Concepts in Biotechnology E-Book

Klaus Buchholz

• Herausgeber: Wiley-VCH
• Kategorie: Fachliteratur
• Sprache: Englisch
• Veröffentlichungsjahr: 2014

Seitenzahl: 908

Beschreibung

Adopting a unique approach, this novel textbook integrates science and business for an inside view on the biotech industry. Peering behind the scenes, it provides a thorough analysis of the foundations of the present day industry for students and professionals alike: its history, its tools and processes, its markets and products. The authors, themselves close witnesses of the emergence of modern biotechnology from its very beginnings in the 1980s, clearly separate facts from fiction, looking behind the exaggerated claims made by start-up companies trying to attract investors. Essential reading for every student and junior researcher looking for a career in the biotech sector.

Leseprobe

Contents

Preface

Abbreviations and Glossary

Part One History

1 Introduction

2 The Early Period to 1850

2.1 Introduction

2.2 Experimental Scientific Findings

2.3 Application

2.4 Theoretical Approaches

References

3 The Period from 1850 to 1890

3.1 Introduction

3.2 Experimental Findings

3.3 Practical Application, Technical Progress and Institutional Development

3.4 Theoretical Approaches

References

4 The Period from 1890 to 1950

4.1 Introduction

4.2 Research – Advances in the Basics of Biotechnology: Experimental Findings

4.3 Technological Development, Progress and Application

4.4 Theoretical Approaches

References

5 Outlook, from 1950 Onwards: Biotechnology – Science or What?

5.1 Introduction

5.2 Traditional Biotechnology and the Dechema Report

5.3 The Changing Focus in Bt in the Usa in the Early 1980s

5.4 Conclusions

References

Part Two The New Paradigm Based on Molecular Biology and Genetics

6 Broadening of Biotechnology through Understanding Life, Genetics and Evolution

References

7 The Beginnings of the New Biotechnology

7.1 Introduction

7.2 The Beginnings of Evolution Theory and Genetics

7.3 The Origin of Recombinant Dna Technology

7.4 Oligonucleotide Synthesis Leads to Protein Engineering

7.5 Synthetic Dna, Reverse Transcriptase: Isolating Genes

7.6 Biodiversity and Gene Mining

7.7 Creating New Diversity by Design or Empirically

7.8 ‘Genetic Fingerprinting’

7.9 Inherited Predisposition to Disease

References

8 Ethical Aspects Related to Genome Research, and Reproductive Medicine

8.1 Negative Public Reaction to Gene Technology

8.2 Ethical Aspects: Animal Cloning and Fertility Research

References

9 Elucidating Protein Structure: The Beginnings of Rational Protein Design

9.1 Cambridge England, the Cradle of Structural Analysis of Macromolecules

9.2 Redesigning the Protein Core

9.3 Redesigning the Protein by Altering Primary Sequence

9.4 Post Translational Modifications

9.5 Total Chemical Synthesis

9.6 Validation of Drug Design Based on the known Structure of the Target

9.7 General Considerations in Drug Development

References

10 The Development of Antibodies as Pharmaceutical Products

10.1 An Introduction to the Immune System

10.2 The Beginnings of Applied Immunology

10.3 Monoclonal Antibodies

10.4 Producing Antibodies via rDNA and Combinatorial Biology

10.5 Affinity Enrichment on Surfaces of Immobilized Target Molecules

10.6 Mice with Human Antibody Gene Repertoires

10.7 Two Severe Setbacks during Clinical Testing

10.8 A Survey of Therapeutic Antibodies

References

11 Hereditary Disease and Human Genome Analysis

11.1 Introduction

11.2 Heredity Studies and Family Counselling

11.3 Early Attempts to Analyse the Human Genome

11.4 The Personalized Genome and Personal Medicine

11.5 Analysing the Effect of the Environment on the Human Genome: Epigenetics

References

12 Transgenic Animals and Plants

12.1 Introduction

12.2 Stem Cells and Gene Targeting

References

13 Extrapolating to the Future

13.1 Summary of the Status Quo

13.2 Insect Control Through ‘Sterile’ Males (Sit)

13.3 The Future of Gene Therapy

13.4 Stem Cell Therapy

13.5 Flash Sequencing Dna: A Human Genome Sequence in Minutes?

13.6 Systems Biology and Looking for ‘Druggable’ Targets

13.7 Synthetic Biology

References

14 Biotechnology and Intellectual Property

14.1 Introduction

14.2 Patents Ensure Growth and Rapid Dissemination of Knowledge

14.3 Owning a Patent does not simply Mean that it can be Implemented: ‘Freedom to Operate’ (Fto)

14.4 Life-forms as ‘Novel Subject Matter’ for Patents?

14.5 Technology ‘State of the Art’: Precedence/Directives, not Fixed by Law

14.6 Who can make Decisions about Public Morality?

14.7 Biotechnology-orientated Directives Guide Patenting Decisions

References

Part Three Application

15 Bioprocess Engineering

15.1 Introduction

15.2 Aspects of Applied Microbiology

15.3 Biocatalysis

15.4 Biochemical Engineering

15.5 Process Sustainability and Ecological Considerations

15.6 Biosystems Engineering, including Omics Technologies

15.7 Outlook and Perspectives

References

16 Industrial Biotechnology

16.1 Introduction

16.2 General Aspects

16.3 Commodities

16.4 Chemicals

16.5 Food Processing and Products

16.6 Environmental Processes

16.7 Summary, Trends and Perspectives

References

17 Pharmaceutical Biotechnology

17.1 Introduction

17.2 Drug Targeting, Discovery Strategies and Development

17.3 Pharmaceuticals Production

17.4 Products, Pharmaceuticals Made by Biotechnology

17.5 Medicinal Techniques, Diagnostics

17.6 Business: Companies and Economic Aspects

References

18 Plant Biotechnology

18.1 Introduction

18.2 Political, Ethical and Biosafety Aspects

18.3 Research and Development

18.4 Application of Modified Plants and Products

18.5 Economic Aspects

18.6 Summary and Outlook

References

Index

Related Titles

Nicolaou, K. C., Montagnon, T.Molecules that Changed the World385 pages2008HardcoverISBN: 978-3-527-30983-2

Sneader, W.Drug DiscoveryA History472 pagesSoftcoverISBN: 978-0-471-89980-8

Ho, R. J. Y., Gibaldi, M.Biotechnology and BiopharmaceuticalsTransforming Proteins and Genes into Drugs576 pages2003SoftcoverISBN: 978-0-471-20690-3

Greenberg, A.The Art of ChemistryMyths, Medicines, and Materials384 pages2003HardcoverISBN: 978-0-471-07180-8

Sapienza, A. M., Stork, D.Leading Biotechnology AlliancesRight from the Start216 pages2001HardcoverISBN: 978-0-471-18248-1

The Authors

Prof. em. Dr. Klaus BuchholzTechnical University BraunschschweigInstitute for Chemical EngineeringHans-Sommer-Str. 1038106 BraunschweigGermany

