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4.7. Summary
4.8. Appendix: Example Regulation of Complex Pathways
Suggestions for Further Reading
Problems

5 MAJOR METABOLIC PATHWAYS

5.1. Bioenergetics
5.2. Glucose Metabolism: Glycolysis and the TCA Cycle
5.3. Respiration
5.4. Control Sites in Aerobic Glucose Metabolism
5.5. Metabolism of Nitrogenous Compounds
5.6. Nitrogen Fixation
5.7. Metabolism of Hydrocarbons
5.8. Biodegradation of Xenobiotics
5.9. Overview of Biosynthesis
5.10. Overview of Anaerobic Metabolism
5.11. Overview of Autotrophic Metabolism
5.12. Summary
Suggestions for Further Reading
Questions

6 HOW CELLS GROW

6.1. Batch Growth

6.1.1. Quantifying Cell Concentration,
6.1.2. Growth Patterns and Kinetics in Batch Culture,
6.1.3. How Environmental Conditions Affect Growth
Kinetics,
6.1.4. Heat Generation by Microbial Growth,
6.2. Quantifying Growth Kinetics
6.2.1. Unstructured Nonsegregated Models,
6.2.2. Models for Transient Behavior,
6.2.3. Cybernetic Models,
6.3. Cell Growth in Continuous Culture
6.3.1. Specific Devices for Continuous Culture,
6.3.2. The Ideal Chemostat,
6.3.3. The Chemostat as a Tool,
6.3.4. Deviations from Ideality,
6.4. Summary
Suggestions for Further Reading
Problems

7 STOICHIOMETRY OF MICROBIAL GROWTH AND

PRODUCT FORMATION

7.1. Coefficients for Atp Consumption and Oxygen

7.2. Stoichiometric Calculations
7.2.1. Elemental Balances,
7.2.2. Degree of Reduction,
7.3. Theoretical Predictions of Yield Coefficients
7.4. Estimation of Elemental Cell Composition
7.5. Stoichiometry by Oxidation-Reduction Half-Reactions
7.6. Thermodynamics of Biological Reactions
7.7. Summary
Suggestions for Further Reading
Problems

8 HOW CELLULAR INFORMATION IS ALTERED

8.1. Evolving Desirable Biochemical Activities Through

Mutation and Selection
8.1.1. How Mutations Occur,
8.1.2. Selecting for Desirable Mutants,
8.2. Natural Mechanisms for Gene Transfer and
Rearrangement
8.2.1. Genetic Recombination,
8.2.2. Transformation,
8.2.3. Transduction,
8.2.4. Episomes and Conjugation,
8.2.5. Transposons: Internal Gene Transfer,
8.3. Genetically Engineering Cells
8.3.1. Basic Elements of Genetic Engineering,
8.3.2. Genetic Engineering of Higher Organisms,
8.3.3. Genome Engineering,
8.4. Genomics
8.4.1. Experimental Techniques,
8.4.2. Computational Techniques,
8.5. Summary
Suggestions for Further Reading
 Problems

Part 2 Engineering Principles for Bioprocesses

9 OPERATING CONSIDERATIONS FOR

BIOREACTORS FOR SUSPENSION AND
IMMOBILIZED CULTURES

9.1. Choosing the Cultivation Method

9.2. Modifying Batch and Continuous Reactors
9.2.1. Chemostat with Recycle,
9.2.2. Multistage Chemostat Systems,
9.2.3. Fed-Batch Operation,
9.2.4. Perfusion Systems,
9.2.5. Membrane Bioreactors,
9.3. Immobilized Cell Systems
9.3.1. Active Immobilization of Cells,
9.3.2. Passive Immobilization: Biological Films,
9.3.3. Diffusional Limitations in Immobilized Cell
Systems,
9.3.4. Bioreactor Considerations in Immobilized Cell
Systems,
9.4. Hybrid Bioreactors: Attached and Suspended Cells
9.5. Solid-State Fermentations
9.6. Summary
Suggestions for Further Reading
Problems

10 SELECTION, SCALE-UP, OPERATION, AND

CONTROL OF BIOREACTORS

10.1. Scale-Up and its Difficulties

10.1.1. Overview of Traditional Reactor Types,
10.1.2. Reactors with Internal Mechanical Agitation,
10.1.3. Bubble Column and Loop Reactor,
10.1.4. Single-Use Bioreactors,
10.1.5. Considerations in Aeration, Agitation, and
Heat Transfer,
10.1.6. Approaches to Scale-Up,
10.1.7. Scale-Down and Microbioreactors,
10.2. Bioreactor Instrumentation and Control
10.2.1. Instrumentation for Measurements of Active
 Fermentation,
10.2.2. Using the Information Obtained,
10.3. Sterilization of Process Fluids
10.3.1. The Kinetics of Death,
10.3.2. Sterilization of Liquids,
10.3.3. Sterilization of Gases,
10.4. Summary
Suggestions for Further Reading
Problems

11 RECOVERY AND PURIFICATION OF PRODUCTS

11.1. Strategies to Recover and Purify Products

11.2. Separation of Insoluble Products
11.2.1. Filtration,
11.2.2. Centrifugation,
11.2.3. Coagulation and Flocculation,
11.3. Cell Disruption
11.3.1. Mechanical Methods,
11.3.2. Nonmechanical Methods,
11.4. Separation of Soluble Products
11.4.1. Liquid–Liquid Extraction,
11.4.2. Aqueous Two-Phase Extraction,
11.4.3. Precipitation,
11.4.4. Dialysis,
11.4.5. Reverse Osmosis,
11.4.6. Ultrafiltration and Microfiltration,
11.4.7. Cross-Flow Ultrafiltration and Microfiltration,
11.4.8. Adsorption,
11.4.9. Chromatography,
11.4.10. Electrophoresis,
11.4.11. Electrodialysis,
11.5. Finishing Steps for Purification
11.5.1. Crystallization,
11.5.2. Drying,
11.6. Integration of Reaction and Separation
11.7. Summary
Suggestions for Further Reading
 Problems

12 BIOPROCESS CONSIDERATIONS IN USING

ANIMAL CELL CULTURES

12.1. Structure and Biochemistry of Animal Cells

12.2. Methods Used for the Cultivation of Animal Cells
12.2.1. Basic Techniques for Animal Cell Culture,
12.2.2. Growth Media,
12.2.3. Growth Dynamics for Animal Cells,
12.3. Bioreactor Considerations for Animal Cell Culture
12.4. Bioreactor Systems for Animal Cell Culture
12.4.1. Nonstirred Reactor Systems,
12.4.2. Systems for Entrapped Cells in Stirred
Reactors,
12.4.3. Suspended Cultures,
12.5. Products of Animal Cell Cultures
12.6. Summary
Suggestions for Further Reading
Problems

13 BIOPROCESS CONSIDERATIONS IN USING PLANT

CELL CULTURES

13.1. Why Plant Cell Cultures?

13.2. Plant Cells in Culture Compared to Microbes
13.3. Bioreactor Considerations
13.3.1. Bioreactors for Suspension Cultures,
13.3.2. Reactors Using Cell Immobilization,
13.3.3. Bioreactors for Organized Tissues,
13.4. Economics of Plant Cell Tissue Cultures
13.5. Summary
Suggestions for Further Reading
Problems

