git | Re oe ETeer vaste Vy eae m eS wl S EneBioreaction Engineering Principles Second Edition Nielsen and John Villadsen snmark and Gunnar Lidén Lund University Lund, Sweden Kluwer Academic/Plenum Publishers New York, Boston, Dordrecht, London, MoscowLibrary of Congress Cataloging-in-Publication Data Nielsen, Jens Hoiniis. Bioreaction engineering principles. —2nd ed /Jens Nielsen and John Villadsen and Gunnar Lid pcm, Includes bibliographical references and index. ISBN 0-306-47349.6 |. Bioreactors, 1. Villadsen, John. I. Lidén, Gunnar. UL. Title. TP248.25.B55 N53 2002 660.6—de21 2002034178 ‘The First Edition of Bioreaction Engineering Principles by Jens Nielsen and John Villadsen was published 1994 by Plenum Press, New York ISBN 0-306-47349-6 ©2003 Kluwer Academic/Plenum Publishers. New York 233 Spring Street, New York. N.Y, 10013, hutpsl/www.wkap.nl! 09876 A CLP. record for this hook is available from the Library of Congress All rights reserved, No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronie, mechanical, photocopying, microfilming, recording, oF otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Printed in the United States of AmericaPreface This is the second edition of the text “Bioreaction Engineering Principles” by Jens Nielsen and John Villadsen, originally published in 1994 by Plenum Press (now part of Kluwer). Time runs fast in Biotechnology, and when Kluwer Plenum stopped reprinting the first edition and asked us to make a second, revised edition we happily accepted. A text on bioreactions written in the early 1990's will not reflect the enormous development of experimental as well as theoretical aspects of cellular reactions during the past decade, In the preface to the first edition we admitted to be newcomers in the field. One of us (JV) has had 10 more years of job training in biotechnology, and the younger author (JN) has now received intemational recognition for his work with the hottest topics of “modem” biotechnology. Furthermore we are happy to have induced Gunnar Lidén, professor of chemical reaction engineering at our sister university in Lund, Sweden to join us as co-author of the second edition. His contribution, especially on the chemical engineering aspects of “real” bioreactors has been of the greatest value. Chapter 8 of the present edition is largely unchanged from the first edition. We wish to thank professor Martin Hjortso from LSU for his substantial help with this chapter. [As was the case for the first edition numerous people helped us by carefully reviewing individual chapters. Professor Lars K Nielsen of University of Queensland was a constant sparring partner, both in Australia and lately as a visiting professor at DTU. The help of Dr. Mats Akesson and of our PhD students, in particular Mikkel Nordkvist, Thomas Grotkjar, Jochen Férster and Morten. ‘Skov Hansen is also gratefully acknowledged. MSc student Rebecca Munk Vejborg was of great help in her careful editing of the final version of the manuscript. [All three authors are chemical engineers by education, and we followed in the footsteps of other chemical engineers who “converted” to biotechnology, but retained their passion for a quantitative treatment of problems from the physical world. One of the greatest innovators of biochemical engineering, professor James E. Bailey was also a chemical engineer by education. We wish to dedicate this book to the memory of this eminent scientist, who was a close colleague and a friend (of the senior author for more than 35 years), and whose work is admired by all three of us. If the pages of this book could inspire some students in the way Jay Bailey inspired hundreds of chemical engineering and biochemical engineering students we could hope for no better reward. John Villadsen and Jens