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parameter dues were considered constant and experjmentally easily accessible. In order to obtain the parameters associated with the glucose fermentation pathway (Y' ,Y , XIG and pl, -). a complete anaembic batch fermentation was carried out using nitmgen sparging. Then the yield coefficients (Y' and Y,,, ) were detennined from a linear X/G regression of glucose or ethanol to œil dry weight and the maximum specific growth rate for glucose fermentation (pl, ,) was evaluated h m a semilogarithmic plot of cd concentration (X) versus the (1). After Y was detennined. Y and a2 were X/G X / G estimated bm aerobic batch fermentation. Sirnilarly, the parameters for the ethanol oxidation pathway (Y,,, . p~, , and as) were obtained fiom the experimental data of oxidative growth on ethanoi. The maximum specinc pwth rate for glucose oxidation ph , was taken from the literanire (CoppeiIa and Dhujati, 1990; Piper and Kirk, 1991). The saturation constants 4, K2 and 4 were obtained from published data (Coppella and Dhu jati, 1990; Sonnleitner and Kappeii, 1986). Lag time rL, was evaluated from experimental data, which is the the required to achieve exponential growth after inoculation. The probabiiity of plasrnid loss @) was determined hm the continuous fermentation data according to the method dexribed in Section 4.3.3. The regdation coefficients of the pacemaker enzyme pools (k, Rh k, and W were esiimated h m the continuous fermentation data and were then adjusted according to published information on yeast rnetabolic pathways (van Dedem and Moo-Young, 1973); Coppella and Dhujati, 1990; Berry and Brown, 1987).
Table 5.1. Summary of Parameters for Recombinant Sacc~omyces cereviskze. Note: N/A = not avalilable Dimension 1 This saidy 1 fiterature - - - - - - - - dimensionless 0.05 NIA g ceug glucose O. 12 O*OS-O. f O, O. 15 g ceWg glucose 0.48 0.49 ,050 g celYg ethmol 0.65 0.53 ,0.72 g ethanolfg dcw 3.35 3.0 unitdg dcw 32-97 NIA unitdg dcw 33.80 NIA h-' 0.38 0.30,0.40-0.45 h-l 0.25 0.25 ,0.25-0.3 ti' 0.06 O. IO
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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
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: 53. Modei Simulation in Batch CuItu
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
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6) Suitable for repeated-batch and
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ecause glucose fermentation dominat
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cells. Tests of the proposed model
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7.2. Recommendations The foUowing f
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APPENDIX A: PLASMID MAP FOR pGAC9 l
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where Bi (Biot number) = - ks De @
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APPENDK C. EFFECTS OF INITIAL GLUCO
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Figure C.2. Effect of Initial Gluco
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Figure C.4 Effect of Initial Glucos
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APPENDLX D. COMPARISON OF ORIGINAL
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(continue)
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(continue)
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(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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