Fermentation microbiology and biotechnology 2011

To that end, the book contains four new chapters: • Functional Genomics • Solid-State Fermentations • Applications of Metabolomics to Microbial Cell Factories • Current Trends in Culturi

BIOLOGICAL SCIENCES & LIFE SCIENCES

Fermentation Microbiology and Biotechnology, Third Edition explores and illustrates the diverse array of meta- bolic 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 fermenta- tion kinetics and dynamics to protein and co-factor engineering.

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:

• 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.

Fermentation Microbiology

and Biotechnology

Edited by

E.M.T El-Mansi • C.F.A Bryce • B Dahhou

S Sanchez • A.L Demain • A.R Allman

Third Edition

Fermentation

Biotechnology Third Edition

Fermentation Microbiology

and

Biotechnology

Third Edition

CRC Press is an imprint of the

Taylor & Francis Group, an informa business

Boca Raton London New York

Fermentation Microbiology

and

Biotechnology

Edited by

E.M.T El-Mansi • C.F.A Bryce • B Dahhou

S Sanchez • A.L Demain • A.R Allman

Third Edition

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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)

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

Chapter 9 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 iso-mers 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, sensor technology, nanobiotechnology, cell and enzyme immobilization, and synthetic biology

bio-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 trum of the book to reflect the multidisciplinary and buoyant nature of this subject area To that end,

spec-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 com-munity, 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 emer-gence 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 tions of new and central concepts are shown in the margin, a feature that is now synonymous with our book

defini-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

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

excit-The Editorial Team

MATLAB® is a registered trademark of The MathWorks, Inc For product information, please contact:

The MathWorks, Inc

3 Apple Hill Drive

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

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 bio-chemistry 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 tinued 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

con-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 con-sultant 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 tions 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 biotech-nology and is currently developing programs in biomedical science for a partner in Singapore

publica-Dr Bryce has served on national and international committees including U.K Deans of Science

(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

audi-tor 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 pec-tic 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 fer-mentation 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 pub-lished 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 “hybrid-ize” 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 exper-tise in-house and throughout the world

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 tory relationships between primary and secondary metabolism in Streptomyces peucetius var cae- sius; he was the first to show that in addition to Glk, an adjacent gene (sco2127) participates in the

regula-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 biotech-nology 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 infor-mation 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 spe-cific 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 ticular 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,

par-the isolation of par-these faults demanded much time because of par-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

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

Hindustan College of Science and Technology

Farha, Mathura, India

Boutaib Dahhou

Centre National de la Recherche ScientifiqueLaboratoire d’Analyse et d’Architecture des Systèmes

Université de ToulouseToulouse, France

Lalitha Devi Gottumukkala

Biotechnology DivisionNational Institute for Interdisciplinary Science and Technology

Council of Scientific and Industrial ResearchTrivandrum, India

Greater Noida, Uttar Pradesh, India

Chris E French

Institute of Cell BiologyUniversity of EdinburghEdinburgh, Scotland, United Kingdom

Brian S Hartley

Grove CottageCambridge, England, United Kingdom

Muhammad Javed

Biocaldol, Ltd

The London Bioscience Innovation Centre

London, England, United Kingdom

Royal College Building

Glasgow, Scotland, United Kingdom

Jens Nielsen

Chalmers University of Technology

Department of Chemical and Biological

Engineering

Gothenburg, Sweden

Ashok Pandey

Biotechnology Division

National Institute for Interdisciplinary Science

and Technology, Council of Scientific and

Industrial Research

Trivandrum, India

Kuniparambil Rajasree

Biotechnology Division

National Institute for Interdisciplinary Science

and Technology, Council of Scientific and

Industrial Research

Trivandrum, India

Gilles Roux

Centre National de la Recherche Scientifique

Laboratoire d’Analyse et d’Architecture des

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 EngineeringMassachusetts Institute of TechnologyCambridge, Massachusetts

Reeta Rani Singhania

Laboratoire de Genie Chimique

et BiochimiqueUniversite Blaise PascalClermont Ferrand, France

Carlos Ricardo Soccol

Biotechnology DivisionFederal University of ParanaCuritiba, Brazil

Raymond Tham

Flinders Centre for Marine Bioprocessing and Bioproducts, School of Medicine

Flinders UniversityAdelaide, Australia

Pamela J Weathers

Department of Biology and Biotechnology

Worcester Polytechnic Institute

Worcester, Massachusetts

Ronnie G Willaert

Department of Structural Biology,

Flanders Institute for Biotechnology

Vrije Universiteit Brussel

and Biotechnology: An

Historical Perspective

E.M.T El-Mansi, Charlie F.A Bryce,

Brian S. Hartley, and Arnold L Demain

“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

CONTENTS

1.1 Fermentation: An Ancient Tradition 11.2 The Rise of Fermentation Microbiology 11.3 Developments in Metabolic and Biochemical Engineering 31.4 Discovery of Antibiotics and Genetic Engineering 51.5 The Rise and Fall of Single-Cell Protein 51.6 Fermentation Biotechnology and the Production of Amino Acids 61.7 Biofuels and the “Evolution” of Biorefineries 61.8 Impact of Functional Genomics, Proteomics, Metabolomics, and Bio-Informatics on

the Scope and Future Prospects of Fermentation Microbiology and Biotechnology 7References 8