Nielsen Gunnar Lidén. BioCentrum-DTU Kemicentrum, Lund UniversityContents List of Symbols Chapter 1. Bioreaction Engineering: From Bioprocess Design to Systems Biology 1.1 The Structure of the Book 1.2. Some Comments on Nomenclature used in the Book 13 A Final Note References Chapter 2. From Cellular Function to Industrial Products 21 Cellular Growth 2.1.1 From Genotype to Phenotype 2.1.2 Transport Processes 2.1.2.1 Free Diffusion 2.1.2.2 Facilitated Diffusion 2.1.2.3 Active Transport 2.1.3 Catabolism 2.1.3.1 Glycolysis 2.1.3.2 TCA Cycle and Oxidative Phosphorylation 2.1.3.3 Fermentative Pathways 2.1.4 Anabolism 2.1.5 Secondary Metabolism 2.1.6 Secreted Proteins 2.2 Biotech Processes — An Overview 2.2.1 Strain Design and Selection 2.2.2 Fermentation Media 2.2.3 Criteria for Design and Optimization 2.2.4 Strain Improvement References, Chapter 3. Biochemical Reactions ~ A First Look 3.1 The Continuous Stirred Tank Reactor 3.2 Yield Coefficients 3.3 Black Box Stoichiometries 3.4 Degree of Reduction Balances 3.5 Systematic Analysis of Black Box Stoichiometries 3.6 Identification of Gross Measurement Errors Problems References Chapter 4. Thermodynamics of Biochemical Reactions 4.1 Chemical Equilibrium and Thermodynamic State Functions 4.1:1 Changes in Free Energy and Enthalpy 4.1.2 Combustion — A Change in Reference State 4.2 Heat of Reaction 4.3 Non-equilibrium Thermodynamics Problems References 10 B 16 20 2 24 25 2 29 30 35 37 37 38 40 4a 2 45 47 47 3 37 B n 88 92 95 95 97 102 103 109 us ngChapter 5. Biochemical Reaction Networks 5.1 Basic Concepts 5.2 Growth Energetics 5.2.1 Consumption of ATP for Cellular Maintenance 5.2.2 Energetics of Anaerobic Processes 5.2.3 Energetics of Aerobic Processes 5.3 Simple Metabolic Networks 5.4 Flux Analysis in Large Metabolic Networks 5.4.1 Use of Measurable Rates 5.4.2 Use of Labeled Substrates 5.4.3 Use of Linear Programming Problems References ‘Chapter 6. Enzyme Kinetics and Metabolic Control Analysis 6.1 Michaelis-Menten and Analogous Enzyme Kinetics 6.2 More Complicated Enzyme Kinetics 6.2.1 Variants of Michaelis-Menten Kinetics 6.2.2 Cooperativity and Allosteric Enzymes 6.3 Metabolic Control Analysis Problems References Chapter 7. Modeling of Growth Kinetics 7.1 Model Structure and Complexity 7.2 A General Structure for Kinetic Models 7.2.1 Specification of Reaction Stoichiometries 7.2.2 Reaction Rates 7.2.3 Dynamic Mass Balances 7.3 Unstructured Growth Kinetics 1.3.1 The Black Box Model 7.3.2 Multiple Reaction Models 7.3.3 The Influence of Temperature and pH 7.4 Simple Structured Models 7.4.1 Compartment Models 7.4.2 Cybernetic Models 17.5 Mechanistic Models 7.5.1 Genetically Structured Models 7.5.2 Single Cell Models 7.6 Morphologically Structured Models 7.6.1 Oscillating Yeast Cultures 7.6.2 Growth of Filamentous Microorganisms Problems References Chapter 8. Population Balance Equations Problems References Chapter 9. Design of Fermentation Processes 9.1 The Stirred Tank Bioreactor 9.1.1 Batch Operation 9.1.2 The Continuous Stirred Tank Reactor 9.1.3 Biomass Recirculation 9.1.4 The Stirred Tank with Substrate Extracted from a Gas Phase Contents 19 ng 124 128 128 132 142 Ist 153 163 m1 179 186 189 190 195 195 201 207 233, 234 235 237 240 240 242 244 245 245 283 261 265 265 274 278 279 289 290 295 300 306 311 315 335 338. 339 340 342 352 359 364Contents 9.1.5 Fed-batch Operation 9.2 The Plug Flow Reactor 9.3 Dynamic Analysis of Continuous Stirred Tank Bioreactors 9.3.1 Dynamic Response of the Reactor for Simple, Unstructured Kinetic Models 9.3.2 Stability Analysis of a Steady State Solution 9.3.3 Dynamics of the Continuous Stirred Tank for a Mixed Microbial Population Problems References Chapter 10. Mass Transfer 10.1 Gas-Liquid Mass Transfer 10.1.1 Models for k, 10.1.2 Interfacial Area and Bubble Behavior 10.1.3 Empirical Correlations for ka 10.1.4 Mass Transfer Correlations Based on Dimensionless Groups 10.1.5 Gas-Liquid Oxygen Transfer 10.1.6 Gas-Liquid Mass Transfer of Components Other than Oxygen 10.2 Mass Transfer to and into Solid Particles. 