Fermentation microbiology and biotechnology 2011

K12604_COVER_PRINT_REV.pdf

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11/23/11

9:26 AM

El-Mansi

BIOLOGICAL SCIENCES & LIFE SCIENCES

Third Edition

Fermentation Microbiology and Biotechnology, Third
Edition explores and illustrates the diverse array of metabolic pathways employed for the production of primary
and secondary metabolites as well as biopharmaceuticals.
This updated and expanded edition addresses the whole
spectrum of fermentation biotechnology, from fermentation kinetics and dynamics to protein and co-factor
engineering.
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M

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CM

The third edition builds upon the fine pedigree of its earlier predecessors and extends
the spectrum of the book to reflect the multidisciplinary and buoyant nature of this
subject area. To that end, the book contains four new chapters:

MY

CY

CMY

K

•
•
•
•

Functional Genomics
Solid-State Fermentations
Applications of Metabolomics to Microbial Cell Factories
Current Trends in Culturing Complex Plant Tissues for the Production of
Metabolites and Elite Genotypes

Organized and written in a concise manner, the book’s accessibility is enhanced
by the inclusion of definition boxes in the margins explaining any new concept or
specific term. The text also contains a significant number of case studies that
illustrate current trends and their applications in the field.
With contributions from a global group of eminent academics and industry experts,
this book is certain to pave the way for new innovations in the exploitation of
microorganisms for the benefit of mankind.

Third
Edition

K12604
ISBN: 978-1-4398-5579-9

90000

9 781439 855799

Fermentation Microbiology and Biotechnology

Fermentation
Microbiology and
Biotechnology

Fermentation
Microbiology
and
Biotechnology

Third Edition
Edited by

E.M.T. El-Mansi • C.F.A. Bryce • B. Dahhou
S. Sanchez • A.L. Demain • A.R. Allman


Fermentation
Microbiology
and
Biotechnology

Third Edition


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Fermentation
Microbiology
and
Biotechnology
Third Edition
Edited by

E.M.T. El-Mansi • C.F.A. Bryce • B. Dahhou
S. Sanchez • A.L. Demain • A.R. Allman

Boca Raton London New York

CRC Press is an imprint of the
Taylor & Francis Group, an informa business


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accuracy of the text or exercises in this book. This book’s use or discussion of MATLAB® software or related products
does not constitute endorsement or sponsorship by The MathWorks of a particular pedagogical approach or particular
use of the MATLAB® software.

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Version Date: 20111007
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A man lives not only his personal life as an individual, but also, consciously
or unconsciously, the life of his epoch and his contemporaries.
Thomas Mann

Professor Dr. Mahmoud Ismael Taha,

A chemist of exactitude and graceful humility (1924–1981)

This edition is dedicated with affection and gratitude to the memory of the late
Professor Dr. Mahmoud Ismael Taha, who ignited in me a lifelong passion
for biochemistry; he often reminded me that Louis Pasteur was a chemist.
E.M.T. El-Mansi
(Editor-in-Chief)


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Contents
Preface...............................................................................................................................................ix
Acknowledgments .............................................................................................................................xi
Editors ............................................................................................................................................ xiii
Contributors ...................................................................................................................................xvii
Chapter 1

Fermentation Microbiology and Biotechnology: An Historical Perspective ............... 1
E.M.T. El-Mansi, Charlie F.A. Bryce, Brian S. Hartley,
and Arnold L. Demain

Chapter 2

Microbiology of Industrial Fermentation: Central and Modern Concepts .................. 9
E.M.T. El-Mansi, F. Bruce Ward, and Arun P. Chopra

Chapter 3

Fermentation Kinetics: Central and Modern Concepts.............................................. 37
Jens Nielsen

Chapter 4

Microbial Synthesis of Primary Metabolites: Current Trends and
Future Prospects .......................................................................................................77
Arnold L. Demain and Sergio Sanchez

Chapter 5

Microbial and Plant Cell Synthesis of Secondary Metabolites and
Strain Improvement .................................................................................................. 101
Wei Zhang, Iain S. Hunter, and Raymond Tham

Chapter 6

Applications of Metabolomics to Microbial “Cell Factories” for
Biomanufacturing: Current Trends and Future Prospects ....................................... 137
David M. Mousdale and Brian McNeil

Chapter 7

Flux Control Analysis and Stoichiometric Network Modeling:
Basic Principles and Industrial Applications ........................................................... 165
E.M.T. El-Mansi, Gregory Stephanopoulos, and Ross P. Carlson