Prof. em. Dr. John CollinsTechnical University BraunschschweigLife Sciences Facultyc/o Helmholtz Centre for Infection Research – HZIAG Directed EvolutionInhoffenstr. 738124 BraunschweigGermany

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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© 2010 WILEY-VCH Verlag & Co. KGaA,Boschstr. 12, 69469 Weinheim, Germany

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Cover Illustration:Production fermenters with kind permission by Roche Penzberg, GermanyCover Adam Design, WeinheimTypesetting Thomson Digital, Noida, IndiaPrinting and Binding betz-druck GmbH, Darmstadt

ISBN: 978-3-527-31766-0

For

Diana and Marie-Christiane

Preface

Over the last century the development of Biotechnology (BT) has followed fascinating pathways to influence ever more aspects of our lives and to provide significant contributions to the improvement of the quality of life. BT flourished in parallel with biological sciences as a result of insights into the molecular details of genetics and the control of biochemical reactions. Following a long-standing tradition, this knowledge was translated by commercial application for human benefit. It enabled biological pathways to be manipulated and even created for the purpose of manufacturing products and developing processes and services on an industrial scale. Historically, controlled fermentation was used to provide efficient storage for food thus enabling a population to survive periods of cold or drought. By the end of the last century biotechnology had developed into a science and engineering discipline in its own right and is considered to be a field of industrial activity with major economic relevance. The applications of BT extend beyond historical tradition, ranging from production of chemicals, bio-fuels and pharmaceuticals to ensuring a continued supply of clean water.

This book reviews the progress of biotechnology over time and highlights the seminal events in this field. It gives an introduction to the main developments, the principles or concepts, and key researchers involved in pioneering work and in conclusion, attempts to extrapolate to further advances expected in the near future. In view of the extensive range of biotechnological activities it was necessary to concentrate on essentials, illustrated with selected examples, as opposed to using an encyclopedic approach. This book is intended to guide the reader through the diverse fields of activity in BT and encourage further reading in the form of books, specialised reviews and original literature as provided in the reference sections. It is envisaged that the readership of this book will include students of biology, biotechnology and biochemical engineering, in addition to scientists and engineers already engaged in or proposing to work in the fields of BT and related disciplines. It may also serve as a broad introduction to BT for other readers who are interested in an overview of the subject, ranging from historical aspects to the latest developments which are largely a result of the accelerated research in molecular biology and bioinformatics that has taken place over the last 20 years.

The historical aspects of BT are discussed in the opening chapters which highlight the role of inquisitiveness and the thirst for knowledge and understanding of natural processes. This involves a discussion of reputation-building, the interplay of economics and business as well as the role of and dependence on theories. We trace the developments in chemistry and physics that became a prerequisite for the study of the chemical nature of the components involved in biological processes such as brewing, wine and bread making. Heated discussions centring on both the vitalist and chemical theories resulted not only in the emergence of theories and paradigms but also in their reversal. The close interaction of scientists, craftsmen and industry together with significant stimulus, promoted continued research.

Pasteur and Koch established the science of microbiology. A few decades later Buchner finally refuted the last metaphysical hypothesis that processes in living cells required a ‘vis vitalis’, a vital factor and following this biochemistry emerged as a new speciality. Biotechnological engineering was based on more precise control of the microbial fermentations involved in food processing including large-scale processes for the manufacture of beer, wine, cheese, bread etc. together with the use of sterile starting materials. This led to the subsequent production of fuels and chemical components for polymers and explosives particularly during war time, and the manufacture of antibiotics and vaccines. This in turn stimulated detailed studies on the manufacture of products from microbial fermentations. By the midtwentieth century, biotechnology had become an accepted speciality.

Basic research in biochemistry, molecular biology and genetics dramatically broadened the field of life sciences and at the same time unified them by the study of genes and their relatedness throughout the evolutionary process. In Part 2 we discuss the development of this fruitful interplay and describe how it broadened the scope of accessible products and services, at the same time making production cheaper, safer, more reproducible and more reliable. Rapid acceleration of gene and protein analysis caused an explosion of data which led to the emergence of bioinformatics. This opened up new avenues for medical analysis that was orientated more towards preventive measures rather than corrective intervention. This is a continuing trend which is substantiated by the prediction that during the next few years affordable analysis of the complete genetic potential of an individual will be available within hours. New areas of research have evolved such as systems biology in which living systems can be successfully modelled as networks of ever-increasing complexity. As the volume of information increases and modelling improves so does the probability that insight into potential targets for pharmaceuticals can be better translated into developing successful medicines. To foster such aims, centres for translational medicine are being founded in many cities where medical schools and hospitals participate in close interactions with basic research institutes.

The understanding of the fundamental programming of animal cells in the developing embryo and in particular the discovery of a small number of proteins capable of guiding stem cell differentiation and even the reprogramming of already differentiated cells, has opened up perspectives for a completely new and very exciting branch of biotechnology in the area of tissue and organ synthesis for regenerative medicine. In combination with advances in fertility medicine this has also led to the cloning of animals and the production of transgenic animals. One aspect of this technology is the use of tissue cloning to produce human tissue cultures as models for inherited disease.

In Part 3 we discuss engineering and applied topics. Biochemical and bioprocess engineering constitute the basis for translating scientific innovation and development into industrial processes. They represent an interdisciplinary field based on molecular biology, biochemistry and engineering disciplines. As a result of the progress in molecular biology, new tools known as the ‘omics’ were developed: genomics, proteomics and metabolomics, to mention only the most common. Biosystems engineering or systems biotechnology, integrates the approaches and the extensive volume of data derived from these specialities and from bioreaction engineering in a ‘holistic’ approach, using bioinformatics tools.

Industrial biotechnology, with its historical roots, continues in diverse industrial fields of activity including food and feed and commodities such as enzymes for use in detergents, bio-fuel and energy production, polymer manufacture and the development and production of many drug constituents, as well as providing services, for example in waste treatment and other processes related to environmental protection.

The approval in 1982 of recombinant human insulin produced in E. coli and developed by Genentech in cooperation with Eli Lilly in the late 1970s, was an historical landmark. By 2006, some 165 biopharmaceuticals had been approved in the EU and/or the USA for human use. This illustrates the emergence and rise of recombinant technologies which constitute the basis of pharmaceutical biotechnology. Today, approximately one in four of all genuinely new drugs currently entering the market is a biopharmaceutical and in 2008 over 400 biopharmaceuticals were in various stages of clinical evaluation. These include hormones, soluble hormone receptors (as hormone antagonists), blood factors, thrombolytics, interferons, monoclonal antibodies, vaccines and therapeutic enzymes. Selected aspects of engineering and production processes together with information relating to their use are discussed in this chapter. Data on industrial development, products, companies and economics are also presented.

