List of Contributors xiii
About the Series Editors xv
1 Introduction and Overview 1
John Villadsen
Part One Fundamentals of Bioengineering 3
2 Experimentally Determined Rates of Bio-Reactions 5
John Villadsen
Summary 5
2.0 Introduction 5
2.1 Mass Balances for a CSTR Operating at Steady State 7
2.2 Operation of the Steady-State CSTR 13
References 16
3 Redox Balances and Consistency Check of Experiments 17
John Villadsen
Summary 17
3.1 Black-Box Stoichiometry Obtained in a CSTR Operated at Steady State 17
3.2 Calculation of Stoichiometric Coefficients by Means of a Redox Balance 20
3.3 Applications of the Redox Balance 23
3.4 Composition of the BiomassX 28
3.5 Combination of Black-Box Models 30
3.6 Application of Carbon and Redox Balances in Bio-Remediation Processes 34
References 38
4 Primary Metabolic Pathways and Metabolic Flux Analysis 39
John Villadsen
Summary 39
4.0 Introduction 39
4.1 Glycolysis 43
4.2 Fermentative Metabolism: Regenerating the NAD+ Lost in Glycolysis 47
4.3 The TCA Cycle: Conversion of Pyruvate to NADH + FADH2, to Precursors or Metabolic Products 50
4.4 NADPH and Biomass Precursors Produced in the PP Pathway 56
4.5 Oxidative Phosphorylation: Production of ATP from NADH (FADH2) in Aerobic Fermentation 57
4.6 Summary of the Biochemistry of Primary Metabolic Pathways 59
4.7 Metabolic Flux Analysis Discussed in Terms of Substrate to Product Pathways 61
4.8 Metabolic Flux Analysis Discussed in Terms of Individual Pathway Rates in the Network 88
4.9 Propagation of Experimental Errors in MFA 94
4.10 Conclusions 96
References 96
5 A Primer to 13C Metabolic Flux Analysis 97
Wolfgang Wiechert, Sebastian Niedenfuhr, and Katharina Noh Summary 97
5.1 Introduction 97
5.2 Input and Output Data of 13C MFA 99
5.3 A Brief History of 13C MFA 101
5.4 An Illustrative Toy Example 102
5.5 The Atom Transition Network 104
5.6 Isotopomers and Isotopomer Fractions 104
5.7 The Isotopomer Transition Network 105
5.8 Isotopomer Labeling Balances 107
5.9 Simulating an Isotope Labeling Experiment 109
5.10 Isotopic Steady State 110
5.11 Flux Identifiability 112
5.12 Measurement Models 113
5.13 Statistical Considerations 114
5.14 Experimental Design 115
5.15 Exchange Fluxes 116
5.16 Multidimensional Flux Identifiability 118
5.17 Multidimensional Flux Estimation 120
5.18 The General Parameter Fitting Procedure 121
5.19 Multidimensional Statistics 123
5.20 Multidimensional Experimental Design 124
5.21 The Isotopically Nonstationary Case 127
5.22 Some Final Remarks on Network Specification 130
5.23 Algorithms and Software Frameworks for 13C MFA 132
Glossary 135
References 137
6 Genome-Scale Models 143
Basti Bergdahl, Nikolaus Sonnenschein, Daniel Machado, Markus Herrgard, and Jochen Forster
Summary 143
6.1 Introduction 143
6.2 Reconstruction Process of Genome-Scale Models 144
6.3 Genome-Scale Model Prediction 147
6.3.1 Mathematical Description of Biochemical Reaction Systems 147
6.3.2 Constraint-Based Modeling 148
6.3.3 Pathway Analysis 148
6.3.4 Flux Balance Analysis 150
6.3.5 Engineering Applications of Constraint-Based Modeling 151
6.4 Genome-Scale Models of Prokaryotes 152
6.4.1 Escherichia Coli 153
6.4.2 Other Prokaryotes 156
6.4.3 Prokaryotic Communities 158
6.5 Genome-Scale Models of Eukaryotes 159