Chapter 8

Enzyme and Cofactor Engineering: Current Trends and Future Prospects

in the Pharmaceutical and Fermentation Industries................................................. 201
George N. Bennett and Ka-Yiu San

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viii

Chapter 9

Contents

Conversion of Renewable Resources to Biofuels and Fine Chemicals:
Current Trends and Future Prospects....................................................................... 225
Aristos A. Aristidou, Namdar Baghaei-Yazdi, Muhammad Javed,
and Brian S. Hartley

Chapter 10 Functional Genomics: Current Trends, Tools, and Future Prospects in the
Fermentation and Pharmaceutical Industries ........................................................... 263
Surendra K. Chikara and Toral Joshi
Chapter 11 Beyond Cells: Culturing Complex Plant Tissues for the Production of
Metabolites and Elite Genotypes ............................................................................. 295
Pamela J. Weathers, Melissa J. Towler, and Barbara E. Wyslouzil
Chapter 12 Cell Immobilization and Its Applications in Biotechnology:
Current Trends and Future Prospects....................................................................... 313
Ronnie G. Willaert
Chapter 13 Biosensors in Bioprocess Monitoring and Control: Current Trends and
Future Prospects ....................................................................................................... 369
Chris E. French and Chris Gwenin
Chapter 14 Solid-State Fermentation: Current Trends and Future Prospects ............................403

Lalita Devi Gottumukkala, Kuniparambil Rajasree, Reeta Rani Singhania,
Carlos Ricardo Soccol, and Ashok Pandey
Chapter 15 Bioreactors: Design, Operation, and Applications ................................................... 417
Anthony R. Allman
Chapter 16 Control of Industrial Fermentations: An Industrial Perspective .............................. 457
Craig J.L. Gershater and César Arturo Aceves-Lara
Chapter 17 Monitoring and Control Strategies for Ethanol Production in
Saccharomyces Cerevisiae ....................................................................................... 489
Gilles Roux, Zetao Li, and Boutaib Dahhou
Appendix: Suppliers List ............................................................................................................. 519
Index .............................................................................................................................................. 521


Preface
I beseech you to take interest in these sacred domains, so expressively called laboratories.
Ask that, there be more and that they be adorned for these are the temples of the future,
wealth and well being.
Louis Pasteur
Microorganisms, free-living and immobilized, are widely used industrially as catalysts in the
biotransformation of many chemical reactions, especially in the production of stereospecific isomers. The high specificity, versatility, and the diverse array of microbial enzymes (proteomes) are
currently being exploited for the production of important primary metabolites including amino
acids, nucleotides, vitamins, solvents, and organic acids, as well as secondary metabolites such as
antibiotics, hypercholesterolemia agents, enzyme inhibitors, immunosuppressants, and antitumor
therapeutics.
Recent innovations in functional genomics, proteomics, metabolomics, bioinformatics, biosensor technology, nanobiotechnology, cell and enzyme immobilization, and synthetic biology
and in silico research are currently being exploited in drug development programs to combat
disease and hospital-acquired infections as well as in the formulation of a new generation of
therapeutics.
The third edition builds upon the fine pedigree of its earlier predecessors and extends the spectrum of the book to reflect the multidisciplinary and buoyant nature of this subject area. To that end,
four new chapters have been commissioned:

•
•
•
•

Functional Genomics
Solid-State Fermentations
Applications of Metabolomics to Microbial Cell Factories
Current Trends in Culturing Complex Plant Tissues for the Production of Metabolites and
Elite Genotypes

More exciting advances and discoveries are yet to be unraveled, and the best is yet to come as we
enter a new era in which the exploitations of microorganisms continue to astonish the world community, especially the use of renewable resources and the generation of new therapeutics to combat
disease are recognized as an urgent need. To that end, Professor Brian S. Hartley predicts the emergence of a new era in which “biorefineries” play a central role in climate control and the balance of
geochemical cycles in our ecosystem.
To aid learning and to make the text more lively and interactive, boxes highlighting the definitions of new and central concepts are shown in the margin, a feature that is now synonymous with
our book.
We very much hope that the third edition will be assimilated and appreciated by those actively
engaged in the pursuit of advancing our field, and to that end, the editor-in-chief wishes to stress
his readiness to receive your feedback, including suggestions by authors who wish to add or
extend the knowledge base of our book, which is becoming increasingly global with every edition.

ix


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Preface

In future editions, our endeavor to keep our readers abreast with recent innovations in this exciting and buoyant field will continue unabatedly.