The potential of transgenic plant biotechnology is to create crops that produce higher yields and are able to grow on less fertile land in order to feed the growing world population. Crops should be resistant to pests and require less chemical treatment, notably with insecticides, fungicides, herbicides and fertilizers, and exhibit low environmental impact. The majority of agricultural scientists are convinced that such crops can be delivered by the exploitation of molecular breeding strategies. Food production has risen considerably over the decades in terms of a ‘Green Revolution’, most notably in developing countries, but the increase in per capita food supply has been small. Hence research in recombinant food production is considered to be a necessary part of the strategies to ensure adequate nutrition. Nevertheless, debates over the risks of the technology have evoked conflicts and created a critical, even negative publicity, particularly in Western Europe.

BT offers in general a sustainable method of production, based mostly on renewable resources with minimal or no waste and by-products that can be recycled or reused, for example as feed components. There are manifold interactions with political, social, economic and environmental issues. Laws, regulations and ethical concerns pertinent to biotechnology are important topics of discussion although there are dramatic differences in legislation between countries. Current efforts are centred around establishing common global regulations including the removal of unfair unilateral advantages and support for health care and economies in developing countries. The regulatory influences which affect how science is carried out and technology is applied are addressed in each chapter. In addition to the underlying scientific concepts, further information is presented in each chapter on the use of products, along with data on industrial activities and production.

The increase in computing power due to the invention and continued development of microchips via nanotechnology has pioneered and driven a revolution in communication during the last three decades. At least one computer, television and mobile telephone have found their place in essentially every home. Biotechnology has also undergone a corresponding development, although perhaps not so immediately identifiable at the level of consumer goods in the shops. There is, however, hardly an area of human activity which has not been affected by the recent biotechnological revolution. We hope that after completing our book, the readers will feel that they have a better understanding of how and why this revolution took place, its roots and its further potential to improve so many aspects of our lives.

Acknowledgements

We are most grateful for comments on the manuscript from a number of friends and colleagues as well others who agreed to have their photographs taken for inclusion in the book. Their comments contributed to the readability of the text, led to the avoidance of certain errors and extended the knowledge base. We, the authors take full responsibility for any remaining mistakes.

In particular thanks are due to Anthony (Tony) C. R. Samson, Karl Simpson, Raimo Franke, Heidi Lloyd-Price, and Erik Pollmann, Ulrich Behrendt, Sonja Berensmeier and Volker Kasche for significant information and relevant advice.

Further valuable information and assistance was contributed by Robert Bud, Arnold Demain, Albert J. Driesel, Reinhard Hehl, Dietmar Hempel, Gerhard Höfle, Hans-Joachim Jördening, Peter Rapp, Jürgen Seibel and Hermann Stegemann. We would also like to thank Frank Weinreich for his wise suggestions during the final stages of converting our manuscript into a book. JC is grateful to the Helmholtz Centre for Infection Research, Braunschweig (HZI; formerly the GBF) for funding the transport costs to international meetings.

This book is dedicated to our wives Diana Buchholz and Marie-Christiane Collins without whose support we could not have completed this project.

Abbreviations and Glossary

Acre

4046m

2

ADM

Archer Daniels Midland (starch producing and converting company, USA)

ADP

adenosine diphosphate

7-ACA

7-Aminocephalosporanic acid

7-ADCA

7-Aminodesoxycephalosporanic acid

6-APA

6-Aminopenicillanic acid

AIChE

American Institute of Chemical Engineers

AMP

adenosine monophosphate

Array CGH

Array

Comparative Genome Hybridization

, for example for comparing (malignant) biopsy material with DNA from normal tissue

ATP

adenosine triphosphate

BAC libraries (BACs)

bacterial artificial chromosome libraries

BHK

baby hamster kidney (cells)

BMP

bone morphogenetic protein

BMS

Bristol Meyers Squibb (USA)

bn

billion

BOD

Biological oxygen demand (of waster water)

BP

Before present

BPTI

Bovine pancreatic trypsin inhibitor

BT

Biotechnology

Bt

Bacillus thuringiensis

C&EN

Chem. Eng. News

CCD

computational cell dynamics

cDNA

copy DNA, reverse transcribed from mRNA

CDR

complementarity-determining region of an antibody

CEPH

Centre d’études des polymorphisms humains, Paris, France (The Centre for the Study of Human Polymorphisms)

CFD

computational fluid dynamics

CFTR

Cystic fibrosis transmembrane conductance regulator

cGMP

current Good Manufacturing Practice

CHO

Chinese hamster ovary (cells)

CIP

clean in place

CMV

Cytomegalie virus

CNV

copy number variation

CP

capsid or coat protein (of virus)

CSF

Colony stimulating factor

Cultivars

cultivated plant varieties

2D

two dimensional

DARPins

Designed Ankyrin Repeat Proteins

2DE

two dimensional electrophoresis

2DE IEF/SDS-PAGE

two dimensional electrophoresis combined with IEF and SDS-PAGE

DGT

direct gene transfer (including particle bombardmet)

DHA

docosahexanoic acid

dm

dry matter

2D-PAGE

two-dimensional gel electrophoresis

2DE IEF/SDS-PAGE

two dimensional electrophoresis method

DH

dehydrogenase

DOE US

Department of Energy, United States of America

DPN

+

diphosphonucleotide (is identical with NAD

+

)

DPNH

hydrogenated diphosphonucleotide (is identical with NADH)

dt/ha

decitonnes (0,1 t) per hectare

€

EURO, 1.40 $ (Oct. 2010, mean)

EBIT

earnings before interest and taxes

E. coli

Escherichia coli

EF

environmental factor

EI

environmental index

ELISA

enzyme linked immunosorbent assay

EMEA

European authority for approval of pharmaceuticals

EP

epothilone

EPA

Environmental Protection Agency (USA)

EPA

eicosapentanoic acid

Epitope

specific region on a protein recognized by an antibody

EPC

European patent convention (5 October 1973)

EPO

European patent office or Erythropoietin

ER

endoplasmatic reticulum

ESC or ES

embryonic stem cells

ESI-MS

electrospray-ionisation mass spectrometry

ESI-TOF MS/MS

electrospray-time of flight-mass spectrometry

EST

expressed sequence tags; short DNA fragments obtained by random sequencing of clones from cDNA libraries

EU

European Union

FAO

Food and Agriculture Organization (USA)

FBA

flux balance analysis

FDA

Food and Drug Administration (USA)

FDP

fructose-1,6-diphosphate

Fluxome

flux distribution of the central metabolic pathways

Ft.

feet (30.5 cm)

Gal

gallon (3,78 L)

GC-MS

coupled gas chromatography-mass spectrometry

GM

genetically modified;

GMO

genetically modified organism

GRAS

generally recognized as safe

GMP

Good Manufacturing Practice

GPCRs

G protein coupled receptors

GSK

GlaxoSmithKline

ha

hectar, 10 000m2

hGH

human growth hormone

HIV

Human immunodeficiency virus

hl

hectoliter (100 l)

HR

hypersensitive response

HTS

high throughput screening

IEF

isoelectric focusing

IFN

interferon

IgG

immune globulin G

IL

interleukin

In.

inch (2.54 cm)

i.v.

intravenous

JACS

Journal Am. Chem. Soc.

