OPTIMIZATION OF ETHANOL YIELD FROM CASSAVA T.Y Nquma (BSc. Hons) Dissertation submitted in fulfillment of the reqeumirents for the degree of Master of Science in Chemical Engineering of the North Wensitv Uersity (Potchefstroom Campus) Supervisor : Prof S. Marx Co-Supervisor : Dr G. Obiero November 2009 Potchefstroom Abstract ABSTRACT The energy crisis and worldwide economic depres hsaiosn highlighted the production of biofuels from agricultural materials as an imtpaonrt national policy. Cassava, a root plant indigenous to Africa, is not cultivatecdo mmercially in South Africa because it is not a staple food source and con staoimnse cyanide components in its raw form. Cassava is mostly grown as a food sumpepnlet by informal households. Cassava roots are rich in starch (approximately )8 a0n%d are therefore an excellent candidate for the production of bio-ethanol in Sho Auftrica. In this research, Cassava roots, which consist mostly of starch, as well haes pteels, which consist of cellulose, were converted to bio-ethanol. As a baseline, tahses Cava starch and the peels were converted to ethanol separately by using traditl iopnraetreatment methods a nd Saccharomyces cerevis iaes yeast. The hydrolysis process for starch wpatism oized with respect to substrate concentration, enzymec ecnotnration, enzyme combination, treatment temperature and pH of the different psrso cseteps. The best fermentation step was determined through fermentation of theim oizpetd starch hydrolysate using the separated hydrolysis and fermentation proceSsHs F)(, the simultaneous saccharification and fermentation process (SSFw) ealls a s a direct fermentation (DF) process from the raw starch usinSgc hwanniomyces occidental(iAs TCC 26076). Cassava roots (starch) and peels (cellulose) wheeren tpretreated and fermented simultaneously using different combinations of emnzeys. A substrate concentration of 20 wt% biomass gave the highest glucose conceonntr aint ithe final hydrolysate, while the best enzyme concentration was found to be 0fo.2r% T ermamyl SC, 0.25% for Spirizyme fuel and 0.1% for Celluclast 1.5L. Thiqeu el faction and saccharification treatment temperature that gave the highest et hyaineoldl were 95°C and 55°C respectively. The best pH for the two hydrolysteisp s was found to be 6 and 4.5 for the liquefaction and saccharification steps resivpelcyt. The optimum pretreatment conditions with a substrate concentration of 20wty%ie lded a final glucose concentration of 141 g-.1L (Yp/s = 0.7 g.g -1) for Cassava starch, 109 g-1. L(Yp/s =0.55g.g-1) for Cassava cellulose (peels) and for the simnuelotaus conversion of both the starch and cellulose, a final glucose concteionntr aof 184 g.L-1 (Yp/s = 0.9 g.g -1) was obtained. It can be concluded from these trse sthual t unpeeled Cassava roots (starch and cellulose) yield a higher final gluc ocsoencentration in the final hydrolysate than converting the cellulose (peenlsd) satarch (peeled roots) separately. ii Abstract This means that it is more productive and econolm toic ause unpeeled Cassava roots with the correct combination of starch and celluel oesnzymes to produce a glucose rich hydrolysate for ethanol production throughm ferntation. The direct fermentation (DF) process yielded the lowest final ethanol conntrcaetion (0.14%) resulting in a yield coefficient (Yp/s) of just 1 %. The SHF process yielded 9.6 % (v(Y/vp)/ s = 0.38 g.g-1) ethanol for Cassava starch and 10.6 % (v/v -p)/s (=Y 0.42 g.g1) for both roots and peels (starch and cellulose) after 48 houmrse fnetration. The SSF process resulted in a final ethanol yield of 7 % (v/v) (pY/s = 0.3g.g -1) for Cassava starch, 4% (v/v) p(/sY = 0.16 g.g-1) for Cassava peels (cellulose) and 10.6% (v/v) -1p/ s (=Y 0.42 g.g) for unpeeled Cassava roots (starch and cellulose).e T rheessults demonstrate that Cassava waste (peels) can be used as an alternative bio mfoar sbsio-ethanol production. However, the SSF process for unpeeled Cassava rreosoutlsts in a higher ethanol yield than processing the peels and starch separate lyth aend combining the hydrolysates only for the fermentation step. It also became envti dthat Cassava containing approximately 85% (g/g) starch is a good feedstfocrk b io-ethanol production in South Africa. Keywords: Cassava, Liquefaction, Saccharification, Fermeionnta, tGlucose, Ethanol iii Opsomming OPSOMMING Die energiekrisis en wêreldwye ekonomiese depr eshseite die produksie van biobrandstof van landboumateriale op die voorgrogneds kuif as ‘n belangrike nasionale beleid. Cassava, ‘n wortelplant inheeamans Afrika, word nie in Suid- Afrika kommersieel verbou nie omdat dit nie ‘n setlavpoedsel is nie en sianiedkomponente bevat in sy rou vorm. Cassava d wmoreestal as ‘n voedingsaanvulling geplant deur informele huishnogusd.i Cassavawortels is ryk aan stysel (ongeveer 80%) en is daarom ‘n uitstekenadned ikdaat vir die produksie van bio-etanol in Suid-Afrika. In hierdie navorsing wdo Cr assavawortels, wat meestal uit stysel bestaan, sowel as die skille, wat meest asl eulliulose bestaan, omgeskakel in bio-etanol. As ‘n basislyn is die Cassavastysel deien skille apart tot etanol omgeskakel deur tradisionele voorbehandelingsmes teondS eaccharomyces cerevis iae as gis te gebruik. Die hidroliseproses vir styse lg ei optimiseer met betrekking tot substraatkonsentrasie, ensiemkonsentrasie, ensmiebminkaosie, behandelingstempe- ratuur en pH van die verskillende prosesseringpsset.a Dp ie beste fermentasiestap is bepaal deur die fermentasie van die geoptimisesetrydse l hidrolisaat deur die gebruik van ‘n afsonderlike hidrolise en fermentasiepro(sSeHsF ), die simultane sakkarifikasie en fermentasieproses (SSF) sowel as ‘n direktee fnetramsieproses (DF) van die stysel deur die gebruik vanS chwanniomyces occidentalis (ATCC 260. 7 6C)assavawortels (stysel) en skille (sellulose) is voorbehandel enfe rgmenteer deur die gebruik van verskillende kombinansies ensieme. ‘n Substraast eknotnrasie van 20 wt% biomassa het die hoogste glukosekombinasie in die finaler ohliisdaat gelewer, terwyl die beste ensiemkonsentrasie geblyk het te wees 0.2% vir aTmeryml SC, 0.25% vir Spirizyme brandstof en 0.1% vir Celluclast 1.5L. Die vervilnogesi en sakkarifikasie- behandelingstemperatuur wat die meeste etanol geer lehwet was 95°C en 55°C onderskeidelik. Die beste pH vir die twee hidreoslitsappe bleik te wees 6 en 4.5 vir die vervloeiing en sakkarifikasiestappe ondersklieki.d e Die optimum voorbehandelingskondisies met ‘n substraatkonsseinet rvaan 20wt% het ‘n finale glukosekonsentraat gelewer van 141- 1g (.YLp/s = 0.7 g.g -1) vir Cassavastysel, 109 g- .L 1 (Yp/s =0.55g.g -1) vir Cassavasellulose (skille) en vir die simuelt aonmskakeling van beide die stysel en sellulose, ‘n finale glukoseskeonntraat van 184 g-.1L (Yp/s = 0.9 g.g-1). Dit kan uit die resultate afgelei word dat onkgiledse Cassavawortels (stysel en sellulose) ‘n hoër finale glukosekonsentraat in fdiniaele hidrolisaat gelewer het as om iv Opsomming die sellulose (skille) en stysel (geskilde wort ealsp)art te gebruik. Dit beteken dat dit meer produktief en ekonomies sal wees om onges kCiladsesavawortels met die korrekte kombinasie van stysel en sellulose-ens ietem geebruik om ‘n glukoseryke hidrolisaat te lewer vir etanolproduksie deur fenrmtaesie. Die direkte fermentasieproses (DF) het die laagste finale elktoansoentraat gelewer (0.14%) wat uitloop of ‘n leweringskoeffisient (pY/s) van net 1 %. Die SHF proses het 9.6 % (v/v) (Yp/s = 0.38 g.g -1) etanol vir Cassavastysel gelewer en 10.6 % ((vY/vp/)s = 0.42 g.g -1) vir beide wortels en skille (stysel en sellulosea) n48 ure van fermentasie. Die SSF proses het uitgeloop op ‘n finale etanolleweringn v7a % (v/v) (Yp/s = 0.3g.g -1) vir Cassavastysel, 4% (v/v) p(/Ys = 0.16 g.g -1) vir Cassavaskille (sellulose) en 10.6% (v/v) (Y -1p/s = 0.42 g.g) vir ongeskilde Cassavawortels (stysel en selelu)l.o Hs ierdie resultate demonstreer dat Cassava-afval (skilleb)r ugike kan word as alternatiewe biomassa vir bio-etanol produksie. Die SSF prosier so nvgeskilde Cassavawortels lewer egter ‘n hoër etanol hoeveelheid as om diilele sekn stysel apart te prosesser en dan die hidrolisate slegs vir die fermentasieseta pk otmbineer. Dit het ook duidelik geword dat Cassava wat omtrent 85% (g/g) styseal tb ‘env goeie voedingstof vir bio- etanolproduksie in Suid-Afrika is. Verworde: Cassava, Vervloeiings, Sakkarifikasie, Fermen,t aGsliuekose, Etanol v Declaration Declaration I Tandokazi Yvonne Nquma hereby declare that I ahme stole author of this dissertation ----------------------------------------- Tandokazi Yvonne Nquma. vi Acknowledgements Acknowledgements “Give thanks in all Circumstances” This work has been made possible by a team of riantsiopni , insights and prayers of distinctive individuals sent by the most sustain minagn in this research God Himself. To God I give thanks, honor and glory for the atyb iHli e gave me to make it through my research on time. Indeed my God is the greaetnegsitn eer. To my wonderful supervisor, Prof Sanette Marx, tIe enxd my unlimited gratitude for the opportunity you gave me to become a bettern tsifcicie researcher. You inspired me as a woman scientist and mostly an in sighted. on To SANERI (CRSES), Thank you so much for your ficnianl support in this work and the opportunity you afforded me to further my car.r ee To Dr George Obiero, thank you for your advices raencdommendations on my work, they have been valuable. To all my lab mates, fellow Masters students anrds opnenel in the School of Chemical and Minerals Engineering, thank you for your inp auntsd help in all aspects in this work. To my mother Mavis, thank you for your support,e lo avnd inspiration you gave me to carry out this work. You are my pride. To my father Jongudumo, thank you for motivating tmo ebetter my career, you are a God given gift in my life. To all my friends, thank you for your support, lo avend understanding when life became sour and reminding me to pray, laugh anody etnhje simple things in life. vii Table of contents TABLE OF CONTENTS TITLE PAGE i ABSTRACT ii OPSOMMING iv DECLARATION vi ACKNOWLEDGEMENTS vii TABLE OF CONTENTS viii NOMENCLATURE xi LIST OF TABLES xii LIST OF FIGURES xiv CHAPTER 1: GENERAL INTRODUCTION Overview……………………………………………………………………..1. 1.1 Background and motivation 1 1.2 Bio-ethanol production from biomass 3 1.3 Hypothesis 4 1.4 Research aim 4 1.5 Research objectives 4 1.6 Scope of investigation 5 1.6.1 Optimization of liquefaction and shaacrcification steps 5 1.6.2 Optimization of ethanol yield 5 1.7 REFERENCES 6 CHAPTER 2: BACKGROUND AND LITERATURE SURVEY O verview 8 2.1 Introduction 8 2.2 Biofuels 9 2.3 Bio-ethanol 10 2.4 Bio-ethanol production 10 2.4.1 Bio-ethanol production from sta-rcichh biomass 11 2.4.2 Enzymatic hydrolysis 12 2.4.3 Fermentation with yeasts 12 2.4.3.S1 accharomyces cerevisiae 13 viii Table of contents 2.4.3.S2 chwanniomyces occidentallis 13 2.4.4 Dry milling process 14 2.4.5 Wet milling process 15 2.4.6 Separate hydrolysis and fermeionnt a(tSHF) 16 2.4.7 Simultaneous saccharification faenrmd entation (SSF) 17 2.4.8 Direct fermentation (DF) 17 2.5 Overview of Cassava 18 2.5.1 Cassava production 18 2.5.2 The Cassava plant 19 2.5.3 Cassava stem and leaves 21 2.5.4 Cassava roots and peels 21 2.5.5 Cassava starch 23 2.6 Potential of Cassava for bioethanol pcrotidoun 24 2.6.1 Energy efficiency of cassava 24 2.6.2 Cassava starch fermentation to ethanol 25 2.7 Optimization of ethanol yield from cavsas a ………25 2.8 REFERENCES 26 CHAPTER 3: EXPERIMENTAL Overview 32 3.1 Materials and chemicals 32 3.1.1 Preparation of yeasts 33 3.1.1.S1 accharomyces cerevis iae 33 3.1.1.S2 chwanniomyces occidenta llis 34 3.1.2 Preparation of raw Cassava 34 3.2 Equipment 35 3.3 Experimental error 37 3.4 Determination of complete starch degriaod na…t …………………………..37 3.5 Experimental procedure…………………………………………………..40 3.6 Analytical procedures 42 3.6.1 Compositional analysis of Cassava...…………………………….42 3.6.2 Determination of moisture content of Cas…sa…va…………………..42 3.6.3 Determination of starch content of Cassava……………………...43 3.6.4 HPLC analysis…………………………………………………………44 3.7 Optimization of liquefaction and saccfhicaarition steps 45 3.7.1 Effect of substrate form onc golsue yield 45 3.7.2 Effect of pH on glucose yield 45 3.7.3 Effect of temperature on gluec oyiseld 45 3.7.4 Effect of biomass load on glsuec oyield 46 3.7.5 Effect of enzyme combination g oluncose yield 46 3.7.6 Effect of enzyme load on gluec oyiseld 47 3.8 Optimization of fermentation step 47 3.8.1 Separate hydrolysis and fertmateionn with S.cerevisiae 47 ix Table of contents 3.8.1.1 Effect of yeaosnt centration on glucose yield 48 3.8.2 Simultaneous saccharificaationnd fermentation wit hS.cerevisiae 48 3.8.3 Direct fermentation wiSth. occidentallis 49 3.9 REFERENCES 5 0 CHAPTER 4: RESULTS AND DISCUSSION Overview 52 4.1 Optimization of liquefaction and sacchiacaritfion steps 52 4.1.1 Effect of substrate form on glsuec oyield 52 4.1.2 Effect of pH on glucose yield 54 4.1.3 Effect of temperature on glucyoiseeld 56 4.1.4 Effect of biomass load on gluc yoiseeld 58 4.1.5 Effect of enzyme combination olunc gose yield 59 4.1.6 Effect of enzyme loading on glsuec oyield 60 4.2 Optimization of fermentation step 61 4.2.1 SHF withS .cerevisiae 61 4.2.2 SSF wi tSh.cerevisiae 64 4.2.2.1 Effect of yeast contcraetnion on glucose yield 64 4.2.2.2 Influence of subst rfaotrem on ethanol yield 65 4.2.3 DF withS . occidentallis 68 4.3 Comparison of bio-ethanol production persosces 69 4.4 Summary of hydrolysis and fermentatiosnu rltes 71 4.5. REFERENCES 72 CHAPTER 5: CONCLUSIONS 5.1 Conclusions 76 APPENDIX A: Calibration curves A.1: Sugar calibration curves 78 A.2: Ethanol calibration curve 81 APPENDIX B: Enzymatic Hydrolysis B 1: Optimization of liquefaction and saccharificoant steps 84 APPENDIX C: Fermentation C.1: Separate hydrolysis and fermentation 88 C.2: Simultaneous saccharification and fermennta tio 89 APPENDIX D: Experimental error D1: Liquefaction 95 D2: Saccharification 96 D3: Separate Hydrolysis and Fermentation 97 x Nomenclature NOMENCLATURE Symbol Description Units σ Standard deviation - Y Yield g.g-1 Y -1p/s Yield product per substrate (Cassava) g .g Y -1g/s Yield glucose per substrate (Cassa va) g.g Yp/g Yield product (Ethanol) per glucose -g1. g Z Z score - ± CONFIDENCE - n Number of samples - Mean (Average) - C Concentration gL-.1 W Weight g/ g.L-1 Acronym Description DDGS- Dried Distiller’s Grain with Solubility SSF Simultaneous Saccharification and Fermentation STDEV Standard deviation GC Gas Chromatography HPLC High Pressure Liquid Chromatography C Celluclast 1.5L T Termamyl SC S Spirizyme Fuel ATCC American Type Culture Collection S. occidentallis Schwanniomyces occidenta llis SHF Separate enzymatic Hydrolysis and Fermentation DF Direct Fermentation S. cerevisia e Saccharomyces cerevis iae xi List of tables LIST OF TABLES Table 1.1: Comparative properties of ethanol with petrnodl adiesel 2 Table 2.1: World utilization of cassava 18 Table 2.2: Cassava production and use in 1993 and prodj etoc t2e020 19 Table 2.3: Percentage composition of Cassava plant 20 Table 2.4: A typical composition of Cassava storage root 23 Table 2.5: General properties of Cassava starch 24 Table 3.1: Materials and chemicals used in this study 2 3 Table 3.2: Equipment used in this study 35 Table 3.3: Error percent and product values for SSF 37 Table 3.4: Starch content of hydrolysate degradation dgu sritnarch 38 h ydrolysis to glucose Table 3.5: Composition of Cassava root peels, unpeeleds aCvas root 42 and peeled root Table 3.6: Composition of Cassava root peels, unpeeleds aCvas root 43 and peeled root Table 3.7: Analysis parameters on the HPLC for Cassavaro hl ysdate and 45 fermentation broth Table 4.1: Final glucose yield for different Cassava fo rdmusring liquefaction 53 Table 4.2: Initial glucose production rate (15 min) foifrf edrent substrate 54 forms Table 4.3: Effect of enzyme treatment on glucose yield 59 Table 4.4: Enzyme loadings in different treatment comtbioinnas 60 Table 4.5: H ydrolysis conditions used during separate hydriso laysnd 62 fermentation (SHF) process Table 4.6: I nfluence of yeast concentration on ethanol yield 64 Table 4.7: I nitial ethanol production rate for different ye acsotncentrations 65 Table 4.8: H ydrolysis and fermentation process conditionsa flol trh ree 66 Cassava substrate forms Table 4.9: E thanol and glucose yield coefficients for the SpSroFc ess 6 8 Table 4.10: C onversion efficiencies for SSF process in wt% 68 Table 4.11: Comparison of glucose and ethanol yields offfe rdeint bio-ethanol 69 production processes xii List of tables Table 4.12: Conclusion and validation of results agaliintesrt ature 71 Table A.1: Preparation (dilutions) of sugar standards 79 Table A.2: Peak areas obtained for each sugar concieont ruasting HPLC 7 9 Table A.3: Preparation of ethanol standard solutions 81 Table A.4: Ethanol peak areas obtained for each stadn sdoalur tion 81 Table B.1: Glucose yield (g.-1g) obtained during liquefaction of different 84 substrate forms of Cassava Table B.2: Glucose yield (g.-1g) obtained at different pH values during 84 liquefaction of unpeeled Cassava roots Table B.3: Glucose yield (g.-1g) obtained during saccharification of unpeeled 85 Cassava roots at differeHn tv palues Table B.4: Glucose yield (g.-1g) obtained during liquefaction of unpeeled 85 C assava roots at different temperatures Table B.5: Glucose yield (g.-1g) obtained during saccharification of unpeeled 85 Cassava roots at differenmt pteratures Table B.6: Glucose yield (g.-1g) obtained during liquefaction of unpeeled 86 Cassava roots at differeionmt bass loadings Table B.7: Glucose yield (g.-1g) obtained during hydrolysis of unpeeled 86 Cassava roots using a 10 wbito%mass loading with and without the addition of Cluecllast 1.5L to the hydrolysis mixture Table B.8: Glucose yield (g.-1g) obtained during hydrolysis of unpeeled 87 Cassava roots using a 20wbito%m ass loading with and without the addition of Cluecllast 1.5L to the hydrolysis mixture Table B.9: Enzyme loadings (wt %) used in different cboinmations in 87 this study Table B.10: Glucose yield (g.-g1) obtained during liquefaction of unpeeled 87 Cassava roots using difnfet reenzyme loadings Table C.1: Ethanol yield (g.-g1) obtained during SHF of Cassava roots by 88 Saccharomyces cerevis ia e Table C.2: Ethanol yield compared to glucose uptaken dgu SriHF of 89 C assava roots bSya ccharomyces cerevis iae xiii List of tables Table C.3: Ethanol yield (g.-g1) and glucose uptake (g-1.)g during SHF of 89 Cassava roots using difnfet ryeeast concentrations Table C.4: Ethanol yield (g.-g1) obtained during SHF of different forms 89 of Cassava roots wSit.h c erevisia e Table C.5: Glucose uptake (g-1.g) during SHF of different substrate forms 90 of Cassava usiSn.g c erevisia e Table C.6: Ethanol yield (g.-g1) compared to glucose uptake (-g1). gduring 90 SHF of Cassava peels us ingg.L -81 S cerevisia e Table C.7: Ethanol yield (g.-g1) and glucose uptake (g-1.)g during SSF of 91 peeled Cassava roots u 8s ign.gL-1 of S. cerevisiae Table C.8: Ethanol yield (g.-g1) and glucose uptake (g-1.)g during SSF of 92 unpeeled Cassava rootnsg u 8s ig.L-1 S. cerevisia e Table C.9: Comparison of ethanol yield (g-1.)g for SHF and SSF 93 Table D.1: Glucose concentration for repeated liquteiofanc experiments at 95 pH 6 and 95°C. Table D.2: Statistical parameters used to calculate the emxpeenrtial error for 95 t h e liquefaction step Table D.3: Glucose concentration for repeated sacfcichatriion experiments 96 at pH 4.5 and 55°C Table D.4: Statistical parameters used to calculaete e txhperimental error for 97 the saccharification step Table D.5: Ethanol yield (g.