Contents

1. What is bioprocess engineering? 2.7 Non earthly/unnatural biological agents 61

2.8 Summary 62
1.1 Biological cycle 1
Problems 63
1.2 Bioprocess engineering applications 2
References 64
1.3 Scales: living organism and

manufacturing 3
3. An overview of chemical
1.4 Green chemistry 4
reaction
1.5 Sustainability 5 analysis
1.6 Biorefinery 5 3.1 Chemical species 65
1.7 Biotechnology and bioprocess 3.2 Chemical reactions 66

engineering 8 3.3 Reaction rates 68

1.8 Mathematics, biology, and engineering 9 3.3.1 Definition of the rate of reaction, rA 69
1.9 The story of penicillin: the dawn 3.3.2 Rate of a single irreversible reaction 71

of bioprocess engineering 9 3.3.3 Rate of an elementary reaction 72

1.10 Bioprocesses: regulatory constraints 12 3.3.4 Rate of a reversible reaction 72
1.11 The pillars of bioprocess kinetics and 3.3.5 Rates of multiple reactions 72

systems engineering 13 3.3.6 Rate coefficients 72

1.12 Summary 14 3.4 Approximate reactions 74
Problems 14 3.5 Stoichiometry 75

References 14 3.6 Yield and yield factor 77

3.7 Reaction rates near equilibrium 79

2. An overview of biological basics 3.8 Energy regularity 83

3.9 Classification of multiple reactions

2.1 Cells and organisms 17
and selectivity 85
2.1.1 Microbial diversity 18
3.10 Coupled reactions 86
2.1.2 How cells are named 18
3.11 Reactor mass balances 88
2.1.3 Prokaryotes 20
3.12 Reaction energy balances 90
2.1.4 Eukaryotes 23
3.13 Reactor momentum balance 96
2.2 Viruses 27
3.14 Ideal reactors 97
2.3 Prions 30
3.15 Bioprocess systems optimization 98
2.4 Stem cell 30
3.16 Summary 100
2.5 Cell chemistry 31
Problems 104
2.5.1 Amino acids and proteins 32

2.5.2 Monosaccharides 37
4. Batch reactor
2.5.3 Disaccharides 42
2.5.4 Polysaccharides 42 4.1 Isothermal batch reactors 109
2.5.5 Phytic acid and inositol 46 4.2 Batch reactor sizing 120
2.5.6 Chitin and chitosan 48 4.3 Nonisothermal batch reactors 123

2.5.7 Lignin 48 4.4 Numerical solutions of batch reactor

2.5.8 Lipids, fats, and steroids 50 problems 127
2.5.9 Nucleic acids, RNA, and DNA 52 4.5 The reactor pinch graph 132
2.6 Cell feed 57 4.6 Summary 135
2.6.1 Macronutrients 58 Problems 136
2.6.2 Micronutrients 60 References 140
2.6.3 Growth media 61

v
vi Contents

5. Ideal flow reactors 7.3.1 Reversible reactions 242

7.3.2 Competitive reactions 243
5.1 Commonly useful parameters 141
7.3.3 Reactions with nbound substrates 243
5.2 Plug flow reactor (PFR) 143
7.3.4 Enzyme-substituted reactions-the
5.3 Continuous stirred tank reactor (CSTR)
ping-pong mechanism 245
and chemostat 150
7.3.5 Bimolecular reactions on
5.4 Multiple reactors 159
allosteric enzymes 246
5.5 Recycle reactors 163 7.4 Enzyme inhibition 248
5.6 PFR with distributed feeding and
7.4.1 Allosteric inhibition 248
withdrawing 166
7.4.2 Competitive inhibition 250
5.6.1 Distributed feed 166 order substrate kinetics
7.5 Higher 253
5.6.2 Membrane reactor 171
7.6 pH effects 258
5.7 Reactive distillation 173
7.7 Temperature effects 260
5.8 PFR or CSTR? 174 7.8 Insoluble substrates and/or
5.9 Steady nonisothermal flow reactors 177
high-enzyme loading 261
5.10 Reactive extraction 183
7.9 Immobilized enzyme systems 262
5.11 Graphic solutions using batch 7.9.1 Methods of immobilization 262
concentration data 184
7.9.2 Electrostatic and steric effects in
5.11.1 Solution of A PFR using batch
immobilized enzyme systems 265
concentration data 185
7.10 Analysis of bioprocess with enzymatic
5.11.2 Solution of A CSTR using batch reactions 265
concentration data 186
7.11 Large-scale production of enzymes 270
5.12 Summary 187
7.12 Medical and industrial utilization
Problems 188 of enzymes 271