14 UTILIZING GENETICALLY ENGINEERED

ORGANISMS

14.1. How the Product Influences Process Decisions

14.2. Guidelines for Choosing Host–Vector Systems
14.2.1. Escherichia Coli,
14.2.2. Gram-Positive Bacteria,
 14.2.3. Lower Eucaryotic Cells,
14.2.4. Mammalian Cells,
14.2.5. Insect Cell–Baculovirus System,
14.2.6. Transgenic Animals,
14.2.7. Transgenic Plants and Plant Cell Culture,
14.2.8. Cell-Free Protein Synthesis,
14.2.9. Comparison of Strategies,
14.3. Process Constraints: Genetic Instability
14.3.1. Segregational Loss,
14.3.2. Plasmid Structural Instability,
14.3.3. Host Cell Mutations,
14.3.4. Growth-Rate-Dominated Instability,
14.4. Avoiding Process Problems in Plasmid Design
14.5. Predicting Host–Vector Interactions and Genetic
Instability
14.6. Regulatory Constraints on Genetic Processes
14.7. Metabolic Engineering
14.8. Synthetic and Systems Biology
14.9. Protein Engineering
14.10. Summary
Suggestions for Further Reading
Problems

15 MEDICAL APPLICATIONS OF BIOPROCESS

ENGINEERING

15.1. Tissue Engineering

15.1.1. What Is Tissue Engineering?,
15.1.2. Tissue-Engineered Skin Replacements,
15.1.3. Chondrocyte Culture for Cartilage
Replacement,
15.2. Gene Therapy Using Viral Vectors
15.2.1. Models of Viral Infection,
15.2.2. Mass Production of Retrovirus,
15.3. Bioreactors
15.3.1. Stem Cells and Hematopoiesis,
15.3.2. Extracorporeal Artificial Liver,
15.3.3. Body-on-a-Chip Systems,
15.4. Summary
 Suggestions for Further Reading
Problems

16 BIOPROCESSES UTILIZING MIXED CULTURES

16.1. Major Classes of Interactions in Mixed Cultures

16.2. Simple Models Describing Mixed-Culture Interactions
16.3. Mixed Cultures in Nature
16.4. Industrial Utilization of Mixed Cultures
16.5. Biological Waste Treatment
16.5.1. Biological Waste-Treatment Processes,
16.5.2. Advanced Wastewater Treatment Systems,
16.5.3. Conversion of Wastewater to Useful Products,
16.6. Summary
Suggestions for Further Reading
Problems

APPENDIX TRADITIONAL INDUSTRIAL

BIOPROCESSES

A.1. Anaerobic Bioprocesses

A.1.1. Ethanol Production,
A.1.2. Lactic Acid Production,
A.1.3. Acetone–Butanol Production,
A.2. Aerobic Processes
A.2.1. Citric Acid Production,
A.2.2. Production of Baker’s Yeast,
A.2.3. Production of Penicillins,
A.2.4. Production of High-Fructose Corn Syrup,
A.3. Bioprocess Technologies: Biofuel and Bioenergy
Production from Biomass
A.3.1. Production of Liquid Fuels,
A.3.2. Production of Gaseous Fuels from Biomass,
A.3.3. Bioelectricity Generation from Wastes Using
Microbial Fuel Cells,
Suggestions for Further Reading

INDEX
 Preface

This third edition of Bioprocess Engineering: Basic Concepts

updates the prior two editions. Although the principles of
bioprocess engineering stated in the first two editions remains
sound, the field has made considerable advances in improving
the productivity, consistency, and safety of bioprocesses.
Some of the major changes in the book come from biology
itself with the development of tools that have greatly increased
our ability to both understand and manipulate cell biology more
effectively and cheaply. On the bioprocess side, tremendous
advances have been made in production of biologicals with
greater emphasis on processes incorporating animal cells. In
particular, productivity in making many heterologous proteins
has increased by several orders of magnitude over the last 16
years. The commercial use of animal cell culture has increased
significantly over this period of time, and plant cell culture is
starting to see commercial applications. Processes with
microbes based on recombinant DNA and related technology
have matured greatly. For any cell type, consistent authentic
posttranslational processing of proteins remains challenging.
The development of the concepts in systems and synthetic
biology is now having an impact on the thinking of bioprocess
engineers.
In this edition, we either introduce or expand discussion of
the following issues:
• The role of small RNAs as regulators
• Cell-free processes
• Biofuels and energy
• Synthetic biology
• Role of genomics
• Development of single-use technology in bioprocesses
• Stem cell technology and utilization
• Use of techniques of microfabrication and
nanobiotechnology as well as 3D printing
• Animal and plant cell biotechnology
• Development in environmental biotechnology
Teaching a subject as broad as bioprocess engineering in one
semester is a difficult challenge, considering the range of
backgrounds students may have. Although many engineering
students have some formal exposure to specific areas of biology,
they often have not had an opportunity to complete courses
from several areas of biology. The biological concepts necessary
for an engineer are described in Part 1, “The Basics of Biology:
An Engineer’s Perspective.” Although this condensed treatment
cannot replace an in-depth knowledge of biochemistry, genetics,
microbiology, and cell biology, it serves as a useful starting
point for a bioprocess professional. On the process side, an
instructor might wish to emphasize Part 2, “Engineering
Principles for Bioprocesses,” dealing with biopharmaceuticals,
bioenergy, or waste treatment, as they decide which topics
would be effective for their students.
We hope this book imparts the excitement of bioprocess
engineering as well as the basic concepts and knowledge to
become a leader in this field.

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ACKNOWLEDGMENTS
The development of this edition has been assisted by
comments of many of our colleagues across the world.
Although a full list is too long to include here, we would like
to acknowledge some specific contributions:
Jeff Chalmers and Brian Pfleger served as technical editors
for the book and provided many useful suggestions and
corrections. Michael Thurston did yeoman’s work as
development editor. Carolyn Lee-Parsons provided detailed
input to Chapter 13 as well as extremely helpful suggestions on
other chapters. Thomas Mansell and Jason Boock provided
detailed input to Chapter 14. Others making significant
contributions to various sections of the book include Kent
Goklen, Itzcoatl Pla, Raymond Ketchum, Harrison Yoon, Taylor
Stevenson, Jeff Varner, and Susan Roberts. Jason Haugh
provided a number of homework problems to us. We would like
to thank Dr. Serkan Eker of Dokuz Eylul University, Izmir,
Turkey, for his expert services in drawing some of the figures in
the book. We also acknowledge the excellent technical support
provided by Brenda Stevens in the preparation of the
manuscript for this book. In addition, many former students
and colleagues have provided useful comments and suggestions.
We thank them, as well as many of our other colleagues, for
their comments and advice.
 About the Authors

Dr. Michael L. Shuler is the Eckert Professor of Chemical

and Biomolecular Engineering and of Biomedical Engineering
at Cornell University. His research has focused on mathematical
models of cell, heterologous protein expression systems,
bioremediation, plant cell tissue culture processes, and “Body-
on-a-Chip” technology for drug development. Many of those
technologies are being used commercially.
Dr. Fikret Kargi is a retired professor of Environmental
Engineering from Dokuz Eylul University in Izmir, Turkey. He
was the former Chair of Environmental Sciences. His recent
research interests include hydrogen gas production from wastes
by fermentation and electrohydrolysis, development of novel
biofilm, and advanced oxidation processes for wastewater
treatment.
Dr. Matthew P. DeLisa is the William L. Lewis Professor of
Engineering in the Robert Frederick Smith School of Chemical
and Biomolecular Engineering at Cornell University. His
research focuses on understanding and controlling the
molecular mechanisms underlying protein biogenesis—folding
and assembly, membrane translocation, and posttranslational
modifications—in the complex environment of a living cell. His
contributions to science and engineering include the invention
of numerous commercially important technologies for
facilitating the discovery, design, and manufacturing of human
drugs and seminal discoveries in the areas of cellular protein
folding and protein translocation.
Part 1: The Basics of Biology:
An Engineer’s Perspective
 1. What Is a Bioprocess Engineer?

We can now manipulate life at its most basic level—the genetic.

