I. J. Dunn, E. Heinzle, J. Ingham, J. E.

Pfenosil

Biological Reaction Engineering

Dynamic Modelling Fundamentals with Simulation Examples Second, Completely Revised Edition

Biological Reaction Engineering. Second Edition. \. J. Dunn. E. Heinzle, J. Ingham, J- E. Pfenosil Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheitn ISBN: 3-527-30759-1

Also of Interest
Ingham, X, Dunn, I. J., Heinzle, E., Pfenosil, J. E.

Chemical Engineering Dynamics An Introduction to Modelling and Computer Simulation

Second, Completely Revised Edition 2000, ISBN 3-527-29776-6

Irving J. Dunn, Elmar Heinzle, John Ingham, Jifi E. Pf enosil

Biological Reaction Engineering

Dynamic Modelling Fundamentals with Simulation Examples Second, Completely Revised Edition

WILEYVCH WILEY-VCH GmbH & Co. KGaA

Dr. Irving J. Dunn ETH Zurich Department of Chemical Engineering CH-8092 Zurich Switzerland Professor Dr. Elmar Heinzle University of Saarland Department of Technical Biochemistry P.O. Box 15 11 50 D-66041 Saarbrucken Germany Dr. John Ingham University of Bradford Department of Chemical Engeering Bradford BD7 1DP United Kingdom Dr.JiriE.Prenosil ETH Zurich Department of Chemical Engineering CH-8092 Zurich Switzerland

This book was carefully produced. Nevertheless, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

First Edition 1992 Second, Completely Revised Edition 2003

Library of Congress Card No.: Applied for. British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library.

Bibliographic information published by Die Deutsche Bibliothek. Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at <http://dnb.ddb.de>.

2003 WILE Y-VCH Verlag GmbH & Co. KGaA, Weinheim All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Printing: Strauss Offsetdruck, Morlenbach Bookbinding: GroBbuchbinderei J. Schaffer GmbH & Co. KG, Griinstadt Printed in the Federal Republic of Germany. Printed on acid-free paper. ISBN 3-527-30759-1

Table of Contents

TABLE OF CONTENTS PREFACE

PART I PRINCIPLES OF BIOREACTOR MODELLING NOMENCLATURE FOR PART I 1 MODELLING PRINCIPLES

V XI
1 3 9

1.1 FUNDAMENTALS OF MODELLING 9 7.7.7 Use of Models for Understanding, Design and Optimization of Bioreactors 9 1.1.2 General Aspects of the Modelling Approach 10 1.1.3 General Modelling Procedure..... 72 1.1.4 Simulation Tools 75 7.7.5 Teaching Applications 75 1.2 DEVELOPMENT AND MEANING OF DYNAMC DIFFEREOTTAL BALANCES 16 1.3 FORMULATION OF BALANCE EQUATIONS ..21 7.5.7 Types of Mass Balance Equations 27 1.3.2 Balancing Procedure 23 1.3.2.1 Case A. Continuous Stirred Tank Bioreactor 24 1.3.2.2 CaseB. Tubular Reactor 24 1.3.2.3 Case C. River with Eddy Current 25 1.3.3 Total Mass Balances 33 1.3.4 Component Balances for Reacting Systems 34 1.3.4.1 Case A. Constant Volume Continuous Stirred Tank Reactor 35 1.3.4.2 Case B. Semi-continuous Reactor with Volume Change 37 1.3.4.3 Case C. Steady-State Oxygen Balancing in Fermentation 38 1.3.4.4 Case D. Inert Gas Balance to Calculate Flow Rates 39 7.5.5 Stoichiometry, Elemental Balancing and the Yield Coefficient Concept.. 40 1.3.5.1 Simple Stoichiometry 40 1.3.5.2 Elemental Balancing 42 L3.5.3 Mass Yield Coefficients 44

