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that the decay rate of enzyme production was higher in the STR bioreactor than in the ALR bioreactor (Figures 4.22 and 4.23). The enzyme concentration feu more rapidly in the STR bioreactor because the M o n of plasmid-bearing œUs in the STR declined faster than in the ALR (Figure 4.24). The agitation was possibly responsible for the increased apparent plasmid instability in the STR bioreactor. When a recombinant E. coli BlpTG201 was grown in a nonselective medium, Huang et aL (1990) observed that the plasmid stability and enzyme productivity decreased with the increase in agitation speed from 50 to 200 rpm. They concluded that a mild agitation rate was favourable for the maintenance of plasmid stability. In the present case, the agitation rate used was high (500 rprn), which might result in a decreased plasmid stabüity in the STR bioreactor. Although the ceil mass was higher in the STR bioreactor, the enzyme production was poorer due to the increased plasmid instability (Fi~gure 4.22 and 4.23). In the STR bioreactors, the energy required for the movement of the fluid is introduced focally at one point in the reactor via a stirrer. The shear stress wiU be greatest near the stimr and dissipate with distance h m it The vigorous fiuid movement and the wide variation of shear force in the reactor may have a detrimental effect on plasmid stability (Huang et al., 1990). In conmt, in the absence of agitation a regular and predictable pattern of bulk circulation generated by injected air exists in the ALR bioreactors. Shear dismiution is homogeneous throughout the ALR bioreactors. nie gentle and approximately wiifom environment is very suitable for M g ceiis. It is me lhat STR bioreactors cm provide higher dissolved oxygen Ievel and generate higher cd
concentration. But this may be more than offset by the increased piasmid instability caused by the agitation. Due to a rehtively low critical dissolved oxygen leveï, ALR bioreactors are able to provide adequate oxygen for the ncombinant yeast. The potentiai use of ALI2 bioreactors in commercial cultivation of recombinant yeast ce& is very promiag. Figure 4.22* Cornparisons of Enzyme, Ceii and Ethawl Concentrations berneen ALR and STR Bioreactors in Continuous Culture Using Originai Strain @ = 0.10 h", T = 30 OC , VVM = 1.0, RPM = 500 and Go= 20.0 @,).
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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
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: B Table 4.5. Metabolic Pathway Anal
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
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F i 6.6. Cornparison of Fnx Cd Mars
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. - O 50 100 150 200 250 nme (hrs)
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Nasi et ai.. 1988) and recombinant
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estimated by using Equation (6.24)
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[a lmmobilked System O Free System
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oxidation pathway. W~th increasing
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O 50 100 150 200 250 300 Tirne (hrs
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epeated batch fermentations, the fe
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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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