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

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.)

observation that led Pasteur into the study of fermentation, in the hope of unraveling the ing reasons behind his observation, which was contrary to stereochemistry and crystallography understandings at the time.

underly-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 reit-erated 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

inter-changeably (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 cal 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

criti-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 estab-lish 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 mented 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

fer-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 mul-tiplying, 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 cals 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

chemi-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

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 sives and, in turn, the need for glycerol

explo-The diversion of carbon flow from alcohol production to glycerol formation was achieved by ing 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 steady-state 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., glyceral-dehyde-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

add-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

1,6-bisphosphate

2 Glyeraldehyde 3-phosphate

Dihydroxyacetone 3-phosphate

Phosphoenol-2 Pyruvate

2 ATP

2 ADP

Bisulphite adduct

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.

While glycerol fermentation was abandoned immediately after the First World War, the

acetone-butanol fermentation process, catalyzed by Clostridium acetobutylicum, flourished Production

lines were modified to accommodate the new approach of “Fill and Spill” (see Box 1.1), which permitted substantial savings in fuels without adversely affecting the output of solvent production during the course of the Second World War However, as soon as the production of organic solvents

as a by-product of the petrochemical industry became economically viable, the acetone-butanol fermentation process was discontinued

1.4 DISCOVERY OF ANTIBIOTICS AND GENETIC ENGINEERING

The discoveries of penicillin in the late 1920s and its antibacterial properties in the early 1940s represent a landmark in the development of modern fermentation biotechnology This discovery,

to a country at war, was both sensational and invaluable However, Penicillium notatum, the

pro-ducing organism, was found to be susceptible to contamination by other organisms, and therefore aseptic conditions were called for Such a need led to the introduction of so-called stirred tank bioreactors, which minimize contamination with unwanted organisms The demand for penicillin prompted a worldwide screen for alternative penicillin-producing strains, leading to the isola-

tion of Penicillium chrysogenum, which produced more penicillin than the original isolate P

notatum P chrysogenum was then subjected to a very intensive program of random

mutagen-esis and screening Mutants that showed high levels of penicillin production were selected and subjected to further rounds of mutagenesis, and so on This approach was successful, as indi-cated by the massive increase in production from less than 1 g l−1 to slightly more than 20 g l−1

of culture

Once the antibacterial spectrum of penicillin was determined and found to be far from universal, pharmaceutical companies began the search for other substances with antibacterial activity These screening programs led to the discovery of many antibacterial agents produced by various mem-bers of the actinomycetes Although the search for new antibiotics is never over, intensive research programs involving the use of genetic and metabolic engineering were initiated with the aim of increasing the productivity and potency of current antibiotics For example, the use of genetic and metabolic engineering has increased the yield of penicillin manyfold

1.5 THE RISE AND FALL OF SINGLE-CELL PROTEIN

The latter part of the 1960s saw the rise and fall of single-cell protein (SCP) production from leum or natural gas A large market for SCP was forecast, as the population in the third world, the so-called underdeveloped countries, continued to increase despite a considerable shortfall in food supply However, the development of SCP died in its infancy, largely due to the sharp rise in the price of oil, which made it economically nonviable Furthermore, improvements in the quality and yields of traditional crops decreased demand for SCP production

petro-Box 1.1 “FILL AND SPILL”

This pattern of fermentation is essentially a “batch fermentation” or “fed-batch fermentation” process in which the organism is allowed to grow, and once product formation has reached the maximum level, the fermentation pot is harvested, leaving some 10% of the total volume

as an inoculum for the next batch This process is repeated until the level of contamination becomes unacceptably high

1.6 FERMENTATION BIOTECHNOLOGY AND

THE PRODuCTION OF AMINO ACIDS

The next stage in the development of fermentation biotechnology was dominated by success in the use of regulatory control mechanisms for the production of amino acids The first breakthrough

was the discovery of glutamic acid overproduction by Corynebacterium glutamicum in the late

1950s and early 1960s, when a number of Japanese researchers discovered that regulatory mutants, isolated by virtue of their ability to resist amino acid analogs, were capable of overproducing amino acids The exploitation of such a discovery, however, was hampered by the induction of degradative enzymes once the extracellular concentration of the amino acid increased beyond a certain level; for example, accumulation of tryptophan induced the production of tryptophanase, thus initiating the breakdown of the amino acid This problem was resolved by the use of penicillin, which, with tryptophan as the sole source of carbon in the medium, eliminated the growing cells (i.e., those capable of metabolizing tryptophan, but not those that were quiescent) Following the addition of penicillin, the mutants that had survived the treatment (3 × 10−4) were further tested Enzymic anal-ysis revealed that one mutant was totally devoid of tryptophanase activity This approach was soon extended to cover the production of other amino acids, particularly those not found in sufficient quantities in plant proteins The successful use of regulatory mutants stimulated interest in the use

of auxotrophic mutants for the production of other chemicals The rationale is that auxotrophic mutants will negate feedback inhibition mechanisms and in turn allow the accumulation of the desired end product For example, an arginine-auxotroph was successfully used in the production of ornithine, while a homoserine-auxotroph was used for the production of lysine

