erumpens MTCC 7317 of cassava fibrous residue (Ka r et al., 2010). However, a moisture co ntent of 90% produced the highest enzyme activ ity (298 U/g) when the thermophi lic strain Thermomyces lanuginosus was used with wheat bran as the solid material support. Activity of less than 150 U/g was obtained when 60% initial moisture content was used d uring the same fermentation (Kunamneni et al., 200 5). When the fermentation was made with a bacterial strain like Bacillus sp. PS 7 for the amylase, the moisture content that produced the best enzyme yield was

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121 60% ; obtained when a ratio of 1:1.5 (wheat bran: tap water) was used (Sodh i et al., 20 05). The same ratio of wheat bran and distilled water (60% moisture ) was obtained as an optimum during fermentation with A. oryzae HS 3 for the production of amyloglucosidase (Singh & Soni, 2001). Figure 2 12 summarizes the optimum moisture contents found for various combinations of microorganism and substrate in enzyme p roduction. Table 2 12 Optimum moisture content [% W/W] for various combinations of microorganism and substrate in enzyme production S/N Microorganism Substrate Optimum Moisture Content for Enzyme Produced Reference Gluco amylase Alpha amylase 1. Aspergillus oryzae BS41 Not reported 60 Kareem et al. (2009) 2. Aspergillus oryzae Cassava fibrous residue and biosludge 60 Pengthamkeerati et al., 2011 3. Aspergillus niger NCIM 1248 Tea waste 60 Selvakuma r et al., 1 998 4. Streptomyce s erumpens MTCC 7317 Cassava fibrous residue 60 Kar et al., ( 2010) 5. Thermomyces lanuginosus Wheat bran 90 Kunamne ni et al., ( 2005 6. Bacillus sp. PS 7 Wheat bran 60 Sodhi et al., ( 2005 7. Aspergillus oryz ae HS 3 Wheat bran 60 Singh & Soni, (2001)

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122 2.2.2.6 Incubation t emperature Temperature is a main factor during SSF for enzyme production, not only because it affects the growth of the microorganism, but because during the production of some thermol abile enzymes, the increasing temperature due to microorganism metabolism can cause its denaturation, lowering the yield of the process. Muller dos Santos et al. (2004) developed a mathematical model that showed the behavior of temperature during SSF for t he production of protease with P. fellatanum in a large scale process with a mixing bioreactor, controlled air flow and water jacket. This system reached a temperature of 48C using one volume of air per total bioreactor volume per minute and cooling water temperature at 30C. However, results showed that only 25% of the enzyme produced retained any activity. On the other hand, using the same air flow rate with water temperature of 14.5C, approximately 87% activity was retained. For A. oryzae the effect o amylase activity when the fermentation process was carried out at 32C, with a sharp decrease at higher te mperatures (Dey & Banerjee, 2012 ). Similar results were obtained by Ellaiah et al. (2002) at an optimum temperature o f 30C for glucoamylase production with A. oryzae SP 3. Francis et al. (2003) also found similar results during optimization of fermentation amylase with spent brewers grains and Bacillus sp. PS 7. Highest productivity was obta ined at 37C with optimum enzyme activity at 60C, which remained very thermostable at 50C during 6 hours (Sodhi et al., 2005). It should be noted that optimum temperatures for the production of the enzyme are not the same as those for enzyme activity. Fo r A. niger amylase activity was obtained at 22C, being negatively affected with higher temperatures (37C and 40C). The best activity of the purified enzyme was obtained at 30C (Varalakshmi et al.,

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123 2009 ). Enzymes produced by thermophi lic mi croorganisms like Thermomyces lanuginosus have better activity during fermentation at higher temperatures. The optimum temperature for amylase production was found to be 50 C (263 U/g) with very high thermostability at this temperature. Optimal enzyme act ivity was found at 60C (Kunamneni et al., 2005), in accordance with the results obtained by Kar et al. (2010) amylase using cassava fibrous residue as a culture medium and the actinomycete strain Streptomyces erumpens MTCC 7317. Table 2 13 summarizes the optimum temperature for enzyme production and ac tivity with various combinations of microorganism and substrate. 2.2.2.7 Inoculums c oncentration The inoculum level is an important factor for the production of amylase. During glucoamylase production with A. niger NCIM 1248, different inoculum concentra tions were used (1, 2, 3, 4, 5%). An inoculums level of 4% produced maximum glucoamylase (198.4 IU/gds at 96 h of fermentation), with no increase in enzyme production at any higher concentration of inoculum (Selvakumar et al., 1998). In the case of A. oryz ae TISTR 3605 with cassava bagasse and biosludge as culture medium, an inoculum level of 3x10 7 amylase activity with 60% initial moisture content and 35C over 48 hours of incubation. The growth curve for this s train of A. oryzae showed an exponential phase of microbial growth during the first 48 hours of incubation followed by a decreasing period after reaching the optimum ( Pengthamkeerati et al., 2012). This same behavior was observed by Dey & Banerjee (2012 ), who amylase activity of 17989 U/gds at 48 hours of incubation followed by reduction o f activity beyond the 48 hours.

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124 Table 2 13 Optimum temperature for enzyme production and activity with various combinations of microorganism and substr ate S/N Microorganism Substrate Optimum Temperature for Enzyme Production and Activity ( o C) Reference Enzyme Production Enzyme Activity 1. P. fellatanum Not reported 14.5 Dos Santos et al. (2004) 2. Aspergillus oryzae Not reported 32 Dey & Banerjee, (2011) 3. A. oryzae SP 3 Not reported 30 Ellaiah et al. (2002) 4. Bacillus sp. PS 7 Spent grains 37 60 Francis et al. ( 2003); (Sodhi et al., 2005). 5. A. niger JGI 24 Not reported 22 30 Varalakshmi et al., ( 2009) 6. Thermomyces lanuginosus Not reported 50 60 Kunamneni et al., ( 2005), 7. Streptomyces erumpens MTCC 7317 Cassava fibrous residue 50 60 Ka r et al. ( 2010) Another strain of A. oryzae BS 41 produced optimum glucoamylase prod uction (980 U/ml) with an inoculum concentration of 4%. A lower concentration could be insufficient for initiating the growth and production of the enzyme during fermentation time (72 hours), and a higher concentration would result in diminished enzyme act ivity due possibly to reduced nutrient availability (Kareem et al., 2009). This inhibitory effect of high levels of inoculum was also seen by Kunamneni et al. (2005) with the highest enzyme production of 267 U/g at an inoculum level of 10% (v/w) and Kumar &

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125 Satyanarayana (2004) during fermentation of wheat bran with the thermophilic mold, Thermomucor indicae seudaticae for glucoamylase production. On the contrary in the study of Anto et al. (2006), the inoculum size showed little effect on enzyme productio n during fermentation with Aspergillus sp. HA 2, obtaining an activity of 264 0.64 U/gds when wheat bran was used as a culture medium, followed by 211.5 1.44 in rice coarse waste and 192.1 1.15 U/gds in medium waste with an optimum inoculum size of 1 0 6 spores/ml. Dey & Banerjee (2012 ) and Sodhi et al. (2005) used an inoculum concentration of 1x10 6 spores/ml for A. oryzae IFO 30103 and 1.5 x10 6 cells/ml for Bacillus sp. PS 7 respectively. 2.2.2.8 F ermentation t ime and pH Fermentation time at a given t emperature for optimum production of enzyme must also be controlled. In the case of Streptomyces erumpens the optimum time at a given temperature was 60 hours, after which enzyme yield decreased (Kar et al., 2010). In the case of Thermomyces lanuginosus the enzyme activity was observed to increase from 24 to 120 hours, reaching a maximum activity of 262 U/g. Beyond the 120 hours, activity decreased, possibly due to denaturation or loss of moisture during the process (Kunamneni et al., 2005). The pH is als o an important factor in fermentation processes, and acts in synergy with other process parameters (Kumar & Satyanarayana, 2004). This became evident in a study Pengthamkeerati et al. amylase production and activity at pH values othe r than 7.0 during fermentation with A. oryzae TISTR 3605. This pH value of 7.0 was also very close to the optimum value obtained by Varalakshmi et al. (2009) for fermentation with A. niger JG24. Some strains of Aspergillus sp. have an optimum value of pH i n the acidic range of 4 6 (Ellaiah et al 2002; Anto et al. 2006; Dey & Banerjee, 2012 )

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126 2.2.3 Bioreactor Designs in Solid State Fermentation Important factors that affect the design and scale up of bioreactors for solid state fermentation (SSF) are th e following: Thermal gradients caused by the lack of heat transfer across surfaces and poor thermal conductivity of the substrate, as well as air flow could impede removal of the metabolic heat produced during the growth of the microorganisms (Mitchell et al., 2000). Transfer of oxygen, nutrients and enzymes affect the substrate characteristics and moisture co ntent (Durand, 2003; Raghavarao e t al., 2003). Operation of mixers where microorganisms or substrates are sensitive to shear forces. Bioreactors for S SF are normally classified by their size or capacity in order to appropriate for laboratory scale, pilot plant, or industrial scale (Durand 2003). Various SSF bioreactor designs commonly used are described briefly below: 2.2.3.1 Packed bed r eactor Packed bed reactors consist of a column with a perforated base, and use forced aeration making it possible to control oxygen supply, temperature and moisture content (Pandey et al., 1994 ; Lu et al., 1998) However, the unidirectional nature of the air flow ca us es an axial temperature gradient which can cause low yields or cell death (Ashley et al., 1999 ; Mitchel et al., 2010). Based on this factor, Lu et al. (1998 ) compared the behavior of a glass packed bed bioreactor using a single layer and a multi layer conf iguration. No significant differences in temperature, residual starch concentration or citric acid production could be found among the layers. The single layer configuration showed a temperature gradient up to 2 C, and the citric acid production was lower than that in the four layer bioreactor when using a strain of Aspergillus niger and kumara as substrate. Mitchel et al. (1999) observed that the behavior of temperature in packed bed

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127 reacto rs using a single layer steadily increase d with height. Their stud y concluded that the bed height must be lower than the height at which the critical temperature is reached during fermentation. Mitchel et al. (2010) modeled the performance of a multi layer configuration with 10 levels, using the N tanks in series approa ch. Sahir et al. (2007) used different operation strategies in order to avoid temperature gradients. Best results were obtained by operating t he reactor as a continuous plug flow, multi layer packed bed, which has a lower rate of heat generation than batch operation. Silva & Yang (1998) found that the performance of a packed bed reactor with forced aeration was superior to that obtained with tray type fermentation reactor 2.2.3.2 Tray bioreactor Tray bioreactors are based on a simple design that evolved from ancient technology. It consists of flat trays that can be perforated or not where the substrate is placed in a thin layer (15 cm maximum) and kept under controlled temperature with humidified air. During fermentation of rice for amylase production wit h Aspergillus oryzae the static solid state fermentation showed that mass transfer is a problem in this type of reactor, even when the bed depth is only several centimeters (Silva & Yang, 1998), and is used in thermostatic chamb ers (Couto and Sanroman, 20 06; Durand, 2003). 2.2.3.3 Drum bioreactor In drum bioreactors, mixing can be carried out continuously or discontinuously by gently rotating the entire cylindrical drum, and controlling the temperature by circulating water in a jacket (Durand, 2003; Kalog eris et al., 1999). Other designs use ribbon shaped baffles for the mixing with rotation of a basket (Raghavarao et al., 2003). In

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128 some cases the continuous agitation can change the characteristics of the medium or cause the damage of the mycelium, and mus t be avoided (Durand, 2003). 2.2.3.4 Fluidized bed bioreactor Fluidized bed designs use forced air in order to maintain continuous agitation to avoid adhesion and aggregation of substrate particles, but the shear forces that develop can cause damage to th e mycelium and affect the final product yield Figure 2 22 is a schematic of a fluidized bed bioreactor (Couto & Sanroman, 2006). Figure 2 22 Schematic of a Fluidized Bed Bioreactor that uses Humidified Air for Pneumatic A gitation ( Photo from Couto & Sanroman, 2006).

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129 2.2.4 Submerged Fermentation (SmF) Submerged fermentation (SmF) is carried out by dispersing the substrate ingredients in an excess of water (Singhania et al., 2010). The c ontrol of temperature and pH is not as complicated as in SSF. On e of the most important variables is oxygen transfer which depends on size and shape of the reactor. The ability to transfer oxygen independently of reactor volume can be quantified by the oxygen transfer coefficient K La (Durand, 2003), which is an importa nt parameter for fermentation process design. Other factors that need to be specified are the medium composition and process conditions that are discussed below. 2.2.4.1 Temperature and pH Various types and strains of microorganism have been studied und er varied temperature and pH conditions to produce amylolytic enzymes. These organisms, each with special characteristics were assessed for enzymes that can be adapted to industrial processes, amylases with high thermo stability (Gupta et al.,2003). Some strains such as Chromohalobacter sp. TVSP 101, produces thermo stable, halo tolerant and alkali amylases. Other strains that were studied inclu de the following: Halobacillus sp. MA 2 (Prakash et al., 2009) Micrococcus sp. NS 211 with the capacity to grow in an acid medium and at high temperatures (Amoozegar et al., 2003), Microbacterium foliorum GA2, a psychrotolerant amylolitic bacteria (Sami e et al., 2012), Bacillus cereus GA6 (Kuddus et al., 2012),

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130 Pseudoalteromonas arctica (Lu et al., 2010), Bacillus sp. (Syu & Chen, 1997; Burhan et al., 2003; Goyal et al., 2005 ; Liu & Xu, 2008), and Streptomyces sp ML 12, a marine actinobacterium isola ted by Sivakumar et al., (2012). The strain Chromohalobacter sp. produced enzyme over a broad range of pH ; from 6.0 to 10 with an optimum of 9.0, at an optimum growth temperature of 37C These optima process conditi ons were also found for Bacillus sp. ANT amylase production (Burhan et al., 2003). Since Chromohalobacter sp. is quite halo tolerant, maximum enzyme production was obtained when the culture medium contained 20% NaCl or 15% KCl. Under these conditions, two isozymes were secreted when g rown with high salt contents from 10 to 30% ( Prakash et al. amylase isoforms were also found during fermentation with Aspergillus oryzae obtaining an increase in concentration of one of these isozymes (AmyB), while the other (AmyA) disappear ed through the culture (Sahnoun et al., 2012). For production of thermo stable enzymes with Micrococcus sp., the highest levels of amylase were found at 85C The microbe was capable of growing at temperatures of 110C and pH from 1.2 to 8.0 with an opti mum pH of 3.5 when starch was used as the carbon source in the culture medium (Samie et al., 2012). On the other hand, Microbacterium foliorum and Bacillus cereus isolated from soil from the Gangotri glacier in western Himalaya India produc ed maximum amylase at 20C. Both strains were pH tolerant with a maximum production of amylase at pH 9 and 10 respectively. With the strain of Bacillus subtilis amylase yield was obtained at pH 7.0 and 50C. Supplementation of the medium with calcium st imulated enzyme production and bacterial growth (Asgher et al., 2007). Konsoula & Liakopoulou Kiriakides (2007)

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131 fermented complex carbohydrates during co galactosidase, obtaining high yields of enzymes when the process was car ried out under pH 7.0 and 40C conditions. Neutral values of pH and optimum temperature of 37C were also observed as the best growth and enzyme production conditions for strains of Bacillus sp. (Syu & Chen, 1997 ; Goyal et al., 2005). 2.2.4. 2 Carbon and n itrogen Aspergillus oryzae glucosidase using different sources of carbon, such as glucose, dextrin, maltose and indigestible dextr in. When maltose or dextrin was used, the production of enzymes was higher than that obtained when glu cose was used as the carbon source. This amylase with B. subtilis when glucose was added to the culture medium along with potato starch (Asgher et al., 2007). Other authors found that for Hallobacillus sp. the maximum excretion of amylolytic enzyme was attained with dextrin (3.2 U/ml) followed by starch (2.4 U/ml). In the case of starch, the production of enzyme was found to be constitutive (Amoozegar et al., 2003). For A. oryzae, the carbon source that showed highest glucosidase and glucoamylase was the indigestible dextrin. The biomass concentration with this carbon source was 35% of that in dextrin culture, which can be useful during industrial processes by improving the viscous fermentati on broth rheology with this microorganism (Sujimoto & Shoji, 2012). Other strains of A. oryzae amylase (Kammoun et al., 2008). Maltose was found to be a good carbon source for fermentation with M. foliorum yielding an amylase production of 4137 units. But, lactose was found to be even better,

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132 producing a maximum yield at 5862 units. However, use of lactose as a carbon source for amylase production with B. cereus produced a lesser yield of 4744 units ( Kuddus et al., 2012). When starch and glycerol were used with Bacillus subtilis IMG 22, a perfect interaction between these variables was found to yield maximum enzyme activity when optimum level s of starch and glycerol were added; the latter w ith the greater effect on enzyme production (Tanyildizi et al., 2005). The use of starch for enzyme production with B. subtilis was also studied by Ko nsoula & Liakopoulou Kiriakides (2007) The authors concluded that soluble starch wa s a good carbon source for enzyme production, while sugars like glucose, maltose or lactose suppressed the production of galactosidase. The alkaline amylase producer Bacillus sp. JCM 9141 yielded maximum amylase activity of 45U amylase/ml with starch as carbon source, and 50U amylase/ml with glucose as a carbon source, showing that f or this strain, starch is not a great inducer for amylase production (Zhang et al., amylase production with Bacillus licheniformis ATCC 9945a were also reported b et al. (2011). Enzyme production was associated with cell growth for Bacillus sp. (Zhang et al., 2004). This same observation was made during fermentation w ith Bacillus subtilis JS 2004 using 1% of waste potato as a carbon source (Asgher et al., 20 07), as well as with Bacillus sp. (Goyal et al., 2005; Liu & Xu, 2008). Agroindustrial wastes like yam peel (Uguro et al., 1997), gruel (wheat grinding by product) (Kammoun et al., 2008) have also been used as carbon sources for submerged fermentation. F or industrial fermentation, the design of the medium is very important, as it affects the cost and the product yield. Sivakumar et al. (2012) used

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133 wheat bran, rice bran and tapioca as carbon sources. They showed that wheat and rice bran enhanced the produc amylase, resulting in 42.412 IU/ml, while tapioca amylase from fermentation of brewery waste water with the strain Aspergillus niger UO 1 was found to increase when s upplemented with starch and the use of casamino acids, yeast extract and peptone (Hernandez et al., 2006). Kuddus et al. (2012) evaluated nitrogen sources, such as casein, glycine, yeast extract, ammonium acetate and ammonium sulfate. Maximum production of amylase was obtained with B. cereus and ammonium acetate, or with M. foliorum and yeast extract Similar results were found with Bacillus subtilis JS 2004 when yeast extract and calcium were added amylas e production (Asgher et al., 2007). On the contrary, during fermentation with B. subtilis IMG 22 amylase production (Tanyildizi et al., 2005). It has also been reported that wheat bran when supplemented with metal ions, carbon and nitrogen sources was the best substrate (among those tested) for the production of amylase and protease (Shivakumar, 2012). 2.2.4.3 Salts The maximum production of enzyme with Halobacillus sp. was observed in a medium with 15% (w/v) NaSO 4 followed by 10% NaCl. When sodium nitrate was added to the medium, no extracellular amylase production was observed (Amoozegar et al., 2003). When potassium nitrate, citrate, potassium chloride and calcium chloride were used with Streptomyces amylase activity were sodium chloride and magnesium sulfate (Sivakumar et al., 2012).