-g1) at different time intervals for five repeated 98 fermentation experiments Table D.6: Statistical parameters used to calculaete e txhperimental error 98 for the liquefaction st e p xiv List of figures LIST OF FIGURES Figure 1.1: 3-d diagram of ethanol 1 Figure 1.2: Different products of biomass 3 Figure 2.1: Overview of conversion route from crops to bio-entohla 9 Figure 2.2: Biomass conversion process 12 Figure 2.3: Process flow diagram of dry milling 15 Figure 2.4: Bloc diagram of the wet milling process 16 Figure 2.5: Process flow diagram of ethanol production frsotmar chy material 17 via SSF Figure 2.6: Principal parts of the Cassava plant 20 Figure 2.7: Typical Cassava storage root and peel 21 Figure 2.8: Cross section of Cassava storage root 22 Figure 3.1: The presence and degradation of starch during flaiqcutieon 38 Figure 3.2: Starch degradation during enzymatic hydrolysis 38 Figure 3.3: Flow diagram of experimental procedure followoerd Cf assava 40 hydrolysis and fermenotna t i Figure 3.4: Flow diagram of experimental procedure followed tfhoer SHF 41 and SSF processes wSi.t hc erevisia e Figure 3.5: Flow diagram of experimental procedure followed tfhoer DF 41 process wiSth. occidentallis Figure 4.1: Influence of Cassava substrate form on g tlhuecose yield during 5 3 liquefaction Figure 4.2: Effect of pH on glucose yield during liquefiaocnt 55 Figure 4.3: Effect of pH on glucose yield during saccharificoant i 55 Figure 4.4: Effect of temperature on glucose yield durlinqgu efaction 57 Figure 4.5: Effect of temperature on glucose yield dursinagc charification 57 Figure 4.6: Influence of biomass load on glucose yield ndgu rliquefaction 58 and saccharification Figure 4.7: The effect of enzyme treatment on glucose yieldh w10itwt% 60 substrate concentration Figure 4.8: T he effect of enzyme treatment on glucose yieldh w20itwt% 60 substrate concentration. xv List of figures Figure 4.9: Influence of enzyme concentration on gluccoosnec entration 61 Figure 4.10: Increase in glucose yield during liquefactino nth e SHF process 62 Figure 4.11: Increase in glucose yield during saccharificatino nth ie SHF 62 p rocess Figure 4.12: Ethanol production in shake flasks Sbayccharomyce s 63 cerevisi auesing the SHF process Figure 4.13: Ethanol production in comparison to glucosea kuep tin shake 63 flasks bSya ccharomyces cerevis iauesing SHF Figure 4.14: Effect of yeast concentration on ethanol yiaefltde r 72 hours 65 using the SSF proc ess Figure 4.15: G lucose uptake during fermentation Sbayc charomyce s 65 cerevisiae in the SSF process Figure 4.16: Increase in glucose concentration during lfiaqcuteion step of 66 the SSF process Figure 4.17: Influence of substrate form on ethanol yield 67 Figure 4.18: Glucose uptake during fermentation of peeelse,l epd roots 68 and unpeeled roots Figure 4.19: Comparison of Ethanol production betweSeSnF , and SHF 69 in shake flasks Figure A.1: Glucose calibration curve 79 Figure A.2: Fructose calibration curve 80 Figure A.3: Sucrose calibration curve 80 Figure A.4: Maltose calibration curve 80 Figure A.5: Maltotriose calibration curve 81 Figure A.6: Ethanol calibration curve 82 Figure A.7: Typical glucose chromatogram obtained from HCP aLnalysis 82 Figure A.8: Typical ethanol chromatogram obtained from HCP aLnalysis 83 Figure A.9: Typical HPLC chromatogram of a fermentation broathm sple 83 Figure C.1: Ethanol yield (g.-g1) compared to glucose uptake (-g1). gduring 91 SHF of Cassava peels u8si ngg.L -1 S. cerevisia e Figure C.2: Ethanol yield (g.-g1) and glucose uptake (g-1.)g during SSF of 92 peeled Cassava roots u 8sgin.Lg-1 of S. cerevisiae Figure C.3: Ethanol yield (g.-g1) and glucose uptake (g-1.)g during SSF of 93 unpeeled Cassava rootnsg u 8sgi .L-1 S. cerevisiae xvi List of figures Figure D.1: Five replicates of increasing glucose yieldri ndgu liquefaction 96 in the SHF process Figure D.2: Five replicates of increasing glucose yieldri ndgu saccharification 97 in the SHF process Figure D.3: Five replicates of ethanol production in sh falakseks by 98 Saccharomyces cerev isuisaieng the SHF process graph of fermentation samples xvii General introduction CHAPTER 1 GENERAL INTRODUCTION “We must develop knowledge optimization initiat ivtoe sleverage our key learnings.” Scott Adams Overview This chapter is divided into three sections, sntagr tini Section 1.1 with the background and motivation to this study, Section 1.2 is on bthaeckground of bio-ethanol. The hypothesis and research aim are in Sections 1. 31 .a4n drespectively. The research objectives are stated in section 1.5 followed bey dthetailed scope of investigation in Section 1.6. 1.1 Background and motivation The worldwide energy crisis and continuous incr eians peetroleum prices has led to alcohol being considered as an alternative to cnotniovneal fuels. Alcohol fuel, which can be produced from Cassava, sugarcane wastet haenrd a ogricultural products, are considered the most promising fuels for the fut ure. Any fuel that could be introduced as an alterna tiov econventional fuel should be evaluated from the aspect of availability, renewlitayb, isafety to the environment, energy balance and cost adaptability to the perafonrcme in existing engines, economy and finally emission (Nag, 2008). The major concse ornf biofuels are related to the energy efficiency of fuel ethanol and its produnc toiof a positive energy income (Agu et al., 1997). Figure 1.1: 3-d diagram of ethanol 1 General introduction In the early years bio-ethanol was not used in mauotboiles due to low energy density, high production cost, and corrosion. However, cthuerr ent shortage of gasoline has made it necessary to substitute ethanol (see F i1g.u1r)e as fuel in spark ignition engines. Ethanol is a renewable fuel that is now widely u sine dmany countries as a power source in vehicles and other internal combustiogni neens. It is currently produced from starch crops as well as feedstock crops ahnedr obtiomass materials that can be converted into fermentable sugars (Nzelibe and oOakgauf, 2007). Ethanol is a renewable fuel which is environmentally more frileyn dthan fossil fuel and can contribute to the reduction of the emission of gsa suech as sulphur dioxide, carbon dioxide and nitrogen oxide that is associated wthiteh utilization of fossil fuels (Cardona and Sanchez, 2007; Dermibas, 2007). Approximately 7% of Cassava produced all over thoer ldw is used by the textile, paper, food and fermentation industries. Anothergr el aconsumer of Cassava is the animal food industry, using about 33% of the woprrldo duction (Pandeey t al., 2000). The comparative properties of ethanol with petrnodl adiesel are shown in Table 1.1 below. Table 1.1: Comparative properties of ethanol with petrol anieds del Property Petrol Diesel Ethanol Specific gravity (15°C) 0.73 0.82 0.79 Boiling point (°C) 30-225 190-280 78.3 Specific heat (MJ/kg) 43.5 43.0 27.0 Heat of vaporization (kJ/kg) 400 600 900 Octane number 91-100 NA NA Catani number Below 15 40-60 Below Cassava M( anihot esculen) t from the Euphorbia family (Euphorbiace aies ) a very important source of carbohydrates, which is noty ornicl h in starch but also in cellulose and hemicellulose (Tonukari, 2004) . sCaavsa is a potential feedstock for ethanol production because of its excellent phyl saicnad chemical characteristics of the starch, high attainable ethanol yields and fleoewd stock costs. Cassava milled flour (also called tapioca flour) is easily and cpolemtely hydrolyzed compared to other starchy flours and therefore its use for ethanold upcr tion is encouraged. There are few reports (Ramasamy and Paramasamy, 2001) coingc ethrne industrial application of Cassava for ethanol production, possibly bec aCusaessava starch has to be 2 General introduction hydrolyzed into fermentable sugars for bioconvenr sinioto ethanol byS accharomyces cerevisiae ,making it time consuming (Ayernoert al., 2002). Cassava roots also contain coumaric acid, which causes harvested rtoo tsspoil within 24 hours after harvesting. The rapid deterioration of Cassavats r oaofter harvesting is the reason why Cassava is not a major export crop (Bayoumeti a l., 2008). If Cassava can be used for ethanol production the roots will have cbheo pped into chips and dried to preserve it long enough for processing into bioa-neothl through fermentation. This additional processing step has thus far limited uistse for the production of bio- ethanol. 1.2 Bio-ethanol production from biomass Biomass can be converted to liquid, solid and guas eporoducts through different processes. The different stages for the converosfi obni omass to these products are shown in Figure 1.2 below. Agricultural Enzymatic residues or acid Solid, and waste, hydrolysis liquid and energy Biological gaseous crops products Figure 1.2: Different products from bioma s(Ns ag, 2008) Bio-ethanol liquid can be produced through fermteionnta of biomass that contains starch and sugar. Starchy tubers such as Cassatv ac atnh be used for bio-ethanol production are important staple foods in most oef dtheveloping countries in the Tropics. They are widely distributed in these ctoriuens and despite their importance, a large proportion of the tubers are lost annudaullye to inadequate and ineffective storage facilities. In terms of energy utilizat ioand process simplicity, enzymatic conversion of the raw Cassava starch is superido rf eaansible (Nweke, 2004). Cassava starch has been fermented with amylase, gluco-asme ylaand ethanol producing organisms, but ethanol yields have been low (Branu emt a l., 1996). Methods used to produce bio-ethanol use more e nfoer geythanol production than the energy in the fuel produced, which is a contradryic teonergy balance and this becomes costly (Braumane t al., 1996). In addition, the concentration of sugfarrosm the 3 General introduction Cassava hydrolysate have a negative impact on tthhaen oel yield, and therefore methods that lower the negative impact of glucons e tohanol yield and are less time consuming should be found to make the productio ne tohfanol from Cassava economical. The methods and alternative organisymesa s(t) that optimize ethanol production conditions, costs and purity are thues mthajor focus for the prosperity of bio-ethanol from Cassava. The fermentation of aCvaas sroots prevents the roots from rapid spoilage after harvest. Although bio-ethapnrool duction has greatly improved through the use of better technologies and a wiadreie tvy of crops, Cassava has received less attention despite its promising prrtoiepse and potential in biofuels (Braumane t al., 1996). 1.3 Hypothesis Alternative methods (SSF), additional amylolytic zyemn es and yeasts (Schwanniomyces caste)l liwi ill reduce the impact of glucose concentrationn thoe ethanol yield and increase the ethanol yield obatbalien from Cassava. 1.4 Research aim The main aim of this resear cwhas to optimize the ethanol production and yield from the Cassava root fermentation process using atlitveern amethods (SSF) with Saccharomyces cerevis iaaes well as direct fermentation usinSgc hwanniomyces castellii/occidentali.s 1.5 Research objectives 1.5.1 Determine the optimum glucose concentratibotna inoable from peeled Cassava roots (starch), Cassava peels (cellulose) and ulendp eCeassava roots (starch and cellulose) during liquefaction. 1.5.2 Determine the optimum glucose concentratibotna inoable from peeled Cassava roots (starch), Cassava peels (cellulose) and ulendp eCeassava roots (starch and cellulose) during saccharification. 1.5.3 Determine the optimum ethanol yield obtaien afbrlom Cassava hydrolysate using a separate hydrolysis and fermentation me, thao d simultaneous 4 General introduction saccharification and fermentation, as well as dt irfecrmentation of Cassava starch. 1.5.4 Determine the cost of ethanol production f rCoamssava. 1.6 Scope of the investigation In the process of defining the investigation, th mreaein objectives and aims for this study came to the fore. 1.6.1 Optimization of liquefaction and saccharification steps The optimum pH, temperature, biomass load, enzyommeb cination and enzyme concentration for the highest final glucose conrcaetinotn in the liquefaction and saccharification hydrolysates were determined bryy invag one parameter at a time while keeping the others constant. The opmtim teumperature, pH, biomass load and enzyme combination was then used to ozpeti mthie fermentation step. Optimization of these parameters was done for tnhpee euled Cassava roots (starch and cellulose). 1.6.2 Optimization of ethanol yield The fermentation of Cassava roots to bio-ethanosl iwnavestigated by varying the yeast concentration during fermentation. Three different processing routes i.e. separater ohlyysdis and fermentation (SHF), simultaneous saccharification and fermeonnta ti(SSF) and direct fermentation (DF) were used to determine the berosct epssing route for the optimal ethanol yield from Cassava. Lastly the SSF process route was used to compaer ed iftfherent Cassava root forms (unpeeled roots, peeled roots and peels ofnolry ) optimal ethanol production. 5 General introduction 1.7 REFERENCES AGU, R. C., AMADIFE, A. E., UDE, C. M., ONYIA, A., OGUE, . O., OKAFOR, M. & EZEJIOFOR, E. 1997. Combined heat treatmendt ancid hydrolysis of Cassava grate waste (CGW) biomass for ethanol pctriodnu. Waste Manageme,n t 17(1): 91-96, 25 Sept. AYERNOR, G.S., HAMMOND, T.K. & GRAFHAM, A. 2002. Teh combination of rice malt and amyloglucosidase for the produnc toiof sugar syrup from Cassava flour. African Journal of Science Technolo, g3y(1): 10-17, June. BAYOUMI, S.A.L., ROWAN, M.G., BLAGBROUGH, I.S., & BEECHING, J.R. 2008. Biosynthesis of scopoletin and scopolin ins sCaava roots during post- harvest physiological deterioration: E-Z-isomeriiosna t stage. Phytochemistr,y 69(17): 2928-2936, 23 Sept. BRAUNMAN, A., KELEKE, S., MALONGA, M., MIAMBI, E., & AMPE, F. 1996. Microbiological and Biochemical Characteriiozna tof a Traditional Lactic Acid Fermentation for (Cassava Flour) ProductiAonp.p lied and environmental microbiology,6 2(8): 2854-2858, 05 Jan . CARDONA, A. C. & SANCHEZ, O.J. 2007. Fuel ethanorlo dpuction: Process design trends and integration opportunitieBsio. resource Technolog, y99(12): 2415-57, 01 Ma r. DERMIBAS, A. H. 2007. Combustion of BiomasEs.n ergy Sources Part A: Recovery, Utilization, and Environmental Effe, c2t9s(6): 549-56, 06 Jan. NAG, A. 2008. Biofuels Refining and Performancen.i tUed States of America: McGraw-Hill: 5-35, 17 Dec. NZELIBE, H. C., & OKAFOAGU, C. U. 2007. Optimizatnio of ethanol production fromG arcinia kola (bitter kola) pulp agro was.t eAfrican Journal of Biotechnology 6, (17): 2033-2037, 05 Sept. 6 General introduction PANDEY, C.R., SOCCOL, P., NIGAM, V.T., VANDENBERGH, EL.P.S., & MOHAN, R. 2000. Biotechnological potential of agirnod-ustrial residues. II. Cassava bagassBei.o resource Technolog y7,4: 81–87, Aug. RAMASAMY, A. & PARASAMY, G. 2001. Production of eathnol from liquefied cassava starch using co-immobilized cells Zoyf momonas mobil isand Saccharomyces diastatic.u Jsournal of Bioscience and Bioengineer,i n9g2(6):560- 564, 20 Jun. TONUKARI, N.J. 2004. Cassava and the future of csht.a Er lectronic Journal of Biotechnolog,y 7 (1): 5-8, 15 Apr. NWEKE, F.I. 2004. “New Challenges in the Cassavan Tsfrormation in Nigeria and Ghana.” EPTD Discussion Paper No. 118: Envireonntm and Production Technology Division, International Food Policy Raersceh Institute (IFPRI), 18 June. United States of America. 7 Background and Literature survey CHAPTER 2 BACKGROUND AND LITERATURE SURVEY "If we could first learn where we are and where waere going, we could be better able to judge what to do and how to do it .” Abraham Lincoln Overview The literature survey is subdivided into variousc tSioens, starting with a brief introduction on bioethanol and Cassava on Secti.o1n. S2ection 2.2 denotes the importance and effect of biofuels followed by a ebthioanol review in Section 2.3. Bioethanol production is extensively outlined in ctSioen 2.4 encompassing subsections on bioethanol production methods angda noisrms. An overview of the Cassava plant, its components and production islin eodu t in Section 2.5. Energy efficiency of Cassava is reviewed in Section 2.l6lo wfoed by the last Section (2.7) on optimization of bioethanol production from Cassa v a. 2.1 Introduction Ethanol from biomass has already been introduc ecdo uinntries such as Brazil, USA and some European countries. In Brazil it is cnutrlyre produced from sugar and in the USA from starch, both at competitive prices. Inc ernet years, there has been an increase in the efficient utilization of industr iaclrops such as Cassava for the production of ethanol (Pandey, 1992; 1994). Ethl aisn ocurrently produced from sugarcane and starch containing materials, wher ec othnversion of starch to ethanol includes a liquefaction step (to make starch soel)u abnl d a hydrolysis step (to produce glucose). The key parameters that most ethanoela rcehs addresses are energy and environmental performance (Ngunyen and Ghweela8, )2. 0 A0 study by Ngunyen and Gheewala (2008) shows that bioethanol produced fCroamssava (E10 fuel), along with its life cycle, reduces certain environmenlotald s. 8 Background and Literature survey Practical investigations have indicated that Caas saasv a perennial starch crop proves to be a beneficial raw material for industrial puroctds such as ethanol (Ayerneotr al., 2002). The Cassava roots specifically produce mthoes t important raw materials, including Cassava flour and Cassava starch, whichhe nw hydrolyzed produces fermentable sugars such as glucose for ethanolu pctriodn (Avancini et al., 2007). The roots are rich in energy due to high carbohtyed craontent. Starch accounts for the highest proportion of the carbohydrate content, imnga kCassava a potential energy reserving plant. The use of Cassava as a sou receth aonf ol for fuel is already being exploited and is very promising. The work of Roebtle a l., (2003) demonstrates direct ethanol production from Cassava starchA bsyp ergillus awamo rai nd Saccharomyces cerevisiaei n a circulating loop bioreactor. Optimization otfh aenol yield from such crops looks also at the experimental design, itesr geyn consumption and production. Optimal production, bio-processing and chemicalc epsrosing of bio-ethanol is greatly influenced by proper selection of crops. The opztaimtioi n of fermentation processes is a crucial tool in bio-ethanol production. The coinmabtion of different steps of an integrated bioprocess into one single unit (SS Fs)e iesn as a prospective optimization tool together with the use of amolytic organismSs. o(ccidentalis/castel)li ifor direct fermentation since no hydrolysis of substrate iqsu irreed (Cardona and Sanchez, 2007). 2.2 Biofuels Biofuels form an important emerging business woirdldew and the amount of biomass feedstock that can be produced for fuels and oethner gy purposes are potentially very large (Hoogwijke t al., 2003). Fermentation of sugars to alcohol is ofn eth e main routes that have been distinguished to pro dbuiocfeuels, mainly by conceiving ethanol (see Figure 2.1) Figure 2.1: Overview of conversion route from crops to bio-entohl a Higher overall energy conversion efficiencies anodw elr overall costs are the key criteria for selecting biofuels for the longer te, rmwhich provides insight into the 9 Background and Literature survey possible barriers to implementation that need t oo vbeercome and the technological improvement options that should be stimulated (Hlianmcke and Faaij, 2006). Brazil and USA are the largest producers of bioa-neothl in the world. It is expected that by 2020, 6.9% of the worldwide transportatifoune l supply will consist of biofuels, depending on the improvement of technioelso.g One of the disadvantages of biofuels is the low energy density compared to edl ieasnd petrol. More than a liter of bio-ethanol is necessary to replace a liter ofo pl;e htrence the production cost of bio- ethanol has to be low in order for it to competeth w pietro l(Bomb et al., 2007). The cost of raw material has a significant impact oen cthost of bio-ethanol production, and by increasing bio-ethanol production from heigtha nol grains and tubers such as Cassava, which are plentiful and less costly, aitn itsicipated that the future progress in biotechnology will decrease the cost of bio-eth apnrolduction (Bombe t al., 2007; Zohreh, 2008) . 