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6.4. As show in Figwe 6.4, the enym concentration in the immobilizedd sysm was higher and deched much slower than the correspoading fke suspension culture. Loss of the recombinant protein expression (glucoamylase) was signincantly reduced in the immobilized c d system Thug enhanad pmductivity and stabùity in production of the recombinant protein was achkved m the immoùibed-ceII-olm airfift bioreactor. O 20 40 60 80 la0 120 14û 16û 1# 200 Time (hrs) Figure 6.2. Continuous Recombinant Yeast Fermentation in Imxnobilki-Ceü-FElm Biomctor Ushg Oiiginel SIraln Temperatme = JO OC, Aeon rate = 1.0 VW, Dihitioü rate = 0.1 h", Go = 20.0 g/L.
Glucoamylase Concentration (units/L) 4 A UI O O O O 3 O Cell, Ethanol and Glucose Concentrations (glL) and PH
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BIOREACTOR STUDIES OF HETEROLOGOUS
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nie University of Waterloo requin%
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ate depended on activities of the p
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Acknowiedgments Fmt of aü, 1 am ve
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2.6. Concluding Rernarks ..........
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5.3.3. Simlilated Results for Prese
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Table 1.1. Table 1.2. Table 1.3. Ta
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Figure 2.1. Figure 3.1. Figure 3.2.
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during Continuous Culture in Airlif
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Figure 5.3. Comparison between Mode
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Cell Systern at Glucose Feed Concen
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a .......... growth ratio (dimensio
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Y, ... ethanol yield for fermentati
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foreign plasmids. One major chalien
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than the denanired, inclusion body
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Table 1.2. Strategies for Enhancing
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Table 1.3. Host Cek, Pfasmids, Gene
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1.4. Immo bilized-Cell Bioreactors
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1. Study the gmwth characteristics,
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plasmid are used; or chromaromal DN
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2.2.1. Genetic Factors 2.2.1.1. Pid
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1- fkquently than a plasmid with Io
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Da Silva and Bailey (1991) used the
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LEU2 or TRPI is cloned into the aux
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eported reduced stabiliity in the s
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on metabolic pathways. In the ferme
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OC), oniy about 35% of the populati
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loss @) is dehned as the probabilit
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+ s Dilution Rate = p' = CI,- - K*+
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concentration couid Iead to coexist
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that factors other than immobiiizat
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earing and plasmid-tke =Ils. Gel be
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productivity irrespective of the mo
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inactive and useless. The problern
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Aerobic fermentation osing iamnobih
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(ranghg h m 1 to 7) on citric acid
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celi fraction decreases monotonicai
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(1987) model aliows for plasmid ins
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affect plasmid stability have ben o
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CEtAPTER 3 MATERIALS AND METHODS 3.
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stiU deleted hm the plasmid. Such p
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1) Reagent solution preparation was
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nonse1ective agar plates, but only
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working Liquid volume. The aeration
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3.5. UNnobilized Ceil Culture 35.1.
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3.52 Cd Lmmobiiization Yeast attach
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3.6. Experimental Error and Reprodu
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-0-Cell(1) +Total CeII (2) 4- Ethan
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* . I I a O 2 4 6 8 10 12 14 Batche
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Cell Maso, Glucose and EthanoI(g1L)
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4.2.2. Batch CuIture in Stirreà-Ta
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[t ~lucoamylase (ALR) -e Glucoamyla
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The finai giucoamyiase concentratio
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Bentley and Kompala, 1989; Satyagal
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160 First ExponenUaI Phase 1 -c ALR
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Figure 4hl. Pathways of Yeast Intem
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fermentation (using nitrogen spargi
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43. Continuous Suspension Culture C
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Figure 4.9. Continuous Suspension C
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1 +Enzyme +Fradlan of PBC +CeIl Mas
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Figures 4.12 and 4.13 show that by
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Fi- 4.13. Cornparison of Glu~iase C
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where 6 = p- - p' + pp*, which is a
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Figure 4-14. Effeds of Dilution Rat
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Impoolsup et ai. (1989a) and Hardji
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Figure 4.16. dB/dt versus B Plots a
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Figure 4.17. Totai Cell Mas at Diff
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another recombinant yeast (impoolsu
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other recombinant yeast under nonse
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4.3.4. Effixt of Dilution Rate On R
Page 141 and 142: Figure 4.18- Etlects of Mluiioa Rat
Page 143 and 144: Number of Generations - - - - - - *
Page 145 and 146: B Table 4.5. Metabolic Pathway Anal
Page 147 and 148: concentration. But this may be more
Page 149 and 150: [+ Enzyme (ALR) + Enzyme (STR) +Fra
Page 151 and 152: necessary for host ce& to grow in t
Page 153 and 154: Glucoamylase ConcentraUon (unltsll)
Page 155 and 156: [a Selective Medium A Nonseiective
Page 157 and 158: 5.1 Introduction CHAPTER 5 MODELING
Page 159 and 160: handle. excess glucose is femented
Page 161 and 162: The carbon fluxes h m the three met
Page 163 and 164: 53. Modei Simulation in Batch CuItu
Page 165 and 166: Table 5.1. Summary of Parameters fo
Page 167 and 168: 1 n Glucose o Cell Mass O Ethano1 A
Page 169 and 170: detennined in the cunent study, the
Page 171 and 172: Figure 5.4. Cornparison between Mod
Page 173 and 174: equations are üsted in Appendix G.