The Editorial Team
MATLAB® is a registered trademark of The MathWorks, Inc. For product information, please
contact:
The MathWorks, Inc.
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Tel: 508 647 7000
Fax: 508 647 7001
E-mail:
Web: www.mathworks.com


Acknowledgments
The third edition builds on the seminal work presented in earlier editions and owes much to the
original and innovative work of our peers and colleagues worldwide. I wish to thank my editorial
team and our distinguished authors for their sound contributions and for being very responsive
throughout; in particular, I wish to thank Brian S. Hartley, whose encouragement and stimulating
discussions across the Internet have been inspirational. It is also befitting to thank my two sons
Adam and Sammy, for their love and the happiness, which they continue to bring to my life.
On behalf of the editorial team and authors, I wish to thank Barbra Norwitz (executive editor)
and Albert Ebinesh (project manager) and their respective teams at CRC Taylor and Francis for
transforming our manuscripts, into a high quality book, which I hope meets with your expectations
as a reader.
The editorial team are only too conscious of mistakes and omissions, which may have crept in
unnoticed; the credit of producing this book is only partly hours, it is the blame that rests totally
with us.
Have a good read.
E.M.T. El-Mansi
Editor-in-Chief

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Editors
Dr. Mansi El-Mansi is a graduate of the University of Assiut, El-Minya,
Egypt (BSc, First Hons and MSc Microbiology). He was intrigued and
fascinated by the versatility of microorganisms and soon realized that
understanding their physiology demanded a clear understanding of their
biochemistry. He made the conscious decision of undertaking his PhD
in the field of microbial biochemistry and was fortunate enough to carry
it out at UCW, Aberystwyth, United Kingdom, under the supervision of
David J. Hopper, whose meticulous approach to experimental design was
a towering influence. During the course of his PhD studies, he became
familiar with the work of Stanley Dagley, the father of microbial biochemistry as we know it, and this in turn galvanized his resolve to further
his understanding of microorganisms at the molecular level.
Immediately after the completion of his PhD, Dr. El-Mansi joined Harry Holms at the Department
of Biochemistry, University of Glasgow, Scotland, and such a happy and stimulating association continued for the best part of a decade, during which their group was the first to clone and show that the
structural gene encoding the bifunctional regulatory enzyme ICDH kinase/phosphatase is indeed a
member of the glyoxylate bypass operon. Soon thereafter, Dr. El-Mansi became acquainted with flux
control analysis and its immense potential in the fermentation and pharmaceutical industries. His
interest in the application of flux control analysis was further stimulated by collaboration with Henrik
Kacser, the founder of metabolic control analysis (MCA) theory, at the University of Edinburgh.
During the course of his employment in Edinburgh, Dr. El-Mansi was the first to postulate and
unravel the role of HS-CoA in the partition of carbon flux among enzymes of central metabolism
during growth of Escherichia coli on acetate. He was also the first to provide evidence supporting
Dan Kosahland’s theory of “ultrasensitivity”. His research activities, which span the best part of
30 years, yielded an extensive list of publications of which four are single-author publications in

peer-reviewed journals.
After 27 years of intensive research and teaching in Scotland, Dr. El-Mansi felt that the time was
right to share his experience with others and to enrich him culturally. He is currently a professor of
biotechnology at Sharda University, Greater Noida, India.
Dr. Charlie Bryce has held posts as Head of the School of Life Sciences
and Dean of the Faculty of Sciences for over 20 years at Edinburgh
Napier University. For the last three years he focused on International
Development for the Faculty of Health, Life and Social Sciences and,
more recently, he extended this to work with the Vice Chancellor on
developing international research and technology transfer links for all
areas of the university. He now acts as an independent, international consultant for program development, learning support material development
including e-learning, continuing professional development, and quality
assessment and audit at the departmental and institutional level.
In the last 30 years, he has published 100 refereed research publications and designed and produced approximately 30 teaching packages in a wide variety of media
formats. He has undertaken an international survey of the biochemistry curriculum for science
and medical students and a pan-European survey of curriculum content for programs in biotechnology and is currently developing programs in biomedical science for a partner in Singapore.
Dr. Bryce has served on national and international committees including U.K. Deans of Science
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Editors