For thousands of years, people have practiced genetic
engineering at the level of selection and breeding. But now it
can be done in a purposeful, predetermined manner with the
molecular-level manipulation of DNA. We now have tools to
probe the mysteries of life in a way unimaginable even a few
decades ago. We are at the doorstep of being able to chemically
synthesize whole genomes and transfer them into a host
bacterium to create semisynthetic life. We now have a whole
host of new techniques to rationally design novel organisms.
With this intellectual revolution emerged new visions and
new hopes: new medicines, semisynthetic organs grown in large
vessels, abundant and nutritious foods, computers based on
biological molecules rather than silicon chips, superorganisms
to degrade pollutants, a wide array of consumer products, and
potentially new sources of energy.
These dreams will remain dreams without hard work.
Engineers will play an essential role in converting these visions
into reality. Biological systems are very complex and beautifully
constructed, but they obey the rules of chemistry and physics
and are susceptible to engineering analysis. Living cells are
predictable, and the processes to use them can be rationally
constructed on commercial scales. Doing this is the job of the
bioprocess engineer.
Probably the reason you are reading this book is your desire
to participate in this intellectual revolution and to make an
important contribution to society. You can do it, but it is not
easy to combine the skills of the engineer with those of the
biologist. This book’s intent is to help you begin to develop
these skills. This book and a one-term course are not enough to
make you a complete bioprocess engineer, but it will help you
form the necessary foundation.
In this chapter, we discuss the origins of bioprocess
engineering, how bioprocess engineering evolved, and the
complementary roles of biologists and engineers. We then
consider the regulatory constraints on bioprocesses and how
they impact the engineer.

1.1. BIOTECHNOLOGY AND

BIOPROCESS ENGINEERING
When new fields emerge from new ideas, old terminology is
usually not adequate to describe these fields. Biotechnology
and what constitutes engineering in this field are best
described with examples rather than single words or short
phrases. However, it is critical to understand the cell’s inner
workings to harness the cellular machinery and engineer the
cell to perform a designated task.
Biotechnology usually implies the use or development of
methods of direct genetic manipulation for a socially desirable
goal. Such goals might be the production of a particular
chemical, but they may also involve the production of better
plants or seeds, or gene therapy, or the use of specially designed
organisms to degrade wastes. The key element for many
professionals is the use of sophisticated techniques outside the
cell for genetic manipulation. Others interpret biotechnology in
a much broader sense and equate it with the technical use of
biology for production of pharmaceuticals, chemicals, food, and
diagnostics; they may include engineering as a subcomponent
of biotechnology.
Many terms are used to describe engineers working with
biotechnology. Bioengineering is a broad title and includes
work on medical, bioprocess, agricultural, and environmental
systems; its practitioners include agricultural, electrical,
mechanical, industrial, environmental, and chemical engineers
as well as others. Biological engineering is similar but
emphasizes applications to plants and animals. Metabolic
engineering is the design of cells with genetically altered
pathways to make small molecules that are often novel for that
cell. This term stresses human design of cells for manufacture of
specialty chemicals. Biochemical engineering has usually meant
the extension of chemical engineering principles to systems
using a biological catalyst to bring about desired chemical
transformations. It is often subdivided into bioreaction
engineering and bioseparations, particularly for production of
biologics, chemicals, and fuels. Biomedical engineering has
been considered to be separate from biochemical engineering,
although the boundary between the two is increasingly vague,
particularly in the areas of cell-surface receptors and animal cell
culture. It focuses on the human body with an emphasis on
application of engineering principles from a variety of
disciplines to design medical devices, synthetic organs, and
novel methods for drug delivery and to develop diagnostics and
instrumentation. Another relevant term is biomolecular
engineering, which has been defined by the National Institutes
of Health as “research at the interface of biology and chemical
engineering and is focused at the molecular level.”
 There is a difference between bioprocess engineering and
biochemical engineering. In addition to chemical engineering,
bioprocess engineering includes the work of mechanical,
electrical, environmental, and industrial engineers to apply the
principles of their disciplines to processes based on using living
cells or subcomponents of such cells. The problems of detailed
equipment design, sensor development, control algorithms, and
manufacturing strategies can utilize principles from these
disciplines. Biochemical engineering is more limited in the
sense that it draws primarily from chemical engineering
principles and broader in the sense that it is not restricted to
well-defined, artificially constructed processes but can be
applied to natural systems.
This book focuses primarily on the application of chemical
engineering principles to systems containing biological
catalysts, but with an emphasis on those systems making use of
biotechnology. The rapidly increasing ability to determine the
complete sequence of genes in an organism offers new
opportunities for bioprocess engineers in the design and
monitoring of bioprocesses. The cell itself is now a designable
component of the overall process.

1.2. DIFFERING APPROACHES TO

RESEARCH FOR BIOLOGISTS AND
ENGINEERS
The fundamental trainings of biologists and engineers are
distinctly different. In the development of knowledge in the
life sciences, unlike chemistry and physics, mathematical
theories and quantitative methods (except statistics) have
played a secondary role. Most progress has been due to
improvements in experimental tools. Results are qualitative,
and descriptive models are formulated and tested.
Consequently, biologists often have incomplete backgrounds
in mathematics but are very strong with respect to laboratory
tools and, more importantly, with respect to the
interpretation of laboratory data from complex systems.
Engineers usually possess a solid background in the physical
and mathematical sciences. Often, a theory leads to
mathematical formulations, and the validity of the theory is
tested by comparing predicted responses to those in
experiments. Quantitative models and approaches, even to
complex systems, are strengths. Biologists are usually adept at
the formation of testable hypotheses, experimental design, and
data interpretation from complex systems. Engineers quite
often are unfamiliar with the experimental techniques and
strategies used by life scientists.
The skills of the engineer and life scientist are
complementary. To convert the promises of molecular biology
into new processes to make new products requires the
integration of these skills. To function at this level, the engineer
needs a solid understanding of biology and its experimental
tools. This book provides sufficient biological background for
you to understand how to apply engineering principles to
biosystems. However, if you are serious about becoming a
bioprocess engineer, you will need to take further courses in
microbiology, biochemistry, and cell biology, as well as more
advanced work in bioengineering. If you already have these
courses, this book can be used for review and to provide a
coherent framework for applications to bioprocessing.

1.3. THE STORY OF PENICILLIN: HOW

BIOLOGISTS AND ENGINEERS WORK
TOGETHER
In September 1928, Alexander Fleming at St. Mary’s Hospital
in London was trying to isolate the bacterium
Staphylococcus aureus, which causes boils. The technique in
use was to grow the bacterium on the surface of a nutrient
solution. One of the dishes had been contaminated
inadvertently with a foreign particle. Normally, such a
contaminated plate would be tossed out. However, Fleming
noticed that no bacteria grew near the invading substance
(see Figure 1.1).
 Figure 1.1. Alexander Fleming’s original plate showing the
growth of the mold Penicillium notatum and its inhibitory
action on bacterial growth. (With permission, from Corbis
Corporation.)