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134 amylase with Bacillus sp. has been reported by se veral workers (Asgher et al., 2007; Konsoula and Liakopoulou Kiriakides, 2007; Kuddus et al., 2012). However, in the case with A. oryzae CBS 819.72, the presence of Ca 2+ amylase production, while magnesium and phosphate where very import ant (Kammoun et al., 2008). 2.2.5 Summary on Enzyme P roduction Production of amylolytic enzymes suitable for industrial use is very important due to the wide range of applications like detergent industry, bakery products, sugar syrups, brewery etc. Because of the differences between these processes, microorganisms isolated from sources as different as glaciers and hot springs or soil and marine sources have been studied in order to obtain enzymes with special characteristics. Studies in search of optimum co mbinations of microorganisms, substrate composition and process conditions revealed there was no one size fits all. Optimum conditions for maximum yields varied widely depending on specific strain of microorganism and substrate composition used in the ferm entation process. Submerged fermentation continues to be the most widely used method of microbial enzyme production in western countries. However, solid state fermentation has gained interest in recent years for fermentation of bulk solids. Novel bioreacto r designs are of interest for researchers because of the necessity to improve mass and heat transfer during fermentation with large amounts of substrate.

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135 CHAPTER 3 PHYSICAL PROPERTIES OF CASSAVA FLOUR MADE FROM SOLAR CONVECTION DRIED CASSAVA CHIPS 3.1 Ba ckground Cassava, Manihot esculenta Crantz sometimes also referred to as Mandioca, Manioc, Tapioca or Yuca is the third largest source of carbohydrate for human food in the world ( Nassar and Ortiz, 2010; Claude and Denis, 1990 ; Phillips, 1982 ). Most of the one cassava producer both in Africa and in the world ( FAO, 2013; Phillips et al., 2004 ; Hillocks, 2002 ). In 2008, Nigeria produced 44.582 million tons of cassava valu ed at over $ 3.312 x 10 9 (Food and Agriculture Organization of the United Nations (FAO), 2010). Haiti is known as a major cassava producer in the South America and Caribbean region (Henry and Hershey, 2002]. The 435,000 tons of cassava produced by Haiti in 2008 ranked her 5 th t); Paraguay (2.219 million t); Columbia (1.803 million t) and Peru (1.172 million t) (FAO, 2010). These nations have been increasing their output mainly due to productivity; cash and subsistence crop profile; tolerance of poor soils and low rainfall; good resistance to pests and diseases; and more than two years harvest window. The long harvest window affords poor farmers not only some protection against famine, but the starchy r oot s underground in the field, they do not need to worry about expensive refrigeration or other food preservation techniques. Perhaps more i mportantly however, is the fact that the cassava crop can be transformed into such products as starch, flour, and ethanol; with huge potential industrial applications (Hakizimana, 2010; Widowati and Hartojo, 2010 ; Johnson et al., 2009; Eneas, 2006; Srinora kutara et al., 2006;

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136 Balagopalan et al., 1988; Berghofer and Sarhaddar, 1988; Srikanta et al., 1987; Matsumoto et al., 1982; Ueda et al., 1981; Lindeman and Rocchiccioli, 1979 ). 3.2 Objectives Constraints that limit the utilization of cassava crop include s helf life and mass. Cassava r oot s are bulky and heavy, and therefore expensive to transpo rt over long distances. The r oot s are also extremely perishable, thus must be either consumed or processed within a few days a fter harvest ( Eneas, 2006 ; Balagopalan e t al., 1988; Kuppuswamy, 1961 ). To overcome these limitations, cassava r oot s would need to be processed near the source of production using appropriate technology, and into products that are less bulky and with longer shelf life such as chips, flour, starc h and sweeteners. The objectives of the work reported in this study were to: 1. Produce dried cassava chips using a prototype solar convection dryer, 2. Produce cassava flour from grinding the chips, 3. Determine and report the following properties of chips and flo ur: a) S orption isotherms and drying curves on the chips, and b) M oisture content, particle size distribution, bulk density, solid particle density, porosity, permeability and specific surface area of the flour. 3.3 Methods and Procedures 3.3.1 Scope of Work T he scope of work undertaken in this study cons isted of a series of efforts to determine basic physical properties for cassava chips and flour. These included sorption isotherms and drying curves on the fresh cassava pulp to produce the dried chips, and moi sture content, particle size distribution, bulk density, solid particle density, porosity, permeability and specific surface area of the flour. The work was carried out in the physical properties laboratory of the Agricultural and B iological Engineering (A BE) D epartment at the University of Florida T he purpose

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137 of this work was to obtain estimates for physical properties of cassava chips and flour th at could be used for design work. 3.3.2 Fresh Cassava Root 3.3.2.1 Sample p reparation Fresh cassava r oot s we re purchased from Brooks Tropicals, Homestead, Florida. Fresh cassava chips were made by peeling the tubers and slicing them into thin chips with approximate thickness of 2 mm using a Hobart Slicer, Model 1712E (Hobart Corporation, Troy, Ohio ) The sliced fresh chips at about 60 % (wb) moisture were spread onto screen mesh trays in a single layer, and placed into a prototype solar convection dryer ; shown in Figure 3 1 designed and fabricated at the Unive rsity of Florida (Schiavone et al., 2013). The chips w ere dried to approximately 10% (db) moisture, and stored in air tight containers until ready for use. Figure 3 1. Prototype Solar Convection Dryer used for Drying Cassava Chip s. Photo courtesy of A. A. Teixeira.

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138 3.3.2.2 Moisture content and water acti vity for sorption isotherms Moisture content was determined by the oven drying method (AOAC, 2000) Pre weighed samples of freshly ground cassava root were placed in a drying oven for 72 hours at 105 o C, after which they were weighed once again to determi ne total moisture removed. Water activity was determined with a water activity meter (AquaLab series 3 TE, Decagon Devices, Inc., Pullman, WA ). A 10 gram sample of freshly ground cassava pulp was placed in the sample cup of the meter after the meter had be en powered up at least 30 min previously, as per manufacturer's directions. After several minutes, when the meter sensed that equilibrium relative humidity had been reached, the values for water activity and the relevant temperature were displayed and then manually recorded. In order to establish the sorption isotherm, a series of pre weighed 10 gr am samples of fresh cassava root were placed in sample cups used for water activity readings. Two sample cups were placed in each of five desic c ator jars maintai ned at different equilibrium relative humidity as shown in Table 3 1. At pre determined time intervals, the cups were removed from jars and weighed. When no further weight change occurred, the samples were removed from their jars for water activity measure ments, and placed in the drying oven to determine their moisture content. The isotherm was established by plotting a graph of moisture content verses water activity 3.3.2.3 Drying curve The thinly sliced cassava chips were dried in a prototype solar conve ction dryer designed and fabricated at the University of Florida (Schiavone et al., 2013). Weigh boa ts containing approximately 100 grams of pre weighed sliced fresh chips were placed onto one of the screen mesh trays in the dryer cabinet to dry over time. The

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139 Table 3 1. Saturated Salt Solutions and Associated Equilibrium Relative Humidity when placed in Desiccator Jars. Saturated Salt Solution Equilibrium Relative Humidity at 22 o C (%) Magnesium Chloride 32.8 Sodium Bromide 57.6 Sodium Chloride 7 5.3 Potassium Bromide 81.8 Potassium Sulfate 97.3 initial moisture content of these chips was determined using the oven dry method (AOAC, 2000) with sister samples of chips. At pre determined time intervals, the weigh boats were removed from the dryer for weight measurement, and returned to the dryer. The drying proce ss was terminated after about 28 hours when no further weight loss was observed. The moisture content at each pre determined time interval was calculated by knowing the initial weight of t he sample and the weight reached at that time interval. The drying curve was established by plotting a graph of moisture content (dry basis) verses time. This work was repeated three times to obtain three replicate drying curves. 3.3.3 Cassava Flour 3.3.3. 1 Sample preparation Samples of cassava flour were made by blending dried chips (10% moi sture db) with Osterizer Designer Cycle Blender (Sunbean Corporation, Fort Lauderdale, Florida) until particle sizes less than 0.5 mm could be obtained. Optimum blende r

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140 operating conditions were chosen by comparing particle size distribution profiles obtained from different combinations of grinding time and blender speed on chips made from two different varieties of cassava (Algodon and Valencia) and two different chip thicknesses (2mm and 4mm). Particle size distributions were obtained using a vibrating stacked sieving machine (Octagon 2000, Endicotts Ltd. London, England). 3.3.3.2 Physical properties Bulk density, solid particle density and porosity were determined by use of a multi pycnometer (Quantachrome, Boynton Beach, FL). Permeability was measured using an air flow apparatus fabricated in shop facilities of the Agricultural and Biological Engineering Department at the University of Florida. This apparatus is illu strated in Figure 3 2. It provided a means by which a sample of cassava flour contained in a Figure 3 2. Air Flow Apparatus for Determination of Permeability cylindrical sample holder of known dimensions could be placed in the path of airflow at known flow rate. By measuring the pressure drop across the sample while air was flowing, the permeability of the sample could be determined as a function of this pressure drop and air flow rate. The cylindrical sample holder was 10cm in length and 2cm in diamete r. The pressure drop across the sample was measured with an inclined

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141 oil manometer, and flow rate was determined with use of an annubar flow measuring device (F.W. Dwyer MFG. Co., Michigan City, Ind iana ) for measurement of flow rate. With the airflow on, t he pressure drop across the annubar was recorded with the same oil manometer to calculate air flow rate, and the pressure drop across the sample of flour was determined. The permeability (K) was calculated from Darcy's equation ( eq. 3 1). Once the permeabi lity was known, the specific surface area of the flour could be estimated as a function of permeability and porosity using the Carmen Kozeny equation ( eq. 3 2). K = (q L) / (A P s ) ( eq. 3 1) K = P 3 / [ 5 S 2 (1 P) 2 ] ( eq. 3 2) W here K = material permeability (m 2 ), q = air flow rate (m 3 /sec), = viscosity of air (1.82E 5 Pa.s), L = length of cylindrical sample holder (m), A = cross sectional area of cylindrical sample holder (m 2 ), P s = pressure across sample (Pa), P = por osity (dimensionless), and S = specific surface area (m 2 /m 3 ). 3.4 Results 3.4.1 Sorption Isotherm and Drying Curves for Fresh Cassava Root Pulp The sorption isotherm for the fresh cassava root pulp is shown in Figure 3 3 The profile is typical of most so rption isotherms, showing that moisture content decreases rapidly with decreasing water activity at relatively high water activity, but slows down dramatically at water activity less than 0.6. A water activity o f 0.6 is well below the water activity at whi ch most yeast and bacterial activity could occur ( Isengard et al. 2011 ). The moisture content at water activity of 0.6 (approximately 10% db) was chosen as target end point for the drying of fresh cassava slices into dried chips.

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142 The three replicate dryi ng curves shown in Figure 3 4 suggest that the target moisture level of 1 0% db could be reached within 25 28 hours of residence time in the solar convection dryer. This was well within the 48 hours actually used for drying the cassava chips in this study. Figure 3 3. Sorption Isotherm of Fresh Cassava Root Pulp Slices for Drying into Chips (Error bars at two standard deviations). 3.4.2 Physical Properties of Cassava Flour Particle size distributions in samples of cassava flour made from grinding dry cass ava chips under various blender operating conditions are shown in Figure 3 5 The particle size distribution profiles shown in the figure were obtained from different 5 7 9 11 13 15 17 19 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Moisture Content [% Dry Basis] Water Activity [Aw]

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143 Figure 3 4. Replicate Drying Curves for Drying Cassava Slices into Chips. Figure 3 5 Particle Size Distribution in Cassava Flour from Grinding Dry C assava Chips under various Blender Operating Conditions. 0 20 40 60 80 100 120 140 0 5 10 15 20 25 30 Moisture Content [% Dry Basis] Time [Hours] Run # 1 Run # 2 Run # 3

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144 combinations of grinding time and blender speed (high or low) on chips made from two different varieties of cassava, Algodon (A) and Valencia (V) and two different chip thicknesses, small (2mm) and large (4mm) Small chips from Valencia r oot s were chosen for the study. The profiles for these small chips for two different blending times at slow spee d appear at the far right side of the figure. The profile obtained from four minutes at low speed produced a sample in which more than 60% of the sample weight consisted of the desired particle size (less than 0.5 mm). These were the grinding conditions ch osen for producing the samples of flour used in this study. Physical properties of moisture content, bulk density, solid particle density, porosity, permeability and specific surface area are summarized in Table 3 2, along with statistics showing number of replicate measurements and minimum and maximum values found among the replicates. Table 3 2. Physical properties of cassava flour with particle size 0.5 mm. Physical Property Mean Value Number of Replicates Minimum Value Maximum Value Moisture Con tent (%db) 12.35 2 12.34 12.37 Bulk Density (kg/m 3 ) 550 2 530 570 Solid Particle Density (kg/m 3 ) 1,480 2 1,442 1,517 Porosity (Dimensionless) 0.636 2 0.630 0.642 Permeability (m 2 ) 1.0 x 10 6 2 1 x 10 6 1 x 10 6 Specific Sur face Area (m 2 /m 3 ) 367 2 367 367

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145 3.5 Summary The m easurements of drying characteristics and physical properties of cassava chips and flour were carried out and reported in this study. These included a sorption isotherm and drying curve on the chips, and moisture content, particle size distribution, bulk density, solid particle density, porosity, permeability and specific surface area of the flour. A prototype solar convection dryer was used to produce the dried cassava chips which were ground into fl our as a means of pr eservation and adding value to the cassava crop. Data on physical properties reported in this study will be most useful to carry out engineering design of storage, handling, and processing systems for the flour thus produced.

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146 CHAPTER 4 SYNERGISTIC ENZYMATIC HYDROLYSIS OF CASSAVA STARCH 4.1 Background Sugar generally refers to a class of chemically related sweet flavored substances, most of which are used as food. They are carbohydrates, composed of carbon, hydrogen and ox ygen There are different types of sugar which include monosaccharides (fructose, galactose, glucose); disaccharides (lactose, maltose, sucrose); and many others (Wik ipedia, 2013 a ; Damodaram et al., 2008). Sucrose can be extracted from plants such as suga rcane and sugar beet while fructose and glucose may be derived from starch by hydrolysis reactions. Global sugar production and consumption has been rising in recent years. In 2009/2010, 2011/2012, and 2012/2013, global sugar production (and consumption) w ere respectively 153.6 x 10 9 kg (153.7 x 10 9 kg); 168.2 x 10 9 kg (160.2 x 10 9 kg); and 174.0 x 10 9 kg (163.0 x 10 9 kg) (USDA, 2013). In 1999, per capita sugar consumption in the world was 24 kg, and this is estimated to reach 25.1 kg by 2015 (FAO, 2002). I n the United States, per capita consumption of refined sugar in 1972 was 46.2 kg. This rose to 71.6 kg in 1999; and 48.8% of the total was corn derived sweeteners (Huntrods et al., 2012; Coulston and Johnson, 2002). Starch is fundamental raw mater ial for production of glucose and fructose sweeteners. These sweeteners are important commercial commodities used extensively in bakery, beverage, confectionery food, pharmaceutical, and allied industries. However, a great proportion of starch used in the world today comes fr om corn/maize ( Johnson et al., 2 005; De Braganca and Fowler, 2004; De Baere, 1999 ). Similarly, m ost sweeteners used are produced from corn starch ( BeMiller, 2009; Gordon, 1999;

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147 Swinkles, 1985 ). The conventional method of glucose sweete ner production requires first extraction of starch from its raw material, followed by two separate unit operations: Liquefaction and Saccharification (Johnson et al., 2009; Berghofer and Sarhaddar, 1988; Solomon, 1978; Aschengreen, 1975 ; Pomeranz et al., 1 964 ). This rather round about procedures impose constr aints and implications in terms of energy; enzyme; labor; equipment; t echnology; time and associated costs. It would be more efficient to hydrolyze the starch in its native raw material and to conduct l iquefaction and saccharification operations simultaneously in the same reactor if possible. It has been stated that sweeteners can be made from any starch and that such sweeteners are essentially identical regardless of which starch is used as the raw ma terial (BeMiller, 2002; Aschengreen et al., 1979). It is also reported that fungal amylases can be used to liquefy starch irrespective of the source of the starch (Witt, 1985). Cassava r oot is said to be richly endowed with good qua lity starch to the exten t of 30 Balagopalan et al., 1988). In addition, Cassava starch has been reported to exhibit vital benefits and advantages over other sources of starch such as wheat, corn, and waxy maize (Central Tuber Crops Research Institute 2010; Juszczak et al., 2003; Larotonda, et al., 2003; Balagopalan, 2002; Lopez Ulibarri and Hall, 1997; Balagopalan et al., 1988; Franco et al., 1988; Vijayagopal et al., 1988; Cereda and Wosiacki, 1985; Moor thy, 1985; Swinkels, 1985) Therefore cassava crop can be an alternative source of starch for production of sweeteners. Attempts have been made to obtain reducing or fermentable sugars from starch sources other than corn. Researchers have used cereal grai ns, seeds, stems and