7.13 Kinetic approximation: why

6. Kinetic theory and reaction kinetics Michaelis-Menten equation works 273

6.1 Elementary kinetic theory 199 7.13.1 Pseudosteady state hypothesis

6.1.1 Distribution laws 201 (PSSH) 277

6.1.2 Collision rate 203 7.13.2 Fast equilibrium step (FES)

approximation 279
6.2 Collision theory of reaction rates 204

6.3 Reaction rate 7.13.3 Modified-fast

equilibrium
analysis/approximation 206
approximation 281
6.3.1 Fastequilibrium step (FES)
7.14 Summary 283
approximation 207

6.3.2 208 Problems 283

Pseudosteady state hypothesis (PSSH)
6.4 Unimolecular reactions 208
6.5 Free radicals 210 8. Chemical reactions on solid surfaces
6.6 Kinetics of acid hydrolysis 211
8.1 Catalysis 291
6.7 Parametric estimation 213
8.2 How does reaction with solid occur? 294
6.8 Summary 220
8.3 Langmuir: theoretical basis of
Problems 221
adsorption kinetics 296
References 227
8.4 Idealization of nonideal surfaces 301

8.4.1 UniLan adsorption isotherm 302

Enzymes 8.4.2 Common empirical approximate
7.1 How enzymes work? 231 isotherms 305

7.2 Simple enzyme kinetics 238

8.5 Cooperative adsorption 309

7.2.1 The fast 8.5.1 Cooperative adsorption of single

equilibrium step
species 311
(FES)assumption 239

7.2.2 The pseudosteady-state 8.5.2 Competitive cooperative adsorption 318

8.5.3 Pore size and surface
hypothesis (PSSH) 239
7.2.3 characterization 319
Specific activity 241
8.6 LHHW: surface reactions with
7.3 Competitive and allosteric enzyme
kinetics 242 rate-controlling steps 320
 Contents vii

8.7 Why rate approximation such as 10. Molecular regulation

LHHW works? 325
8.8 Chemical reactions on nonideal 10.1 Single substrate reactions 402

surfaces based on the distribution 10.1.1 Catalysis of a homosteric enzyme 403

of interaction energy 334

10.1.2 Catalysis of an allosteric enzyme 411
10.2 "Unimolecular" reactions 413
8.9 Cooperative catalysis 336
8.9.1 Unimolecular reactions 336
10.2.1 Homosteric dimeric enzyme 413

8.9.2 Bimolecular reactions 10.2.2 Homosteric enzymes 416

338
8.10 Kinetics of reactions surfaces 10.3 Bimolecular reactions 418
on

where the solid is either 10.3.1 Multisited enzymes 418

a product
reactant 10.3.2 Enzyme substitution 419
or 339
8.11 Decline of surface 10.4 The simplest polymorph: a bimorph
activity: catalyst
deactivation 341 of two monomeric forms of the
same 419
8.12 Summary 341 enzyme
Problems 10.4.1 An indifferent bimorph or
345
References 349 morpheein 420
10.4.2 A ligand-stabilized bimorph 421
10.4.3 A lignd-induced bimorph 422
Protein-ligand interactions
10.4.4 A simplistic kinetic polymorph 423
9.1 Multifunctionalization of proteins 352 10.5 Kinetics of polymorphic catalysis 426
9.2 Covalently bound oligomers 353 10.5.1 Substrate-induced polymorph 426
9.3 Noncovalent assembly of protein 357 10.5.2 Substrate-stabilized polymorph 427
9.4 Protein assembly via domain 10.5.3 Substrate-indifferent polymorph 429
swapping 362 10.6 Multimolecular reactions on
enzymes
9.5 Coexistence of protein oligomer with ligand-specific active centers 432
mixtures 365 10.6.1 Simplistic allosteric enzyme 433
9.6 Three simplistic models of enzyme 10.6.2 Dimers with parallel allosteric
interactions 368 sites 434
9.6.1 The MWC model 368 10.6.3 allosteric enzyme
Catalysis on