VI 1.3.5.4 1.3.6 1.3.6.1 1.3.6.2 1.3.7 1.3.6.3 2

Table of Contents Energy Yield Coefficients Equilibrium Relationships General Considerations Case A. Calculation of pH with an Ion Charge Balance Energy Balancing for Bioreactors..... Case B. Determining Heat Transfer Area or Cooling Water Temperature 45 46 46 47 49 52 55 .....55 .....56 57 ....58 60 63 67

BASIC BIOREACTOR CONCEPTS 2.1 INFORMATION FOR BIOREACTOR MODELLING... 2.2 BIOREACTOR OPERATION 2.2.7 Batch Operation 2.2.2 Semicontinuous or Fed Batch Operation..... 2.2.3 Continuous Operation 2.2.4 Summary and Comparison

BIOLOGICAL KINETICS

3.1 ENZYME KINETICS 68 3.1.1 Michaelis-Menten Equation 68 3.1.2 Other Enzyme Kinetic Models 73 3.1.3 Deactivation 76 3.1.4 Sterilization 76 3.2 SIMPLE MICROBIAL KINETICS 77 3.2.1 Basic Growth Kinetics 77 3.2.2 Substrate Inhibition of Growth 80 3.2.3 Product Inhibition 81 3.2.4 Other Expressions for Specific Growth Rate 81 3.2.5 Substrate Uptake Kinetics 83 3.2.6 Product Formation 85 3.2.7 Interacting Microorganisms ....86 3.2.7.1 Case A. Modelling of Mutualism Kinetics..... 88 3.2.7.2 Case B. Kinetics of Anaerobic Degradation 89 3.3 STRUCTURED KINETIC MODELS ..........91 3.3.1 Case Studies 93 3.3.1.1 Case C. Modelling Synthesis of Poly-B-hydroxybutyric Acid (PHB) 93 3.3.1.2 Case D. Modelling of Sustained Oscillations in Continuous Culture 94 3.3.1.3 Case E. Growth and Product Formation of an Oxygen-Sensitive Bacillussubtilis Culture 97 4 BIOREACTOR MODELLING 4.1 GENERAL BALANCES FOR TANK-TYPE BIOLOGICAL REACTORS 4.1.1 The Batch Fermenter. 4.1.2 The Chemostat 4.1.3 The Fed Batch Fermenter 4.1.4 Biomass Productivity 4.1.5 Case Studies 101 101 103 104 1 06 109 109

Table of Contents 4.1.5.1 Case A. Continuous Fermentation with Biomass Recycle 4.1.5.2 Case B. Enzymatic Tanks-in-series Bioreactor System 4.2 MODELLING TUBULAR PLUG FLOW BIOREACTORS 4.2.1 Steady-State Balancing 4.2.2 Unsteady-State Balancing for Tubular Bioreactors 5 MASS TRANSFER

