Modern biotechnology n mosier, m ladisch (wiley sons, 2009)

MODERN BIOTECHNOLOGY
Connecting Innovations in Microbiology
and Biochemistry to Engineering
Fundamentals

Nathan S. Mosier
Michael R. Ladisch

A JOHN WILEY & SONS, INC., PUBLICATION



MODERN BIOTECHNOLOGY



MODERN BIOTECHNOLOGY
Connecting Innovations in Microbiology
and Biochemistry to Engineering
Fundamentals

Nathan S. Mosier
Michael R. Ladisch

A JOHN WILEY & SONS, INC., PUBLICATION


Copyright © 2009 by John Wiley & Sons, Inc. All rights reserved
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Library of Congress Cataloging-in-Publication Data:
Mosier, Nathan S., 1974Modern biotechnology : connecting innovations in microbiology and biochemistry to engineering
fundamentals / Nathan S. Mosier, Michael R. Ladisch.
p. cm.
Includes index.
ISBN 978-0-470-11485-8 (cloth)
1. Biotechnology. I. Ladisch, Michael R., 1950- II. Title.
TP248.2.M675 2009
660.6–dc22

2009001779
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1


CONTENTS

Preface
Acknowledgments
List of Illustrations
1

xv
xvii
xix

Biotechnology

1

Introduction
The Directed Manipulation of Genes Distinguishes the New
Biotechnology from Prior Biotechnology
Growth of the New Biotechnology Industry Depends on
Venture Capital
Submerged Fermentations Are the Industry’s Bioprocessing
Cornerstone
Oil Prices Affect Parts of the Fermentation Industry

1

Growth of the Antibiotic/Pharmaceutical Industry
The Existence of Antibiotics Was Recognized in 1877
Penicillin Was the First Antibiotic Suitable for Human
Systemic Use
Genesis of the Antibiotic Industry
Other Antibiotics Were Quickly Discovered after the
Introduction of Penicillin
Discovery and Scaleup Are Synergistic in the Development of
Pharmaceutical Products
Success of the Pharmaceutical Industry in Research, Development,
and Engineering Contributed to Rapid Growth but Also
Resulted in Challenges
Growth of the Amino Acid/Acidulant Fermentation Industry
Production of Monosodium Glutamate (MSG) via Fermentation
The Impact of Glutamic Acid Bacteria on Monosodium
Glutamate Cost Was Dramatic
Auxotrophic and Regulatory Mutants Enabled Production of
Other Amino Acids
Prices and Volumes Are Inversely Related
Biochemical Engineers Have a Key Function in All Aspects of
the Development Process for Microbial Fermentation

2
3
10
10
11
11
12

12
13
15

15
16
17
17
17
19
21

v


vi

2

CONTENTS

References

22

Homework Problems

24

New Biotechnology

27

Introduction

27

Growth of the Biopharmaceutical Industry
The Biopharmaceutical Industry Is in the Early Part of Its
Life Cycle
Discovery of Type II Restriction Endonucleases Opened a New
Era in Biotechnology
The Polymerase Chain Reaction (PCR) Is an Enzyme-Mediated,
In Vitro Amplification of DNA

28

Impacts of the New Biotechnology on Biopharmaceuticals, Genomics,
Plant Biotechnology, and Bioproducts
Biotechnology Developments Have Accelerated Biological
Research
Drug Discovery Has Benefited from Biotechnology Research
Tools
The Fusing of Mouse Spleen Cells with T Cells Facilitated
Production of Antibodies
Regulatory Issues Add to the Time Required to Bring a New
Product to Market
New Biotechnology Methods Enable Rapid Identification of
Genes and Their Protein Products
Genomics Is the Scientific Discipline of Mapping, Sequencing, and

Analyzing Genomes
Products from the New Plant Biotechnology Are Changing the
Structure of Large Companies that Sell Agricultural Chemicals
Bioproducts from Genetically Engineered Microorganisms
Will Become Economically Important to the
Fermentation Industry

3

31
33
33
34
35
36
36
36
39
39
42

43

References

45

Homework Problems

47

Bioproducts and Biofuels

49

Introduction

49

Biocatalysis and the Growth of Industrial Enzymes
Glucose Isomerase Catalyzed the Birth of a New Process for
Sugar Production from Corn
Identification of a Thermally Stable Glucose Isomerase and an
Inexpensive Inducer Was Needed for an Industrial Process
The Demand for High-Fructose Corn Syrup (HFCS) Resulted
in Large-Scale Use of Immobilized Enzymes and Liquid
Chromatography

49
51
53

53


CONTENTS

Rapid Growth of HFCS Market Share Was Enabled by LargeScale Liquid Chromatography and Propelled by Record-High
Sugar Prices
Biocatalysts Are Used in Fine-Chemical Manufacture

