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................. TABLE OF CONTENTS ............. .. ....................................................... AB STRACT .. . . iv Acknowledgments .................................................................................................... vii ... List of Tables .............................................................................................................. m List of Figures ............................ . ............................................................................ xv ... NOMENCLATURE ...................................... . xxui .................................................... CHAPTER 1 . UV'T'RODUCTION ................................................................................. 1 1.1. Importance of Recombinant Saccharomyces cerevisioe ................................ 2 1.2. Instability of Recombinant Yeast ................................................................... 4 1.3. Investigations of Imrnobiiized Recombinant Yeast Systems .......................... 7 1.4. Im mo bilized-Ce11 Bioreactors ...................................................................... 10 1 .5 . Objectives ........................................................................................... II CHAPTER 2 . LITERATURE REVTEW ..................................................................... 13 2.1. Yeast Plasmid Vectors ............................*.................. ....,, 13 2.2. Factors Affecting Plasmid Stabüity in Recombinant S . cerevisiae ................. 15 2.2.1. Genetic Factors ................................................................................ 16 2.2.2. Environmental Factors ............ ,.l ........................................... 2 4 2.3. Plasmid Stability in Immobiüzed CeUs ...................................................... 3 5 2.3. I . Effects of Immobilization ......................... ., ............................... 35 1.3.2. Possible Stabilizing Mechanisms ....................................................... 40 2.4. hrno bilization Methods and immobilized Ce11 Bioreactors .......................... 41 2.5. Modehg of Recombinant Yeast .............. . ................................ 4 6
2.6. Concluding Rernarks ...................................................................... 5 1 CHAPTER 3 . MATERIALS AND METHODS ................................................... 5 4 3.1. Recombinant S . cerevkiae and Culture Media ............................................. 54 3.1.1. Microorganism and Plasmid .......................................................... 54 3.1.2. Compositions of Culture Media ................................................... 55 3.2. Sinin Screening under Double Selection Pressure ...................................... 55 3.3. Anaiytical Methods .............. . .... . ....................................................... 57 3.3.1. CeU Concentration ......................... ....... ......................................... 57 3.3.2. Glucose measurement ............................................................... 5 7 3.3.3. Ethano1 anaiysis ............................................................................... 59 3.3.4. Glucoamylase assay ..................... . ........................................... 59 3.3.5. Detenination of Fraction of Plasrnid- Bearing Cells ....................... 59 3.35. Determination of Maximum Specific Growth Rares .......................... 60 3.4. Fiee Suspension Culture .............................................................................. 60 3.4.1. Batch Fermentation ...................................................................... 61 3.4.2 . Continuous Fermentation ................................................................ 62 3.4.3. Repeated Batch Fermentation .......................................................... 63 3.5. Immobilized Cell Culture ........................ ., ................................................ 64 3.5.1. Structure of the Immobilized-CeU-Film Airlift Bioreactor ................. 64 3.52. Ceii immob~tion ................... ., ................................................. 66 3.5.3. Continuous Fermentation with Immobilized Cells ................... . .... 66 3.5.4. Repeated Bath Fermentation with Immobilized Cek ....................... 67
Page 1 and 2: BIOREACTOR STUDIES OF HETEROLOGOUS
Page 3 and 4: nie University of Waterloo requin%
Page 5 and 6: ate depended on activities of the p
Page 7: Acknowiedgments Fmt of aü, 1 am ve
Page 11 and 12: 5.3.3. Simlilated Results for Prese
Page 13 and 14: Table 1.1. Table 1.2. Table 1.3. Ta
Page 15 and 16: Figure 2.1. Figure 3.1. Figure 3.2.
Page 17 and 18: during Continuous Culture in Airlif
Page 19 and 20: Figure 5.3. Comparison between Mode
Page 21 and 22: Cell Systern at Glucose Feed Concen
Page 23 and 24: a .......... growth ratio (dimensio
Page 25 and 26: Y, ... ethanol yield for fermentati
Page 27 and 28: foreign plasmids. One major chalien
Page 29 and 30: than the denanired, inclusion body
Page 31 and 32: Table 1.2. Strategies for Enhancing
Page 33 and 34: Table 1.3. Host Cek, Pfasmids, Gene
Page 35 and 36: 1.4. Immo bilized-Cell Bioreactors
Page 37 and 38: 1. Study the gmwth characteristics,
Page 39 and 40: plasmid are used; or chromaromal DN
Page 41 and 42: 2.2.1. Genetic Factors 2.2.1.1. Pid
Page 43 and 44: 1- fkquently than a plasmid with Io
Page 45 and 46: Da Silva and Bailey (1991) used the
Page 47 and 48: LEU2 or TRPI is cloned into the aux
Page 49 and 50: eported reduced stabiliity in the s
Page 51 and 52: on metabolic pathways. In the ferme
Page 53 and 54: OC), oniy about 35% of the populati
Page 55 and 56: loss @) is dehned as the probabilit
Page 57 and 58: + s Dilution Rate = p' = CI,- - K*+
Page 59 and 60:
concentration couid Iead to coexist
Page 61 and 62:
that factors other than immobiiizat
Page 63 and 64:
earing and plasmid-tke =Ils. Gel be
Page 65 and 66:
productivity irrespective of the mo
Page 67 and 68:
inactive and useless. The problern
Page 69 and 70:
Aerobic fermentation osing iamnobih
Page 71 and 72:
(ranghg h m 1 to 7) on citric acid
Page 73 and 74:
celi fraction decreases monotonicai
Page 75 and 76:
(1987) model aliows for plasmid ins
Page 77 and 78:
affect plasmid stability have ben o
Page 79 and 80:
CEtAPTER 3 MATERIALS AND METHODS 3.