VII 110 112 113 113 775 117

5.1 MASS TRANSFER IN BIOLOGICAL REACTORS 117 5.7.7 Gas Absorption with Bioreaction in the Liquid Phase 777 5.1.2 Liquid-Liquid Extraction with Bioreaction in One Phase 778 5.1.3 Surface Biocatalysis 778 5.7.4 Diffusion and Reaction in Porous Biocatalyst 779 5.2 INTERPHASEGAS-LIQUID MASS TRANSFER 119 5.3 GENERAL OXYGEN BALANCES FOR GAS-LIQUID TRANSFER 123 5.3.1 Application of Oxygen Balances 725 5.3.1.1 Case A. Steady-State Gas Balance for the Biological Uptake Rate 125 5.3.1.2 Case B. Determination of KLa Using the Sulfite Oxidation Reaction 126 5.3.1.3 Case C. Determination of Kj^a by a Dynamic Method 126 5.3.1.4 Case D. Determination of Oxygen Uptake Rates by a Dynamic Method 128 5.3.1.5 Case E. Steady-State Liquid Balancing to Determine Oxygen Uptake Rate.. 129 5.3.1.6 Case F. Steady-State Deoxygenated Feed Method for KJJI 130 5.3.1.7 Case G. Biological Oxidation in an Aerated Tank 131 5.3.1.8 Case H. Modelling Nitrification in a Fluidized Bed Biofilm Reactor 133 5.4 MODELS FOR OXYGEN TRANSFER IN LARGE SCALE BIOREACTORS 137 5.4.1 Case Studies for Large Scale Bioreactors 7 39 5.4.1.1 Case A.Model for Oxygen Gradients in a Bubble Column Bioreactor 139 5.4.1.2 Case B.Model for a Multiple Impeller Fermenter 140 6 DIFFUSION AND BIOLOGICAL REACTION IN IMMOBILIZED BIOCATALYST SYSTEMS 145 6.1 EXTERNAL MASS TRANSFER 6.2 INTERNAL DIFFUSION AND REACTION WITHIN BIOCATALYSTS ..... 6.2.1 Derivation of Finite Difference Model for Diffusion-Reaction Systems. 6.2.2 Dimensionless Parameters from Diffusion-Reaction Models 6.2.5 The Effectiveness Factor Concept. 6.2.4 Case Studies for Diffusion with Biological Reaction 6.2.4.1 Case A. Estimation of Oxygen Diffusion Effects in a Biofilm 6.2.4.2 Case B. Complex Diffusion-Reaction Processes (Biofilm Nitrification).... 7 AUTOMATIC BIOPROCESS CONTROL FUNDAMENTALS 7.1 7.2 ELEMENTS OF FEEDBACK CONTROL TYPES OF CONTROLLER ACTION 7.2.7 On-OffControl 7.2.2 Proportional (P) Controller 7.2.3 Proportional-Integral (PI) Controller 146 149 151 754 755 757 157 157 161 161 163 163 764 765

VIII 7.2.4 Proportional-Derivative (PD) Controller 7.2.5 Proportional-Integral-Derivative (PID) Controller 7.3 CONTROLLER TUNING 7.3.1 Trial and Error Method 7.3:2 Ziegler-Nichols Method. 7.3.3 Cohen-Coon Controller Settings 7.3.4 Ultimate Gain Method 7.4 ADVANCED CONTROL STRATEGIES 7.4.1 Cascade Control 7.4.2 Feed Forward Control 7.4.3 Adaptive Control 7.4.4 Sampled-Data Control Systems 7.5 CONCEPTS FOR BIOPROCESS CONTROL 7.5.7 Selection of a Control Strategy 7.5.2 Methods of Designing and Testing the Strategy REFERENCES

Table of Contents 166 167 169 769 769 170 777 172 772 173 774 774 175 776 7 78 181 181 184

REFERENCES CITED IN PART I RECOMMENDED TEXTBOOKS AND REFERENCES FOR FURTHER READING PART II

DYNAMIC BIOPROCESS SIMULATION EXAMPLES AND THE BERKELEY MADONNA SIMULATION LANGUAGE. 191

SIMULATION EXAMPLES OF BIOLOGICAL REACTION PROCESSES USING BERKELEY MADONNA

193

8.1 INTRODUCTORY EXAMPLES 193 8.7.7 Batch Fermentation (BATFERM) 793 8.7.2 ChemostatFermentation (CHEMO) 799 8.1.3 Fed Batch Fermentation (FEDBAT) 204 8.2 BATCH REACTORS 209 8.2.7 Kinetics of Enzyme Action (MMKINET) 209 8.2.2 Lineweaver-Burk Plot (LINEWEAV) .....272 8.2.3 Oligosaccharide Production in Enzymatic Lactose Hydrolysis (OLIGO) 215 8.2.4 Structured Model for PHB Production (PHB) ....279 8.3 FED BATCH REACTORS 224 8.3.1 Variable Volume Fermentation (VARVOL and VARVOLD) 224 8.3.2 Penicillin Fermentation Using Elemental Balancing (PENFERM) 230 8.3.3 Ethanol Fed Batch Diauxic Fermentation (ETHFERM) 240 8.3.4 Repeated Fed Batch Culture (REPFED) 245 8.3.5 Repeated Medium Replacement Culture (REPLCUL) 249 8.3.6 Penicillin Production in a Fed Batch Fermenter (PENOXY) 253 8.4 CONTINUOUS REACTORS 257 8.4.7 Steady-State Chemostat (CHEMOSTA) 257 8.4.2 Continuous Culture with Inhibitory Substrate (CONINHIB) 267 8.4.3 Nitrification in Activated Sludge Process (ACTNITR) 267

