Preface ......................................................... v
Chapter 1. Industrial Microbiology
1.1 Introduction ............................................... 1
1.2 Process fermentation ....................................... 2
1.3 Application of fermentation processes ...................... 4
1.4 Bioprocess products ........................................ 5
1.4.1 Biomass ............................................. 5
1.4.2 Cell products ....................................... 6
1.4.3 Modified compounds (biotransformation) .............. 6
1.5 Production of lactic acid .................................. 6
1.6 Production of vinegar ..................................... 7
1.7 Production of amino acids (lysine and glutamic acid) and
insulin .................................................... 8
1.7.1 Stepwise amino acid production ...................... 8
1.7.2 Insulin ............................................. 9
1.8 Antibiotics, production of penicillin ...................... 9
1.9 Production of enzymes ..................................... 10
1.10 Production of baker's yeast ............................... 12
References ................................................ 12
Chapter 2. Dissolved Oxygen Measurement and Mixing
2.1 Introduction .............................................. 14
2.2 Measurement of dissolved oxygen concentrations ............ 14
2.3 Batch and continuous fermentation for production of SCP ... 15
2.3.1 Analytical methods for measuring protein content
of baker's yeast (SCP) ............................. 16
2.3.2 Seed culture ....................................... 17
2.4 Batch experiment for production of baker's yeast .......... 17
2.5 Oxygen transfer rate (OTR) ................................ 18
2.6 Respiration quotient (RQ) ................................. 19
2.7 Agitation rate studies .................................... 19
2.8 Nomenclature .............................................. 21
References ..................................................... 21
Chapter 3. Gas and Liquid System (Aeration and Agitation)
3.1 Introduction .............................................. 22
3.2 Aeration and agitation .................................... 22
3.1 Effect of agitation on dissolved oxygen ................... 23
3.4 Air sparger ............................................... 23
3.5 Oxygen transfer rate in a fermenter ....................... 24
3.5.1 Mass transfer in a gas-liquid system ............... 25
3.6 Mass transfer coefficients for stirred tanks .............. 26
3.7 Gas hold-up ............................................... 28
3.8 Agitated system and mixing phenomena ...................... 28
3.9 Characterisation of agitation ............................. 28
3.10 Types of agitator ......................................... 29
3.11 Gas-liquid phase mass transfer ............................ 30
3.11.1 Oxygen transport ................................... 33
3.11.2 Diameter of gas bubble formed D0 ................... 35
3.12 Nomenclature .............................................. 42
References ................................................ 43
3.13 Case study: oxygen transfer rate model in an aerated
tank for pharmaceutical wastewater ........................ 43
3.13.1 Introduction ....................................... 44
3.13.2 Material and method ................................ 46
3.13.3 Results and discussion ............................. 47
3.13.4 Conclusion ......................................... 48
3.13.5 Nomenclature ....................................... 48
References ................................................ 49
3.14 Case study: fuel and chemical production from the water
gas shift reaction by fermentation processes .............. 50
3.14.1 Introduction ....................................... 50
3.14.2 Kinetics of growth in a batch bioreactor ........... 51
3.14.3 Effect of substrate concentration on microbial
growth ............................................. 55
3.14.4 Mass transfer phenomena ............................ 58
3.14.5 Kinetic of water gas shift reaction ................ 61
3.14.6 Growth kinetics of CO substrate on Clostridium
ljungdahlii ........................................ 65
3.14.7 Acknowledgements ................................... 65
3.14.8 Nomenclature ....................................... 66
References ................................................ 67
Chapter 4. Fermentation Process Control
4.1 Introduction .............................................. 69
4.2 Bioreactor controlling probes ............................. 71
4.3 Characteristics of bioreactor sensors ..................... 72
4.4 Temperature measurement and control ....................... 72
4.5 DO measurement and control ................................ 74
4.6 pH/Redox measurement and control .......................... 76
4.7 Detection and prevention of the foam ...................... 77
4.8 Biosensors ................................................ 79
4.9 Nomenclature .............................................. 80
References ................................................ 80
Chapter 5. Growth Kinetics
5.1 Introduction .............................................. 81
5.2 Cell growth in batch culture .............................. 81
5.3 Growth phases ............................................. 82
5.4 Kinetics of batch culture ................................. 83
5.5 Growth kinetics for continuous culture .................... 84
5.6 Material balance for CSTR ................................. 89
5.6.1 Rate of product formation .......................... 90
5.6.2 Continuous culture ................................. 90
5.6.3 Disadvantages of batch culture ..................... 91
5.6.4 Advantages of continuous culture ................... 91
5.6.5 Growth kinetics, biomass and product yields,
YX/S and YP/S ....................................... 91
5.6.6 Biomass balances (cells) in a bioreactor ........... 93