J&J

Johnson & Johnson

LC-MS

liquid chromatography-mass spectrometry

LD (LOD score)

linkage disequilibrium in population genetics

LRR

leucine-rich-repeat proteins, for example ankyrin

mAB

monoclonal antibody

MALDI-TOF-MS

Matrix-Assisted-Laser-Desorption/Ionization – Time-Of-Flight-Mass-Spectrometry

MDR

multi drug resistant

MFA

metabolic flux analysis

MI

mass Index

miRNAs

micro RNAs

mn

million

Mtoe

million tons oil equivalents

Mw

molecular weight, molar mass

m-Arrays

micro-arrays

NAD

Nicotinamide-adenine-dinucleotide

NADH

hydrogenated NAD

NBF

new BT firm

NCE

new chemical entity

NGOs

non governmental organizations

NIH

National Institutes of Health (USA)

NK cells

natural killer cells

NMR

nuclear magnetic resonance

NRRL

Northern Regional Research Laboratory (USA)

NSO

mouse myeloma derived mammalian cells

ON

oligonucleotides

ORF

open reading frame

OS

oligosaccharides

OTA

Office of Technology Assessment (USA)

PAGE

polyacrylamide gel electrophoresis

PAT

process analytical technology

PDO

1,3-propanediol

PDR

pathogen-derived resistance

PEG

polyethylene glycol

PEGylation

attachment of polyethylene glycol

PET

positron emission tomography

PHB

polyhydroxybutyrate (a polyester)

pI

Ionic strength (logarithmic scale)

Plastids

Intracellular organelles, e.g., chloroplasts that have their own double stranded DNA

pO

2

oxygen partial pressure

Pound

453 g

PR

pathogenesis related

PR

plant disease resistance

PS

iPS and piPS Pluripotent stem cells, induced pluripotent stem cells, protein-induced pluripotent stem cells

PSTI

Human pancreatic secretory trypsin inhibitor

QTL

quantitative trait locus

QM

quality management

R

resistance (genes)

rasiRNAs

repeat-associated small interfering RNA.s

R&D

research and development

rDNA

recombinant DNA

rDNA technologies

recombinant DNA technologies

rh

recombinant human

RNAi

interfering RNA, RNA interference

rPC

real time PCR

rRNA

ribosomal RNA

$

US $, corresponding to 0,71 € (Oct. 2010, mean)

SAGE

serial analysis of gene expression

SDA

stearidonic acid

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEC

size exclusion chromatography

SIP

sterilization in place

siRNA

small interfering RNA

shRNA

short hairpin RNA

SNP

single nucleotide polymorphism

STR

stirred tank reactor

SUB

single use bioreactor

t/a

tonnes per year

™

trade mark

TNF

Tumor necrosis factor

tPA

Tissue plaminogen activator

Translation capacity

the number of times a transcript is translated.

USDA

US Department of Agriculture

US$

US dollar (see $)

YAC libraries.

yeast artificial chromosome. libraries

Part OneHistory

1

Introduction

Historical events in early biotechnology comprise fascinating discoveries, such as yeast and bacteria as living matter being responsible for the fermentation of beer and wine. The art of Biotechnology emerged from agriculture and animal husbandry in ancient times through the empirical use of plants and animals which could be used as food or dyes, particularly where they had been preserved by natural processes and fermentations. Improvements were mostly handed on by word of mouth, and groups that maintained these improvements had a better chance of survival during periods of famine and drought. In such processes alcohol was produced and provided highly acceptable drinks such as beer and wine, or acids were formed which acted as the preservative agent in the storable food produced.

In this book we follow how the study of the chemical nature of the components involved in biotechnology first became possible subsequent to the development of chemistry and physics. Serious controversies about the theories both vitalist and chemical, resulted in the reversal of theories and paradigms; significant interaction with and stimulus from the arts and industries prompted the continuing research and progress. Last but not least, it was accepted that the products produced by living organisms should not be treated differently from inorganic materials. Pasteur’s work led to the abandonment of the idea which had been an anathema to exact scientific enquiry in the life sciences, namely ‘spontaneous generation’. He established the science of microbiology by developing pure monoculture in sterile medium, and together with the work of Robert Koch the experimental criteria required to show that a pathogenic organism is the causative agent for a disease were also recognised. Several decades later Buchner disproved the hypothesis that processes in living cells required a metaphysical ‘vis vitalis’ in addition to what was necessary to understand general chemistry. Enzymes were shown to be the chemical basis of bioconversions. Biochemistry emerged as a new speciality prompting dynamic research in enzymatic and metabolic reactions. However, the structure of proteins was not established until more than 40 years later.

The requirements for antibiotics and vaccines to combat disease, and chemical components required for explosives particularly in war time, stimulated exact studies in producing products from microbial fermentations. By the mid-twentieth century, Biotechnology was becoming an accepted speciality with courses being established in the life sciences departments of several Universities.

Basic research in Biochemistry and Molecular Biology dramatically widened the field of life sciences and at the same time unified them considerably by the study of genes and their relatedness throughout the evolutionary process. The scope of accessible products and services expanded significantly. Economic input accelerated research and development, by encouraging and financing the development of new methods, tools, machines, and robots. The discipline of ‘New Biotechnology’, one of the lead sciences which resulted from an intimate association between business and science, is still the subject of critical public appraisal in many Western countries due to a particular lack of confidence in the notion that improved quality of life will prevail in spite of commercial interest.

The study of the history of science, and specifically that of Biotechnology, should go beyond documenting and recording events and should ideally contribute to an understanding of the motives and mechanisms governing the dynamics of sciences; the role of inquisitiveness in gaining new knowledge and insights into nature, of reputation-building, of the interplay between economics and business; the role of and dependance on theories, the role of analytical and experimental, as well mathematical methods, and more recently computation and robotics.

In the first part of this book the emphasis is on scientific discoveries and results, theories, technical development, the creation of leading paradigms, and on their decay. Industrial problems and political issues, and their influence on and correlation with developments in science are also addressed.

Although biotechnology has historical roots, it continues to influence diverse industrial fields of activity, including food and feed and commodities, for example polymer manufacture, providing services such as environmental protection, biofuel and energy including biofuel cells, and the development and production of many of the most effective drugs.

2

The Early Period to 1850

2.1 Introduction

Fermentation has been of great practical and economic relevance as an art and handicraft for thousands of years, yet, in the absence of analytical tools, there was initially no understanding of the changes in constituents that took place. There were serious controversies regarding the vitalistic or chemical nature of these processes. The vitalists believed that mysterious events and forces would be involved such as spontaneous generation of life and a specific vital force, while the chemical school, with Liebig at its head, was in contrast, convinced that only chemical decay processes took place, denying that any living organism was involved in fermentation. Nevertheless most of the phenomena that are relevant to the understanding of the role of microorganisms were noted. It was only during the following period, from 1855 onwards, that Pasteur proposed a theory of fermentation which discredited the hypothesis of spontaneous generation as well as that of Liebig and his school.

