bioreaction engineering principles

a Cell age h a Specific interfacial area m2per m3of medium ad Specific interfacial area m2per m3of gas–liquid dispersion acell Specific cell surface area m2per gram dry weight A Matrix o

Bioreaction Engineering Principles

John Villadsen l Jens Nielsen l Gunnar Lide´n

Bioreaction Engineering Principles

Third Edition

John Villadsen

Department of Chemical

and Biochemical Engineering

Technical University of Denmark (DTU)

Lyngby, Denmark

jv@kt.dtu.dk

Gunnar Lide´nDepartment of Chemical EngineeringLund University

Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011931856

# Springer Science+Business Media, LLC 2011

All rights reserved This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York,

NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis Use in connection with any form of information storage and retrieval, electronic adaptation, computer software,

or by similar or dissimilar methodology now known or hereafter developed is forbidden.

The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject

to proprietary rights.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

With very little hesitation we accepted the offer.

Since 2003 the book has been used as course-book, in European universities andalso in North and South America, in the Far East, and in Australia We wished notonly to revise the text, but also to write a book that would appeal to students at thebest universities, at least until 2020 In short courses given at major Biotechcompanies we have also found that some of the material in the previous editionscould be used right away to give the companies a better understanding of theirprocesses and to propose better design of their reactors This acceptance of the book

by the industrial community prompted us to include even more examples relevantfor design of processes and equipment in the industry The changes that have beenmade since the second edition are outlined in the first, introductory chapter of thepresent edition

Our initial enthusiasm to embark on a complete revision of the text was mollified

by the duties imposed on two of us (J.N and G.L.) in handling large researchgroups and with the concomitant administration One of us (J.V.) had much moretime available in his function as senior professor, and he became the main respon-sible person for the work during the almost 2 years since the start of the project.But we are all happy with the result of our common efforts – “Tous pour un, unpour tous.”

Some chapters have been read and commented by our colleagues Special thanksare owed to Prof John Woodley for commenting on Chaps 2 and 3, and to Prof.Alvin Nienow for long discussions concerning the right way to present Chap 11.The former Ph.D students, Drs Mikkel Nordkvist and Thomas Grotkjær havekindly given comments to many of the chapters

v

We also thank Ph.D student Saeed Sheykshoaie at Chalmers University whoredrew many of the figures in the last rush before finishing the manuscript Ph.D.student Jacob Brix at DTU has often assisted J.V with his extensive knowledge of

“how to handle the many tricks of Word.”