2.3 Bio-ethanol Bio-ethanol is a liquid transportation fuel madeo mfr renewable resources or plant biomasses such as agricultural wastes, corn, g grarains,ses, sugar cane, straw, wood based waste such as newsprint, woodchips, and macatnuurifng waste materials. Ethanol is a clean burning fuel that lowers ove grarellen house gas emissions (as the biomass absorbs carbon dioxide as it grows), cnosn ta ihigh percentage of oxygen (35%) and therefore produces more complete fuel bcuosmtion. Ethanol can be blended with petroleum, integrates into existinge l fduelivery systems and provides energy security by reducing our reliance on fosfuseil ls. The new Flexible Fuel Vehicles currently available operate on E85 eth abnaosled fuels with a content of 85% ethanol and 15 % petroleumG o(ettemoeller and Goettemoeller, 20).0 7Most vehicles on the road use petrol and diesel as their fuet lf,o bsusil fuels are limited. Combustion of petroleum-based fuels increases net emissiocna robfo n dioxide, different toxic and volatile compounds that are responsible for thelt h ehaazards and pollutions such as benzene, toluene and xylene (With recent advannc ethse i biotechnology sector, it is plausible to convert biomass into high quality egnye cr arriers such as liquid fuels (Larsone t al., 1993). 10 Background and Literature survey 2.4 Bio-ethanol production Ethanol can be manufactured using a dry mill or mweillt process, where dry milling entails the fermentation of starch into sugarse, r awfthich it is distilled into alcohol. The distinct difference between the two processe sth ei initial treatment of the biomass. Srinorakutareat al., (2004) reported that in the s2t c1entury the production of ethanol from abundant, low cost agricultural protds uisc growing due to the certainty that it reduces the cost of ethanol production haansd global environmental benefit. Bio-ethanol production had increased sharply froemss lthan 1 GL in 1975 to 49.8 GL in 2006 (Nag, 2008). Sugar crops constituted 60% of bio-ethanol prodounc tin 2000, but its share decreased to 47% in 2006 in Brazil, while the ufs set aorch crops for the production of bio-ethanol increased from 39% to 53% in 2006. Tinhcere ase in the use of starch crops for the production of bio-ethanol is preddic teo increase towards 2015 (Nag, 2008). 2.4.1 Bio-ethanol production from starch-rich biomass Ethanol is produced largely through biochemical tahnedrmo-chemical processes (see Figure 2.2). Biomass is generally pretreated beyc hmanically cleaning and sizing the biomass, and then destroying its cell structurem atok e it more accessible to further chemical and biological treatment. The starch poaf rtbiomass is converted by hydrolysis to monomeric sugars such as glucose. e mThonomeric sugars are fermented to ethanol, which is further purified adnedhydrated for industrial use (Hamelinck and Faaij, 2006). Several agricultuprraol ducts can be used as the raw material for ethanol production, such as sugarc aricne ,and Cassava. Among these commercial crops, Cassava, simply transformed iteod dcrhips, is recommended as the most suitable raw material for ethanol productiDone r(mibas, 2007). 11 Background and Literature survey Figure 2.2: Biomass conversion proce(sDse rmibas, 2007) 2.4.2 Enzymatic hydrolysis The thermostableα- amylases (endoα-- 1, 4-glucanase) are used in starch hydrolysis to break the starch bonds and thus release the sftoarr chhy drolysis to simple sugars by β-glucosidase. The major end products from the na cotifo α-amylase on starch are dextrins. The glucoamylases anβd-a mylases (exo-α -1, 4-glucanase) are used in saccharification of liquefied starch producing golsuec, maltose, maltotriose, maltotetraose, maltopentaose and maltohexaoseth, eayn da re produced bAys pergillus niger. The pullulanases and isoamylases (enαd-o1-, 6-glucanase) are used for debranching starch into complete fermentable su sguacrsh as glucos (eHema et al., 2006). Fermentation of the simple sugars to ethanol thhro uygeast takes approximately 72 hours. The pH should not dropo wbe l4.5 becauseα -amylase denatures at low pH-values (Aktinson and Mavitu1n9a9, 1). Higher ethanol yields and low concentrations of by-products are obtaiante md ild process conditions. 2.4.3 Fermentation with yeasts An increase in the utilization and production ohf aentol through the fermentation of starchy materials has aggravated the intensive arrcehs e into improving the 12 Background and Literature survey conventional fermentation procedures through opztaimtioi n. Non-renewable, commercial bacterial and fungal amylases are uosre dth ef common liquefaction and saccharification of starch substrates. Howeveerr, et hare a considerable number of cost effective amylolytic yeasts that produce t hoewirn amylases for starch conversion and are also capable of fermentation (Wilson angdle Idnew, 1982). Yeasts have been the most commonly used micro-organisms for ethapnrodl uction, mainly because they are a species which can produce ethanol a ms athine fermentation product (Lin and Tanaka, 2008). 2.4.3.1S accharomyces cerevisiae S. cerevisia eis the most well-known and widely used yeast oxno hse sugars such as glucose and the disaccharide sucrose because ith hea asbility to grow on these sugars. This yeast is tolerant to ethanol up t%o 1o5f its concentration in the fermentation broth, but it cannot ferment pentossuegsa rs such as xylose (Saha, 2003). It offers advantages over other yeasts in the bnivoecrosion of sugars. Under excess carbon conditions, its metabolic flux to ethano hl aisrdly affected by the presence of oxygen (Lagunas, 1979) and it is able to grow u nhdiegrhly anaerobic conditions (Visser, 1995). The main restriction oSf. cerevisia eis its inability to convert relatively inexpensive polysaccharide-rich subsetsr,a tsuch as starchy materials to fermentable sugars (Hahn-Hagerdeat l al., 1994). The fermentation step is usually performed in an open vessel that is mechanicaliltya taegd and coil refrigeratedS. . cerevisiae is added and it immediately consumes the glucno sthee i hydrolysate. The product is continually fed to a distillation colum ton recover ethanol as an ethanol rich mixture with water (Nag, 2008). 2.4.3.2S chwanniomyces caste/lolici cidentalis Schwanniomyces castelalilis o known asS chwanniomyces occidenta hlisas the ability to synthesize alpha amylase and two gluco-amylanszey mees, that is, it can both hydrolyze and ferment Cassava starch (Wilson angdle dInew, 1982; Poonam and Dalel, 1995). The three enzymes producedS b. yc astelli ihave a common optimum pH of 6 and optimum temperature of 60°C. The twluoc og-amylase enzymes oSf. castellii/occidentali sdiffer only with their molecular weights and th eriar tes of hydrolysis of the carbon substrates such as ma, ltgolusecose, glycogen and dextr in . The excretion and biosynthesis of the amylases frthoem yeast depends and is 13 Background and Literature survey influenced by the composition of the culture me dIita .is useful in the direct fermentation of starch to as much as 4 % (w/v) neothl a(Wilson and Ingledew, 1982). S. castelli i is used as a solid culture and the ethanol prodd ubcye the organism is continuously recuperated in a cold trapper tank. 2.4.4 Dry milling process In the dry milling process (see Figure 2.3) thei ng/truaber is ground into fine pulp or flour. The grain is processed without separation thoef starch from the fiber components (Avancinei t al., 2007). The flour or meal is slurred with watoe rf ot rm a mash. The slurry is directly further processed iqbuye lfaction and saccharification and the starch in the mixture is converted to sugarrosu tghh the use of water and enzymes at high temperatures (55-95°C). The mash is proecde isns a high-temperature cooker to reduce bacteria levels ahead of fermentatione. mThash is cooled and transferred to fermentors where yeast is added and the converosf iosnu gar to ethanol and carbon dioxide (CO2) begins (Wyman, 1996). During the fermentation process, the mash is aegdi tatnd kept cool to facilitate the activity of the yeast. After fermentation, the rletisnug broth is transferred to distillation columns where the ethanol is separ afrteodm the remaining silage. The ethanol is concentrated using conventional distitoilnla and then dehydrated in a molecular sieve system (Wayman, 1996). The alc ophroodl uct at this stage is called anhydrous ethanol (pure, without water) and is oaxpipmrately 200 proof. Ethanol that will be used for fuel is denatured, or madefit ufonr human consumption, with a small amount of petrol (2-5%) added at the ethapnlaonl t. The silage is sent through a centrifuge that separates the coarse grain from s othluebles. The solubles are then concentrated to about 30% solids by evaporatiosnu, ltrieng in Condensed Distillers Solubles (CDS). The coarse grain and the syru pt haerne dried together to produce dried distiller grains with solubles (DDGS), a h igqhuality and nutritious livestock feed. Carbon dioxide is given off in great quaensti tiduring fermentation and many ethanol plants collect, compress, and sell it fsoer iun other industries. In addition, the fuel cell industry has developed re-formulators t thuase ethanol as a source of hydrogen. The C2O is also captured and sold for use in carbonatoinftg dsrinks and beverages and the manufacture of dry ice (Wyma9n6, )1. 9 14 Background and Literature survey Figure 2.3: Process flow diagram of dry millin(gW yman, 1996) . 2.4.5 Wet milling process During the wet milling process the grain is sepeadr aint to starch and fiber components by soaking or steeping the grain in water and afocrid 2 4 to 48 hours. The slurry produced is processed through degermers to se pthaera gterain germ. The oil from the germ is extracted while the remaining fiber, glu atennd starch components are further segregated by centrifugation, screening and hyodnroicc l separators. The steeping liquid is concentrated in an evaporator. This conntrcaeted product, heavy steep water, is co-dried with the fiber component and is thenld saos gluten feed to the livestock industry. The starch and any remaining water frthoem m ash can then be processed in one of three ways: fermented into ethanol, dried saonld as dried starch or processed into syrup. The fermentation process for ethan ovle irsy similar to the dry mill process (Wyman, 1996). The wet milling process is descdr iibne Figure 2.4 15 Background and Literature survey Figure 2.4: Block diagram of the wet milling proce(sWs yman, 1996). The conversion of starchy biomass to ethanol haesn baen important focus during recent years. There are two techniques availablreld wiode for the conversion of the sugar content of starch to ethanol. These are nthzey meatic hydrolysis and acid hydrolysis processes. 2.4.6 Separate enzymatic hydrolysis and fermentatnio (SHF) Enzymatic hydrolysis of starch to dextrins is thirest fstep (called liquefaction) in this method, where the resulting dextrins are then crotendv eto glucose in a second hydrolysis step (called saccharification). Sacicfihcartion is done at an optimum temperature between 50°C and 65°C. The major vdaisnatadge of SHF is that the sugar released from hydrolysis inhibits enzymev aitcy tibecause glucose is a strong inhibitor for β-glucosidase. The activity oβf- glucosidase reduces by 75% at a level of 3g.L-1 of glucose. Contamination is another problemaatict ofr for SHF because hydrolysis is a lengthy process and a dilute sonlu tiof sugar has a risk of contamination by competing external micro-organi,s emvsen at optimum temperatures of 45-50°C (Philippidis and Smith, 1995). 16 Background and Literature survey 2.4.7 Simultaneous saccharification and Fermentatnio (SSF) Simultaneous saccharification and fermentationn i se tahanol production method that has been reported to give high ethanol yields aenqdu ires minimum amounts of enzyme because end-product inhibition from glucaonsde cellobiose sugars formed during enzymatic hydrolysis is relieved by yeasrmt fentation (McMillan, 1999). This is an advantage related to the process of etharnoodlu pction from different feedstocks that was first described by Takageit al., (1997). During the simultaneous saccharification and fermentation process, the menaztiyc degradation of starch is combined with the fermentation of glucose from hoylydsris of starch by yeasts into ethanol (see Figure 2.5). This method does neodt nsequential processes, because the glucose is converted to ethanol as soon as fiot rimed from dextrin in the hydrolysate. The main advantage of this methodts islo iwer energy consumption and a lower content of non-glucosidic impurities, retisnugl in better ethanol production (Mojovic et al., 2006). Figure 2.5: Process flow diagram of ethanol production fromr cshtay material via SSF (Cardona and Sanchez, 2007). 2.4.8 Direct Fermentation (DF) Bio-ethanol production has been greatly improvedro utghh the use of better technologies and a wide variety of crops. Cassavsa r ehceived less attention despite its promising properties and potential in biofu e Plsr.oduction costs tend to be high for 17 Background and Literature survey ethanol production since the enzymes have to blieze udt ithroughout. However, direct fermentation through the use oSf chwanniomyces castellii/occidentaliast a temperature of 30° Cis a method that does not require hydrolysis of Cthaessava starch (Wilson and Ingledew, 1982). 2.5 Overview of Cassava The main non-food uses of Cassava are animal fnede ds ta rch. Approximately 5.5- 6% of world production goes into starch for indiuasl tpr rocesses such as alcohol and 10% and more of the total production is classifaiesd lo st as waste (see Table 2.1). Table 2.1: World utilization of Cassava (Data presented as earc epntage of total production)( Cock, 1985) . Area Human Animal feed Industrial use Export Waste consumption and starch Africa 50.8 1.4 a a 9.5 World 33.8 11.5 5.5 7.0 10.0 a Less than 1% In the years between the two World Wars, alcohosl wpraoduced from Cassava in Brazil and Australia, but that declined becauseth oe f availability of cheap supplies of petroleum products. There has been renewed int tienr epsroducing alcohol (ethanol) from this neglected crop because of its potentoiar l bfio-ethanol prosperity (Cock, 1985). It is a particularly attractive source faolrc ohol production since it can be grown on marginal land (less favored agriculturraela as) and need not compete for land used for food crops. Therefore development imopf roved technology for conversion of Cassava starch to ethanol should eanlshoance the net energy ratio as production yields of Cassava rises (Cock, 1985). 2.5.1 Cassava production In South Africa Cassava is considered a minor c Irto ips .mainly used as a subsistence crop by resource poor farmers. Plant breederso,n aogmrists and molecular biologists have made substantial improvements in Cassava sy ideuldring the past years and continue to do so. Genetic characterization anpdp minag has revealed some insights in the molecular nature of Cassava (Fregetn ael ., 2003). The growth of roots and tubers accounts for nearly 122 million metric townisth most of the increase being 18 Background and Literature survey Cassava, 80 million metric tons (66%) of the to t aTl.otal cassava production is projected to reach 168 million tons by 2020 basne dth oe current production rate (see Table 2.2). Moreover, with the increasing demand aestablishment of starch- utilizing industries in developing countries, threo dpuction of Cassava will increase beyond expectation (Scoettt al., 2000), thus making Cassava a prospective crop because of its high yields and low costs. Table 2.2: Cassava production and use in 1993 and project e2d0 2to0 (Scotte t al., 2000) Country/region Area Yield Production Total use (million ha) (mt/ha) (million mt) (million mt) 1993 2020 1993 2020 1993 2020 1993 2020 Sub-Saharan Afric a 11.9 15.9 7.4 10.6 87.8 168.6 87.7 168.1 Latin America 2.7 2.7 11.3 15.6 30.3 41.7 30.3 42.9 South-east Asi a 3.5 3.5 12.1 13.7 42.0 48.2 18.9 24.4 India 0.2 0.2 23.6 28.4 5.8 7.0 5.7 7.3 Other South Asia 0.1 0.1 9.4 13.5 0.8 1.3 0.9 1.4 China 0.3 0.3 15.1 20.2 4.8 6.5 5.1 6.4 Other East Asi a na na na na na na 1.8 1.9 Developing 18.8 22.9 9.2 12.0 172.0 274.7 152.0 254.6 Developed …. …. 12.1 14.7 0.4 0.4 20.7 20.5 World 18.8 22.9 9.2 12.0 172.7 275.1 172.7 275.1 2.5.2 The Cassava plant Cassava was earlier classified as two specMie su,l tissima Phol andM . aipi Phol. These two species with varying cyanogenic gluco sciodnecentrations have recently been classified as being the same speMcie. se,s culent aCrantz. It is the only species of 98 species in the Euphorbiaceae family that iidse wly cultivated for food and industrial benefit. Cassava is a cultigen thagt ionraited in Brazil and is grown as an annual worldwide. It is a source of low cost cahrybdorates with Brazil being the largest producer, followed by Thailand and Africcaonu ntries such as Nigeria and South Africa, and in Africa its production continsu teo increase. It is a starch crop that can grow and produce high yields in areas ew hmeor st crops such as maize cannot grow or produce well. It can tolerate drhotu agnd can be grown on soils of pH 4.0 to 8.0 with low nutrient capacity and still preosnd well to irrigation, use of fertilizers and high rainfall, but not flooding (No get al., 2005). This crop is grown for its enlarged starch-filled roots with the lesa vceontaining a high level of protein 19 Background and Literature survey and stems rich in potassium. The main productCs aosfs ava are Cassava chips for human consumption, starch for ethanol productiodn paenllets normally used for animal feed. Cassava is propagated vegetatively as clones trhea t diafferent cultivars with institutional code names. Classification of cualtrisv is mostly based on pigmentation and shape of the leaves, stems and roots. Cusl ticvoamr monly vary in yield, root diameter and length, harvest time and temperatduarep tation. All kinds of Cassava cultivars have potential for ethanol production dtou ethe fact that industrial uses of Cassava include manufacturing of products suchl caosh oal from its starch (Cock, 1985). The principal parts of the mature Cassaavnat palre leaves, stem, and roo (tsee Figure 2.6) and their composition is expressed paesr caentage of the whole plant (see Table 2.3). Leaves Main stem/stalk Fibrous roots Tuberous/ storage roots Figure 2.6: Principal parts of the cassava pla nt Table 2.3: Percentage composition of Cassava p l(aNngto et al., 2005) Plant part Composition Leaves 6% Stem 44% Roots 50% 20 Background and Literature survey 2.5.3 Cassava stem and leaves Cassava leaves contain high levels of protein (aagvee r30.5%), vitamins and micronutrients, but low amounts of carbohydratetsh wsitarch as the major proportion of the leaf carbohydrate. The amylose contenht eo fl et af starch has been reported to range from 19-24%, with the crude fiber low in croanst to the stem that has higher levels of the fiber (Tewee t al., 1976). The leaves are mainly used as a potherb, sometimes for animal feed and for compost (Viljoaennd Laurie, 2006). 