Growth of Renewable Resources as a Source of Specialty Products
and Industrial Chemicals
A Wide Range of Technologies Are Needed to Reduce Costs
for Converting Cellulosic Substrates to Value-Added
Bioproducts and Biofuels
Renewable Resources Are a Source of Natural Plant Chemicals
Bioseparations Are Important to the Extraction, Recovery, and
Purification of Plant-Derived Products

4

vii

55
56
58

59
63
64

Bioprocess Engineering and Economics

65

Bioseparations and Bioprocess Engineering

66

References

67

Homework Problems

71

Microbial Fermentations

73

Introduction

73

Fermentation Methods
Fermentations Are Carried Out in Flasks, Glass Vessels,
and Specially Designed Stainless-Steel Tanks

75

Microbial Culture Composition and Classification
Microbial Cells: Prokaryotes versus Eukaryotes
Classification of Microorganisms Are Based on Kingdoms
Prokaryotes Are Important Industrial Microorganisms
Eukaryotes Are Used Industrially to Produce Ethanol,
Antibiotics, and Biotherapeutic Proteins
Wild-Type Organisms and Growth Requirements in
Microbial Culture
Wild-Type Organisms Find Broad Industrial Use

Microbial Culture Requires that Energy and All Components
Needed for Cell Growth Be Provided

78
78
81
81

Media Components and Their Functions (Complex and
Defined Media)
Carbon Sources Provide Energy, and Sometimes
Provide Oxygen
Complex Media Have a Known Basic Composition but a
Chemical Composition that Is Not Completely Defined
Industrial Fermentation Broths May Have a High Initial Carbon
(Sugar) Content (Ethanol Fermentation Example)
The Accumulation of Fermentation Products Is Proportional to
Cell Mass in the Bioreactor

75

82
83
83
86
86
86
89
91
92

viii

CONTENTS

A Microbial Fermentation Is Characterized by Distinct Phases
of Growth
Expressions for Cell Growth Rate Are Based on Doubling
Time
Products of Microbial Culture Are Classified According to
Their Energy Metabolism (Types I, II, and III Fermentations)
Product Yields Are Calculated from the Stoichiometry of
Biological Reactions (Yield Coefficients)
The Embden–Meyerhof Glycolysis and Citric Acid Cycles Are
Regulated by the Relative Balance of ATP, ADP, and AMP
in the Cell

5

6

93
94
96
102

104

References

105

Homework Problems

108

Modeling and Simulation

111

Introduction

111

The Runge–Kutta Method
Simpson’s Rule
Fourth-Order Runge–Kutta Method
Ordinary Differential Equations (ODEs)
Runge–Kutta Technique Requires that Higher-Order Equations
Be Reduced to First-Order ODEs to Obtain Their Solution
Systems of First-Order ODEs Are Represented in Vector Form

112
112
113
115

Kinetics of Cell Growth
Ks Represents Substrate Concentration at Which the Specific

Growth Rate Is Half Its Maximum

117

Simulation of a Batch Ethanol Fermentation
Ethanol Case Study

122
123

Luedeking–Piret Model

127

Continuous Stirred-Tank Bioreactor

128

Batch Fermentor versus Chemostat

132

References

133

Homework Problems

135

Aerobic Bioreactors

141

Introduction

141

Fermentation Process
Fermentation of Xylose to 2,3-Butanediol by Klebsiella
oxytoca Is Aerated but Oxygen-Limited
Oxygen Transfer from Air Bubble to Liquid Is Controlled by
Liquid-Side Mass Transfer

144

115
116

120

144
153


CONTENTS

7

8

ix

Chapter 6 Appendix: Excel Program for Integration of
Simultaneous Differential Equations

159

References

161

Homework Problems

162

Enzymes

165

Introduction

165

Enzymes and Systems Biology

165

Industrial Enzymes

166

Enzymes: In Vivo and In Vitro

167

Fundamental Properties of Enzymes

169

Classification of Enzymes

170

Sales and Applications of Immobilized Enzymes

172

Assaying Enzymatic Activity
Enzyme Assays

173
181

Batch Reactions

187

Thermal Enzyme Deactivation

187

References

192

Homework Problems

195

Enzyme Kinetics

199

Introduction

199

Initial Rate versus Integrated Rate Equations
Obtaining Constants from Initial Rate Data Is an Iterative
Process

200

Batch Enzyme Reactions: Irreversible Product Formation
(No Inhibition)
Rapid Equilibrium Approach Enables Rapid Formulation of
an Enzyme Kinetic Equation
The Pseudo-Steady-State Method Requires More Effort to Obtain
the Hart Equation but Is Necessary for Reversible Reactions

204
207
207
209

Irreversible Product Formation in the Presence of Inhibitors
and Activators

210

Inhibition
Competitive Inhibition
Uncompetitive Inhibition
(Classical) Noncompetitive Inhibition
Substrate Inhibition

212
213
214
216
217


x

9

CONTENTS

Example of Reversible Reactions
Coenzymes and Cofactors Interact in a Reversible Manner

