Rajiv Dutta

Fundamentals of Biochemical Engineering

Rajiv Dutta

Fundamentals of
Biochemical Engineering

[iJ ~ Springer
Ane Books India
Author:
Professor Rajiv Dutta
Deputy Director & Head
Amity Institute of Biotechnology
Lucknow, India
E-mail: rajivd@lko.amity.edu

ISBN 978-81-8052-202-4 Ane Books India

ISBN 978-3-540-779fYJ-l Springer Berlin Heidelberg New York

Library of Congress Control Number: 2008920288

Springer is a part of Springer Science + Business Media

springer.com

Ane Books India, 2008

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I n affectionate reverance
this 600t is dedicated
to my
Lord and 9Ylaster
qaneslia
Preface

The biotechnology and chemical engineering students at various

Indian Universities and engineering institutions are required to take
up the 'Biochemical Engineering' course either as an elective or a
compulsory subject. This book is written keeping in mind the need
for a text book on the aforesaid subject for students from both
engineering and biology backgrounds. The main feature of this book
is that it contains s'olved problems, which help the students to
understand the subject better.
The book is divided into three sections: Enzyme mediated
bioprocess, whole cell mediated bioprocess and the engineering
principle in bioprocess
In the first section of this book, the brief introduction about
biochemical engineering is given in chapter 1. The second chapter
deals with basics of enzyme reaction kinetics. The third chapter deals
with an important aspect in enzyme bioprocess Le. immobilization of
enzyme and its kinetics. Chapter 4 is concerned about the industrial
bioprocess involving starch and cellulose.
The second section of this book is related to whole cell mediated
bioprocess. Chapter 5 introduces students with basics of
microbiology and cell culture techniques for both plant and animal
cells. The area emerged as most important because of latest
development in pharmaceutical industry. Chapter 6 deals with cell
kinetics and fermenter design. The kinetics analysis based on
structure and unstructured model is also included. In chapter 7,
genetic engineering aspects were explained, and genetic stability
problems and important engineering aspects of genetically modified
cells were also addressed.
The third section deals with engineering principles of bioprocess.
The chapter 8 deals with sterilization process and its engineering
considerations (up stream process). The ninth chapter includes
agitation and aeration in cellular growth and its impact on designing
the bioreactors. The last chapter deals with brief introduction to
down stream processing.
viii Preface

I wish to thank to Neeta, my wife and Ayuushi and Anukritii, my

daughters for their untiring support in realizing this mission. This
book would not have existed without the untiring effort of Miss
Pragati Sahay, Lecturer, AlB and my Ph.D. students, who did all the
proof reading jobs. I will fail in my duties if I don/t acknowledge
professional studies (M.E. Biotechnology) students of BITS, Pilani
who helped my in preparing internet notes while teaching this
course which later took shape of this book. I wish to extend my
sincere thanks to Mr. Aseem K. Chauhan, Additional President,
R~EF, mother organization of Amity University Uttar Pradesh
(APUP) and Maj Gen KK Ohri, AVSM (Retd), Director General of
AUUP-Lucknow Campus for this constant encouragements for the
mIssion to complete. I am also thankful to all my colleagues at Amity
Institute of Biotechnology (AlB), Lucknow helping me at different
levels for the success of this mission.

Rajiv Dutta
Contents

Preface vii
Chapter 1: Introduction 1.1
1.1 Biotechnology 1
1.2 Biochemical Engineering 2
1.3 BiologicalProcess 5
1.4 Definition of Fermentation 6
1.5 Problems 7
1.6 References 7

Chapter 2: Enzyme Kinetics 8

2.1 Introduction 8
2.1.1 Nomenclature of Enzymes 8
2.1.2 Commercial Applications of Enzymes 9
2.2 Simple Enzyme Kinetics 11
2.2.1 Michaelis-Menten Approach 14
2.2.2 Briggs-Haldane Approach 16
2.2.3 Numerical Solution 19
2.3 Evaluation of Kinetic Parameters 22
2.4 Enzyme Reactor with Simple Kinetics 28
2.4.1 Batch or Steady-State Plug-Flow Reactor 29
2.4.2 Continuous Stirred-Tank Reactor 30
2.5 Inhibition of Enzyme Reactions 31
2.5.1 Competitive Inhibition 32
2.5.2 Noncompetitive Inhibition 33
2.6 Other Influences on Enzyme Activity 34
2.6.1 Effect of pH 35
2.6.2 Effect of Temperature 37
x Contents