Table of Contents

IX

8.4.4 Tubular Enzyme Reactor (ENZTUBE) 272 8.4.5 Dual Substrate Limitation (DUAL) 275 8.4.6 Dichloromethane in a Biofllm Fluidized Sand Bed (DCMDEG) 280 8.4.7 Two-Stage Chemostat with Additional Stream (TWOSTAGE) 286 8.4.8 Two Stage Culture with Product Inhibition (STAGED) 290 8.4.9 Fluidized Bed Recycle Reactor (FBR) 295 8.4.10 Nitrification in a Fluidized Bed Reactor (NITBED)... 299 8.4.11 Continuous Enzymatic Reactor (ENZCON) 305 8.4.12 Reactor Cascade with Deactivating Enzyme (DEACTENZ) 308 8.4.13 Production ofPHB in a Two-Tank Reactor Process (PHBTWO) 314 8.5 OXYGEN UPTAKE SYSTEMS 318 8.5.1 Aeration of a Tank Reactor for Enzymatic Oxidation (OXENZ) 318 8.5.2 Gas and Liquid Oxygen Dynamics in a Continuous Fermenter (INHIB) 321 8.5.3 Batch Nitrification with Oxygen Transfer (NITRIF) 327 8.5.4 Oxygen Uptake and Aeration Dynamics (OXDYN) 331 8.5.5 Oxygen Electrode for Kia (KLADYN, KLAFIT and ELECTFIT) 335 8.5.6 Biofiltration Column with Two Inhibitory Substrates (BIOFILTDYN). 342 8.5.7 Optical Sensing in Microtiter Plates (TITERDYN and T1TERB1O) 349 8.6 CONTROLLED REACTORS 354 8.6.1 Feedback Control of a Water Heater (TEMPCONT) 354 8.6.2 Temperature Control of Fermentation (FERMTEMP) 358 8.6.3 Turbidostat Response (TURBCON) 363 8.6.4 Control of a Continuous Bioreactor, Inhibitory Substrate (CONTCON)367 8.7 DIFFUSION SYSTEMS ....371 8.7.1 Double Substrate Biofilm Reaction (BIOFILM) 377 8.7.2 Steady-State Split Boundary Solution (ENZSPLIT).... 377 8.7.3 Dynamic Porous Diffusion and Reaction (ENZDYN).... 383 8.7.4 Oxygen Diffusion in Animal Cells (CELLDIFF) 388 8.7.5 Biofilm in a Nitrification Column System (NITBEDFILM) 393 8.8 MULTI-ORGANISM SYSTEMS ..400 8.8.1 Two Bacteria with Opposite Substrate Preferences (COMMENSA) 400 8.8.2 Competitive Assimilation and Commensalism (COMPASM) 406 8.8.3 Stability of Recombinant Microorganisms (PLASMID) 411 8.8.4 Predator-Prey Population Dynamics (MIXPOP) 417 8.8.5 Competition Between Organisms (TWOONE) 422 8.8.6 Competition between Two Microorganisms in a Biofilm (FILMPOP). 425 8.8.7 Model for Anaerobic Reactor Activity Measurement (ANAEMEAS).... 433 8.8.8 Oscillations in Continuous Yeast Culture (YEASTOSC) 441 8.8.9 Mammalian Cell Cycle Control (MAMMCELLCYCLE) 445 8.9 MEMBRANE AND CELL RETENTION REACTORS 451 8.9.1 Cell Retention Membrane Reactor (MEMINH) 451 8.9.2 Fermentation with Pervaporation (SUBTILIS) 455 8.9.3 Two Stage Fermentor With Cell Recycle (LACMEMRECYC) 464 8.9.4 Hollow Fiber Enzyme Reactor for Lactose Hydrolysis (LACREACT). 470 8.9.5 Animal Cells in a Fluidized Bed Reactor (ANIMALIMMOB) 477