1.7 BIOFuELS AND THE “EVOLuTION” OF BIOREFINERIES

In the last decade, fermentation biotechnology has taken a leap forward in consequence of the rising price of oil and international concern about global warming Brazil was a pioneer since the 1970s

in developing bioethanol production from its huge sugarcane industry and alongside it an expanding industry for production of ethanol-utilizing cars; as a result, today about 80% of Brazilian vehicles are fueled by > 20% bioethanol–gasoline mixtures

The United States, the largest consumer of gasoline, has expanded its program and increased its bioethanol production from corn by some sixfold over the last decade, thus superseding Brazil’s production from sugarcane However, this is still only a fraction of U.S gasoline consumption Ethanol production from corn is much less efficient than from sugarcane, so subsidies are required

to market it as a 15% ethanol–gasoline blend, which can be used in conventional car engines without modification Nevertheless, the rocketing price of oil is close to making unsubsidized corn bioetha-nol competitive with gasoline Corn prices are also rising, but the increasing animal feed value of the fermentation residues (distillers’ dried grains, or DDGs) almost compensates

There is therefore a worldwide trend to increase conventional bioethanol production, even if subsidies or tariffs are required and although the carbon footprint of the fuel is only marginally better than that of gasoline The production of bioethanol or the capacity to make it appears to have

a buffering capacity against further increases in oil prices There are many situations in which bioethanol production represents a logical alternative to farming subsidies, such as wheat produc-tion in the European Union There is, however, valid opposition to “first-generation” bioethanol as

a sustainable biofuel on grounds of minimal reduction of global warming and competition with food supply Fortunately, fermentation biotechnology has an answer We harvest at best 20% of what farmers grow and eat only a fraction of that Agricultural residues such as corn stover, cereal straws, and palm oil wastes are lignocellulosic biomass that could dwarf current bioethanol produc-tion Harvested factory residues, such as sugarcane bagasse, corn cobs, wheat bran, palm oilcake, and so on represent an immediate opportunity Lignocellulosic residues alone could yield sufficient bioethanol to fuel all the cars in the world

As we will be discussing in Chapter 9, these residues are composed mostly of bundles of long cellulose fibers (40–50% dry weight) waterproofed by a coat of lignin (15–25%) and embedded in a loose matrix of hemicelluloses (25–35%) Most R&D has been directed to cellulose utilization since

it can be hydrolyzed to glucose and fermented by yeasts However, this is a slow and/or intensive process, so cellulosic ethanol is not competitive with corn or wheat bioethanol, let alone cane bioethanol In contrast, hemicelluloses are easily hydrolyzed to a mixture of C5 and C6 sugars, most of which cannot be fermented by yeasts Many microorganisms can ferment these sugars, but produce lactic acid rather than ethanol Therefore, much effort has gone into genetic manipulation

energy-to divert carbon flux from lactate production energy-to bioethanol formation

Notable among such microorganisms are thermophilic Geobacilli found naturally in compost

heaps and/or silage They can be engineered to produce ethanol from hemicellulosic sugars with yields equivalent to those from yeast fermentations of starch sugars They have the additional advan-tages of extremely rapid continuous fermentations at high temperatures in which ethanol vapor can

be removed continuously from the broth Hence, although such fermentations have not yet been commercialized, calculations indicate that the production cost will be well below that of cane bio-ethanol or gasoline, so the scene is set for the evolution of biorefineries

By analogy with the emergence of petrochemicals from processing the by-products of oil refineries, new biochemicals will emerge from the processing of biomass in biorefineries Sugarcane, for example, could yield sugar or ethanol from the juice, ethanol from the hemicel-lulosic pith, waxes from the external rind, plus heat and electricity from efficient combustion of the lignocellulosic fibers (KTC-Tilby 2011) Alternatively, the fibers could be used directly for packaging or building board or for paper production after ethanol extraction of the lignin The lignin extract could make an efficient biodiesel or give rise to a new range of bioaromatics to compete with those currently derived from oil The residual stillage from ethanol distillation has high animal feed value

Another advantage of such biorefineries is that they would be self-contained units built close to existing food-processing plants, such as sugar refineries, flour mills, or oil-processing plants Since rape seed or palm oils are already used for biodiesel production, an intriguing proposal to use the by-product glycerol together with hemicellulosic sugars for high-yield bioethanol production would produce biorefineries to rival oil refineries in producing both fuels from a single raw material The scene is therefore set for the evolution of biorefineries

1.8 IMPACT OF FuNCTIONAL GENOMICS, PROTEOMICS,

METABOLOMICS, AND BIO-INFORMATICS ON THE

SCOPE AND FuTuRE PROSPECTS OF FERMENTATION

MICROBIOLOGY AND BIOTECHNOLOGY

Modern biotechnology, a consequence of innovations in molecular cloning and overexpression in the early 1970s, has started in earnest in the early 1980s after the manufacture of insulin, with the first wave of products hitting the market in the early 1990s During this early period, the biotechnology companies focused their efforts on specific genes/proteins (natural proteins) that were of well-known therapeutic value and typically produced in very small quantities in normal tissues Later, monoclonal antibodies became the main products of the biopharmaceutical industry