Fleming’s genius was to realize that this observation was

meaningful and not a “failed” experiment. Fleming recognized
that the cell killing must be due to an antibacterial agent. He
recovered the foreign particle and found that it was a common
mold of the Penicillium genus (later identified as Penicillium
notatum). Fleming nurtured the mold to grow and, using the
crude extraction methods then available, managed to obtain a
tiny quantity of secreted material. He then demonstrated that
this material had powerful antimicrobial properties and named
the product penicillin. Fleming carefully preserved the culture,
but the discovery lay essentially dormant for over a decade.
World War II provided the impetus to resurrect the
discovery. Sulfa drugs have a rather restricted range of activity,
and an antibiotic with minimal side effects and broader
applicability was desperately needed. Howard Florey and Ernst
Chain of Oxford decided to build on Fleming’s observations.
Norman Heatley played the key role in producing sufficient
material for Chain and Florey to test the effectiveness of
penicillin. Heatley, trained as a biochemist, performed as a
bioprocess engineer. He developed an assay to monitor the
amount of penicillin made so as to determine the kinetics of the
fermentation, developed a culture technique that could be
implemented easily, and devised a novel back-extraction
process to recover the very delicate product. After months of
immense effort, they produced enough penicillin to treat some
laboratory animals.
Eighteen months after starting on the project, they began to
treat a London bobby for a blood infection. The penicillin
worked wonders initially and brought the patient to the point of
recovery. Most unfortunately, the supply of penicillin was
exhausted, and the man relapsed and died. Nonetheless, Florey
and Chain had demonstrated the great potential for penicillin, if
it could be made in sufficient amount. To make large amounts
of penicillin would require a process, and for such a process
development, engineers, in addition to microbial physiologists
and other life scientists, would be needed.
The war further complicated the situation. Great Britain’s
industrial facilities were already totally devoted to the war.
Florey and his associates approached pharmaceutical firms in
the United States to persuade them to develop the capacity to
produce penicillin, since the United States was not at war at that
time.
 Many companies and government laboratories, assisted by
many universities, took up the challenge. Particularly
prominent were Merck, Pfizer, Squibb, and the USDA Northern
Regional Research Laboratory in Peoria, Illinois.
The first efforts with fermentation were modest. A large
effort went into attempts to chemically synthesize penicillin.
This effort involved hundreds of chemists. Consequently, many
companies were at first reluctant to commit to the fermentation
process beyond the pilot-plant stage. It was thought that the
pilot-plant fermentation system could produce sufficient
penicillin to meet the needs of clinical testing, but large-scale
production would soon be done by chemical synthesis. At that
time, U.S. companies had achieved a great deal of success with
chemical synthesis of other drugs, which gave the companies
significant control over the drug’s production. The chemical
synthesis of penicillin proved to be exceedingly difficult. (It was
accomplished in the 1950s, and the synthesis route is still not
competitive with fermentation.) However, in 1940 fermentation
for the production of a pharmaceutical was an unproved
approach, and most companies were betting on chemical
synthesis to ultimately dominate.
The early clinical successes were so dramatic that in 1943 the
War Production Board appointed A. L. Elder to coordinate the
activities of producers to greatly increase the supply of
penicillin. The fermentation route was chosen. As Elder recalls,
“I was ridiculed by some of my closest scientific friends for
allowing myself to become associated with what obviously was
to be a flop—namely, the commercial production of penicillin by
a fermentation process” (from Elder, 1970). The problems
facing the fermentation process were indeed very formidable.
The problem was typical of most new fermentation
processes: a valuable product made at very low levels. The low
rate of production per unit volume would necessitate very large
and inefficient reactors, and the low concentration (titer) made
product recovery and purification very difficult. In 1939 the
final concentration in a typical penicillin fermentation broth
was one part per million (ca. 0.001 g/l). Furthermore, penicillin
is a fragile and unstable product, which places significant
constraints on the approaches used for recovery and
purification.
Life scientists at the Northern Regional Research Laboratory
made many major contributions to the penicillin program. One
was the development of a corn steep liquor–lactose-based
medium. This medium increased productivity about tenfold. A
worldwide search by the laboratory for better producer strains
of Penicillium led to the isolation of a Penicillium chrysogenum
strain. This strain, isolated from a moldy cantaloupe at a Peoria
fruit market, proved superior to hundreds of other isolates
tested. Its progeny have been used in almost all commercial
penicillin fermentations.
The other hurdle was to decide on a manufacturing process.
One method involved the growth of the mold on the surface of
moist bran. This bran method was discarded because of
difficulties in temperature control, sterilization, and equipment
size. The surface method involved growth of the mold on top of
a quiescent liquid medium. The surface method used a variety
of containers, including milk bottles, and the term “bottle plant”
indicated such a manufacturing technique. The surface method
gave relatively high yields but had a long growing cycle and was
very labor intensive. The first manufacturing plants were bottle
plants because the method worked and could be implemented
quickly.
However, it was clear that the surface method would not
meet the full need for penicillin. If the goal of the War
Production Board was met by bottle plants, it was estimated
that the necessary bottles would fill a row stretching from New
York City to San Francisco. Engineers generally favored a
submerged tank process. The submerged process presented
challenges in terms of both mold physiology and tank design
and operation. Large volumes of absolutely clean, oil- and dirt-
free sterile air were required. What were then very large
agitators were required, and the mechanical seal for the agitator
shaft had to be designed to prevent the entry of organisms. Even
today, problems of oxygen supply and heat removal are
important constraints on antibiotic fermenter design.
Contamination by foreign organisms could degrade the product
as fast as it was formed, consume nutrients before they were
converted to penicillin, or produce toxins.
In addition to these challenges in reactor design, there were
similar hurdles in product recovery and purification. The very
fragile nature of penicillin required the development of special
techniques. A combination of pH shifts and rapid liquid–liquid
extraction proved useful.
Soon, processes using tanks of about 10,000 gal were built.
Pfizer completed in less than 6 months the first plant for
commercial production of penicillin by submerged fermentation
(Hobby, 1985). The plant had 14 tanks, each of 7000-gal
capacity. Large-scale fermenters (typically 50,000 to 100,000 l)
now used in antibiotic fermentations are shown in Figures 1.2
and 1.3.
 Figure 1.2. Series of large-scale antibiotic fermenters.
(Courtesy of Pfizer, Inc.)

Figure 1.3. Inside view of a large antibiotic fermenter.

(From Trends in Biotechnology 3(6), 1985. Used with
permission of Elsevier Science Publishers.)

By a combination of good luck and hard work, the United

States had the capacity by the end of World War II to produce
enough penicillin for almost 100,000 patients per year. The
impact of penicillin was immediate, and the advent of
antibiotics saved many lives. Although the first impact was in
war zones, the overall effect was critical globally to fighting
many diseases (see Figure 1.4).
 Figure 1.4. First impact of penicillin in war zones. (WW II
Museum, New Orleans, LA.)

This accomplishment required a high level of

multidisciplinary work. For example, Merck realized that men
who understood both engineering and biology were not
available. Merck assigned a chemical engineer and
microbiologist together to each aspect of the problem. They
planned, executed, and analyzed the experimental program
jointly, “almost as if they were one man” (Silcox in Elder, 1970).
Progress with penicillin fermentation has continued, as has
the need for the interaction of biologists and engineers. From
1939 to now, the yield of penicillin has gone from 0.001 g/l to
over 50 g/l of fermentation broth. Progress has involved better
understanding of mold physiology, metabolic pathways,
penicillin structure, methods of mutation and selection of mold
genetics, process control, and reactor design (see Figure 1.5).
 Figure 1.5. Schematic of penicillin production process.

Before the penicillin process, almost no chemical engineers

sought specialized training in the life sciences. With the advent
of modern antibiotics, the concept of a bioprocess engineer was
born. The penicillin process also established a paradigm for
bioprocess development and biochemical engineering. This
paradigm still guides much of our profession’s thinking. The
mindset of bioprocess engineers was cast with the penicillin
experience. It is for this reason that we have focused on the
penicillin story rather than on an example for production of a
protein from a genetically engineered organism. Although many
parallels can be made between the penicillin process and our
efforts to use recombinant DNA, no similar paradigm has yet
emerged from our experience with genetically engineered cells.
We must continually reexamine the prejudices the field has
inherited from the penicillin experience.
It is you, the student, who will best be able to challenge these
prejudices.

