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cuitanes of E. coli BZ18@TG 201) without selection pressure, J. Bucte~oL.165, 871- 877. De Taxis du Pot. P., A~atid* Y.. Barbotin. LN. and Thomas, D. (1987). Piasmid stabiity in irnmobilUed and £iee recombinant E. coli N105(pK.K223-200): importance of oxygen diffusion. growth rate. and plasrnid copy nurnber, Appl. Env. Microbiof., 53, 1548- 1555. Dhuiscer, P.. Barbotin, J-N., and Thomas, D. (1984), Cultm and bioconversion use of plasinid-harboring suain of imrno bilized E. coli. Appl. Microbiol. Biotecki~i., 20.87-93. DiBiasio. D. and Sadonini, C. (1986), Stability of continuous culture with recombinant organism Ann. N. Y. Acad. Sci.. 469, 11 1-147. Edlin, G.. Tait, R.C.. and Rodrigues, RL. (1984). A bacteriophage h. cohaive ends (cos) DNA fragment enhances the fimess of plasmid-containing bacteria growing in energy- lirnited chemostats, BiolTechnol.. 2,25 1-254. El-Sayed, A.H. M.M.. Mahrnoud, W.M.. and Coughh. R.W. (1990). Production of dextransucrase by Leuconostoc mesenteroides immobilized in cakium-alginate beads: 1. Batch and fed-batch fermentations, Biotechnol. Bioeng., 36,338-345.
Ejiofor, A. O., Chisti, Y., and Moo-Young, M. (1996), Fed-batch production of baker's yeast using millet (Pennisetm ryphoides) flou hydrolysate as the carbon source, J. M. Microbiol., 16, 102-109. Emenck, A.W., Bertoiam, B.L., Ben-Bassat, A., White, TJ., and Konrad, M.W. (1984), Expression of a p-lactamase prcproinsulin fusion protein in E. coli, BioTTechnol., 2, 165- 168. Fan, L.S.. Fujie, K., Long, T.R., and Tang, W.T. (1987). Characteristics of draft tube gas- liquid-solid Buidired bed bioreactor with immobW iiving cells for phenol degradation, Biotechnol. Biorng., 30,498-504. Flickinger, M.C. and ROUX. M.P. (1993), Sustaining protein synthesis in the ab~nce of npid ceii division: An investigation of plamid-encoded protein expression in E. coli during very slow growth. Biotechnol. Prog., 9,555-572. Fu, I.. Wion, D.B. and Shuler, M.L. (1993). Continuous, high level, production and excretion of a plasmid-cncoâed protein by Escherichia in a two-stage chemostat, Biotechnol. Bioeng., 41,937-946. Futcher, A.B. and Cox, B.S. (1 984), Copy number and the stability of 2 pm ele-based arcificial plamiids of S. cerevkiae, J. Bacteriol.. 157.283-290.
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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
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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
Page 231 and 232: Figure C.4 Effect of Initial Glucos
Page 233 and 234: APPENDLX D. COMPARISON OF ORIGINAL
Page 235 and 236: (continue)
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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
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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