In the mid-1980s, Thomas Roderick coined the term genomics

to describe the discipline of mapping, sequencing, and analyzing

genomic DNA with the view to answering biological, medical, or

industrial questions (Jones 2000) Recent advances in functional

genomics, stimulated by the Human Genome Project and

com-puter software technology, led many biotechnologists to venture

from the traditional in vivo and in vitro research into the in silico

Functional genomics is a discipline of

biotech-nology that attempts to exploit the vast wealth

of data produced by genome-sequencing projects A key feature of functional genomics

is their genome-wide approach, which ably involves the use of a high-throughput approach.

invari-approach, thus establishing a new science in the shape of bio-informatics In this invari-approach, biologists employ computers to store, retrieve, analyze, and compare a given sequence of DNA or protein with those stored in data banks from other organisms Microbial biotechnologists were

micro-quick to realize that the key to successful commercialization of a given sequence relied on the development of an innovative meth-odology (bio-informatics) that facilitates the transformation of a given sequence into a diagnostic tool and/or a therapeutic drug, thus bridging the gap between academic research and commer-cialization

The new innovations in functional genomics—proteomics, metabolomics, and bio-informatics—will certainly play a major role in transforming our world in an unparalleled way, despite political and ethical controversies

In this book, we have addressed the multidisciplinary nature

of this subject and highlighted its many fascinating aspects in the hope that we are providing a stepping stone in its progress

As we enter a new era in which the use of renewable resources for the production of desirable end products is recognized as an urgent need, fermentation microbiology and biotechnology have

a central role to play

Stephenson, M 1949 Bacterial Metabolism London: Longmans, Green.

Proteomics is a new discipline that focuses

on the study and exploitation of proteomes

A proteome is the complete set of proteins

expressed by a given organism under certain

conditions.

Metabolomics is a new discipline that focuses

on the study and exploitation of

metabo-lomes A metabolome refers to the complete

set of primary and secondary metabolites as

well as activators, inhibitors, and hormones

that are produced by a given organism under

certain conditions It is noteworthy, however,

that it is not currently possible to analyze the

entire range of metabolites by a single

ana-lytical method.

Fermentation: Central

and Modern Concepts

E.M.T El-Mansi, F Bruce Ward, and Arun P Chopra

CONTENTS

2.1 Introduction 102.2 Chemical Synthesis of Bacterial Protoplasm or Biomass 112.2.1 Central and Intermediary Metabolism 112.2.2 Anaplerotic Pathways 122.2.3 Polymerization and Assembly 132.2.4 Biomass Formations 132.2.5 Logarithms 142.2.5.1 Use of Semilogarithmic Graph Paper 142.3 Growth Cycle 142.3.1 Lag Phase 162.3.2 Exponential Phase 172.3.2.1 Metabolic Interrelationship between Nutrient Limitations

and Specific Growth Rate (μ) 192.3.3 Stationary Phase and Cell Death 212.3.4 Maintenance and Survival 222.4 Diauxic Growth 242.5 Growth Yield in Relation to Carbon and Energy Contents of Growth Substrates 242.6 Fermentation Balances 262.6.1 Carbon Balance 262.6.2 Redox Balance 262.7 Efficiency of Central Metabolism 262.7.1 Impact of Futile Cycling on the Efficiency of Central Metabolism 262.7.2 Impact of Metabolite Excretion on the Efficiency of Central Metabolism 272.8 Continuous Cultivation of Microorganisms 282.8.1 Types of Continuous Cultures 292.8.2 Principles and Theory of Continuous Cultures 292.8.2.1 Microbial Growth Kinetics in Continuous Cultures 292.8.2.2 Interrelationship between Growth Rate (µ) and Dilution Rate (D) 302.8.2.3 Efficiency and Productivity of Fermentation Processes 312.9 Current Trends in the Fermentation and Pharmaceutical Industry 312.9.1 “Quiescent Cell Factory”: A Novel Approach 322.9.2 Applications of Batch-Fed Two-Stage Fermentation in the Production of

Biopharmaceuticals: A Robust Approach 32

“ et le rêve de toute cellule: devenir deux cellules”.

Jacques Monod, 1970 2.1 INTRODuCTION

Microorganisms play a central role in the production of a wide range of primary and secondary metabolites, industrial chemicals, enzymes, and antibiotics The diversity of fermentation processes may be attributed to many factors, including the high surface-to-volume ratio and the ability to utilize a wide spectrum of carbon and nitrogen sources The high surface-to-volume ratio supports

a very high rate of metabolic turnover: for example, the yeast Saccharomyces cerevisiae has been

reported to be able to synthesize protein by several orders of magnitudes faster than plants On the other hand, the ability of microorganisms to adapt to different metabolic environments makes them capable of utilizing inexpensive renewable resources such as wastes and by-products of the farming and petrochemical industries as the primary carbon source Industrially important microorganisms include bacteria, yeasts, molds, and actinomycetes

While the metabolic route through which glucose is converted to pyruvate, glycolysis, is versally conserved among all organisms, microorganisms differ from eukaryotes in their ability

uni-to process pyruvate through a diverse array of routes (Figure 2.1), giving rise uni-to a multitude of