2.5.4 Cassava roots and peels The tuberous roots covered with a brown outer b(paerke ls), grow in clusters with a cream white interior containing high starch con.t e nTthe root is usually from 1-4 inches in diameter and 8-15 inches long. The p ceoenlstain toxic hydrocyanic acid, which is eliminated during cooking (gelatinizatio. nF)igure 2.7 shows a typical cassava starch root (a) and the peels (b) tha t caolnstoain starch, making Cassava a potential producer of ethanol for energy and ennvmiroental performance. a) Cassava storage root b) Cassava peels Figure 2.7: a) Typical Cassava storage root; b) Cassava p eels Cassava roots mature to harvest within 8 to 24 hmso notf planting, depending on cultivar and climate. The root is circular in csro ssection (see Figure 2.8), consisting of three principal parts transversely (Negto a l., 2005). • The periderm: It is the outer most layer of thet ,r ocomposed mainly of dead cork cells that seal the surface of the root. • The cortex: A layer beneath the periderm • The starchy flesh: The central surrounding portoiof nt he root packed with starch grains 21 Background and Literature survey Figure 2.8: Cross-section of Cassava storage r(oOobt adinae t al., 2006). Cassava wastes such as the peels constitute 20o-3f 5th%e weight of the tuber and have approximately 61 % (w/w) starch content. Cqounesently, a large amount of Cassava peel waste is generated annually (Obaedt inaal. , 2006). However, the possibility of using Cassava peels for ethanol upcrotidon has not been given much consideration, but a study by Adesaneyta al., (2008) on milled Cassava peels as substrate for ethanol production revealed that aCvaas speel starch can be readily degraded either by amylolytic organisms up to 0m.8g8.m l-1 reducing sugar and more with enzymes approximately (57g-1.)L. The starchy flesh comprises up to 80% to 90% of the root, and that includes all the compotsn ein Table 2.2 below. The composition of Cassava roots is shown in Tableb 2e.l2o w, amylose and amylopectin are between 24-35% on a dry weight basis, makinsgs aCvaa a predominantly starchy food with starch having the highest percentage popf roaximately 64-78% of the carbohydrates (Ngeot al., 2005). The Cassava root system is distinguished by va raioduvsentitious root types such as fibrous roots (FRs) (see Figure 2.6 above) thato rabb swater and mineral salts providing support function and storage roots (S Rwsh)i,ch accumulate starch as a reserve compound. Cassava roots also contain seu, cmroasltose, glucose and fructose in limited amounts. The storage roots are richa ilnc icum, phosphorus and vitamin C, but low in protein and minerals, except for the lsp etehat contain more protein and vitamins than is found in the root flesh. The mrainl econtent of the dry bark (periderm) is higher than that of the cortex. 22 Background and Literature survey Table 2.4: A typical composition of Cassava storage r(oNogto et al., 2005; Tonukari, 2004) Constituent Composition Moisture 60-70% (wet basis) Carbohydrates 24-35% Fiber (cellulose) 1-2% Protein 1-2% Other 3% 2.5.5 Cassava starch Cassava contains 64-84% of the purest renewabulera nl aptolymer (starch )on a dry basis, with a low quantity of other constituentcsh s uas proteins and fiber, thus making it useful for a wide range of applications, inclnugd ibiofuels (Lasztity, 1999). Fresh Cassava roots were found to contain 38.7% dry mr atnted of the 38.7%, 31 % consisted of starch and soluble carbohydrates (lE awnedl Wiley, 1893). Further experiments done by Ewell and Wiley (1893) to dmetineer the yield of air dry starch that can be obtained from Cassava roots reveaalet d2 6th% of the carbohydrates were pure starch with trace amounts of nitrogenous miaal.t eAr study on the viscosity of Cassava starch done by Lasztity (1999) indicateadt Cthassava starch contains a high concentration of amylopectin compared to amylos0e: 2(08), which gives it a high viscosity but low potential for retro-gradation, kminag resulting in good freeze-thaw stability. Cassava is a low priced carbohydrated sfetoeck for ethanol fermentation with good susceptibility to acid and/or enzymatyicd rholysis even at low temperatures as shown in Table 2.5 below, which gives Cassaavrac hs tproperties. Cassava is a relatively cheap source of raw material containain hgi gh concentration of starch (dry- matter basis) that can equal the properties of febrye dother starch crops, general properties of Cassava are given in Table 2.5. 23 Background and Literature survey Table 2.5: General properties of Cassava star(cLha sztity 1999). PROPERTIES VALUE Chemical composition (%) - Protein 0.15-0.30 - Fat 0.0-0.1 - Ash 0.10-0.15 Granule size (µm by SEM image analysis) 3-34 Amylose content( % by HPSEC) 17-23 Swelling power 85°C (0.1g in 15ml distilled water) 40-62% Degree of hydrolysis (%, using 1% each oαf 25-60 amylase and gluco-amylase at 37°C, 48 hrs) 2.6 Potential of Cassava for bio-ethanol productio n The use of Cassava for ethanol production has trleyc aetntracted attention because it can be cultivated on marginal land where other sc rcoapnnot be grown successfully and because it is not considered to be a staplde sfouoch as maize, wheat and r i c e. Cassava roots are rich in energy due to the higrbho chaydrate content of the roots, which makes Cassava a suitable biomass sourcei of-oert hbanol production (Ague t al., 1997) 2.6.1 Energy efficiency of Cassava In most studies, including an energy analysis byd gHeo, (2002), it is concluded that the net energy value of Cassava bio-ethanol proiodnu cist greater than that of maize bio-ethanol production. One reason for the net goaf iennergy during the production of bio-ethanol from Cassava roots is the higher yoief ldC assava per hectare (12 ton/ha), which is much higher than that of maize. In adodni,t iCassava cultivation requires less fertilizer (100 kg.-h1) than maize (144 kg.h-1a). A study conducted in China by Hu et al., (2004) concluded that bio-ethanol produced frCoams sava is both energy and renewable energy efficient in converting a solide regny source into a liquid energy source, since the solar energy trapped in Cassisa vgar e ater than the energy used in the industrial processing of Cassava (Zheatn agl ., 2003) . 24 Background and Literature survey 2.6.2 Cassava starch fermentation to ethanol Bio-ethanol has already been introduced as a torartnastpion fuel in countries like Brazil, the United States and some European coeusn. t rIin Brazil it is produced from cane sugar and in the United States from maizec hs.t aBrio-ethanol can be produced from either lignocellulose or starch. Commerciaiol -ebthanol production from lignocellulose material is hindered due to techcnon-eomic considerations, while bio- ethanol production from starch is widely used in smt obio-ethanol producing countries. Furthermore, high ethanol yields froimgn olcellulosic material require complete hydrolysis of both the cellulose and hemlluiclose materials, followed by efficient fermentation of all sugars in the biom.a sTshe bioconversion of Cassava starch into ethanol can be done in three ways. fTirhste is fermentation through hydrolysis of starch by the method of separate menaztiyc hydrolysis and fermentation (SHF), while the second method is simultaneous hsaarcifcication and fermentation (SSF) where the sugars is utilized as soon a sf oitr mised (Johne t al., 2006). The third method is the use of amylolytic yeast for the dti refecrmentation of starch that eliminates hydrolysis through solid state fermeionnta. t The amylolytic yeast Schwanniomyces caste lfloiir direct fermentation of starch is considere db eto suitable for the direct fermentation of starch (Meat al., 2000; Onere t al., 2005). 2.7 Optimization of ethanol yield from Cassava Optimization of ethanol yield is concerned with theenergy consumption during production. A combination of different steps in tphreoduction process of bio-ethanol into one single unit is seen as a prospective oizpatitmion tool, while the immediate continuous removal of ethanol from the biotransfaotrimon process is another opportunity to increase product yield, while alseod urcing product costs (Cardona and Sanchez, 2007). In the earlier works of Mullis aNnedSmith, (1984) the economic evaluation of starchy materials as feedstock for- ebthi anol production on a small scale was done to exasperate on the cost affeyc otifv bitio-ethanol. 25 Background and Literature survey 2.8 REFERENCES ADESANYA, O. A., OLUYEMO, K.A., JOSIAH, S.J., AESAYNA, R.A., SHITU, L.A.J., OFUSORI, D.A., BANKOLE, M.A., & BABALOLA, G.B. 2008. Ethanol Production by Saccharomyces Cerevisiae from Ca ssPaeveal Hydrolysat.e The Internet Journal of Microbiology 5, (1), Feb AGU, R. C., AMADIFE, A. E., UDE, C. M., ONYIA, A., OGUE, . O., OKAFOR, M. & EZEJIOFOR, E. 1997. Combined heat treatment acnid haydrolysis of Cassava grate waste (CGW) biomass for ethanol productWiona.s te Manageme,n 1t 7(1): 91- 96, 25 Sept. AKTINSON, B. & MAVITUNA, F. 1991. Upstream procesnsgi. Biochemical Journal of Engineering and Biotechnolo:g 8y34–835, Apr. AVANCINI, S.R.P., FACCIN, G.L., VIEIRA, M.A., ROVARIS, A.A., PODESTA, R., TRAMONTE, R. & De SOUZA, N.M.A. 2007. Cassavtaa rsch fermentation wastewater: Characterization and preliminary tolxoigciocal studies. Food and Chemical Toxicolog,y 45:2273-2278, 01 Dec. AYERNOR, G.S., HAMMOND, T.K. & GRAFFHAM, A. 2002 .T he combination of rice malt and amyloglucosidase for the produnc toiof sugar syrup from cassava flour. African Journal of Science and Technol,o 3g(y1): 10-17, 01 Jan. BOMB, C., McCORMICK, K., DERWAARDER, E., & KÅBERGE,R T. 2007. Biofuels for transport in Europe: lessons from Gaenrmy and the UKE. nergy Policy , 35:2256–2267, Apr. CARDONA, A. C. & SANCHEZ, O.J. 2007. Fuel ethanporol duction: Process design trends and integration opportunitieBsi.o resource Technolog, y99(12): 2415-57, 01 Mar. COCK, J.H. 1985. Cassava: a basic energy sou rtchee intropics.S cience, 218(4574): 755-762, 19 Nov. DERMIBAS, A. H. 2007. Combustion of BiomassE.n ergy Sources Part A: Recovery, Utilization, and Environmental Effe, c2t9s(6): 549-56, 06 Jan. 26 Background and Literature survey EWELL, E. E. & WILEY. H. W. 1893. Some products coaf ssava.J ournal of the American Chemistry Socie t1y5, :78–82. FREGRENE, M.A., SUAREZ, M., MKUMBIRA, J., KULEMBEKA, H., NDEDYA, E., KULAYA, A., MITCHELL, S., GULLBERG, U., ROSLING, H., DIXON, A.G., DEAND, R., & KRESOVICH, S. 2003. Simple sequencpee raet marker diversity in cassava landraces: genetic diversity and diffearetionnti in an asexually propagated crop. Theoretical and Applied Genet,ic 1s07(6): 1083-1093. GOETTEMOELER, A. & GOETTEMOELER, J. 2007. Sustailnea bEthanol. 1st ed. Prairie Oak. HAHN-HADERDAL, B., JEPPSON, H., SKOOG, K. & PRIORB, .A. 1994. Biochemistry and physiology of xylose fermentatbioyn y easts.E nzyme Microbiology Technology ,16: 933-943, HAMELINCK, C.N., & FAAIJ, A.P.C. 2006. Outlook fo ardvanced biofuels. Energy Policy, 34:3268–3283. HEMA, A., TRIVEDI, U.B. & PATEL, K.C. 2006. Glucaomylase production by solid-state fermentation using rice flake manufrainctgu waste products as substrate. Bioresource Technology9,7 : 1161–1166, Jul. HODGE, C. 2002. Ethanol use in US gasoline shouel db abnned, not expandeOd.il Gas Journal ,100: 20-28, Sept. HOOGWIJK, M., FAAIJ, A., Van Den BROEK, R., BERNDE, SG., GIELEN, D. & TURKENBURG, W. 2003. Exploration of the ranges ohfe tglobal potential for biomass for energyB. iomass Bioenerg, y25: 119–133, Aug. HU, Q., SOMMERFELD, M., JARVIS, E., GHIRARDI, M., OPSEWITZ, M., SEIBERT, M. & DARZINS, A. 2004. Microalgal triacyllygcerols as feedstocks for biofuel production: perspectives and advanPcelasn. t Journal,5 4: 621–639, May. 27 Background and Literature survey JOHN, R.P., NAMPOOTHIRI, K.M. & PANDEY, A. 2006. imSultaneous saccharification and fermentation of cassava baeg faosrs l (+) lactic acid production. Applied Biochemistry Biotechnolog 1y3,4: 263–272, Dec. LAGUNAS, R. 1979. Energetic irrelevance of aerosbisio for S.cerevisia egrowing on sugars. M olecular Cell Biochemistry 2,7(3): 139-146, Mar. LARSON, E.D. & WLLIAMS, R.H. 1993. Biomass plantoanti energy systems and sustainable development. (In: J. Goldemberg and. JTo.Bhansson, EditorsE,n ergy as an Instrument for Socio-Economic Developm).e nNt ew York: United Nations Development Programme: 91–106, Nov LASZTITY, R. 1999. Cereal chemistry. Hungary: Akamdiaei Kiado: 11-51. LIN, Y. & TANAKA, S. 2008. Ethanol fermentation fmro biomass resources: current state and prospec tAs.pplied Microbiology Biotechnolog, y69: 627–642, Apr. MA, Y. J., Lin, L. L., CHIEN, H. R. & HSU, W. H. 2000. Efficient utilization of starch by a recombinant strain Soaf ccharomyces cerevis iaperoducing glucoamylase and isoamylaseB. iotechnology Applied Biochemis,t r3y1:55-59, 31 Feb. McMILLAN, J.D. NEWMAN, M.M. TEMPLETON D.W. & MOHAGHEGHI, A. 1999. Simultaneous saccharification and co-fermtieont aof dilute-acid pretreated yellow poplar hardwood to ethanol using xylose-fenrmting Zymomonas mobil.i s Applied Biochemistry and Biotechnolo: g7y7–79, Mar. MOJOVIC, L., NIKOLIC, S., RAKIN, M. & VUKASINOVIC, M. 2006. Production of bioethanol from corn meal hydrolyzateFsu.e l, 85 (12-13): 1750-1755, Oct. MULLIS, J.T. & NeSMITH, C. 1984. Integrated etha nporloduction and utilization system for small farmsB. iomass Journa l6, : 155–166, 08 Jul. 28 Background and Literature survey NAG, A. 2008. Biofuels Refining and Performancen. itUed States of America: McGraw-Hill: 5-35, 17 Dec. NGO, T. D., INGER, L. & NGUNYEN, T. M. 2005. Interrocpping cassava (Manihot esculenta Crantz) with Flemingia (Flemingia macryollpah); effect on biomass yield and soil fertility. L ivestock Research for Rural Developm. e1n7t(6), Feb. NGUNYEN, T.L.T. & GHWEEWALA , S.H. 2008. Life cyc leassessment of fuel ethanol from cassava in ThailanIndt.e rnational Journal of LCA, 13(2): 147-154, Jun. OBADINA, A.O., OYEWOLE, O.B., SANNI, .L. O. & ABIOLA, S. S. 2006. Fungal enrichment of cassava peels proteiAnfsr.ic an Journal of Biotechnolog, y5 (3): 302- 304, May. ONER, E.T. STEOHEN, G. O. & KYRDAR, B. 2005. Procdtiuon of Ethanol from Starch by Respiration-Deficient RecombinanSt a ccharomyces cerevisi aAe.pplied and environmental microbiolog7y,1 (10): 6443–6445, 25 Nov. PANDEY, A. 1994. Solid State Fermentation; An rovview in solid state fermentation. New Delhi, India: Wiley. 3-10 PANDEY, A. 1992. Recent developments in solid s tafetermentation. Process Biochemistry,2 7: 109-117, Jul. PHILIPPIDIS, G.P. & SMITH, T.K. 1995. Limiting faocrts in the simultaneous saccharification and fermentation process for corsnioven of cellulosic biomass to fuel ethanol. A pplied and Biochemical Biotechnolog5y1,/ 52:117–124, Sept. POONAM, N. & DALEL, S. 1995. Enzymes and microbisayl stems involved in starch processingE.n zyme and Microbial technolog 1y7, : 770-777, 17 Feb. ROBLE, N. 2003. Development of production systemosr rfaw cassava starch bioconversion in novel bioreactors with cells immiliozbed in loofa (Luffa cylindrica) sponge. Japan: University of Tsukuba (Thesis-PhD). 29 Background and Literature survey SAHA, B.C. 2003. Hemicellulose conversioJno.u rnal of Industrial Microbiology and Biotechnology 3, 0:279–291. SCOTT, G, J., ROSEGRANT, M. W. & RINGLER, C. 200R0o. ots and Tubers for the 21st Century: Trends, Projections, and Policpyti oOns. United States of America: 1-71. SRINORAKUTARA, T., SUESAT, C., PITIYONT, B., KITPRECHAVANIT, W. & CATTITHAMMANIT, S. 2004. Utilization of Waste form Cassava Starch Plant for Ethanol Production (.Joint International Conference on “Sustainable rEgyn eand Environment (SEE), Hua Hin. Thailand. p. 773-7 6). TAKAGI, M., ABE, S., SUZUKI, S., EMERT, G.H. & YATA, N. 1977. A method for production of ethanol directly from celluloses inug cellulase and yeast. (Proceedings of Bioconversion Symposium. Swedenh:iD, pe l551–71). TEWE, O.O., JOB, T.A., LOOSLI, J.K. & OYENUGA, V.A 1.976. Composition of two local cassava varieties and the effect of psrsoicneg on their hydrocyanic acid content and nutrient utilization by the raNti.g erian Journal of Animal Production3, (2): 60-66, Jun. TONUKARI, N.J. 2004. Cassava and the future of csht.a rElectronic Journal of Biotechnolog,y 7 (1): 5-8, 15 Apr. VILJOEN, J.C. & LAURIE S.M. 2006. Results obtainefrdo m a baseline study on cassava in South Africa. Agricultural Research Ccoilu-Rnoodeplaat and Ornamental Plant Institute. Technical report. Pretoria. 1-50. VISSER, W. 1995. Oxygen requirements of fermeneta tyiveast. Netherlands: Delft university of Technology. (Thesis-PhD). WILSON, J.J. & INGLEDEW, W.M. 1982. Isolation andh acracterization of Schwanniomyces alluvi usamylolytic enzymes. Applied and Environmental Microbiology, 44: 301–307, Aug. 30 Background and Literature survey WYMAN, C.E. 1996. Ethanol production from lignocuelollsic biomass: Handbook on Bioethanol Production and Utilization: 1-18. ZHANG, C. HAN, W.J., PU, G.Q. & WANG, C.T. 2003. fLei cycle economic analysis of fuel ethanol derived from cassava inu thSwoest China.R enewable and Sustainable Energy Revie7w:, 353-66, 18 Mar. ZOHREH, A. 2008. Ethanol and glucose tolerance Mo.fi ndicus in aerobic and anaerobic conditions. Sweden: University College Bofras. (Thesis-M.Sc.) 5-40. 31 Experimental CHAPTER 3 EXPERIMENTAL “Normal people ... believe that if it isn’t brokedno,n 't fix it. Engineers believe that if it isn’t broken, it doesn't have enough features y et.” Scott Adams Overview In this chapter the experimental work done and reimxpeental procedures followed in this study is discussed in detail. The chapter is subdivided tihnrteoe sections. The first Section (3.1) gives an overview of the materials used and raw materialp aprraetion. The equipment used in this investigation is discussed in Section 3.2, showthineg e xperimental setup. The experimental error and starch degradation process are shown in Se c3t.i3o nand 3.4 respectively and the experimental procedure follows in Section 3.5. lAytnicaal procedures are detailed in Section 3.6 together with the compositional analysis of Cas scauvltaivar used in this study. Optimization of the enzymatic hydrolysis of cassava roots is dissecdu sin detail in Section 3.7 followed by the optimization of fermentation steps in Section 3.8. 3.1 MATERIALS AND CHEMICALS All materials, chemicals, enzymes and microorgans isumsed in this study are listed in Table 3.1. All chemicals and enzymes were used without anoyr pprui rification. 32 Experimental Table 3.1: Material and chemicals used in this study Chemicals Description Supplier Purity Glucose Glucose calibration curve Saarchem (Merck) 99.0% Fructose Fructose calibration curve Sigma Aldrich 99.0% Sucrose Sucrose calibration curve Sigma Aldrich 99.0% Maltose Maltose calibration curve Sigma Aldrich 95.0% Maltotriose Maltotriose calibration Sigma Aldrich 95.0% curve Ethanol Ethanol calibration curve Rochelle chemlsi ca 99.9% Calcium Hydroxide pH operation Saarchem (Merck) 95.0% Sulphuric acid pH operation Labchem 98.0% Iodine Starch test Sigma Aldrich - Yeast extract Yeast growth media Sigma Aldrich - Malt extract Yeast growth media Sigma Aldrich - Peptone Yeast growth media Sigma Aldrich - Glycerol Yeast storage Sigma Aldrich - Magnesium Sulfate Yeast growth media Sigma Aldrich 99.0% Heptahydrate Potassium phosphate Yeast growth Sigma Aldrich 99.0% monobasic Urea Yeast growth Sigma Aldrich 98.0% Enzymes Termamyl SC α-Amylase enzyme Novozymes, South Africa - mixture Spirizyme Fuel Gluco-amylase enzyme Novozymes, South Africa - mixture Celluclast 1.5L cellulase enzyme mixture Novozy,m Seosuth Africa - Micro-organisms S. cerevisiae Bakers’ yeast for Anchor Yeast, South - fermentation Africa S. castellii/occidentallis Amylolytic yeast for ATCC, USA - fermentation Materials Cassava roots Raw feedstock for ethanoAl gricultural Research - production Council 33 Experimental 3.1.1 Preparation of yeasts 3.1.1.1S accharomyces cerevisiae S. cerevisia ewas revived from the dormant state by using thrme efentation broth (medium for fermentation of hydrolysate) as growth medium foern tminutes before use in the batch fermentation. 3.1.1.2S chwanniomyces occidentalis/castellii Freeze dried organisms were rehydrated with s twerailte r (water) and plated on malt extract agar and growth was observed after two days. Stock rceuslt uwere made in 15% glycerol for long term storage. Schwanniomyces occidentalis/cast eAllTiiCC 26706 was preserved and stored on glyceoroclk st at 4°C; it was subcultured on malt extract agatre psl afor 72hrs at 32°C, from which an inoculum was prepared. The yeast was grown at 45°C, 150nrp ma ilt extract broth (YM broth) containing 0.5 g.L-1 MgSO .4 7H2O, 0.5 g.L -1 (NH4)2HPO4, 1.5 g.L -1 yeast extract, 5 g-.1L glucose, 1.5 g.