(chairman), U.K. Interest Group on Education in Biotechnology (chairman), Education Group of
the Biochemical Society (chairman), vice president of the European Federation of Biotechnology
(EFB), EFB Task Group on Education and Mobility (chairman), EFB Task Group on Innovation
(chairman), secretary general of the Association for Higher Education in Biotechnology
(HEduBT, the body that oversees the operation of the Eurodoctorate in Biotechnology), specialist
adviser to several British Council and COSTED Projects, member of the Editorial Board of New

Biotechnology, and former executive editor of the journal Bioinformatics. He has acted as an auditor and a subject reviewer for the Quality Assurance Agency (QAA) and for the Scottish Funding
Council (SFC) and has also undertaken audit/assessment work in Bangladesh and in Hong Kong.
He currently chairs accreditation panels for the Forensic Science Society and has also undertaken
research project evaluation for the Commission of the European Communities (CEC).
Dr. Arnold Demain, research fellow in microbial biochemistry at
the Charles A. Dana Research Institute for Scientists Emeriti of Drew
University in Madison, NJ, is an icon synonymous with excellence in the
fields of industrial microbiology and biotechnology. Born in Brooklyn,
New York City, in 1927, he was educated in the New York public school
system and received his BS and MS in bacteriology from Michigan State
University in 1949 and 1950, respectively. He obtained his PhD on pectic enzymes in 1954 from the University of California, having divided
his time between the Berkeley and Davis campuses. In 1956, he joined
Merck Research Laboratories at Rahway, NJ, where he worked on fermentation microbiology, β-lactam antibiotics, flavor nucleotides, and
microbial nutrition. In 1965, he founded the Fermentation Microbiology
Department at Merck and directed research and development on processes for monosodium glutamate,
vitamin B12, streptomycin, riboflavin, cephamycin, fosfomycin, and interferon inducers. In 1969, he
joined MIT, where he set up the Fermentation Microbiology Laboratory. Since then, he has published extensively on enzyme fermentations, mutational biosynthesis, bioconversions, and metabolic
regulation of primary and secondary metabolism. His success is evident in a long list of publications
(over 530), 14 books of which he is co-editor or co-author, and 21 U.S. patents. His ability to “hybridize” basic studies and industrial applications was recognized by his election to the presidency of
the Society for Industrial Microbiology in 1990, membership in the National Academy of Sciences
in 1994, the Mexican Academy of Sciences in 1997, and in the Hungarian Academy of Science in
2002. In recognition of his outstanding contribution to our current understanding in fermentation
microbiology and biotechnology, he has been awarded honorary doctorates from the University of
Leon (Spain), Ghent University (Belgium), Technion University (Israel), Michigan State University
(United States), Muenster University (Germany), and Drew University (United States).
Dr. Anthony (Tony) R. Allman, a graduate of the University of
Liverpool (BSc, PhD), has been a member of the Institute of Biology
and the Society of General Microbiology for more than 25 years.
He began his career at Glaxo, where he spent six years carrying out
research into the development of subunit bacterial vaccines. During

that time, he became acquainted with fermentors and their applications.
Subsequently, he acted as a specialist in this area for the U.K. agent
of a major European fermentor manufacturer. When Infors U.K. was
established in 1987, Tony joined the new company as Product Manager
(later Technical Director) and in 2002, he became Fermentation
Product Manager for the Swiss parent company, Infors AG. His work
involves providing technical support, training, and application expertise in-house and throughout the world.


Editors

xv

Dr. Allman is well known among research and industrial communities for having a passion for
making fermentation accessible to the wider public. His “extracurricular activities” of devising
practical workshops and giving lectures on fermentation technology speak volumes about the active
pursuit of this aim.
Dr. Sergio Sanchez, born in Mexico City, Mexico, received his
MD in 1970 and his PhD in 1973, both from the National University
of Mexico. After two postdoctoral research fellowships at the U.S.
Department of Agriculture in Peoria, IL, and then at the MIT,
Cambridge, MA, Dr. Sanchez began his career as a researcher at the
Institute of Biomedical Research, National University of Mexico and
in 1984 he co-founded the first Biotechnology Department at the same
University. He has served several times as a head of that department
and more recently as technical secretary of the same institute.
As a professor of industrial microbiology, Dr. Sanchez published
extensively and his work is characterized by a sustained level of
important discoveries in several areas of industrial microbiology,
including research on the interrelation between the role of glutathione and the amino acid transport