Page 175 and 176: a dilution rate at which the giucoa
Page 177 and 178: [ o Expetfmental data -Simuiated mt
Page 179 and 180: The effixts of dilution rate on the
Page 181 and 182: Ttme (hrs) 1 a Enzyme A Phsrnid-Bea
Page 183 and 184: It may take several generations for
Page 185 and 186: understanding of the nature of the
Page 187 and 188: On the other hand. the net accumula
Page 189 and 190: Differentiating Equation (6.20) res
Page 191: Enyme collcentration m the t>ioreac
Page 195 and 196: 50 100 150 200 Time (hrs) 1 O Free
Page 197 and 198: F i 6.6. Cornparison of Fnx Cd Mars
Page 199 and 200: . - O 50 100 150 200 250 nme (hrs)
Page 201 and 202: Nasi et ai.. 1988) and recombinant
Page 203 and 204: estimated by using Equation (6.24)
Page 205 and 206: [a lmmobilked System O Free System
Page 207 and 208: oxidation pathway. W~th increasing
Page 209 and 210: O 50 100 150 200 250 300 Tirne (hrs
Page 211 and 212: epeated batch fermentations, the fe
Page 213 and 214: Glucoamylase Concentration (unitslL
Page 215 and 216: 6) Suitable for repeated-batch and
Page 217 and 218: ecause glucose fermentation dominat
Page 219 and 220: cells. Tests of the proposed model
Page 221 and 222: 7.2. Recommendations The foUowing f
Page 223 and 224: APPENDIX A: PLASMID MAP FOR pGAC9 l
Page 225 and 226: where Bi (Biot number) = - ks De @
Page 227 and 228: APPENDK C. EFFECTS OF INITIAL GLUCO
Page 229 and 230: Figure C.2. Effect of Initial Gluco
Page 231 and 232: Figure C.4 Effect of Initial Glucos
Page 233 and 234: APPENDLX D. COMPARISON OF ORIGINAL
Page 235 and 236: (continue)
Page 237: (continue)
Page 240 and 241: (continue)
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E.2. Batch Fermentation with Recomb
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(continue)
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(continue) tfhJ 0.5 10.5 12.75 12.5
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(continue)
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E.3.2. Experimental Data (Parker an
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Run No: CALE (Selected Strain. D =
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Run No: CALR 5 (Original Suain, D =
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Run No: CSTR3 (Selected Suain, D =
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(continue)
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Run No: CICR2 (Selected Suain, D =
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Run No: CiCR4 (Selected Suain, D =
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Eh. Repeated Batch Culture in hmobi
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APPENDIX F. C CODE FOR THE PROPOSED
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* the initial data */ /* gening the
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* getting the founh term in R-K met
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I*Plasrnid-bearing ceU concentratio
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* Func tion C 1 */ îloat C l(float
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APPEMIM G. EXAMPLES OF MAPLE V CODE
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REFERENCES Ataai. MM. and Shuler. M
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Caunt, P.. hpoolsup, A.. and Greenf
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cuitanes of E. coli BZ18@TG 201) wi
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Gabrieisen. O.S. et aL (1990), Effi
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Huang, C-T., Peretti, S.W., and Bry
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Kim, B.G. and Shuler, M.L. (1990a),
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Lee, S.B.. Ryu, D.D.Y., seigel, R,
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Nasri, M., Sayadi. S.. Barbotin, J.
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Pin, S.J. and Kmwski. W.M. (1970),
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Seo, I-H. and Bailey. J.E. (1985).
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Tumer. B.G., Avgerinos G-C.? Melnic
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Yu, J. and Tang, X (Editor, 1991),
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