-L1 malt extract and 2.5 g-.1L peptone pH 5.5 (Srinorakutaerat al., 2004). The concentration oSf. occidentalis/castell iui sed was 10% (v/v) of fermentation sample (Saelti mal ., 2008). 3.1.2 Preparation of raw Cassava Three forms of Cassava were used in this proj.eec.t ,u inpeeled roots (starch and cellulose), peels (cellulose) and peeled roots (starch). The rootrse wweashed thoroughly by hand to remove any soil residues. After washing, the roots were pe eulseidng a knife and chipped/sliced with knives. Some roots were not peeled; they were only wasnhde ds laiced. Some Cassava chips were oven dried at 60°C for 24 hours and some were driedh ein stun for 3 days. All the Cassava chips were milled into fine flour and sieved with a +1m.5m screen. 34 Experimental 3.2 Equipment All equipment used in this study is listed in Ta 3b.le2. Table 3.2: Equipment used in this stu dy Equipment Description Model number Supplier Hammer mill for model TRF-70 Trapp milling of Cassava chips HPLC for analysis model 1200 Series Agilent of hydrolysate and Technologies fermentation broth Oven used to heat model 276 Scientific distilled water for liquefaction as well as to keep the liquefaction mixture 95°C. Shaker incubator model FSIE-SPO Labcon used to keep the 8-35 saccharification temperature at 55°C and fermentation broth at 30°C while agitating constantly. Centrifuge was Rotilabo-mini- Carl Roth used for separating centrifuge the fermentation samples to liquid and solids 35 Experimental Mass balance was Model ZSP-250 Scientech used for weighing the material used in this study. pH meter used to Model HI 99161 Hanna Instruments measure pH during all the experiments Moisture analyzer Model HR 83 Mettler-Toledo used for moisture analysis of the materials Spectrophotometer Model 20- Thermo Fischer used to measure 4001/UV-VIS Scientific absorbance of unhydrolyzed and hydrolyzed sample s Glassware used for 1000mL with DIN Duran group liquefaction, thread GL 45 saccharification and fermentation experiments The analysis equipment used in this study are: 1. HPLC 2. Moisture Analyzer 3. Spectrophotometer 36 Experimental 3.3 Experimental error The experimental error associated was determinre eda fcoh separate hydrolysis and fermentation step. The details of the calculations can be fo iunn dAppendix D. The experimental error associated with each of the process steps utiilniz ethdis investigation is listed in Table 3.3. Table 3.3: Error percent and product values for SHF Process step Error % Liquefaction 2 Saccharification 5 Fermentation 5 3.4 Determination of complete starch degradation The ability of iodine to bind amylose has been u tsoe dunderstand a variety of structural and functional aspects of starch in food systems. aInrc sht granules, the linear amylose polymer binds a significantly higher proportion of iodine thane dso the branched amylopectin molecule. After molecular dispersion, the amylose-iodine bindingil itay bis commonly used to quantify the amylose content of starches from various botansiocuarl ces ((Morrison andL aignelet, 198)3. The presence of starch in this study was determ binye da 0.05 mol 2I (12.69g 2I + 20g KI) solution. A drop of the iodine solution was inolactued in the slurry before and after hydrolysis. A deep purple/blue color would be an indicationt hoef presence of starch, dark red indicates dextrins and light brown to clear color indicaticnogm plete hydrolysis of the starch (see Figure 3.1). The iodine solution was used to determin ael l ifthe starch in the Cassava sample used for liquefaction and saccharification has been redutoc egdlu cose. 37 Experimental Before hydrolysis After liquefaction After saccharification (Starch) (Dextrins) (Glucose) Figure 3.1: The presence and degradation of starch duringe lfiaqcution The degradation of starch to reducing sugars wallso wfeod using a colorimetric method (Morrison andL aignelet, 198)3. An UV-VIS spectrophotometer (see Table 3.2) wusaesd at a wavelength of 620nm to determine the absorbancieo doaf ted hydrolysate samples. A blank solution consisting of water and iodine solution sw uased to standardize the absorbance measurements (Saibaene and Seetharaman, 2008)r.b Aanbcseo measured is proportional to the starch concentration of the sample tested. Theo rbaabnsce of hydrolyzed starch will thus decrease from the original non-hydrolyzed starcluht isoon. Table 3.4: Starch content of hydrolysates degradation duritnagrc sh hydrolysis to glucose Hydrolysis Time(hrs) process Contro l Hydrolysate 0 - 0.9 3.9 1.5 Liquefaction 0.4 2.4 48 Saccharificatio n 2.3 0.6 38 Experimental 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0 20 40 60 Time(hrs) Figure 3.2: Starch degradation during enzymatic hydrolysis (♦ - Control, - Hydrolysate ) The results from Table 3.4 and Figure 3.1 confirhme pt resence of starch in the hydrolysis process. The results also indicate that starch d weagsraded to glucose in this study, because the absorbance decreases with time and a definite ceh iann cgolour of the hydrolysate was observed during this period. the degradation of the stabryc hth e enzymes used, by observing the physical properties of starch determination and the valuf ethse o absorbance it clearly shows a decrease in the starch present as hydrolysis succeeds. Ther ocl osnht ows an increase in the starch concentration, which is probably reason for thee anbces of enzymes to degrade the starch. One factor in the increase of starch concentrationh ien ctontrol is that the Cassava granules in the slurry were swelling from exposing more starch. Othne contrary, the hydrolysate was immediately hydrolyzed by the enzymes as the s twaracsh exposed more and more. 39 Absorbance (620nm) Experimental 3.5 Experimental procedure 40 Experimental The experimental procedure is also shown schemllay tiicna Figure 3.4 and 3.5. In Figure 3.4 the SSF and SHF Sby. cerevisia eare presented and Figure 3.5 shows the procedure for direct fermentation bSy. occidentali.s Peeling, washing and slicing Drying of peels and Milling of of roots Unpeeled, peeled roots Peeled roots Unpeeled root and peel ss Starch presence - Iodine Enzymatic hydrolysis: Starch degradation - iodine Ethanol yield Vacuum determination distillation SSF/SHF with S. cereevvisisiaiaee HPLC analysis of hydrolysate And fermentation broth Starch content determination Figure 3.4: Flow diagram of experimental procedure followed tfhoer SHF and SSF processes with S. cerevisiae Drying of peels, Milling of Peeling, washing and slicing unpeeled root s Unpeeled roots, peeled ro ots of roots and and peel s Peeledr oots Starch presence -Iodine Direct Fermentation Ethanol yield with S. occidentali s determination HPLC analysis o f fermentation brot h Figure 3.5: Flow diagram of experimental procedure followed tfhoer DF process with S. occidentalis 41 Experimental 3.6 Analytical procedures 3.6.1 Compositional analysis of Cassava A complete compositional analysis of the Cassavltaiv caur used in this study was done by the South African Grain Laboratory for Cthaes sava peeled roots (starch), Cassava peels (cellulose) and unpeeled Cassavsa ( srotaortch and cellulose). The results of the composition analysis done by the LS AaGccording to the AACC standard methods are presented in Table 3.5. Table 3.5: Composition of Cassava root peels, unpeeled Cas rsoaovt aand peeled root Component Composition (wt %) Unpeeled Cassava Cassava peeled Cassava peels root root Moisture 9.5 10 9.2 Protein 2.5 2.7 5.1 Starch 81 82 67 Fat 0.6 0.8 1.1 Ash 2.5 2.5 7.0 Crude Fibre 3.9 2.0 11 Total 100 100 100 The main components were found to be starch, pnr,o tfeait, ash, crude fiber and moisture. The main component of all the samplebsm situted for analysis was found to be starch in a range of 67 to 83 wt%. These vacluoerrse spond to the findings of Aryee et al., (2006). The moisture and starch content are einfcluing factors in hydrolysis and fermentation for ethanol product iTonh.e moisture content lowers the rate of reaction because the higher the moistunrete cnot, the higher the viscosity of the feedstock and the more difficult it is for the emnzeys to reach the starch particles. The starch content determines how much fermentabler s u(galucose) can be expected as well as the expected ethanol yield. The higher stthaer ch content, the higher the glucose and ethanol yield will be. 3.6.2 Determination of moisture content of Cassava The initial moisture content (%) of raw Cassavat sr oaos well as the moisture content of the dried and milled Cassava flour was determd infoer this study. The initial moisture content of the raw Cassava roots werer mdeinted by drying a wet slice of unpeeled Cassava root with a known initial massa ni no ven at 60°C for 24 hours. 42 Experimental The percentage weight loss during this time wasc uclatled as the initial moisture content of the root. Two methods were used for euenlepd Cassava roots as a comparison to other work done by other researcohne rssp ecifically unpeeled Cassava roots moisture content. One single method was stheed ufor the three forms in this study for comparison of the three feedstock formpesc isfic in this study. The moisture content of dried and milled Cassava starch, Cas pseaevla and unpeeled Cassava roots were determined by the AACC 44-15A standard met huosdin,g a Mettler-Toledo HR 83 Halogen moisture analyzer. Each sample (2g) hweaste d at 130°C ± 1°C for one hour and the recorded weight loss was calculat ethde a isnitial moisture content of the samples. The result of the moisture analyses ise ng ivn Table 3.6. However the glucose yields were not calculated by the moistcuornet ent percentage but rather glucose yield were influenced by the moisture cnotn. te Table 3.6: Composition of Cassava root peels, unpeeled Cas rsoaovt aand peeled root Sample Method Moisture content (w/w) Raw unpeeled Cassava roots Oven dried 55-62% Dried, milled Cassava starch AACC 44-15A 10% Dried, milled Cassava peels AACC 44-15A 9.2% Dried, milled Cassava roots AACC 44-15A 9.5% Moisture content of a Cassava sample is a funcotfi othne cultivar, the planting season as well as the soil type and fertilizer used. Thaex im um reported moisture content in a Cassava sample was 72wt% (Negt oa l., 2005) on a wet basis. This result is similar to that of (Ngoe t al., 2005 and Tonukari, 2004) in their respective kw onr Cassava intercropping and the future of Cassava starch.u Tkaorni (2004) stated a moisture content of 70% and Ngeot al., (2005) approximately the same amount. 3.6.3 Determination of starch content of Cassava The starch content in starch rich biomass can bter mdeined by hydrolysis of the starch to glucose and indirectly determining thaer csht content with Equation 3.1 by determining the final glucose concentration in hthyed rolysate before fermentation (Brunt et al., 1998). C is the concentration of glucose (saem spollution), 0.93 is the conversion factor taken from Brunet al., (1998) for glucose to starch and W is the 43 Experimental weight of sample to be used for fermentation inL -(1g).. The amount of starch calculated with Equation 3.1 is the equivalent anmt oouf starch in the aliquot use d. C × 0.93 % Starch( g/100g) = ×100 [3.1] W The starch content of the Cassava sample usedis i ns ttuhdy calculated with Equation 3.1 by hydrolysis was determined to be between 6a9n%d 87 % (w/w) of the aliquot used. The starch content determined through hysdirso lwy ith Equation 3.1 of the Cassava cultivar used in this study is the same r oordf magnitude as the starch content of 31 Cassava varieties determined by A reyte eal., (2006), who reported general starch content between 67% and 88% (wT/wh)e. r esults in this study are also similar to the results reported by Srinorakuteatr aa l., (2004) who reported a starch content of between 61.84-69.90% (w/w). The sugoanrt ecnt from HPLC was used to determine the starch content by calculation. Tohleo rcimetric measurement was to determine that all the starch had been conver ted. 3.6.4 HPLC analysis After liquefaction, saccharification and fermenotant,i samples were filtered through a 0.2µm syringe filter and analyzed using the High Pemrfaonrce Liquid Chromatography (see Table 3.2). Before analys tihs eo fhydrolysate and fermentation broth calibration curves were prepared from the nqtiutaative determination of the available sugars in Cassava using the HPLC. A rcaatiloibn curve for the quantitative determination of the weight percentage ethanol hine tfermentation broth was prepared from known samples of ethanol concentnr autisoing HPLC. The method used for analyzing and processing the standards t haen dexperimental data on the HPLC as well as the calibration are curves discdu sins edetail in Appendix A. The areas of the peaks, resulting from the analysisth eo f concentration injected on the HPLC, were used to determine the composition of sthuegars and ethanol by converting the measured areas mass/volume usin gg rtahdeient of the calibration curves. Reproducibility of the analysis of the samples gu stihne HPLC was confirmed by injection of each sample three times. The peakse wdeisrtinct and no significant 44 Experimental overlapping of the peaks occurred. The analysisd ictions for the two columns used in the HPLC are given in Table 3.7. Table 3.7: Analysis parameters on the HPLC for Cassava hydsraotely and fermentation broth Parameters Shodex Zorbax Mobile phase 100% HPLC grade water 75% Aceton:i t2ri5le% wate r Column temperature 80°C 60°C Detector temperature 55°C 50°C Flow rate 1.00ml/min 1.00ml/min Inj. Volume 10µl 10µl Run time 20 min 10 min 3.7 Optimization of liquefaction and saccharificatoi n steps Enzymatic hydrolysis was done employing the met huosdesd by Ayernore t al., (2002) and Mojovice t al., (2006) with modifications . 3.7.1 Effect of substrate form on glucose yield The effect of substrate form on the amount of gsluec othat can be produced by liquefaction and saccharification was investigabteyd u sing three different forms of the Cassava root. Cassava peels, unpeeled roo ptse aenledd roots were investigated for glucose production capability before and during .S STFhe peels, peeled root and unpeeled roots were thoroughly washed, sun driedd m ainlled to a +1.5mm sieve size. The substrate concentration was 20wt% (200g mCillaesds ava + 800mL water) for all the forms of Cassava. Liquefaction was carriedu osuint g 7µ L.g-1 of Termamyl SC at a pH of 6, a temperature of 95°C one hour. Theco gsleu yield for each of the Cassava forms was recorded at various time intervals antde r acfompletion of the enzyme hydrolysis. The results of this investigation sahreo wn in Section 4.1.1. 3.7.2 Effect of pH on glucose yiel d The effect of pH on glucose concentration was itnigvaetsed for unpeeled Cassava roots using a substrate concentration of 17% anrydi nvga the pH between 5.5, 6 and 45 Experimental 6.5 during the liquefaction step. The liquefactiotinm e was 90 minutes at a temperature of 95°C using an oven and a Termamy ld SosCage of 7µ l.g-1. The liquefaction step was followed by saccharificatiwointh 7.5 µl.g-1 Spirizyme fuel at a temperature of 55°C for 48 hours in a shaker intcourb at 150rpm. The effect of varying the pH on the final glucose concentratioans winvestigated by varying the pH for the saccharification step between 4 and 5.5n. trCool samples (no enzyme) were used for both processes. The results of this tinigvaetsion can be found in Section 4.1.2. 3.7.3 Effect of temperature on glucose yield The effect of temperature on the glucose yield ndgu rtihe liquefaction step was investigated on Cassava unpeeled roots by carroyuint gth e liquefaction in an oven at a pH of 6 for 90 minutes using Termamyl SC -1µ (l.7g ) and varying the liquefaction temperature between 85°C and 95°C. The effect mopf eterature on the glucose yield during saccharification was investigated by cargry oinut the saccharification step at a pH of 4.5 in an incubator shaker (150 rpm) usingir izSypme fuel (7.5 µl.g-1) and varying the temperature between 55°C and 65°C. nAtr ocol sample (no enzyme) was used for each set of experiments. The resultsh ios f int vestigation can be found in Section 4.1.3 . 3.7.4 Effect of biomass load on glucose yield The effect of biomass load on the glucose concteionntr aduring liquefaction and saccharification was investigated by using a 10 ,w ta%nd a 20 wt% substrate concentration of milled unpeeled Cassava rootse. sTahme concentration of enzyme was used in the liquefaction and saccharificatitoenp ss for all substrate concentrations. Liquefaction was performed at a temperature of 9 w5°itCh a pH of 6 using Termamyl SC (7 µl.g-1) for one hour and the saccharification step wasrf oprmeed at a temperature of 55°C and a pH of 4.5 with Spirizyfmuel (7.5 -1µl.g ) and Celluclast 1.5L (4 µl.g-1) for four hours. The hydrolysis was performed inu raDn bottles in a shaker incubator at 170rpm. The results of thvise sintigation are presented in Section 4.1.4. 46 Experimental 3.7.5 Effect of enzyme combination on glucose conctreation The effect of different enzyme combinations durliinqgu efaction and saccharification was investigated by using different combination sT eorfmamyl SC, Celluclast 1.5 L and Spirizyme fuel during the hydrolysis steps.l l eMdi unpeeled Cassava roots (100g) were mixed with distilled water up to 500ml totaol luvme in Duran bottles. In all instances, liquefaction was performed at a temupreer aotf 95°C with a pH of 6 using Termamyl SC (7µ l.g-1) for one hour. The saccharification step was opremref d at a temperature of 55°C with a pH of 4.5 for 4 hoursi,n ug either only Spirizyme Fuel (7.5 µl.g-1) or both Spirizyme fuel (7.5µ l.g-1) and Celluclast 1.5L (4 -1µ l.g ). The results of this investigation are presented in iSone c4t.1.5. 3.7.6 Effect of enzyme load on glucose yield Unpeeled milled Cassava roots (100g) was mixed 4w0it0hml of distilled water (20wt %). The mixture was hydrolyzed with various contcraetnions of Termamyl Sc per gram Cassava (7µ l.g-1, 5 -1 -1µl.g , 2µl.g ) for liquefaction at a pH of 6 and at a temperature of 95°C for 1hour, Spirizyme fuel prearm g Cassava (7µ.5l.g-1, 5.5µl.g-1, 2.5µl.g-1) and Celluclast 1.5L per gram Cassavaµ l.g(4-1, 2µl.g-1, 1µl.g-1) for saccharification at a pH of 4.5 and a temperatufr e5 5o°C for four hours. The hydrolysates were filtered for analysis. The rtess oufl this investigation are presented in Section 4.1.6. 3.8 Optimization of fermentation step 3.8.1 Separate hydrolysis and fermentation withS . cerevisiae Termamyl SC, Spirizyme fuel and Celluclast 1.5L 7(7.5, and -1µ L.g Cassava) were used in the liquefaction and saccharification s trepsspectively. Four grams of bakers’ yeast was used for fermentation because literastugreg ests 150g in 20L hydrolysate for bakers’ yeast to effect the fermentation. Theea syt was first subjected to the hydrolysate to be used for fermentation for ten umteins so as to revive it from the dormant state (see Section 3.1.1) All samples were liquefied (production of dextrinw)i th 7µl termamyl per gram Cassava in Duran bottles at a temperature of 9p5H°C 6, for 60 minutes in an oven. 47 Experimental Samples were collected, filtered and analyzed flourc ogse concentration with the HPLC. All samples were subjected to saccharificna t(iporoduction of glucose) with a mixture of 7µl Spirizyme fuel per gram Cassava anµdl C4elluclast per gram Cassava in Duran bottles at a temperature of 55°C, pH o4r.5 4 f8 hours in a shaker at 150 rpm. Samples were collected, filtered and analyzed flourc ogse concentration with the HPLC. Cassava hydrolysates from all samples obtained ftrhoem two-step hydrolysis of the Cassava unpeeled roots were subjected to etharnmoel nfetation byS accharomyces cerevisiae under anaerobic conditions. Four grams of anchaokre rb’s yeast was used for fermentation in Duran bottles at a temperaotuf r3e0 °C and pH 4 for 120 hours in a shaker at 70 rpm. Samples of the fermented broetrhe wcollected on an hourly basis for three hours, and during the first 24hours ormf fentation and thereafter every 24 hours. The results of the SHF process for etharnoodl upction are presented in Section 4.2.1. 