Page 81 and 82:
stiU deleted hm the plasmid. Such p
Page 83 and 84:
1) Reagent solution preparation was
Page 85 and 86:
nonse1ective agar plates, but only
Page 87 and 88:
working Liquid volume. The aeration
Page 89 and 90:
3.5. UNnobilized Ceil Culture 35.1.
Page 91 and 92:
3.52 Cd Lmmobiiization Yeast attach
Page 93 and 94:
3.6. Experimental Error and Reprodu
Page 95 and 96:
-0-Cell(1) +Total CeII (2) 4- Ethan
Page 97 and 98:
* . I I a O 2 4 6 8 10 12 14 Batche
Page 99 and 100:
Cell Maso, Glucose and EthanoI(g1L)
Page 101 and 102:
4.2.2. Batch CuIture in Stirreà-Ta
Page 103 and 104:
[t ~lucoamylase (ALR) -e Glucoamyla
Page 105 and 106:
The finai giucoamyiase concentratio
Page 107 and 108:
Bentley and Kompala, 1989; Satyagal
Page 109 and 110:
160 First ExponenUaI Phase 1 -c ALR
Page 111 and 112:
Figure 4hl. Pathways of Yeast Intem
Page 113 and 114:
fermentation (using nitrogen spargi
Page 115 and 116:
43. Continuous Suspension Culture C
Page 117 and 118:
Figure 4.9. Continuous Suspension C
Page 119 and 120:
1 +Enzyme +Fradlan of PBC +CeIl Mas
Page 121 and 122:
Figures 4.12 and 4.13 show that by
Page 123 and 124:
Fi- 4.13. Cornparison of Glu~iase C
Page 125 and 126:
where 6 = p- - p' + pp*, which is a
Page 127 and 128:
Figure 4-14. Effeds of Dilution Rat
Page 129 and 130:
Impoolsup et ai. (1989a) and Hardji
Page 131 and 132:
Figure 4.16. dB/dt versus B Plots a
Page 133 and 134:
Figure 4.17. Totai Cell Mas at Diff
Page 135 and 136:
another recombinant yeast (impoolsu
Page 137 and 138:
other recombinant yeast under nonse
Page 139 and 140:
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 and 192:
Enyme collcentration m the t>ioreac
Page 193 and 194:
Glucoamylase Concentration (units/L
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)
Page 242 and 243:
E.2. Batch Fermentation with Recomb
Page 244 and 245:
(continue)
Page 246 and 247:
(continue) tfhJ 0.5 10.5 12.75 12.5
Page 248 and 249:
(continue)
Page 250 and 251:
E.3.2. Experimental Data (Parker an
Page 252 and 253:
Run No: CALE (Selected Strain. D =
Page 254 and 255:
Run No: CALR 5 (Original Suain, D =
Page 256 and 257:
Run No: CSTR3 (Selected Suain, D =
Page 258 and 259:
(continue)
Page 260 and 261:
Run No: CICR2 (Selected Suain, D =
Page 262 and 263:
Run No: CiCR4 (Selected Suain, D =
Page 264 and 265:
Eh. Repeated Batch Culture in hmobi
Page 266 and 267:
APPENDIX F. C CODE FOR THE PROPOSED
Page 268 and 269:
* the initial data */ /* gening the
Page 270 and 271:
* getting the founh term in R-K met
Page 272 and 273:
I*Plasrnid-bearing ceU concentratio
Page 274 and 275:
* Func tion C 1 */ îloat C l(float
Page 276 and 277:
APPEMIM G. EXAMPLES OF MAPLE V CODE
Page 278 and 279:
REFERENCES Ataai. MM. and Shuler. M
Page 280 and 281:
Caunt, P.. hpoolsup, A.. and Greenf
Page 282 and 283:
cuitanes of E. coli BZ18@TG 201) wi
Page 284 and 285:
Gabrieisen. O.S. et aL (1990), Effi
Page 286 and 287:
Huang, C-T., Peretti, S.W., and Bry
Page 288 and 289:
Kim, B.G. and Shuler, M.L. (1990a),
Page 290 and 291:
Lee, S.B.. Ryu, D.D.Y., seigel, R,
Page 292 and 293:
Nasri, M., Sayadi. S.. Barbotin, J.
Page 294 and 295:
Pin, S.J. and Kmwski. W.M. (1970),
Page 296 and 297:
Seo, I-H. and Bailey. J.E. (1985).
Page 298 and 299:
Tumer. B.G., Avgerinos G-C.? Melnic
Page 300:
Yu, J. and Tang, X (Editor, 1991),
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