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O 5 10 15 20 25 30 35 40 Tirne (hrs) Figure C.S. Effect of initial Glucose Concentration on Fraction of Plasmid-Bedng in Batch Culture.
APPENDLX D. COMPARISON OF ORIGINAL AND SELECTED STRAINS IN SHAKE FLASK EXPERIMENTS Table D.1. Comparison of Cell and Enzyme Concentrations between Original and Selected Strains in Shake Flask Experimenîs (YPG medium. T=30 OC, RPM=2SO and GO= 20.0 &). 1 1 Original Shin 1 Selected Strain Tri piicates 24 hour 36 hour 48 hour 24 hour 36 hour 48 hour # 1 Enz. Con. (u nits/L) 24.7 1 99.61 133.03 39.13 135.9 1 226.52 # 2 Enz. Con. (unis) 23.89 82.37 135.91 38.30 140.03 230.34 # 3 Enz. Con. (units/L) 26.77 96.86 I 142.09 41.19 133.03 223.46 # 1 CeU Mas 3.07 (fi) 5.84 8.14 3.0 1 5.93 7.69 # 2 Ce11 Mass C,& 3.02 6.04 8.19 3.08 5.66 7.9 1 # 3 CeU Mass (.du 3.05 6.02 8.24 3.06 5.88 7.53 Table D.2. Comparison of Specific Enzyme Activity (unitdg ceIl) between Original and Selected Strains in Shake Flask Experirnents (YPG medium, T=30 T, RPM350 and Ge 20.0 fi). 1 1 Original Strai9n 1 Selected S train 1
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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
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Figure 4.18- Etlects of Mluiioa Rat
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Number of Generations - - - - - - *
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B Table 4.5. Metabolic Pathway Anal
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concentration. But this may be more
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[+ Enzyme (ALR) + Enzyme (STR) +Fra
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necessary for host ce& to grow in t
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Glucoamylase ConcentraUon (unltsll)
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[a Selective Medium A Nonseiective
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5.1 Introduction CHAPTER 5 MODELING
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handle. excess glucose is femented
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The carbon fluxes h m the three met
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53. Modei Simulation in Batch CuItu
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Table 5.1. Summary of Parameters fo
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1 n Glucose o Cell Mass O Ethano1 A
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detennined in the cunent study, the
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Figure 5.4. Cornparison between Mod
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equations are üsted in Appendix G.
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a dilution rate at which the giucoa
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[ o Expetfmental data -Simuiated mt
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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: Figure C.4 Effect of Initial Glucos
Page 235 and 236: (continue)
Page 237: (continue)
Page 242 and 243: E.2. Batch Fermentation with Recomb
Page 246 and 247: (continue) tfhJ 0.5 10.5 12.75 12.5
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 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
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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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