3.8.1.1 Effect of yeast concentration on ethanol eylid The effect of yeast concentration on the final neothl ayield was investigated using unpeeled Cassava roots with a 20wt % biomass lonad uasing different concentrations oSf . cerevisia efor the fermentation (8g.-1L, 5g.L-1 and 3g.L-1). The fermentation broths were filtered for ethanol ansiasl.y The results of this investigation are presented in Section 4.2.2.1. 3.8.2 Simultaneous saccharification and fermentatnio (SSF) Simultaneous saccharification and fermentation pwearsfo rmed using three forms of Cassava i.e. peeled Cassava roots, Cassava rolso ta pnede unpeeled Cassava roots. A substrate concentration of 20wt% Cassava was uosr eadll fexperiments. SSF was performed the same way for all three Cas sfoarvms. The process was carried out in 1L duran bottles. Pretreatment oef three Cassava slurries with Termamyl SC at a pH of 6 and a temperature of 9w5a°Cs performed for one hour prior to SSF. A starting batch size of 20wt% (1 0C0agssava + 400g distilled water) of the pretreated slurry was used for ethanol fermtieont aand was suitable for the 1L 48 Experimental duran bottle. The yeast concentration was setg .aLt- 18 of the total sample (batch sizes), resulting in the amount of yeast addedg b 4egin. The yeast and the enzymes (Spirizyme fuel 7µl.g-1 and Celluclast 1.5L 4µ l.g-1) were added simultaneously, but not until the pH had been adjusted to 4.5 by 0.1H 2MS O4 and the temperature had reached 30°C. Samples were taken at time interfvoarl s 72 hrs under careful monitoring of the process. Samples were filtererdo utghh micro filters and later analyzed with HPLC (Norgard, 2004). The resultsth oef SSF process can be found in Section 4.2.2. 3.8.3 Direct fermentation with S. occidentali s Direct fermentation of Cassava roots was done w20itwh t% substrate concentration. The slurry was inoculated with 1% peptone and wuatso calaved at a temperature of 121°C for 15min. The peels were prepared the same w aTyh.e mixture was inoculated with 1% peptone. After the heat pretreatment, the slurrsy iwnaoculated with 25 ml of the 24 hour old inoculum at a pH of 4.5 and was procesinse ad shaker at 150rpm, at a temperature of 37°C. Samples were taken for 7 danadys were centrifuge, filtered and HPLC analyzed. The method used for the direct efenrtmation with S. occidentali s was adapted from Rojaent al., (2007). 49 Experimental 3.9 REFERENCES ARYEE, F.N.A., ODURO, I., ELLIS, W.O. & AFUAKWA, J.. 2006. The physicochemical properties of flour samples frome rtohots of 31 varieties of cassava. Food Control,1 7: 916-922. ASSOCIATION FOR CLINICALCHEMISTRY (AACC). 1999. “Suoth African Grain Laboratory, S.A. AYERNOR, G.S., HAMMOND, T.K. & GRAFFHAM, A. 2002 .T he combination of rice malt and amyloglucosidase for the produnc toiof sugar syrup from cassava flour. African Journal of Science and Technol,o 3g(y1): 10-17, 01 Jan. BRUNT, K., SANDERS, P. & ROZAMA, T. 1998. The enzaymtic determination of starch in food, feed and raw materials of the sht ainrdcustry.S tarch, 50: 413–419. MOJOVIC, L., NIKOLIC, S., RAKIN, M. & Vukasinovic,M . 2006. Production of bioethanol from corn meal hydrolyzateFsu.e l, 85 (12-13): 1750-1755, Oct. MORRISOM, W. & LAIGNELET, B. 1983. An improved corliometric procedure for determining apparent and total amylose in cereadl oatnher starches. J o urnal of Cereal Science 1, :9–20. NGO, T. D., INGER, L. & NGUNYEN, T. M. 2005. Interrocpping cassava (Manihot esculenta Crantz) with Flemingia (Flemingia macryollpah); effect on biomass yield and soil fertility. L ivestock Research for Rural Developm. e1n7t(6), Feb. NORGAD, J. 2004. Ethanol Production from Biomass O-p timization of Simultaneous Saccharification and Fermentation Rweithspect to Stirring and Heating, Department of Chemical Engineering, Lund Institouft eT echnology. ROJAN, P.J, RAJEEV, K., SUKUMARAN, K., NAMPOOTHIR IM, . & Pandey, A. 2007. Statistical optimization of simultaneous shacricfication and l (+)-lactic acid 50 Experimental fermentation from cassava bagasse using mixed recu oltfu lactobacilli by response surface methodologyB. iochemical Engineering Journa3l.6 (3): 262-267. SAELIM, K., YAOWALUK, D. & ARAN, H. 2008. Saccharicf ation of cassava starch byS accharomycopsis fibuligerias olated from Loog-Pang (rice cake starter). Journal of Science and Technolog3y0. ( 1): 65-71. SAIBENE, D. & SEETHARAMAN, K. 2008. Use of iodines aa tool to understand wheat starch pasting propertieSsta. rch-stark,e 60(1): 1-7. SRINORAKUTARA, T., SUESAT, C., PITIYONT, B., KITPRECHAVANIT, W. & CATTITHAMMANIT, S. 2004. Utilization of Waste form Cassava Starch Plant for Ethanol Production (.Joint International Conference on “Sustainable rEgyn eand Environment (SEE), Hua Hin. Thailand. p. 773-76). TONUKARI, N.J. 2004. Cassava and the future of csht.a rElectronic Journal of Biotechnolog,y 7 (1): 5-8, 15 Apr. 51 Results and discussion CHAPTER 4 RESULTS AND DISCUSSION “Creativity is allowing yourself to make mistakeAsr.t is knowing which ones to kee p” Unknown Overview All the hydrolysis and fermentation experiments ew edrone according to the procedures described in chapter 3. In this ch,a pthter results of the optimization of the enzymatic hydrolysis of Cassava are presenntedd daiscussed. The chemical composition of Cassava is given in Section 4.1c. t iSone 4.2 focuses on the results of the enzymatic hydrolysis experiments, after whihceh rtesults of the fermentation are documented and discussed extensively in Sectio.n 4S.u3mmary of results for this work are tabled in Section 4.4 4.1 Optimization of liquefaction and saccharificatoi n steps 4.1.1 Effect of substrate form on glucose yield The effect that substrate form has on the finalc ogslue yield and rate of glucose production was investigated by liquefying threef edriefnt forms of Cassava (Cassava starch, Cassava peels and unpeeled Cassava rcocootsrd) iang to the method described in Section 3.7.1. Figure 4.1 and Table 4.1 illutset rtahat Cassava peels can be readily degraded by enzymes to glucose, but that a lowuecro gsel yield is obtainable than for the Cassava starch and unpeeled Cassava rootsa.v Ca apsesels have almost the same glucose yield as the Cassava starch. This is od uthee t fact that Cassava peels consist of two layers (periderm and cortex), which resuinlt sa high starch and cellulose content (77wt %). Furthermore, Sulphuric acid wuasse d to operate the pH during liquefaction, the acid may have hydrolyzed the ucloeslle in the peels since cellulose can be broken down through acid hydrolysis by surlipch acid at high temperatures of 90ºC which were used during liquefaction and ab o vTeh.e combined starch and cellulose content of the peels is close to thec hst acrontent of Cassava starch (82wt %). Moreover during the liquefactions step, onley tshtarch was liquefied though not 52 Results and discussion completely liquefied, most possibly the starch cahtetad to the peels were more accessible for liquefaction by the enzymes thant tjhues peels and the swelling of the starch in the presence of water can hinder the meensz yfrom getting the starch particles. 0.1 0.08 0.06 0.04 0.02 0 0 20 40 60 80 Time (min) Figure 4.1: Influence of Cassava substrate form on the gluc ose yield during liquefactio n (♦ - Peels, - peeled Cassava roots, -unpeeled Cassava roots) The glucose yield increases with time for all Cavsas faorms as the starch is converted to dextrin and glucose by the added enzymes. Thel gfilnuacose yield for the three different substrate forms are summarized in Tab.1le 4 Table 4.1: Final glucose yield for different Cassava forms indgu rliquefaction Substrate form Final glucose yield (g.-g1) Cassava starch 0.05 Cassava peels 0.04 Unpeeled Cassava roots 0.09 The unpeeled Cassava roots yielded a much highuecro sgel yield than the peels or the starch samples even though the starch contentl l fothre a three samples are in the same order of magnitude. The unpeeled roots containh bthoet peels with the two epidermal layers (Cellulose) with higher fiber content (3.4)7 t%han the peeled roots (2.01%) and the starch. The acid (Sulphuric acid) was alsod ufsoer pH operation for unpeeled Cassava roots, the acid hydrolysis of cellulose lwikaesly to take place hence a glucose yield similar to the sum of the glucose yield foere pls and Cassava starch was obtained. The difference in glucose yield for the unpeeleds sCaava roots and the sum of the peels 53 Glucose yield (g.g-1) Results and discussion and starch glucose yield is higher than the expeenrtimal error (see Section 3.3). The yield per mass of biomass for the unpeeled Casrsoaovtas was obtained with the same amounts of enzymes per gram of biomass as for etheels pand the Cassava starch. This means that the mixture of peels and starch in tnhpee euled roots were better utilized/converted to produce glucose than for rsaetpea hydrolysis of the two forms of biomass. This is evident when the glucose proodnu crtai tes for the liquefaction of the different substrate forms are compared. The buebsst trsate form for glucose production from Cassava is thus unpeeled Cassava roots. The initial rate of glucose production for the teh redifferent substrate forms were calculated using Equation 4.1 and the values aersee pnrted in Table 4.2 dCglucose [4.1] rglucose = dt Table 4.2: Initial glucose production rate (15 min) for diffeenrt substrate forms Substrate form Production rate (r) (g.g-1.min-1) Unpeeled root s 0.0015 peeled root s 0.0007 peels 0.0002 The initial rate (0.001 5g.g-1.min-1) of glucose production for the unpeeled roots is much faster than the other two substrate forms hw haicve rates of 0.0007 g-1..gmin-1 and 0.0002 g.-g1.min-1 for unpeeled roots and peels respectively. Threc hs tias bound by the peels which have a higher cellulose compto annedn thus not as accessible to enzyme attack as pure starch granules, resulti nag lionw glucose production rate when comparing pure starch and the unpeeled roxottu rmei of starch and cellulose. The density of the latter is lower due to the pnrecsee of the cellulose pieces in the starch. A lower density facilitates the easy asc coefs enzymes to both the cellulose and starch components, resulting in a very highc ogslue production rate. Starch swells in the presence of water, forming agglomeesr atht at are not easily accessible for conversion to glucoseK, o( molprasert and Ofoli, 200 7b)y the enzymes and thus a lower glucose production rate is obtained for thuere p starch substrate than for the unpeeled roots. 54 Results and discussion 4.1.2 Effect of pH on glucose yield The effect of pH on the production of glucose dgu rinthe liquefaction and saccharification was investigated according tom theeth ods discussed in Section 3.7.2. The change in glucose concentration with a chan gpeH i during the liquefaction step is presented in Figure 4.2. 0.06 0.05 0.04 0.03 0.02 0.01 0 0 20 40 60 80 100 Time (min) Figure 4.2: Effect of pH on glucose yield during liquefaction (♦ - control, - pH 6,  - pH 6.5, - pH 5.5) 1.2 1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25 30 Time (h) Figure 4.3: Effect of pH on glucose yield during saccharificoant i (♦ - control, - pH 4.5, - pH 4,  - pH 5) The effect of pH on the enzyme activity and gluc ocosencentration (Figure 4.2 and 4.3) indicates that theα -amylase (Termamyl) is active at pH 6-6.5 and cittsiv aity declines at pH 5.5. The optimal pH for liquefact iwoans found to be 6. The glucose 55 Glucose yield (g.g-1) Glucose yield (g.g-1) Results and discussion yield initially increases with time for all samp.l e sAccording to the manufacturer’s specification sheet, the optimum treatment time sftoarrch with Termamyl SC is 60 minutes. From Figure 4.2 it can be seen that a6f0te rminutes, the control samples have the lowest glucose yield and the sample tdre at ea pH of 6 has the highest glucose yield. The increase in glucose yield hfoer ctontrol sample is most probably due to the acid hydrolysis of starch caused by hSuurlipc acid during pH operation. The fact that all samples treated with Termamyl hSaCve a higher glucose yield than the control sample after 60 minutes indicates tthea ta ddition of Termamyl SC to the samples had a positive effect on the glucose yaienld the activity of the enzyme is thus confirmed. The final glucose for samples tetrde aat a pH of 6 and 6.5 increased after 60 minutes and was approximately the samteh ea te nd of the liquefaction process (90 minutes) while the glucose yield foer sthample treated at a pH of 5 as well as the glucose yield of the control decreasleigdh tly after 60 minutes. This suggests that the best pH to use during liquefna cisti othus a pH of 6. The effect of pH on the glucose yield during sacricfihcation is presented in Figure 4.3. It can be seen that the glucose yield of ctohnet rol sample remained constant, while the glucose yield of all the samples treawteitdh Spirizyme fuel increased with time, thus confirming the activity of the enzyme c tonvert dextrin into glucose. At the end of the saccharification process, all sasm ptrleated at different pH values yielded approximately the same glucose yield. Tmhiesans that the pH had no significant effect on the glucose yield during shaacrcification for the pH range investigated in this study. This result is in accordance with the work of Ob(o2h0 08). The optimum pH for saccharification was found to be 4.5 (Figure 4T.6h)i.s result is in agreement with the work of Srinorakutarae t al., (2004) who used the same pH for saccharificatinodn a obtained 75% glucose concentration. 4.1.3 Effect of temperature on glucose yield The effect of temperature on the glucose yield ndgu ri liquefaction and saccharification was investigated according tom theeth od discussed in Section 3.7.3. The influence of temperature on glucose yield dgu rliiqnuefaction and saccharification is shown in Figure 4.4 and 4.5 respectively. 56 Results and discussion 0.06 0.05 0.04 0.03 0.02 0.01 0 0 20 40 60 80 Time (min) Figure 4.4: Effect of temperature on glucose yield during liquefaction (♦ - 95°C, - 90°C, - 85°C) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 Time (h) Figure 4.5: Effect of temperature on glucose yield during saccharification (♦ - 65°C,  - 60°C, - 55°C) From Figure 4.4 it can be seen that all sampleast etdre at different temperatures initially has the same production rate up to apipmroaxtely 35 minutes. After 35 minutes the production of glucose increases fafostre sra mples treated at 95°C than at the other temperatures. Glucose production draallsyt icdecreases from 0.05g-1. gto 0 after 60 minutes for samples treated at 85°C wthiele g lucose yield only levels off at approximately 75 minutes for samples treated atC 9. 5 °The extra 25 minutes of increased glucose production for samples treat e9d5 °aCt results in a much higher 57 Glucose yield (g.g-1) Glucose yield (g.g-1) Results and discussion final glucose concentration than for samples trde atte 85°C and 90°C. This result corresponds to the manufacturer’s specificatione ts fhoer Termamyl SC. From Figure 4.5 it can be seen that all treatmemntp teratures initially have high production rates and that the saccharification tiroena cis almost complete after 4 hours. The highest final glucose yield was obtda infoer a treatment temperature of 55°C. According the error analysis in appendix Dth.2e difference between the temperatures was insignificant and therefore thwee slot temperature was chosen for energy conservation. The best temperature to ursineg d saccharification of Cassava roots with Spirizyme Fuel is thus 55°C. 4.1.4 Effect of biomass load on glucose yield The effect of biomass load on the glucose yield winavsestigated according to the methods described in Section 3.7.4. The effecbti omf ass load on glucose yield can be seen in Figure 4.6. 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 6 Time (h) Figure 4.6: Influence of biomass load on glucose yield duriqnuge lfaction and saccharification (♦ - 10wt% substrate, - 20wt% substrate ) From Figure 4.6 it can be seen that during liquteiofanc (up to one hour) the glucose yield for a biomass load of 10wt% is higher tharn afo biomass load of 20wt%. With more biomass present, the viscosity of the mixitsu rhei gher and thus the biomass is not that easily accessible to the enzymes. The rl ovwiscosity for a biomass load of 10wt% also results in a faster conversion of dnesx trito glucose during the saccharification step, as can been seen at a tfi mtweo o hours in Future 4.6. After approximately four hours, enough biomass/dextrianss bheen converted to glucose so 58 Glucose yield (g.g-1) Results and discussion that the effect of viscosity on glucose productiso ns tarting to become limited and thus the enzymes can now access all of the avea ildaebxltrins/biomass for conversion to glucose. At the end of the hydrolysis proce sis citlear that a 20wt% biomass load gives a higher final glucose yield. The result nis aigreement to work done by Aggarwal et al., (2001) that reported an optimum biomass loa2d5 owf t% of Cassava in their study. 4.1.5 Effect of enzyme combination on glucose yie ld The effect of enzyme combination on the glucoseld ywieas studied according to the procedure described in Section 3.7.5. The purpoof steh is investigation is to verify whether the addition of Celluclast 1.5L to the hoylydsris step would have a significant influence on the final glucose yield. The effecf t aodding Celluclast 1.5L to the hydrolysis step is summarized in Table 4.3 The glucose yield from enzymatic hydrolysis (5 hso) uor f 20 wt% and 10 wt% Cassava flour was 178 g-1. L(0.9 g.g-1), and 91g.L-1 (0.91 g.g-1) respectively with three enzymes (Termamyl SC, Spirizyme fuel and Celluc 1la.5stL) compared to 156 g-.1L (0.78 g.g-1) and 83 g.L-1 (0.83g.g-1) respectively (see Figure 4.7 and 4.8), for two enzymes (Termamyl SC and Spirizyme fuel) unders tahme e hydrolysis conditions of pH and Temperature. The results are in agreemethn tt hwei results reported by Sriroth et al., (2000). The use of additional enzymes also hetlop erded uce the viscosity of the sample solution and improved starch hydrolysis. Table 4.3: Effect of enzyme treatment on glucose y ield Cassava Glucose yield (g.g-1) roots and Termamyl SC Termamyl SC peels (wt %) Spirizyme fuel Spirizyme fuel Celluclast 1.5L 10 0.83 0.91 20 0.78 0.90 59 Results and discussion 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 6 Time (h) Figure 4.7: The effect of enzyme treatment on glucose yiehld 1 w0iwt t% substrate concentration (♦ - Without Celluclast, - With Celluclast) 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 6 Time (h) Figure 4.8: The effect of enzyme treatment on glucose yiethld 2 w0iwt% substrate concentration. (♦ - Without Celluclast, - With Celluclast) 4.1.6 Effect of enzyme loading on glucose yield The effect of enzyme loading on the final glucosie ldy after hydrolysis was investigated with the methods described in Sect3io.7n. 6. The concentration of enzymes used is summarized in Table 4.4 60 Glucose yield (g.g-1) Glucose yield (g.g-1) Results and discussion Table 4.4: Enzyme loadings in different treatment combinat ions Loading Termamyl SC Spirizyme fuel Celluclast (µL.g-1) (µL.g-1) ( -1µL.g ) Loading 1 7 5 2 Loading 2 7.5 5.5 2.5 Loading 3 4 2 1 The results of hydrolysis of a 20wt% biomass lolaudrr ys with the different enzyme loadings is presented in Figure 4.8. 