5.6.7 Material balance in terms of substrate in
a chemostat ........................................ 94
5.6.8 Modified chemostat ................................. 95
5.6.9 Fed batch culture .................................. 96
5.7 Enzyme reaction kinetics .................................. 97
5.7.1 Mechanisms of single enzyme with dual substrates ... 99
5.7.2 Kinetics of reversible reactions with dual
substrate reaction ................................ 105
5.7.3 Reaction mechanism with competitive inhibition .... 106
5.7.4 Non-competitive inhibition rate model ............. 107
5.8 Nomenclature ............................................. 128
References ............................................... 129
5.9 Case study: enzyme kinetic models for resolution of
racemic ibuprofen esters in a membrane reactor ........... 130
5.9.1 Introduction ...................................... 130
5.9.2 Enzyme kinetics ................................... 130
5.9.3 Enzyme kinetics for rapid equilibrium system
(quasi-equilibrium) ............................... 135
5.9.4 Derivation of enzymatic rate equation from rapid
Equilibrium assumption ' ......................... 135
5.9.5 Verification of kinetic mechanism ................. 138
References ............................................... 140
Chapter 6. Bioreactor Design
6.1 Introduction ............................................. 142
6.2 Background to bioreactors ................................ 143
6.3 Type of bioreactor ....................................... 143
6.3.1 Airlift bioreactors ............................... 144
6.3.2 Airlift pressure cycle bioreactors ................ 145
6.3.3 Loop bioreactor ................................... 145
6.3.1 Stirred tank bioreactors .......................... 145
6.5 Bubble column fermenter .................................. 149
6.6 Airlift bioreactors ...................................... 150
6.7 Heat transfer ............................................ 151
6.8 Design equations for CSTR fermenter ...................... 154
6.8.1 Monod model for a chemostat ....................... 154
6.9 Temperature effect on rate constant ...................... 158
6.10 Scale-up of stirred-tank bioreactor ...................... 159
6.11 Nomenclature ............................................. 168
References ............................................... 169
Chapter 7. Downstream Processing
7.1 Introduction ............................................. 170
7.2 Downstream processing .................................... 170
7.3 Filtration ............................................... 173
7.3.1 Theory of filtration .............................. 174
7.4 Centrifugation ........................................... 175
7.4.1 Theory of centrifugation .......................... 176
7.5 Sedimentation ............................................ 178
7.6 Flotation ................................................ 180
7.7 Emerging technology for cell recovery .................... 180
7.8 Cell disruption .......................................... 181
7.9 Solvent extraction ....................................... 182
7.9.1 Product recovery by liquid-liquid extraction ...... 183
7.9.2 Continuous extraction column process, rotating
disk contactors ................................... 184
7.10 Adsorption ............................................... 185
7.10.1 Ion-exchange adsorption ........................... 185
7.10.2 Langmuir isotherm adsorption ...................... 186
7.10.3 Freundlich isotherm adsorption .................... 186
7.10.4 Fixed-bed adsorption .............................. 186
7.11 Chromatography ........................................... 187
7.11.1 Principle of chromatography ....................... 189
7.12 Nomenclature ............................................. 197
References ............................................... 198
Chapter 8. Immobilization of Microbial Cells for the
Production of Organic Acid and Ethanol
8.1 Introduction ............................................. 199
8.2 Immobilised microbial cells .............................. 200
8.2.1 Carrier binding ................................... 200
8.2.2 Entrapping ........................................ 200
8.2.3 Cross-linking ..................................... 202
8.2.4 Advantages and disadvantages of immobilised
cells ............................................. 202
8.3 Immobilised cell reactor experiments ..................... 202
8.4 ICR rate model ........................................... 203
8.3 Nomenclature ............................................. 206
References ............................................... 206
8.6 Case study: ethanol fermentation in an immobilised
cell reactor using Saccharomyces cerevisiae .............. 206
8.6.1 Introduction ...................................... 207
8.6.2 Materials and methods ............................. 209
8.6.3 Results and discussion ............................ 215
8.6.4 Conclusion ........................................ 220
8.6.5 Acknowledgement ................................... 221
8.6.6 Nomenclature ...................................... 221
References ............................................... 222
8.7 Fundamentals of immobilisation technology, and
mathematical model for ICR performance ................... 222
8.7.1 Immobilisation of microorganisms by covalent
bonds ............................................. 222
8.7.2 Oxygen transfer to immobilised microorganisms ..... 223
8.7.3 Substrate transfer to immobilised microorganisms .. 223
8.7.4 Growth and colony formation of immobilised
microorganisms .................................... 224
8.7.5 Immobilised systems for ethanol production ........ 227
Reference ................................................ 227
Chapter 9. Material and Elemental Balance
9.1 Introduction ............................................. 228
9.2 Growth of stoichiometry and elemental balances ........... 229