‘Natural’ processes would have been identified and adopted by human populations as soon as food hoarding became of interest. Water-tight vessels, such as pots and animal skins would have aided these processes as compared to drying, salting and smoking. This would have allowed the spontaneous discovery of processes for making beer, wines, yoghurt, and sauerkraut from fruit juices, milk, and vegetables. ‘Natural’ fermentations would have been discovered spontaneously but have only been documented in more recent history, although such practices presumably predate writing by many thousands of years.

Barley, which is the basic raw material for beer preparation but not for bread making, was the first cereal to be cultured about 12 500 years BP (= before present), and was grown 6000 years before bread became a staple food; the first document on food preparation was written by the Sumerians 6000 years ago and describes the technique of brewing. A new theory by Reichholf [1] claims that mankind formed settlements after the discovery of fermentation and used the alcohol produced by this process for indoctrination into a cult or for purposes of worship [1, p. 265–269, 259–264].1) Thus beer and wine manufacture form the roots of Biotechnology practices which were developed in ancient times. The vine is assumed to originate from the Black and Caspian seas, and to have been cultivated in India, Egypt and Israel during this early period. In Greek mythology gods such as Dionysos or Bacchus, granted the availability of wine (Figure 2.1) and the birthplace of Dionysos is believed to have been in the Indian mountain Nysa (Hindukusch) [2, p. 441], [3, p. 591–595], [4, Vol. 12, p. 1, 2].

Figure 2.1 Olympus, Nectar Time (Dionysos: god’s thunder – the miracle of wine formation) [5].

Written documentation on beer and wine manufacture which form the roots of Biotechnology can be traced back in ancient history: about 3500 BC brewers in Mesopotamia manufactured beer following established recipes [6]. In 3960 BP King Osiris of Egypt is assumed to have introduced the production of beer from malted cereals [7,p. 1001]. In Asia, fermentation of alcoholic beverages has been documented since 4000 BP, and fermentation starters2) are estimated to have been produced about 6000 BP by the daughter of the legendary king of Woo, known as the Goddess of rice- wine in Chinese culture [8, p. 38, 39,45]. Soya fermentation was established in China around 3500 BP. Around about 2400 BP, Homer described in The Iliad the coagulation of milk using the juice produced from figs which contains proteases. Pozol, a nonalcoholic fermented beverage, dates back to the Maya culture in Yucatan, Mexico [9]. There is a mythical report that Quetzalcoatl, a Toltec king of the tenth century, was seduced by demons to drink wine with his servants and his sister so that they became drunk and addicted to desire and pleasure; later Quetzalcoatl set fire to himself as an act of repentance and was resurrected as a king on another planet [10].

Tacitus reports that the Germans have a history of beer-making [11, p. 299]. The famous German law of 1516 on brewing has its origins in Bavaria. The medieval tradition of brewing can be traced back through the literature, such as the first books by the ‘Doctor beider Rechte’ Johannes Faust, who wrote five books on the ‘divine and noble art of brewing [12, Vol. 2, p. 409, 410].

Thus in the absence of detailed knowledge of the process, fermentation became a rational method of utilizing living systems. The fermentation of tobacco and tea were also established in ancient times. These fermentations were presumably initially adopted as fortuitous processes. In fact all of them utilize living systems, however, the early users of fermentation had no understanding of neither the origins of the essential organisms involved nor their identities nor the way in which they predominated over possible contaminants. A most significant step was the description of tiny ‘animalcules’ in drops of liquids, which Leeuwenhook observed with his microscope (about 1680, the year in which he became a member of the Royal Society in London). This, however, was not seen in the context of, or correlated to fermentation. Stahl in his 1697 book Zymotechnika Fundamentals (the Greek ‘zyme’, meaning yeast) explored the nature of fermentation as an important industrial process, where by zymotechnica was used as a descriptor for the scientific study of such processes [13].

Various descriptions were associated with ‘bad air’ in marshy districts. In 1776 A. Volta observed the formation of ‘combustible air’ (methane, ‘hidrogenium carbonatrum’, as analysed by Lavoisier in 1787) from sediments and marshy places in lake Lago Maggiore in Italy. He noted that ‘This air burns with a beautiful blue flame…’ [14]. The first enzymatic reactions, then considered to be fermentative in nature, were observed by the end of the eighteenth century. Thus the liquefaction of meat by the gastric juices was noted by Spallanzani as early as 1783 [15], the enzymatic hydrolysis of tannin was described by Scheele in 1786 [16], and Irvine detected starch hydrolysis in the aqueous extract of germinating barley in 1785 [17, p. 5]. Such processes or reactions were distinguished from simple ‘inorganic’ reactions on the basis that the reaction could be stopped by heat denaturation of the ‘organic’ components.

In addition to providing pleasure, beer and wine manufacture have become economically important because for several thousand years dating back to the early economy of Mesopotamia and Egypt, it has been a major source of tax revenue. The manufacture of alcoholic beverages developed into major industrial activities during the nineteenth century, and further fermentation processes were embraced enthusiastically in order to widen the horizon for new business opportunities, the expression of which was the foundation of numerous research institutes in several European countries during the nineteenth century.

2.2 Experimental Scientific Findings

2.2.1 Alcoholic Fermentation

Figure 2.2 Lavoisier’s apparatus for the investigation of fermentation. In the first vessel A the material to be fermented, for example sugar, and beer yeast is added to water of a weight determined exactly; the foam formed during fermentation is collected in the following two vessels B and C; the glass tube h holds a salt, for example nitrate, or potassium acetate; this is followed by two bottles (D, E) containing an alkaline solution which absorbs carbon dioxide, and only air is collected in the last bottle F. This device allowed determination ‘with high precision’ of the weight of the substances undergoing fermentation and formed by the reaction. [20, p. 139, 140, Planche X].

From the end of the eighteenth century efforts to find a solution to the fermentation problem bear witness to the attempt to approach and explain this phenomenon either as the result of the activity of living organisms or as purely interactions of chemical compounds. However from the mid-1830s evidence began to accumulate to indicate that biological aspects formed the basis of fermentation [21, p. 24].