220
223

King–Altman Method

225

Immobilized Enzyme
Online Databases of Enzyme Kinetic Constants

234
236

References

237

Homework Problems

238

Metabolism

243

Introduction

243

Aerobic and Anaerobic Metabolism
Glycolysis Is the Oxidation of Glucose in the Absence of Oxygen
Oxidation Is Catalyzed by Oxidases in the Presence of O2,
and by Dehydrogenases in the Absence of O2
A Membrane Bioreactor Couples Reduction and Oxidation
Reactions (R-Mandelic Acid Example)
Three Stages of Catabolism Generate Energy, Intermediate
Molecules, and Waste Products
The Glycolysis Pathway Utilizes Glucose in Both Presence
(Aerobic) and Absence (Anaerobic) of O2 to Produce Pyruvate
Glycolysis Is Initiated by Transfer of a High-Energy Phosphate
Group to Glucose
Products of Anaerobic Metabolism Are Secreted or Processed
by Cells to Allow Continuous Metabolism of Glucose by
Glycolysis
Other Metabolic Pathways Utilize Glucose Under Anaerobic
Conditions (Pentose Phosphate, Entner–Doudoroff, and
Hexose Monophosphate Shunt Pathways)
Knowledge of Anaerobic Metabolism Enables Calculation of
Theoretical Yields of Products Derived from Glucose
Economics Favor the Glycolytic Pathway for Obtaining
Oxygenated Chemicals from Renewable Resources

245
245

Citric Acid Cycle and Aerobic Metabolism
Respiration Is the Aerobic Oxidation of Glucose and Other

Carbon-Based Food Sources (Citric Acid Cycle)
The Availability of Oxygen, under Aerobic Conditions,
Enables Microorganisms to Utilize Pyruvate via the Citric
Acid Cycle
The Citric Acid Cycle Generates Precursors for Biosynthesis of
Amino Acids and Commercially Important Fermentation
Products
Glucose Is Transformed to Commercially Valuable Products via
Fermentation Processes: A Summary
Essential Amino Acids Not Synthesized by Microorganisms
Must Be Provided as Nutrients (Auxotrophs)

246
247
248
249
250

253

255
257
258
259
260

260

264
264

267


CONTENTS

The Utilization of Fats in Animals Occurs by a Non–
Tricarboxylic Acid (TCA) Cycle Mechanism
Some Bacteria and Molds Can Grow on Hydrocarbons or
Methanol in Aerated Fermentations (Single-Cell Protein
Case Study)
Extremophiles: Microorganisms that Do Not Require Glucose,
Utilize H2, and Grow at 80–100 °C and 200 atm Have
Industrial Uses
The Terminology for Microbial Culture Is Inexact: “Fermentation”
Refers to Both Aerobic and Anaerobic Conditions While
“Respiration” Can Denote Anaerobic Metabolism

10

11

xi

267

269

270

271

Metabolism and Biological Energetics

272

References

272

Homework Problems

273

Biological Energetics

277

Introduction

277

Redox Potential and Gibbs Free Energy in Biochemical Reactions

277

Heat: Byproduct of Metabolism

286

References

292

Homework Problems

293

Metabolic Pathways

295

Introduction
Living Organisms Control Metabolic Pathways at Strategic and
Operational Levels
Auxotrophs Are Nutritionally Deficient Microorganisms that
Enhance Product Yields in Controlled Fermentations (Relief
of Feedback Inhibition and Depression)
Both Branched and Unbranched Pathways Cause Feedback
Inhibition and Repression (Purine Nucleotide Example)
The Accumulation of an End Metabolite in a Branched Pathway
Requires a Strategy Different from that for the Accumulation
of an Intermediate Metabolite

295

Amino Acids
The Formulation of Animal Feed Rations with Exogeneous
Amino Acids Is a Major Market for Amino Acids
Microbial Strain Discovery, Mutation, Screening, and
Development Facilitated Introduction of Industrial, Aerated

Fermentations for Amino Acid Production by Corynbacterium
glutamicum
Overproduction of Glutamate by C. glutamicum Depends
on an Increase in Bacterial Membrane Permeability
(Biotin-Deficient Mutant)

305

296

296
299

301

306

308

309


xii

CONTENTS

A Threonine and Methionine Auxotroph of C. glutamicum Avoids
Concerted Feedback Inhibition and Enables Industrial Lysine
Fermentations
Cell (Protoplast) Fusion Is a Method for Breeding Amino Acid

Producers that Incorporate Superior Characteristics of Each
Parent (Lysine Fermentation)
Amino Acid Fermentations Represent Mature Technologies

12

310

312
313

Antibiotics
Secondary Metabolites Formed During Idiophase Are Subject
to Catabolite Repression and Feedback Regulation
(Penicillin and Streptomycin)
The Production of Antibiotics Was Viewed as a Mature
Field Until Antibiotic-Resistant Bacteria Began to Appear
Bacteria Retain Antibiotic Resistance Even When
Use of the Antibiotic Has Ceased for Thousands
of Generations
Antibiotic Resistance Involves Many Genes
(Vancomycin Example)