1 What Is This Book About? 1

1.1 Note on Nomenclature 5

2 Chemicals from Metabolic Pathways 7

2.1 The Biorefinery 8

2.1.1 Ethanol Production 9

2.1.2 Production of Platform Chemicals in the Biorefinery 14

2.2 The Chemistry of Metabolic Pathways 17

2.2.1 The Currencies of Gibbs Free Energy and of Reducing Power 18

2.2.2 Glycolysis 22

2.2.3 Fermentative Metabolism: Oxidation of NADH in Anaerobic Processes 26

2.2.4 The TCA Cycle: Provider of Building Blocks and NADH/FADH2 30

2.2.5 Production of ATP by Oxidative Phosphorylation 33

2.2.6 The Pentose Phosphate Pathway: A Multipurpose Metabolic Network 36

2.2.7 Summary of the Primary Metabolism of Glucose 38

2.3 Examples of Industrial Production of Chemicals by Bioprocesses 41

2.3.1 Amino Acids 42

2.3.2 Antibiotics 45

2.3.3 Secreted Proteins 49

2.4 Design of Biotech Processes: Criteria for Commercial Success 50

2.4.1 Strain Design and Selection 51

2.4.2 Criteria for Design and Optimization of a Fermentation Process 52

2.4.3 Strain Improvement 54

vii

2.5 The Prospects of the Biorefinery 56

Problems 58

References 60

3 Elemental and Redox Balances 63

3.1 The Continuous, Stirred Tank Reactor 65

3.1.1 Mass Balances for an Ideal, Steady-State Continuous Tank Reactor 69

3.2 Yield Coefficients 71

3.3 Black Box Stoichiometries 76

3.4 Degree of Reduction Balances 78

3.4.1 Consistency Test of Experimental Data 86

3.4.2 Redox Balances Used in the Design of Bioremediation Processes 92

3.5 Systematic Analysis of Black Box Stoichiometries 96

3.6 Identification of Gross Measurement Errors 100

Problems 110

References 117

4 Thermodynamics of Bioreactions 119

4.1 Chemical Equilibrium and Thermodynamic State Functions 120

4.1.1 Changes in Free Energy and Enthalpy 120

4.1.2 Free Energy Changes in Bioreactions 124

4.1.3 Combustion: A Change in Reference State 128

4.2 Heat of Reaction 129

4.2.1 Nonequilibrium Thermodynamics 135

4.2.2 Free Energy Reclaimed by Oxidation in the Electron Transfer Chain 137

4.2.3 Production of ATP Mediated byF0
F1ATP Synthase 142

Problems 145

References 149

5 Biochemical Reaction Networks 151

5.1 Basic Concepts 152

5.1.1 Metabolic Network with Diverging Branches 157

5.1.2 A Formal, Matrix-Based Description of Metabolic Networks 166

5.2 Growth Energetics 172

5.2.1 Consumption of ATP for Cellular Maintenance 172

5.2.2 Energetics of Anaerobic Processes 175

5.2.3 Energetics of Aerobic Processes 180

5.3 Flux Analysis in Large Metabolic Networks 184

5.3.1 Expressing the Rate of Biomass Formation 186

5.3.2 The Network Structure and the Use of Measurable Rates 187

5.3.3 The Use of Labeled Substrates 199

Problems 206

References 212

6 Enzyme Kinetics and Metabolic Control Analysis 215

6.1 Enzyme Kinetics Derived from the Model of Michaelis–Menten 217

6.2 More Complicated Enzyme Kinetics 221

6.2.1 Variants of Michaelis–Menten Kinetics 222

6.2.2 Cooperativity and Allosteric Enzymes 227

6.3 Biocatalysis in Practice 232

6.3.1 Laboratory Studies in Preparation for an Industrial Production Process 233

6.3.2 Immobilized Enzymes and Diffusion Resistance 238

6.3.3 Choice of Reactor Type 243

6.4 Metabolic Control Analysis 244

6.4.1 Definition of Control Coefficients for Linear Pathways 245

6.4.2 Using Connectivity Theorems to Calculate Control Coefficients 249

6.4.3 The Influence of Effectors 257

6.4.4 Approximate Methods for Determination of theCJi 258

Problems 265

References 268

7 Growth Kinetics of Cell Cultures 271

7.1 Model Structure and Complexity 272

7.2 A General Structure for Kinetic Models 275

7.2.1 Specification of Reaction Stoichiometries 275

7.2.2 Reaction Rates 277

7.2.3 Dynamic Mass Balances 278

7.3 Unstructured Growth Kinetics 279

7.3.1 The Monod Model 280

7.3.2 Multiple Reaction Models 289

7.3.3 The Influence of Temperature and pH 297

7.4 Simple Structured Models 300

7.4.1 Compartment Models 301

7.4.2 Cybernetic Models 311

7.5 Derivation of Expression for Fraction of Repressor-free Operators 315

7.6 Morphologically Structured Models 327

7.6.1 Oscillating Yeast Cultures 331

7.6.2 Growth of Filamentous Microorganisms 334

7.7 Transport Through the Cell Membrane 341

7.7.1 Facilitated Transport, Exemplified by Eukaryotic Glucoside Permeases 342

7.7.2 Active Transport 345

Problems 348

References 353

8 Population Balance Equations 359

Problems 378

References 381

9 Design of Fermentation Processes 383

9.1 Steady-State Operation of the STR 386

9.1.1 The Standard CSTR with vf ¼ ve ¼ v 387

9.1.2 Productivity in the Standard CSTR 390

9.1.3 Productivity in a Set of Coupled, Standard CSTR’s 394

9.1.4 Biomass Recirculation 399

9.1.5 Steady-State CSTR with Substrates Extracted from a Gas Phase 405

9.2 The STR Operated as a Batch or as a Fed-Batch Reactor 407

9.2.1 The Batch Reactor 408

9.2.2 The Fed-Batch Reactor 411

9.3 Non-steady-State Operation of the CSTR 419

9.3.1 Relations Between Cultivation Variables During Transients 419

9.3.2 The State Vector [s, x, p] in a Transient CSTR Experiment 422

9.3.3 Pulse Addition of Substrate to a CSTR Stability of the Steady State 425

9.3.4 Several Microorganisms Coinhabit the CSTR 429

9.3.5 The CSTR Used to Study Fast Transients 436

9.4 The Plug Flow Reactor 439

9.4.1 A CSTR Followed by a PFR 441

9.4.2 Loop Reactors 443

Problems 448

References 458

10 Gas–Liquid Mass Transfer 459

10.1 The Physical Processes Involved in Gas to Liquid Mass Transfer 460

10.1.1 Description of Mass Transfer Usingkla 462

10.1.2 Models forkl 465

10.1.3 Models for the Interfacial Area, and for Bubble Size 466

10.2 Empirical Correlations forkla 474

10.3 Experimental Techniques for Measurement of O2Transfer 482

10.3.1 The Direct Method 482

10.3.2 The Dynamic Method 484

10.3.3 The Sulfite Method 485

10.3.4 The Hydrogen Peroxide Method 486

10.3.5 kla Obtained by Comparison with the Mass Transfer Coefficient of Other Gases 488

Problems 490

References 495

11 Scale-Up of Bioprocesses 497

11.1 Mixing in Bioreactors 498

11.1.1 Characterization of Mixing Efficiency 499

11.1.2 Experimental Determination of Mixing Time 502

11.1.3 Mixing Systems and Their Power Consumption 505

11.1.4 Power Input and Mixing for High Viscosity Media 514

11.1.5 Rotating Jet Heads: An Alternative to Traditional Mixers 520

11.2 Scale-Up Issues for Large Industrial Bioreactors 527

11.2.1 Modeling the Large Reactor Through Studies in Small Scale 527

11.2.2 Scale-Up in Practice: The Desirable and the Compromises 535

Problems 541

References 545

Index 547

List of Symbols

Symbols that are defined and used only within a particular Example, Note, orProblem are not listed It should be noted that a few symbols are used for differentpurposes in different chapters For this reason, more than one definition may applyfor a given symbol

a Cell age (h)

a Specific interfacial area (m2per m3of medium)

ad Specific interfacial area (m2per m3of gas–liquid dispersion)

acell Specific cell surface area (m2per gram dry weight)

A Matrix of stoichiometric coefficients for substrates, introduced in (7.2)b(y) Breakage frequency (h
1)

B Matrix of stoichiometric coefficients for metabolic products,

introduced in (7.2)

ci Concentration of theith chemical compound (kg m
3)

ci Saturation concentration of theith chemical compound (kg m
3)

c Vector of concentrations (kg m
3)