Table of Contents

APPENDIX: USING THE BERKELEY MADONNA LANGUAGE.. 483

9.1 9.2 A SHORT GUIDE TO BERKELEY MADONNA SCREENSHOT GUIDE TO BERKELEY MADONNA 483 488

10 ALPHABETICAL LIST OF EXAMPLES 11 INDEX

497 499

Preface
Our goal in this textbook is to teach, through modelling and simulation, the quantitative description of bioreaction processes to scientists and engineers. In working through the many simulation examples, you, the reader, will learn to apply mass and energy balances to describe a variety of dynamic bioreactor systems. For your efforts, you will be rewarded with a greater understanding of biological rate processes. The many example applications will help you to gain confidence in modelling, and you will find that the simulation language used, Berkeley Madonna, is a powerful tool for developing your own simulation models. Your new abilities will be valuable for designing experiments, for extracting kinetic data from experiments, in designing and optimizing biological reaction systems, and for developing bioreactor control strategies. This book is based on part of our successful course, "Biological Reaction Engineering", which has been held annually in the Swiss mountain resort of Braunwald for the past twenty five years and which is now known, throughout European biotechnology circles as the "Braunwald Course". More details can be found at our website www.braunwaldcourse.ch. Modelling is often unfamiliar to biologists and chemists, who nevertheless need modelling techniques in their work. The general field of biochemical reaction engineering is one that requires a very close interdisciplinary interaction between applied microbiologists, biochemists, biochemical engineers, engineers and managers; a large degree of collaboration and mutual understanding is therefore important. Professional microbiologists and biochemists often lack the formal training needed to analyze laboratory kinetic data in its most meaningful sense, and they may sometimes experience difficulty in participating in engineering design decisions and in communicating with engineers. These are just the very types of activity required in the multi-disciplinary field of biotechnology. Chemical engineering's greatest strength is its well-developed modelling concepts, based on mass and energy balances, combined with rate processes. Biochemical engineering is a discipline closely related to conventional chemical engineering, in that it attempts to apply physical principles to the solution of biological problems. This approach may be applied to the measurement and interpretation of laboratory kinetic data or as well to the design of large-scale fermentation, enzymatic or waste treatment processes. The necessary interdisciplinary cooperation requires the biological scientists and chemical engineers involved to have at least a partial understanding of each other's field. The purpose of this book is to provide the mathematical tools necessary for the quantitative analysis of biological kinetics and other biological process phenomena. More generally, the mathematical modelling

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methods presented here are intended to lead to a greater understanding of how the biological reaction systems are influenced by process situations. Engineering science depends heavily on the use of applied theory, quantitative correlations and mathematics, and it is often difficult for all of us (not only the biological scientist) to surmount the mathematical barrier, which is posed by engineering. A mistake, often made, is to confuse "mathematics" with the engineering modelling approach. In modelling an attempt is made to analyze a real and possibly very complex situation into a simplified and understandable physical analog. This physical model may contain many subsystems, all of which still make physical sense, but which now can be formulated as mathematical equations. These equations can be handled automatically by the computer. Thus the engineer and the biologist are freed from the difficulties of mathematical solution and can tackle complex problems that were impossible before. Models, however, still have to be formulated and one of the most important tools of the biochemical engineer, in this operation, is the use of material balance equations. Though it may not be easy for the microbiologist to fully appreciate the importance of differential equations, mass balance equations are not so difficult to understand, since the first law of conservation, namely that matter can neither be created nor destroyed, is fundamental to all science. Mass balances, when combined with kinetic rate equations, to form simple mathematical models, can be used with very great effect as a means of planning, conducting and analyzing experiments. Models are especially important as a means of obtaining a better understanding of process phenomena. A rational approach to experimentation and design requires a considerable knowledge of the system, which can really only be achieved by means of a mathematical model. This book attempts to demonstrate this by way of the many detailed examples. The contribution made to biotechnology by the biochemical engineering modelling approach is especially important because the basic procedure can be developed from a few fundamental principles. An aim of this book is to demonstrate that you do not have to be an engineer to learn modelling and simulation. The basic concepts of the material balance, combined with biological and enzyme kinetics, are easily applied to describe the behavior of well-stirred tank and tubular fermenters, mixed culture dynamics, interphase gas-liquid mass transfer and internal biofilm diffusional limitations, as demonstrated in the computer examples supplied with this book. Such models, when solved interactively by computer simulation, become much more understandable to non-engineers. The Berkeley Madonna simulation language, used for the examples in this book, is especially suitable because of its sophisticated computing power, interactive facility and ease of programming. The use of this digital simulation programming language makes it possible for the reader, student and teacher to experiment directly with the model, in the classroom or at the desk. In this way it is possible to immediately determine the influence of changing various