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148 tubers in various experiments. Nakamura and Park (1975) characterized some physico chemical properties of cassava starch. Their work demonstrated that fermented cassava starch is more soluble in water, and also less viscous when heated than unfermented starch. Kearsley and Nketsia Tabiri (1979) conducted hydrolysis experiments with commercial starches as well as whole grains, tubers and roots of rice, wheat, potato, and cassava. They concluded that it was possible to produce glucose sy rups from a wide variety of starch containing crops. Aschengreen et al., ( 1979 ) liquefied, saccharified and isomerized starch es from sources other than maize including potato and tapioca Greenfield and Brooks (1982) investigated the effect of size reduct ion operations on liquefaction, saccharification and fermentation of cassava roots and stems to ethanol. They discovered that the resulting bimodal particle size which were readily hydrolysable. Hoehn et al., (1983), produced high fructose syrups from Jerusalem artichoke tubers. Monma et al. (1989) successfully digested starches from potato, rice and sago. Gorinstein and Lii (1992) subjected amaranth, cassava and potato star ches to enzymatic hydrolysis. Their report revealed that cassava starch is less resistant to thermostable alpha amylase than amaranth and potato starches. These cited works clearly demonstrated that reducing and fermentable sugars can be produced from vari ous starch sources other than corn. However, a major limitation of conventional production of glucose sweetener is the two separate operations of liquefaction and saccharification. Liquefaction is the first stage whereby starch is gelatinized by heat treat ment and thermostable alpha amylase partially hydrolyses the starch to maltodextrins. Saccharification commences in the

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149 second stage where the partially hydrolyzed starch is converted to glucose by the action of glucoamylase. This protocol requires more pr ocess vessels, piping, and associated equipment; conditioning for enzyme systems; and gelatinization is energy intensive. It would be advantageous to deploy alpha amylase and glucoamylase simultaneously and carry out both liquefaction and saccharification processes inside the same reactor vessel as a single unit operation. This option is simple, and more time, energy, equipment and labor efficient. It is termed synergism and has been studied by several authors. Gauss et al., (1976) patented a process for al cohol production by simultaneous saccharification and fermentation of cellulosic materials with cellulase enzyme and alcohol producing microorganism. Wankhede and Ramteke (1982) studied the synergistic digestibility of native starches by various enzymes. S ubstrates studied included Ragi ( Eleusine coracana ), Foxtail millet ( Sataria italic a), Jowar ( Sorghum vulgare ) and Rice ( Oryza sativa ). The study revealed that where one enzyme system hydrolyzed native starch by 35.58%, synergistic action hydrolyzed 71.20% The works of Fujii et al., (1981), Fujii and Kawamura (1985), and Fujii et al., ( 1988 ) illuminated synergistic hydrolysis mechanism of alpha amylase and glucoamylase. According to their model, alpha amylase catalyzes endo wise random cleavage of large mo lecules on the surface of starch granules. The splitting action produces maltodextrins and non reducing sugars that blanket the granule, inhibiting alpha amylase from further attacks on the substrate. At this juncture, glucoamylase peels off the blanket of maltodextrins, hydrolyzing them to glucose and thereby exposing interior glycosidic bonds of the substrate to new surface attacks by alpha amylase. After the molecular weight of the

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150 substrate decreases to about 5000, the action of alpha amylase can be neg lected and the rate of formation of glucose is governed by the kinetics of glucoamylase (Fujii and Kawamura, 1985). The cooperative action between alpha amylase and glucoamylase constitutes synergism and results in enhanced hydrolysis yields and increased enzyme activity, perhaps making the energy intensive gelatinization pretreatment process unnecessary. Franco et al., (1989) demonstrated increased levels of reducing sugars (glucose and maltose) in hydrolysis systems subjected to synergistic actions of al pha amylase and glucoamylase. Rattanachomsri et al., (2009) reported increased fermentable sugars (glucose and xylose) and alcohol production from simultaneous multi enzyme saccharification and yeast fermentation. The process was said to be energy efficien t by obviating the pre gelatinization step. Arasaratnam and Balasubramaniam (1993), observed that synergistic h ydrolysis of dry milled starch was more efficient than wet milled starch; and that optimum ratio of glucoamylase to alpha amylase was 1.8 AGU (A myloglucosidase Unit) /1.0 KNU (Kilo Novo Unit) reported that the synergistic effect of alpha amylase and glucoamylase hydrolysis of starch is a function of many variables including substrate composition, concentration, and degree of polymerization, as well as reaction duration. The synergistic technique was applied to production of lactic acid. Linko and Javanainen (1996) claimed 98% yield of l actic acid when 130 g/L starch was s imultaneous ly liquefied, saccharified and fermented. Lactic acid concentration of 162 g/L was reported Similarly, Anuadha et al. (1999), reported higher yields of 1.2 1 g/L.hour lactic acid production for simultaneous sy stems over conventional two step processes. Liakopoulou Kyriakides et al. (2001),

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151 investigated the synergistic effects of different combinations of alpha amylase and glucoamylase from various sources. Their report showed that the combination of Bacillus li cheniformis alpha amylase and Rhizopus mold glucoamylase gave the highest substrate conversion of 76% at 50 o C; and that combination of Bacillus licheniformis alpha amylase and Aspergillus niger glucoamylase enzymes retained initial activity after incuba tion at 60 o C. Another important constraint to conventional glucose sweetener production is the time, energy, and labor intensive starch extraction process. Most of the work reported in literature on glucose production used model systems of soluble or com mercial starches. Very few authors have tried the possibility of using direct conversion technique. Direct conversion procedure involves hydrolyzing native starch in raw materials with minimal value adding processing history. Lages and Tannenbaum ( 1978 ), c onducted laboratory scale batch operations for the production of glucose from tapioca meal (also called farinha de mandioca). Tapioca meal is the flour produced after rasped peeled cassava roots are toasted and dried. The authors reported virtual 100% conv ersion of the meal after 24 36 h ou rs of hydrolysis. This compares to 12 24 h ou rs required for 100% conversion of pure cassava starch. Menezes et al., (1978) hydrolyzed cassava root slurry with alpha amylase and glucoamylase in conventional two stage h ydrolysis procedure. However, fungal cellulase was synergistically added at the saccharification stage. The authors reported not only increased rate of sugar formation and starch conversion, but also improved rheological properties. Kea rsley and Nketsia Ta biri ( 1979 ), performed direct hydrolysis of native starches in grains, roots and tubers with alpha and beta amylase enzymes. The results for alpha amylase experiments shown

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152 here in Table 4 1 indicated that unprocessed native starches compared favorably wi th commercial starches, especially in total starch conversion. Table 4 1. Some Results of Alpha Amylase Hydrolysis of Starch from Sources other than corn (Kearsley and Nketsia Tabiri, 1979) Starch Source Dextrose Equivalent (DE) of Hydrolysate at 24 H ou r s Percent Starch Conversion at 24 H ou rs Commercial Starches Maize 43 98 Rice 41 96 Wheat 44 94 Potato 40 94 Whole Grain A ( Milled Large Particles) Maize 22 92 Rice 36 94 Wheat 33 100 Whole Grain B ( Milled Small Particles) Maize 28 89 Ric e 41 93 Wheat 38 99 Whole Tubers and Roots (Minced Particles) Potato 30 86 Cassava 38 100 Sreekantiah and Rao (1980), used mixed culture fermentation, enzyme saccharification, and acid liquefaction to produce ethyl alcohol from fresh potatoes, dehy drated tapioca, and dehydrated sweet potatoes. They reported corresponding fermentation efficiencies of 68%, 81%, and 75% for the respective substrates. Maeda et

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153 al. (1982), subjected dried cassava chips slurry to alpha amylase liquefaction. After a clarif ication and centrifugation step aided by various enzymes, the supernatant fluid was continuously saccharified by immobilized glucoamylase gel. The authors reported glucose content of resulting product to be 94.4%. Srikanta et al. (1987), reported product ion of ethanol from fresh cassava roots, flour and starch. After liquefaction of 20% solids content substrates with alpha amylase, saccharification was executed with moldy bran or commercial glucoamylase before fermentation with yeast inoculation. Their re sult indicated that after 4 hou rs of saccharification at 60 o C, the hydrolysis scheme yielded 9 10% reducing sugars which on fermentation, generated alcohol fermentation efficiencies of 95 to 98% among the different substrates. Berghofer and Sarhaddar ( 1988 ), investigated glucose production directly from fresh and dried cassava roots. The authors reported conversion yield of 88 89% of the overall starch content of raw material (fresh unpeeled tuber) compared to 82 87% conversion through conventiona l starch extraction processes. Ghildyal et al., (1989), performed comparative economic analysis of the production of high fructose syrup from cassava chips and extracted cassava starch. They reported that the unit cost for production of 10 t of high fruct ose syrup per day was US $ 0.51/kg and US $ 0.46/kg respectively for cassava chips and extracted cassava starch. They noted that the gain in lower expenses on cassava chips raw material as compared to extracted cassava starch was upset by higher capital in vestment on plant and machinery; as well as higher operating expenses on process steam, activated carbon, and purification requirements for cassava chips products. The authors concluded that direct utilization of cassava chips does not offer any economic

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154 a dvantage. Johnson et al., (2009), applied the direct conversion technique to production of glucose and high fructose syrup from cassava roots and sweet potato tubers. They used various enzymes and reported glucose yields of 22 25% and 14.0 15.7% for ca ssava roots and sweet potato tubers respectively. Fructose yields were reported to be 8.36 9.78% for cassava, and 5.2 6.0% for sweet potato. Total starch conversion to glucose was reported to be higher with cassava at 95 98%; compared to 88 92% wit h sweet potato. However, the authors used the conventional two step procedure of liquefaction followed by saccharification, except that one of the enzymes used; Stargen TM 001, had both alpha amylase and glucoamylase activities. From extensive literature search, it is observed that there is dearth of information on direct conversion technique on the one hand; and on synergistic procedure on the other hand, especially where glucose sweetener is the desired end product. Most of the reported work was done for the production of alcohol or lactic acid, where reducing sugar such as glucose is a transition item to be fermented to the desired end product. 4.2 Objectives Based on the foregoing, the objectives of the work undertaken in this study were to: 1. C ombine b oth direct conversion and synergistic techniques in the production of glucose sweetener, using cassava crop as the raw materials source. 2. Demonstrate the conversion of starch to glucose sweetener from synergistic hydrolysis with commercial enzymes on the fo llowing substrates: a) Commercially available refined cassava starch. b) Flour produced from solar convection dried cassava chips. c) Freshly ground cassava root pulp

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155 3. Estimate reaction kinetics parameters such as first order rate constant and Arrhenius activation energy f r om enzyme hydrolysis of each substrate at two different temperatures. 4.3 Materials and Methods 4.3.1 Enzymes The enzymes used in this study are presented in Table 4 2. Table 4 2. Statistics of Enzymes used in the Research Work Enzyme Manufacture r Product Name Type of Enzyme Source of Enzyme Enzyme Activity Bio Cat Inc. Fungal Amylase Alpha amylase Fungal: Aspergillus oryzae 100,000 SKB/g. One SKB Unit is the number of grams of soluble starch dextrinized, in the presence of excess beta amylase per hour under the assay conditions Glucoamylase Glucoamylase Fungal: Aspergillus nige r 1000 AG/g. One AG Unit is the amount of glucoamylase that will liberate 0.1 mol of p nitrophenol per minute from PNPG solution under the assay condition s BioSun Alpha Amylase Alpha Amylase Bacterial: Bacillus subtilis 5 x 10 6 BAAU/g. One BAAU Unit is amount of enzyme that breaks down 5.26 mg of Lintner starch per hour under the assay conditions Starch Clear ase Glucoamylase Fungal: Aspergillus ni ge r 200 GAU/g One GAU Unit is the amount of enzyme that breaks down 0.1 mole of p nitrophenol per minute from a substrate of PNPG under the assay conditions. DSM Validase BAA 1000L Alpha Amylase Bacterial: Bacillus subtilis 1 x 10 6 MWU/g. One MWU Unit is the amount of enzyme activity that will dextrinize 1 mg of soluble starch to a defined blue color in thirty minutes under the assay conditions Validase GA 400L Glucoamylase Fungal: Aspergillus nige r 400 GAU/mL. One GAU Unit is the amount of e nzyme ac tivity that will liberate 1 g of reducing sugar as D glucose per hour under the assay conditions

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156 Table 4 2 Continue d. Statistics of Enzymes used in the Research Work Enzyme Manufacturer Product Name Type of Enzyme Source of Enzyme En zyme Activity Genencor Spezyme Fred Alpha amylase Bacterial: Bacillus licheniformis 17400 LU/g. One LU Unit is the measure of the digestion time required to produce a color change with iodine solution, indicating a definite stage of dextrinization of star ch substrate under the assay conditions Optidex L 400 Glucoamylase Fungal: Aspergillus niger 350 GAU/g. One GAU Unit is the amount of enzyme that will liberate 1 g of reducing sugar as glucose from soluble starch substrate per hour under the assay condit ions. Gensweet SGI Glucose Isomerase Bacterial: Streptomyces rubiginosus 3000 GIU/g. One GIU Unit is the amount of enzyme that will convert one micromole of glucose to fructose per minute under the assay conditions. Novozymes Liquozyme Supra Alpha a mylase Bacterial: Bacillus licheniformis 135 KNU/g Dextrozyme DX Glucoamylase & Pullulanase Fungal: Aspergillus niger and Bacterial: Bacillus subtilis 170 AGU/g & 340 NPUN/g Sweetzyme IT Glucose Isomerase Bacterial: Streptomyces murinus 400 IGIU D/g These enzymes were graciously donated by their respective manufacturers. After initial trials of preliminary studies, Bacterial alpha amylase (Activity: 5 x 10 6 BAAU/g) and fungal glucoamylase (Activity: 1000 AG/g) were used synergistically in all experim ents. 4.3.2 Substrates Three types of cassava substrates: Root, Flour and Starch were used in all experiments.

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157 4.3.2.1 Roots Fresh roots were purchased commercially from Brooks Tropicals, Homestead, Florida. The roots were coated with food wax and supplie d refrigerated in 18 kg boxes. Each consignment or batch was promptly received and maintained under refrigeration (< 10 o C) until utilized. 4.3.2.2 Flour produced in our laboratories from solar dried chips of the fresh roots. 4.3.2.3 Starch Cassava starch was purchased from commercial source; My Spice Sage, Yonkers, New York. The starch was supplied in 25 kg packaging and was stored under o C, 38% RH). 4.3.3 Sample Preparation 4.3.3.1 Root Fresh cassava roots were retrieved from refrigeration, plac ed on a cutting board (Figure 4 1A), and peeled with a kitchen knife (Figure 4 1B). The peeling process involved carefully removing the peel (pellicle (periderm or epidermis) and cortex) from the parenchymatous tissue. The exposed parenchyma was cut into slices of about 10 cm long x 3 cm wide x 3 cm deep dime nsions. Subsequently, the slices were rasped into shreds with a kitchen grater (Figure 4 1C). The shreds were pulverized into smooth paste with a blender. Two types of blender were used. Figure 4 1D: Osterizer Designer Cycle Blender (Sunbean Corporation, F ort Lau derdale, Florida), and Figure 4 1E: SmartPower Cuisinart Blender, Model CPB 300 (Intertek Group, London, England). One

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158 blender was used when the other required a period of cooling off. The paste was constituted with distilled water to obtain a 10% ( w/w) suspension. A B C D E F G H I Figure 4 1. Size Reduction Devices used in Sample Preparation s A) Cutting Board B) Knives C) Grater D) Blender 1 E) Blender 2 F) Slice O Matic G) Hobart Slicer H) Solar Convection Dryer I) Vibrat ing Sieving Machine All photos courtesy of the author.

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159 4.3.3.2 Flour Depending on size of root, either a kitchen knife (Figure 4 1B) or one of two slicers: Slice O Matic Slicer (TeleBrands, Fairfield, New Jersey; Figure 4 1F) or Hobart Slicer, Model 171 2E (Hobart Co rporation, Troy, Ohio; Figure 4 1G) was used for slicing. Peeled roots were sliced into approximately circular chips of 0.5 cm thickness. The sliced chips were subsequently dried to less than 10% moisture content dry basis using a solar natura l convection dryer developed at the University of Florida (S chiavone et al., 2013; Figure 4 1H). It took about 1.5 to 3 days to achieve required drying, depending on loading density, solar radiation and other weather and operating conditions. Dried chips w Sieve No. 325) with the aid of a vibrating stacked sieving machine: Octagon 2000; Serial No. OCT 2000/2 340 96 (Endocotts Lim ited, London, England; Figure 4 1I). Flour produce d was constituted with distilled water to achieve a 10% (w/w) suspension. 4.3.3.3 Starch The commercial cassava starch purchased was constituted with distilled water to achieve a 10% (w/w) suspension. 4.3.4 Reactor The batch reactors used for hydrolysi s ex periments are shown in Figure 4 2A. They were 1000 mL capacity Pyrex Griffin Beakers (Fisher Scientific Inc., Waltham Massachusetts). The beakers were mounted in a precision microprocessor controlled water bath, Model 285 (Thermo Scientific Inc., Walth am Massachusetts). The system was coupled to a Phipps and Bird Stirrer (Phipps and Bird Inc., Richmond, Virginia). A typical setup of an experimental run of the reactors, water bath and s tirrer is presented in Figure 4 2B.

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1 60 A B Figure 4 2. React or Devices and Systems for Hydrolysis Experiments A) Reactor Beakers for Hydrolysis Experiments B) Typical Setup of a Test Run System with Reactors, Water Bath and Stirrer Photos courtesy of the author. 4.3.5 Analytical Methods 4.3.5.1 Proximate composit ion, starch, and sugar analysis Pro ximate composition, starch, and sugar profile analyses were conducted by ABC Research Laboratories, Gainesville, Florida. Samples of cassava root, flour from solar dried chips, and commercial starch were delivered to ABC Research Laboratories sealed in transparent plastic sachets and bowls. Aliquots of selected hydrolysate samples were delivered in sanitary laboratory vials under refrigerated conditions. Reports from ABC Research Laboratories indicated that the f ollowing a ssay methods (Table 4 3) were used, with modifications where appropriate.

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161 Table 4 3. Assay Methods us ed by ABC Research Labor atories for Analysis of Samples S/N Component Assay Method 1 Ash AOAC 942.05, 920.153 2 Calories By Computation 3 Carbohydrate By Computation 4 Crude Fiber AOAC 978.10 5 Fat AOAC 948.15 6 Moisture AOAC Moisture 7 Protein AOAC 981.10 8 Starch AOAC 996.11 9 Total Pectin JBT 6 th Edition # 23; AES Bulletin 570 1 Sugars 10 Fructose AOAC 977.20 MOD 11 Glucose AOAC 977.20 M OD 12 Lactose AOAC 977.20 MOD 13 Maltose AOAC 977.20 MOD 14 Sucrose AOAC 977.20 MOD 4.3.5.2 Glucose Analysis Glucose is a monosaccharide carbohydrate because it cannot be broken down to any simpler carbohydrate molecule via hydrolysis. Therefore, gl ucose like any other monosaccharide is called a simple sugar. Glucose is also classified as an aldose. An aldose is a monosaccharide whose carbonyl function is an aldehyde (known as the aldehydo group). A second group of monosaccharides are called ketoses. A ketose is a monosaccharide whose carbonyl function is a ketone (the keto group). Oxidation is the loss of electrons while reduction is the gain of electrons. In a chemical reaction, aldoses can readily give up electrons to an oxidizing agent which accep ts electrons from the aldose. In such a reaction, the oxidizing agent is reduced and the aldose is oxidized by acting as reducing agent. Aldoses are therefore referred to as reducing sugars.