Cij Concentration control coefficient of thejth intermediate with respect to

the activity of theith enzyme

CJ

i Flux control coefficient with respect to the activity of theith enzymeC* Matrix containing the control coefficients defined in (6.34)

db Bubble diameter (m)

df Thickness of liquid film (m)

dmean Mean bubble diameter (m)

dmem Lipid membrane thickness (m)

ds Stirrer diameter (m)

dSauter Mean Sauter bubble diameter (m), given by (10.18)

D Dilution rate (h
1), given by (3.1)

Dmax Maximum dilution rate (h
1)

Dmem Diffusion coefficient in a lipid membrane (m2s
1)

Deff Effective diffusion coefficient (m2s
1)

xiii

Di Diffusion coefficient of theith chemical compound (m2s
1)

e0 Enzyme concentration (g enzyme L
1)

Eg Activation energy of the growth process in (7.28)

E Mixing efficiency, defined in (11.1)

E Elemental matrix for all compounds

Ec Elemental matrix for calculated compounds

Em Elemental matrix for measured compounds

f(y,t) Distribution function for cells with propertyy in the population (8.1)

F Variance–covariance matrix

g Gravity (m s
2)

G Gibbs free energy (kJ mol
1)

G0 Gibbs free energy at standard conditions (kJ mol
1)

DGci Gibbs free energy of combustion of the ith reaction component

f Gibbs free energy of formation at standard conditions (kJ mol
1)

Gr Grashof number, defined in Table 10.6

h Test function, given by (3.54)

h(y) Net rate of formation of cells with propertyy upon cell division (cells

h
1)

h+(y) Rate of formation of cells with propertyy upon cell division (cells h
1)

h
(y) Rate of disappearance of cells with propertyy upon cell division (cells

h
1)

HA Henry’s constant for compound A (atm L mol
1)

DHci Enthalpy of combustion of theith reaction component (kJ mol
1)

DH0

f Enthalpy of formation (kJ mol
1)

I Identity matrix (diagonal matrix with 1 in the diagonal)

J Jacobian matrix (9.102)

k0 Enzyme activity (g substrate [g enzyme]
1h
1)

ki Rate constant (e.g., kg kg
1h
1)

kg Mass transfer coefficient for gas film (e.g., mol atm
1s
1 m
2)

kl Mass transfer coefficient for a liquid film surrounding a gas bubble

(m s
1)

kla Volumetric mass transfer coefficient (s
1)

ks Mass transfer coefficient for a liquid film surrounding a solid particle

(m s
1)

Ka Acid dissociation constant (mol L
1)

Kl Overall mass transfer coefficient for gas–liquid mass transfer (m s
1)

mATP Maintenance-associated ATP consumption (moles ATP [kg DW]
1

NA Aeration number, defined in (11.14)

Nf Flow number, defined in (11.6)

Np Power number, defined in (11.10)

p Extracellular metabolic product concentration (kg m
3)

pA Partial pressure of compound A (e.g., atm.)

p(y,y*,t) Partitioning function (8.5)

P Dimensionless metabolic product concentration

P Permeability coefficient (m s
1)

P Power input to a bioreactor (W)

Pg Power input to a bioreactor at gassed conditions (W)

P Variance–covariance matrix for the residuals, given by (3.48)

Pe Peclet number, defined in Table 10.6

qtA Volumetric rate of transfer of A from gas to liquid (mol L
1h
1)

qx Volumetric rate of formation of biomass (kg DW m
3h
1)

q Volumetric rate vector (kg m
3h
1)

qt Vector of volumetric mass transfer rates (kg m
3h
1)

Q Number of morphological forms

Q Heat of reaction (kJ mol
1)

Qt Fraction of repressor-free operators, given by (7.47)

Q2 Fraction of promotors being activated, given by (7.53)

Q3 Fraction of promoters, which form complexes with RNA polymerase, in

(7.55)

ri Specific reaction rate for speciesi (kg [kg DW]
1h
1)

r Enzymatic reaction rate (Chap 6) (g substrate L
1h
1)

rATP Specific ATP synthesis rate (moles of ATP [kg DW]
1h
1)

r Specific reaction rate vector (kg [kg DW]
1h
1)

rs Specific substrate formation rate vector (kg [kg DW]
1h
1)

rp Specific product formation rate vector (kg [kg DW]
1h
1)

rx Specific formation rate vector of biomass constituents (kg [kg DW]
1

h
1)

r(y,t) Vector containing the rates of change of properties, in (8.2)

R Gas constant (¼8.314 J K
1mol
1)

R Recirculation factor (Sect 9.1.4)

R Redundancy matrix, given by (3.41)

Rr Reduced redundancy matrix

Re Reynolds number, defined in Table 10.6

s Extracellular substrate concentration (kg m
3)

s Extracellular substrate concentration vector (kg m
3)

sf Substrate concentration in the feed to the bioreactor (kg m
3)

S Dimensionless substrate concentration

DS Entropy change (kJ mol
1K
1)

Sc Schmidt number, defined in Table 10.6

Sh Sherwood number, defined in Table 10.6

t Time (h)

tc Circulation time (s) (11.7)

tm Mixing time (s) (11.3)

T Temperature (K)

T Matrix in (5.11) TT, the transform of T, is the stoichiometric matrix

T1 Matrix corresponding to calculated fluxes (5.12)

T2 Matrix corresponding to measured rates (5.12)

ub Bubble rise velocity (m s
1)

ui Cybernetic variable, given by (7.36)

us Superficial gas velocity (m s
1)

u Vector containing the specific rates of the metamorphosis reaction

(kg kg
1h
1)

v Liquid flow (m3h
1)

ve Liquid effluent flow from the reactor (m3h
1)

vf Liquid feed to the reactor (m3h
1)

vg Gas flow (m3h
1)

vi Flux of internal reactioni in metabolic network (kg [kg DW]
1 h
1)

vpump Impeller induced flow (m3s
1) (11.6)

v Flux vector, i.e., vector of specific intracellular reaction rates (kg [kg

DW]
1h
1)