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 6 Time (h) Figure 4.9: Influence of enzyme concentration on glucose cotrnacteionn ( - Loading 1, - Loading 2,♦ - Loading 3) Enzyme combination 1 gave a better glucose yie.l9d2 (50 g.g-1) followed by loading 3 and loading 2 though the difference was insignniftic, agiven a 5% error. The lower glucose yield at higher enzyme loading can be bauttterid to the fermentation of by- products when more enzymes are present, resunlti nlegs si glucose being produced. This corresponds with was reported by Ku Ismeat ial l., (2008). 4.2 Optimization of fermentation step For the study on glucose consumptionS bayc charomyces cerevis,ia 2e0wt% substrate concentration was used, and the concentration wydarso lhyzed with 0.7% T, 0.75% S & 0.4% C. 61 Glucose yield (g.g-1) Results and discussion 4.2.1 Separate hydrolysis and fermentation withS . cerevisiae Separate hydrolysis and fermentation was carriet dw oituh a 20 wt % biomass loading according to the method described in Section 3 . 8C.o1n. ditions used are summarized in Table 4.5. Table 4.5: Hydrolysis conditions used during separate hydriosl yasnd fermentation (SHF) proces s Process pH T (°C) Termamyl SC Spirizyme fuel Celluclast ( L.g-1) ( L.g-1µ µ ) (µL.g-1) Liquefaction 6 95 7 - - Saccharification 4.5 55 - 7.5 4 The increase in glucose yield during liquefactionnd asaccharification is shown in Figure s 4.10 and 4.11 respectively. 0.1 0.08 0.06 0.04 0.02 0 0 20 40 60 80 Time (min) Figure 4.10: Increase in glucose yield during liquefaction t hine SHF process 1.2 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 6 7 Time (h) Figure 4.11: Increase in glucose yield during saccharificat ionn the SHF process 62 Glucose yield (g.g-1) Glucose yield (g.g-l) Results and discussion The glucose rich hydrolysate from hydrolysis waosc iunlated withS cerevisia efor ethanol production. Figure 4.12 shows the ethanieoldl yduring fermentation and the ethanol yield in comparison to the glucose uptaukrein dg fermentation are shown in Figure 4.13. The initial ethanol concentration )( vin/v the broth was 9.6%. 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 Time(h) Figure 4.12: Ethanol production in shake flasks by Saccharom cyeceresvisiae using the SHF process From Figure 4.12 it can be seen that a final avee reathganol concentration of 75g-1.,L corresponding to a yield coefficient (Y) of 0.38g.g-1p/s (gram of ethanol per gram biomass) was reached after only 24 hours. Theia li ngitlucose yield in the 20wt% biomass load hydrolysate was 0.92-1g. . gThe ethanol yield per gram of glucosea p(/Yg) was 0.4g.g-1. 1 0.8 0.6 0.4 0.2 0 0 20 40 60 80 Time (h) Figure 4.13: Ethanol production in comparison to glucose uptiank seh ake flasks by Saccharomyces cerevisiae using S HF (♦ - Ethanol, - Glucose) From Figure 4.13 it can be seen that the final ogsluec yield in the fermentation broth was 0.006g.-g1. Equivalent amounts of ethanol and carbon diox(CidOe2 ) are formed 63 Ethanol Yield (g.g-1) Ethanol yield (g.g-1) Results and discussion during the fermentation reaction. Theoretically k 1g0 of sugar will produce 5.1 kg of ethanol and 4.9 kg of carbon dioxide (Salle, 19 9T3h)e.refore if 40 % of the available glucose in the initial hydrolysate was utilized ftohre production of ethanol as the fermentation results above show, theoretically, roaxpipmately 38% must have been utilized for the production of C2O. From a mass balance on glucose it can then be said that approximately 21.4% of the glucose wailisz eudt for yeast production. The same analogy was used by Yusaku et al., (2004) osntu day focusing o nethanol fermentation of raw cassava starch wRith izopus ko jiin a gas circulation type fermentor. These results are in agreement wit hr ethsuelts reported by Srinorakutara et al., (2004) for the production of ethanol from Cassainv aa fermentor. In other studies (Thailand Institute of Scientific and Teoclhongical Research, 2002) a maximum ethanol concentration of only 5% after 6o9u rhs of fermentation was reported. It can be thus be concluded that opatimtioinz of the hydrolysis steps had a positive effect on the final ethanol yield in thsitsu dy. 4.2.2 Simultaneous saccharification and fermentatnio with S. cerevisia e 4.2.2.1 Influence of yeast concentration on ethan oyileld The influence of yeast concentration on ethanoldl ydieuring the SSF was investigated on a 20wt% biomass load of unpeeled Cassava rococtosr daing to the methods presented in Section 3.8.1.1. The process condsi tuiosed to investigate the influence of yeast concentration on the ethanol yield duSrinSgF is given in Table 4.6. Table 4.6: Influence of yeast concentration on ethanol y ield Process T (°C) pH Termamyl Spirizyme fuel Celluclast Yeast ( L.g-1) ( L.g-1) ( L.g-1µ µ µ ) (g.L-1) Liquefaction 95 6 7 - - - Saccharification 30 4.5 - 7.5 4 8,5,3 and Fermentation The ethanol yield and glucose yield during fermteionnta using SSF process for a 20wt% biomass load of unpeeled Cassava and diftf eyreanst concentrations are presented in Figures 4.14 and 4.15 respectively 64 Results and discussion 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 Time (h) Figure 4.14: Effect of yeast concentration on ethanol yield ra 7ft2e hours, using the SSF proces s (♦ - 8 g.L-1, - 5g.L-1 ,  - 3g.L-1) 0.8 0.6 0.4 0.2 0 0 20 40 60 80 Time (h) Figure 4.15: Glucose uptake during fermentation by Saccharom cyecreesvisiae in the SSF proce ss (♦ - 8 g.L-1,  - 5g.L-1,  - 3g.L-1) From Figures 4.14 and 4.15 it can be seen thath ael l yeast concentrations used resulted in approximately the same ethanol yieTldh.e initial ethanol production rates for the different yeast concentrations used are ng ivn Table 4.7 Table 4.7: Initial ethanol production rate for different yea csotncentration s Yeast Concentration (g.L-1) Initial ethanol production rate 3 0.025 5 0.029 8 0.035 From Table 4.7 it can be seen that the initial neothl aproduction rate is faster at the higher yeast concentration. This is expected sminocree yeast organisms will convert the glucose faster, but in the end, given enoumghe tihe same ethanol yield should be 65 Glucose yield (g.g-1) Ethanol yield (g.g -1) Results and discussion reached. In this study all three chosen yeast ecnotnractions resulted in almost complete utilization of glucose, implying that 3-g1 .Lyeast is sufficient to convert all available glucose formed in the SSF process toe tbhioa-nol within 72 hours. 4.2.2.2 Influence of substrate form on ethanol yide l Simultaneous saccharification and fermentation wcasrr ied out with a 20wt% biomass loading according to the methods presein tSede ction 3.8.2. The hydrolysis and fermentation process conditions for all threaes sCava substrate forms (Cassava starch, Cassava peels and unpeeled Cassava rroeo gtsiv) ean in Table 4.8. Table 4.8: Hydrolysis and fermentation process conditionsa flol rth ree Cassava substrate forms Process T (°C) pH Termamyl Spirizyme fuel Celluclast Yeast ( L.g-1) ( L.g-1 -1 -1µ µ ) (µL.g ) (g.L ) Liquefaction 95 6 7 - - - Saccharification 30 4.5 - 7.5 4 8 and Fermentation The increase in the glucose concentration duriqnuge lfiaction in the SSF process is presented in Figure 4.16 0.1 0.08 0.06 0.04 0.02 0 0 20 40 60 80 Time (min) Figure 4.16: Increase in glucose concentration during liquefoanc tsi tep of the SSF process (♦ - Peels, - peeled Cassava roots, -unpeeled Cassava roots) From Figure 4.17 it can be seen that the unpeealesds aCva roots gave the highest final ethanol yield. The higher ethanol yield for unpeede lCassava roots compared to Cassava starch and Cassava peels is due to ther hgigluhcose production during liquefaction (see Table 4.8). 66 Glucose yield (g.g-1) Results and discussion The hydrolysate for liquefaction was inoculated hw Sit . cerevisia eas well as saccharification enzymes. The influence of subtes tfroarm on the final ethanol yield and glucose uptake during SSF at a yeast concieont roaft 8g.L-1 is presented in Figures 4.17 and 4.18 respectively. 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 Time (h) Figure 4.17: Influence of substrate form on ethanol yield (♦ - Peels, - peeled Cassava roots, -unpeeled Cassava roots) 1 0.8 0.6 0.4 0.2 0 0 20 40 60 80 Time (h) Figure 4.18: Glucose uptake during fermentation of peels, pde reoleots and unpeeled roots (♦ - Peels, - peeled Cassava roots, -unpeeled Cassava roots) The results graphically presented in Figure 4.1e7 saurmmarized together with yield coefficients for the SSF process in Table 4.9. 67 Glucose during SSF(g.-g1) Ethanol yield (g.g-1) Results and discussion Table 4.9: Ethanol and glucose yield coefficients for the SpSroFc ess Substrate Glucose a Ethanol form Y -1 b -1 c -1 dp/s ( g.g ) Yp/s ( g.g ) Yp/g ( g.g ) Yp/s ( ml.g-1) Y e 1 p/g ( ml.g ) Unpeeled 0.90 0.42 0.45 0.53 0.58 roots Peeled roots 0.70 0.30 0.40 0.40 0.50 (starch) Peels 0.55 0.16 0.30 0.20 0.37 (cellulose) a gram glucose per gram Cassava subs t rate; b gram ethanol per gram glucose hydrolysate c gram ethanol per gram Cassava subs trate d mL ethanol per gram glucose hydrolysate e mL ethanol per gram Cassava substrate The conversion efficiencies for the SSF proces sg iavreen in Table 4.10. Table 4.10: Conversion efficiencies for SSF process in wt% Substrate Substrate Glucose Glucose Glucose Total form utilized for utilized for utilized for utilized for glucose glucose ethanol CO2 cell growth conversion production production production and other products Unpeeled roots 90% 45% 43% 11.4% 0.994 Peeled roots 70% 40% 38% 20.8% 0.988 Peels 55% 30% 29% 40.5% 0.995 From Table 4.10 it can be seen that the unpeelesdsa Cva roots performed better than the other substrate forms in terms of conversiofinc ienf cy for substrate to glucose, as well as glucose conversion to ethanol. The toitnaal l f glucose to products is approximately the same for all the substrate fo rm Tsh.is implies that more by- products were formed during the fermentation ofe p Curassava starch and Cassava peels than for the unpeeled Cassava roots. Thnet ifidcaetion of the formed by- products fell outside the scope of this investiogna.t i In conclusion it can be said that unpeeled Cassava roots are the best substratet ofo urmse for the production of bio- ethanol and that a 45% conversion of substrateio t-oe tbhanol can be achieved. 4.2.3 Direct fermentation with S. occidentalis Direct fermentation with unpeeled Cassava rootsh wS.it occidentallisw as done with 20wt% biomass loading according to the procedursec rdibeed in Section 3.8.3. The final ethanol yield obtained was only 0.0025-1g. .g The final yield was too low to be 68 Results and discussion economically feasible; therefore this productions wnaot investigated further. From this it could be concluded that direct fermenta tiso nnot an ideal method for bio- ethanol production from starch crop that requirder hoylysis. 4.3 Comparison of bio-ethanol production processes The different production processes (SHF, SSF an)d fDorF the production of bio- ethanol from Cassava roots were quantitatively caormedp to select the best process to use. The ethanol yield for the three differentc persoses is compared in Figure 4.19. The ethanol yield obtained from DF process wasl otowo t o show on Figure 4.19. 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 Time (h) Figure 4.19: Comparison of ethanol production betweSeSnF , and SHF in shake flasks (- SSF, - SHF) From Figure 4.19 it can be seen that although HthFe pSrocess initially produces more ethanol faster, the final ethanol yield for the S pSroFcess is higher. The final ethanol yields and conversion efficiencies for the threoec persses investigated is presented in Table 4.11. Table 4.11: Comparison of glucose and ethanol yields of difnfet rbeio-ethanol production processes Process Yp/g (g.g -1) Yp/s (g.g -1) SHF 0.92 0.38 SSF 0.90 0.43 DF - 0.03 From Table 4.11 it can be seen that although thFe pSrHocess produced more glucose from the same amount of biomass, more of the gelu cwoass converted to ethanol in the SSF process, making the latter economicallye m aottractive and promising. The 69 Ethanol yield (g.g-1) Results and discussion better conversion of glucose to ethanol in SSF epsrso cis because the end-product inhibition from glucose formed during enzymatic hroyldysis is relieved by yeast fermentation. 4.4 Summary of hydrolysis and fermentation results A comparative summary of all results obtained iins tshtudy is presented in Table 4.12. The Table shows that this work has beend avtaeldi through other sources of similar work. The different forms of Cassava habvee n used for ethanol production, but peels has not been done extensively hences ttuhdisy shared a focus on that as well. Therefore this table strives to reveal howuc mh work has been done on Cassava ethanol production. This study adds value to trhees epnt work as well as it might be seen as an expansion of it. 70 Results and discussion Table 4.12: Conclusion and validation of results against litteurrae Theoretical/ Literature Nquma et al., 2009 (this study) Moisture content Unpeeled root- 60-72% (wet basis6) 1% (wet basis)- unpeeled root (Ngo et al. 2005 and Tonukari, 2004) 9.2% (db)- unpeeled root 9.2-9.7wt% (db)- peels peels – 8.50wt% (db) 10.2wt% (db)- peeled root peeled root-------- Starch content (db) 70-88% (Aryee et al., 2006 and 83% - unpeeled root (unpeeled roots) (Lasztity,1999) 51-67% - peels ( peels) 61% (Obadinae t al., 2006) 70-82% -peeled root (peeled roots) --- Glucose Yield 60-93% (Ejiofore t al. 1996) 60-92%, (0.9;0.6 g.-1g ) – conversion (Yp/s) (Krzysztoef t al., 2007) unpeeled roots (unpeeled roots) ------ Peeled roots 70% (0.7 g. -g1 )- peeled roots (peels) 55% (0.55g. -g1 )- peels Ethanol Yield coefficient 0.45 ml.g-1 ( Drapchoe t al., 2008) 0.53ml. g-1 (SSF); 0.4 ml. -g1 (Yp/s) 0.41 g.g-1 0. 42 g.g-1 – unpeeled roots (peeled and unpeeled --- roots) 0.3 g.g-1 – peeled roots (peels) 0.16 g.g-1 –peels Ethanol Yield coefficient 0.51 g. g-1 (Ejiofor et al., 1996) 0.41 g.g-1 (SHF); 0.45 g. -g1 (Yp/g) -------- (SSF); - unpeeled roots (peeled and unpeeled roots) 0.4 g. g-1 – peeled roots (peels) 0.3 g.g-1 –peels Ethanol percent Roots-8-12% (Atthasampunneat al., 9.8% (SHF); 10.6% (SSF); - (peeled and unpeeled 1987) unpeeled roots roots) Peels-1.05% ( Adesanyeat al., 2008) (peels ) 7%- peeled roots 4% -peels Substrate concentration 25-30% (Aggarwale t al., 2001); (Ku 20% Ismail et al., 2008) Enzyme concentration 0.3% Term, 0.2 % Spir ( Ku Ismaeilt 0.2% Term, 0.25 % Spir and al., 2008) 0.1% Cell Yeast concentration 10g.L-1 -5 g.L-1 ( Norgard, 2008) 3 g.-L1 Optimum pH 6 (liq), (Oboh, 2008). 4.5 (Sacc) 6 (liq), 4.5 (Sac) (Srinorakutarae t al., 2004) Optimum Temperature 100-85 (liq), 55-65 (Sac) (Ejiofoer t 95 (liq), 55 (Sac) (°C) al. 1996 and Srinorakutareat al. 2004) Efficient enzyme Termamyl, Spirizyme and CellulaseT ermamyl SC, Spirizyme Fuel combination (Sriroth et al,. 2000) and Celluclast 1.5L 71 Results and discussion 4.5 REFERENCES ADESANYA, O. 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Thermo- enzymatic Hydrolysis of Cassava Starch αb-ya mylase and amyloglucosidase. (Proceedings of MUCET-2008, Malaysia:Perlis). LASZTITY, R. 1999. Cereal chemistry. Hungary: Akamdiaei Kiado: 11-51. McMILLAN, J.D. NEWMAN, M.M. TEMPLETON D.W. & MOHAGHEGHI, A. 1999. Simultaneous saccharification and co-fermtieont aof dilute-acid pretreated yellow poplar hardwood to ethanol using xylose-fenrmting Zymomonas mobil.i s Applied Biochemistry and Biotechnolo: g7y7–79, Mar. MORRISOM, W. & LAIGNELET, B. 1983. An improved corliometric procedure for determining apparent and total amylose in cereadl oatnher starches. J o urnal of Cereal Science 1, :9–20. NGO, T. D., INGER, L. & NGUNYEN, T. M. 2005. Interrocpping cassava (Manihot esculenta Crantz) with Flemingia (Flemingia macryollpah); effect on biomass yield and soil fertility. L ivestock Research for Rural Developm. e1n7t(6), Feb. NORGAD, J. 2004. Ethanol Production from Biomass O-p timization of Simultaneous Saccharification and Fermentation Rweithspect to Stirring and Heating, Department of Chemical Engineering, Lund Institouft eT echnology. 73 Results and discussion OBADINA, A.O., OYEWOLE, O.B., SANNI, .L. O. & ABIOLA, S. S. 2006. Fungal enrichment of cassava peels proteiAnfsr.ic an Journal of Biotechnolog, y5 (3): 302- 304, May. OBOH, G. 2005. Isolation and characterization ofy lamse from fermented cassava (Manihot esculentaC rantz) wastewate. rAfrican Journal of Biotechnolog, y4(10), 1117-1123. OCLOO, F. C. K. & AYENOR, G. S. 2008. Physical, mchiceal and microbiological changes in alcoholic fermentation of sugar syruopm f rcassava flourA. frican Journal of Biotechnolog,y 7 (2): 164-168. OMEMU, A.M., AKPAN, I., BANKOLE, M.O. & TENIOLA, O.D. 2005. Hydrolysis of raw tuber starches by amylase Aosfp ergillus nigerA M07 isolated from the soil. African Journal of Biotechnolog, y4: 19-25. SAIBENE, D. & SEETHARAMAN, K. 2008. Use of iodin aes a tool to understand wheat starch pasting propertieSsta. rch-stark,e 60(1): 1-7. SALLE, A. J. 1993. The Permaculture Book of Ferment and Human Nuntr itio Tyalgum: Tagari. SCHENCK, F.W. 2002. Starch hydrolysates - An oveewrv. i Intenational Sugar Journal, 104, 82-89. SRIROTH, K., CHOLLAKUO, R., CHOTNEERANAT, S., PIYAHCOMKWAM, K. & OATES, C.G. 2000. Processing of Cassava wastre imfoproved biomass utilization. Bioresource Technolog, y71: 63-69. SRINORAKUTARA, T., SUESAT, C., PITIYONT, B., KITPRECHAVANIT, W. & CATTITHAMMANIT, S. 2004. Utilization of Waste form Cassava Starch Plant for Ethanol Production (.Joint International Conference on “Sustainable rEgyn eand Environment (SEE), Hua Hin. Thailand. p. 773-76). 74 Results and discussion THAILAND INSTITUTE OF SCIENTIFIC AND TECHNOLOGICAL RESEARCH (TISTR). 2002. The improvement on ethanol qualtihtye: Royal Chitralada Project. Ministry of Science and Technology, Thailand :Baonkg.k TONUKARI, N.J. 2004. Cassava and the future of csht.a rElectronic Journal of Biotechnolog,y 7 (1): 5-8, 15 Apr. WEBB, F.C. 1964. Biochemical Engineering, D.Van tNraonsd Ltd. London. YUSAKU, F., MASAFUMI, O., and SEINOSUKE, U. 2004.t hEanol fermentation of raw cassava starch with Rhizopus koji in a gas uclairtcion type fermentor. Biotechnology and Bioengineerin2g7, (8): 1270-1273, 18 Feb. 75 Conclusions CHAPTER 5 CONCLUSIONS 5.1 Conclusions This study was undertaken to investigate the opztaimtioin of ethanol production from enzymatic pretreatment of the Cassava roots. Fhroism s tudy some conclusions and recommendations can be made. • In this dissertation it was shown that Cassavas r ohoatve a high starch content that can be enzymatically utilized for the prodounc tiof glucose that can be converted to bio-ethanol. • All Cassava root material used in this study hamdo ais ture content of between 9 and 10 wt% (dry basis). • The starch content of all Cassava roots used sin s thuidy was determined to be between 69 wt% and 87wt%. • Iodine solution was used successfully in this s tutod ydetermine complete conversion of starch to dextrins during liquefanc.t io • It was shown conclusively in this study that suabtset rform has a significant influence on the glucose produced during liqueofanc, tiand that unpeeled Cassava roots are the best substrate form to use. • It was found that pH significantly influences thiqeu lefaction step, but not the saccharification. • Liquefaction of Cassava unpeeled roots should bnee daot a pH of 6 for optimum glucose production. 76 Conclusions • It was found that temperature significantly influcend the amount of glucose produced during hydrolysis, and that a temperatuorf e 95°C during liquefaction and a temperature of 55°C during sarcicfihcation will produce the highest glucose yield. • A 20 wt% biomass load resulted in a higher glucyoieseld during liquefaction and saccharification. • Enzyme loading had a significant influence on tihnea l fglucose yield during hydrolysis and it was found that an enzyme loadoifn 2g µL.g-1 Termamyl SC, 2.5 -1µL.g Spirizyme fuel and 1µ L.g-1 Celluclast 1.5L resulted in the highest glucose yield. • It was shown in this study that the addition of luCcelal st 1.5L to the hydrolysis step had a positive effect on the glucose yielda ionbetd. • Direct fermentation of unpeeled Cassava roots pcreod ua very small amount of ethanol. • The SHF process showed a better conversion of bsiso mtoa glucose, while the SSF process showed better conversion of glucoesteh aton ol. • It was shown that a yeast concentration of onlyL -31 gis. sufficient to convert the available glucose to ethanol in 72 hours. • An ethanol yield of 45wt% (g ethanol per g subset)r awtas achieved with the SSF process, resulting in a production potenti a5l 8o0fL of ethanol per ton of unpeeled Cassava roots. 