A thorough introduction to the basics of bioengineering, with a focus on applications in the emerging "white" biotechnology industry.
As such, this latest volume in the "Advanced Biotechnology" series covers the principles for the design and analysis of industrial bioprocesses as well as the design of bioremediation systems, and several biomedical applications. No fewer than seven chapters introduce stoichiometry, kinetics, thermodynamics and the design of ideal and real bioreactors, illustrated by more than 50 practical examples. Further chapters deal with the tools that enable an understanding of the behavior of cell cultures and enzymatically catalyzed reactions, while others discuss the analysis of cultures at the level of the cell, as well as structural frameworks for the successful scale-up of bioreactions. In addition, a short survey of downstream processing options and the control of bioreactions is given.
With contributions from leading experts in industry and academia, this is a comprehensive source of information peer-reviewed by experts in the field.
Table of Contents:
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 Niedenführ, and Katharina Nöh 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 Herrgård, and Jochen Förster
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 α-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 Kjøbsted 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 Grotkjær
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
About the Author :
John Villadsen is Professor in the Department of Chemical and Biochemical Engineering at the Technical University of Denmark (DTU) in Lyngby. In his early career he worked at the Danish spray drier company NIRO Atomizer in Sao Paulo (Brasil), as Professor of Chemical Engineering at the University of Houston, Texas (USA) and headed the Danish Center for Bioprocess Engineering at DTU 1985 -2001. Since 1985 he focused on the commercial use of industrially relevant microorganisms, specifically in the field of microbial physiology applied to lactic bacteria, yeast and filamentous fungi and he helped to develop novel routes for the production of bulk chemicals by fermentation. For many years he has been consultant to Bio-industrial companies in Europe and in the USA. Among his many awards is "The Novozymes prize for Bioengineering research" instituted in 2015 in his name.
Sang Yup Lee is Distinguished Professor at the Department of Chemical and Biomolecular Engineering at the Korea Advanced Institute of Science and Technology (KAIST). He is currently the Director of the Center for Systems and Synthetic Biotechnology, Director of the BioProcess Engineering Research Center, and Director of the Bioinformatics Research Center. He has published more than 500 journal papers, 64 books and book chapters, and more than 580 patents (either registered or applied). He received numerous awards, including the National Order of Merit, the Merck Metabolic Engineering Award, the ACS Marvin Johnson Award, Charles Thom Award, Amgen Biochemical Engineering Award, Elmer Gaden Award, POSCO TJ Park Prize, and HoAm Prize. He currently is Fellow of American Association for the Advancement of Science, the American Academy of Microbiology, American Institute of Chemical Engineers, Society for Industrial Microbiology and Biotechnology, American Institute of Medical and Biological Engineering, the World Academy of Science, the Korean Academy of Science and Technology, and the National Academy of Engineering of Korea. He is also Foreign Member of National Academy of Engineering USA. He is currently honorary professor of the University of Queensland (Australia), honorary professor of the Chinese Academy of Sciences, honorary professor of Wuhan University (China), honorary professor of Hubei University of Technology (China), honorary professor of Beijing University of Chemical Technology (China), and advisory professor of the Shanghai Jiaotong University (China). Lee is the Editor-in-Chief of the Biotechnology Journal and Associate Editor and board member of numerous other journals. Lee is currently serving as a member of Presidential Advisory Committee on Science and Technology (Korea).
Jens Nielsen is Professor and Director to Chalmers University of Technology (Sweden) since 2008. He obtained an MSc degree in Chemical Engineering and a PhD degree (1989) in Biochemical Engineering from the Technical University of Denmark (DTU) and after that established his independent research group and was appointed full Professor there in 1998. He was Fulbright visiting professor at MIT in 1995-1996. At DTU, he founded and directed the Center for Microbial Biotechnology. Jens Nielsen has published more than 350 research papers, co-authored more than 40 books and he is inventor of more than 50 patents. He has founded several companies that have raised more than 20 million in venture capital. He has received numerous Danish and international awards and is member of the Academy of Technical Sciences (Denmark), the National Academy of Engineering (USA), the Royal Danish Academy of Science and Letters, the American Institute for Medical and Biological Engineering and the Royal Swedish Academy of Engineering Sciences.
Professor Gregory Stephanopoulos is the W. H. Dow Professor of Chemical Engineering at the Massachusetts Institute of Technology (MIT, USA) and Director of the MIT Metabolic Engineering Laboratory. He is also Instructor of Bioengineering at Harvard Medical School (since 1997). He received his BS degree from the National Technical University of Athens and his PhD from the University of Minnesota (USA). He has co-authored approximately 400 research papers and 50 patents, along with the first textbook on Metabolic Engineering. He has been recognized by numerous awards from the American Institute of Chemical Engineers (AIChE) (Wilhelm, Walker and Founders awards), American Chemical Society (ACS), Society of industrial Microbiology (SIM), BIO (Washington Carver Award), the John Fritz Medal of the American Association of Engineering Societies, and others. In 2003 he was elected member of the National Academy of Engineering (USA) and in 2014 President of AIChE.