1.4. BIOPROCESSES: REGULATORY

CONSTRAINTS
To understand the mindset of a bioprocess engineer, you
must understand the regulatory climate in which many
bioprocess engineers work. The U.S. FDA (Food and Drug
Administration) and its equivalents in other countries must
insure the safety and efficacy of medicines. For bioprocess
engineers working in the pharmaceutical or biotechnology
industry, the primary concern is not reduction of
manufacturing cost (although that is still a very desirable
goal), but the production of a product of consistently high
quality in amounts to satisfy the medical needs of the
population.
Consider briefly the process by which a drug obtains FDA
approval. A typical drug undergoes 6.5 years of development
from the discovery stage through preclinical testing in animals.
Human clinical trials are conducted in three phases. Phase 1
clinical trials (about 1 year) are used to test safety; typically 20
to 80 volunteers are used. Phase II clinical trials (about 2 years)
use 100 to 300 patients, and the emphasis is on efficacy (i.e.,
whether it helps the patient) as well as further determining
which side effects exist. Compounds that are still promising
enter phase III clinical trials (about 3 years) with 1000 to 3000
patients. Since individuals vary in body chemistry, it is
important to test the range of responses in terms of both side
effects and efficacy by using a representative cross section of the
population. Data from clinical trials is presented to the FDA for
review (about 18 months). If the clinical trials are well designed
and demonstrate statistically significant improvements in
health with acceptable side effects, the drug is likely to be
approved. Even after this point, there is continued monitoring
of the drug for adverse effects. The whole drug discovery-
through-approval process takes 12 to 15 years on the average
and costs up to about $5 billion (in 2013). Only one in ten drugs
that enter human clinical trials receives approval. Recent FDA
reforms have decreased the time to obtain approval for life-
saving drugs in treatment of diseases such as cancer and AIDS,
but the overall process is still lengthy.
This process greatly affects a bioprocess engineer. FDA
approval is for the product and the process together. There are
tragic examples in which a small process change has allowed a
toxic trace compound to form or become incorporated in the
final product, resulting in severe side effects, including death.
For example, heparin contaminated with oversulfated
chondroitin sulfate caused significant problems. Thus, process
changes may require new clinical trials to test the safety of the
resulting product. Since clinical trials are very expensive,
process improvements are made under a limited set of
circumstances. Even during clinical trials it is difficult to make
major process changes.
Drugs sold on the market or used in clinical trials must come
from facilities that are certified as good manufacturing practice
(GMP). GMP concerns the actual manufacturing facility design
and layout, the equipment and procedures, training of
production personnel, control of process inputs (e.g., raw
materials and cultures), and handling of product. The plant
layout and design must prevent contamination of the product
and dictates the flow of material, personnel, and air. Equipment
and procedures must be validated. Procedures include not only
operation of a piece of equipment but also cleaning and
sterilization. Computer software used to monitor and control
the process must be validated. Off-line assays done in
laboratories must satisfy good laboratory practices (GLP).
Procedures are documented by standard operating procedures
(SOP).
The GMP guidelines stress the need for documented
procedures to validate performance: “Process validation is
establishing documented evidence which provides a high degree
of assurance that a specific process will consistently produce a
product meeting its predetermined specifications and quality
characteristics” and “There shall be written procedures for
production and process-control to assure that products have the
identity, strength, quality, and purity they purport or are
†
represented to possess.”
†
Guidelines on General Principles of Process Validation, Federal Register of May
11, 1987 (52 FR 17638).

The actual process of doing validation is often complex,

particularly when a whole facility design is considered. The FDA
provides extensive information and guidelines, which are
updated regularly. Bioprocess engineers involved in
biomanufacturing for pharmaceuticals need to consult these
sources. For example, the recent introduction of disposable
bioreactors requires considerations of how FDA regulations can
be applied. However, certain key concepts do not change. These
concepts are written documentation, consistency of procedures,
consistency of product, and demonstrable measures of product
quality, particularly purity and safety. These tasks are
demanding and require careful attention to detail. Bioprocess
engineers often find that much of their effort is to satisfy these
regulatory requirements.
The key point is that process changes cannot be made
without considering their significant regulatory impact.

SUGGESTIONS FOR FURTHER

READING

Synthetic Biology
GIBSON, D. G., ET AL., Creation of a Bacterial Cell Controlled by a
Chemically Synthesized Genome, Science 329(5987): 52–56,
2010.

History of Penicillin
ELDER, A. L., ed., The History of Penicillin Production, Chem.
Eng. Prog. Symp. Ser. 66 (#100), American Institute of
Chemical Engineers, New York, 1970.
HOBBY, G. L., Penicillin. Meeting the Challenge, Yale University
Press, New Haven, CT, 1985.
LAX, E., The Mold in Dr. Florey’s Coat: The Story of the
Penicillin Miracle, Henry Holt & Company, New York, New
York, 2004.
MOBERG, C. L., Penicillin’s Forgotten Man: Norman Heatley,
Science 253(5021): 734–735, 1991.

Regulatory Issues
AGALLOCO, J. P., AND CARLETON, F. J., Validation of
 Pharmaceutical Processes, 3d ed., CRC Press, Boca Raton,
FL, 2007.
KISHIMOTO, T. K., ET AL., Contaminated Heparin Associated with
Adverse Clinical Events and Activation of the Contact
System, N. Engl. J. Med. 358(23): 2457–2567, 2008.
MULLARD, A., New Drugs Cost US $2.6 Billion to Develop, Nat.
Rev. Drug Discov. 13: 877, 2014.
NAGLAK, T. J., KEITH, M. G., AND OMSTEAD, D. R., Validation of
Fermentation Processes, Biopharm. (July/Aug.): 28–36,
1994.
RAO, G., MOREIRA, A., AND BRONSON, K., Disposable
Bioprocessing: The Future Has Arrived, Biotechnol. Bioeng.
102: 348–356, 2009.

Education
LEE-PARSONS, C. W. T., Biochemical Engineering Taught in the
Context of Drug Discovery to Manufacturing. Chem. Eng.
Edu. 39(Pt 3): 208–221, 2005.

QUESTIONS
1.1. What is GMP, and how does it relate to the regulatory
process for pharmaceuticals?
1.2. When the FDA approves a process, it requires validation
of the process. Explain what validation means in the FDA
context.
1.3. Why does the FDA approve the process and product
together?
1.4. Carefully specify the differences among biotechnologist,
bioprocess engineer, and bioengineer.
1.5. What is achieved by submerged fermentation in
penicillin production versus surface fermentation?
 2. An Overview of Biological Basics

A basic understanding of biology is essential to the biochemical

engineer. Here we present selected aspects of basic biology at a
level that will allow the student to function as a biochemical
engineer. Although specific courses in biochemistry, genetics,
microbiology, and cell biology provide much more depth, most
engineering students do not have an opportunity to take three
or four courses in the biological sciences, and some have no
opportunity to take any of these courses. Thus, we have selected
the parts of microbiology, biochemistry, genetics, and cell
biology most directly relevant to bioprocesses to emphasize
throughout chapters 2 to 8 and present this material with
examples of application to bioprocesses.