V Volume (m3)

Vd Total volume of gas–liquid dispersion (m3) (10.16)

Vg Dispersed gas volume (m3) (10.16)

Vl Liquid volume (m3)

Vy Total property space (8.2)

wi Cybernetic variable, given by (7.47)

x Biomass concentration (kg m
3)

X Dimensionless biomass concentration

Xi Concentration of theith intracellular component (kg [kg DW]
1)

X Vector of concentrations of intracellular biomass components (kg [kg

DW]
1)

y Property state vector

Yij Yield coefficient ofj from i (kg j per kg of i or C-mol of j per kg of i)

YxATP ATP consumption for biomass formation (moles of ATP [kg DW]
1)

Zi Concentration of theith morphological form (kg [kg DW]-1)

Greek Letters

aji Stoichiometric coefficients for substratei in intracellular reaction j

bji Stoichiometric coefficient for metabolic producti in intracellular reaction j_g Shear rate (s
1)

gji Stoichiometric coefficient for intracellular component i in intracellularreactionj

_g Shear rate (s
1), defined in (11.24)

G Matrices containing the stoichiometric coefficients for intracellular biomasscomponents

d Vector of measurement errors in (3.43)

D Matrix for stoichiometric coefficients for morphological forms

e Gas holdup (m3of gas per m3of gas–liquid dispersion)

 Internal effectiveness factor, defined in (9) of Note 6.2

pi Partial pressure of compoundi (atm)

y Dimensionless time

ki Degree of reduction of theith compound

m Specific growth rate of biomass (h
1)

mmax Maximum specific growth rate (h
1)

mq Specific growth rate for theqth morphological form (kg DW [kg DW]
1h-1)

rcell Cell density (kg wet biomass [m
3cell])

rl Liquid density (kg m
3)

s Surface tension (N m
1)

s2 Variance

t Space time in reactor (h)

t Shear stress (N m
2), defined in (11.25)

tp Tortousity factor, used in (6.23)

Fn Thiele modulus for reaction of ordern (2) and (5) in Note 6.2

Fgen Generalized Thiele modulus, Note 6.2

c(X) Distribution function of cells (8.8)

AcCoA Acetyl co-enzyme A

ADP Adenosine diphosphate

AMP Adenosine monophosphate

ATP Adenosine triphosphate

CoA Coenzyme A

DHAP Dihydroxy acetone phosphate

DNA Deoxyribonucleic acid

Ec Energy charge

EMP Embden–Meyerhof–Parnas

FAD Flavin adenine dinucleotide (oxidized form)

FADH Flavin adenine dinucleotide (reduced form)

FDA Food and Drug Administration

HAc Acetic acid

HLac Lactic acid

LAB Lactic acid bacteria

MCA Metabolic control analysis

MFA Metabolic Flux Analysis

NAD+ Nicotinamide adenine dinucleotide (oxidized form)

NADH Nicotinamide adenine dinucleotide (reduced form)

NADP+ Nicotinamide adenine dinucleotide phosphate (oxidized form)NADPH Nicotinamide adenine dinucleotide phosphate (reduced form)

PEP Phosphoenol pyruvate

RNA Ribonucleic acid

mRNA Messenger RNA

rRNA Ribosomal RNA

tRNA Transfer RNA

List of Examples

Chapter 3

3.1 Anaerobic yeast fermentation 813.2 Aerobic growth with ammonia as nitrogen source 823.3 Anaerobic growth of yeast with NH3as nitrogen source

and ethanol as the product 833.4 Biomass production from natural gas 833.5 Consistency analysis of yeast fermentation 873.6 Citric acid produced byAspergillus niger 913.7 Design of an anaerobic waste water treatment unit 943.8 Anaerobic yeast fermentation with CO2, ethanol, and glycerol

as metabolic products 973.9 Production of lysine from glucose with acetic acid as byproduct 983.10 Calculation of best estimates for measured rates 1033.11 Application of the least-squares estimate 1073.12 Calculation of the test function h 1073.13 Error diagnosis of yeast fermentation 108Chapter 4

4.1 Thermodynamic data for H2O 1234.2 Equilibrium constant for formation of H2O 1234.3 Free energy changes of reactions in the EMP pathway 1254.4 Calculation ofDGcfor ethanol combustion at 25C, 1 atm 129

4.5 Heat of reaction for aerobic growth of yeast 1324.6 Anaerobic growth on H2and CO2to produce CH4 134

Chapter 5

5.1 Analysis of the metabolism of lactic acid bacteria 1595.2 Anaerobic growth ofSaccharomyces cerevisiae 1615.3 Aerobic growth ofSaccharomyces cerevisiae 164

xxi

5.4 Production of butanol and acetone by fermentation 1705.5 Growth energetics for cultivation ofLactococcus lactis 1785.6 Energetics ofBacillus clausii 1825.7 Metabolic Flux Analysis of citric acid fermentation

byCandida lipolytica 1935.8 Analysis of the metabolic network inS cerevisiae

during anaerobic growth 1965.9 Identification of lysine biosynthesis 2005.10 Analysis of a simple network 204Chapter 6