2.10 Microbial Fermentations and The Production of Biopharmaceuticals 332.10.1 Production of Insulin: A Case Study 332.10.1.1 History and Background 332.10.1.2 Cloning and Commercial Production of Insulin 332.10.2 Protein Engineering of Insulin 342.10.2.1 Fast-Acting Insulin Analogs 342.10.2.2 Long-Acting Insulin Analogs 34Summary 35References 35

Glucose Glycolysis

H 3 C

Alcoholic fermentation

Proplonic fermen tation

ferm entation

Lactic acid

OH Homolactic-acid O

Pyruvic acid O

FIGuRE 2.1 Diversity of fermentation pathways among microorganisms.

different end products Such diversity has been fully exploited by fermentation technologists for the production of fine chemicals, organic solvents, and dairy products.

Prokaryotic organisms differ from yeast and fungi as well as other eukaryotes in a number

of ways, including cell structure and growth cycle For example, while DNA is ized within the nucleus in eukaryotes, it is neatly folded within the cytoplasm in prokaryotes The site of oxidative phosphorylation represents yet another example; while oxidative phosphorylation

compartmental-is associated with mitochondria in eukaryotic organisms, oxidative phosphorylation in prokaryotes

is associated with cytoplasmic membranes Furthermore, the newly synthesized DNA molecules in prokaryotes need no special assembly to form a chromosome, as the DNA is already attached to the bacterial membrane, thus ensuring its successful segregations into two daughter cells Any treat-ment that dislodges the DNA or compromises the attachment of DNA to the cytoplasmic membrane may lead to failure in segregation of DNA This was the scientific bases on which sodium dode-

cyle sulphate (SDS) was used to cure large endogenous plasmids from Klebsiella pneumoniae and

Pseudomonas putida (El-Mansi et al 2001)

2.2 CHEMICAL SYNTHESIS OF BACTERIAL PROTOPLASM OR BIOMASS

In addition to carbon, nitrogen, phosphate, potassium, sulfur,

irons, and magnesium, the chemical input required for growth

also include trace elements The necessity for such a multitude of

inputs is paramount as it is required for enzymic activities The

contribution of each additive to biomass and product formation

can be assessed quantitatively

2.2.1 C entral and I ntermedIary m etabolIsm

Throughout Section 2.2, we will be addressing the function of enzymes of central and intermediary metabolism (Figure 2.2) with respect to their role in bacterial growth, and as such it would be help-ful if we were to appreciate the functional role of each

During growth on a glucose minimal medium under aerobic conditions, Escherichia coli

catabolizes glucose through glycolysis and the Krebs cycle (Figure 2.2) to bring about its formation to biosynthetic precursors; there are 12 in total, over half of which are phosphorylated adenosine-5’-triphosphate (ATP) and reducing powers in the shape of NADH, NADPH, and FADH2

trans-as well trans-as esterified CoA derivatives (e.g., succinyl CoA and malonyl CoA) In this process a whole host of different reactions, the fueling reactions (Figure 2.3) are coordinated in a precise manner

to ensure successful adaptation and survival of the organism Once the biosynthetic precursors, ATP, and reducing powers are generated, the organism employs anabolic enzymes to convert those biosynthetic precursors into monomers (building blocks) in the shape of various sugars, amino acids, and nucleotides The monomers are then polymerized into polymers (macromolecules), which, in turn, are assembled into different structures or organelles and thence to biomass (Figure 2.3) The central metabolic pathways (glycolysis, the pentose phosphate pathway, and the Krebs cycle) fulfill

both catabolic (from cata, a Greek word for breakdown) and anabolic (from ana, a Greek word for buildup) functions and as such may be referred to as amphibolic pathways.

Although the central metabolic pathways are highly conserved in all organisms, microorganisms

display a great deal of diversity even within a single species such as E coli in response to growth

conditions As can be seen from Figure 2.2, microorganisms are capable of utilizing a wide range

of substrates and afford a different entry point for each into central metabolism As the point of entry into the central metabolic pathways of the Krebs cycle vary from one substrate to another, the makeup of the enzymic machinery necessary for metabolism changes accordingly (Guest and

Russell 1992) For example, during growth on acetate or fatty acids, E coli expresses uniquely the

anaplerotic sequence of glyoxylate bypass (Cozzone 1998)

While central metabolism (glycolysis, the pentose phosphate pathway, and the Krebs cycle) is concerned with the breakdown of growth substrates and their conversion to the

12 biosynthetic precursors, ATP, and ing powers, intermediary metabolism focuses

reduc-on the creduc-onversireduc-on of those biosynthetic cursors to monomers and their subsequent polymerization and assembly into polymers.

pre-2.2.2 a naplerotIC p athways

During growth on glucose or other acetogenic substrates (i.e., those that support flux to acetate excretion), metabolites of the Krebs cycle, namely, α-ketoglutarate, succinyl CoA, and oxaloac-etate (Figure 2.2), are constantly being withdrawn for biosynthesis It follows, therefore, that such intermediates must be replenished; otherwise, the cycle will grind to a halt The reactions that fulfill

such a function are known as anaplerotic, a Greek word for replenishing, pathways The makeup