6.5.1 Saccharomyces Cerevisiae 160
6.5.2 Other Unicellular Eukaryotes 164
6.5.3 Other Multicellular Eukaryotes 166
6.6 Integration of Polyomic Data into Genome-Scale Models 169
6.6.1 Integration of Transcriptomics and Proteomics Data 170
6.6.2 Metabolomics Data 171
6.6.3 Integration of Multiple Omics 172
Acknowledgment 172
References 173
7 Kinetics of Bio-Reactions 183
John Villadsen
Summary 183
7.1 Simple Models for Enzymatic Reactions and for Cell Reactions with Unstructured Biomass 184
7.2 Variants of Michaelis-Menten and Monod kinetics 189
7.3 Summary of Enzyme Kinetics and the Kinetics for Cell Reactions 201
7.4 Cell Reactions with Unsteady State Kinetics 203
7.5 Cybernetic Modeling of Cellular Kinetics 211
7.6 Bioreactions with Diffusion Resistance 213
7.7 Sequences of Enzymatic Reactions: Optimal Allocation of Enzyme Levels 221
References 230
8 Application of Dynamic Models for Optimal Redesign of Cell Factories 233
Matthias Reuss
Summary 233
8.1 Introduction 233
8.2 Kinetics of Pathway Reactions: the Need to Measure in a Very Narrow Time Window 235
8.2.1 Sampling 238
8.2.2 Quenching and Extraction 240
8.2.3 Analysis 241
8.2.4 Examples for Quantitative Measurements of Metabolites in Stimulus-Response Experiments 242
8.3 Tools for In Vivo Diagnosis of Pathway Reactions 245
8.3.1 Modular Decomposition of the Network: the Bottom-Up Approach 247
8.4 Examples: The Pentose-Phosphate Shunt and Kinetics of Phosphofructokinase 247
8.4.1 Kinetics of the Irreversible Reactions of the Pentose-Phosphate Shunt 247
8.4.2 Kinetics of the Phophofructokinase I (PFK1) 252
8.5 Additional Approaches for Dynamic Modeling Large Metabolic Networks 256
8.5.1 Generalized Mass Action 259
8.5.2 S-Systems Approach 260
8.5.3 Convenience Kinetics 260
8.5.4 Log-Lin and Lin-Log Approaches 260
8.6 Dynamic Models Used for Redesigning Cell Factories. Examples: Optimal Ethanol Production in Yeast and Optimal Production of Tryptophan in E. Coli 268
8.6.1 Dynamic Model 269
8.6.2 Metabolic Control (Sensitivity) Analysis 270
8.6.3 Synthesis Amplification of Hexose Transporters 271
8.6.4 Objective Function 273
8.6.5 Optimal Solutions 275
8.6.6 Flux Optimization of Tryptophan Production with E. Coli 276
8.7 Target Identification for Drug Development 280
References 285
9 Chemical Thermodynamics Applied in Bioengineering 293
John Villadsen
Summary 293
9.0 Introduction 293
9.1 Chemical Equilibrium and Thermodynamic State Functions 296
9.2 Thermodynamic Properties Obtained from Galvanic Cells 305
9.3 Conversion of Free Energy Harbored in NADH and FADH2 to ATP in Oxidative Phosphorylation 310
References 317
Part Two Bioreactors 319
10 Design of Ideal Bioreactors 321
John Villadsen
Summary 321
10.0 Introduction 321
10.1 The Design Basis for a Once-Through Steady-State CSTR 322
10.2 Combination of Several Steady-State CSTRs in Parallel or in Series 329
10.3 Recirculation of Biomass in a Single Steady-State CSTR 332
10.4 A Steady-State CSTR with Uptake of Substrates from a Gas Phase 338
10.5 Fed-Batch Operation of a Stirred Tank Reactor in the Bio-Industry 340
10.6 Loop Reactors: a Modern Version of Airlift Reactors 349
References 355
11 Mixing and Mass Transfer in Industrial Bioreactors 357
John Villadsen
Summary 357
11.0 Introduction 357