Important findings based on well-designed experiments were published by Schwann and Cagniard-Latour in 1837 and 1838. They showed independently that yeast is a microorganism, an ‘organized’ body, and that alcoholic fermentation is linked to living yeast. In 1838 Cagniard-Latour reported that ‘In the year VIII (1799–1800) the class of physical and mathematical sciences of the Institute (Institut de France) had proposed for the subject (of fermentation) a prize (for solving) the following question: ‘What are the characters in vegetable and animal matter….’ ‘I have undertaken a series of investigations but proceeding otherwise than had been done. That is by studying the phenomena of this activity by the aid of a microscope. … This attempt … was useful since it has supplied several new observations with the following principal results: 1. That the yeast of beer (this ferment of which one makes so much use and which for this reason was suitable for examination in a particular manner) is a mass of little globular bodies able to reproduce themselves, consequently organized, and not a substance simply organic or chemical, as one supposed. 2. That these bodies appear to belong to the vegetable kingdom and to regenerate themselves into two different ways. 3. That they seem to act on a solution of sugar only as long as they are living. From which one can conclude that it is very probably by some effect of their vegetable nature that they disengage carbonic acid from this solution and convert it into spirituous liquor…. I will add that the question formerly proposed by the Institut appears now to be solved… I have communicated (the results) to the Philomatic Society during the years 1835 and 1836.’ [22].

Schwann [23] first reported his experiments concerning spontaneous generation to the Annual Assembly of the Society of German naturalists and Physicians, held in Jena in September 1836. He demonstrated that provided the air was heated neither mould nor infusoria appeared in an infusion of meat and that the organic material did not decompose and become putrid. Schwann perceived that these experiments did not support those of the proponents of spontaneous generation. They could be explained on the basis that air normally contained germs (Keime). Schwann concluded that alcoholic fermentation was promoted by proliferating yeast organisms which he classified as sugar fungi, derived from ‘Zuckerpilz’ or sugar fungus [21, p. 26]. From experiments with known poisons he drew the conclusion that ‘a plant was probably the organism to be expected.’…

‘With microscopic examination of beer yeast there appear the known granules which form the ferment; most of them hanging together in rows…. Frequently also one sees …small granule seated sideways as the foundation of a new row that is without doubt a plant’. ‘Besides sugar a nitrogen-containing body is necessary. One must therefore picture the vinous fermentation as the decomposition which is so brought about that the sugar fungus draws from the sugar and a nitrogen-containing body the substances necessary for its own nutrition and growth. Whereby the elements of these bodies not entering the fungus (probably amongst several other substances) combine preferentially to alcohol’.

‘Beer yeast is made up almost entirely of these fungi…. These grow visibly under the microscope, so that already after to1 hour one can observe the increase in volume of a very small granule which sits on a larger one…. It is highly probable that the latter by its development causes the phenomena of fermentation’. [23].

Also independently, Kützing [24] performed microscopic investigations on yeast and ‘mother of vinegar’ (‘Essigmutter’, in acetic acid fermentation). He confirmed the thesis of living organisms both with respect to yeast and the ‘vegetabile’ organisms (vegetabilische Organismen) active in acetic acid fermentation (‘Essigmutter’) and also recognized nucleation in yeasts. Turpin [25] refers to Cagniard-Latour, dealing with ‘organisation, vegetation, reproduction and growth of yeast,… repeating his observations carefully’. The aim of these recent publications is to ‘…elucidate this mysterious process from a microscopic-physiological viewpoint’. The author’s investigations were carried out in a large brewery and took into account the procedures of inoculation and growth of yeast. He had no doubts concerning the ‘vegetable organized existence’ that circumscribes the nature of living entities (‘vegetabile organisierte Existenz’). He described his procedure in extensive detail, including taking samples from the technical vessel at various times. The observations included inoculation by spores or reproducing bodies (‘Sporen oder reproduktiven Körpern’), and growth of spheres. Turpin concluded that fermentation is a purely physiological process, ‘beginning and ending with the existence of infusoria plants or animals…’ (living species).

A fermentation process that was different from alcoholic fermentation was described by Gay-Lussac and Pelouze [26]. It was used to isolate, purify and characterize lactic acid (later called lactic acid fermentation). Mixed fermentations were described by Gaultier de Claubry [27] and by Schill [28]. Schill refers to a considerable number of earlier studies on milk fermentation from 1754 onwards. Fermentation occurred both with and without the addition of ferment (in some cases yeast, in others cheese).

In his book on chemical technology Knapp [11] described the current technology for beer and wine fermentation and presented a detailed description of growing yeast including figures showing the increase in the number of yeast cells, similar to ‘primitive plant cells’, over time and several generations. He also noted some details concerning the cell wall and an internal, protein-like substance. He concluded that yeast is not a non-living precipitate but an organized being (‘Wesen’) of the ‘lowest type’, an initial, early state (‘Anfangsstufe’) of plants [11, p. 277]. Knapp came to the conclusion that no one of the ‘…hypotheses (Ansichten)… is up to now accepted as unequivocal truth’ [11, p. 271]. A detailed review of the work of the scientists mentioned is given by Barnett [29].

2.2.2 ‘Unformed, Unorganized Ferments’

Unformed or unorganized ferments had characteristics that were obviously different from those of yeast: it was definitely not living matter; the substances were water soluble, they could be precipitated and thus isolated. They are enzymes in today’s terms, as first proposed by Kühne [30]. Interestingly several isolated ferments, such as diastase (amylases and glucamylases in today’s terminology), could be characterized to a considerable extent. Their nature however remained unknown and even obscure.

Payen and Persoz [31] investigated in detail and with high precision, the action of extracts of germinating barley known as diastase, on starch and formulated some basic principles of enzyme action (see also [17, p. 5]):

the active principle can be isolated by precipitation and thus purified

it is water soluble

small amounts of the preparation were able to liquefy large amounts of starch

the material was thermolabile, that is it loses its (catalytic) potential when boiled.

Payen and Persoz [31] also described in detail the isolation and purification of diastase from germinating barley by the procedures of maceration, pressing, filtration and repeated precipitation with alcohol. The precipitated ferments could not be crystallized, remained amorphous and chemically undefined. The fact that no definite chemical composition could be established was in contrast to the fact that the products which were sugars, as obtained by Guerin-Varry [32] who established the chemical composition [C12H28O14] (although not fully correct), could be crystallized, for example glucose.

The first industrial processes that used enzymes were established from the 1830s onwards in France based on Payen’s work (see Section 2.3).3)

In 1830 Robiquet and Boutron observed that an extract from bitter almonds which was named ‘emulsin’ by Liebig and Wohler in 1837, hydrolysed a glycoside, amygdaline [17, p. 5]. Schwann [34] precipitated the active principle from mucus ‘as an individual substance’ and named it pepsin; he also characterized its action in precipitating casein [21, p. 22]. ‘By all these reactions the digestive principle is characterized as an individual substance, to which I have given the name pepsin’ [34].

2.3 Application

Handicraft and art became a source for, and a subject of scientific investigation. ‘To Stahl… science was the basis of technology,… providing key ideas,… the basis … of that important German industry of Gärungskunst – the art of brewing’. ‘His concern was with its chemical interpretation’. Bud considers Stahl’s ‘Zymotechnica Fundamentalis’ (published in 1697) ‘to be the founding text of biotechnology’ [13].

The application of fermentation as well as the use of unformed ferments (the enzyme diastase) proceeded pragmatically during the early nineteenth century despite the fact that no commonly accepted theory had been established. Several books on chemical technology gave detailed and advanced information on the procedures of beer, wine, bread and acetic acid production and although based on ample technical experience, the discussion of the theoretical approaches to the fermentation phenomena relied in part on mysterious and contradictory background information. Nevertheless both handicraft and industrial fermentation represented important and successful production processes.