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XIII

operating parameters on the bioreactor performance - a real learning experience. The simulation examples serve to enforce the learning process in a very effective manner and also provide hands-on confidence in the use of a simulation language. The readers can program their own examples, by formulating new mass balance equations or by modifying an existing example to a new set of circumstances. Thus by working directly at the computer, the no-longer-passive reader is able to experiment directly on the bioreaction system in a very interactive way by changing parameters and learning about their probable influence in a real situation. Because of the speed of solution, a true degree of interaction is possible with Berkeley Madonna, allowing parameters to be changed easily. Plotting the variables in any configuration is easy during a run, and the results from multiple runs can be plotted together for comparison. Other useful features include data fitting and optimization. In our experience, digital simulation has proven itself to be absolutely the most effective way of introducing and reinforcing new concepts that involve multiple interactions. The thinking process is ultimately stimulated to the point of solid understanding.

Organization of the Book

The book is divided into two parts: a presentation of the background theory in Part I and the computer simulation exercises in Part II. The function of the text in Part I is to provide the basic theory required to fully understand and to make full use of the computer examples and simulation exercises. Numerous case studies provide illustration to the theory. Part II constitutes the main part of this book, where the simulation examples provide an excellent instructional and self-learning tool. Each of the more than fifty examples is self-contained, including a model description, model equations, exercises, computer program listing, nomenclature and references. The exercises range from simple parameter-changing investigations to suggestions for writing a new program. The combined book thus represents a synthesis of basic theory and computerbased simulation examples. Quite apart from the educational value of the text, the introduction and use of the Berkeley Madonna software provides the reader with the considerable practical advantage of a differential equation solution package. In the appendix a screenshot guide is found concerning the use of the software. Part I: "Principles of Bioreactor Modelling" covers the basic theory necessary for understanding the computer simulation examples. This section presents the basic concepts of mass balancing, and their combination with kinetic relationships, to establish simple biological reactor models, carefully presented in a way that should be understandable to biologists. In fact, engineers may also find this rigorous presentation of balancing to be valuable.

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In order to achieve this aim, the main emphasis of the text is placed on an understanding of the physical meaning and significance of each term in the model equations. The aim in presenting the relevant theory is thus not to be exhaustive, but simply to provide a basic introduction to the theory required for a proper understanding of the modelling methodology. Chapter 1 deals with the basic concepts of modelling, the basic principles, development and significance of differential balances and the formulation of mass and energy balance relationships. Emphasis is given to physical understanding. The text is accompanied by example cases to illustrate the application of the material. Chapter 2 serves to introduce the varied operational characteristics of the various types of bioreactors and their differing modes of operation, with the aim of giving a qualitative insight into the quantitative behavior of the computer simulation examples. Chapter 3 provides an introduction to enzyme and microbial kinetics. A particular feature of the kinetic treatment is the emphasis on the use of more complex structured models. Such models require much more consideration to be given to the biology of the system during the modelling procedure, but despite their added complexity can nevertheless also be solved with relative ease. They serve as a reminder that biological reactions are really infinitely complex. Chapter 4 is used to derive general mass balance equations, covering all types of fermentation tank reactors. These generalized equations are then simplified to show their application to the differing modes of stirred tank bioreactor operation, discussed previously and which are illustrated by the simulation examples. Chapter 5 explains the basic theory of interfacial mass transfer as applied to fermentation systems and shows how equations for rates of mass transfer can be combined with mass balances, for both liquid and gas phases. A particular extension of this approach is the combination of transfer rate and material balance equations to models of increased geometrical complexity, as represented by large-scale air-lift and multiple-impeller fermenters. Chapter 6 treats the cases of external diffusion to a solid surface and internal diffusion combined with biochemical reaction, with practical application to immobilized biocatalyst and biofilm systems. Emphasized here is the conceptual ease of handling a complex reaction in a solid biocatalyst matrix. The resulting sets of tractable differential-difference equations are solved by simulation techniques in several examples. Chapter 7 describes the importance of control and summarizes control strategies used for bioreaction processes. Here the fundamentals of feedback control systems and their characteristic responses are discussed. This material forms the basis for performing the many recommended control exercises in the simulation examples. It also will allow the reader-simulator to develop his or her own control models and simulation programs.