9.6.2 The KNF model 370 with n-pairs of parallel sites 436
9.6.3 The morpheein model 370 10.7 Parallel allosteric competitive interactions 439
9.7 Protein-ligand interactions 370 10.7.1 Simplistic allosteric enzyme 439
9.8 Single ligand species versus enzymes 10.7.2 Dimers with parallel allosteric sites 441
with two identical sites 374 10.7.3 Interactive
enzymes with pairs of
9.9 Single-ligand species on a homosteric
parallel n-homosteric sites 444
enzyme 377 10.8 Summary 446
9.10 Sequential single ligand species on Problems 448
allosteric enzymes 382 References 451
9.11 Single-ligand species on random-access
allosteric enzymes 384 11. Cell metabolism
9.12 Multiple different ligand-specific active
centers 11.1 The central dogma 454
386
11.2 DNA replication: preserving and
9.12.1 Simple allosteric enzyme 387
9.12.2 Dimers with parallel allosteric propagating the cellular message 455

sites 11.3 Transcription: sending the message 457

388
9.12.3 Parallel interaction 11.4 Translation: message to product 462
389
9.12.4 Uniallosteric interaction 391 11.4.1 Genetic code: universal message 462
11.4.2 Translation: how the
9.13 Competitive multiligand interactions machinery
on homosteric enzymes 392 works 463

9.13.1 Two-site homosteric enzyme 392 11.4.3 Posttranslational processing:

9.13.2 n-site homosteric
enzyme 394 making the product useful 465

9.14 11.5 Metabolic regulation 467

Summary 396
Problems 397 11.5.1 Genetic-level control: which

References 398 proteins aresynthesized? 467

11.5.2 Metabolic pathway control 472
viii Contents

11.6 How a cell senses its extracellular 12.4.2 Complimentary DNA or cDNA 524
environment? 478 12.4.3 Cloning genes into a plasmid 524

11.6.1 Mechanisms to transport small 12.4.4 Polymerase chain reaction 524

molecules across cellular 12.4.5 Vectors and plasmids 524
membranes 479 12.5 Applications of genetic engineering 529

11.6.2 Role of cell receptors in metabolism 12.6 The product and process decisions 531

and cellular differentiation 482 12.7 Host-vector system selection 532

11.7 Major metabolic pathways 483 12.7.1 Escherichia coli 533
11.7.1 Bioenergetics 483 12.7.2 Gram-positive bacteria 534

11.7.2 Glucose metabolism: glycolysis 12.7.3 Lower eukaryotic cells 534

and the TCA cycle 486 12.7.4 Mammalian cells 535
11.7.3 Metabolism of common plant 12.7.5 Insect cell-baculovirus system 536
biomass-derived 12.7.6 Transgenic animals 537

monosaccharides 491 12.7.7 Transgenic plants and plant

11.7.4 Fermentative pathways 491 cell culture 537
11.7.5 Respiration 494 12.7.8 Comparison of strategies 538
11.7.6 Control sites in aerobic glucose 12.8 Regulatory constraints on genetic
metabolism 495 processes 538
11.7.7 Metabolism of nitrogenous 12.9 Metabolic engineering 540

compounds 496 12.10 Protein engineering 542

11.7.8 Nitrogen fixation 496 12.11 Summary 542

11.7.9 Metabolism of hydrocarbons 496 Problems 543
11.7.10 Interrelationships of metabolic References 544

pathways 497
11.8 Overview of biosynthesis 498
13. How cells grow
/

11.9 Overview of anaerobic metabolism 500

11.10 Overview of autotrophic metabolism 501 13.1 biomass
Quantifying 545
11.11 Overall kinetic assymptote: 13.1.1 Cell number density 546
the Monod equation 503 13.1.2 Cell mass concentration 546
11.12 Summary 506 13.2 Batch 548
growth patterns
Problems 508 13.3 Biomass yield 550
References 511 13.4 Approximate growth kinetics and
Monod equation 554

13.5 Cell death rate 557

Evolution and genetic engineering 13.6 Cell maintenance and endogenous
12.1 Mutations 513 metabolism 559

12.1.1 What causes genetic mutations? 514 13.7 Product yield 563
12.1.2 Types of mutations 515 13.8 Oxygen demand for aerobic
12.1.3 Large-scale mutations 517 microorganisms 564