9.3 Energy balance for continuous ethanol fermentation ....... 230
9.4 Mass balance for production of penicillin ................ 231
9.5 Conservation of mass principle ........................... 234
9.5.1 Acetic acid fermentation process .................. 238
9.5.2 Xanthan gum production ............................ 241
9.5.3 Stoichiometric coefficient for cell growth ........ 243
9.6 Embden-Meyerhoff-Parnas pathway .......................... 244
References ............................................... 251
Chapter 10. Application of Fermentation Processes
10.1 Introduction ............................................. 252
10.2 Production of ethanol by fermentation .................... 252
10.1 Benefits from bioethanol fuel ............................ 253
10.4 Stoichiometry of biochemical reaction .................... 253
10.5 Optical cell density ..................................... 253
10.6 Kinetics of growth and product formation ................. 254
10.7 Preparation of the stock culture ......................... 254
10.8 Inoculum preparation ..................................... 255
10.9 Seed culture ............................................. 255
10.10 Analytical method for sugar analysis .................... 257
10.10.1 Quantitative analysis ............................ 257
10.11 Analytical method developed for ethanol analysis ........ 257
10.12 Refractive index determination .......................... 257
10.13 Measuring the cell dry weight ........................... 257
10.14 Yield calculation ....................................... 258
10.15 Batch fermentation experiment ........................... 258
10.16 Continuous fermentation experiment ...................... 258
10.17 Media sterilisation ..................................... 261
10.18 Batch experiment ........................................ 261
10.18.1 Optical cell density, ethanol and carbohydrate
concentration .................................... 261
10.18.2 Continuous ethanol fermentation experiment ....... 261
10.19 Expected results ........................................ 261
References ............................................... 262
Chapter 11. Production of Antibiotics
11.1 Introduction ............................................. 263
11.2 Herbal medicines and chemical agents ..................... 263
11.3 History of penicillin .................................... 264
11.4 Production of penicillin ................................. 265
11.5 Microorganisms and media ................................. 266
11.6 Inoculum preparation ..................................... 266
11.7 Filtration and extraction of penicillin .................. 268
11.8 Experimental procedure ................................... 269
11.9 Fermenter description .................................... 269
11.10 Analytical method for bioassay and detecting
antibiotic ............................................... 269
11.11 Antibiogram and biological assay ........................ 269
11.12 Submerged culture ....................................... 270
11.12.1 Growth kinetics in submerged culture ............. 270
11.13 Bioreactor design and control ........................... 272
11.14 Estimation for the dimension of the fermenter ........... 273
11.15 Determination of Reynolds number ........................ 275
11.16 Determination of power input ............................ 275
11.17 Determination of oxygen transfer rate ................... 277
11.18 Design specification sheet for the bioreactor ........... 278
References .............................................. 278
Chapter 12. Production of Citric Acid
12.1 Introduction ............................................. 280
12.2 Production of citric acid in batch bioreactor ............ 280
12.2.1 Microorganism ..................................... 281
12.3 Factors affecting the mold growth and fermentation
process .................................................. 281
12.4 Starter or seeding an inoculum ........................... 283
12.5 Seed culture ............................................. 283
12.6 Citric acid production ................................... 283
12.7 Analytical method ........................................ 284
12.7.1 Cell dry weight ................................... 284
12.7.2 Carbohydrates ..................................... 285
12.7.3 Citric acid ....................................... 285
12.8 Experimental run ......................................... 285
References ............................................... 286
Chapter 13. Bioprocess Scale-up
13.1 Introduction ............................................. 287
13.2 Scale-up procedure from laboratory scale to plant scale .. 287
13.2.1 Scale-up for constant KLa ......................... 289
13.2.2 Scale-up based on shear forces .................... 290
13.2.3 Scale-up for constant mixing time ................. 290
13.3 Bioreactor design criteria ............................... 293
13.3.1 General cases ..................................... 293
13.3.2 Bubble column ..................................... 293
13.4 CSTR chemostat versus tubular plug flow .................. 298
13.5 Dynamic model and oxygen transfer rate in activated
sludge ................................................... 312
13.6 Aerobic wastewater treatment ............................. 325
13.6.1 Substrate balance in a continuous system .......... 327
13.6.2 Material balance in fed batch ..................... 328
13.7 Nomenclature ............................................. 330
References ............................................... 331
Chapter 14. Single-Cell Protein
14.1 Introduction ............................................. 332
14.2 Separation of microbial biomass .......................... 333
14.3 Background ............................................... 333
14.4 Production methods ....................................... 334
14.5 Media preparation for SCP production ..................... 335