314

References

320

Homework Problems

323

Genetic Engineering: DNA, RNA, and Genes

331

Introduction

331

DNA and RNA
DNA Is a Double-Stranded Polymer of the Nucleotides:
Thymine, Adenine, Cytosine, and Guanine
The Information Contained in DNA Is Huge
Genes Are Nucleotide Sequences that Contain the Information
Required for the Cell to Make Proteins
Transcription Is a Process Whereby Specific Regions of the
DNA (Genes) Serve as a Template to Synthesize Another
Nucleotide, Ribonucleic Acid (RNA)
Chromosomal DNA in a Prokaryote (Bacterium) Is
Anchored to the Cell’s Membrane While Plasmids Are in
the Cytoplasm
Chromosomal DNA in a Eukaryote (Yeast, Animal or Plant
Cells) Is Contained in the Nucleus
Microorganisms Carry Genes in Plasmids Consisting of Shorter
Lengths of Circular, Extrachromosomal DNA
Restriction Enzymes Enable Directed In Vitro Cleavage of
DNA
Different Type II Restriction Enzymes Give Different Patterns

of Cleavage and Different Single-Stranded Terminal
Sequences
DNA Ligase Covalently Joins the Ends of DNA Fragments

332

314
317

318
318

332
332
333

333

333
334
334
337

339
341


CONTENTS

DNA Fragments and Genes of ≤150 Nucleotides Can Be

Chemically Synthesized if the Nucleotide Sequence Has Been
Predetermined
Protein Sequences Can Be Deduced and Genes Synthesized
on the Basis of Complementary DNA Obtained from
Messenger RNA
Genes and Proteins
Selectable Markers Are Genes that Facilitate Identification of
Transformed Cells that Contain Recombinant DNA
A Second Protein Fused to the Protein Product Is Needed to
Protect the Product from Proteolysis (β-Gal-Somatostatin
Fusion Protein Example)
Recovery of Protein Product from Fusion Protein Requires
Correct Selection of Amino Acid that Links the Two Proteins
(Met Linker)
Chemical Modification and Enzyme Hydrolysis Recover an
Active Molecule Containing Met Residues from a Fusion
Protein (β-Endorphin Example)
Metabolic Engineering Differs from Genetic Engineering by
the Nature of the End Product

13

xiii

342

343
344
344

346

347

347
348

References

349

Homework Problems

350

Metabolic Engineering

355

Introduction

355

Building Blocks

359

l-Threonine-Overproducing Strains of E. coli K-12
Genetically Altered Brevibacterium lactoferrin Has Yielded
Improved Amino Acid–Producing Strains

Metabolic Engineering May Catalyze Development of New
Processes for Manufacture of Oxygenated Chemicals
Gene Chips Enable Examination of Glycolytic and Citric
Acid Cycle Pathways in Yeast at a Genomic Level
(Yeast Genome Microarray Case Study)
The Fermentation of Pentoses to Ethanol Is a Goal of
Metabolic Engineering (Recombinant Bacteria and
Yeast Examples)
Metabolic Engineering for a 1,3-Propanediol-Producing
Organism to Obtain Monomer for Polyester Manufacture
Redirection of Cellular Metabolism to Overproduce an
Enzyme Catalyst Results in an Industrial Process for
Acrylamide Production (Yamada–Nitto Process)

359

References

377

Homework Problems

379

360
362

362

364

370

373


xiv
14

CONTENTS

Genomes and Genomics

385

Introduction
Human Genome Project
Deriving Commercial Potential from Information Contained
in Genomes
The Genome for E. coli Consists of 4288 Genes that Code
for Proteins
DNA Sequencing Is Based on Electrophoretic Separations
of Defined DNA Fragments
Sequence-Tagged Sites (STSs) Determined from
Complementary DNA (cDNA) Give Locations of Genes
Single-Nucleotide Polymorphisms (SNPs) Are Stable Mutations
Distributed throughout the Genome that Locate Genes More
Efficiently than Do STSs
Gene Chip Probe Array

385

385

Polymerase Chain Reaction (PCR)
The Polymerase Chain Reaction Enables DNA to Be
Copied In Vitro
Thermally Tolerant DNA Polymerase from Thermus
aquaticus Facilitates Automation of PCR
Only the 5'-Terminal Primer Sequence Is Needed to Amplify
the DNA by PCR
The Sensitivity of PCR Can Be a Source of Significant
Experimental Error
Applications of PCR Range from Obtaining Fragments of
Human DNA for Sequencing to Detecting Genes Associated
with Diseases