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Part II, "Dynamic Bioprocess Simulation Examples and the Berkeley Madonna simulation language" comprises Chapter 8, with the computer simulation examples, and Chapter 9, which gives the instructions for using Madonna, Each example in Chapter 8 includes a description of its physical system, the model equations, that were developed in Part I, and a list of suggested exercises. The programs are found on the CD-ROM. These example exercises can be carried out in order to explore the model system in detail, and it is suggested that work on the computer exercises be done in close reference to the model equations and their physical meaning, as described in the text. The exercises, however, are provided simply as an idea for what might be done and are by no means mandatory or restrictive. Working through a particular example will often suggest an interesting variation, such as a control loop, which can then be programmed and inserted. The examples cover a wide range of application and can easily be extended by reference to the literature. They are robust and are well tested by a variety of undergraduate and graduate students and by also the 350 participants, or so, who have previously attended the Braunwald course. In tackling the exercises, we hope you will soon come to share our conviction that, besides being very useful, computer simulation is also fun to do. For the second edition, the text was thoroughly revised and some of our earlier, less relevant material was omitted. On the other hand, a number of new examples resulting mainly from the authors' latest research and teaching work were added. There was also an opportunity in this new edition to eliminate most of the past errors and to avoid new ones as much as possible. Most importantly, the examples have been rewritten in Berkeley Madonna, which all of our reader-simulators will greatly appreciate. Our book has a number of special characteristics. It will be obvious, in reading it through, that we concentrate only on those topics of biological reaction engineering that lend themselves to modelling and simulation and do not attempt to cover the area completely. Our own research work is used to illustrate theoretical points and from it many simulation examples are drawn. A list of suggested books for supplementary reading is found at the end of Chapter 6, together with the list of cited references. The diversity of the simulation examples made it necessary to use separate nomenclature for each. The symbols used in Chapters 1 - 6 are defined at the end of Part I. The authors' four nationalities and three mother tongues, made it difficult to settle on American or British spelling. Somehow we like "modelling" better than "modeling". We are confident that the book will be useful to all life scientists wishing to obtain an understanding of biochemical engineering and also to those chemical and biochemical engineers wanting to sharpen their modelling skills and wishing to gain a better understanding of biochemical process phenomena. We hope that teachers with an interest in modelling will find this to be a useful textbook for undergraduate and graduate biochemical engineering and biotechnological courses.

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Acknowledgements
A major acknowledgement should be made to the excellent pioneering texts of R. G. E. Franks (1967 and 1972) and also of W. L. Luyben (1973), for inspiring our interest in digital simulation. We are especially grateful to our students and to the past-participants of the Braunwald course, for their assistance in the continuing development of the course and of the material presented in this book. Continual stimulus and assistance has also been given by our doctoral candidates, especially at the Chemical Engineering Department, ETH-Zurich, as noted throughout the references. We are grateful to and have great respect for the developers of Berkeley Madonna and hope that this new version of the book will be useful in drawing attention to this wonderful simulation language.