systems and the production of penicillin in Penicillium chrysogenum. He also explored the regulatory relationships between primary and secondary metabolism in Streptomyces peucetius var. caesius; he was the first to show that in addition to Glk, an adjacent gene (sco2127) participates in the
process of carbon catabolite repression in this organism.
He was elected as the first president to the Mexican Society for Biotechnology and Bioengineering
(MSBB), and in recognition of his contributions, the MSBB has established the Sergio Sanchez
award to recognize the best thesis research project for pre- and postgraduate students in biotechnology and bioengineering in Mexico. In 1986, he co-founded the Postgraduate Biotechnology
Programme at the National University of Mexico, being its first coordinator.
Currently, he is an editor on the editorial board of Applied Microbiology and Biotechnology and
the Editor-in-Chief of BioTecnología, an international journal published by the Mexican Society for
Biotechnology and Bioengineering.
Dr. Boutaib Dahhou, a graduate of (PhD 1980) of Paul
Sabatier University–Toulouse III, France, has been addressing
various issues of supervision, control, and modeling of linear
and nonlinear systems. In his studies, Dr. Dahhou adopted a
new strategy consisting of adding a road base or a block of
supervision in which one can exploit all of the available information. He further developed this process by modifying the
layer immediately above than the adaptive loop, which is the
layer of supervision of the control; at that level, the signals
evolved from adaptation and feedback loops are used as signal identifiers to recognize specific physiological situations and to act on the parameters of the algorithms of control and
estimation.
He recognized that, in a given system, significant signals have to be identified to test its validity
on the basis of certain preset criteria. Violation of these criteria triggers the start of a second task
by the supervisor. For example, in biotechnological processes, these anomalies can be related to
specific biological reaction or ascribed to the operation of actuators or sensors.
Currently, the detection and isolation of faults in the dynamic of nonlinear systems is of particular interest. Addressing this aspect, Dr. Dahhou developed new algorithms on the basis of the
adaptive observers that made possible the instantaneous detection of any fault. On the other hand,


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Editors

the isolation of these faults demanded much time because of the procedure of parameter adaptation.
To resolve this problem, he is currently developing a new approach of isolation that is based on the
parameter intervals.
Dr. Dahhou has successfully supervised 18 PhD students and published more than 72 articles in
international journals and 130 communications in international congresses.


Contributors
César Arturo Aceves-Lara
Université de Toulouse; UPS, INSA, INP,
LISBP; Toulouse, France and INRA
UMR792, Ingénierie des Systèmes
Biologiques et des Procédés
Toulouse, France
Anthony R. Allman
Infors U.K., Ltd.
Rigate, England, United Kingdom
Aristos A. Aristidou
Bioprocess Development
Centennial, Colorado
Namdar Baghaei-Yazdi
Biocaldol, Ltd., The London Bioscience
Innovation Centre
London, England, United Kingdom
George N. Bennett
Department of Biochemistry and Cell Biology
Rice University
Houston, Texas
Charlie F.A. Bryce

Edinburgh Napier University
Edinburgh, Scotland, United Kingdom
Ross P. Carlson
Department of Chemical and Biological
Engineering
Center for Biofilm Engineering
Montana State University
Bozeman, Montna
Surendra K. Chikara
Xcelrislabs, Ltd.
Ahmedabad, India
Arun P. Chopra
Department of Biotechnology
Hindustan College of Science and Technology
Farha, Mathura, India

Boutaib Dahhou
Centre National de la Recherche Scientifique
Laboratoire d’Analyse et d’Architecture des
Systèmes
Université de Toulouse
Toulouse, France
Lalitha Devi Gottumukkala
Biotechnology Division
National Institute for Interdisciplinary Science
and Technology
Council of Scientific and Industrial Research
Trivandrum, India
Arnold L. Demain
Research Institute for Scientists Emeriti

Drew University
Madison, New Jersey
E.M.T. El-Mansi
Department of Biotechnology, School of
Medical Sciences and Research
Sharda University
Greater Noida, Uttar Pradesh, India
Chris E. French
Institute of Cell Biology
University of Edinburgh
Edinburgh, Scotland, United Kingdom
Craig J.L. Gershater
Institute of Continuing Education
University of Cambridge
Cambridge, England, United Kingdom
Chris Gwenin
School of Chemistry
Bangor University
Wales, United Kingdom
Brian S. Hartley
Grove Cottage
Cambridge, England, United Kingdom
Iain S. Hunter
Department of Pharmaceutical Sciences
University of Strathclyde
Glasgow, Scotland, United Kingdom
xvii


xviii

Muhammad Javed
Biocaldol, Ltd.
The London Bioscience Innovation Centre
London, England, United Kingdom
Toral Joshi
Xcelrislabs, Ltd.
Ahmedabad, India
Zetao Li
Electrical Engineering College
Guizhou University
Guiyang, Guizhou, People’s Republic of China
Brian McNeil
Institute of Pharmacy and Biomedical Sciences
Royal College
Strathclyde University
Glasgow, Scotland, United Kingdom