Fructose phosphate

Glucose phosphate

6-Pentose phosphate

4-Serine, glycine, cystine 3-Phosphoglycerate

Aromatic amino acids

Fructose 1,6-bisphosphate

ADP ATP Glucose

pyruvate

Phosphoenol-Pyruvate Acetyl-CoA

Valine, leucine, alanine

Lactate Fatty acids

Acetate Aromatic &

branched amino acids Oxaloacetate

Malate

Acetyl-CoA Glyoxylate

Isocitrate

CO2Fumarate

Succinate

Methionine, valine, isoleucine

Porphyrins

Histidine

glutarate

α-Keto-Glutamate, glutamine, proline, arginine Succinyl-

Tyrosine, phenylalanine

Aspartate, asparagine, threonine, isoleucine, methionine,

ADP ATP

1

5 6

FIGuRE 2.2 An overview of the metabolic pathways of central and intermediary metabolism employed by

E coli for the conversion of glucose and other substrates to biosynthetic precursors; indicated by heavy-dotted arrows, ATP, and reducing powers Entry of substrates other than glucose into central metabolism is indicated

by dashed arrows Key enzymes are as follows: 1, glucose–phosphoenolpyruvate (PEP) phosphotransferase system; 2, the pentose phosphate pathway; 3, pyruvate kinase; 4, PEP carboxylase; 5, PEP carboxytranspho- sphorylase; 6, pyruvate carboxylase; 7, isocitrate lyase; 8, malate synthase; 9, malic enzyme; and 10, PEP carboxykinase.

of the anaplerotic enzymes differs from one phenotype to another, in other words, it is substrate dependent For example, during growth on glucose, phosphoenolpyruvate carboxylase, pyruvate carboxylase, and phosphoenolpyruvate carboxytransphosphorylase (Figure 2.2) represent the full complement of anaplerotic enzymes that may be used in full or in part depending on the organism

under investigation During growth on acetate, however, E coli employs the glyoxylate bypass operon

enzymes, namely, isocitrate lyase and malate synthase, as an anaplerotic sequence Interestingly,

however, while the enzymes of the glyoxylate bypass in E coli form an operon and include the

bifunctional regulatory enzyme isocitrate dehydrogenase kinase/phosphatase, these enzymes are

not organized in the same way in Corynebacterium glutamicum.

2.2.3 p olymerIzatIon and a ssembly

Following transcription through the activity of RNA polymerase, mRNA is translated into protein

by ribosomes In this process, ribosomes attach to mRNA together with co-factors, enzymes, and complementary tRNA, thus forming a polysome and, in turn, initiating the synthesis of polypep-tides Polysomes are one of the most abundant organelles in growing cells; each polysome contains approximately 20 subunits of ribosomal RNA (Ingraham et al 1983)

2.2.4 b Iomass F ormatIons

The rate of product formation in a given industrial process, a significant parameter, is directly related

to the rate of biomass formation, which, in turn, is influenced directly or indirectly by a whole host

of different environmental factors (e.g., oxygen supply, pH, temperature, and accumulation of itory intermediates) It is, therefore, important that we are able to describe growth and production in quantitative terms The study of growth kinetics and growth dynamics involves the formulation and

inhib-Lipid

Inclusion bodies

Cell envelope

Flagella Pili

ribosomes

Poly-Biomass

Cytosol

Lipopolysaccharide Glycogen Peptidoglycan

Protein

RNA

DNA

Amino acids (20)

Nucleotides (8)

Nucleotide

Sugars (25)

Fatty acids (8)

FIGuRE 2.3 A diagrammatic representation of the reactions involved in the conversion of glucose and

simple salts to biomass of E coli (This figure is a slight modification of Ingraham, J.L., Neidhardt, F.C., and Schaechter, M., A Molecular Approach, Sinauer Associates, Inc., Sunderland, MA, 1990, reproduced with the

kind permission of Sinauer Associates, Inc., Sunderland, MA, 1990.)

use of differential equations (for more details, see Chapter 3) While the mathematical derivation

of these equations is beyond the scope of this chapter, it is important that we understand how such equations can be used to further our understanding of microbial growth in general and its impact on product formation (yield) in particular

In microbiology we generally deal with very large numbers, and for convenience we express these numbers as multiples of 10 raised to an appropriate power; for example, 1 million (1,000,000)

is written as 1 × 106

2.2.5 l ogarIthms

Logarithms, otherwise known as logs, are very useful mathematical functions in both microbiology

and biochemistry, especially in measuring important parameters such as growth, death, and enzyme kinetics

The logarithm of a number is the exponent to which we must raise a base to obtain that number

The exponent is also known as the index or the power For example, consider the number 1000 and

the base 10; the number 10 is raised to the power 3 to obtain 1000; it follows that the logarithm to the base 10 of 1000 is 3 The base of a logarithm is conventionally written as a subscript For example, the logarithm to the base 10 is written as log10 and the logarithm to the base e is written as loge If a log is given without a subscript, it denotes a logarithm to the base 10 Logarithms to the base e (loge)

are also called natural logarithms, which may also be written as In.