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162 However, under alkaline conditions, ketoses are isomerized to al doses and can thus act as reducing sugars (BeMiller and Huber, 2008). A reducing sugar may therefore be defined as a sugar that contains the free aldehydo carbonyl group or is capable of forming such a free group. Examples of reducing sugars a re: arabinose xylose (pentoses); galactose, glucose, mannose (h exoses). Pentose s have five carbon atoms while h substituent on the anomeric hydroxyl group of a sugar prevents the sugar from undergoing the equilibrium between ring forms and the open Gregory, 2012; Personal Communication). Consequently, such a sugar does not have a free or reducing end; and so is not reactive. Sucrose is not a reducing sugar because sucrose does n ot have a free carbonyl group or reducing end. Both anomeric carbons on sucrose are in glycosidic linkage. 4.3.5.3 Determination of Reducing Sugar One analytical technique used to determine reducing sugars is the 3,5 dinitrosalicylic acid (DNS) method. It was developed by James Sumner and coworker in 1921 to estimate reducing sugars in diabetic patients (Sumner, 1924; Sumner and Graham, 1921). The DNS method tests for the presence of free carbonyl (aldehydo) group in a sample. This is achieved by the oxidat ion/reduction reaction pathway. When 3,5 dinitrosalicylic acid is reacted with a reducing sugar, aldehydo group is oxidized while 3.5 dinitrosalicylic acid is reduced. The reaction is such that the nitrate group (NO 2 ) in 3,5 dinitrosalicylic acid is reduce d to the amino group (NH 2 ). The reduction product is called 3 amino 5 nitrosalicylic acid Figure 4 3 represents classica l stoichiometry of the reaction scheme. 3 amino 5 nitrosalicylic acid is a colored aromatic

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163 compound that absorbs light strongly at 540 nm. The absorbance can be read with a spectrophotometer and used to estimate the quantity of reducing sugar in a sample. + C 6 H 12 O 6 C 7 H 4 O 7 C 7 H 6 N 2 O 5 1 mole Glucose 1 mole 3,5 Dinitrosalicylic acid 1 mole 3 Amino 5 nitrosalicylic acid 180 g 228 g 198 g Fi gure 4 3. Stoichiometry for the Detection of Reducing Sugars as Glucose through Reactions with 3,5 dinitrosalicylic acid (DNS) Reagents The DNS technique has been adopted, modified, adapted and utilized in various materials, products, and applications. Sumner (1935) applied it to the determination of saccharase activity. Bernfeld (1955) suggested the technique be used to estimate alpha and beta amylases activities In 1959, Gail Miller modified the reagent to minimize the loss of su gar in the assay (Miller, 1959); and in 1973 Lindsay applied the assay to the measurement of reducing sugars in potatoes (Lindsay, 1973 ) In the work presented here, the DNS Reagent was composed as follows (Lindsay, 19 73; Bernfeld, 1955) : 1 g of 3,5 Dinitrosalicylic acid 20 cm 3 of 2M Sodium Hydroxide [2M = Mix 2 g NoaH with 25 cm 3 distilled water] 50 cm 3 of Distilled water 30 g of Sodium Potassium Tartrate Tetrahydrate (Rochelle Salt) Add distilled water to bring volume to 100 cm 3

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164 In the assay for reducing sugars as glucose, 1 mL of relevant hydrolysate or control sample (starch, flour or root) was reacted with 2 mL of DNS reagent in a 10 mL test tube. The test tube containing the mixture was heated for 10 minut es in boiling water and then cooled to room temperature. After addition of 2 mL of distilled water, the absorbance of the reaction product was read at 570 nm with a spectrophotometer (Evolution 60, Model No. 840 189600, Thermo Fisher Scientific Inc., Walth am Massachusetts; Figure 4 4A or Milton Roy Analytical Spectrophotometer, Model No. 335402, Milton Roy Analytical Products Rochester, New York; Figure 4 4B). A blank sample was prepared in the same manner but without the hydrolysate. The blank was used to zero the spectrophotometers. The percentage of reducing sugar (as glucose) in each sample was estimated by reference t o a calibration curve (Figure 4 5); established with reagent grade D Glucose A B Figure 4 4. Spectrophotometers used for Measureme nt of Absorbance in Samples Reacted with DNS Reagent in Order to Determine Reducing Sugars as Glucose A) Evolution 60 Spectrophotometer B) Milton Roy Spectrophotometer Photos courtesy of the author.

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165 Figure 4 5 Calibration Curve used for Estimation o f Reducing Sugars (As Glucose ) in the 3, 5 dinitrosalicylic acid (DNS) Analytical Method The Absorbance was read with Spectrophotometers at 570 nm with 1 cm path length cuvette cells. ( Graph is showing the m ean of six replications with error bars at two s tandard deviations ) 4.3.6 Experimental Procedure The experiments were conducted in batches and at two temperatures: 60 o C and 37 o C. Total reaction mass for each batch was 800 g and the repartition of components was as follows: Substrate, 10%; Enzyme, 0.015%; Distilled water made up the balance. The two enzymes applied synergistically were bacterial alpha amylase (BAA) and fungal glucoamylase (FGA) The ratio of enzyme app lication was 3 : 1 (FGA : BAA ). A typical sequence of experimental run at 60 o C required preheating the water bath to the operating temperature. The desired substrate was constituted in the reactor beaker and y = 0.5048x R = 0.9995 0 0.2 0.4 0.6 0.8 1 1.2 0 0.5 1 1.5 2 2.5 Absorbance Glucose [mg/mL]

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166 mass at time zero (T0). Mass of the be aker with sample was then measured and recorded (Ohaus Adventure Scale, Model AR 5120, Ohaus Corporation, P ine Brook, New Jersey; Figure 4 6A). A Figure 4 6. Scal es used for Mass Measurements A) Ohaus Scale; Precision at 0.1 g B) Mettler Scale; Precision at 0.001 g Photos courtesy of the author. The weighed reactor was carefully positioned in the water bath and covered with aluminum foil sheet that had a slit. The flat blade stirrer was inserted through the slit and installed so as not to touch any side or bottom of the beaker. A mercury in glass thermometer was also installed into the reactor beaker for temperature tracking. Finally, a fabricated steel mass was placed over the reactor as a ballast, to insure stability. The stirrer was then star ted to operate at 180 5 rpm. On attainment of the operating temperature, in this case 60 o C, the two enzymes were carefully measured out, introduced into the reactor simultaneously, and hydrolysis proceeded. Measurement of the mass of enzymes to three d ecimal places was achieved with Mettler Scale, Model PE 160 (Mettler T oledo, Columbus, Ohio; Figure 4 6B). Samples were withdrawn for analysis at predetermined time intervals {T1: 5 minutes (0.083 hours); T2: 10 minutes B

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167 (0.166 hours); T3: 15 minutes (0.25 hours); T4: 30 minutes (0.5 hours); T5: 45 minutes (0.75 hours); T6: 60 minutes (1.0 hour); T7: 90 minutes (1.5 hours); T8: 120 minutes (2.0 hours); T9: 180 minutes (3.0 hours); T10: 240 minutes (4.0 hours); T11: 480 minutes (8.0 hours); T12: 720 minutes ( 12.0 hours); and T13: 1440 minutes (24.0 hours)}. At each sampling time, the stirrer was stopped. The stirrer and thermometer were disengaged. The reactor beaker with substrate sample was then withdrawn from the water bath, wiped dry with a clean cloth and reweighed. The water lost by evaporation was replaced with distilled water at 60 o C. A separate beaker with distilled water was maintained in the water bath for this purpose. The replaced evaporated water was thoroughly stirred into the reaction mass wi th a metal spatula to achieve a homogeneous hydrolysate. At this juncture, all necessary measurements (pH, soluble (Eppend orf, Hamburg, Germany; Figure 4 7) into a test t ube. The test tube with aliquot Figure 4 7. Pipettes used in Sample withdrawals and Analysis Photo courtesy of the author. was capped and immediately immersed into boiling water for thirty minutes to inactivate enzyme activities. Boiled samples w ere stored under refrigeration until assayed (within

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168 48 hours) for reducing sugars as glucose using the DNS methodology. A control reactor without enzyme input was incubated and sampled in a similar way. The mass of the beaker and remaining substrate samp le was weighed again and recorded. The beaker was returned to the water bath; the stirrer, thermometer, aluminum foil sheet and steel ballast were reinstalled and timing for the experiment was continued when the reaction mass temperature reached 60 o C. Th e time spent in the sampling process was discounted. In other words, only the actual time the substrate sample was maintained at the operating temperature was computed into sampling time requirements. This sampling scheme was applied to all the sampling ti mes; including 24 hours and 96 hours for 60 o C and 37 o C experiments respectively. No other control was imposed on the system. No cofactors were added and no buffers were added. The pH of reaction mass was as obtained by mixing substrate sample with dist illed water. However, hydrolysate pH was routinely assessed at selected sampling time intervals with Orion 3 Star portable pH meter, serial no. A 11326 (Thermo Fisher Scientific Inc., W altham, Massachusetts; Figure 4 8A). Soluble solids were occasionally m onitored with Figure 4 8. Monitoring Devices for pH, and Soluble Solids A) pH Meter B) Table Top Refractometer C) Portable Refractometer Photos courtesy of the author. A B C

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169 the aid of refractometers; Abbe Mark II, Model # 10480 (Leica In c., Buffalo, New York, Figure 4 8B). The described scheme was replicated three times for each substrate at 60 o C. The same protocol was adopted at 37 o C. However, at 37 o C each experiment was run for 96 hours. Therefore additional hydrolysate samples were withdrawn for glucose assay at T14: 2880 minutes (48 hours); T15: 4320 minutes (72 hours); and T16: 5760 minutes (96 hours). Table 4 4 highlights the experimental design executed. Table 4 4. Experimental Design for Synergistic Enzymatic Hydrolysis of Different Cas sava Substrates Temperature [ o C] Substrate Replication Operating pH Substrate Concentration Enzyme Concentration Enzyme Type and Proportion 60 for 24 H ou rs Commercial Cassava Starch S1 @ 60 As obtained by mixing substrate with distilled wate r 10 % 0.015 % Fungal Glucoamylase 75 % PLUS Bacterial Alpha Amylase 25 % S2 @ 60 S3 @ 60 Flour from solar dried cassava chips F1 @ 60 F2 @ 60 F3 @ 60 Fresh Cassava Root Pulp R1 @ 60 R2 @ 60 R3 @ 60 37 for 96 H ou rs Commercial Cassava Starch S1 @ 37 As obtained by mixing substrate with distilled water 10 % 0.015 % Fungal Glucoamylase 75 % PLUS Bacterial Alpha Amylase 25 % S2 @ 37 S3 @ 37 Flour from solar dried cassava chips F1 @ 37 F2 @ 37 F3 @ 37 Fresh Cassava Root Pulp R1 @ 37 R2 @ 37 R3 @ 37

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170 All the hydrolysate remaining in the reactor at the end of each experiment: 24 hours for 60 o C and 96 hours for 37 o C were pooled together, clar ified by sedimentation, and concentrated to various soluble solids content using Precision vacuum evaporator Model Cat. No. 65486 (Precision Scientific Compa ny, Chicago, Illinois; Figure 4 9). Figure 4 9. Vacuum Evaporator Used for Glucose Syrup Concent ration Photo courtesy of the author. 4.3.7 Mass Balance Stoichiometry To determine the efficiency of hydrolysis in terms of the rate and absolute conversion of starch to glucose, the law of conservation of matter (Antoine Laurent de

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171 Lavoisier ; 26 August 1743 8 May 1794) was applied Since the enzymes only catalyzed the hydrolysis, they did not go into the hydrolysis equation. Therefore the hydrolysis stoichiometry was derived for cassava starch reaction with water to produce glucose. The stoichi ometry as presented in Figure 4 10 revealed that 1 gram of starch yields 1.1111 gram of glucose. This was the basis for all mass balance computations. Cassava Starch Water Glucose + [C 6 H 10 O 5 ] n = 1 H 2 O C 6 H 12 O 6 1 Mole of Starch 1 Mole o f Water 1 Mole of Glucose 162 g 18 g 180 g Figure 4 10 Mass Balance Stoichiometry for Enzymatic Hydrolysis of Different Cassava Substrates Image of water and glucose structure courtesy of Wikipedia. 4.3.8 Reaction Kinetics Mathematical models capable o f simulating the process of enzyme hydrolysis are very useful because they are able to predict processing outputs in response to different input variables, such as substrate concentration and hydrolysis time. The kinetic model most widely used to describe enzymatic hydrolysis is the Michaelis Menten rate

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172 expression (Shuler and Kargi, 2002). Well designed batch reactors with cell free enzymes are typically operated as homogeneous systems in which the enzyme and substrate are well mixed to ensure uniform dist ribution of temperature, pH, and concentration throughout the reactor. Under these conditions, process design and operation rely essentially on the reaction rate or enzyme kinetics. Conventional methods for determining kinetic parameters for the Michaelis Menten rate expression are used to obtain the reaction rate as a function of substrate concentrations ( Zhou et al., 200 3 ) and then perform a graphical method of data analysis or a direct linear pl ot (Cavaille and Combes, 1995), such as the Lineweaver Burk plot (Doran, 1995). However, it is difficult to find a proper model for an enzymatic reaction that obeys inhibition kinetics. In addition, a kinetic model obtained in this way is only valid for an enzyme reaction under predetermined optimal process conditi ons. Two well known mathematical models based on the Michaelis Menten rate expression were used to show the complexity of describing these types of enzymatic reactions. Model 1 is for enzyme hydrolysis in which the reaction rate is a function of substrate concentration only. Most studies of animal and vegetable protein hydrolysis showed inhibition by the substrate (Kristinsson and Rasco, 2000 ; Van Den Heuvel and Beeftink, 1988 ), as well as uncompetitive inhibition in the case of vegetable proteins, which wa s assumed for model 1 (Marquez Moreno and Fernando Cuadrado, 1993). Model 2 reflects the more complex situation in which the reaction rate is a function of both substrate and active enzyme concentrations. In the work undertaken in this study, the enzyme r eaction was carried out under predetermined optimal process conditions of enzyme and substrate concentrations.

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173 Under these conditions only the substrate composition changes with time as the enzyme hydrolysis proceeds. This type of reac tion can be describe d by first order kinetics, which was the type of model chosen for this work. 4.3.9 Statistical Analysis Data were analyzed by descriptive statistics (Ott and Longnecker 2010; Montgomery, 2009). Means, standard deviations, correlation coefficients, etc. wer e computed using Microsoft Office Excel Software Package 2007. The excel software was also used to plot graphs, derive and display equations, as well as generate constants. 4.4 Results and Discussion 4.4.1 Compositional analysis of different substrates R esults from laboratory analyses on the composition of each subs trate are summarized in Table 4 5. Aside from differences in moisture content which were approximately 11 %, 8 % and 61 % respectively for commercial cassava starch, flour from solar dried cass ava chips and ground fresh cassava root pulp these data show that pure starch accounted for more than 80% total solids in the commercial starch, about 75% of total solids in the cassava flour, and 29% of t otal solids in the cassava root pulp These differ ences dictated the maximum glucose concentration that could be reached when hydrolyzing substrates made with the same weight percent of substrate ingredient. 4.4.2 Enzyme h ydrolysis at 60C Profiles of glucose concentration over time during enzyme hydrolys is at 60C of commercial cassava starch, flour from solar dried cassava chips and ground fresh cassava root pulp are shown in Figures 4 11, 4 12 and 4 13, respectively. All three

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174 profiles reveal a classic exponential decay of unaccomplished glucose convers ion over time, with more than 90%, 80% and 60% conversion of available starch accomplished within the first four hours of hydrolysis respectively for commercial cassava starch, flour from solar dried cassava chips and ground fresh cassava root pulp. The ma ximum glucose concentrations reached reflected the quantity of pure starch available in each of the substrates. The substrate made with 10% pure commercial starch converted to virtually 10% glucose, while substrates made with 10% flour or ground root pulp contained only 8% or 7% starch to begin with. Table 4 5. Compositional Analysis of Different Cassava Substrates S/N Chemical Component [% Mass of Sample; Otherwise stated] Substrate Commercial Cassava Starch Flour Produced from Solar Convectio n Dried Cassava Chips m) Fresh Cassava Root 1 Ash NE NE 0.78 2 Carbohydrate 37.73 3 Fat 0.29 4 Fiber (Crude) 1.04 5 Protein 1.64 6 Moisture 11.20 7.86 60.52 7 Starch 80.16 74.60 29.08 Sugars 8 Fructose ND [DL = 0.1] 0.644 0.267 9 Glucose 0.958 0.414 10 Lactose ND [DL = 0.1] ND [DL = 0.1] 11 Maltose 0.293 ND [DL = 0.1] 12 Sucrose 5.56 1.46 13 Energy (Calories) [Cal/100 g] NE NE 160.09 14 Total Pectin [mg GA/kg] 314 2 478 349 NE: Not Evaluated ; ND: Not Detected ; DL: Detection Limit

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175 Figure 4 11 Profile of Glucose Content Vs Time during Enzyme Hydrolysis of Commercial Cassava Starch at 60 o C (Mean of three replications ; and error bars of standard deviations ) Figure 4 12 Profile of Glucose Con tent Vs Time du r ing Enzyme Hydrolysis of Flour Produced from Solar Dried Cassava Chips at 60 o C (Mean of three replications ; and error bars of standard deviations ) 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 18 Glucose Content [g/100 g] Time [Hours] 0 0.5 1 1.5 2 2.5 3 3.5 4 0 2 4 6 8 10 12 14 16 18 Glucose Content [g/100 g] Time [Hours]

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176 Figure 4 13 Profile of Glucose Content Vs Time du r ing Enzyme Hydrolysis of Ground Fres h Cassava Root Pulp at 60 o C (Mean of three replication s ; and error bars of standard deviations ) Maximum starch conversions reached after 24 hours with each sub strate are presented in Table 4 6. A comparison of all three glucose time profiles is shown as a family of curves on one graph in Figure 4 14. A similar comparison using bar graphs to represent percent starch conversion to glucose with each substrate at various points in time during hydrolysis is shown in Figure 4 15. The results are similar to tho s e of Srikanta et al. (1987) who also reported greater conversion with pulp than with flour Table 4 6 Maximum Starch Converted to Glucose in Different Cassava Substrates after 24 hou rs of Enzyme Hydrolysis at 60 o C Substrate Starch Conversion [%] Commercial Starch 96 Flour from Solar Dried Chips 27 Ground Fresh Root Pulp 53 0 0.5 1 1.5 2 2.5 3 3.5 4 0 2 4 6 8 10 12 14 16 18 Glucose Content [g/100 g] Time [Hours]