12.2 Selection 518 13.9 Autotrophic growth 566

12.2.1 Natural selection 518 13.10 Effect of environmental conditions 567

12.2.2 Artificial selection (selection 13.10.1 Effect of temperature 567

of mutants with useful 13.10.2 Effect of pH 568

mutations) 519 13.10.3 Effect of redox potential 568

12.3 Natural mechanisms for gene transfer 13.10.4 Effect of electrolytes and
and rearrangement 520 substrate concentration 569
12.3.1 Genetic recombination 521 13.11 Heat generation by microbial growth 569

12.3.2 Transformation 521 13.12 Overview of cell growth kinetic

12.3.3 Transduction 522 models 570

12.3.4 Episomes and conjugation 523 13.12.1 Unstructured growth models 572

12.3.5 Transposons: internal gene 13.12.2 Simple growth rate model:

transfer 523 Monod equation 572
of 13.12.3 Modified Monod equation with
12.4 Techniques genetic engineering 523
12.4.1 Gene synthesis 523 growth inhibitors 574
 Contents ix

13.12.4 Multiple limiting substrates 576 15.5.1 Segregational instability 690

13.12.5 Simplest reaction network 15.5.2 Plasmid structural instability 691

model 577 15.5.3 Host cell mutations 691
13.12.6 Simplest metabolic pathway 577 15.5.4 Growth-rate-dominated instability 691

13.12.7 Cybernetic models 578 15.5.5 Considerations in plasmid design

13.12.8 Computational systems biology 580 to avoid process problems 692
13.13 Selective substrate uptake kinetics 580 15.5.6 Host-vector interactions and
13.14 Summary 586 genetic instability 694
Problems 588 15.6 Mixed cultures 699
References 592 15.6.1 Major classes of interactions
in mixed cultures 699

14. Cell cultivation 15.6.2 Interactions of two species

fed on the same limiting
14.1 Batch culture 593
substrate 701
14.2 Continuous culture 597
15.6.3 Interactions of two mutualistic
14.2.1 Chemostat devices for continuous
species 704
culture 597
15.6.4 Industrial applications of
14.2.2 The ideal chemostat 598
mixed cultures 705
14.2.3 Cell composition change in
15.6.5 Mixed culture in nature 706
chemostat 606
15.7 Sustainability of mixed culture 706
14.2.4 The chemostat as a tool 608
15.7.1 Predator and prey interactions 706
14.3 Choosing the cultivation method 608
15.7.2 Lokka-Volterra model a simplified

-
14.4 Chemostat with recycle 610
predator-prey interaction model 709
14.5 Multistage chemostat systems 614
15.8 Summary 711
14.6 Waste water treatment process 622
Problems 711
14.7 Immobilized cell systems 626

14.7.1 Active immobilization of cells 626

14.7.2 Passive immobilization— 16. Combustion, reactive hazard,
biological films 628 and bioprocess safety
14.8 Solid substrate fermentations 630
14.9 Fed-batch 632
16.1 Biological hazards 720
operations
16.2 Identifying chemical reactivity hazards 723
14.9.1 Theoretical considerations 634
16.2.1 Chemical hazard labeling 723
14.9.2 Ideal isothermal fed-batch
16.2.2 Chemical reactivity hazard 729
reactors 637
16.3 Heat, flames, fires, and explosions 732
14.9.3 Isothermal pseudosteady state 16.4 Probabilities, redundancy, and
fed-batch growth 640
worst-case scenarios 734
14.10 Summary 647
16.5 Chain reactions 734
Problems 649
16.6 Autooxidation and safety 736

and
16.6.1 A simple model of
15. Sustainability stability autooxidation 737

15.1 Feed stability of a CSTR 660 16.6.2 Spoilage of food 738

15.1.1 Multiple steady states (MSS) 661 16.6.3 Antioxidants 738
15.1.2 Stability of steady state 663 16.7 Combustion 738
Effect of feed parameters
15.1.3 on MSS 665 16.7.1 Hydrogen oxidation 739
15.2 Thermal stability of a CSTR 675 16.7.2 Chain branching reactions 741
15.3 Approaching steady state 681 16.7.3 Alkane oxidation 742
15.4 Catalyst instability 687 16.7.4 Liquid alkane oxidation 742
15.4.1 Fouling 687 16.7.5 Thermal ignition 742

15.4.2 Poisoning 687 16.7.6 Thermal and chemical

15.4.3 Sintering 687 autocatalysis 744

15.4.4 Catalyst activity decay 688 16.8 Premixed flames 745

15.4.5 Spent catalyst regeneration 688 16.8.1 Stability of a tube flame 745
15.5 Genetic instability 688 16.8.2 Premixed burner flames 746
x Contents