10.2.1 Extemal Mass Transfer 10.2.2 Intraparticle Diffusion Problems References Chapter 11. Scale-Up of Bioprocesses 11.1 Scale-up Phenomena 11.2 Bioreactors 11.2.1 Basic Requirements and Reactor Types 11.2.2 The Stirred Tank Bioreactor 11.3 Physical Processes of Importance for Scale-Up 11.3.1 Mixing 113.2 Power Consumption 113.3 Heat Transfer 11.3.4 Scale-Up Related Effects on Mass Transfer 113.5 Rheology of Fermentation Broths 11.3.6 Flow in Stirred Tank Reactors 11.4 Metabolic Processes Affected by Scale-up 11.5 Scale-up in Practice Problems References Index 367 372 380 380 388, 397 409 420 423 425 428 430 438 442 448 433, 436 456 460 469 474 a7 471 478 478 480 482 432 486 491 495, 496 sot 508 510 514 317 519List of Symbols ‘Symbols that are defined and used only within a particular Example, Note, or Problem are not listed. It should be noted that a few symbols are used for different purposes in different chapters. For this reason ‘more than one definition may apply for a given symbol. Cell age (h) Specific interfacial area (m? per m’ of medium) Specific interfacial area (m? per m’ of gas-liquid dispersion) Specific cell surface area (m‘ per gram dry weight) Matrix of stoichiometric coefficients for substrates, introduced in Eq. 7.2 Breakage frequency (h"') Biot number, given by Eq. (10.59) Matrix of stoichiometric coefficients for metabolic products, introduced in Eq. 7.2 Concentration of the ith chemical compound (kg m™*) Saturation concentration of the ith chemical compound (kg m) ‘Vector of concentrations (kg m”) Concentration control coefficient of the jth intermediate with respect to the activity of the ith enzyme Flux control coefficient with respect to the activity of the ith enzyme Matrix containing the control coefficients [defined in Eq. (6.44)] Bubble diameter (m) ‘Thickness of liquid film (m) Mean bubble diameter (m) Lipid membrane thickness (m) Stirrer diameter (m) Mean Sauter bubble diameter (m), given by Eq. (10.18) Dilution rate (h”), given by Eq. (3.1) Maximum dilution rate (h') Diffusion coefficient in a lipid membrane (m* s"!) Effective diffusion coefficient (m* s') Diffusion coefficient of the ith chemical compound (m’ s'') Damkihler number, given by Eq. (10.37) Enzyme concentration (g enzyme 1) Activation energy of the growth process in Eq. (7.27) Elemental matrix for all compounds Elemental matrix for calculated compounds Elemental matrix for measured compounds Distribution function for cells with property y in the population, Eq. (8.1) Variance-covariance matrix Gravity (ms) Gibbs free energy (kJ mole") Gibbs free energy at standard conditions (kJ mole") Gibbs free energy of combustion of the ith reaction component (kJ mole")List of Symbols Gibbs free energy of denaturation (kJ mole"), Eq, (7.28) Gibbs free energy of combustion of the ith reaction component at standard conditions (kJ mole) Gibbs free energy of formation at standard conditions (kJ mole") Grashof number, defined in Table 10.6 Test function, given by Eq. (3.52) Net rate of formation of cells with property y upon cell division (cells br’) Rate of formation of cells with property y upon cell division (cells h") Rate of disappearance of cells with property y uponcell division (cells h) Henry's constant for compound A (atm L mole") Enthalpy of combustion of the ith reaction component (kJ mole”) Enthalpy of formation (kJ mole’') Identity matrix (diagonal matrix with 1 in the diagonal) Jacobian matrix, Eq. (9.102) Enzyme activity (g substrate [z enzyme] b') Rate constant (e.g. kg