Contributors

Ka-Yiu San
Department of Bioengineering
Rice University
Houston, Texas
Sergio Sanchez
Departamento de Biología Molecular y
Biotecnología, Instituto de Investigaciones
Biomédicas, Universidad Nacional
Autónoma de México
México City, México

Gregory Stephanopoulos
Department of Chemical Engineering
Massachusetts Institute of Technology
Cambridge, Massachusetts

David M. Mousdale
beÒcarta Ltd.
Royal College Building
Glasgow, Scotland, United Kingdom

Reeta Rani Singhania
Laboratoire de Genie Chimique
et Biochimique
Universite Blaise Pascal
Clermont Ferrand, France

Jens Nielsen
Chalmers University of Technology
Department of Chemical and Biological
Engineering
Gothenburg, Sweden

Carlos Ricardo Soccol
Biotechnology Division
Federal University of Parana
Curitiba, Brazil

Ashok Pandey
Biotechnology Division
National Institute for Interdisciplinary Science

and Technology, Council of Scientific and
Industrial Research
Trivandrum, India

Raymond Tham
Flinders Centre for Marine
Bioprocessing and Bioproducts,
School of Medicine
Flinders University
Adelaide, Australia

Kuniparambil Rajasree
Biotechnology Division
National Institute for Interdisciplinary Science
and Technology, Council of Scientific and
Industrial Research
Trivandrum, India

Melissa J. Towler
Department of Biology and
Biotechnology
Worcester Polytechnic Institute
Worcester, Massachusetts

Gilles Roux
Centre National de la Recherche Scientifique
Laboratoire d’Analyse et d’Architecture des
Systèmes
Université de Toulouse
Toulouse, France

F. Bruce Ward
Institute of Cell Biology
University of Edinburgh,
Darwin Building
Edinburgh, Scotland, United Kingdom


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Contributors

Pamela J. Weathers
Department of Biology and Biotechnology
Worcester Polytechnic Institute
Worcester, Massachusetts

Barbara E. Wyslouzil
William G. Lowrie Department of Chemical
and Biomolecular Engineering
Ohio State University
Columbus, Ohio

Ronnie G. Willaert
Department of Structural Biology,
Flanders Institute for Biotechnology
Vrije Universiteit Brussel
Brussels, Belgium

Wei Zhang

Flinders Centre for Marine Bioprocessing and
Bioproducts, School of Medicine
Flinders University
Adelaide, Australia


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Microbiology
1 Fermentation
and Biotechnology: An
Historical Perspective
E.M.T. El-Mansi, Charlie F.A. Bryce,
Brian S. Hartley, and Arnold L. Demain
CONTENTS
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8

Fermentation: An Ancient Tradition.........................................................................................1
The Rise of Fermentation Microbiology .................................................................................. 1
Developments in Metabolic and Biochemical Engineering ..................................................... 3
Discovery of Antibiotics and Genetic Engineering .................................................................. 5
The Rise and Fall of Single-Cell Protein .................................................................................5

Fermentation Biotechnology and the Production of Amino Acids .......................................... 6
Biofuels and the “Evolution” of Biorefineries .......................................................................... 6
Impact of Functional Genomics, Proteomics, Metabolomics, and Bio-Informatics on
the Scope and Future Prospects of Fermentation Microbiology and Biotechnology ............... 7
References ..........................................................................................................................................8
“Dans le champ de l’observation, le hasard ne favorise que les esprits préparés.”
Louis Pasteur, 1854

1.1

FERMENTATION: AN ANCIENT TRADITION

Fermentation has been known and practiced by humankind since prehistoric times, long before
the underlying scientific principles were understood. That such a useful technology should arise
by accident will come as no surprise to those people who live in tropical and subtropical regions,
where, as Marjory Stephenson put it, “every sandstorm is followed by a spate of fermentation in
the cooking pot” (Stephenson 1949). For example, the productions of bread, beer, vinegar, yogurt,
cheese, and wine were well-established technologies in ancient Egypt (Figures 1.1 and 1.2). It is an
interesting fact that archaeological studies have revealed that bread and beer, in that order, were the
two most abundant components in the diet of ancient Egyptians. Everyone, from the pharaoh to the
peasant, drank beer for social as well as ritual reasons. Archaeological evidence has also revealed
that ancient Egyptians were fully aware not only of the need to malt the barley or the emmer wheat
but also of the need for starter cultures, which at the time may have contained lactic acid bacteria
in addition to yeast.