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177 F igure 4 14 Comparison Profil e of Glucose Content Vs. Time during Enzyme Hydrolysis of Commercial Cassava Starch, Flour Produced from Solar Dried Cassava Chips an d Ground Fresh Cassava Root Pulp at 60 o C (Means of three replications ; and error bars of standard deviations ) Figure 4.15 : Conversion Pro file of Enzyme Hydrolysis of Starch to Glucose in Different Cassava Substrates at 60 o C (Values were estimated from three replications of each substrate and e rror bars are for standard deviations ) 0 2 4 6 8 10 12 0 5 10 15 20 Glucose Content [g/100 g] Time [Hours] Mean Root Mean Flour Mean Starch 0 20 40 60 80 100 120 Starch Conversion [%] Time [Hours] Fresh Root Pulp Solar Flour Commercial Starch 0.25 1.5 4.0 24.0

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178 4.4.3 Enzyme hydrolysis at 37 C Profiles of glucose concentration over time during enzyme hydrolysis at 37 C of commercial cassava starch, flour produced from sola r dri ed cassava chips and ground fresh cassava r oot pulp are shown in Figures 4 16, 4 17 and 4 18, respectively. Figure 4 16 Profi le of Glucose Content Vs. Time du r ing Enzyme Hydrolysis of Commercial Cassava Starch at 37 o C Figure 4 17 Profile of Glucose Content Vs Time du r ing Enzyme Hydrolysis of Flour Produced from Solar Dried Cassava Chips at 37 o C 0 2 4 6 8 10 12 0 20 40 60 80 100 120 Glucose Content [g/100 g Sample] Time [Hours] 0 1 2 3 4 5 6 7 8 0 20 40 60 80 100 120 Glucose Content [g/100 g Sample] Time [Hours]

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179 Figure 4 18 Profile of Glucose Content Vs Time du r ing Enzyme Hydrolysis of Ground Fresh Cassava Root Pulp at 37 o C All three profiles als o reveal a classic exponential decay of unaccomplished glucose conversion over time, but much more slowly than at 60 C. At 37 C hydrolysis must proceed for approximately 72 hours (three days) in order to reach 80 90% conversion of available starch t o glucose. Again, the maximum glucose concentrations reached after 96 hours of hydrolysis reflected the quantity of pure starch originally present in each of the subst rates, and are given in Table 4 7. Table 4 7 Maximum Starch Converted to Glucose in Dif ferent Cassava Substrates after 96 hou rs of Enzyme Hydrolysis at 37 o C Substrate Starch Conversion [%] Commercial Starch 100 Flour from Solar Dried Chips 72 Ground Fresh Root Pulp 55 0 1 2 3 4 5 6 0 20 40 60 80 100 120 Glucose Content [g/100 g Sample] Time [Hours]

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180 A comparison of all three glucose time profiles is shown as a family of curves on one graph in Figure 4 19. A similar comparison using bar graphs to represent percent starch conversion to glucose with each substrate at various points in time during hydrolysis is shown in Figure 4 20. Figure 4 19 Compar ison Profil e of Glucose Content Vs. Time during Enzyme Hydrolysis of Commercial Cassava Starch, Flour Produced from Solar Dried Cassava Chips and Ground Fresh Cassava Root Pulp at 37 o C Figure 4 20 Conversion Pro file of Enzyme Hydrolysis of Starch to Glucose in Different Cassava Substrates at 37 o C 0 2 4 6 8 10 12 0 50 100 150 Glucose Content [g/100 g Sample] Time [Hours] Root Flour Starch 0 20 40 60 80 100 120 Starch Conversion [%] Time [Hours] Fresh Root Solar Flour Commercial Starch 1 4 48 72 96

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181 4.4.4 Reaction Kinetics of Enzyme Hydrolysis As noted earlier, all glucose time profiles revealed a classic exponential decay of unaccomplished glucose conversion over time. All experiments were carried ou t using the same substrate and enzyme concentrations. Under these conditions, any differences in performance of hydrolysis with different substrates would be noted in the rate of reaction. Therefore, the exponential decay of unaccomplished glucose conversi on over time could be treated as a first order biochemical reaction that could be described by first order reaction kinetics. These kinetics can be quantified by reporting numerical values for first order rate constants (k) at different temperatures, and the Arrhenius activation energy (E A ) describing the temperature dependency of the rate constants. 4.4.4.1 First order rate constants The first order rate constants were taken from the slopes of straight lines obtained on a semi log graph of unaccomplishe d glucose conversion over time. The unaccomplished glucose conversion was expressed mathematically as the difference between maximum glucose concentration reached [C max ] and the g l ucose concentration at any point in time, [C]. Semi log plots of unaccomplis hed glucose conversion over time for all three substrates at 60C are presented in Figure 4 21, and at 37C in Figure 4 22. First order rate constants [k] were taken from the slope of the straight line of best fit by linear regression for each substrate a nd are listed in Table 4 8 and Table 4 9 for hydrolysis at 60C and 37C respectively.

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182 Figure 4 21 Semi log Plot of Unaccomplished Glucose Conversion over Time for Estimation of First Order Reaction Rate Constants for Enzyme Hydrolysis of Starch w ith Different Cassava Substrates at 60 o C Figure 4 22 Semi log Plot of Unaccomplished Glucose Conversion over Time for Estimation of First Order Reaction Rate Constants for Enzyme Hydrolysis of Starch with Different Cassava Substrates at 37 o C 3.5 3 2.5 2 1.5 1 0.5 0 0.5 1 1.5 0 0.5 1 1.5 2 2.5 Ln [Cmax C] Time [Hours] Fresh Root Solar Flour Commercial Starch 0.5 0 0.5 1 1.5 2 2.5 0 20 40 60 80 Ln [Cmax C] Time [Hours] Fresh Root Solar Flour Commercial Starch

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183 Tab le 4 8 Reaction Rat e Constant (k ) Correlation Coefficient (R 2 ) and Regression Equation of First Order Reaction f or Enzyme Hydrolysis of Different Cassava Substrates at 60 o C. Substrate k Value [H ou r 1 ] R 2 Equation Commercial Starch 1.1017 0 .9920 y = 1.1017x + 1.2825 Solar Flour 1.3198 0.9986 y = 1.3198x 0.4222 Fresh Root 0.8164 0.9397 y = 0.8164x 0.0949 Table 4 9. Reaction Rat e Constant (k ) Correlation Coefficient (R 2 ) and Regression Equation of First Order Reaction f or Enzyme Hydrolysis of Different Cassava Substrates at 37 o C. Substrate k Value [H ou r 1 ] R 2 Equation Commercial Starch 0.0250 0.9886 y = 0.025x + 2.1476 Solar Flour 0.0205 0.9953 y = 0.0205x + 1.5058 Fresh Root 0.0232 0.9921 y = 0.0232x + 1.4363 4.4.4.2 Activation energy The Arrhenius activation energy (E A ) describing the temperature dependency of the rate constants was taken from the slope of the straight line obtained on an Arrhenius plot of the natural log of rate con stant against reciprocal absolute temperature. Arrhenius plots for each substrate are shown in Figure 4 23, and the resulting activation

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184 energies for each substrate are listed in Table 4 10. A full report of the reaction kinetics for each of the three subs trates is summarized in Table 4 11. Figure 4 23. Semi log Plot of First Order Reaction Rate Constants versus Reciprocal Absolute Temperature for Estimation of Arrhenius Activation Energy for Enzyme Hydrolysis of Cassava Starch at 37 o C and 60 o C Tab le 4 10 Activation Energy ( E A ) and Regression Equations for Estimating the Arrhenius Temperature Dependency of Rate Constant for Enzyme Hydrolysis of Different Cassava Substrates Substrate E A [kJ/mol] Equation Commercial Starch 143.067 y = 17208 x + 51.720 Solar Flour 157.392 y = 18931x + 57.070 Fresh Root 134.562 y = 16185x + 48.353 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0.5 0.00295 0.003 0.00305 0.0031 0.00315 0.0032 0.00325 Ln k 1/T [1/K] Fresh Root Solar Flour Commercial Starch

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185 Table 4 11. Reaction Kinetics Parameters for Enzyme Hydrolysis of Starch in Different Cassava Substrates Substrate Reaction Rate Constant; k [H ou r 1 ] Activation Energy; E A [kJ/mol] 37 o C 60 o C Commercial Starch 0.0250 1.1017 143.067 0 Solar Flour 0.0205 1.3198 157.392 0 Fresh Root 0.0232 0.8164 134.562 0

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186 CHAPTER 5 ANAEROBIC DIGESTION OF CASSAVA WASTE FOR PRODUCTION OF B IOGAS AND AMYLOLYTIC ENZYMES 5.1 Background As a result of climate change and other environmental issues, inevitable depletion of fossil fuels supply, sustainability, renewability and biodegradability concerns, energy security, balance of trade and foreign exchange matters, as well as associated socio economic implications, considerable efforts are being deployed globally to the development of renewable energy sources (Nigam and Sin gh, 2011; Demirbas, 2009, International Energy Agency (IEA), 2009; Demirbas, 2007; Lin and Tanaka, 2006; Wyman et al., 2005; Lynd and Wang, 2004; Wyman, 1999; Lynd et al., 1991; Menezes, 1982). To this effect, biomass in its various forms appears to be a major energy source ( Bloomberg New Energy Finance, 2012; Nigam and Singh, 201 1; Renewable Energy Policy Network for the 21 st Century (REN21) 2011; Ruiz et al., 2011; Demirbas, 2009; Herrera, 2006; Lin and Tanaka, 2006; Ragauskas et al., 2006 ). However, the use of edible parts of food crops (e.g. sugarcane, corn/maize, soy bean, pal m oil) for biofuel (e.g. bioethanol, biodiesel) production has raise d ethical concerns about diversion of food to fuel production (Pullammanappallil, 2013 ; Ch eng and Timilsina, 2011 ; Glover et al. 2010; Godfray et al., 2010; Thomson et al., 2010; Fargione et al., 2008; Searchinger et al., 2008; The Royal Society, 2008 ). Therefore, a reasonable alternative is biofuel production from agricultural residues and wastes, as well as from microalgae, and lignocellulosic biomass originating from non food sources. Cassava wastes such as peels are generated during the processing and production of numerous cassava based food products like casabe, chickwangue, farinha de mandioca, fufu, fuku, gaplek, gari, konkonte, landang, and peujeum. The

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187 cassava peel is reported to constitute 15 19 % fresh weight of the cassava root (Gomez and Valdivieso, 1983; Gomez et al., 1985). With manual peeling, the peel can constitute 20 35 % of the total weight of the root (Ekundayo, 1980). More than 60 % of global cassava production is processed as food for human consumption ( Tonukari, 2004). In 2011, world cassava output was 252.20 x 10 9 kg and most of it was produced by developing countries (FAO, 2013). Therefore enormous organic waste is generated from cassava processi ng. This waste c ould potentially be a good feed stock for anaerobic digestion and fermentation processes for the production of biogas and hydrolytic enzymes. Agricultural residues such as cassava peel, cowpea was te, plantain peel, rice bran; yam peel, etc have been rep orted as suitable fermentation substrates for the production of enzymes (Adeniran and Abiose, 2011; Adeniran et al., 2010; Adeniran and Abiose, 2009; Kareem et al., 2009; Silva et al., 2009; Alva et al., 2007; Swain and Ray, 2007; Uguru et al., 1997; Sani et al., 1992). Also, cassava stem residues, waste water and peel are reported to have been used in biogas production ( Zhu et al., 2013, Gao et al., 2012; Cuzin et al., 1992). However, the author did not identify any report that evaluated simultaneous produ ction of biogas and enzymes from cassava waste. It will appear to be a win and win again situation if it is possible to us e cassava peel waste in anaerobic digestion to produce biogas that could be used as a source of energy on the one hand; and also to ex tract a natural cocktail of hydrolytic enzymes from the post digestion broth on the other hand. This study explored these possib ilities. 5.2 Objectives The objectives of the work reported in this study were to:

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188 Determine the proportions of cassava root co mponents. These include the peel (periderm /epidermis or outer coat and cortex), the parenchymatous tissue, the head and the tail ends. Demonstrate the feasibility of anaerobic digestion of cassava waste by performing experiments with cassava peels and rep orting performance indicators such as: methane yield, chemical oxygen demand ( COD ) pH, etc. E xplore feasibility of extracting from the post anaerobic digestion broth active enzymes that are capable o f hydrolyzing cassava st a rch D etermine the activity of any extracted enzyme 5.3 Materials and Methods 5.3.1 Cassava Roots Fresh cassava r oots were purchased from Brooks Tropicals, Homestead, Florida. The roots were coated with food wax and supplie d refrigerated in 18 kg boxes. Upon receipt, e ach consignmen t or batch was promptly received and maint ained under refrigerated storage (< 10 o C) until utilized. 5.3.2 Reactor Th e reactor used in this study constituted mesophilic anaerobic digester. The digester vessel was fabricated from a Pyrex glass jar about 0. 2540 m high from base to shoulder and 0.1651 m in diameter (Figure 5 1 b) The neck was approximately 0.0889 3 ; of which 0.003 m 3 was utilized in each batch of the experiments. The reactor was charged from top through the neck opening. The openi ng was capped with air and liquid tight closure system that consisted of Pyrex lid, Teflon O ring and Stainless steel clamp. The vessel was fitted with ports at top of the lid and on the side close to the bottom for biogas transfer and leachate withdrawal respectively. The reactor vessel was incubated a t mesophilic temperature (36 3 o C) in a converted household refrigerator

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189 chamber, heated with a 65 W Sylvania light bulb (Sylvania lighting Services Corporation, Danvers, Massachusetts). The bulb was contr olled by BFG thermostat, model #6910796 (Whirlpool Corporation, Benton Harbor, Michigan). Biogas production was monitored with a positive displacement type gas meter that was connected from outside of the incubator chamber. The meter device consisted of a transparent PVC U tube that was filled with orange colored anti freeze solution; float switch (W W Grainger, Lake Forest, Illinois); solenoid valve (Fabco Air Inc., Gainesville, Florida); Dayton off time delay relay model 6X153E (W W Grainger, Lake Forest, Illinois); and a counter (Redington C ounters Inc., Windsor, Connecticut ). The design assembly of the anaerobic reactor digest er and biogas measurement system is shown in Figure 5 1 Figu re 5 1 Anaerobic Digestion System Design

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190 5.3.3 Inoculum A bou t 0.003 m 3 of mixed liquor from an active municipal solid waste fed mesophilic lab scale digester was used as inoculum to start up the first experiment. Sub s equently, mixed liquor from previous experiment was used as inoculum for next experiment 5.3.4 B ulking Agent To prevent compaction of peel waste in the digester about 1 .5 kg of lava rocks was used as bulking agent. The rocks which were irregularly shaped and about 2.5 cm in average dimension were obtained from a landscaping supplier. 5.3.5 Sample Preparation 5.3.5.1 Root components p roportion For each experimental run, between 1780 g to 2828 g of fresh cassava roots was retrieved from r efrigeration and used. Roots were weigh ed with heavy duty shipping scale serial # HD 150 (My Wei gh, Phoenix, Ariz ona, Figure 5 2 ) Deliberate efforts were Figure 5 2 M y Weigh Shipping Scale used to w eigh out Cassava Roots

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191 made to include for each batch, small, medium and large size roots. The measured roots were placed on a cutting board shown in Figure 5 3 A, and then peeled with a kitchen knife (Figure 5 3 B ). A B Figure 5 3 Implements used to Peel Cassava Roots and Prepare the Peel ings for Experiment s. A) Cutting Board B) Peeling Knives Photos courtesy of author. The peeling process involved t wo steps; first, carefully cutting off the head and tail ends of each roo t such that the main edible portion (the parenchyma) was not affected. Secondly, the peel (periderm /epidermis or bark/ outer coat and cortex) was separated from the parenchymatous tis sue, again making sure that the parenchyma was not affected. The root components: Head and Tail ends; Periderm /Epidermis ; Cortex; and Parenchyma were subsequently weighed separately to determine root mass proportions. The masses were thereafter added and compared to the initial mass of roots to estimate the lost mass. Figure 5 4 shows the different components and the types, shapes and sizes of roots encountered. The described opera tion s were replicated four times.

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192 A D Figure 5 4 Components of Cassava Roo t. A), B), C), D) and F) Head & Tail Ends Shown E) and G) Periderm /Epidermis Cortex and Parenchyma Shown Photos B, C, D and E courtesy of google.com images; Photo G courtesy of bing.com/images The remainder of the photos courtesy of author. E Head Tail Parenchyma Periderm Coat Cortex Head Tail Tail Head Head Tail G: Parenchyma Periderm Coat Cortex Head Tail F C B

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193 5.3.5.2 Anaerobic diges tion samples For anaerobic digestion experiments, the cortex which was 0.203 to 0.224 cm thick was cut into pieces of 2.5 cm average dimension. Similarly, the head and tail ends, as well as the periderm or epidermis was reduced to pieces of about 2.5 cm average dimensions. These cut up three root components (Head and Tail ends, Periderm /epidermis and Cortex) were comb ined, mixed thoroughly and used as substrates in all the anaerobic digestion experiments. The substrate as prepared was 10 o C) refrigeration until required. 5.3.6 Anaerobic Digestion Procedure Two h und red grams of substrate was charg ed for each batch of anaerobic digestion experiment. Freshly prepared substrate was used or previously frozen sample was thawed overnight before utilization. To start the loading process, the substrate was divided into two e qual portions of one hundred grams each; and the 1.5 kg lava rock bulking agent was divided into three equal portions of five hundred grams each. The reactor vessel cap was then disengaged and one portion of the bulking agent ( five hundred grams ) layered o n the bottom of the vessel. The first one hundred grams portion of substrate was spread on the rocks. Then the second portion of five hundred grams of lava rocks was layered over the substrate and the remaining one hundred grams portion of substrate spread on the rocks. Finally, the remaining five hundred grams of lava rocks was layered over the substrate. The first run was inoculated with 3000 cm 3 of mixed culture inoculums and 35 g of soda ash (sodium bicarbonate) was added to buffer against pH changes. T he digester cap was replaced and clamped tight;

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194 digester placed in the incubator chamber; biogas lines were connected ; the incubator door was closed ; and anaerobic digestion allowed to proceed. When gas production capacity was depleted (no biogas produced in two or more consecutive days), the process was interrupted and stopped. The cap was opened and the second batch (200 g) of substrate was loaded from the top. Similarly, the third and fourth batch es of 200 g substrate each was charged at the end of the second and third run s respectively Each subsequent run was therefore initiated by the post digestion broth of the preceding experiment; no additional inoculums or buffer were added. At the end of the fourth run, all the contents of the digester were disch arged and the digestion broth and substrate digestate (residue) were analy zed for enzyme activity. For the fifth experiment, 200 g of substrate was prepared and layered as described for run number one above. The rema ining broth from the first four runs wa s made up to 3000 cm 3 with fresh ino culums and used f o r inoculation ; no additional buffer was added. Each batch of experiment was operated in a bat c h mode; as a single stage, leach bed, and unmixed system. The digester contents were not agitated 5.3.7 An alysis 5.3.7.1 Biogas c omposition The methane and carbon dioxide composition of biogas produced was analyzed with Gas Chromatograph (GC) Series 580 (Gow Mac Instrument Co., Bethlehem, Pennsylvania ) that was coupled with Gas Partitioner Model 1200 (Fisher Scientific Inc., Waltham, Massachusetts). The GC was equipped with thermal conductivity detector and calibrated with a mixture of nitrogen (N 2 ), methane (CH 4 ), and carbon dioxide (CO 2 ) in the volume ratio of 25:45:30 [N 2 :CH 4 :CO 2 ]. The GC analytical system that is being described and was used in the experimental work is shown in Figure 5 5.