2.2.5.1 use of Semilogarithmic Graph Paper

A semilogarithmic (semilog) graph paper is a graph paper that has an arithmetic scale on one axis

and a logarithmic scale on the other Although, it may appear very strange, it is user friendly and

is very useful as it allows you to plot your data directly without having to calculate logs On the logarithmic scale, there are a number of cycles Each cycle represents a change in data of an order

of magnitude, a factor of 10; any set of factors of 10 can be used, but remember that there is no zero

on the log scale

In plotting your data on semilog paper, the independent variable is plotted on the x-axis (abscissa), while the dependent variable is plotted on the y-axis (ordinate) of the graph For example, in deter-

mining the mean generation time (T) of a given organism under certain conditions, time is the

independent variable and is plotted on the x-axis, while the number of cells, which increases nentially during the course of the logarithmic phase of growth, is plotted on the y-axis However,

expo-it is noteworthy to remember that in determining the minimum inhibexpo-itory concentration (MIC)

of antibiotics, the drug dosage increases in an exponential fashion and as such the log scale here becomes the x-axis

2.3 GROWTH CYCLE

Now let us consider the basic equation used to describe microbial growth:

dx dt

ax b

Equation 2.1 implies that the rate of biomass (x) formation changes as a function of time (t) and that the rate of change is

directly proportional to the concentration of a particular factor

(a) such as growth substrate or temperature but is inversely portional to the concentration of another factor (b) such as inhibi- tors In Equation 2.1, both a and b are independent of time t, and

pro-While growth kinetics focuses on the

mea-surement of growth rates during the course

of fermentation, growth dynamics relates the

changes in population (biomass) to changes

in growth rate and other parameters (e.g., pH

and temperature) To unravel such intricate

interrelationships, the description of growth

kinetics and growth dynamics relies on the

use of differential equations.

the proportionality factor in Equation 2.1 can in effect be ignored In the early stages of any tation process, the increase in biomass is unrestricted, and, as such, the pattern of growth follows an autocatalytic first-order reaction (autocatalytic growth) up to a

fermen-point where either side of Equation 2.1 becomes negative,

result-ing in autocatalytic death

During batch fermentation, a typical pattern of growth curve,

otherwise known as the growth cycle, is observed (Figure 2.4)

Clearly, a number of different phases of the growth cycle can be

differentiated These are

6 Accelerated death phase

7 Exponential death phase

8 Death or survival phase

The changes in the specific growth rate (μ) as the organism progresses through the growth cycle can also be seen in Figure 2.4

We shall now describe the metabolic events and their implications in as far as growth, vival, and productivity are concerned Naturally, the scenario begins with the first phase of the growth cycle

sur-The term autocatalytic growth is generally

used to indicate that the rate of increase in biomass formation in a given fermentation

is proportional to the original number of cells present at the beginning of the process, thus reflecting the positive nature of growth.

The term growth cycle is used to describe

the overall pattern displayed by isms during growth in batch cultures It is noteworthy that such a cycle is by no means

microorgan-a fundmicroorgan-amentmicroorgan-al property of the bmicroorgan-acterimicroorgan-al cell, but rather a consequence of the progressive decrease in food supply or accumulation of inhibitory intermediates in a closed system

to which no further additions or removals are made.

0.8 0.4 0.0

–1 ()

I II III IV V VI VII VIII

1.0 0.8 0.6 0.4

0.2

FIGuRE 2.4 Typical pattern of growth cycle during the growth of microorganisms in batch cultures; the

vertical dotted lines and the roman numerals indicate the changes in specific growth rate (μ) throughout the cycle.

2.3.1 l ag p hase

In this phase, the organism is simply faced with the challenge of adapting to the new environment While adaptation to glucose as a sole source of carbon appears to be relatively simple, competition with other carbon sources, although complex, is resolved in favor of glucose through the operation

of two different mechanisms, namely, catabolite inhibition and catabolite repression

Adaptation to other carbon sources, however, may require the induction of a particular set of enzymes that are specifically required to catalyze transport and hydrolysis of the substrate (e.g., adaptation to lactose) or to fulfill anaplerotic as well as regulatory functions, as is the case in adap-tation to acetate as the sole source of carbon and energy Irrespective of the mechanisms employed for adaptation, the net outcome at the end of the lag phase is a cell that is biochemically vibrant (i.e., capable of transforming chemicals to biomass)

Entering a lag phase during the course of industrial fermentation is not desirable as it is very costly and as such should be avoided The question of whether a particular organism has entered a

lag phase in a given fermentation process can be determined graphically by simply plotting log n

(biomass) as a function of time, as shown in Figure 2.5

Note that the transition from the lag phase to the exponential phase involves another phase: the acceleration phase, as described in Figure 2.4 This difficulty can be easily overcome by extrapolat-ing the lag phase sideways and the exponential phase downward as shown, with the point of inter-ception (L) taken as the time at which the lag phase ended What is also interesting about the graph

5.0 4.0 3.0 2.0

1.0 0.8 0.6 0.4 Optical density (OD)0.2 0.1

0.05 0.04 0.02 0.01

log n – log n0log n

L t

log n0

FIGuRE 2.5 Graphical determination of the lag phase and the number of viable cells at the onset of batch

fermentation; as the exponential phase is extrapolated downward, it intercepts the extrapolated line of the lag phase and the ordinate, respectively (see text for details).