6.1 Analysis of enzymatic reaction data 2256.2 Competition of two substrates for the same enzyme 2306.3 Determination of NADH in cell extract using a

cyclic enzyme assay 2306.4 Lactobionic acid from lactose 2336.5 Kinetics for lactobionic acid synthesis applied to

an immobilized enzyme 2426.6 Illustration of Metabolic Control Analysis using

analytical expressions forri 2546.7 Calculation of the flux control coefficient at a reference

state by large deviations 2606.8 Elasticities and flux control coefficients determined by

the lin-log method 2616.9 Determination ofE and CJfrom transients

in a steady-state chemostat 262Chapter 7

7.1 Steady-state chemostat described by the Monod model

with sterile feed 2827.2 Steady-state chemostat described by the Monod model

including maintenance 2907.3 An unstructured model describing the growth of

Saccharomyces cerevisiae 2927.4 Extension of the Sonnleitner and Ka¨ppeli model to

describe protein production 2967.5 Analysis of the model of Williams 3057.6 Two-compartment model for lactic acid bacteria 3067.7 A model for diauxic growth 3227.8 A simple morphologically structured model describing

plasmid instability 3297.9 A simple morphologically structured model for the growth

of filamentous microorganisms 3377.10 Transport of glucose to a yeast cell by facilitated diffusion 3437.11 Free diffusion of organic acids across the cell membrane 346

Chapter 8

8.1 Specification of the partitioning function

and the breakage frequency 3638.2 Population balance for recombinantEscherichia coli 3678.3 Age distribution model forSaccharomyces cerevisiae 3698.4 Population model for hyphal elements 373Chapter 9

9.1 Biomass and product concentrations for Monod kinetics

with maintenance 3889.2 Design of a robust waste water treatment plant 3959.3 Design of cell recirculation system 4039.4 Design of a recirculation system – with maintenance requirement 4049.5 Design of an integrated lactic acid production unit 4049.6 Optimal design of a single cell production 4069.7 Design of a fed-batch process for baker’s yeast production 4169.8 A step change ofsffor constantD 4219.9 Transients obtained after a change of dilution rate

fromD0toD 4239.10 Competing microbial species 4319.11 Reversion of a desired mutant to the wild type 4349.12 A steady-state CSTR followed by a PFR 4429.13 Design of a loop reactor for single cell production 444Chapter 10

10.1 The oxygen requirement of a rapidly respiring yeast culture 46010.2 Requirements forkla in a laboratory bioreactor 46210.3 Bubble size and specific interfacial area in an agitated vessel 47210.4 Derivation of empirical correlations forkla

in a laboratory bioreactor 475Chapter 11

11.1 Mixing time in a baffled tank reactor 50111.2 Macro- and micro-mixing of a liquid 50111.3 Measuring a pulse response using the pH rather than [H+] 50511.4 Power required for liquid mixing and for gas

dispersion at the sparger 51211.5 Calculation of mixing time in Stirred Tank Reactors 51411.6 Rheological characterization of xanthan solutions 518

11.7 A two-compartment model for oxygen transfer

in a large bioreactor 53011.8 Regimen analysis of penicillin fermentation 53311.9 Scale-up of a 600-L pilot plant reactor to 60 m3

for unaerated mixing 53511.10 Oxygen transfer to a 60 m3industrial reactor 537

List of Tables

Chapter 2

2.1 Twelve sugar-based building blocks suggested

by Werpy and Petersen (2004) 152.2 Precursor metabolites and some of the building blocks

synthesized from the precursors 392.3 Composition ofE coli cells grown at 37C on a glucose

minimal medium at a specific growth raterx¼ m ¼ 1.04 g cell

formed per gram cell per hour and the corresponding

requirements for ATP and NADPH 402.4 Measured concentrations of AMP, ADP, and ATP

in a continuous culture ofLactococcus lactis 402.5 Typical complex media used in the fermentation industry 422.6 The 20 physiologically important (L-) amino acids

and their net-chemical formula 432.7 Four classes of antibiotics 462.8 Pros and cons of different production organisms for

recombinant proteins 52Chapter 3

3.1 Average composition ofS cerevisiae 733.2 Elemental composition of biomass for several microbial species 743.3 Values of the w2distribution 107

Chapter 4

4.1 Concentrations (at pH ¼ 7) of intermediates and of cofactors

of the EMP pathway in the human erythrocyte 1264.2 ApproximateDGRvalues for the EMP pathway reactions

in the human erythrocyte 127

xxv

4.3 Heat of combustion for various compounds

at standard conditions 1314.4 Single-electrode potential for electronacceptors 140Chapter 5

5.1 Experimentally determined values ofYxATPandmsfor various

microorganisms grown under anaerobic conditions

with glucose as the energy source 1765.2 Calculated values of the requirements for NADPH for

biomass synthesis and the amount of NADH formed in

connection with biomass synthesis 1825.3 Fluxes through key reactions in the metabolic network during

anaerobic growth ofS cerevisiae and using different models 197Chapter 6

6.1 Enzymatic rate datar at four levels of s and p 2266.2 Reconstruction the reaction ratesR1andR2using measurements

of (s1/s0,s/s0) 264

Chapter 7

7.1 Compilation ofKsvalues for growth of different microbial cells

on different sugars 2827.2 Different unstructured kinetic models with one limiting substrate 2847.3 “True” yield and maintenance coefficients for different

microbial species growing at aerobic growth conditions 2907.4 Model parameters in the Sonnleitner and Ka¨ppeli model 2957.5 Model parameters in mmax(T), (7.29) forKlebsiella pneumoniae

and forEscherichia coli 2987.6 Characteristics of microbial growth on truly

substitutable substrates 312Chapter 9

9.1 Advantages and disadvantages of different reactor types

and of different operating modes of the reactors 384Chapter 10

10.1 Henry’s constant for some gases in water at 25C 463

10.2 Parameter values for power law correlation of specific

interfacial areaa 47210.3 Data for a sparged, mechanically mixed pilot plant bioreactor 473