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195 Figure 5 5 Gas Chromatograph (GC) System used to Estimate the Methane and Carbon dioxide Content s of Biogas Generate d by Anaerobic Digestion of Cassava Peel Waste Photo courtesy of author. In operation, the GC system was turned on and allowed thirty minutes of come up time to attain operating temperature of 50 o C. Thereafter about 20 mL of biogas sample was drawn with a syringe and injected into the gas partitio ner. The GC automatically performs analysis; gas chromatograms were processed and stored with SP 4290 Integrator (Spectra Physics, Santa Clara, California).

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196 5.3.7.2 Soluble chemical oxygen demand (sCOD) The soluble chemical oxygen demand (sCOD) was anal yzed for daily leachate samples drawn from the digester using Hach COD reagent kit (Hach Company, Loveland, Colorado). A bout 4 mL of leachate was withdrawn from the digester and centrifuged for ten minutes at 2500 rpm with Marathon Micro H centrifuge (Fish er Scientific Inc., Waltham, Massachusetts; Figure 5 6 A). The supernatant was Figure 5 6 Laboratory Equipment used in sCOD analysis of Leachate from Anae robic Digestion of Cassava Peel Waste A) Marathon Micro H Centrifuge B) Hach s COD Reactor C) Hach sCOD Colorimeter Photos courtesy of the author. Maidstone, Kent, UK) The filtrate was diluted twenty times and 2 mL pipetted into Hach COD reagent vials (with A B C

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197 the range: 20 1500 mg/L (2 150 ppm) COD). The vials were digested (incubated) for two hours at 150 o C in the COD reactor model # 45600 00 (Hach Company, Loveland, Colorado; Figure 5 6 B). Subsequently, the COD of the leachate was read with DR/890 colorimeter (Hach Company, Loveland, Colorado; Figure 5 6 C), after zeroing with a blank. 5.3.7.3 pH The pH of digester broth was monitored about the same time daily with Orion 3 Star pH Bench top (Thermo Scientific Inc., Waltham Massachusetts). Assay technique involved wi thdrawing 20 mL of broth in a plastic vial. The pH meter was turned on and warmed for thirty minutes. Meter probe which had been properly calibrated with neutral and acidic standard media was rinsed with distilled water and wiped dry with lint free kimwipe s (Kimberly Clark Corporation, Irving Texas). The probe was subsequently inserted into the broth and held until the meter gave a stable reading. 5.3.7.4 Total solids (TS), volatile solids (VS), and other p roperties Total solids (TS) and volatile solid s ( VS) constituents of the pe e l waste (feedstock) were determined gravimetrically. About 100 g of sample was dried for 72 Hrs at 105 o C using Isotemp Oven Model 350 G (Fisher Scientific Inc., Waltham, Massachusetts). The dried sample was then burnt in Iso temp Muffle Furnace (Fisher Scientific Inc., Waltham, Massachusetts) at 550 o C for 2 Hrs. From the masses of t he sample, dried sample, ash residue, sample pan, etc., TS, VS, and other properties were compu ted. The formulae used for computations and methan e yield an alysis are

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198 presented in Table 5 1. The procedure described was executed for each batch or replication of the experiment. Table 5 1. Formu lae for Methane Yield Analysis and for Determination of Properties of Cassava Peel Waste used as Feedstock/S ubstrate in Anaerobic Digestion for Biogas and Enzyme Production Parameter Symbol Parameter ID Unit Computational Formula A Mass of Dish and Sample Dried for 72 Hrs at 105 o C g B Mass of Dish Alone g C Mass of Wet Sample and Dish g D Mass of Dish and Sample Burnt for 2 Hrs at 550 o C g E Cumulative Methane Production L F Batch of Feedstock ( Peel Waste ) Charge in the Digester g G Total Solids % H Volatile Solids % I Volatile Solids Proportion g VS J Fixed Solids % K Moisture Content ; Wet Basis % L Moisture Content ; Dry Basis % M Methane Yield Per Feedstock Charge L/g N Methane Yield Per Volatile Solids L/g VS O Methane Production Efficiency %

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199 5.3.8 Enzyme Extraction and Enzyme Activity The work performed included extraction of enzymes as well as testing the activity of extracted enzymes. 5.3.8.1 Enzyme extraction It has been noted that the location of extracellular enzymes impacts the capacity to degrade substrate (Parawira et al., 2005). Depending on whether the enzyme is released into the surrounding environment or attached to the cell surface, the enzyme could be described as cell free or cell associate d (Priest, 1984). In the work carried out in this study, three divisions o f enzyme were identified ( Zhang et al., 2007). These were cell free enzyme; cell associated enzyme; and biofilm associated enzyme. 5.3.8.1.1 Cell free enzyme This is the enzyme that has been secreted into the environment and is potentially solubilized in the leachate broth. To extract this class of enzyme, 10 mL of leachate was withdrawn into a plastic vial and centrifuged at 3000 g for ten minutes with International Clinical Centrifuge, Model CL 96327H 3 (Fisher Scientific Inc., W altham, Massachusetts; Fi gure 5 7). The supernatant was used as source of cell free enzyme. Figure 5.7: Clinical Centrifuge used for Enzyme Extraction Photo courtesy of the author.

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200 5.3.8.1.2 Cell associated enzyme The cell associated enzyme is presumed to be attached to the s urface cells of organisms that are freely suspended in leachate liquor (broth). On centrifugation, suspended cells are separated from the liquor into a pellet; carrying the attached enzymes with them. The pellet is harvested and the cell associated enzyme extracted from it thus: Wash the pellet resulting from cell fre e enzyme extraction two times. For e ach washing, centrifuge with 10 mL of potassium dihydrogen phosphate buffer (pH 7.0; 0.1 mol L 1 ) for two minutes at 3000 g Suspend the washed pellet in 2 mL of sodium acetate buffer (pH 6.0; 0.05 mol L 1 ). Then centrifuge the suspension at 3000 g for ten minutes. The supernatant is collected as the source of cell associated enzyme. 5.3.8.1.3 Biofilm associated enzyme In the milieu of anaerobic digestion some microbes are attached to the surface of the substrate as biofilm. Those enzymes that are attached to microbes that themselves are attached to the substrate are called biofilm associated enzymes. A biofilm associated enzyme is neither in solution in the leachate broth (cell free enzyme) nor attached to the surface of organisms that are suspended in the leachate broth (cell associated enzyme). Biofilm associated enzyme was extracted with a procedure similar to that used for cell associated enzyme. Abo ut 5 g of substrate was taken out of the anaerobic digester (or 5 g of digestate was taken at the end of a batch anaerobic digestion op eration) The substrate/digestate was washed twice. Each washing process was carried out with 10 mL of potassium dihydro gen phosphate buffer (pH 7.0; 0.1 mol L 1 ) ; by centrifuging for two minutes at 3000 g Washed substrate/digestate was suspended in 2 mL of sodium acetate buffer (pH 6.0; 0.05 mol L 1 ). The suspension was centrifuged at 3000 g for ten minutes. The supernat ant was then collected as the source of biofilm associated enzyme.

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201 5.3.8.2 Enzyme activity The determination of enzymatic activity was carried out for each of the three divisions of enzyme: cell free; cell associated; and biofilm associated. Specifically, amylase activity was estimated by method of Bernfeld (Bernfeld 1955), but with modifications where appropriate Assay system used to determine activity for each enzyme division contained 0.5 mL of the cr ude enzyme solution and 0.5 mL o f 1 % commercial ca ssava starch. The 1 % starch may be used as is; not buffered to estimate overall amylolytic activity. The starch may be buffered with disodium hydrogen phosphate buffer (pH 6.9 ; 0.1 mol L 1 ) to estimate amylase activity (Adeniran et al., 2010; Zhan et al., 2007). Alternatively the 1 % starch solution may be buffered with sodium acetate buffer (pH 4 ; 0.1 mol L 1 ) to estimate glucoamylase activity (Adeniran et al., 2010; de Moraes et al., 1999; Zanin and De Moraes, 1998). The mixture was then incubated at 37 o C for thirty minutes and the released reducing sugar (as glucose) estimated with the 3,5 dinitrosalicylic acid (DNS) procedure. The DNS methodology was defined and discussed in chapter four of this d issertation. One unit of enzyme activity (EU) was def ined as the amount of the enzyme that (EU/ The protein content of each enzyme solution was estimated by the modified Lowr (Thermo Fisher Scientific Inc., 2011; Lowry et al., 1951), taking liquid bovine serum albumin or BSA (Thermo Scientific, Rockford Illinois) as the standard.

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202 5.4 Results and Discussion 5.4.1 Cassava Root Components Proportions Table 5 2 shows th e proportion of cassava root components on fresh mass basis. The major human food component, the parenchyma, constituted the largest mass with the average value of 81.16 % and range of 80.72 % to 81.75 %. The cortex was second with the range of 12.76 % to 13.33 % and mean value of 13.11 %. The periderm or epidermis, also known as the bark or outer coat averaged 3.31%; while head and tail ends had a mean value of 1.71 %. Since the peel consists of cortex and periderm, its mean proportion of cassava fresh ro ot was computed to be 16.42 %. This value is s imilar to the work of Gomez and Valdivieso (1983) and Gomez et al., (1985) who Table 5 2 Mass Distribution of Cassava Root Components S/N Component Mass Proportion [% Fresh Mass] Run No. 1 Run No. 2 R un No. 3 Run No. 4 Mean Edible Portion 1 Parenchyma 81.30 81.75 80.88 80.72 81.16 Waste Portion 2 Cortex 13.33 12.76 13.24 13.11 13.11 3 Periderm 3.51 3.19 3.38 3.15 3.31 4 Head & Tail Ends 1.35 1.18 1.79 2.54 1.71 Lost Portion 5 Lost Mass 0.51 1.12 0.71 0.48 0.71 6 Total Waste = Sum of waste portion = 18.13 % reported cassava root peel proportions of 15 % 18 % and 15 % 19 % respectively. Dufour (1988), reported relative contributions of the bark (periderm) and c ortex fractions

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203 to root fresh weight of 1.6 % and 14 3 % respectively. Furthermore, it has also been reported in the literature that after 4 months to 10 months of cultivation, the cortex constituted about 15 % of total fresh weight of root (Cooke and de l a Cruz, 1982). An interesting root component presented in Table 5 2 is termed lost mass. It was discovered that the sum of the masses of conventional root components (parenchyma, cortex, periderm, and head and tail ends) was less than the total mass of roo t that was started out with. The lost mass may be due to evaporative loss of moisture, micro and nano particles that may have drifted away, volati li zation and loss of t he wax coating, etc. The lost mass concept was therefore introduced to account for these irrecoverable l osses. Consequently, the lost mass was discounted, and the total waste associated with the peeling process was reported to be the sum of cortex, periderm, and head and tail ends. The sum of these waste components, and therefore the total wa ste, averaged 18.13 % of the fresh mass of cassava root. 5.4.2 Composition of Cassava Peel Waste The characteristics of the cassava peel waste are presented in Table 5 3. The mean moisture content of the peel wastes was 66.79 % wet basis. The values r anged from 63.14 % for run number one to 73.33 % for run number two. Total solids composition averaged 33.19 % and on average, 96.37 % of the solids was volatile. The range for total solids varied from 26.66 % (run number two) to 36.85 % (run number one); while that for volatile solids varied from 95.38 % (run number four) to 96.90 % (run number three) Furthermore, the dry weight mean was 66.36 g, ranging from 53.32 g for run number two to 73.70 g for run number one, while for the fixed solids, the range w as from 3.09 % for run number three to 4.61 % for run number four, with the mean value of 3.61 % ( Table 5 3 )

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204 Table 5 3. Substrate Characteristics and Bio Methane Potential s of Mesophilic Anaerobic Digestion of Cassava Peel Waste S/N Variable Parameter Run Number One Run Number Two Run Number Three Run Number Four Mean 1 Mass of Wet Substrate Charge [g] 200 200 200 200 200 2 Moisture Content, Wet Basis [%] 63.14 73.33 63.21 67.50 66.79 3 Moisture Content, Dry Basis [%] 171.26 275.0 0 171.82 207.69 206.44 4 Total Solids [%] 36.85 26.66 36.78 32.50 33. 19 5 Dry Weight of Solids [g] 73.70 53.32 73.56 65.00 66.36 6 Volatile Solids [%] 96.81 96.42 96.90 95.38 96.37 7 Volatile Solids Proportion [gVS] 71.35 51.42 71.30 62.00 64.01 8 Organic Load [gVS/ L ] 23.78 17.13 23.75 20.66 21.33 9 Fixed Solids [%] 3.18 3.57 3.09 4.61 3.61 10 Cumulative Methane Production [L] 14.21 15.94 12.71 19.11 15.49 11 Methane Yield Per Substrate Charge [L /g] 0.0710 0.0797 0.0635 0.0955 0.0774 12 Methane Yield Per Volatile Solids Proportion [L @ STP /gVS] 0.1991 0.3099 0.1782 0.3082 0.2488 13 Residence Time [Days] 30.37 24.93 19.35 33.28 26.98 14 Biogasification Efficiency [%] 56.89 88.55 50.91 88.06 71.10

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205 5.4.3 Biog asification of Cassava Peel Wa s te The profiles of cumulative methane yield versus time for the four runs are presented in Figure 5 8 Run number two had the highest performance followed by run Figure 5 8. Methane Yield of Mesophilic Anaerobic Digestion of Cassava Peel Waste number four. Run number one B iogasification was initiated with mixed liquor from an anaerobic digester treating municipal solid waste It is quite possible that the organisms were not adapted to wastes from cassava processing. This may explain the long lag time of six days that it took to commence methanogenesis and biogas production in run number one However, eight days later, on day fourteen from date of start up, methane production rate peaked at 2.48 L per day. After about ninet een days from commencement of the experiment, methane production dropped to 0.12 L per day. Gas production capacity was eventually exhausted by the thirtieth day when gas production rate dropped to zero. Run number two was initiated with the now active bro th from run number one It can be seen from Figure 5 8 that there was no lag time in commencing 0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0 10 20 30 40 Methane Yield [L @ STP/gVS] Time Elapsed [Days] Run No. One Run No. Two Run No Three Run No. Four

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206 biogas production. Methanogenesis started quickly and gas production rate peaked on day seven at 2.14 L per day. Biogas production thereafter slowly tapered off and was exhausted by day twenty five with the cumulative methane yield of 0.31 L @ STP /gVS. Residence time for the batch operations (the time from when the digester was charged or loaded to the time when no biogas was produced in two or more consecutive days) ranged from 19.35 days for run number three to 33.28 days for run number four. The mean residence time was 26.98 days. The mean cumulative methane yield for all the runs was 0.25 L @ STP @STP /gVS for run number three to 0.31 L @ STP / gVS for run number two and four ; Table 5 3. The range and mean results of cumulative methane yield for cassava peel waste are in accord with what have been reported in the literature as biochemical methane potential for biomass and waste f eedstocks such as sorghum, napiergrass, poplar, sugarcane, willow, municipal solid waste, as well as spent sugar beet processing by products (Koppar and Pullammanappallil, 2008; Polematidis et al., 2008; Hutnan et al., 2001, 2000; Chynoweth et al., 1993; Stoppok and Buchholz, 1985; Frostell et al., 1984). 5.4.4 Soluble Chemical Oxygen Demand (sCOD) Profiles of Soluble Chemical Oxygen Demand (sCOD) of Mesophilic Anae robic Digestion of Cassava Peel waste for run number one and run numbe r three are shown in Figure 5 9 The day one sCOD for run number one was 2440 mg/L. Ten days later, on day eleven of the experiment, sCOD for run number one peaked at 14340 mg/L. On that peak date, the cumulative methane production, cumulative methane production rate, and cum ulative methane yield for run number one were respectively 2.56 L, 1.27 L per day, and 0.0359 L @ STP/gVS. The sCOD thereafter tapered down and

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207 Figure 5 9. Profiles of Soluble Chemical Oxygen Demand (sCOD) for Mesophilic Anaerobic Digestion of Cassava Pe el Waste approached the value of 2840 mg/L by day nineteen of the run. Run number three started with sCOD of 2140 mg/L on the loading day. Six hours later the value was 3540 mg/L, and by the next day (day one) sCOD for r un number three rose to 6700 mg/L. The sCOD value continued the rise and peaked on day seven at 19320 mg/L. On that peak date of sCOD of run number three, the cumulative methane production, cumulative methane production rate, and cumulative methane yield were respectively 2.61 L, 0.528 L pe r day, and 0.0366 L @ STP /gVS. The sCOD thereafter tapered down and approached the value of 2220 mg/L at the end of the run. A careful stud y of Figures 5 8 and 5 9 revealed some interesting phenomena. First, the cumulative methane yield for runs one and th ree were similar on the days the sCOD peaked. These were 0.0359 L @ STP /gVS (on day eleven) and 0.0366 L @ STP /gVS (on day 7) respectively for ru n number one and run number three; Figure 5 9 0 5000 10000 15000 20000 25000 0 5 10 15 20 25 sCOD [mg/L] Elapsed Time [Days] Run Number One Run Number Three