2.1. MICROBIAL DIVERSITY

Life is very tenacious and can exist in extreme environments.
Living cells can be found almost anywhere that water is in
the liquid state. The right temperature, pH, and moisture
levels vary from one organism to another.
Organisms from extreme environments (extremophiles)
often provide the human race with important tools for processes
to make useful chemicals and medicinals. They are also key to
the maintenance of natural cycles and can be used in the
recovery of metals from low-grade ores or in the desulfurization
of coal or other fuels. The fact that organisms can develop the
capacity to exist and multiply in almost any environment on
earth is extremely useful.
Some cells can grow at –20°C (in a brine to prevent freezing),
while others can grow at 120°C (where water is under high
enough pressure to prevent boiling). Cells that grow best at low
temperatures (below 20°C) are usually called psychrophiles,
and those with temperature optima in the range of 20° to 50°C
are mesophiles. Organisms that grow best at temperatures
greater than 50°C are thermophiles.
Most organisms grow around neutral pH (6 to 8) and are
called neutrophiles. There are some, however, that have pH
optima far from neutrality, preferring pH values down to 1 or 2
(acidophiles) or well above pH 9 (alkaliphiles). Some organisms
can grow at both low pH values and high temperatures.
Although most organisms can grow only where water activity is
high, others can grow on barely moist solid surfaces or in
solutions with high salt concentrations (halophiles).
 Some cells require oxygen for growth and metabolism. Such
organisms can be termed aerobic. Other organisms are
inhibited by the presence of oxygen and grow only
anaerobically. Some organisms can switch the metabolic
pathways to allow them to grow under either circumstance.
Such organisms are facultative.
Often, organisms can grow in environments with almost no
obvious source of nutrients. Some cyanobacteria (formerly
called blue-green algae) can grow in an environment with only a
little moisture and a few dissolved minerals. These bacteria are
photosynthetic and can convert CO2 from the atmosphere into
the organic compounds necessary for life. They can also convert
N2 into NH3 for use in making the essential building blocks of
life. Such organisms are important in colonizing nutrient-
deficient environments.
Not only do organisms occupy a wide variety of habitats, but
they also come in a wide range of sizes and shapes. Spherical,
cylindrical, ellipsoidal, spiral, and pleomorphic cells exist.
Special names are used to describe the shape of bacteria. A cell
with a spherical or elliptical shape is often called a coccus
(plural, cocci); a cylindrical cell is a rod or bacillus (plural,
bacilli); a spiral-shaped cell is a spirillum (plural, spirilla).
Some cells may change shape in response to changes in their
local environment.
Thus, organisms can be found in the most extreme
environments and have a wondrous array of different shapes,
sizes, and metabolic capabilities. This great diversity provides
the engineer with an immense variety of potential tools, many
of which have been exploited. Take, for example, the heat-
resistant enzyme Taq DNA polymerase, an enzyme which is
derived from a thermophilic bacterium named Thermus
aquaticus. Taq has become one of the most important enzymes
in molecular biology because of its use in a DNA amplification
technique known as the polymerase chain reaction (PCR;
discussed in Chapter 8, “How Cellular Information Is Altered”).

2.1.1. Naming Cells

How cells are named is complicated by the bewildering
variety of organisms present. A systematic approach to
classifying these organisms is an essential aid to their
intelligent use. Taxonomy is the development of approaches
to organize and summarize our knowledge about the variety
of organisms that exist. Although knowledge of taxonomy
may seem remote from the needs of the engineer, it is
necessary for efficient communication among engineers and
scientists working with living cells. Taxonomy can also play a
critical role in patent litigation involving bioprocesses.
While taxonomy is concerned with approaches to
classification, nomenclature refers to the actual naming of
organisms. For microorganisms, we use a dual name (binary
nomenclature). The names are given in Latin, or are Latinized.
A genus is a group of related species, and a species includes
organisms that are substantially alike. A common gut organism
that has been well studied is Escherichia coli. Escherichia is the
genus and coli the species. When writing a report or paper, it is
common practice to give the full name when the organism is
first mentioned but in subsequent discussion to abbreviate the
genus to the first letter. In this case, we would use E. coli.
Although organisms that belong to the same species all share
the same major characteristics, there are subtle and often
technologically important variations within species. An E. coli
used in one laboratory may differ from that used in another.
Thus, various strains and substrains are designated by the
addition of letters and numbers. For example, E. coli B/r A will
differ in growth and physiological properties from E. coli K12.
Now that we know how to name organisms, we could
consider broader classification up to the level of kingdoms.
There is no universal agreement on how to classify
microorganisms at this level. Such classification is rather
arbitrary and need not concern us. However, we must be aware
that there are two primary cell types: eucaryotic and
procaryotic. The primary difference between them is the
presence or absence of a membrane around the cell’s genetic
information.
Procaryotes have a simple structure with a single
chromosome. Procaryotic cells have no nuclear membrane and
no organelles, such as the mitochondria and endoplasmic
reticulum. Eucaryotes have a more complex internal structure,
with more than one chromosome (DNA molecule) in the
nucleus. Eucaryotic cells have a true nuclear membrane and
contain mitochondria, endoplasmic reticulum, Golgi apparatus,
and a variety of specialized organelles. We describe each of
these components in Section 2.1.4. A detailed comparison of
procaryotes and eucaryotes is presented in Table 2.1. Structural
differences between procaryotes and eucaryotes are discussed
later.
 TABLE 2.1. A Comparison of Procaryotes with Eucaryotes

There is further distinction of subgroups within procaryotes.

Evidence suggests that a common or universal ancestor gave
rise to three distinctive branches of life: eucaryotes, eubacteria
(or “true” bacteria), and archaebacteria. Table 2.2 summarizes
some of the distinctive features of these groups. All of the genes
of whole organisms, known as the genome, can now be readily
sequenced and this information is having a great impact on our
understanding of how these families evolved and are related.

TABLE 2.2. Primary Subdivisions of Cellular Organisms

That Are Now Recognized

Viruses cannot be classified under any of these categories, as

they are not free-living organisms. Let’s consider first some of
the characteristics of these rather simple “organisms.”