It is important to note that inoculation was used for yeast fermentation in industrial processes. Thus Lampadius [35] describes the utilization of yeast from the previous year’s fermentation for the current industrial production of wine in Saxony, and Knapp [11] reported the common practice of inoculation in brewing.

Work on technological issues was equally important and even dominated over that on the basic aspects from the beginning of the nineteenth century. This is apparent from the large amount of text that was devoted to fermentation processes in the books of the time on technology and chemical engineering ([36, 380 ff., based on [37], [38, 75 ff.], [11, 249 ff.], [39, 74 ff.], [2,195 ff.]). From these books the close relationship of technology, industry and scientific research was also obvious. Knapp [11, p. 367] stated that ‘no fermentation has for industry, and notably for agriculture, such an importance, or weight, as alcoholic fermentation because the production of all alcoholic beverages, of wine, beer,…has this fermentation as a basis’. He considered scientific and practical and industrial interests to be equally important.

In his book on technology Poppe [38, p. 387] introduces the chapter on beer with enthusiastic characterization, as a marvellous, refreshing, caloric and healthy wineous drink. Knapp [11, p. 333–349] mentions that brewing was carried out in Germany at the level of a handicraft, in vessels (Bottichen) of 1000–2000 1 in volume, whereas in the UK it was carried out on an industrial scale in large factories with major investment of capital; the fermenters were up to 240 000 l in volume (Figure 2.3). In the many details of the process, he states that the normal process proceeds by inoculation with yeast and procedures which did not employ inoculation were in danger of failure. Furthermore the breweries always produced a surplus of yeast which was then supplied to the baking and distilling industries. Thus the growth of yeast became an obvious, even economically important fact, making Liebig’s theory obsolete.

It is estimated that in Germany in 1840 about 22.7 million hectolitres of alcohol were produced. Wagner enumerates 42 different beer specialities, including Bavarian and English products with their different characteristics, such as alcohol content. Thus beer, and more particularly fermentation products including wine, acetic and lactic acids, became most relevant contributors to the national economies.

Figure 2.3 A brewing vessel as used in the UK and Belgium. The substrate is preheated in part B of the vessel where water vapour is introduced via the tubes rr; fermentation takes place subsequently in part A (the next substrate being preheated in B); part A is equipped with a stirrer dd (held by aa) which is equipped with chains that agitate the sediments on the bottom [11, p. 332].

An acetic acid fermentation process known as ‘fast acetic acid manufacture’ (‘Schnellessigfabrikation’) was developed by Schützenbach in 1823. It worked, remarkably, with active acetic acid bacteria (of course not recognized at that time) immobilized on beechwood chips. The process was carried out in vessels made of wood, some 1–2m wide and 2–4m high, and was aerated to oxidize the substrate alcohol. First acetic acid was introduced to wet the wood chips, and alcohol was then continuously added and oxidized. The process took only 3 days in contrast to the classical fermentation that required several weeks. Since the bacteria were immobilized on the wood chips, a period of slow growth phase was not needed so that the oxidation of the alcohol took place immediately (Figure 2.4) [40, p. 514], [2, Vol. 2, p. 480–498].

Figure 2.4 Acetic acid fermentation using immobilized bacteria. The vessel was equipped with sieve plates in positions D and B. Space A was filled with beechwood chips (on which the bacteria were immobilized). A 6–10% alcohol solution was added from the top to a solution containing 20% acetic acid and beer (containing nutrients). Air for oxidation was introduced through holes in a position above B, the temperature was maintained at 20–25 °C. The product containing 4–10% acetic acid was continuously removed via position E [40, p. 514].

Figure 2.5 Process for dextrin manufacture showing the reaction vessel (a), filter (b), reservoir (c) and concentration unit (d) [2, Table XXV, in Vol. 1].

In his description of the chemical technology Wagner [39, p. 365/6], as did Poppe [38], enumerates three different fermentations: alcoholic, with yeast as the ferment, lactic and acetic acid fermentation, and putrefaction. The nature of the ferments is still not considered to be clear, either as an organized (living) body (‘Wesen’) such as yeast or a proteic substance (‘Körper’) undergoing decay. Alcoholic fermentation (‘geistige Gährung’) is the basis of both wine and beer production and baking [39].

The first industrial processes using an unformed ferment (enzyme), diastase, were established from the 1830s onwards in France and were based on Payen’s work (Figure 2.5). Payen and Persoz [31, p. 74–78] had presented details for the production of dextrin on a large scale (500 kg) using 6–10% diastase. The product, dextrin, was used in bakeries, as well as for the production of beer and wines from fruits [11, 39].

2.3.1 Education

As a result of the economic relevance of fermentation, education in this field was established from the time it originated in the nineteenth century. The schools for commerce in the German speaking countries began to combine knowledge obtained from the arts with scientific knowledge. In Germany, the first agricultural high school was founded in 1806 and in 1810 became part of the new Berlin University. A further 20 agricultural schools were subsequently established up until 1858 in German speaking countries, amongst them schools in Vienna and Braunschweig.4) In France, Boussigault founded a private agricultural research laboratory in 1835 and in London Lawes and Gilbert founded their laboratory in 1842. The brewers of Bohemia asked the director of the schools of engineering in Prague to establish an institute to train experts for their industry. As a consequence, in 1818 the pharmacist J. J. Steinmann offered the first course in fermentation chemistry, presumably the first worldwide. Balling, his successor, introduced a science-based but nevertheless practically orientated course in brewing [6, p. 21–24].

2.4 Theoretical Approaches

Beginning in the time of the renaissance during the sixteenth century, handicraft and art became a source of science. The transition from handicraft and art to science has been analysed in detail by Böhme et al. [42], who refer for example to Powers’ experimental philosophy of 1633 (p. 7), and by Mittelstrass [43, p. 167–179] in the context of ‘La Nuova Scienza’ with reference to eminent artists, scientists and engineers such as Brunelleschi, Leonardo da Vinci and Tartaglia. The academies founded subsequently, the Royal Society in London in 1662 and the Académie des Sciences in Paris in 1666, accepted and introduced observation, experimentation and measurement as the basis of scientific work. This also led to the accumulation of an empirical knowledge base that was described in the form of laws [42, p. 13–128, p. 136–139]. The traditional processes used in arts and handicraft that were developed in ‘workshops’ (‘Werkstöatten’) together with technical phenomena including baking and brewing, also became sources of empirical knowledge [42, p. 188–190]. With respect to biotechnology, Bud [13] considers Stahl’s ‘Zymotechnica Fundamentalis’ (published in 1697) ‘to be the founding text’. ‘To Stahl … science was the basis of technology, … providing key ideas, … the basis … of that important German industry of Gärungskunst – the art of brewing’. ‘His concern was with its chemical interpretation’. [13]. However, when Lavoisier later laid the foundations of modern chemistry, Stahl’s theories were considered to be incorrect.