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208 Secondly, methane production rate peaked on day fourteen for run number one and on day ten for run number three; Figure 5 8 It is interesting to note that these dates were respectively three days after sCOD peaked. It may appear like maximum methane production rate is preceded by three days of sCOD peak. These might b e relevant data for optimal design of mesophilic anaerobic digesters/ reactors for cassava peel waste and similar biomass. 5.4.5 Total Solids ( Dry Matter ) and Volatile Solids Reduction The total dry matter and volatile solids reduction and their computatio nal scheme are presented in Table 5 4. After four runs, the 254.8 g of dry matter loaded had only 76.08 g remaining; thus 70.14 % (178.72 g) was digested. Similarly, out of 245.55 g of volatile solids loaded, 71.06 g remained; translating to 71.06 % volat ile solids reduction or the digestion of 174.49 g of volatile solids, Table 5 4. The percent volatile solids reduction corresponded with the Biogasification efficiency of the cassava peel waste presented earlier in Table 5 3. Table 5 4. Dry Matter and Vol atile Solids Reduction during Anaerobic Digestion of Cassava Peel Waste. S/N Variable Parameter Parameter ID and Computing Formula Parameter Value 1 Dry matter added during the four runs A 254.8 g 2 Dry matter remaining at the end of four runs B 76.08 g 3 Total dry matter (solids) reduction C = [(A B)/A]*100 70.14 % 4 Volatile solids added during the four runs D 245.55 g 5 Volatile solids remaining at the end of four runs E 71.06 g 6 Volatile solids reduction F = [(D E)/D]*100 71.06 %

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209 5.4.6 Enzyme Activity The activities of enzymes isolated as a consequence of anaerobic digestion of cassava peel was te are presented in Table 5 5. When the experiment was performed with cut up pieces of whole unpeeled cassava roots, higher activities were obtained. The data are shown in Table 5 6. It appears like the production of amylolytic enzymes Table 5 5. The Activities of Enzymes Produced by Anaerobic Digestion of Cassava Peel Waste (Mean of duplicate measurements ). Time of Enzyme Extraction [Days] Enzyme Activ ity [ EU = ( ) ] Cell Free Enzyme Cell Associated Enzyme Biofilm Associated Enzyme 1 29.58 2 29.58 2.50 3 29.18 2.64 46 (Last Day) 13.93 0.79 9.17 Table 5 6. The Activities of Enzymes Produced by Anaerobic Digestion of Pieces of Whole Unpeeled Cassava Roots (Mean of duplicate measurements ). Time of Enzyme Extraction [Days] Enzyme Activity Cell Free Enzyme Cell Associated Enzyme Biofilm Associated Enzyme 2 68.93 2.37 26.08 3 48.79 2.7 0 13.66 4 46.28 4.55 10.10

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210 will require adding the parenchyma component of the root in the feedstock. Peels alone may not be good enough for production of high activity amylolytic enzymes. The s protein) of the enzymes are summarized in Table 5 7. Despite the wide differences in the total activities of the enzymes presented in Table 5 5 and Table 5 6, the specific activities of the enzymes are similar (Table 5 7). This similarity may be attributed to the prote in concent ration in each enzyme solution Table 5 7. Sp e cific Activities of Enzymes Produced by Anaerobic Digestion of Cassava Root Materials (Mean s of duplicate measurement s) S/N Enzyme System Specific Activity [ EU/ protein] 1 Cell Free Enzyme 0.011 0.044 2 Cell Associated (Pellet) Enzyme 0.012 0.033 3 Biofil m Associated Enzyme 0.016 0.065 5.5 Dividends and Applications of Anaerobic Digestion of Cassava Peel Waste Apart from the enzymes that could be derived or harvested from anaerobic digestion of cassava peel waste, other benefits include the biogas generated and the fertilizer value of the post digestion effluent. In this section, we analyze the biomethane ( energy ) potential and applications of t he biogas as well as the fertilizer value of the effluent. 5.5.1 Biomethane Potential and Applications of Biogas M ass balance analysis for processing one tonne (1000 kg) of cassava roots and the anaero bic digestion of generated waste are presented in Fig ure 5 10. At 18.13 % peeling waste capacity, 181.30 kg peel waste wa s generated and subjected to

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211 Figure 5 10. Mass Balance for Anaerobic Digestion of Peel Waste from one tonne (1000 kg) of fresh Cassava R oot anaerobic digestion. About 81.16 % (811.6 g) of the fresh root mass was realized as root pulp or edible parenchyma, while the balance of 0.71 % (7.1 g) represented the lost mass; Table 5 2. The parenchyma could be used to produce chips, flour, starch, glucose, etc. However, anaerob ic digestion of the peel waste generated 10.44 kg (14621 L) of methane with estimated energy value of 581.25 MJ computed as follows. One T onne of Cassava Roots (1000 kg) Peeling Operation Parenchyma: Root Pulp (811.60 kg) Anaerobic Digestion Operation Biogas (42.26 kg) 31.82 kg CO 2 (16199.29 L) 10.44 kg CH 4 (14621 L ) Peel Waste (181.30 kg M o isture (121.09 kg) Dry Matter (60.21 kg) 58.02 kg VS 2.18 kg Ash Effluent (139.01 kg 121.09 kg Liquid 17.92 kg Dry Matter 15.74 kg VS 2.18 kg Ash

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212 The high heating value (heat of combustion) and molecular weight of methane mole (Doran, 1995). Therefore the 10.44 kg of methane will yield: {[(890.8 kJ/g mole)/(16 g/g mole)] x [(10.44 kg CH 4 )(1000 g/kg CH 4 )]} = 581247 kJ or 581.25 MJ of thermal energy. This energy could have at least two possible applications. a. Boil Water: Th e energy [Q (kJ)] required to boil a given mass of water [M (kg)] can be estimated with the equation: Q = MC p v 1) where C p is specific heat of water (4.187 kJ/kg o difference ( o v is the latent heat of vaporization of water (2256.9 kJ/kg). From equation 5 1, to boil 1 kg of water initially at 25 o C, with T = 100 25 or 75 o C requires: {(1 kg x 4.187 kJ/kg o C x 75 o C) + (1 kg x 2256.9 kJ/kg)} = 2570.925 kJ. Consequently, the water that could be boiled with 581247 kJ from the anaerobic digestion of the cassava peel waste is (581247 kJ/2570.925 kJ) x 1 kg = 226.08 kg. b. Generate Electricity: Alternatively, the 581247 kJ (581.247 MJ) energy value of the biomethane can be transformed to electricity A t the conversio n factor of 0.2778 kWH e /MJ and conver sion efficiency of 40 %, the electricity obtained wou ld be {(581.247 MJ x 0.2778 kWH e /MJ) x (40/100)} = 64.588 kWH e This electric energy could be used to light homes in a rural village and enable children to do home work and read/study at night. The Figure 5 11 shows the mass balance for anaerobic digesti on of one tonne (1000 kg) of cassava peel waste. The composition of the fresh peel waste as shown in Figure 5 11A consisted of moisture: 66.8 % (668 kg), volatile solids: 32 % (320 kg), and ash: 1.2 % (12 kg). After the peel waste was subjected to anaerobi c digestion, the results were carbon dioxide, 55 % (176 kg), methane, 18 % (57.6 kg), and residual organic matter, 27 % (86.4 kg); Figure 5 11B. This amount of methane could boil 1247.34 kg of water or generate 356.35 kWH e To achieve this quantity of peel waste would require processing approximately 5515.72 kg of fresh cassava roots.

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213 Figure 5 11. Mass Balance for Anaerobic Digestion of 1 tonne (1000 k g) of Cassava Peel Waste A) Components d istribution of the fresh Cassava Peel Waste before anaerob ic digestion. B) Repartition of Volatile Solids after anaerobic digestion. 5.5.2 Fertilizer Value Potential of Effluent from Mesophilic Anaerobic Digestion of Cassava Peel Waste The liquid effluent from anaerobic digestion of the cassava peel waste was ana lyzed to determine its value as a source of organic fertilizer if applied on farm land. Moisture (668 kg = 66.8%) Volatile Solids (320 kg = 32.0%) Ash (12 kg = 1.2%) A Carbon Dioxide (176.0 kg = 55%) Methane (57.6 kg = 18%) Residual Organic Matter (86.4 kg = 27%) B

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214 The analyses for Nitrogen (N) Phosphorus (P) and Pota ssium (K) and other constituents were conducted according to the methodologies presented in Table 5 8. Table 5 8 Methodologies used for the Analysis of Fertilizer Components of Post Anaerobic Digestion Li quor. (All the fertilizer component analyses were carried out by the University of Florida Institute of Food and Agricultural Sciences Analytical Services Laborato ries) S/N Fertilizer Components Analytical Methods and Instruments 1 Ammonia EPA 350.1 on a Continuous Flow Autoanalyzer. Alpkem Flow IV (O I Analytical, College Station, TX 77842) 2 Nitrate + Nitrite EPA 353.2 on a Continuous Flow Autoanalyzer. Alpk em Flow IV (O I Analytical, College Station, TX 77842) 3 Total Kjeldahl Nitrogen EPA 351.2 on a Continuous Flow Autoanalyzer. A2 Analyzer (Astoria Pacific International, Clackamas, OR 97015) 4 Metals EPA 200.7 on an Inductively Coupled Plasma Spectro photometer (ICP). Spectro Arcos (Spectro Analytical Instruments, Mahwah, NJ 07430) 5 Ortho Phosphorus EPA 365.1 on an AQ2 Discrete Analyzer (Seal Analytical, Mequon, WI 53092) 6 Total Phosphorus EPA 365.1 on a Continuous Flow Autoanalyzer. Alpkem Flo w IV (O I Analytical, College Station, TX 77842) The results showed that each liter of effluent contained 572.7 mg, 30890 g, and 1066 mg of ( N ) ( P ) and ( K ) respectively. It h as been estimated that 600 kg of NPK (15: 15: 15) fertilizer per hectare i s required to produce 25 tonnes of cassava roots ( http://cassavabiz.org/agroenterprise/farming.htm [Accessed 10/17/ 2013 ] ). It is also reported that the fertilizer nutrients are available to plants as elemental nitrogen for ( N );

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215 as P 2 O 5 for (P) ; and as K 2 O for (K) ( http://en.wikipedia.org/wiki/NPK_rating [Accessed 10/27/2013]). B ased on these data, we compute that 24 kg of NPK (15: 15: 15) fertilizer is required to produce one tonne of cassava roots; with the 15 % of (N) supplied or available as elemental ni trogen ; the 15 % of (P) available as P 2 O 5 ; and the 15 % of (K) available as K 2 O. 5.5.2.1 Nitrogen The nitrogen requirement is estimated as: N itrogen = 15 % [ 24 k g ( elemental N ) /t cassava root] = 3.6 kg ( N ) /t cassava root. Nitrogen that can be obtained fro m digestion is estimated as: From effluent analysis, ( N ) concentration = 572.7 mg/L. Thus for the 3 L of digester volume, ( N ) = 3 L (572.7 mg/L) = 1718.1 mg. Nitrogen fertilizer value of the effluent can now be estimated. Note that 1718.1 mg is mass of (N) in the dry ma t ter of 4 experimental runs. Since the dry ma t ter was 254.8 g, the mass of (N) per unit dry ma t ter = 1718.1 mg ( N ) /254.8 g dry Wt. x (1 g/1000 mg) = 0.00674 g ( N ) /g dry Wt. However, for 1tonne of roots, only 60.21 k g dry matter was generat ed. This dry matter was fed the digester, see mass balance schematics ; Figure 5 10. Consequently, potential (N) = 0.00674 g ( N ) /g dry Wt x (60.21 kg dry Wt.) = 0.4058 kg (N) But 3.6 kg (N) is required to produce one tonne of cassava roots. Therefore propor tion of (N) met by anaerobic digestion = 0.4058 kg (N)/ 3.6 kg (N) x (100) = 11.27 %

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216 5.5.2.2 Phosphorus The phosphorus requirement is estimated as: Phosphorus = 15 % [ 24 kg ( P 2 O 5 ) /t cassava root] = {[ 3.6 kg ( P 2 O 5 ) /t cassava root] x [62 kg (elemental P)/142 kg ( P 2 O 5 )]} = 1.5718 kg ( P ) /t cassava root. Phosphorus that can be obtained from digestion is estimated as: From effluent analysis, ( P ) concentration = 30890 g /L Thus f or the 3 L of digester volume, ( P ) = 3 L ( 30890 g /L ) = 92670 g or 92.67 mg. Phosp horus fertilizer value of the effluent can now be est imated. The 92.67 mg is mass of (P) in the dry matter of 4 experimental runs. Since the dry matter was 254.8 g the mass of (P ) per unit dry matter = 92.67 mg (P) /254.8 g dry Wt. x (1 g/1000 mg) = 0.00 0363 g (P) /g dry Wt. However, for 1tonne of roots, only 60.21 kg dry matter was generated. This dry matter was fed the digester, see mass balance schematics; Figure 5 10. Consequently, potential (P ) = 0.000363 g (P) /g dry Wt x (60.21 kg dry Wt.) = 0.021 8 9 8 k g (P ) But 1.5718 kg (P ) is required to produce one tonne of cassava roots. Therefore proportion of (P ) met by anaerobic digestion = 0.02189 8 k g (P )/ 1.5718 kg (P ) 1. 40 % 5.5.2.3 Potassium The potassium requirement is estimated as: Potassium = 15 % [24 kg ( K 2 O) /t cassava root] = {[3.6 kg ( K 2 O) /t cassava root] x [78 kg (elemental K)/94 kg (K 2 O )]}

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217 = 2.9872 kg (K) /t cassava root. Potassium that can be obtained from digestion is estimated as: From effluent analysis, ( K ) concentration = 1066 mg/L T hus f or the 3 L of digester volume, ( K ) = 3 L ( 1066 mg/L ) = 3198 mg. Potassium fertilizer value of the effluent can be estimated thus : We now have 3198 mg of (K) in the dry matter of 4 experimental runs. Since the dry matter was 254.8 g, the mass of ( K ) per unit dry matter = 3198 mg ( K )/254.8 g dry Wt. x (1 g/1000 mg) = 0.0 1255 g ( K )/g dry Wt. However, for 1tonne of roots, only 60.21 kg dry matter was generated. This dry matter was fed the digester, see mass balance schematics; Figure 5 10. Consequently potential ( K ) = 0.01255 g ( K )/g dry Wt x (60.21 kg dry Wt.) = 0. 755696 kg ( K ) But 2 9872 kg ( K ) is required to produce one tonne of cassava roots. Therefore proportion of ( K ) met by anaerobic digestion = 0. 7556 9 6 kg ( K )/ 2 9 8 72 kg ( K ) 30 % The organic fertilizer generation capability of anaerobic digestion of cassava peel waste is summarized in Table 5 9 Obviously, anaerobic digestion can comfortably satisfy at least one quarter of the potassium requirement.

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218 Table 5 9 Organic Fertilizer Potential of Anaerobic Digestion of Cassava Peel Waste S/N Organic Fertilizer Element Quantity Required [kg/tonne of Cassava Root Produced] Quantity Generated by Anaerobic Digestion of Cassava Peel Waste [kg/tonne of Cassava Root Pr ocessed] Proportion of Fertilizer Requirement Met [%] 1 Nitrogen (N) 3.60 0.406 11.30 2 Phosphorus (P) 1.57 0.022 1.40 3 Potassium (P) 2.99 0.756 25.30

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219 CHAPTER 6 CONCLUSIONS AND RE COMMENDATIONS 6.1 Conclusions The researches conduc ted for this doctoral dissertation demonstrated several value added potentials for the cassava crop. Namely dehydration of cassava root parenchyma to produce chips using solar convection dryer; production of cassava flour from the solar convection dried ca ssava chips ; glucose sweetener production by s imultaneous and s ynergistic enzymatic hydrolysis of native unprocessed cassava starch ; as well as anaerobic digestion of cassava waste to produce biomethane and amylolytic enzymes. Based on these demonstrations the foll ow ing conclusions can be drawn. 1 Fresh cassava roots can be processed into flour from solar convection dried cassava chips. The drying process does not require any payment for fuel or electricity. 2 The characterized physical properties of the chips and flour such as sorption isotherm, drying curve, moisture content, particle size distribution, bulk density, solid particle density, porosity, permeability and specific surface area ar e beneficial to the execution of engineering design of storage, handl ing, and processing systems 3 Simultaneous and synergistic enzymatic hydrolysis of cassava starch is possible. In this technique, liquefaction and saccharification processes are executed as a single unit operation. 4 The s imultaneous and s ynergistic applicati on of fungal gluco amylase (FGA) and bacterial alpha amylase (BAA) at ratio of 3:1 (FGA:BAA), yielded 96 %,

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220 27 %, and 53 % conversion of starch to glucose at 60 o C after 24 hou rs of hydrolysis respectively for commercial cassava starch, flour produced fro m solar convection dried cassava chips and fresh cassava root pulp. 5 At 37 o C operating temperature and 96 hours incubation time, the synergistic hydrolysis proto col achieved 100 %, 72 %, and 55 % conversion of starch to glucose respectively for commerci al cassava starch, flour produced from solar convection dried cassava chips, and fresh cassava root pulp. 6 Glucose sweetener can be obtained by direct conversion of cassava starch substrate. With this technique cassava root pulp or flour can be hydrolyzed directly to glucose without going through the expensive, time and energy intensive starch extraction process 7 The simultaneous/synergistic and direct conversion approaches minimize energy, equipment and technical inputs, as well as the overall cost hithe rto required for t he production of sweeteners by first extracting the starch from cassava root. 8 F irst order kinetic parameters (rate constant along with the Arrhenius activation energy) were determined for the enzyme hydrolysis of each substrate at the tw o different temperatures 9 Cassava root processing wastes such as peels can be converted to bio methane via anaerobic digestion. The methane is a clean and renewable energy that could reduce dependence on fossil fuels and thus mitigate associated adverse e nvironmental impacts.