14.6 Analytical methods ....................................... 336
14.6.1 Coomassie-protein reaction scheme ................. 336
14.6.2 Preparation of diluted BSA standards .............. 336
14.6.3 Mixing of the coomassie plus protein assay
reagent ........................................... 337
14.6.4 Standard calibration curve ........................ 337
14.6.5 Standard calibration curve for starch ............. 337
14.7 SCP processes ............................................ 338
14.8 Nutritional value of SCP ................................. 339
14.9 Advantages and disadvantages of SCP ...................... 340
14.10 Preparation for experimental run ........................ 341
References ............................................... 341
Chapter 15. Sterilisation
15.1 Introduction ............................................. 342
15.2 Batch sterilisation ...................................... 342
15.3 Continuous sterilisation ................................. 343
15.4 Hot plates ............................................... 344
15.5 High temperature sterilisation ........................... 345
15.6 Sterilised media for microbiology ........................ 345
15.6.1 Sterilisation of media for stoke cultures ......... 347
15.6.2 Sterilisation of bacterial media .................. 347
15.6.3 Sterilise petri dishes ............................ 347
15.7 Dry heat sterilisation ................................... 348
15.8 Sterilisation with filtration ............................ 348
15.9 Microwave sterilisation .................................. 349
15.10 Electron beam sterilisation ............................. 349
15.11 Chemical sterilisation .................................. 349
References .................................................... 350
Chapter 16. Membrane Separation Processes
16.1 Introduction ............................................. 351
16.2 Types of membrane ........................................ 351
16.2.1 Isotropic membranes ............................... 352
16.2.2 Anisotropic membranes ............................. 353
16.2.3 Ceramic, metal and liquid membranes ............... 353
16.3 Membrane processes ....................................... 354
16.4 Nature of synthetic membranes ............................ 357
16.5 General membrane equation ................................ 360
16.6 Cross-flow microfiltration ............................... 362
16.7 Ultrafiltration .......................................... 365
16.8 Reverse osmosis .......................................... 367
16.9 Membrane modules ......................................... 369
16.9.1 Tubular modules ................................... 369
16.9.2 Flat-sheet modules ................................ 369
16.9.3 Spiral-wound modules .............................. 371
16.9.4 Hollow-fibre modules .............................. 371
16.10 Module selection ........................................ 373
16.11 Membrane fouling ........................................ 376
16.12 Nomenclature ............................................ 377
References .............................................. 378
16.13 Case study: inorganic zirconia 7-alumina-coated
membrane on ceramic support ............................. 378
16.13.1 Introduction ..................................... 379
16.13.2 Materials and methods ............................ 385
16.13.1 Preparation of PVA solution ...................... 385
16.13.3 Results and discussion ........................... 387
16.13.4 Conclusion ....................................... 388
16.13.5 Acknowledgements ................................. 388
References ............................................... 388
Chapter 17. Advanced Downstream Processing in Biotechnology
17.1 Introduction ............................................. 390
17.2 Protein products ......................................... 391
17.3 Cell disruption .......................................... 392
17.4 Protein purification ..................................... 393
17.4.1 Overview of the strategies ........................ 393
17.4.2 Dye-ligand pseudo-affinity adsorption ............. 394
17.5 General problems associated with conventional
techniques ............................................... 394
17.6 Fluidised bed adsorption ................................. 395
17.6.1 Mixing behaviour in fluidised/expanded beds ....... 396
17.7 Design and operation of liquid fluidised beds ............ 397
17.7.1 Hydrodynamic characterisation of flow in
fluidised/expanded beds and bed voidage ........... 397
17.7.2 Minimum fluidisation velocity of particles ........ 398
17.7.3 Terminal settling velocity of particles ........... 399
17.7.4 Degree of bed expansion ........................... 401
17.7.5 Matrices for fluidised bed adsorption ............. 402
17.7.6 Column design for fluidised bed adsorption ........ 403
17.8 Experimental procedure ................................... 404
17.9 Process integration in protein recovery .................. 404
17.9.1 Interfaced and integrated fluidised bed/expanded
bed system ........................................ 405
17.10 Nomenclature ............................................ 407
References .............................................. 407
17.11 Case study: process integration of cell disruption and
fluidised bed adsorption for the recovery of labile
intracellular enzymes ................................... 409
17.11.1 Introduction ..................................... 409
17.11.2 Materials and methods ............................ 410
17.11.3 Results and discussion ........................... 411
17.11.4 Conclusion ....................................... 413
17.11.5 Acknowledgement .................................. 414
References ............................................... 414
Appendix ...................................................... 416
Index ......................................................... 418
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