10.4 Parameter values for the empirical correlation 47510.5 Data for a standard laboratory bioreactor

with two Rushton turbines 47610.6 Some important dimensionless groups for mass

transfer correlations 47910.7 Literature correlations for the Sherwood number,Sh 48010.8 Solubility of oxygen in pure water at anoxygen

pressure pO¼ 1 atm 48110.9 Solubility of oxygen at 25C and p

O¼ 1 atm in variousaqueous solutions 48310.10 Molecular diffusivityDAof solutes in dilute

pilot plant bioreactor 53411.5 Scale-up by a factor 125 from pilot plant reactor

to industrial reactor 534

List of Notes

Chapter 3

3.1 Time-dependent output with constant values of input variables 663.2 How to treat ions in the black box model 773.3 BOD as a unit of redox power 933.4 Variance–covariance matrix of the rate estimates 1033.5 Calculation of the variance–covariance matrix from the errors

in the primary variables 106Chapter 4

4.1 On the proper use of thermodynamic data from tables 1394.2 50 years of controversy about the chemiosmotic hypothesis

may now be resolved 143Chapter 5

5.1 Comparison of the method based on the net fluxesV,

and the method based on the total set

of internal fluxesv 1695.2 Calculation of the total ATP consumption for maintenance 1755.3 Biomass equation in metabolic network models 1865.4 Sensitivity analysis of the stoichiometric matrices 1895.5 Linear dependency in reaction stoichiometries 1915.6 Measurement of13C-enrichment 202

Chapter 7

7.1 Model complexity 2747.2 The genesis of the Monod Model 2877.3 Stable and unstable RNA 3037.4 What should be positioned in the active compartment

of a simple structured model? 3037.5 Derivation of expression for fraction of repressor-free operators 3207.6 Mechanistic parameters in the protein synthesis model 3247.7 Relation betweenToscand the dilution rate in continuous culture 333

Chapter 8

8.1 Determination of the total number of cells from a

substrate balance 3598.2 General form of the population balance 360Chapter 9

9.1 Comparison of the productivity of a fed-batch and a continuous

baker’s yeast process 4189.2 Sampling in the Buziol et al system and extraction of metabolites 439Chapter 10

10.1 Calculation of maximum stable bubble diameter using

the statistical theory of turbulence 46910.2 Derivation of and use of the relationSh = 2 for a sphere

in stagnant medium 480Chapter 11

11.1 Sheer stress as a tensor property 51511.2 In the design of RJH: Can power inputP

be scaled with medium volumeV? 52411.3 Mixing with stationary jets 525

Chapter 1

What Is This Book About?

Looking back to the introductory Chap 1 of the first and second editions ofBioreaction Engineering Principles, we still find that the schematic representation

of topics in Fig.1.1adequately describes what we believe should be the focus of thethird edition

Throughout the text we emphasize that the design of bioprocesses should bebased on modeling of the system A quantitative approach in which fundamentalresults from the biosciences is combined with core disciplines from the engineeringsciences will lead to the best design of the bioprocess

It is our sincere hope that the book will find readers from the combinedcommunity of microbiologists, biochemists, and chemical engineers The com-bined expertise of these scientists constitutes what could be called thebiochemicalengineer In many universities, Departments of Chemical Engineering have beenrenamed as Departments of Chemical and Biochemical Engineering in realization

of the need for engineers and scientists who are able to tackle the challenge oftransforming the astounding discoveries of the biosciences into new industrialprocesses which will make an impact on all levels of our daily lives The formation

of biochemical engineers is by no means an easy task, since the qualities of two verydifferent academic cultures, Bioscience and Chemical Engineering Science, must

be combined without losing the essential qualities of either of the two

Both the student of Chemical Engineering and the student of one of theBiosciences will be trained to recognize new possibilities within their field

of study and to exploit their inventiveness This results in academic papers thatgive new fundamental insights and in patents for new procedures or processes Butthe approach of the chemical engineer is – generally speaking – that of modeling theprocess, and thereafter set into motion a recursive process of experimental studiesand further modeling analysis The bioscientist with a much broader basis in factualknowledge and laboratory techniques is – again generally speaking – more likely topursue deep studies of a particular system, uncovering layer after layer of the Truthabout this specimen of Life

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The authors of the present textbook have taught the subject of bioreactionengineering to many generations of students, both in their home universities and

in courses at foreign universities The classes have invariably been composed ofstudents with either a strong background in Chemical Engineering – Transportphenomena, Unit operations, and Reaction Engineering – or with an extensivetraining in experimental techniques in the Biochemistry laboratory, underpinned

by studies of heavy tomes on Biochemistry and Biology Through comments to thefirst and second edition of our textbook we have learnt to appreciate the difficulties

of many biostudents in tackling model building and even rather simple cal topics We have also learnt that “true” chemical engineering students have little

mathemati-or no appreciation of the hard wmathemati-ork it takes to learn even the fundamentals ofmetabolism and molecular biology

The biostudentsmust learn to apply mathematics and numerical simulation, atleast up to a certain level Otherwise the methodologies used in Metabolic FluxAnalysis, Metabolic Control Analysis, and Data Analysis will remain the preroga-tive of physicists and engineers Similarly, the engineering studentsmust spend time

to become familiar with the core disciplines of the biosciences Otherwise, theirmodeling studies will be faulted by the Bioscience community, and their biorelatedpapers, published in secondary journals, will have little or no real value for society

In this third edition ofBioreaction Engineering Principles, we have tried to buildbridges between the two communities Chapter2gives a short, but in our opinionsufficient introduction to metabolism in microorganisms, and through the followingchapters we build further on this basis Our short primer in cell physiology can in nosense replace the detailed picture of physiology and biology offered by full sizetextbooks on these topics, but the student will be encouraged to improve his status

by consulting these texts

Bioreaction Engineering Principles

Steady state balances

Stoichiometry Energetics Reaction rates

Fig 1.1 Topics of bioreaction engineering (adapted from first edition (1993) of the book)