11.1 Definitions of Mixing Processes 358
11.2 The Power Input P Delivered by Mechanical Stirring 362
11.3 Mixing and Mass Transfer in Industrial Reactors 367
11.4 Conclusions 372
References 376
Part Three Downstream Processing 379
12 Product Recovery from the Cultures 381
Sunil Nath
Summary 381
12.0 Introduction 381
12.1 Steps in Downstream Processing and Product Recovery 383
12.2 Baker's Yeast 383
12.3 Xanthan Gum 384
12.4 Penicillin 385
12.5 ?-A Interferon 386
12.6 Insulin 390
12.7 Conclusions 391
References 391
13 Purification of Bio-Products 393
Sunil Nath
Summary 393
13.1 Methods and Types of Separations in Chromatography 394
13.2 Materials Used in Chromatographic Separations 396
13.3 Chromatographic Theory 398
13.4 Practical Considerations in Column Chromatographic Applications 399
13.5 Scale-Up 401
13.6 Industrial Applications 402
13.7 Some Alternatives to Column Chromatographic Techniques 403
13.8 Electrodialysis 403
13.9 Electrophoresis 404
13.10 Conclusions 407
References 407
Part Four Process Development, Management and Control 409
14 Real-Time Measurement and Monitoring of Bioprocesses 411
Bernhard Sonnleitner
Summary 411
14.1 Introduction 411
14.2 Variables that should be Known 414
14.3 Variables Easily Accessible and Standard 415
14.4 Variables Requiring More Monitoring Effort and Not Yet Standard 422
14.4.1 Biomass 422
14.4.2 Products and Substrates 427
14.5 Data Evaluation 433
References 434
15 Control of Bioprocesses 439
Jakob Kjobsted Huusom
Summary 439
15.1 Introduction 439
15.2 Bioprocess Control 440
15.2.1 Design Variables in Bioreactor Control 443
15.2.2 Challenges with Respect to Control of a Bioreactor 450
15.3 Principles and Basic Algorithms in Process Control 450
15.3.1 Open Loop Control 450
15.3.2 Feed-forward and Feedback Control 451
15.3.3 Single-Loop PID Control 452
15.3.4 Diagnostic Control Strategies 456
15.3.5 Plant-Wide Control Design 458
References 460
16 Scale-Up and Scale-Down 463
Henk Noorman
Summary 463
16.1 Introduction 463
16.2 Description of the Large Scale 465
16.2.1 Mixing 468
16.2.2 Mass Transfer 472
16.2.3 CO2 Removal 475
16.2.4 Cooling 475
16.2.5 Gas-Liquid Separation 476
16.3 Scale Down 480
16.3.1 One-Compartment Systems 482
16.3.2 Two-Compartment Systems 484
16.4 Investigations at Lab Scale 485
16.4.1 Gluconic Acid 485
16.4.2 Lipase 486
16.4.3 Baker's Yeast 488
16.4.4 Penicillin 490
16.5 Scale Up 491
16.6 Outlook 494
References 495
17 Commercial Development of Fermentation Processes 499
Thomas Grotkjaer
Summary 499
17.1 Introduction 499
17.2 Basic Principles of Developing New Fermentation Processes 501
17.3 Techno-economic Analysis: the Link Between Science, Engineering, and Economy 506
17.3.1 Value Drivers and Production Costs of Fermentation Processes 506
17.3.2 Assessment of New Fermentation Technologies 519
17.3.3 Assessment of Competing Petrochemical Technologies 526
17.4 From Fermentation Process Development to the Market 528
17.4.1 The Value Chain of the Chemical Industry 530
17.4.2 Innovation and Substitution Patterns in the Chemical Industry 534
17.5 The Industrial Angle and Opportunities in the Chemical Industry 537
17.6 Evaluation of Business Opportunities 540
17.7 Concluding Remarks and Outlook 542
Acknowledgment 543
References 543
Index 547
