Fundamental Bioengineering

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.

• List of Contributors xiiiAbout the Series Editors xv1 Introduction and Overview 1John VilladsenPart One Fundamentals of Bioengineering 32 Experimentally Determined Rates of Bio-Reactions 5John VilladsenSummary 52.0 Introduction 52.1 Mass Balances for a CSTR Operating at Steady State 72.2 Operation of the Steady-State CSTR 13References 163 Redox Balances and Consistency Check of Experiments 17John VilladsenSummary 173.1 Black-Box Stoichiometry Obtained in a CSTR Operated at Steady State 173.2 Calculation of Stoichiometric Coefficients by Means of a Redox Balance 203.3 Applications of the Redox Balance 233.4 Composition of the BiomassX 283.5 Combination of Black-Box Models 303.6 Application of Carbon and Redox Balances in Bio-Remediation Processes 34References 384 Primary Metabolic Pathways and Metabolic Flux Analysis 39John VilladsenSummary 394.0 Introduction 394.1 Glycolysis 434.2 Fermentative Metabolism: Regenerating the NAD+ Lost in Glycolysis 474.3 The TCA Cycle: Conversion of Pyruvate to NADH + FADH2, to Precursors or Metabolic Products 504.4 NADPH and Biomass Precursors Produced in the PP Pathway 564.5 Oxidative Phosphorylation: Production of ATP from NADH (FADH2) in Aerobic Fermentation 574.6 Summary of the Biochemistry of Primary Metabolic Pathways 594.7 Metabolic Flux Analysis Discussed in Terms of Substrate to Product Pathways 614.8 Metabolic Flux Analysis Discussed in Terms of Individual Pathway Rates in the Network 884.9 Propagation of Experimental Errors in MFA 944.10 Conclusions 96References 965 A Primer to 13C Metabolic Flux Analysis 97Wolfgang Wiechert, Sebastian Niedenführ, and Katharina Nöh Summary 975.1 Introduction 975.2 Input and Output Data of 13C MFA 995.3 A Brief History of 13C MFA 1015.4 An Illustrative Toy Example 1025.5 The Atom Transition Network 1045.6 Isotopomers and Isotopomer Fractions 1045.7 The Isotopomer Transition Network 1055.8 Isotopomer Labeling Balances 1075.9 Simulating an Isotope Labeling Experiment 1095.10 Isotopic Steady State 1105.11 Flux Identifiability 1125.12 Measurement Models 1135.13 Statistical Considerations 1145.14 Experimental Design 1155.15 Exchange Fluxes 1165.16 Multidimensional Flux Identifiability 1185.17 Multidimensional Flux Estimation 1205.18 The General Parameter Fitting Procedure 1215.19 Multidimensional Statistics 1235.20 Multidimensional Experimental Design 1245.21 The Isotopically Nonstationary Case 1275.22 Some Final Remarks on Network Specification 1305.23 Algorithms and Software Frameworks for 13C MFA 132Glossary 135References 1376 Genome-Scale Models 143Basti Bergdahl, Nikolaus Sonnenschein, Daniel Machado, Markus Herrgård, and Jochen FörsterSummary 1436.1 Introduction 1436.2 Reconstruction Process of Genome-Scale Models 1446.3 Genome-Scale Model Prediction 1476.3.1 Mathematical Description of Biochemical Reaction Systems 1476.3.2 Constraint-Based Modeling 1486.3.3 Pathway Analysis 1486.3.4 Flux Balance Analysis 1506.3.5 Engineering Applications of Constraint-Based Modeling 1516.4 Genome-Scale Models of Prokaryotes 1526.4.1 Escherichia Coli 1536.4.2 Other Prokaryotes 1566.4.3 Prokaryotic Communities 1586.5 Genome-Scale Models of Eukaryotes 1596.5.1 Saccharomyces Cerevisiae 1606.5.2 Other Unicellular Eukaryotes 1646.5.3 Other Multicellular Eukaryotes 1666.6 Integration of Polyomic Data into Genome-Scale Models 1696.6.1 Integration of Transcriptomics and Proteomics Data 1706.6.2 Metabolomics Data 1716.6.3 Integration of Multiple Omics 172Acknowledgment 172References 1737 Kinetics of Bio-Reactions 183John VilladsenSummary 1837.1 Simple Models for Enzymatic Reactions and for Cell Reactions with Unstructured Biomass 1847.2 Variants of Michaelis–Menten and Monod kinetics 1897.3 Summary of Enzyme Kinetics and the Kinetics for Cell Reactions 2017.4 Cell Reactions with Unsteady State Kinetics 2037.5 Cybernetic Modeling of Cellular Kinetics 2117.6 Bioreactions with Diffusion Resistance 2137.7 Sequences of Enzymatic Reactions: Optimal Allocation of Enzyme Levels 221References 2308 Application of Dynamic Models for Optimal Redesign of Cell Factories 233Matthias ReussSummary 2338.1 Introduction 2338.2 Kinetics of Pathway Reactions: the Need to Measure in a Very Narrow Time Window 2358.2.1 Sampling 2388.2.2 Quenching and Extraction 2408.2.3 Analysis 2418.2.4 Examples for Quantitative Measurements of Metabolites in Stimulus–Response Experiments 2428.3 Tools for In Vivo Diagnosis of Pathway Reactions 2458.3.1 Modular Decomposition of the Network: the Bottom-Up Approach 2478.4 Examples: The Pentose-Phosphate Shunt and Kinetics of Phosphofructokinase 2478.4.1 Kinetics of the Irreversible Reactions of the Pentose-Phosphate Shunt 2478.4.2 Kinetics of the Phophofructokinase I (PFK1) 2528.5 Additional Approaches for Dynamic Modeling Large Metabolic Networks 2568.5.1 Generalized Mass Action 2598.5.2 S-Systems Approach 2608.5.3 Convenience Kinetics 2608.5.4 Log–Lin and Lin–Log Approaches 2608.6 Dynamic Models Used for Redesigning Cell Factories. Examples: Optimal Ethanol Production in Yeast and Optimal Production of Tryptophan in E. Coli 2688.6.1 Dynamic Model 2698.6.2 Metabolic Control (Sensitivity) Analysis 2708.6.3 Synthesis Amplification of Hexose Transporters 2718.6.4 Objective Function 2738.6.5 Optimal Solutions 2758.6.6 Flux Optimization of Tryptophan Production with E. Coli 2768.7 Target Identification for Drug Development 280References 2859 Chemical Thermodynamics Applied in Bioengineering 293John VilladsenSummary 2939.0 Introduction 2939.1 Chemical Equilibrium and Thermodynamic State Functions 2969.2 Thermodynamic Properties Obtained from Galvanic Cells 3059.3 Conversion of Free Energy Harbored in NADH and FADH2 to ATP in Oxidative Phosphorylation 310References 317Part Two Bioreactors 31910 Design of Ideal Bioreactors 321John VilladsenSummary 32110.0 Introduction 32110.1 The Design Basis for a Once-Through Steady-State CSTR 32210.2 Combination of Several Steady-State CSTRs in Parallel or in Series 32910.3 Recirculation of Biomass in a Single Steady-State CSTR 33210.4 A Steady-State CSTR with Uptake of Substrates from a Gas Phase 33810.5 Fed-Batch Operation of a Stirred Tank Reactor in the Bio-Industry 34010.6 Loop Reactors: a Modern Version of Airlift Reactors 349References 35511 Mixing and Mass Transfer in Industrial Bioreactors 357John VilladsenSummary 35711.0 Introduction 35711.1 Definitions of Mixing Processes 35811.2 The Power Input P Delivered by Mechanical Stirring 36211.3 Mixing and Mass Transfer in Industrial Reactors 36711.4 Conclusions 372References 376Part Three Downstream Processing 37912 Product Recovery from the Cultures 381Sunil NathSummary 38112.0 Introduction 38112.1 Steps in Downstream Processing and Product Recovery 38312.2 Baker’s Yeast 38312.3 Xanthan Gum 38412.4 Penicillin 38512.5 α-A Interferon 38612.6 Insulin 39012.7 Conclusions 391References 39113 Purification of Bio-Products 393Sunil NathSummary 39313.1 Methods and Types of Separations in Chromatography 39413.2 Materials Used in Chromatographic Separations 39613.3 Chromatographic Theory 39813.4 Practical Considerations in Column Chromatographic Applications 39913.5 Scale-Up 40113.6 Industrial Applications 40213.7 Some Alternatives to Column Chromatographic Techniques 40313.8 Electrodialysis 40313.9 Electrophoresis 40413.10 Conclusions 407References 407Part Four Process Development, Management and Control 40914 Real-Time Measurement and Monitoring of Bioprocesses 411Bernhard SonnleitnerSummary 41114.1 Introduction 41114.2 Variables that should be Known 41414.3 Variables Easily Accessible and Standard 41514.4 Variables Requiring More Monitoring Effort and Not Yet Standard 42214.4.1 Biomass 42214.4.2 Products and Substrates 42714.5 Data Evaluation 433References 43415 Control of Bioprocesses 439Jakob Kjøbsted HuusomSummary 43915.1 Introduction 43915.2 Bioprocess Control 44015.2.1 Design Variables in Bioreactor Control 44315.2.2 Challenges with Respect to Control of a Bioreactor 45015.3 Principles and Basic Algorithms in Process Control 45015.3.1 Open Loop Control 45015.3.2 Feed-forward and Feedback Control 45115.3.3 Single-Loop PID Control 45215.3.4 Diagnostic Control Strategies 45615.3.5 Plant-Wide Control Design 458References 46016 Scale-Up and Scale-Down 463Henk NoormanSummary 46316.1 Introduction 46316.2 Description of the Large Scale 46516.2.1 Mixing 46816.2.2 Mass Transfer 47216.2.3 CO2 Removal 47516.2.4 Cooling 47516.2.5 Gas–Liquid Separation 47616.3 Scale Down 48016.3.1 One-Compartment Systems 48216.3.2 Two-Compartment Systems 48416.4 Investigations at Lab Scale 48516.4.1 Gluconic Acid 48516.4.2 Lipase 48616.4.3 Baker’s Yeast 48816.4.4 Penicillin 49016.5 Scale Up 49116.6 Outlook 494References 49517 Commercial Development of Fermentation Processes 499Thomas GrotkjærSummary 49917.1 Introduction 49917.2 Basic Principles of Developing New Fermentation Processes 50117.3 Techno-economic Analysis: the Link Between Science, Engineering, and Economy 50617.3.1 Value Drivers and Production Costs of Fermentation Processes 50617.3.2 Assessment of New Fermentation Technologies 51917.3.3 Assessment of Competing Petrochemical Technologies 52617.4 From Fermentation Process Development to the Market 52817.4.1 The Value Chain of the Chemical Industry 53017.4.2 Innovation and Substitution Patterns in the Chemical Industry 53417.5 The Industrial Angle and Opportunities in the Chemical Industry 53717.6 Evaluation of Business Opportunities 54017.7 Concluding Remarks and Outlook 542Acknowledgment 543References 543Index 547