Matrix algebra is necessary to understand, even on a superficial level, theconcepts discussed in papers on Systems Biology, and we introduce matrix-basedMetabolic Flux Analysis in Chap.5 But the subject is introduced slowly aftersimple networks have been studied by methods that require very little beyond basicalgebra Similarly, at the end of Chap.3, methods that are necessary to evaluate thequality of experimental data are discussed, but only after many examples whereelemental and redox balances are used to analyze steady-state rate data and toderive the stoichiometry of bioreactions When in Chap.6reactions catalyzed byimmobilized enzymes are treated, one must apply some basic concepts fromtransport phenomena Diffusion into the pellets is treated with the help of detailedexamples The objective is to give the reader an understanding of the topic which is

on the par with that given in standard texts on Chemical Reaction Engineering.The book has more than 100 examples and notes The Examples will help thereader to better understand the text while topics which extend the text, sometimes

by pointing to applications in different areas, are treated in Notes The teaching ofthe examples is further extended into the Problem sections of each chapter Some ofthe examples and problems are simple illustrations of the text, while others aredetailed quantitative studies relevant for building up a further understanding of bio-reactions, both for use in scientific studies and for design of equipment The designexamples often refer to the collaboration of the authors with major bio-techcompanies – suitably reformulated if necessary Hopefully these design examplesdemonstrate that Bio-reaction Engineering, by combining core subjects fromChemical Engineering and the Bio-Sciences, contributes to secure bioprocesses aprominent place in the process industry

For the casual reader of the text the Examples and Notes may give quickintroduction to the philosophy of the textbook Therefore lists of examples andnotes are given right after the list of Contents Also included is a list of the Tables

In the tables the student may find many valuable and often used data, just as theexamples will be useful for a reader who wishes to obtain information concerning aparticular research topic

The third edition is in fact more like a complete rewriting of the second editionthan a revision of the edition from 2003 In Chap.2, we have decided to give abroad introduction to all the final products that can be derived from sugar andindirectly also from lignocellulosic biomass The intention is to make the studententhusiastic about the whole area of bioreactions while at the same time introducingthe primary metabolism of microorganisms bit by bit We hope that the review ofthe potential of industrial production based on renewable materials is an unbiasedand fair account of this fascinating area

In the new edition, Chaps.3and4introduce concepts of value for tion studies There is no reason why the environmental engineer should not see hisproblems in the light of a more general discussion of mass balances treated with theaid of elemental and redox balances, and with suitable reference to the thermody-namics of the ecosystems

bioremedia-Chapter 6 has now developed into a general overview of enzyme-catalyzedsystems Both the fundamental aspects of enzyme kinetics and the application of

free or immobilized enzymes in scientific studies and in industrial practice arecovered The last Sect.6.4of the chapter deals with Metabolic Control Analysis,

a concept used in strain improvement by targeted changes in the metabolicpathways The results of this section can be used equally well to optimize ordinaryenzyme processes with several enzymatic reactions

In Chap.7, a central chapter of the book, the reader is guided from the simplestunstructured models for cell kinetics through to the fascinating molecular levelkinetics that is involved in catabolite repression and in transport through the cellmembrane

Chaps.9 11deal with the design of bioreactions The ideal (i.e., well mixed)reactor is the subject of Chap.9, and while some material from the second editionhas been left out the chapter now deals also with new reactor types, and theimportant fed-batch mode of operation is treated in more detail with respect toboth chemical and mass-transfer limited reactions Chapter11now offers a hands-

on introduction to the problems of scale-up which in general are caused by quate mixing of the medium The purpose is not to give a manual on mixingequipment and the empirical foundation for design of such equipment Rathersome general concepts of mixing are highlighted, for example, that the quality ofmixing may be improved by clever design, but the power input to any mixingsystem basically determines the mixing time through application of the samerelations derived from fluid dynamics

inade-A one-semester course based on our text should include Chap.2, Sect.3.1–3.5

and Sect.5.1–5.3.2 This will give the students enough competence to derivestoichiometries from carefully examined experimental data and from simple net-work models Many design problems – especially those in which the economicfeasibility of a proposed process is to be determined – can be treated with thisbackground in biochemistry and bioenergetics

The design of steady-state stirred tank reactors and fed-batch reactors in Chap.9is,

of course essential, and Chaps.10and11give an input from textbooks in chemicaland mechanical engineering that will make the design of real bioreactors trustworthy.Chaps.6 and8might be included in a two-semester course, or they could beused as basis for short courses on enzyme reactions and on population-basedmodels for bioreactions (essentially the basis is the same as that used for a course

in reactive dissolution or precipitation of solids)

Finally Chap.4 is a short introduction to Chemical Thermodynamics Verysimple calculations useful for, e.g., heat-exchanger design are treated, but theinsight that thermodynamics will give in the feasibility of pathway reactions and

in the functioning of essential life processes is, indeed valuable in its own right

In conclusion, it is seen that the contents of the book follow the main route inanalysis of bioreactions that was indicated in Fig.1.1

The authors wish for all those who spend time with our text that someuseful experience is the reward for their efforts, be they students or industrialemployees

1.1 Note on Nomenclature

The nomenclature used in this edition is the same as that used in previous editionsand shown in the “List of Symbols.” Deviations from the listed symbols mayappear, but only if the meaning of a shorter nomenclature is quite clear

