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glucoamylase concentrations and htion of pIasmid-bedg aUs were foUowed during chemostat cultures. 2) Continuous suspension fermentations ming different glucose feedùg concentrations (5, 10, 20 fi) at a nxed dilution rate (D = 0.10 KI) to investigate glucose eEect and obtain quantitative lcnowledge about the metabolic pathways of the recombinant yeast 3) Continuous suspension fermentations asing YNB selective medium (medium I) at a fixed dilution rate (D = 0.10 h-') to examine the effect of selection pressure. 3.4.3. Repeated-Batch Fermentation Repeated barch fermentation experiments were designed to investigate plasmid stability and recombinant protein pmductivity in this reactor operation mode. A 120 ml inoculum, prepared in shake flasks in YNB selective medium, was txsed to inocdate the airüft bioreactor fîlled with 1080 ml of nohîelective YPG medium (1.2 L total working liquid volume). After inoculation, the fermentation proceeded in the batch mode for 24 hours. The culture fluid was pumped out hm the bioreactor and about 120 ml was retained as the inocuiwn for the next batch fermentation. The next batch fermentation was Üiitiated by pumping the 1080 mi of h h YPG nonselective medium into the bioreactor. This procedure was repeated every 24 hours. The npeated-batch fermentation was performed for 2 weeks. During this period, cell, glucose, ethanol and glucoamylase concenaations and fraction of plaunid-bearing ceh were measured.
3.5. UNnobilized Ceil Culture 35.1. Structure of lmmabilized-Cd-FiIm Airiift Biomador In view of the limitations of the other types of immo~-cell bioreactors. a novel immobilized-cefl-fiIm airlift bioreactor was devised, The immobilized-cell-fiZm airlift bioreactor and cage for supporting conon ~10th are show in Fig. 3.1. The same split column airlift bioreactor as in the k e suspension cultore was used as a culture vessel. Its inner diameter is 6.5 cm and its aspect ratio is 7.0. A 6.5~28.5 cm banle was set in the middle of the column to forrn the riser and downcomer. Two corton cloth cages of half column (diameter = 6.0 cm, height = 26 cm) were placed on both sides of the baffle. The Cotton cloth was fimd ont0 the 2mm stainless steel win mesh in the cages to form double-Iayer cloth nùns The disuince between the cloth films was 7.0 mm. The total cloth area in the bioreactor available for cd amchment was 1566.5 cm2. The liquid volume in the bioreactor was 1100 ml. The clearance bewn the sparger and the bottom of baffie was 6.5 cm. The distance be-n the Liquid swface and the top of the banie was 6.5 cm. Dissolved oxygen (DO) and pH were monitored during the fermentation process by using appropnate electrodes. Temperature was controiied at 30 O C by circulating heated water h m a water bath through a jacket around the bioreactor. When the Eitexed steriie air was injected into the bioreactor through the sparger. steady liquid circulation was achievcd throughout the nactor due to the bulk density difference between riser and downcorner. providing good rnixing and mas transfer. In the case of fia suspension culture, the cages were taken out hm the bioreactor.
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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
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: working Liquid volume. The aeration
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
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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
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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 @
Page 227 and 228:
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 =
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
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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
Page 286 and 287:
Huang, C-T., Peretti, S.W., and Bry
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Kim, B.G. and Shuler, M.L. (1990a),
Page 290 and 291:
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),
Page 296 and 297:
Seo, I-H. and Bailey. J.E. (1985).
Page 298 and 299:
Tumer. B.G., Avgerinos G-C.? Melnic
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Yu, J. and Tang, X (Editor, 1991),
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