2.6.3 Effect of Shear 37

2.7 Experiment: Enzyme Kinetics 38
Objectives: 38
Materials: 38
Calibration Curve for Glucose Assay: 38
Experiment Procedures: 39
Data Analysis: 39
2.8 Nomenclature 39
Subscript 40
2.9 Problems 40
2.10 References 48

Chapter 3: Immobilized Enzyme 50

3.1 Immobilization Techniques 50
3.1.1 Chemical Method 50
3.1.2 Physical Method 52
3.2 Effect of Mass-Transfer Resistance 53
3.2.1 External Mass-Transfer Resistance 54
3.2.2 Internal Mass-Transfer Resistance 56
3.2.3 Effective Diffusivities in Biological Gels 63
3.3 Nomenclature 66
3.4 Problems 67
3.5 References 69

Chapter 4: Industrial Applications of Enzymes 70

4.1 Carbohydrates 70
4.1.1 Monosaccharides 70
4.1.2 Disaccharides 73
4.1.3 Polysaccharides 74
4.2 Starch Conversion 76
4.3.1 Lignocellulosic Materials 78
4.3.2 Cellulose Pretreatment and Hydrolysis 80
4.3.3 Cellulases 81
4.3.4 Kinetics of Enzymatic Hydrolysis of Cellulose 81
 Contents xi

4.4 Experiment S6
4.4.1 Reducing Sugar Analysis 86
Materials: 86
Dns Reagent Preparation: 86
Assay Procedures: 87
4.4.2 Enzyme Assay: Filter Paper Activity 87
Materials: 87
Assay Procedures: 87
4.4.3 Enzymatic Hydrolysis of Cellulose (Materials) 88
Procedures: 88
4.5 Nomenclature 89
Subscript 89
4.6 Problems 89
4.7 References 90

Chapter 5: Cell Cultivations 92

5.1 Microbial Cell Cultivations 92
5.1.1 Microbial Cells 92
5.1.2 Bacteria 95
5.1.3 Fungi 98
5.1.4 Culture Media 99
5.1.5 Example of Penicillin Production 101
5.2 Animal Cell Cultivations 102
5.2.1 Animal Cells 103
5.2.2 Growth Media 105
5.2.3 Monoclonal Antibodies 105
5.3 Plant Cell Cultivations 109
5.3.1 Plant Cells 110
5.3.2 Types of Plant Tissue Culture 112
5.3.3 Culture Media 114
5.3.4 Secondary Metabolite Production 115
5.4 Cell Growth Measurement 116
5.4.1 Measurement of Cell Number 117
5.4.2 Measurement of Cell Mass 118
xii Contents

5.4.3 Indirect Methods 119

5.5 Cell Immobilization 120
5.6 Experiments 122
5.6.1 Yeast Gro wth Curve 122
Procedures: 122
5.6.2 Plant Cell Growth Curve Materials; 123
Procedures: 123
5.6.3 Plant Cell Immobilization Materials: 124
Procedures: 124
5.7 References 124
Suggested Reading 126

Chapter 6: Cell Kinetics and Fermenter Design 127

6.1 Introduction 127
6.2 Definitions 128
6.3 Growth Cycle for Batc!). Cultivation 129
6.3.1 Lag Phase 130
6.3.2 Exponential Growth Phase 131
6.3.3 Factors Affecting the Specific Growth Rate 133
6.3.4 Stationary and Death Phase 135
6.4 Stirred-tank Fermenter 135
6.4. 1 Batch or Plug-Flow Fermenter 137
6.4.2 Ideal Can tin uous Stirred-tank Fermen ter 140
6.4.3 Evaluation of Monad Kinetic Parameters 143
6.4.4 Productivity of CSTF 146
6.~.5 Comparison of Batch and CSTF 148
/