401

388
390
391
394

394
398

402
403
404
405

405

Conclusions

406

References

407

Homework Problems

409

Index

411


PREFACE

Biotechnology has enabled the development of lifesaving biopharmaceuticals, deciphering of the human genome, and production of bioproducts using environmentally friendly methods based on microbial fermentations. The science on which
modern biotechnology is based began to emerge in the late 1970s, when recombinant microorganisms began to be used for making high-value proteins and peptides
for biopharmaceutical applications. This effort evolved into the production of some
key lifesaving proteins and the development of monoclonal antibodies that subsequently have provedn to be effective molecules in the fight against cancer. In the
late 1980s and early 1990s biotechnology found further application in sequencing
of the human genome, and with it, sequencing of genomes of many organisms
important for agriculture, industrial manufacture, and medicine.
The human genome was sequenced by 2003. At about the same time the
realization developed that our dependence on petroleum and other fossil fuels was

beginning to have economic consequences that would affect every sector of our
economy as well as the global climate. Modern biotechnology began to be applied
in developing advanced enzymes for converting cellulosic materials to fermentable
sugars. The process engineering to improve grain-to-ethanol plants and the rapid
buildout of an expanded ethanol industry began. This provided the renewable liquid
fuels in small but significant quantities.
Thus biology has become an integral part of the engineering toolbox through
biotechnology that enables the production of biomolecules and bioproducts using
methods that were previously not feasible or at scales previously thought impossible.
We decided to develop this textbook that addresses modern biotechnology in engineering. We started with the many excellent concepts described by our colleagues
by addressing bioprocess engineering and biochemical engineering from a fundamental perspective. We felt that a text was needed to address applications while at
the same time introduce engineering and agriculture students to new concepts in
biotechnology and its application for making useful products. As we developed the
textbook and the course in which this textbook has been used, the integration of
fundamental biology, molecular biology, and some aspects of genetics started to
become more common in many undergraduate curricula. This further expanded the

xv


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PREFACE

utility of an application-based approach for introducing students to biotechnology.
This book presents case studies of applications of modern biotechnology in the
innovation process that has led to more efficient enzymes and better understanding
of microbial metabolism to redirect it to maximize production of useful products.
Scaling up biotechnology so that large quantities of fermentation products could be
produced in an economic manner is the bridge between the laboratory and broader

society use.
Our textbook takes the approach of giving examples or case studies of how
biotechnology is applied on a large scale, followed by discussion of fundamentals
in biology, biochemistry, and enzyme or microbial reaction engineering. Innovations
in these areas have occurred at an astounding rate since the mid-1990s. The current
text attempts to connect the innovations that have occurred in molecular biology,
microbiology, and biochemistry to the engineering fundamentals that are employed
to scale up the production of bioproducts and biofuels using microorganisms and
biochemical catalysts with enhanced properties.
The approach that we take treats microorganisms as living biocatalysts, and
examines how the principles that affect the activity of microorganisms and enzymes
are used in determining the appropriate scaleup correlations and for analyzing performance of living and nonliving biocatalysts on a large scale. Our textbook will
hopefully provide the basis on which new processes might be developed, and sufficient background for students who wish to transition to the field and continue to
grow with the developments of modern biotechnology industry. While we cannot
hope to teach all the fundamentals that are required to cover the broad range of
products that are derived using biotechnology, we do attempt to address the key
factors that relate engineering characteristics to the basic understanding of biotechnology applied on a large scale.
October 7, 2008

Nathan Mosier and Michael Ladisch


ACKNOWLEDGMENTS

We wish to thank our family, colleagues, and Purdue University for giving us the
time to focus on developing an organized approach to teaching the broad set of
topics that define biotechnology. This enabled us to transform our teaching into a
format that others may use to lecture and to gain from our experience. Special
thanks go to Carla Carie, who worked diligently on preparing drafts of manuscripts,
and assisted with the many processes involved in finalizing the manuscript for publication. We thank Dr. Ajoy Velayudhan for his development of the Runge-Kutta

explanation and our many students, especially Amy Penner and Elizabeth Casey,
for inputs and suggestions as well as assisting with making improvements in the
various sections of the book. We also thank Craig Keim and Professor Henry Bungay
(from RPI) for contributions to the Runge-Kutta code. We also thank the Colleges
of Agriculture and Engineering, and specifically Dr. Bernie Engel, head of the Agricultural and Biological Engineering Department, and Professor George Wodicka,
head of the Weldon School of Biomedical Engineering, for granting us the flexibility
to complete this textbook and for providing encouragement and resources to assist
us in this process.
One of the authors (Michael Ladisch) wishes to convey his appreciation to the
heads of the Agricultural and Biological Engineering Department and Weldon School
of Biomedical Engineering at Purdue University for facilitating a partial leave of
absence that is enabling him to work as Chief Technology Officer at Mascoma
Corporation. As CTO, he is a member of the team building the first cellulose ethanol
plant. It is here that some of the lessons learned during the teaching of this material
are being put into practice.
Most of all, we would like to thank the students in our mezzanine-level course
ABE 580 (Process Engineering of Renewable Resources) with whom we developed
the course materials. Their enthusiasm and success makes teaching fun, and keeps
us feeling forever young. We also wish to thank John Houghton from the U.S.
Department of Energy Office of Biological and Environmental Research for his
review of a draft of this textbook and his helpful comments and suggestions.

xvii



LIST OF ILLUSTRATIONS

Figures
1.1.