16.8.3 Diffusion flames 746 Bioreactor and

18. design operation
16.8.4 Laminar and turbulent
18.1 Bioreactor selection 819
flames 747
18.2 Reactor operational mode selection 823
16.9 Heat generation 747
16.9.1 Radiation 748
18.3 Aeration, agitation, and heat transfer 825

16.9.2 Flammability limits 748

18.4 Scale-up 828

18.5 Scale-down 830

16.10 Gasification and pyrolysis 748
18.6 Bioinstrumentation and controls 830
16.10.1 Pyrolysis 750
16.10.2 Coke and
18.7 Sterilization of process fluids 831
charcoal 752
18.7.1 Batch thermal sterilization 832
16.10.3 The campfire or charcoal grill 753
18.7.2 Continuous thermal sterilization 835
16.10.4 Solid wood or coal combustion 753
18.7.3 Sterilization of liquids 841
16.10.5 Gasification and Fisher-Tropsch
18.7.4 Sterilization of gases 842
technology 756
18.7.5 Ensuring sterility 842
16.11 Solid and liquid explosives 760
16.12 Explosions and detonations 761
18.8 Aseptic operations and practical
16.13 Reactor safety
considerations for bioreactor
762
16.14 768 system construction 843
Summary
Problems 769
18.8.1 Equipment, medium transfer
and flow control 843
18.8.2 Stirrer shaft 844
17. Mass transfer effects: immobilized and
18.8.3 Fermenter inoculation and
heterogeneous reaction systems
sampling 844
17.1 How transformation occurs in a 18.8.4 Materials of construction 845

heterogeneous system? 773 18.8.5 Sparger design 845

17.2 Molecular diffusion and mass transfer 18.8.6 Evaporation control 845
rate 775 18.9 Effect of imperfect mixing 845

17.3 External mass transfer 777 18.9.1 Compartment model 846

17.4 Are kinetic constants of microbial 18.9.2 Surface adhesion model 847
growth dependent on cell size? 783 18.10 Summary 851

17.5 Reactions in isothermal porous Problems 853

catalysts 785 References 856

17.5.1 Asymptote of effectiveness factor
and generalized Thiele modulus 786 19. Real reactors and residence
17.5.2 Isothermal effectiveness factor time distributions
for KA = Q 788
19.1 Real reactors 857
17.5.3 Isothermal effectiveness factor
19.2 Residence-time distribution function 861
for KA -> oo 789
17.5.4 Effectiveness factor for isothermal
19.2.1 RTD determination with a pulse
790 input 862
porous catalyst
17.6 Mass transfer effects in nonisothermal
19.2.2 RTD determination with a step
input 863
porous particles 794
19.2.3 RTD determination with any
17.7 Encapsulation immobilization 801
17.8 Combined
tracer inputs 864
external and internal mass
19.2.4 Characteristics of the RTD 865
transfer effects 803
19.3 Residence-time distributions of ideal
17.9 The shrinking core model 806
17.9.1 Time reactors 869
required to completely
19.3.1 RTDs in batch reactors and PFRs 870
dissolve a porous slab full of
19.3.2 RTD in a CSTR 870
fast-reactive materials 807
17.9.2 Time 19.3.3 RTD in an ideal laminar-flow
required to completely
dissolve full tubular reactor 871
a porous sphere
of fast-reactive materials 19.3.4 A comparison of RTDs in ideal
808
17.10 reactors 872
Summary 809
19.4 Tubular dispersion reactor 877
Problems 812
19.5 PFR with partial distributed
References 816
feed/withdraw and recycle 879
 Contents xi

882 20.5 Plackett-Burman design 896

19.6 Summary
Problems 882 20.6 Taguchi experimental design 899

20.7 Central composite design 904

20.8 Box-Behnken design 908

Design of experiment
20.9 Doehlert design 916
20.1 Experiment 885 918
20.10 Superlative box design
20.1.1 Controlled experiments 886
20.11 Quality impartial design 923
20.1.2 Observational study: natural
20.12 DOE for cubic and quartic response
or field experiments 887 923
models
888
20.2 Adaptive design 20.13 Independent variable versus
20.3 Factorial design, L1 891 929
normalized factor
20.4 Fractional factorial design 893 930
20.14 Summary
20.4.1 Linear 893
response
20.4.2 Response surface 894 Index 935
20.4.3 Non-interacting systems 895