77 Appendix APPENDIX A CALIBRATION CURVES Overview In this Appendix the preparation of calibration vceusr for the determination of sugar concentration and ethanol concentration in a Cas sfearvmentation broth using high performance liquid chromatography (HPLC) is preesde.n t The Appendix is subdivided into two sections. The results for rtehdeu cing sugar calibration curves are given in Section A.1. The tabulated results andib rcatlion curve for ethanol is given in Section A.2. The Chromatograms for the sugadrs eatnhanol are shown in Section A.3. In order to use high performance liquid chromatopghrya as an analytical tool, a calibration curve is required. Standard mixturfe ds ifoferent glucose concentration are analyzed with HPLC and the response area of thke fpoer aeach concentration used is recorded. The areas of the peaks are then ploatgteadin st the concentration used to obtain each of the peaks and a straight line tiesd f ito the data to obtain an equation that can be used to determine the glucose conctieont rian any Cassava fermentation broth. y = mx + b m: gradient x: Amount (concentration) y: Height (area) Where m is the gradient, for every sample, y isw kn oand thus x can be calculated. The same principle holds for all the calibrationrv ceus used in this study. A.1 Sugar calibration curves The fermentable sugars available in Cassava tharet wpreepared as standards are; glucose, sucrose, fructose, maltose and malto.t ri oTshee calibration curves (slope) were used to determine the amount/yield of sugraerse pnt in a specific hydrolysate 78 Appendix and fermentation broth, reported in chapter 4. Tchoencentration prepared for standards are concentrations that were expectebde to btained during and after hydrolysis. The amount of water and sugar used rteop apre the standard sugar mixtures for the calibration curves are given inb lTea A.1. Table A.1: Preparation (dilutions) of sugar standar ds Concentration Volume of sugar Volume of water Final Volume (ml) (g.L-1) (ml) (ml) 200 2 0 2 100 1 1 2 50 1 1 2 10 0.4 1.6 2 5 1 1 2 1 0.4 1.6 2 Table A.2: Peak areas obtained for each sugar concentratioing u HsPLC Concentration Glucose Fructose Maltose Sucrose Maltotriose (g.L-1) 1 8.09E+04 7.89E+0 4 1.06E+05 7.23E+0 4 9.04E+04 5 3.85E+05 2.66E+0 5 4.33E+05 3.71E+0 5 4.06E+05 10 6.76E+05 4.73E+0 5 8.28E+05 7.96E+0 5 7.99E+05 50 3.79E+06 2.90E+0 6 3.95E+06 3.90E+0 6 3.96E+06 100 7.76E+06 5.82E+0 67.83E+06 7.78E+0 6 7.98E+06 200 1.54E+07 1.30E+0 71.47E+07 1.53E+0 7 1.56E+07 1.8E+07 1.6E+07 y = 77039x 1.4E+07 R² = 0.9999 1.2E+07 1.0E+07 8.0E+06 6.0E+06 4.0E+06 2.0E+06 0.0E+00 0 50 100 150 200 250 Concentration (g.L-1) Figure A.1: Glucose calibration curv e 79 Peak Area Appendix 1.4E+07 1.2E+07 y = 63336x R² = 0.9963 1.0E+07 8.0E+06 6.0E+06 4.0E+06 2.0E+06 0.0E+00 0 50 100 150 200 250 Concentration (g.L-1) Figure A.2: Fructose calibration curv e 1.8E+07 1.6E+07 y = 76823x R² = 0.9999 1.4E+07 1.2E+07 1.0E+07 8.0E+06 6.0E+06 4.0E+06 2.0E+06 0.0E+00 0 50 100 150 200 250 Concentration (g.L-1) Figure A.3: Sucrose calibration curv e 1.6E+07 y = 74698x 1.4E+07 R² = 0.9985 1.2E+07 1.0E+07 8.0E+06 6.0E+06 4.0E+06 2.0E+06 0.0E+00 0 50 100 150 200 250 Concentration (g.L-1) Figure A.4: Maltose calibration curv e 80 Peak Area Peak Area Peak Area Appendix 1.8E+07 1.6E+07 y = 78404x 1.4E+07 R² = 0.9998 1.2E+07 1.0E+07 8.0E+06 6.0E+06 4.0E+06 2.0E+06 0.0E+00 0 50 100 150 200 250 Concentration (g.L-1) Figure A.5: Maltotriose calibration curv e A.2 Ethanol calibration curve The ethanol concentrations in each of the standsoalrudt ions used were determined with HPLC. The concentration prepared for stand aarrdes concentrations that were expected to be obtained after fermentation of tuhgea rss. The amount of water and ethanol used to prepare each of the standard osnoslu itsi given in Table A.3. Table A.3: Preparation of ethanol standard solutions Concentration Volume of ethanol Volume of water Final Volume (ml) (g.L-1) (ml) (ml) 150 2 0 2 100 0.7 1.3 2 50 1 1 2 25 1 1 2 15 0.8 1.2 2 7.5 1 1 2 3.75 1 1 2 Table A.4: Ethanol peak areas obtained for each standard sioonlu t Concentration (g.L-1) Area 15 5.70E+05 25 9.30E+05 50 2.00E+06 100 3.66E+0 6 150 5.56E+0 6 81 Peak Area Appendix 6.E+06 y = 37149x 5.E+06 R² = 0.9986 4.E+06 3.E+06 2.E+06 1.E+06 0.E+00 0 20 40 60 80 100 120 140 160 Ethanol Concentration (g.L-1) Figure A.6: Ethanol calibration curv e A.3 Sugar and ethanol chromatograms Figure A.7: Typical glucose chromatogram obtained from HPLCl yasnias 82 Peak Area Appendix Figure A.8: Typical ethanol chromatogram obtained from HPLC laysnias Ethanol Sucrose Glucose Figure A.9: Typical HPLC chromatogram of a fermentation broathm sple 83 Appendix APPENDIX B ENZYMATIC HYDROLYSIS Overview In this Appendix the glucose yields obtained du reingzymatic hydrolysis of Cassava for different values of the manipulated variableres laisted. The results given in this Appendix is graphically presented and discussecdh ainp ter 4 of this dissertation. B.1 Optimization of liquefaction and saccharificatoi n steps The glucose yield is recorded ags/ sY (g.g -1) (gram glucose per gram Cassava). B.1.1 Effect of substrate form on glucose yield Table B.1: Glucose yield (g.-g1) obtained during liquefaction of different substetr a forms of Cassav a Substrate form Time Peels Peeled roots Unpeeled roots (minutes) (Cellulose) (Starch) (Starch and Cellulose) 0 0.0240 0.0250 0.0075 10 0.0250 0.0260 0.0150 15 0.0265 0.0360 0.0300 30 0.0315 0.0360 0.0450 45 0.0325 0.0375 0.0800 60 0.0400 0.0500 0.0900 B.1.1 Effect of pH on glucose yield Table B.2: Glucose yield (g.-g1) obtained at different pH values during liquefaocnt iof unpeeled Cassava roo ts Time (min) Control pH 6.5 pH 6 pH 5.5 0 0.0036 0.0060 0.0066 0.0060 15 0.0036 0.0084 0.0126 0.0270 30 0.0186 0.0216 0.0300 0.0336 45 0.0246 0.0366 0.0318 0.0378 60 0.0240 0.0312 0.042 0.0372 75 0.0288 0.0300 0.0432 0.0402 90 0.0102 0.0186 0.0498 0.0486 84 Appendix Table B.3: Glucose yield (g.-g1) obtained during saccharification of unpeeled Cassava roots at different pH valu es Time(h) Control pH 5.5 pH 4.5 pH 4 0 0.0102 0.0186 0.0498 0.0486 1 0.1320 0.7620 0.7680 0.7980 2 0.0900 0.9420 0.8580 0.8040 3 0.078 0.9180 0.9180 0.8640 4 0.1200 0.9300 0.8580 0.7920 8 0.132 0.9600 0.8400 0.8520 12 0.162 0.9300 0.8640 0.8640 24 0.330 0.8020 0.9720 0.9420 28 0.306 0.8580 0.8880 0.8520 B.1.3 Effect of temperature on glucose yield Table B.4: Glucose yield (g.-g1) obtained during liquefaction of unpeeled Cassava roots at different temperatur es Time (min) 95°C 90°C 85°C 0 0.0025 0.0055 0.0070 15 0.0140 0.0105 0.0125 30 0.0200 0.0250 0.0230 60 0.0535 0.0350 0.0500 Table B.5: Glucose yield (g.-g1) obtained during saccharification of unpeeled Cassava roots at different temperatu res Time (h) 65°C 60°C 55°C 0 0.063 0.000 0.0415 1 0.713 0.325 0.6400 2 0.750 0.765 0.7150 3 0.760 0.790 0.7650 4 0.820 0.840 0.7750 28 0.870 0.800 0.8500 48 0.850 0.830 0.9050 85 Appendix B.1.4 Effect of biomass load on glucose yield Table B.6: Glucose yield (g.-g1) obtained during liquefaction of unpeeled Cassava roots at different biomass loadin gs Time(min) 10wt% 20wt% 0 0.01 0.010 15 0.04 0.040 30 0.05 0.045 45 0.05 0.075 60 0.11 0.085 120 0.66 0.470 180 0.71 0.710 240 0.75 0.775 300 0.83 0.890 B.1.5 Effect of enzyme combination on glucose yie ld Table B.7: Glucose yield (g.-g1) obtained during hydrolysis of unpeeled Cassava roots using a 10wt% biomass loading with and with tohue addition of Celluclast 1.5L to the hydrolysis mixtur e Time(min) Without Celluclast 1.5 L With Celluclast 1.5L 0 0.01 0.01 15 0.04 0.03 30 0.05 0.04 45 0.05 0.04 60 0.11 0.12 65 0.46 0.56 73 0.54 0.57 88 0.57 0.63 90 0.64 0.62 120 0.66 0.65 180 0.71 0.65 240 0.75 0.69 300 0.83 0.91 86 Appendix Table B.8: Glucose yield (g.-g1) obtained during hydrolysis of unpeeled Cassava roots using a 20wt% biomass loading with and with tohue addition of Celluclast 1.5L to the hydrolysis mixtur e Time(min) Without Celluclast 1.5L With Celluclast 1.5L 0 0.010 0.010 15 0.040 0.030 30 0.045 0.045 45 0.075 0.016 60 0.085 0.085 65 0.330 0.525 73 0.365 0.575 88 0.375 0.780 90 0.410 0.815 120 0.470 0.835 180 0.710 0.860 240 0.775 0.865 300 0.780 0.890 B.1.6 Effect of enzyme loading on glucose yield Table B.9: Enzyme loadings (wt %) used in different combinnast iion this stud y Combination Termamyl SC Spirizyme Fuel Celluclast 1.5 L Combo 1 0.2 0.25 0.1 Combo 2 0.5 0.55 0.2 Combo 3 0.7 0.75 0.4 Table B.10: Glucose yield (g.-g1) obtained during liquefaction of unpeeled Cassava roots using different enzyme loadin gs Time(h) Combo 1 Combo 2 Combo 3 0 0.010 0.005 0.005 0.25 0.030 0.015 0.015 0.5 0.045 0.020 0.020 0.75 0.080 0.040 0.035 1 0.085 0.060 0.050 1.08 0.575 0.750 0.750 1.25 0.780 0.760 0.760 1.5 0.815 0.770 0.790 2 0.835 0.810 0.795 3 0.860 0.870 0.860 4 0.865 0.890 0.890 5 0.890 0.860 0.924 87 Appendix APPENDIX C FERMENTATION Overview In this Appendix the ethanol and glucose yield evas lu(data) during fermentation will be given. The manipulative variable which was ftehrem entation route for the optimization of ethanol yield are reported in dfaotram with respect to Section C.1, SHF and Section C.2 SSF. The ethanol yield is rdeecdo ras Yg/s (g.g -1) of cassava. C.1 Separate Hydrolysis and Fermentation Table C.1: Ethanol yield (g.g-1) obtained during SHF of Cassava roots by Saccharomyces cerevisiae Time (h) Y (g.g-1) 0 0.031 0.25 0.190 0.5 0.200 1 0.190 2 0.285 3 0.275 24 0.380 48 0.390 72 0.380 96 0.395 120 0.375 88 Appendix Table C.2: Ethanol yield compared to glucose uptake during SoHf CFassava roots by Saccharomyces cerevisiae Time (h) Ethanol (g.g-1) glucose (g.g-1) 0 0.031 0.8250 0.25 0.190 0.4600 0.5 0.200 0.3550 1 0.190 0.3550 2 0.285 0.1825 3 0.275 0.1900 24 0.380 0.0150 48 0.390 0.0185 72 0.380 0.0080 96 0.395 0.0065 120 0.375 0.0060 C.2 Simultaneous Saccharification and Fermentation Table C.3: Ethanol yield (g.g-1) and glucose uptake (g-1.)g during SHF of Cassava roots using different yeast concentrations Ethanol (g.g-1) Glucose (g.g-1) Time (h) 8 g.L-1 5 g.L-1 3 g.L-1 8 g.L-1 5 g.L-1 3 g.L-1 0 0.025 0.0200 0.0175 0.620 0.625 0.6200 0.5 0.040 0.0450 0.0300 0.555 0.735 0.5850 1 0.050 0.0525 0.0400 0.560 0.550 0.5900 2 0.055 0.0550 0.0500 0.565 0.545 0.6100 3 0.140 0.1100 0.0700 0.390 0.520 0.5700 4 0.155 0.1450 0.1300 0.460 0.445 0.5250 8 0.205 0.2075 0.1700 0.320 0.220 0.3250 24 0.280 0.3350 0.2100 0.155 0.040 0.0200 48 0.395 0.3800 0.4000 0.010 0.010 0.0085 72 0.420 0.415 0.4250 0.065 0.005 0.0030 Table C.4: Ethanol yield (g.g-1) obtained during SHF of different forms of Cass ava roots with S. cerevisiae Time Peels Peeled Cassava Unpeeled Cassava (h) (Cellulose) (Starch) (Starch and Cellulose) 0 0.017 0.020 0.025 0.5 0.052 0.055 0.040 1 0.06 0.055 0.050 2 0.075 0.060 0.055 3 0.050 0.070 0.140 4 0.065 0.085 0.155 8 0.110 0.130 0.210 24 0.130 0.140 0.280 48 0.150 0.270 0.395 72 0.160 0.300 0.420 89 Appendix Table C.5: Glucose uptake (g-.1g) during SHF of different substrate forms of Casas av using S. cerevisiae Time Peels Peeled Cassava Unpeeled Cassava (h) (Cellulose) (Starch) (Starch and Cellulose ) 0 0.550 0.710 0.9200 0.5 0.330 0.630 0.5600 1 0.260 0.540 0.5600 2 0.120 0.490 0.5700 3 0.120 0.390 0.3900 4 0.060 0.290 0.4600 8 0.055 0.190 0.3200 24 0.027 0.120 0.1600 48 0.008 0.015 0.0100 72 0.005 0.012 0.0065 Table C.6: Ethanol yield (g.g-1) compared to glucose uptake (-g1). gduring SHF of Cassava peels using 8 g-1. LS cerevisiae Time Ethanol yield Glucose uptake (h) (g.g-1) (g.g-1) 0 0.017 0.550 0.5 0.052 0.330 1 0.060 0.260 2 0.075 0.120 3 0.050 0.120 4 0.065 0.060 8 0.110 0.055 24 0.130 0.027 48 0.150 0.008 72 0.160 0.005 90 Appendix 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 70 80 Time (hr) Figure C.1: Ethanol yield (g.g-1) compared to glucose uptake (-g1). gduring SHF of Cassava peels using 8 g-1. LS cerevisiae Table C.7: Ethanol yield (g.g-1) and glucose uptake (g-1.)g during SSF of peeled Cassava roots using 8 g-1. Lof S. cerevisiae Time Ethanol yield Glucose uptake (h) (g.g-1) (g.g-1) 0 0.020 0.7050 0.5 0.055 0.6250 1 0.055 0.5400 2 0.060 0.4850 3 0.070 0.3900 4 0.085 0.2900 8 0.125 0.1900 24 0.140 0.1200 48 0.270 0.0150 72 0.30 0.0115 91 Yield (g.g-1) Appendix 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 70 80 Time (hr) Figure C.2: Ethanol yield (g.g-1) and glucose uptake (g-1.)g during SSF of peeled Cassava roots using 8 g-1. Lof S. cerevisiae Table C.8: Ethanol yield (g.g-1) and glucose uptake (g-1.)g during SSF of unpeeled Cassava roots using 8 g-1. LS. cerevisiae Time Ethanol yield Glucose uptake (h) (g.g-1) (g.g-1) 0 0.025 0.9200 0.5 0.040 0.5600 1 0.050 0.5600 2 0.055 0.5700 3 0.140 0.3900 4 0.155 0.4600 8 0.210 0.3200 24 0.280 0.1600 48 0.395 0.0100 72 0.42 0.0065 92 Yield (g.g-1) Appendix 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 70 80 Time (hr) Figure C.3: Ethanol yield (g.g-1) and glucose uptake (g-1.)g during SSF of unpeeled Cassava roots using 8 g-1. LS. cerevisiae Table C.9: Comparison of ethanol yield (g-1.)g for SHF and SSF Time Ethanol yield(g.g-1) (hr) SSF SHF 0 0.025 0.030 0.5 0.040 0.190 1 0.050 0.200 2 0.055 0.275 3 0.140 0.285 24 0.280 0.380 48 0.395 0.390 72 0.420 0.380 93 Yield (g.g-1) Appendix APPENDIX D EXPERIMENTAL ERROR Overview In this Appendix the experimental error values a(d) atduring liquefaction, saccharification and fermentation will be given. eT Ahppendix is subdivided into three Sections with respect to Section D.1, liqcuteiofan, Section D.2 saccharification and fermentation in Section D.3. The experimental error was done according to thlloew foing principles to validate this study. Average ( ) - The arithmetic mean, and is calculated by ad dai nggroup of numbers and then dividing by the count of those numbers. Z score (Z) -The z score for an item, indicates how far an dw hinat direction, that an item deviates from its distribution's mean, expereds sin units of its distribution's standard deviation. The equation (2) is used fomr psleas less than 100 (small samples), where TINV returns the inverse of theis ttr-idbution, which is used in the hypothesis testing of small sample data sets. STDEV (σ) - The standard deviation is the unit of measuret moef nthe z-score. It allows comparison of observations from differenrtm naol distributions, which is done frequently in research (see equation 1) Confidence Limit (±) - Returns a value that you can use to construccot nafi dence interval for a population mean. The confidence rivnatel is a range of values. Your sample mean,, is at the center of this range and the rang±e CisO NFIDENCE (see equation 3) 94 Appendix Experimental error - The approximation error in the data is the discrepancy between an exact value and some approximation. (tsoe iet equation 4) (1) (2) (3) (4) D.1 Liquefaction The experimental error for the liquefaction steps wdaetermined by repeating the liquefaction of raw Cassava starch at a pH of 6 aa ntedmperature of 95 °C five times. The glucose concentration (g-1.)g obtained for each time interval for the five raetpeed experiments are listed in Table D.1. The stataisl tpicarameters used to calculate the experimental error are listed in Table D.2. Thec ogslue concentration determine at each time interval for the five repeated liquefoanc tiexperiments is graphically presented in Figure D.1. Table D.1: Glucose concentration for repeated liquefactione erximpents at pH 6 and 95°C. Time (min) Sample 1 Sample 2 Sample 3 Sample 4 Salme p5 0 0.0155 0.015 0.0155 0.014 0.014 15 0.025 0.026 0.029 0.0275 0.026 30 0.048 0.0505 0.0505 0.0535 0.051 45 0.07 0.0765 0.074 0.0705 0.075 60 0.079 0.0805 0.0795 0.0795 0.081 Table D.2: Statistical parameters used to calculate the exmpenrital error for the liquefaction step Sample Final Glucose Concentration 1 0.079 2 0.0805 3 0.0795 4 0.0795 5 0.081 Mean 0.0799 Standard deviation 0.0008 Confidence limit (95%) 0.002 Experimental error 2.36% 95 Appendix 0.1 0.08 0.06 0.04 0.02 0 0 20 40 60 80 Time(min) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Figure D.1: Five replicates of increasing glucose yield durliinqgu efaction in the SHF process D.2 Saccharification The experimental error for the saccharificationp swteas determined by repeating the saccharification of a liquefied Cassava hydsraotley at a pH of 4.5and a temperature of 55°C five times. The glucose cotnrcaetinon (g.g-1) obtained for each time interval for the five repeated experims eanrte listed in Table D.3. The statistical parameters used to calculate the emxpeenrtial error are listed in Table D.4. The glucose concentration determine at eamche tiinterval for the five repeated saccharification experiments is graphyi cparellsented in Figure D.2. Table D.3: Glucose concentration for repeated saccharifica teioxnperiments at pH 4.5 and 55°C. Five replicates of samples Time(h) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 0 0.079 0.0805 0.0795 0.0795 0.081 0.3 0.875 0.905 0.915 0.905 0.905 0.7 0.91 0.915 0.915 0.895 0.89 1 0.85 0.85 0.865 0.855 0.865 2 0.865 0.885 0.89 0.89 0.89 3 0.885 0.905 0.905 0.905 0.91 4 0.905 0.92 0.93 0.915 0.93 6 0.915 0.93 0.93 0.935 0.925 8 0.93 0.94 0.93 0.93 0.935 24 0.925 0.935 0.93 0.93 0.935 28 0.925 0.935 0.93 0.905 0.93 48 0.935 0.89 0.9 0.935 0.93 96 glucose yield (g.g-1) Appendix Table D.4: Statistical parameters used to calculate the exmpenrital error for the saccharification step Sample Final Glucose Concentration 1 0.935 2 0.89 3 0.9 4 0.935 5 0.93 Mean 0.918 Standard deviation 0.021 Confidence limit (95%) 0.049 Experimental error 5.36 % 1 0.8 0.6 0.4 0.2 0 0 20 40 60 Time(h) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Figure D.2: Five replicates of increasing glucose yield dursinagc charification in the SHF process D.3 Separate Hydrolysis and Fermentation The experimental error for the fermentation steps wdaetermined by repeating the fermentation step using a yeast concentration .oLf -18 gfive times. The ethanol yields (g.g-1) for each experiment at different time intervarlse alisted in Table D.5. The statistical parameters used to calculate the emxpeenrtial error is listed in Table D.6. The ethanol yield for each experiment at each tinmter val for the five repeated fermentation experiments are graphically preseinnt eFdig ure D.3 . 97 Glucose yield (g.g-1) Appendix Table D.5: Ethanol yield (g.g-1) at different time intervals for five repeated fermentation experiments Time (min) Sample 1 Sample 2 Sample 3 Sample 4 Salme p5 0 0.031 0.031 0.031 0.031 0.031 0.25 0.245 0.1765 0.1775 0.2 0.17 0.5 0.23 0.195 0.195 0.2 0.175 1 0.19 0.19 0.19 0.18 0.19 2 0.215 0.35 0.255 0.295 0.32 3 0.275 0.3 0.27 0.26 0.275 24 0.365 0.38 0.385 0.385 0.375 48 0.38 0.395 0.39 0.39 0.39 72 0.375 0.39 0.395 0.395 0.375 96 0.38 0.395 0.405 0.4 0.395 120 0.385 0.375 0.37 0.365 0.38 Table D.6: Statistical parameters used to calculate the exmpenrital error for the liquefaction ste p Sample Final ethanol yield 1 0.385 2 0.375 3 0.37 4 0.365 5 0.38 Mean 0.375 Standard deviation 0.008 Confidence limit (95%) 0.018 Experimental error 4.85 % 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 50 100 150 Time(h) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Figure D.3: Five replicates of ethanol production in shake kflsa sby Saccharomyces cerevisiae using the SHF process graph of fermieonnt astamples 98 Ethanol yield (g.g-1)