1.2.
1.3.

2.1.
2.2.
2.3.
2.4.

2.5.
3.1.
3.2.
3.3.

3.4.
3.5.
3.6.
4.1.
4.2.
4.3.

4.4.

Example of Process Improvements of Biotechnology Product and Impact
on Cost
Hierarchy of Values Represented as a Log–Log Plot of Price as a Function of Volume for Biotechnology Products
Log–Log Plot of Concentration as a Function of Selling Price for Small
and Large Molecules; and Products Used in a Range of Applications
from Food to Therapeutic
Conceptual Representation of Biotechnology Industry Life Cycle
Cash Flows for Amgen During Its Early Growth

One Common Way to Genetically Engineer Bacteria Involves the Use of
Small, Independently Replicating Loops of DNA Known as Plasmids
To Produce Monoclonal Antibodies, Antibody-Producing Spleen Cells
from a Mouse that Has Been Immunized Against an Antigen Are Mixed
with Mouse Myeloma Cells
A Mouse Spleen Cell and Tumor Cell Fuse to Form a Hybridoma
Unit Operations of a Biorefinery
Schematic of Pretreatment Disrupting Physical Structure of Biomass
Schematic Diagram of Combined Immobilized Enzyme Reactor and
Simulated Moving-Bed Chromatography for Producing 55% HighFructose Cor Syrup (HFCS)
Trends in Sugar Prices and Consumption
Chart Showing Industrial Chemicals Derived from Starches and Sugars
Chart Showing Products Derived from Renewable Sources of Fats and
Oils
Schematic Diagram of Incubator-Shaker Used for Shake Flask Culture
of Microbial Cells
Picture of a Laboratory Fermentor Showing Major Components
Diagram of an Instrumented Fermentor for Aerated Fermentation of
Products Generated under Sterile Conditions in a Closed, Agitated
Vessel
Schematic Representations of a Eukaryote and a Prokaryote and Woese
Family Tree Showing Relationship between of One-Celled Life and
Higher Organisms

xix


xx
4.5.
4.6.

4.7.
4.8.
4.9.
4.10.

4.11.
4.12.

5.1.
5.2.
5.3.
5.4.
5.5.
5.6.
5.7.
5.8.
5.9.
6.1.
6.2.
6.3.
6.4.
6.5.

6.6.
6.7.
6.8.
6.9.
6.10.

LIST OF ILLUSTRATIONS

Overlap of pH Optima for Hydrolysis and Fermentation Are Needed
for Efficient Simultaneous Saccharification and Fermentation (SSF)
Schematic Illustration of Several Phases of Growth Showing Cell Mass
Concentration
Linearized (SemiLog) Plot of Cell Mass as a Function of Time
Comparison of Linear and Semilog Plots of Cell Mass versus Time from
Fermentation
Schematic Representation of Characteristic Cell Mass, Product, and
Sugar Accumulation for Types I and II Fermentations
Schematic Representations of the Three Stages of Catabolism, Glycolysis, Citric Acid Cycle, and Products from Pyruvate Anaerobic Metabolism of Pyruvate by Different Microorganisms that Do Not Involve the
Citric Acid Cycle
Schematic Representation of Curves for Characteristic Cell Mass,
Product, and Sugar Accumulation
Characteristic Cell Mass, Product, and Sugar Accumulation for Type III
Fermentation Where the Product Is Not Produced Until an Inducer Is
Added
Schematic Diagram of Numerical Integration by Simpson’s Rule
Schematic Representation of Inverse Plot of Monod Equation that May
Be Used to Represent Microbial Growth Data
Concentration of Substrate and Cells as a Function of Time
Schematic Representation of Definition of Ks
Inhibitory Effect of Ethanol on Specific Ethanol Production by Saccharomyces cerevisiae
Process Flow Diagram for Molasses Fermentation System
Graphical Representation of Luedeking-Piret Model
Schematic Representation of a Continuous Stirred-Tank Bioreactor
(CSTB)
Biomass as a Function of Dilution Rate
Change in Xylose and 2,3-Butanediol Concentration as a Function of
Time

Accumulation of Cell Mass and Protein as a Function of Time
Changes in Dissolved Oxygen as % Saturation, CO2, Oxygen Uptake
Rate, and Respiratory Quotient
Schematic Representation of Xylose Metabolism in Klebsiella oxytoca
during Oxygen-Limited Growth
Plot of Simulation of 2,3-Butanediol Fermentation Showing Cell Mass,
Substrate Concentration, and Product Accumulation as a Function of
Time
Schematic Representation of an Air Bubble in a Liquid
Rate of Oxygen Absorption as a Function of Concentration Gradient in
Liquid Phase
Schematic Representation of Measuring Holdup H Based on Differences
in Fluid Level in Tanks with and without Aeration
Oxygen Transfer Coefficient as a Function of Oxygen Diffusion
Correlation of Power Number as a Function of Reynolds Number for
Flat-Blade Turbine in a Baffled Reactor


LIST OF ILLUSTRATIONS

6.11.
6.12.
7.1.
7.2.
7.3.
7.4.
7.5.
7.6.
7.7.
7.8.