kg" h'') ‘Mass transfer coefficient for gas film (e.g, mole atm" sm) Mass transfer coefficient for a liquid film surrounding a gas bubble (ms!) Volumetric mass transfer coefficient (s") ‘Mass transfer coefficient for a liquid film surrounding a solid particle (m s") ‘Acid dissociation constant (moles L") ‘Overall mass transfer coefficient for gas-liquid mass transfer (m s') Partition coefficient Equilibrium constant Michaelis constant (g 1"), Eq. (6.1) Amount of biomass (kg) Degree of mixing, defined in Eq. (11.1) Maintenance-associated ATP consumption (moles ATP [kg DWJ' h') ‘Maintenance-associated specific substrate consumption (kg [kg DW]" h) ‘The nth moment of a one-dimensional distribution function, given by Eq. (8.9) ‘Number of cells per unit volume (cells m*), Eq. (8.1) Stirring speed (s') ‘Aeration number, defined in Eq. (11.9) Flow number Power number, defined in Eq. (11.5) Extracellular metabolic product concentration (kg m°) Partial pressure of compound A (e.g. atm) Partitioning function, Eq. (8.5) Productivity of species i in a chemostat (c.g. kgm” h) Dimensionless metabolic product concentration Permeability coefficient (ms) Power input to a bioreactor (W) Power input to a bioreactor at gassed conditions (W) \Variance-covariance matrix for the residuals, given by Eq. (3.46) Peclet number, defined in Table 10.6 ‘Volumetric rate of transfer of A from gas to liquid (moles L"' h'') Observed volumetric formation rate of A (kg m” h''), Eq. (10.45) Volumetric rate of formation of biomass (kg DW m “py ‘Volumetric rate vector (kg m” h) ‘Vector of volumetric mass transfer rates (kg m” h')List of Symbols Q Q Q, xii ‘Number of morphological forms Heat of reaction (kJ mole") Fraction of repressor-free operators, given by Eq. (7.52) Fraction of promotors being activated, given by Eq. (7.58) Fraction of promoters, which form complexes with RNA polymerase, in Eq. (7.60) Specific reaction rate (kg [kg DW]' h') Enzymatic reaction rate (Chapter 6) (g substrate L" hi) Specific ATP synthesis rate (moles of ATP [kg DW]" h'') Specific reaction rate vector (kg [kg DW]" h) Specific substrate formation rate vector (kg [kg DW]' h'') Specific product formation rate vector (kg {kg DW] h") Specific formation rate vector of biomass constituents (kg [kg DW] h'') ‘Vector containing the rates of change of properties, in Eq. (8.2) Gas constant (=8.314 J K mole") Recirculation factor Redundancy matrix, given by Eq, (3.39) Reduced redundancy matrix Reynolds number, defined in Table 10.6 Extracellular substrate concentration (kg m*) Extracellular substrate concentration vector (kg m”) Substrate concentration in the feed to the bioreactor (kg m”) Dimensionless substrate concentration Entropy change (kJ mole" K') ‘Schmidt number, defined in Table 10.6 ‘Sherwood number, defined in Table 10.6 Time (h) Circulation time (8) Mixing time (s) Temperature (K) Total stoichiometric matrix Stoichiometric matrix corresponding to non-measured rates in rows of T Stoichiometric matrix corresponding to known rates of T” Bubble rise velocity (m s") Cybernetic variable, given by Eq. (7.41) Superficial gas velocity (ms'') Vector containing the specific rates of the metamorphosis reaction (kg kg b') Liquid flow (m’ bh) Liquid effluent flow from the reactor (m° h') Liquid feed to the reactor (m° h') Gas flow (m'h') Flux of reaction i (kg [kg DWJ" h"') Impeller induced flow (m’ s" ) Flux vector, i.e. vector of specific intracellular reaction rates (kg [kg DWJ" h") Volume (m’) Total volume of gas-liquid dispersion (m*) Dispersed gas volume (m’) Liquid volume (m*) Total property space, Eq. (8.2) Cybernetic variable, given by Eq. (7.42) Biomass concentration (kg m”) Dimensionless biomass concentrationxiv Greek Letters a, Bs ss aa be moe 0 Ney 8 «i “ Has He Peet a o & % ® Deen wo Abbreviations