2.1.2. Viruses
Viruses are very small and are obligate parasites of other
cells, such as bacterial, yeast, plant, and animal cells. Viruses
cannot capture or store free energy and are not functionally
active except when inside their host cells. The sizes of viruses
vary from 30 to 200 nanometers (nm). Viruses contain either
DNA (DNA viruses) or RNA (RNA viruses) as genetic
material. DNA stands for deoxyribonucleic acid, and RNA is
ribonucleic acid; we will soon discuss these molecules in
more detail. In free-living cells, all genetic information is
contained in the DNA, whereas viruses can use either RNA or
Another random document with
no related content on Scribd:
 C’est une des caractéristiques du génie de Henri Poincaré qu’il
réunit un prodigieux esprit d’invention à un esprit critique
extrêmement aiguisé. Sa critique semble même aller parfois
jusqu’au scepticisme; il contemplait sans tristesse les ruines des
théories. Alors que d’autres constatent avec regret que certaines
idées ne s’accommodent plus aux faits, et commencent par penser
que ceux-ci ont été mal vus ou mal interprétés, Poincaré a plutôt une
tendance contraire, bien qu’elle se soit peut-être atténuée dans les
dernières années. Ainsi un jeune physicien ayant cru jadis pouvoir
s’inscrire contre la célèbre expérience de Rowland, d’après laquelle
une charge électrique en mouvement produit un champ magnétique
conformément à la théorie de Maxwell, cette annonce ne parut pas
étonner Poincaré. Nul n’eut moins que lui la notion statique d’une
science se reposant sur quelques conquêtes définitives, et c’est ce
qui explique que plusieurs se soient crus autorisés à tirer de certains
de ses écrits, où il poussait sa tendance critique presque jusqu’au
paradoxe, des conclusions sur la vanité de la Science contre
lesquelles il dut protester.
Quelques Préfaces des Leçons de Poincaré ont vivement attiré
l’attention. Dans l’Introduction du Livre Électricité et Optique, il
discute ce qu’on doit entendre par «interprétation mécanique d’un
phénomène». Cette interprétation est ramenée d’après lui à la
possibilité de la formation d’un système d’équations de Lagrange
avec un certain nombre de paramètres que
l’expérience atteint directement et permet de mesurer. Dans ces
équations figurent l’énergie cinétique et une fonction des forces
. Cette possibilité étant supposée, on pourra toujours déterminer
masses (masses visibles ou cachées) et leurs coordonnées (
) fonctions des (en prenant assez grand), de manière
que la force vive de ce système de masses soit égale à l’énergie
cinétique figurant dans les équations de Lagrange.
L’indétermination est ici très grande, et c’est précisément là qu’en
veut venir Poincaré, dont la conclusion est que, s’il y a une
explication mécanique, il y en a une infinité. Il faut avouer, dirons-
nous, que cette indétermination est même trop grande, car on perd
complètement de vue les corps en présence. Ainsi, suivant les
formes qu’auront l’ensemble des masses partiellement
indéterminées , on n’aura pas nécessairement dans la suite les
mêmes mouvements; il pourra, par exemple, y avoir ou non des
chocs. Que devient aussi la répartition des forces réelles dans les
systèmes en partie fictifs auxquels on est ainsi conduit?
Dans la Préface de sa Thermodynamique, Poincaré, voulant
descendre en quelque sorte jusqu’au fond du principe de la
conservation de l’énergie, conclut que «la loi de Meyer est une forme
assez souple pour qu’on puisse y faire rentrer presque tout ce qu’on
veut». Il semble à la vérité un peu effrayé de sa conclusion, car il
ajoute plus loin qu’il ne faut pas «pousser jusqu’à l’absolu». Nous
retrouverons cet esprit hypercritique, si j’ose le dire, clans certains
écrits philosophiques de Poincaré.
Poincaré, sans cesse curieux de nouvelles théories et de
nouveaux problèmes, ne pouvait manquer d’être attiré par
l’Électromagnétisme qui tient une si grande place dans la Science de
notre époque. On ne saurait trop admirer avec quelle sûreté et
quelle maîtrise il repense les diverses théories, les faisant ainsi
siennes. Il leur donne parfois une forme saisissante, comme quand,
dans l’exposition de la théorie de Lorentz, il distingue entre les
observateurs ayant les sens subtils et les observateurs ayant les
sens grossiers. La considération, bien personnelle à Poincaré, de ce
qu’il appelle «la quantité de mouvement électromagnétique», la
localisation de celle-ci dans l’éther et sa propagation avec une
perturbation électromagnétique sont venues rétablir d’importantes
analogies. Le Mémoire sur la dynamique de l’électron, écrit en 1905,
restera dans l’histoire du principe de la relativité; le groupe des
transformations de Lorentz, qui n’altèrent pas les équations d'un
milieu électromagnétique, y apparaît comme la clef de voûte dans la
discussion des conditions auxquelles doivent satisfaire les forces
dans la nouvelle dynamique. La nécessité de l’introduction dans
l’électron de forces supplémentaires, en dehors des forces de liaison
est établie, ces forces supplémentaires pouvant être assimilées à
une pression qui régnerait à l’extérieur de l’électron. Poincaré
montre encore quelles hypothèses on peut faire sur la gravitation
pour que le champ grafivique soit affecté par une transformation de
Lorentz de la même manière que le champ électromagnétique.
 On sait l’importance qu’a prise aujourd’hui le principe de la
relativité, dont le point de départ est l’impossibilité, proclamée sur la
foi de quelques expériences négatives, de mettre en évidence le
mouvement de translation uniforme d’un système au moyen
d’expériences d’optique ou d’électricité faites à l’intérieur de ce
système. En admettant, d’autre part, que les idées de Lorentz et ses
équations électromagnétiques sont inattaquables, on a été conduit à
regarder comme nécessaire le changement de nos idées sur
l’espace et sur le temps; espace et temps ( ) n’ont plus
leurs transformations séparées et entrent simultanément dans le
groupe de Lorentz. La simultanéité de deux phénomènes devient
une notion toute relative; un phénomène peut être antérieur à un
autre pour un premier observateur, tandis qu’il lui est postérieur pour
un second. Les mathématiciens, intéressés par un groupe de
transformations qui transforment en elle-même la forme quadratique
( = vitesse de la lumière) se sont livrés à
d’élégantes dissertations sur ce sujet et ont sans doute contribué à
la popularité du principe de relativité. A d’autres époques, on eût
peut-être, avant de rejeter les idées traditionnelles de l’humanité sur
l’espace et le temps, passé au crible d’une critique extrêmement
sévère les conceptions sur l’éther et la formation des équations de
l’électromagnétisme; mais le désir du nouveau ne connaît pas de
bornes aujourd’hui. Les objections ne manquent pas cependant, et
d’illustres physiciens, comme Lord Kelvin et Ritz, sans parler des
vivants, ont émis des doutes très motivés. La Science assurément
ne connaît point de dogmes, et il se peut que des expériences
positives précises nous forcent un jour à modifier certaines idées
devenues notions de sens commun; mais le moment en est-il déjà
venu?
Poincaré voyait le danger de ces engouements, et, dans une
conférence sur la dynamique nouvelle, il adjurait les professeurs de
ne pas jeter le discrédit sur la vieille Mécanique qui a fait ses
preuves. Et puis, il a vécu assez pourvoir les principaux
protagonistes des idées nouvelles ruiner partiellement au moins leur
œuvre. Dans tout ce relativisme, il reste un absolu, à savoir la
vitesse de la lumière dans le vide, indépendante de l’état de repos
ou de mouvement de la source lumineuse. Cet absolu va
probablement disparaître, les équations de Lorentz ne représentant
plus qu’une première approximation. Les plus grandes difficultés
viennent de la gravitation, au point que certains théoriciens de la
Physique croient ne pouvoir les lever qu’en attribuant de l’inertie et
un poids à l’énergie, d’où en particulier la pesanteur de la lumière. Si
Poincaré avait vécu, il eût sans doute été conduit à rapprocher des
vues actuelles son essai de 1905 sur la gravitation. Au milieu des
incertitudes qui se présentent aujourd’hui en électro-optique, son
esprit lumineux va nous manquer singulièrement. Il faut avouer que
dans tout cela les bases expérimentales sont fragiles, et peut-être
Poincaré eût-il suggéré des expériences apportant un peu de
lumière dans cette obscurité.
Un des derniers travaux de Poincaré a été une discussion
approfondie de la théorie des quanta, édifiée par Planck, d’après
laquelle l’énergie des radiateurs lumineux varierait d’une manière
discontinue. De ce point de vue «les phénomènes physiques, dit
Poincaré, cesseraient d’obéir à des lois exprimables par des
équations différentielles, et ce serait là sans aucun doute la plus
grande révolution et la plus profonde que la philosophie naturelle ait
subie depuis Newton». Quelque grande, en effet, que doive être
cette révolution, il est permis toutefois de remarquer que des
circonstances plus ou moins analogues se sont déjà présentées.
Ainsi, dans un gaz à la pression ordinaire, ou peut parler de pression
et l’on peut appliquer les équations différentielles de la dynamique