1.2 THE RISE OF FERMENTATION MICROBIOLOGY
With the advent of the science of microbiology, and in particular fermentation microbiology, we
can now shed light on these ancient and traditional activities. Consider, for example, the age-old
1

2

Fermentation Microbiology and Biotechnology, Third Edition

FIGuRE 1.1 Bread making as depicted on the wall of an ancient Egyptian tomb dated c. 1400 bc. (Reprinted
with the kind permission of the Fitzwilliam Museum, Cambridge, England.)

FIGuRE 1.2 Grape treading and wine making as depicted on the walls of Nakhte’s tomb, Thebes, c. 1400 bc.
(Reprinted with the kind permission of AKG, London, England/Erich Lessing.)

technology of wine making, which relies upon crushing grapes (Figure 1.2) and letting nature take
its course (i.e., fermentation). Many microorganisms can grow on grape sugars more readily and
efficiently than yeasts, but few can withstand the osmotic pressure arising from the high sugar
concentrations. Also, as sugar is fermented, the alcohol concentration rises to a level at which only
osmotolerant, alcohol-tolerant cells can survive. Hence inhabitants of ancient civilizations did not
need to be skilled microbiologists in order to enjoy the fruits of this popular branch of fermentation
microbiology.
In fact, the scientific understanding of fermentation microbiology and, in turn, biotechnology
only began in the 1850s, after Louis Pasteur had succeeded in isolating two different forms of
amyl alcohol, of which one was optically active (L, or laevorotatory) while the other was not.
Rather unexpectedly, the optically inactive form resisted all of Pasteur’s attempts to resolve it
into its two main isomers, the laevorotatory (L) and the dextrorotatory (D) forms. It was this


Fermentation Microbiology and Biotechnology: An Historical Perspective

3

observation that led Pasteur into the study of fermentation, in the hope of unraveling the underlying reasons behind his observation, which was contrary to stereochemistry and crystallography

understandings at the time.
In 1857, Pasteur published the results of his studies and concluded that fermentation is associated
with the life and structural integrity of the yeast cells rather than with their death and decay. He reiterated the view that the yeast cell is a living organism and that the fermentation process is essential
for the reproduction and survival of the cell. In his paper, the words cell and ferment are used interchangeably (i.e., the yeast cell is the ferment). The publication of this classic paper marks the birth
of fermentation microbiology and biotechnology as a new scientific discipline. Guided by his critical and unbiased approach to experimental design, Pasteur was able to confidently challenge and
reject Liebig’s perception that fermentation occurs as a result of contact with decaying matter. He
also ignored the well-documented view that fermentation occurs as a result of “contact catalysis,”
although it is possible that this concept was not suspect in his view. The term “contact catalysis”
probably implied that fermentation is brought about by a chain of enzyme-catalyzed reactions. In
1878, Wilhelm Kühne (1837–1900) was the first to use the term enzyme, which is derived from the
Greek word ενζυμον (“in leaven”) to describe this process. The word enzyme was used later to refer
to nonliving substances such as pepsin, and the word ferment used to refer to chemical activity
produced by living organisms.
Although Pasteur’s interpretations were essentially physiological rather than biochemical, they
were pragmatically correct. During the course of his further studies, Pasteur was also able to establish not only that alcohol was produced by yeast through fermentation but also that souring was a
consequence of contamination with bacteria that were capable of converting alcohol to acetic acid.
Souring could be avoided by heat treatment at a certain temperature for a given length of time. This
eliminated the bacteria without adversely affecting the organoleptic qualities of beer or wine, a
process we now know as pasteurization.
A second stage in the development of fermentation microbiology and biotechnology began in
1877, when Moritz Traube proposed the theory that fermentation and other chemical reactions are
catalyzed by protein-like substances and that, in his view, these substances remain unchanged at
the end of the reactions. Furthermore, he described fermentation as a sequence of events in which
oxygen is transferred from one part of the sugar molecule to another, culminating in the formation
of a highly oxidized product (i.e., CO2) and a highly reduced product (i.e., alcohol). Considering the
limited knowledge of biochemistry in general and enzymology in particular at the time, Traube’s
remarkable vision was to prove 50 years ahead of its time.
In 1897 Eduard Buchner, two years after Pasteur died, discovered that sucrose could be fermented to alcohol by yeast cell-free extracts and coined the term “zymase” to describe the enzyme
that catalyses this conversion. The term “zymase” Is derived from the Greek word “zymosis”, which
means fermentation. In 1907, he received the Nobel Prize in Chemistry for his biochemical research