Major progress in the understanding of fermentation was made by Schwann, Cagniard-Latour and others in the 1830s and had its source and origin of interest in practical fermentation not in the observation of natural processes. The investigations of Cagniard-Latour as well as those of Turpin (see Section 2.2.1), were undertaken in a brewery thus making it clear that the origin of the scientific problem was rooted in an industrial process. Extensive microscopic investigations in breweries demonstrated that microorganisms were the source of fermentation. However, Schwann and Cagniard-Latour, were not able to establish a commonly accepted theory, even if the experimental basis was broad and scientifically strong. This was in contrast to Pasteur who was able to establish his theory during the 1860s; – the question is why? One strong counter-current which delayed arrival at the correct conclusion (the vitalist view) was the opposition by the chemical school, first and foremost, led by Liebig and Berzelius.

2.4.1 The School of Vitalism

Early theories: Schwann, Cagniard-Latour: living and growing cells

An exciting debate was in progress which referred to the questions of whether

fermentation was due to living organisms and a vital factor? – the vitalist view,

or was it a purely chemical process? – the viewpoint of the chemical, notably Liebig’s, school. Furthermore, the vitalists discussed the question of whether fermentation was a

spontaneous phenomenon, due to spontaneous generation of living organisms, or

if an agent, the addition of a ferment (inoculation) was necessary to initiate it, as had been shown experimentally by Schwann;

a living force, the

vis vitalis

, governs the activity and reactions observed in fermentation and surpasses the pure chemical forces such as affinity.

As to the origin of ferments, Gay-Lussac had postulated a ‘generatio spontanea’, a spontaneous generation, in 1810, the hypothesis that a continuing – rather mysterious – chain of events should be the cause of spontaneous generation (of organisms) ([44, p. 130], summary by [45]). Typical controversial points of view may be illustrated by the subsequent citations: Some vital factor, ‘le principe vital’, was considered to be an important principle in the chemical processes associated with the synthesis of materials isolated from living matter: ‘All simple bodies in nature are subject to the action of two powers, of which one, that of attraction, tends to unite the molecules of bodies one with another, while the other, produced by caloric, forces them apart… A certain number of these simple bodies in nature are subject to a third force, to that caused by the vital factor, which changes, modifies and surpasses the two others, and whose limits are not yet understood’ [46]. Similarly rather mysterious concepts were summarized in an early book on technology. ‘Fermentation is seen as a – at a time and under circumstances spontaneous – occurring mighty movement in a liquid of different compounds …, which is due to the fact that several compounds act in harmony with each other, others in opposition to each other, so that the first attract, the latter reject each other’ [38, p. 229]. A vital factor may be considered as an incoherence, or inconsistency in theories of the new science of chemistry, based on Lavoisier’s [20] and others’ experimental and empirical foundations as well as the theory of affinities by Berthollet [47, p. 2] which states that every substance reacts according to its affinity and quantity. Non-physical phenomena were not considered.

Schwann [23] and Cagniard-Latour [22] published important basic findings based on well-designed experiments. Both presented a theory of fermentation in terms of the vitalist approach essentially corresponding to that of Pasteur which would be put forward about two decades later. The fact that fermentation depends on inoculation was shown by conclusive experiments undertaken by Schwann [23]. The growth of yeast had been definitively observed by Schwann, Cagniard-Latour and Turpin. Inoculation with yeast in brewing was common practice (Section 2.3). ‘Now, that these fungi are the cause of fermentation, follows, first, from the constancy of their occurrence during the process; secondly, from the cessation of fermentation under any influence by which they are known to be destroyed,…, a phenomenon which is met with only in living organisms.’ (citation from [44, p. 139]). These findings were not accepted by the leading chemists of the time, Berzelius, Liebig and Wöhler (see below).

The myth of ‘living force’ continued to be discussed and Kützing [24] confirmed that it created organic mass (which was a living body in his terminology) from inorganic substances which corresponded to the hypothesis of spontaneous generation. He developed and advanced his theory on the vital force (‘organisierende Lebenskraft’), based on numerous observations on yeast and acetic acid bacteria (‘Essigmutter’) which he had published since 1834, and on the ‘generatio spontanea’ (‘Urbildung organischer Materie, Gebilde’) that had been proposed by Gay-Lussac. Kützing concluded from his findings, that ‘… two forces, the organizing living force (Lebenskraft) and the chemical affinity (chemische Verwandschaft) are in operation’. Quevenne [48] also believed that fermentation resulted from the ‘secret of living force’.5) In his book on ‘chemical technology’ ([11], p. 271) which was of a high scientific and technical standard, Knapp supported the concepts of the leading chemists (see below) in stating that ferments are not living bodies, but he nevertheless underlined the fact that inoculation and growth of yeast was common practice in breweries.

The vitalist theory created a strong empirical foundation broadly based on observations from technical processes and scientific investigations together with the experimental observations of several scientists working independently. This provided convincing arguments which constituted the vitalist theory of fermentation [29].

However, in today’s terms there were several irrational arguments in the vitalist theoretical concept:

the ‘

vis vitalis’

, a mysterious vital force, was assumed to be essential, responsible for, and dominant in the reactions observed during fermentation; this argument was disproved by Buchner’s work in 1897;

the

‘generatio spontanea

’, spontaneous generation (Urzeugung) of living (micro-) organisms, occurred in fermentations where the starting materials were solutions of chemical compounds (not living matter, in contrast to the common practice of inoculation).

6)

Pasteur’s work of around 1860 disproved this second thesis.

It was much later that it was proposed anew that life (at least once) had been generated spontaneously and autonomously and this inevitably became seen as an emergent property of inorganic matter (see [49]). In 1929 Haldane assumed that this property had been established 3.5 billion years ago in the ‘primordial soup’ of organic molecules that had been formed by the energetic reactions between water and the components of the prebiotic atmosphere ([50], see also [51]). Later similar assumptions led to the Urey-Miller experiment in 1953 and subsequent investigations [52]. Spontaneous generation was considered to be a singular event or a series of a few rare events. The mechanistic basis and details still cannot be traced in detail and remain unknown, but are assumed to be based on known chemical principles.7)

Unformed ferments (enzymes) had been identified and characterized and even used in industrial processes, but their chemical nature remained obscure as they could not be crystallized.8) Progress resulting from studies on enzymes stagnated from 1840 onwards for several decades, almost certainly due to the two opposing theories put forward by Liebig (decomposition hypothesis) and Pasteur (vis vitalis), a situation which discouraged scientific work on ferments.

2.4.2 The Chemical School

The conclusion drawn from the investigations of Cagniard-Latour, Schwann, Kützing and others that yeast was the organism accountable for alcoholic fermentation was severely criticized by Berzelius. He saw in this hypothesis the detrimental expression of a philosophical influence on science (Naturphilosophie)