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221 10 The biomethane may be used to heat/boil water or generate electricity. The electrical power can help pupils in villages d o their homework and study at n i ght. 11 Processing one tonne (1000 kg) of fresh cassava root creates 181.30 kg of peel waste. The waste can be digested in anaerobic reactor to generate 10.44 kg (14621 L) of methane with energy value of 581.25 MJ. This thermal energy can boil 226.08 kg of water which was initially at 25 o C. Alternatively; the thermal energy could be used to generate 64.58 kWH e for illumination or motive power applications. 12 The post digestion effluent can be used as source of organic fertilizer to supplement the nitrogen (at 11 %) and potassium (at 25 %) requirements for cassava cultivation. 13 The anaer obic digestion of cassava waste demonstrated viability of digester reactor operation with the following performance indices. Organic load of 20 gVS/m 3 (gram volatile solids/cubic meter); residence time of 27 days; bio methane yield of 0.25 L/gVS (liter/ gr am volatile solids); and bio methane production efficiency of 71 %. 14 It is possible to obtain amylolytic (starch hydrolyzing) enzymes from mesophilic anaerobic digestion of cassava waste. 15 Extracellular enzymes we re located in the three divisions defined a s cell free (leachate) enzyme; cell associated (pellet) enzyme; and biofilm associated enzyme

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222 16 Beta a mylase and Glucoamylase activities were identified in the cell free, cell associated and biofilm associated portions of the digester substrate. 17 Anaerobi c digestion of pieces of whole unpeeled cassava root resulted in much higher activities than those obtained with the peel waste alone. 18 The highest activity was found in the cell free portion, followed by the biofilm associated portion. The cell associat ed portion had minimal activity. 19 The activity of the cell free enzyme was 1.5 to 3.0 t imes higher than that of the biofilm associated enzyme at 13 93 EU to 68.93 EU and 9.17 EU to 26.08 EU respectively for the cell free and biofilm associated enzymes 20 Th e specific activities (EU/ cell free, cell associated and biofilm associated were found to be similar. 21 The processes demonstrated can be accommodated by developing nations to foster appropriate technologies for cottage industries, encourage t he use of indigenous raw materials, enable local capacity building, as well as promote economic empowerment 6.2 R ecommendations for Future Work This is an exciting endeavor in that we are engaged in a new research frontier of simultaneous and synergistic hydrolysis reaction s as well as mesophili c anaerobic digestion of agricultural and biological ma te rials The results of our research can be applied by people with low income or financial resources and little or no technical skills anywhere in the world fo r the production of flour, sweeteners biomethane and enzymes that could be used by the households and industries However, in order to maximize the value adding potentials of these endeavors, optimization of the research demonstrations are urged. Areas to be considered for further/future work include:

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223 Development and implementation of purification system for the digester derived enzymes Evaluation of activity and stability of the digester derived e nzymes at different temperatures. Evalua tion of the c apability of the digester derived enzymes to hydrolyze native un extracted starch in cassava flour and root pulp. To u nderstand and characterize the relationship, if any, between biomethane production, enzyme production and enzyme activity. Develop ment o f an economic model with comprehensive plant design and cost benefit analysis for a closed loop system that simultaneously produces biomethane and amylolytic enzymes. The enzymes would be used for hydrolysis of cassava starch for the production of sweetene rs while the methane would be used to heat both the anaerobic digester and the hydrolysis reactors.

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224 APPENDIX A PHOTOGRAPHS OF SOME MATERIALS AND EQUIPMENT USED AND THE PRODUCTS CREATED A 1. Solar Convection Dryer All the pho tos courtesy of the author unless otherwise indicated.

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225 A 2. Cassava C hips that were Dried by Solar Convection Dryer

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226 A 3. Flo ur from Cassava C hips that were Dried by Solar Convection Dryer

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227 A 4. Electric C onvection O ven

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228 A 5 Cassava Chips Dried by Electric Convection Oven

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229 A 6 Flour from Cassava C hips that were Dried by E lectric Convection Oven

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230 A 7. Freeze Dryer

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231 A 8. Freeze Dried Cassava Chips

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232 A 9. Cassava Flour Produced from Freeze Dried Cassava C hips

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233 A 10. Cassava C hips that were Dried by D ifferent Drying M ethods

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234 A 11 Flour from Cassava Chips that were Dried by D ifferent Drying M ethods

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235 A 12. Microphotograph of Cassava Flour

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236 A 13 Glucose Syrup [27.7 o Brix] Produced by Synergistic Enzymatic Hydrolysis of C assava S ubstrates (Commercial S tarch, Flour, and Fresh R oots]

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237 A 14 Glucose Syrup [39.3 o Brix] P ro duced by Synergistic Enzymatic H yd rolysis of Cassava Substrates (Commercial Starch, Flour, and Fresh R oots]

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238 A 15 Glucose Syrup [42 o Brix] Produced by Synergisti c Enzymatic H yd rolysis of Cassava Substrates (C omme rcial Starch, Flour, and Fresh R oots]

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239 A 16 Glucose Syrup [53 o Brix] P ro duced by Synergistic Enzymatic H ydrolysis of Cassava Substrates (Commercial Starch, Flour, and Fresh R oots]

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240 A 17 Glucose Syrup of Various Soluble S olids [ o Brix] Produced by S yner gistic Enzymatic Hydrolysis of C assava Substrates (Commercial Starch, Flour, and Fresh R oots]

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241 A 1 8 Various Packaging Options for Value Added Cassava P roducts : Chips; Flour, Glucose S yrup

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242 A 19 ASTM Standard Sieves Used for Particle Size A nalysis

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243 A 20 Incubators for Anaerobic Digestion Reactors

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244 A 21 Mesophilic Anaerobic Digester Incubating at 26 3 o C

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24 5 A 2 2 Cylinders for Carrier Gases used with GC E quipment (background) for Biogas A nalysis

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246 A 23 Electric Range and Pots Used to Concentrate Glucose S yrups

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247 A 24 Mason Jars U sed for Packaging and Canning Cassava P roducts

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248 A 25 Walk in F reezer Used to Freeze S amples at 20 o C

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249 A 26 Vortex Machines Used to H omogeniz e Research Samples for A nalysis

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250 A 27 Rack, Test Tubes and S tir P late/ Heater U sed to Hold and Prepare Sample s for A nalysis

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251 A 28 D esiccator Jars and Cassava Samples Used for S orption Isotherm Experiments

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252 A 29 Weights U se d as Ballast to Stabilize Research Samples under C entrifugal Force F ield

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253 A 30. Components of Cassava Root Lost Mass: 0.71 % Heads & Tails: 1 .71 % Periderm: 3.31 % Cortex: 13.11 % Parenchyma: 81.16 %

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254 A 31. Heads & Tails of Cassava Root

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255 A 32 Periderm/Epidermis (Bark or Outer Coat) of Cassava Root

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256 A 33. Cortex o f Cassava Root

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257 A 34. Parenchyma (Pulp) of Cassava Root; Frozen Slices

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258 A 3 5 Cassava Root Peeling Waste (Heads & Tails, Periderm and Cortex) used as Feedstock/Substrate in Anaerobic Digestion Experiments for Biomet hane and Enzyme Production

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259 A 36. C ell Free Enzyme Derived from Anaerobic Digestion of Cassava Waste

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260 A 37. Cell Associated (Pellet) Enzyme Derived from Anaerobic Digestion of Cassava Waste

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261 A 38. Biofilm Associated Enzyme Derived from Anaerobic Digestion of Cassava Waste

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262 A 39. PhD Candidate & Graduate Committee Members [Front row from Right to Left: Dr Wade Yang, Member; Dr Arthur A Teixeira, Chairman; Sammy Aso, PhD Candidate. Back row from left to right: Dr Bruce A Welt, Mem ber; Dr Robert P Bates, Member; Dr Spyros A Svoronos, Member, and Dr Pratap C Pullammanappallil, Member

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263 A 40. Graduate Committee Members

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264 A 41 nalysis

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265 APPENDIX B RECOMMENDED REFERENCES FOR FUTHER READING B 1. Catalytic Efficiency http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Complex_Reactions/Catalytic_ efficiency_of_enzymes [Accessed Friday 27th April 2012] B 2. Enzyme Kinetics http://graphpad.com/curvefit/introduction63.htm [Accessed Friday 27th April 2012] http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html [Accessed Friday 27th April 2012] http://class.fst.ohio state.edu/fst605/605p/Enzymekinetics.pdf [Accessed Friday 27th April 2012] recommended B 3. Ultrahigh Pressure and C assava G, A.T Fonseca, AT, and Ferreira, JMF. ( 1998 ) Processing of porous cordierite bodies by starch consolidation Materials research bulletin, 33 (10 ) : 1439 1448 Cereda, MP and Mattos, MCY. ( 1996 ) Linamarin The Toxic Compound of Cassava. Journal of Venomous Animals and Toxins 2 (1): version ISSN 0104 7930 Che L, Li D, Wang L zkan N, Chen XD and Mao Z. ( 2007 ) Effect of High Pressure Homogenization on the Structure of Cassava Starch. International Journal of Food Properties, 10 (4): 911 922. Kasemwong K, Ruktanonchai, UR, Srinuanchai, W, Itthisoponkul, T and Sriroth, K. ( 2011 ) Effect of high pressure microfluidization on the structure of cassava starch granule. Starch Strke, 63 (3) : 160 170, March 2011 B 4. Pulsed UV Light and Cassava Bertolini AC Mestres C Colonna P ( 2000 ) Rheological properties of acidified and UV irradiated starches Starch/Strke 52 : 340 4 Bertolini AC Mestres C Colonna P Raffi J ( 2001 A) Free radical formation in UV and gamma irradiated cassava sta rch Carbohydrate Polymers, 44 : 269 71

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266 Bertolini AC Mestres C Raffi J Bulon A Lerner D Colonna P ( 2001B) Photodegradation of cassava and corn starches J ournal of Agricultural and Food Chemistry, 49 : 675 82 Bhat, R and Karim, AA. ( 2009 ) Impact of Radiation Processing on Starch. Comprehensive Reviews in Food Science and Food Safety, 8 (2) : 44 58. Also available at: http://onlinelibrary.wiley.com/doi/10.1 111/j.1541 4337.2008.00066.x/full#b30 Franco, CML, Ogawa, C, Rabachini, T, Rocha, TS, Cereda, MP and Jane, J. ( 2010 ) Effect of lactic acid and UV irradiation on the cassava and corn starches. Brazilian Archives of Biology and Technology 53 (2): HTML Version Zucca C ( 1953 ) Mucinolytic action of ultraviolet rays on som e polysaccharides 1st. Botan. University Lab. crittoge Pavia Atti Vol. 10 85 p. B 5. Gam ma Irr adiation [Flours a nd Starches ] Greenwood CT Mackenzie C ( 1963 ) The irradiation of the starch Part I: the properties of potato starch and its components after irradiation with high energy electron Die Starke 15 : 359 63 Kume T Tamura N ( 1987 ) C hange in digestibility of raw starch by gamma irradiation Starch 39 : 71 4 Adeil Pietranera MS Narvaiz P ( 2001 ) Examination of some protective conditions on technological properties of irradiated food grade polysaccharides Radiation Physics and Chemist ry, 60 : 195 201 Rombo GO Taylor JRN Minnaar A ( 2001 ) Effect of irradiation, with and without cooking of maize and kidney bean flours, on porridge viscosity and in vitro starch digestibility Journal of the Science of Food and Agriculture, 81 : 497 502 B 6. Ultrasounds in Cassava Boonapatcharoen N, Meepian K, Chaiprasert P and Techkarnjanaruk S. ( 2007 ) Molecular Monitoring of Microbi al Population Dynamics During Operational Periods of Anaerobic Hybrid Reactor Treating Cassava Starch Wastewater Microbial Ecology 54 (1 ): 21 30 Gao, W, Lin, X, Lin, X, Ding, J, Huang, X and Wu, H. ( 2011 ) Preparation of nano sized flake carboxymethyl cassava starch under ultrasonic irradiation. Carbohydrate Polymers 84 (4 ): 1413 1418. Hao, X, Gu, C, Liu, J, Song, W and Chen, S. ( 2007 ) Study on Properties and Preparation Conditio ns of Cassava Amylodextrins with Acid alcohol Media. Food Science December 2007.

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267 Nit ayavardhana, S, Rakshit, SK, Grewell, D, Leeuwen, JH and Khanal, SK. ( 2008 ) Ultrasound pretreatment of cassava chip slurry to enhance sugar release for subsequent ethanol production. Biotechnology and Bioengineeri ng 101 (3): 487 496. Nitayavardhana, S, Shrestha, P, Rasmussen, ML, Lamsal, BP, Leeuwen, JH and Khanal, SK. ( 2010 ) Ultrasound improved ethanol fermentation fro m cassava chips in cassava based ethanol plants. Bioresource Technology 101 (8 ): 2741 2747 Xiaomi ng, C, Kaimian, L, Jianxiang, T, Jianqiu, Y, Xiaojing, L and Feijie, L. ( 2012 ) Response Surfa ce Methodology on Ultrasonic Extraction Technology of Total Coumarin from Cassava Peel. Transactions of the Chinese Society for Agricultural Machinery 1, 027 (Ja nuary 2012). OR? {Jianxiang, C. X. L. K. T., & Feijie, Y. J. L. X. L. (2012). Response Surface Methodology on Ultrasonic Extraction Technology of Total Coumarin from Cassava Peel. Transactions of the Chinese Society for Agricultural Machinery 1 027.} B 7. Statistics References Box, GEP ( 1999 ) Statistics as a Catalyst to Learning by Scientific Method P art II A Discussion. Journal of Q uality Technology Vol. 31, No.1 Pp 16 29. Box, GEP and Liu, PYT ( 1999 ) Statistics as a Catalyst to Learning by Scientific Method Part I -An Example. Journal of Quality Technology Vol. 31, No.1 Pp 1 15. Box, GEP and Wilson, KB. ( 1951 ) On the Experimental Attainment of Optimum Conditions. Journal of the Royal Statistical Society Series B (Methodological), Vol. 13, No. 1. Pp 1 45. Hill, WJ and Hunter, WG. ( 1966 ) A Review of Response Surface Methodology: A Literature Survey. Te chnometrics Vol. 8 No. 4, Pp. 571 590. Li, W, Du, W and Liu, DH. ( 2007 ) Optimization of whole cell catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology. Journal of Molecular Catalysis B: Enzymatic Vol. 45 Nos. 3 4, Pp. 122 127. Mead, R and Pike, DJ. 1975. A biometrics invited paper. A review of response surface methodology from a biometric viewpoint Biometrics Vol. 31 No. 4, Pp. 803 851. Mon tgomery, DC. ( 2009 ) Design and Analysis of Experiments, 7 th Edition. John Wiley and Sons, Inc.

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268 Myers, RH, Khuri, AI and Carter, WH. ( 1989 ) Response surface methodology: 1966 1988. Technometrics Vol. 31 No. 2 Pp. 137 157. Myers, RH, Montgomery, DC, V ining, GG, Borror, CM and Kowalski, SM. ( 2004 ) Response surface methodology: A retrospective and literature survey. Journal of Quality Technology Vol. 36 No. 1. Pp 53 77. Naveena, BJ, Altaf, Md, Bhadrayya, K, Madhavendra, SS and Reddy, G. ( 2005 ) Direc t fermentation of starch to 1(+) lactic acid in SSF by Lactobacillus amylophilus GV6 using wheat bran as support and substrate: medium optimization using RSM. Process Biochemistry Vol. 40 No. 2, Pp. 681 690. Noordin, MY, Venkatesh, VC, Sharif, S, Eltin g, S and Abdullah, A. ( 2004 ) Application of response surface methodology in describing the performance of coated carbide tools when turning AISI 1045 steel. Journal of Materials Processing Technology Vol. 145 No.1, Pp. 46 58. B 8. Fermentation/Hydrol ysis Papers Adney, WS, Rivard, CJ, Shiang, M and Himmel, ME. ( 1991 ) Anaerobic Digestion of Lignocellulosic Biomass and Wastes. Cellulases and Related Enzymes. Applied Biochemistry and Biotechnology Vol. 30, No. 2, Pp. 165 183. Balan V, Bals B, Chundawat SPS, Marshall D and Dale BE. ( 2009 ) Lignocellulosic Biomass Pretreatment Using AFEX. In Biofuels, Methods and Protocols. Vol. 581, Pp 61 77. Jonathan R. Mielenz (Ed.). Eveleigh, DE, Mandels, M, Andreotti, R and Roche, C. ( 2009 ) Measurement of saccharifying cellulo se. Biotechnology for Biofuels 2:21 doi: 10.1186/1754 6834 2 21. Also available online at: http://www.biotechnologyforbiofuels.com/content/2/1/21 Ghose, TK. ( 1987 ) Measurement of Cellulase Activities. Pure & Applied Chem istry 59 ( 2 ), 257 268. Gomec CY Kim, M, Ahn, Y and Speece, RE. ( 2002 ) The role of pH in mesophilic anaerobic sludge solubilization. Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering Vol. 37 No. 10, Pp 1871 1878 Liaw, WC, C hen, CS, Chang, WS and Chen, KP. ( 2008 ) Xylitol production from rice straw hemicellulose hydrolyzate by polyacrylic hydrogel thin films with immobilized candida subtropicalis WF79. Journal of Bioscience and Bioengineering Vol. 105 No. 2, Pp. 97 105. Ol sson, L and Hahn Hgerdal, B. ( 1996 ) Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microbial Technology 18 ( 5 ), 312 331.

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322 BIOGRAPHICAL SKETCH Samuel NwaneLe Aso [Sammy ] was born in the Village of Elelenwo, R ivers State, Nigeria. H e started elementary education in the then St. Marks School Elelenwo. His elementary education was interrupted by the Nigerian civil war from 1966 to 1969. In 1970 he resumed elementary education and received the first sc hool leaving certificate in 1971. Sammy enrolled into County Grammar School Ikwerre/Etche in 1972 for secondary education; graduating in 1976 with the West African Examinations Council (WAEC) certificate for secondary education. In the fall of 1977 he enro lled into Utah State University, Logan, but transferred to The University of Tennessee, Knoxville, in the summ er of 1978. Sammy received his b December 1981 and enrolled into t he m degree program in the spring of 1982 at the same institution. He received the Master of Science degree with concentration in food engineering in August 1983. Sammy immediately returned to Nigeria and on completion of National Youth Service Cor ps (NYSC) program in 1984, served as the general manager of Erijoy Nigeria Limited, a multi branch hotel/catering firm situated in Port Harcourt, Nigeria. He joined Rivers State University of Science and Technology (RSUST) in 1991 as assistant lecturer. Sa mmy rose through the ranks and became a senior lecturer in food engineering at RSUST by 2006. In the spring of 2010, He commenced this Doctor of Philosophy degree program in food engineering at the University of Florida. The research interests of Sammy inc lude life cycle assessment (LCA) of food engineering systems; food irradiation; ohmic food processing; high pressure food processing; heat pipe technology; and the engineering of consumables (especially food)

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323 for extraterrestrial body surface and micro gra vity environments such as Space Shuttle Orbiters, International Space Station, Lunar and Martian Outposts, Asteroid Mining Bases, as well as other ETI and Planet Surface habitats. Sammy is a deep space enthusiast and lover of space mementos.