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the au's ability to complete the necessary repairs, hence increasing the Lilcelihood of loss and mutation. Figure 2.1. Ceii Cycle of Budding Yeast (Eïjortso and Baiiey, 1984). CUL Although the foregoing nasonhg does explain the effect of dilution rate on plasmid stability in selective media, it does not explain that effect in nonselective media An alternative possible explanation fdîows. For continuous cuiture in selective media, increase in dilution rate raises the specinc growth rate of plasmid-harboring ce* according to the weil known Monod eqttation:
+ s Dilution Rate = p' = CI,- - K*+S ' however. plasmid-free ce& do not grow, or grow very slowly, because of the seledon pressure. Thenfore. the growth ratio (a = p-/p+) decreases as the dilution rate is increased, so the plasmid is more stable* III the case of nonseleztive media. because the growth rate of recombinants is addy âffected by the borden of plasmids and the expression of recombinant DNA (Moo-Young et aL, 1996). piasmici-fke ce& iisually have a growth advanrage. In general, the growth ratio (a) exceeds unity, ranging over 1.0 to 2.0 (hanaka and Aiba, 198 1). Increaring dilution rate in nonselective media even cm Mer raise the growth ratio (a) becaw the plasmid-behg cells are less able to respond to the dilution rate changes Therefore. it is not surprising that plasmid stability decreases with increasing dilution rate in the nonseleaive media In fact, in continuous culture of recombinant cells, it is impossible to achieve steady state in nonselective media if the growth ratio (a) is more than 1. The initial plasruid-bearing ceii population is sure to be taken over by the plasmid-£ree ceik eventuaily. 222.5. Bioreactor Operation Modes Fermentation pmcesses are usuaIly openited as batch, fed-biitch, or continnous cdtore. The modes of operation cm infiuenœ the plasmid stability and the productivity of expressed products. In batch culture, plasmids are nlatively stable because the culture periods are generally short, hence, the nomber of generations since inoculation is maiL Because of long growth periods. conthrious culture of recombinant cells suffers hm
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BIOREACTOR STUDIES OF HETEROLOGOUS
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nie University of Waterloo requin%
Page 5 and 6: ate depended on activities of the p
Page 7 and 8: Acknowiedgments Fmt of aü, 1 am ve
Page 9 and 10: 2.6. Concluding Rernarks ..........
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: loss @) is dehned as the probabilit
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
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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
Page 125 and 126:
where 6 = p- - p' + pp*, which is a
Page 127 and 128:
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
Page 171 and 172:
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
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Ttme (hrs) 1 a Enzyme A Phsrnid-Bea
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It may take several generations for
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understanding of the nature of the
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On the other hand. the net accumula
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Differentiating Equation (6.20) res
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Enyme collcentration m the t>ioreac
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Glucoamylase Concentration (units/L
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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)
Page 237:
(continue)
Page 240 and 241:
(continue)
Page 242 and 243:
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 =
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
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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
Page 278 and 279:
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
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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