7.9.
7.10.
7.11.
7.12.
7.13.
7.14.
7.15.
8.1.
8.2.
8.3.

8.4.
8.5.
8.6.
8.7.
8.8.
8.9.
8.10.
8.11.

xxi

Gassed Power as a Function of Ungassed Power, Turbine Configuration,
and Air (Gas) Volumetric Throughput
Power Number as a Function of Reynolds Number for an Agitated Tank
with Six-Blade Turbine and Four Baffles
Schematic Representations of Immobilized Enzymes
Representation of Three-Point Attachment of a Substrate to a Planar
Active Site of an Enzyme
Bond Specificity of β-Glucosidase

Illustration of Peptide Bond Cleavage Sites for Chymotrypsin and
Trypsin
Relative Velocity (v/Vmax) as a Function of Substrate Concentration for
Different Values of Km
Percentages of % Relative and Residual Enzymatic Activity as a Function of Temperature and Time, Respectively
Schematic Illustration of Anson Assay
Absorption Spectra of NAD+ and NADH for 44 mg/ml Solution for a
1 cm Path Length
Coupled Assay for Hexokinase Activity and Assay of an NADH- or
NADPH-Dependent Dehydrogenase
Calibration Curve for Enzymatic Analysis
Schematic Diagram of Principal Components of the Original Beckman
Glucose Analyzer
MutaRotation TimeCourse for Glucose
Oxidative Stability of Subtilisins, with Comparison of Wild Type to
Leu-222 Variant
The Polypeptide Chain of Lysozyme From Bacteriophage T4 Folds into
Two Domains
First-Order Deactivation Curve for Cellobiase from Trichoderma
viride
Examples of Lineweaver–Burke Plots for Competitive Inhibition
Timecourse of Cellobiose Hydrolysis by Endoglucanase
Double-Reciprocal Lineweaver–Burke Plot with Range of Substrate
Concentrations Chosen to Be Optimal for Determination of Km and Vmax;
Double-Reciprocal Plot Where the Range of Substrate Concentration S
Is Higher than Optimal and Reaction Velocity V Is Relatively Insensitive
to Changes in S
Illustration of Hofstee or Eadie Plot of Rectangular Hyperbola and
Hanes Plot of Rectangular Hyperbola
A Schematic Illustration of Pseudo-Steady State Assumption

Schematic Diagram of Competitive Inhibition Where I3 > I2 > I1
Schematic Representation of Replot of Slope as a Function of Inhibitor
Concentration
Schematic Representation of Uncompetitive Inhibition for I3 > I2 > I1
Schematic Diagram of Replot of Inhibitor Effect
Schematic Diagram Showing Pattern for Noncompetitive Inhibition
Where Inhibitor Concentrations Follow the Order I3 > I2 > I
Schematic Diagram of Curve for Substrate Inhibition with Respect to
Slope B


xxii
8.12.
9.1.
9.2.
9.3.
9.4.
9.5.
9.6.
9.7.
9.8.
9.9.
9.10.
9.11.
9.12.
9.13.
9.14.
9.15.
9.16.
9.17.

9.18.
9.19.

9.20.
9.21.
9.22.
9.23.
9.24.
9.25.
9.26.
HP9.4.
10.1.
10.2.
10.3.

LIST OF ILLUSTRATIONS

Schematic Representation of Membrane Reactor
Diagrammatic Representation of Some of the Metabolic Pathways in a
Cell
Structures of Important Energy Transfer Molecules in the Cell
Metabolism Follows Catabolic (Energy-Generating) and Anabolic (Synthesizing) Pathways Connected through Amphobilc Pathways
Oxidases Catalyze the Oxidization of Compounds Using O2; Ethanol
Dehydrogenase Uses NAD+ to Oxidize Ethanol to Acetaldehyde
NADH Acts as a Reducing Agent in the Synthesis of β-Lactam for the
Synthetic Production of Antibiotics
An In Vitro Membrane Bioreactor to Generate Precursors for the Synthetic Production of Antibiotics
Structure of Acetyl-CoA
Simplified Diagram of Three Stages of Catabolism
First Half of Glycolysis Where α-d-Glucose Is Phosphorylated and