6.5 ,Multiple Fermenters Connected in Series 149

6.5.1 CSTF and PFF in Series 149
6.5.2 Multiple CS TFs in Series 151
6.6 Cell Recycling 154
6.6.1 PFF with Cell Recycling 155
6.6.2 CSTF with Cell Recycling 157
6.7 Alternative Fermenters 159
6.7.1 Column Fermenter 159
6.7.2 Loop Fermenter 161
 Contents xiii

6.8 Structured Model 162

6.8.1 General Structured Model 162
6.8.2 Two-Compartment Model 164
6.9 Nomenclature 167
Subscript 167
6.10 Problems 168
6.11 References 174
Chapter 7: Genetic Engineering 176
7.1 DNA and RNA 176
7.2 Cloning of a Gene 178
7.2.1 Understanding of DNA Sequences 179
7.2.2 The Joining of DNA Molecules 181
7.3 Stability of Recombinant Microorganisms 183
7.3.1 Fermentation Kinetics of the Recombinant
Cultures 184
7.3.2 Continuous Stirred-tank Fermenter (CSTF) 187
7.3.3 Methods of Stabilization 189
7.4 Genetic Engineering of Plant Cells 189
7.4.1 Gene Manipulation 191
7.4.2 Transformation 194
7.5 Nomenclature 194
Subscript 195
7.6 Problems 195
7.7 References 196

Chapter 8: Sterilization 197

8.1 Sterilization Methods 197
8.2 Thermal Death Kinetics 198
8.3 Design Criterion 199
8.4 Batch Sterilization 200
8.5 Continuous Sterilization 203
8.6 Air Sterilization 208
8.7 Nomenclature 216
8.8 Problems 218
8.9 References 220
xiv Contents

Chapter 9: Agitation and Aeration 221

9.1 Introduction 221
9.2 Basic Mass-Transfer Concepts 223
9.2. 1 Molecular Diffusion in Liquids 223
9.2.2 Mass-Transfer Coefficient 225
9.2.3 Mechanism of Mass Transfer 228
9.3 Correlation for Mass-Transfer Coefficient 229
9.4 Measurement of Interfacial Area 233
9.5 Correlations for a and D32 234
9.5.1 Gas Sparging with No Mechanical Agitation 234
9.5.2 Gas Sparging with Mechanical Agitation 234
9.6 Gas Hold-Up 236
9.7 Power Consumption 237
9.8 Determination of Oxygen-Absorption Rate 240
9.8.1 Sodium Sulfite Oxidation Method 242
9.8.2 Dynamic Gassing-out Technique 244
9.8.3 Direct Measurement 244
9.8.4 Dynamic Technique 245
9.9 Correlation for KLa 246
9.9.1 Bubble Column 246
9.9.2 Mechanically Agitated Vessel 246
9.10 Scale-Up 247
9.10.1 Similitude 247
9.10.2 Criteria of Scale-Up 251
9.11 Shear-Sensitive Mixing 253
9.12 Nomenclature 254
SUBSCRIPT 256
9.13 Problems 256
9.14 References 258

Chapter 10: Downstream Processing 261

10.1 Introduction 261
10.2 Solid-Liquid Separation 262
10.2.1 Filtration 262
10.2.2 Centrifugation 265
 Contents xv

10.3 Cell Rupture 266

10.4 Recovery 267
10.4.1 Extraction 268
Single-stage Extraction 269
Multistage Extraction 270
10.4.2 Adsorption 274
Adsorption Isotherm 276
Adsorption Operation 277
10.5 Purification 282
10.5.1 Precipitation 282
10.5.2 Chromatography 282
10.5.3 Electrophoresis 284
10.5.4 Membrane Separation 285
10.6 Nomenclature 288
10.7 Problems 290
10.8 References 291
Suggested Reading 292