ADP AMP ATP CoA DNA E List of Symbols Concentration of the ith intracellular component (kg [kg DW]') Vector of concentrations of intracellular biomass components (kg (kg DW]") Property state vector Yield coefficient of j from i (kg j per kg of i or C-mole of j per kg of i ATP consumption for biomass formation (moles of ATP [kg DW]") Concentration of the ith morphological form (kg [kg DW]') Stoichiometric coefficients for substrate iin intracellular reaction j Stoichiometric coefficient for metabolic product i in intracellular reaction i Shear rate (s'') Stoichiometric coefficient for intracellular component j in intracellular reaction j Matrices containing the stoichiometric coefficients for intracellular biomass components Vector of measurement errors in Eq, (3.41) Matrix for stoichiometric coefficients for morphological forms Gas holdup (m° of gas per m’ of gas-liquid dispersion) Porosity of a pellet Elasticity coefficients, defined in Eq. (6.37) Vector of residuals in Eq. (3.44) Matrix containing the elasticity coefficients Dynamic viscosity (kg ms") Internal effectiveness factor, defined in Eq, (10.46) Partial pressure of compound i (atm) Dimensionless time Degree of reduction of the ith compound ‘The specific growth rate of biomass (h"') The maximum specific growth rate (h'!) The specific growth rate for the qth morphological form (kg DW [kg DW]" h' ) Cell density (kg wet biomass [m’* cell]) Liquid density (kg m*) Surface tension (N m") Variance Space time in reactor (h) Shear stress (Nm?) Tortuosity factor, used in Eq, (10.43) Thiele modulus, given by Eq. (10.49) Generalized Thiele modulus, given by Eq, (10.55) Distribution function of cells, Eq (8.8) Adenosine diphosphate Adenosine monophosphate Adenosine triphosphate Coenzyme A Deoxyribonucleic acid Energy chargeList of Symbols EMP FAD FADH, FDA F6P GTP Gor MCA NAD+ NADH NADP+ NADPH PEP PP PSS PTS PYR P/O ratio RNA mRNA rRNA tRNA RQ RSP TCA uQ Embden-Meyerhof-Parnas Flavin adenine dinucleotide (oxidized form) Flavin adenine dinucleotide (reduced form) Food and Drug Administration Fructose-6-phosphate Guanosine triphosphate Glucose-6-phosphate Metabolic control analysis Nicotinamide adenine dinucleotide (oxidized form) Nicotinamide adenine dinucleotide (reduced form) Nicotinamide adenine dinucleotide phosphate (oxidized form) Nicotinamide adenine dinucleotide phosphate (reduced form) Phosphoenol pyruvate Pentose phosphate Protein synthesizing system Phosphotransferase system Pyruvate Number of molecules of ATP formed per atom of oxygen used in the oxidative phosphorylation Ribonucleic acid Messenger RNA Ribosomal RNA. Transfer RNA Respiratory quotient Ribose-5-phosphate Tricarboxylic acid UbiquinoneBioreaction Engineering Principles Jens Nielsen, John Villadsen, and Gunnar Lidén Eee CERCA ia Ric MND oem eT Geta NF ARCOM NC clei Lee TCS eden Un eels) OES T Antica acim Ree MeN Rr trial biological processes today is a prerequisite for both the design of new RUE aecemec tauren erelgeleers Ca Cee DN CUeN en eI aR Re aan Sanu LUR CCN Roum eres oie ecm egies een cmt ict Rm oa Ream nr Mean Cec Ces ea Cole) Prete Mea Se inet Pee RM a Ce UCM MCR Ur Laer UCC Cece) Cr iekeot tM eum meee tae MR eC) jolism is the starting point in the treat em Meg emec Cac CMO CR CCRT cna ae oma a rar POE eSe Cure me Caress AC acm eke rere eS Wet eee Seen acento als ber teees ecole aligned acre ae TAN Semeur mcr ete ne tr ime Moe ie ment is treated in chapters concerning mass transfer and design of bio ele Mae VAR esse ye aer Cet Reet ees TTremayat The book combines, in a rather unique way, a quantitative treatment of TN aeRO ERC Te uae Tec Colm a Co Tet en aM reese ieee Cee Ue Rn Mkt Cae a PME Rn ea Mc ele aa Cat N O-30b-47349-b 35 90000 9N780306"473494! na i KLUWER ACADEMIC / em Odea] lal eto) 03