and his discovery of cell-free fermentation. In the early 1900s, the views of Pasteur were modified
and extended to stress the idea that fermentation is a function of a living, but not necessarily multiplying, cell and that fermentation is not a single step but rather a chain of events, each of which is
probably catalyzed by a different enzyme.

1.3 DEVELOPMENTS IN METABOLIC AND
BIOCHEMICAL ENGINEERING
The outbreak of the First World War provided an impetus and a challenge to produce certain chemicals that, for one reason or another, could not be manufactured by conventional means. For example,
there was a need for glycerol, an essential component in the manufacture of ammunition, because
no vegetable oils could be imported due to the naval blockade. German biochemists and engineers
were able to adapt yeast fermentation, turning sugars into glycerol rather than alcohol. Although


4

Fermentation Microbiology and Biotechnology, Third Edition

this process enabled the Germans to produce in excess of 100 tons of glycerol per month, it was
abandoned as soon as the war was over because glycerol could be made very cheaply as a by-product
of the soap industry. There was also, of course, a dramatic drop in the level of manufacture of explosives and, in turn, the need for glycerol.
The diversion of carbon flow from alcohol production to glycerol formation was achieved by adding sodium bisulfite, which reacts with acetaldehyde to give an adduct that cannot be converted to
alcohol (Figure 1.3). Consequently, NADH accumulates intracellularly, thus perturbing the steadystate redox balance (NAD+:NADH ratio) of the cell. The drop in the intracellular level of NAD+ is
accompanied by a sharp drop in the flux through glyceraldehyde-3-phosphate dehydrogenase,
which in turn allows the accumulation of the two isomeric forms of triose phosphate (i.e., glyceraldehyde-3-phosphate and dihydroxyacetone-3-phosphate). Accumulations of the latter together with
high intracellular levels of NADH trigger the expression of glycerol-3-phosphate dehydrogenase,
which in turn leads to the diversion of carbon flux from ethanol production to glycerol formation,
thus restoring the redox balance within the cells by regenerating NAD+ (Figure 1.3). Although this
explanation is with the hindsight of modern biochemistry, the process can be viewed as an early
example of metabolic engineering.
Following the First World War, research into yeast fermentation was largely influenced by
the work of Carl Neuberg and his proposed scheme (biochemical pathway) for the conversion of

sugars to alcohol (alcohol fermentation). Although Neuberg’s scheme was far from perfect and
proved erroneous in many ways, it provided the impetus and framework for many scientists at
the Delft Institute, who vigorously pursued research into oxidation/reduction mechanisms and
the kinetics of product formation in a wide range of enzyme-catalyzed reactions. Such studies
were to prove important in the development of modern biochemistry as well as fermentation
biotechnology.

Fructose 1,6bisphosphate

Glucose

2 ADP
2 ATP

2 ATP

2 ADP
2 Glyeraldehyde
3-phosphate

2 Phosphoenolpyruvate
2 NADH, H+

2 Pyruvate
2 Acetaldehyde

2 NAD+

×

Bisulphite

2 Alcohol

Dihydroxyacetone
3-phosphate
NADH, H+
1
NAD+
Glycerol, 3-phosphate
2

Bisulphite
adduct

Glycerol

FIGuRE 1.3 Diversion of carbon flux from alcohol production to glycerol formation in the yeast
Saccharomyces cerevisiae. Note that the functional role of bisulfite is to arrest acetaldehyde molecules, thus
preventing the regeneration of NAD+ as a consequence of making alcohol dehydrogenase redundant. To
redress the redox balance (i.e., the NAD+:NADH ratio), S. cerevisiae diverts carbon flow (dashed route) toward
the reduction of dihydroxyacetone-3-phosphate to glycerol-3-phosphate, thus regenerating the much-needed
NAD+. The glycerol-3-phosphate thus generated is then dephosphorylated to glycerol.