Broken Down into a Three-Carbon Molecule
Second Half of Glycolysis
The Product of Glycolysis (Pyruvate) Is Further Processed to Ethanol in
Order to Recycle NADH to NAD+ to Allow Glycolysis to Continue
The Product of Glycolysis (Pyruvate) Is Further Processed to Lactate in
Order to Recycle NADH to NAD+ to Allow Glycolysis to Continue
Overall Stoichiometry of Lactic Acid Fermentation from Glucose
Formic Acid Fermentation Showing Electron Transfer Driven by External Reduction of Formate
Succinic Acid Fermentation
Partial Diagram for Glucose Monophosphate Pathway
Partial Diagram of Entner–Doudoroff Pathway
Metabolic Pathway for the Mixed-Acid Fermentation of
Bifidobacterium
Minimum Economic Values of Ethanol and Ethylene Derived by Fermentation of Glucose to Ethanol Followed by the Catalytic Dehydration
of Ethanol to Ethylene
Simplified Representation of Citric Acid Cycle
Conversion of Phosphoenolpyruvate (PEP) to Oxalacetate
Conversion of Pyruvate to Oxalacetate
Properties, Structures, and Nomenclature for Uncharged Amino Acids
Properties, Structures, and Nomenclature for Charged Amino Acids, and
Uncharged Polar Amino Acids
Glycerol Forms the Backbone for Triglyceride Fats
Pathways for Growth of Microorganisms on Fat and n-Alkanes, and
Oxidation of Fat
Central and Anaplerotic Pathways and Regulation Patterns in Glutamic
Acid Bacteria
Pathway Showing Glycolysis and Products from Anaerobic Metabolism
of Pyruvate by that Do Not Involve the Citric Acid Cycle
Structures Representing ATP, ADP, and AMP; and Partial Representation of ATP Synthase
Equilibrium Reaction between Glyceraldehyde 3-Phosphate and Dihydroxyacetone Phosphate

LIST OF ILLUSTRATIONS

xxiii

The Redox Reaction for NAD+ to NADH
The Redox Reaction for FAD+ to FADH
Cell Mass and Heat Generation by Klebsiella fragilis
Rate of Heat Production and Total Heat Produced as Function of
Oxygen Consumption; and Rate of Heat Production and Total Heat
Produced as Function of CO2 Generation
11.1.
Intermediate Metabolite P of an Unbranched Pathway Is the Product in
Controlled Fermentation
11.2.
Supplementation of Metabolite
in Fermentation Crosses Cell Membrane of an Auxotrophic Cell
11.3.
Intermediate Metabolite P of a Branched Pathway Is the Product in
Controlled Fermentation
11.4.
Metabolic Control for the Production of Purine Nucleotides
11.5.
End Metabolite of Pathway 1 Represents the Desired Product P in Controlled Fermentation
11.6.
Branched Metabolic Pathway with Complex Feedback Inhibition
11.7.
Inhibition of Amino Acid Production by Analog Compound
11.8.

Culture Screening for Desired Auxotrophs
11.9.
Isomerization of d-Methionine to l-Methionine by a Two-Step EnzymeCatalyzed Process
11.10.
Overproduction of Glutamate by Limiting the Expression of αKetoglutarate Dehydrogenase
11.11.
Synthesis of Biotin
11.12.
Auxotrophs for Producing Threonine and Methionine
11.13.
Cell Fusion for Developing Lysine-Producing Microorganism
11.14.
Metabolic Pathway for the Production of Penicillin from Amino Acid
Precursors in Penicillium chrysogenum with Feedback Inhibition by
Lysine of Homocitrate Synthetase
11.15.
Benzyl Penicillin Is Synthesized from Two Amino Acids
11.16.
Streptomycin Is Synthesized from Sugars
11.17.
Fermentation Timecourse for Penicillin Production
HP11.9.1. Antibiogram—Graphical Representation Mapping Susceptibility of Different Microorganisms to Antibacterial Drugs
HP11.9.2. Molecular Logic of Vancomycin Resistance
12.1.
Unique Cleavage Sites for pBR322
13.1.
Metabolic Reprogramming Inferred from Global Analysis of Changes in
Gene Expression
13.2.
Metabolic Pathways to 1,2- and 1,3-Propanediol from Dihydroxyacetone (DHAP), a Common Intermediate in Sugar Metabolism

13.3.
Schematic Representation of Separation Sequence for FermentationDerived 1,2-Propanediol
13.4.
Effect of Acrylamide on the Activity of Nitrile Hydratases from Pseudomonas chlororaphis B23 and Brevibacterium R312
14.1.
Genetic Map of Drosophila Chromosome 2L Showing Location of
Alcohol Dehydrogenase with DNA Sequence
14.2.
Graphical Illustration of Gel Electrophoresis of DNA
14.3.
Southern Blotting of DNA Fragments Separated by Gel Electrophoresis
14.4.
Schematic Illustration of Single-Nucleotide Polymorphisms
14.5.
Schematic Representation of Oligonucleotide Array
10.4.
10.5.
10.6.
10.7.