As in previous editions, we insist on using the symbolr for the specific rate ofbioreactions (e.g., in units of kg converted (kg biomass h) 1) since the cell is thereactor, and IUPAC defines the rate per kg (or m3) reactor by the letterr In massbalances for bioreactors the volumeV of the reactor vessel is introduced We use theletter q to designate the rate of the reaction per unit of V The nomenclature isintroduced in Sect.3.1.1together with the basic mass balances for the reactor

To designate the specific growth rate of biomass we shall use both the standardsymbol m (in units of, e.g., kg biomass produced (kg biomass h) 1) orrx, a usefulnomenclature when several specific rates are discussed together

Yield coefficients (in units of, e.g (kg/kg)) are designated byYijwhere indexj isthe compound in the numerator andi is the compound in the denominator Thisdefinition is explained in Sect.3.2

Chapter 2

Chemicals from Metabolic Pathways

For the past 80–90 years petroleum and natural gas have served as raw materials forthe majority of the finished products of our daily lives After World War II theseraw materials decisively substituted coal, and they have been the foundation of anenormous increase in material wealth and welfare throughout the World

A few basic raw materials, petroleum, natural gas,þS from oil or natural gas,and O2þ N2from air, generate first primary (or platform) chemicals, next second-ary (commodity) chemicals, then intermediates, and finally the finished products ofvirtually all industries that provide consumer goods

The aromatic fraction of petroleum deliversplatform chemicals, such as ene, ethyl benzene, cyclohexane, and cumene These are used to synthesizesec-ondary chemicals such as styrene, adipic acid, caprolactam, acetone, andterephthalic acid; and these in turn are raw materials for thepolymer industry thatproduces textiles, packaging for food products, appliances, and communicationequipment (pencils, inks, computer casings, optical fiber) The aliphatic fraction

propyl-of petroleum contains other platform chemicals (iso-butylene, butadiene, etc.) thatsupplement the aromatic fraction to produce intermediates for the abovementionedindustries Thetransportation sector directly receives consumer goods from C5to

C14aliphatic compounds, while products such as antifreeze and gasoline additivesare derived by chemical processing of petroleum platform chemicals

Natural gas and cracked naphtha deliver other platform chemicals (ethylene,propylene, CO/H2, NH3) for the solvent industry (methanol, ethanol, ethyleneglycol, etc.), for the polymer industry (formaldehyde, polyethylene, polypropylene,PVC), and forfertilizers

Together the platform chemicals from petroleum and natural gas are combined

to give most of the products of the health and hygiene industry, the housingindustry, and the exploding recreation industry Except for the input from themining and the forest industry, and the inorganic platform chemicals (such ascement and phosphates) it is, indeed, hard to imagine the modern world withoutthe crucial input from oil and gas

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It is, however, known to every observant citizen that we have to prepare for a differentworld, a future in which petroleum can only be used as a raw material for a few platformchemicals (mostly aromatics), and where even natural gas will run scarce The use of thecalorific value of oil and gas for heating and cooling, or for producing electricity willneed to be sharply reduced, if not banned The transportation sector will have to findsolutions where gasoline is substituted by other means of vehicle propulsion.

This is the challenge for all modern societies, and at the same time it is likely

to be the most brilliant opportunity for science in the 21st century

In the following, the special role of the biorefinery in this quest for newsolutions to the raw material challenge will be outlined

2.1 The Biorefinery

By analogy with the role of the oil refinery, where the raw materials, such aspetroleum, natural gas, S, O2and N2, are converted in a series of chemical steps

to consumer goods, the role of the biorefinery is to convert raw materials originating

in the agricultural sector intothe same final consumer goods

Food and feed products are, and will remain the primary products of agriculture.Nevertheless, for many years to come a certain fraction of the primary productsfrom agriculture, such as sugar from beets or cane and starch from grain, potatoesand other storage compounds of plants will be processed into chemicals andtransportation fuels – a striking example is the conversion of almost 40% of theenormous cane sugar production of Brazil into bioethanol The main driver for thisdevelopment has previously been surplus production of primary agriculturalproducts in the Americas and in Europe This has resulted in very costly programswhere farmers were sometimes subsidized to reduce production As long as in someparts of the world the production capacity for primary agricultural products remainsmuch higher than the market can absorb, nonfood utilization, also for biofuels, willhave a considerable and positive socioeconomic benefit

The future lies in additional utilization of the huge quantities of waste productsfrom agriculture and forestry – straw, corn cobs, sugarcane bagasse, and forestindustry residues – supplemented by household and other waste products of modernsociety Typically, one ton of straw is produced per ton of grain

Hence,cellulose, hemicelluloses, and lignin, collectively known as losic biomass, will become an important raw material for the biorefinery

lignocellu-Cellulose is an unbranched, crystalline microfibril constructed from 7,000 to15,000a-Dglucose molecules Embedded in hemicelluloses it acts as reinforcementlike iron rods in concrete to give strength to cell walls Hemicelluloses are compos-ite, branched polysaccharides, polymerized from 500 to 3,000D-C5sugar units to

an amorphous structure of xylan, arabinoxylan, glucomannan, and others In trast to cellulose, hemicelluloses are relatively easily hydrolyzed by acid treatment

con-or by enzymes to fcon-orm C5monomers, with the C5sugarD-xylose being the mostabundant pentose derived from many materials

Lignin is an amorphous hydrophobic polymer into which the cellulose microfibrilsare embedded It adds chemical resistance to the network of hemicelluloses andcellulose, but has a number of other functions such as regulating the flow of liquid

in the living plant The building blocks of the very complex lignin polymerchange from one lignocellulosic biomass to another, but generally they are of aromaticcharacter, dominated by derivatives of phenyl propane Figure 2.1a shows threecommercially valuable products that can be obtained by hydrolysis of lignin