in Figure 2.5 is that if one continues to extrapolate the exponential phase downward, then the point

at which the ordinate is intersected gives the number of cells that were viable and metabolically active at the point of inoculation

The question of whether a lag (L) has occurred during the course of the fermentation process and

for how long can be easily determined by

logn logn log

−

2 (2.2)

where n is the total number of cells after a given time (t) since the start of fermentation, n0 is the

number of cells at the beginning of fermentation, and T is the organism’s mean generation time

(doubling time) Equation 2.2 describes the exponential growth, taking into consideration a lag phase in the process In Section 2.3.2, we shall describe the exponential phase in general and the derivation of Equation 2.2 in particular

2.3.2 e xponentIal p hase

In this phase, each cell increases in size, and providing that conditions are favorable, it divides into two, which, in turn, grow and divide; and the cycle continues During this phase, the cells are capable of transforming the primary carbon source into biosyn-

thetic precursors, reducing power and energy, which is generally

trapped in the form of ATP, phosphoenolpyruvate (PEP), and

proton gradients The biosynthetic precursors thus generated are

then channeled through various biosynthetic pathways for the biosynthesis of various monomers (amino acids, nucleotides, fatty acids, and sugars) that, in turn, are polymerized to give the required polymers (proteins, nucleic acids, ribonucleic acids, and lipids) Finally, these polymers are assem-bled in a precise way, and the cell divides to give the new biomass characteristic of each organism (Figure 2.3) The time span of each cycle (cell division) is known as generation time or doubling

time, but because we generally deal with many millions of cells in bacterial cultures, the term mean

generation time (T) is more widely used to reflect the average generation times of all cells in the

culture Such a rate, providing conditions are favorable, is fairly constant

If a given number of cells (n0) is inoculated into a suitable medium and the organism was allowed

to grow exponentially, then the number of cells after one generation is 2n0; at the end of two

gen-erations, the number of cells becomes 4n0 (or 22n0) It follows, therefore, that at the end of a certain

number of generations (Z), the total number of cells equals 2 Z n0 If the total number of cells, or its

log value, at the end of Z generations is known, then

log n  = log n0 + Z log 2 (2.4)

To determine the number of cell divisions, that is, the number of generations (Z) that have taken

place during fermentation, Equation 2.4 can be modified to give

If during the course of a particular fermentation, a lag time has been demonstrated, Equation 2.6 can be modified to take account of this observation The modified equation is

While Equation 2.8 describes the exponential phase of growth in pure terms, Equation 2.9, however, takes into consideration the existence of a lag phase in the process

The exponential scale (i.e., 2, 4, 8, 16, and so on) demonstrated in Figure 2.6 obeys Equations 2.8

for logarithmic growth Note the different position of log n0 to that cited in Figure 2.5

Although the use of log to the base 2 has the added advantage of being able to determine the

number of generations relatively easily, because an increase of one unit in log 2n corresponds to one

generation, the majority of researchers continue to use log to the base 10 In this case, the slope of the line (Figure 2.6) equals μ/2.303 The relationship between T and the specific growth rate (μ) can

be described mathematically by

ln 2 = μT (2.10)

1.0 20 40 60

0.8 0.6 0.4 Optical density (OD)0.2

0.1 10

8 6 4 2

FIGuRE 2.6 Graphical determination of the mean generation time (T) during batch fermentation; note the

different position of log n with respect to that in Figure 2.2.

intracel-population (N) represents, therefore, the rate of growth of each individual cell in the intracel-population

multiplied by the total number of cells Such a rate can be described mathematically by this ferential equation:

dif-dN

dt = µN (2.13)

This equation implies that the rate of new biomass formation is directly proportional to the cific growth rate (μ) of the organism under investigation and the number of cells (N) This pattern

spe-of growth may be referred to as autocatalytic, a term described in this chapter If Equation 2.13

describes the exponential phase correctly, then a straight line should be obtained when ln N is

plotted as a function of time (t) During the exponential phase, it is generally assumed that all cells

are identical, and as such the specific growth rate (μ) of individual cells equals that of the whole

population Equation 2.13 can therefore be rearranged to take account of the fact that dN/dt is portional to the number of cells (N) and that the specific growth rate (μ) is a proportionality factor

The specific growth rate (μ) is usually expressed in terms of units per hour (h–1)

Although Equations 2.9 and 2.10 describe the exponential phase satisfactorily, in some

fermenta-tions, as is the case during growth of E coli on sodium acetate (El-Mansi unpublished results), the

organism fails to maintain a steady state for any length of time and so μ falls progressively with time until the organism reaches the stationary phase Such a drop in growth rate (μ) can be accounted for

by a whole host of different factors, including nutrient limitations, accumulation of inhibitors, and/

or the crowding factor (i.e., as the population increases in number, μ decreases)

2.3.2.1 Metabolic Interrelationship between Nutrient

Limitations and Specific Growth Rate (μ)

To account for the effect of nutrient limitations on growth rate (μ), Monod modified Equation 2.14

so that the effect of substrate concentration (limitations) on μ could be assessed quantitatively Monod’s equation is

+

m S