des fluides; il n’en est plus de même dans un gaz raréfié, où il n’est
plus possible de parler de pression. Il faudra peut-être nous résigner
à faire usage, suivant les limites entre lesquelles nous étudions une
catégorie de phénomènes, de représentations analytiques
différentes, si pénible que puisse être cette sorte de pluralisme pour
ceux qui rêvent d’unité. Mais c’est là encore le secret de l’avenir, et il
serait imprudent d’affirmer qu’on ne trouvera pas quelque biais
permettant de rétablir dans nos calculs la continuité.
 V.
Les nombreux écrits de Poincaré, sur ce qu’on appelle la
philosophie des sciences, ont fait connaître son nom à un public très
étendu. Nous entrons ici dans un autre domaine que celui des
recherches proprement scientifiques, et je n’ai pas l’intention
d’étudier à fond cette partie de son œuvre. Il y est tout d’abord
singulièrement difficile de se rendre compte de l’originalité de telle
ou telle étude; ainsi, dans ses écrits sur l’hypothèse dans la Science,
Poincaré s’est rencontré plus d’une fois avec divers auteurs, mais
l’illustration de son nom, consacrée par tant de découvertes
mathématiques, donnait à ses opinions une autorité particulière. La
forme en ces questions est aussi de grande importance. La phrase
concise de Poincaré, allant droit au but, parfois avec une légère
pointe de paradoxe, produit une singulière impression; on est un
moment subjugué, même quand on sent qu’on n’est pas d’accord
avec l’auteur. Mainte page de Poincaré a produit sur plus d’un
lecteur un vif sentiment d’admiration en même temps qu’une sorte
d’effroi et d’agacement devant tant de critique.
On a parlé quelquefois de la philosophie de Poincaré. En fait,
penseur indépendant, étranger à toute école, Poincaré ne chercha
jamais à édifier un système philosophique, comme un Renouvier, un
Bergson ou même un William James. Il a écrit des livres de
«Pensées», où savants et philosophes trouvent ample matière à
réflexions. Il n’est esclave d’aucune opinion, pas même de celle qu’il
a émise antérieurement, et il sera un jour intéressant de suivre
certaines variations de la sa pensée, où l’on voit quelque peu
s’atténuer ce qu’on a appelé son nominalisme. Il fut ainsi conduit à
expliquer certaines affirmations qui, prises trop à la lettre, avaient été
mal comprises et utilisées dans un dessein dont il n’avait aucun
souci.
 Si l’on voulait toutefois caractériser d’un mot les idées de
Poincaré, on pourrait dire que sa philosophie est la philosophie de la
commodité. Dans quelques unes de ses pages, le mot commode
revient constamment et constitue le terme de son explication.
D’aucuns pensent qu’il faudrait donner les raisons de cette
commodité, et, parmi eux, les plus pressants sont les biologistes
toujours guidés par l’idée d’évolution. La commodité résultera pour
eux d’une longue adaptation, et, ainsi approfondie, deviendra un
témoignage de réalité et de vérité. A l’opposé des évolutionnistes,
d’autres ne voient que l’esprit humain tout formé et sa fonction la
pensée. A certaines heures au moins, Poincaré fut de ces derniers,
et cet idéalisme lui a inspiré des pages d’une admirable poésie qui
resteront dans la littérature française; telle cette dernière page de
son Livre sur la valeur de la Science, qui débute par ces mots «Tout
ce qui n’est pas pensée est le pur néant». Entre des doctrines si
différentes toute communication est impossible, et l’on arrive à se
demander si l’on peut discuter de l’origine des plus simples notions
scientifiques, sans avoir à l’avance une foi philosophique à la
formation de laquelle auront d’ailleurs concouru d’autres éléments
que des éléments proprement scientifiques.
Pour ne pas rester uniquement dans les généralités, arrêtons-
nous un moment sur les principes de la Géométrie. Poincaré part
d’un esprit humain, dans lequel l’idée de groupe préexiste et
s’impose comme forme de notre entendement. L’esprit, après un
travail d’abstraction aboutissant aux premiers concepts de la
Géométrie (point, droite, etc.), cherche à exprimer les rapports de
position des corps; il le fait au moyen de l’idée de groupe, prenant le
groupe le plus commode et le plus simple qui est le groupe de la
géométrie dite euclidienne. Les propriétés géométriques ne
correspondent, pour Poincaré, à aucune réalité; elles forment un
ensemble de conventions que l’expérience a pu suggérer à l’esprit,
mais qu’elle ne lui a pas imposées. L’évolutionniste dont je parlais
plus haut voit là de grandes difficultés, non pas seulement pour la
raison banale que la dualité ainsi posée entre l’esprit et le milieu
extérieur est contraire à sa doctrine, mais parce que, cherchant à
retracer la genèse des origines de la Géométrie dans l’espèce
humaine, il lui paraît impossible de séparer l’acquisition des notions
géométriques et celles des notions physiques les plus simples, la
Géométrie ayant dans des temps très anciens fait partie de la
Physique. Sans changer l’ensemble de ces notions, on ne peut,
semble-t-il, remplacer le groupe euclidien par un autre, et les
exemples cités de transport d’un homme dans un autre milieu (où
cet homme commencerait par mourir) sont plus pittoresques que
probants. On retombe ainsi, sous un autre point de vue, sur les
idées de Gauss qui considérait comme un fait expérimental que la
courbure de notre espace est nulle, et regardait, contrairement à
Poincaré, que la géométrie euclidienne est plus vraie que les
géométries non euclidiennes. Il y a sans doute bien des hypothèses,
ne disons pas des conventions, en Géométrie. C’en est une par
exemple, oubliée quelquefois, que notre espace est simplement
connexe. Peu importe quelle est la connexité de l’espace, quand on
se borne à envisager une partie assez petite, celle-ci s’étendît-elle
jusqu’aux lointaines nébuleuses, mais il pourrait en être autrement
quand on considère l’espace dans son ensemble.
Tous les esprits élevés trouveront, dans l’œuvre philosophique et
littéraire de Poincaré, matière à longues réflexions, soit qu’ils se
laissent convaincre par sa dialectique, soit qu’ils cherchent des
arguments contraires. Certaines pages sont d’une austère grandeur,
comme celle où la pensée est qualifiée d’«éclair au milieu d’une
longue nuit». Non moins suggestive est la parenthèse ouverte un
peu avant «étrange contradiction pour ceux qui croient au temps»,
où l’on est presque tenté de voir un demi-aveu. Les inquiétudes
qu’on peut concevoir au sujet de la notion même de loi furent-elles
jamais exprimées avec plus de profondeur que dans l’étude sur
l’évolution des lois? J’ai déjà fait allusion au prétendu scepticisme de
Poincaré. Non, Poincaré ne fut pas un sceptique; à certaines heures,
il fut pris, comme d’autres, d’angoisse métaphysique, et il sut
éloquemment l’exprimer. Mais tournons le feuillet, et le savant,
confiant dans l’effort de l’esprit humain pour atteindre le vrai, nous
apparaît dans des pages admirables sur le rôle et la grandeur de la
Science. Les plus belles peut-être forment cet hymne à l’Astronomie
qu’il faudrait faire lire aux jeunes gens à une époque où tend à
dominer le souci exclusif de l’utile. Aucune des préoccupations de
notre temps ne fut d’ailleurs étrangère au noble esprit de Poincaré;
c’est ce dont témoigne une de ses dernières études sur la morale et
la science, où l’argumentation est irréprochable, si par morale on
entend la morale impérative de Kant.
On ne ferme pas sans tristesse ces volumes d’un contenu si riche
et dont quelques parties auraient été l’objet de nouveaux
développements, si la plume n’était tombée des mains de leur
auteur. Tous ceux qui ont le culte de la Science pure et
désintéressée ont été douloureusement émus par sa mort
prématurée, mais ce sont surtout les sciences mathématiques qui
sont cruellement frappées par cette disparition. Poincaré fut, avant
tout, un profond mathématicien, qui, pour la puissance d’invention,
est l’égal des plus grands. L’heure n’est pas venue de porter un
jugement définitif sur son œuvre que le temps grandira encore, ni de
le comparer aux plus célèbres géomètres du siècle dernier: peut-être
Henri Poincaré fût-il encore supérieur à son œuvre?
[1] On sait qu’un savant Finlandais M. Sundmann vient de donner
une solution complète du problème des trois corps. Il serait injuste
de ne pas reconnaître que les travaux antérieurs de Poincaré ont
eu une grande influence sur les recherches de l’astronome
d’Helsingfors. J’ai fait une étude des Mémoires de M. Sundmann
dans un article récent de la Revue générale des Sciences (15
octobre 1913) et dans le Bulletin des Sciences Mathématiques
(octobre 1913).
[2] C’est en approfondissant cette idée, et en ne craignant pas de
comprendre dans son analyse le cas des chocs que M.
Sundmann est arrivé à une solution du problème de trois corps
(voir la note ci-dessus).

53717 Paris,—Imprimerie Gauthier-Villars, 55, quai des Grands-

Augustins.

AU LECTEUR
L’orthographe d’origine a été conservée et n’a pas été harmonisée, mais les
erreurs clairement introduites par le typographe ou à l’impression ont été
corrigées.
Une table des matières a été ajoutée pour faciliter la tâche du lecteur.
 *** END OF THE PROJECT GUTENBERG EBOOK L'ŒUVRE DE
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