Historia de La Biotecnologia-1

Appl Microbiol Biotechnol (2013) 97:37473762
DOI 10.1007/s00253-013-4768-2
MINI-REVIEW
The rootsa short history of industrial microbiology

and biotechnology
Klaus Buchholz & John Collins
Received: 20 December 2012 / Revised: 8 February 2013 / Accepted: 9 February 2013 / Published online: 17 March 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Early biotechnology (BT) had its roots in fascinating discoveries, such as yeast as living matter being
responsible for the fermentation of beer and wine. Serious
controversies arose between vitalists and chemists, resulting
in the reversal of theories and paradigms, but prompting
continuing research and progress. Pasteurs work led to the
establishment of the science of microbiology by developing
pure monoculture in sterile medium, and together with the
work of Robert Koch to the recognition that a single pathogenic organism is the causative agent for a particular
disease. Pasteur also achieved innovations for industrial
processes of high economic relevance, including beer, wine
and alcohol. Several decades later Buchner, disproved the
hypothesis that processes in living cells required a metaphysical vis vitalis in addition to pure chemical laws.
Enzymes were shown to be the chemical basis of bioconversions. Studies on the formation of products in microbial
fermentations, resulted in the manufacture of citric acid, and
chemical components required for explosives particularly in
war time, acetone and butanol, and further products through
fermentation. The requirements for penicillin during the
Second World War lead to the industrial manufacture of
penicillin, and to the era of antibiotics with further antibiotics, like streptomycin, becoming available. This was
followed by a new class of high value-added products,
K. Buchholz (*)
Institute for Chemical Engineering,
Technical University of Braunschweig, Hans-Sommer Str. 10,
38106 Braunschweig, Germany
e-mail: k.buchholz@tu-bs.de
J. Collins
Life Science Faculty, c/o Helmholtz Centre
for InfectionResearch - HZI, AG Directed Evolution,
Technical University of Braunschweig, Inhoffenstr. 7,
38124 Braunschweig, Germany
e-mail: tojohncollins@gmail.com
mainly secondary metabolites, e.g. steroids obtained by

biotransformation. By the mid-twentieth century, biotechnology was becoming an accepted specialty with courses
being established in the life sciences departments of several
universities. Starting in the 1970s and 1980s, BT gained the
attention of governmental agencies in Germany, the UK,
Japan, the USA, and others as a field of innovative potential
and economic growth, leading to expansion of the field.
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 the foundation of new
companies. The discipline of New Biotechnology became
one of the lead sciences. Although biotechnology has historical roots, it continues to influence diverse industrial fields of
activity, including food, feed and other commodities, for
example polymer manufacture, biofuels and energy production, providing services such as environmental protection, and
the development and production of many of the most effective
drugs. The understanding of biology down to the molecular
level opens the way to create novel products and efficient
environmentally acceptable methods for their production.
Keywords Biotechnology . History . Fermentation theories .
Industrial microbiology . Genetic techniques . Biotech
companies
Introduction
Fermentation has been of great practical and economic
relevance as a handicraft for thousands of years, notably
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the production of beer, wine and bread. The written first

document was by the Sumerians 6,000 years ago and describes the technique of brewing (Bud 1993). Beer and wine
manufacture was economically so important in ancient
Mesopotamia and Egypt that it became a major source of
tax revenue. Soya fermentation was established in China
around 3500 BP. Due to its great practical relevance alcoholic fermentation was of major technical as well as scientific interest. Controversies over basic concepts, e.g.
vitalism versus materialism in chemistry and biology,
resulted in the establishment, and reversal of theories and
paradigms, but finally lead to scientific rationalisation of
causality, and continuous technical progress, resulting in
the emergence of BT.
The early period till 1850fermentation mysteries

Leeuwenhook, about 1680, had observed, with the aid of his
microscope, tiny animalcules in droplets of liquids, which
he, however, did not associate with fermentation. Then, in
the second half of the eighteenth century Spallanzani undertook microscopic investigations of many specimens, including sperm and microbial growth. By the end of the
eighteenth and beginning of the nineteenth centuries, respectively, Lavoisier and Gay-Lussac had elaborated quantitative correlations for alcoholic fermentation, without
giving explanations for the process underlying it. From the
mid-1830s evidence began to accumulate which pointed to
the biological nature of fermentation. Based on welldesigned experiments, Schwann (1837) and CagniardLatour (1838) independently showed that yeast is a microorganism, an organized body, and that alcoholic fermentation is linked to living yeast. Both observed the yeast of beer
being little globular bodies able to reproduce themselves,
excluding spontaneous generation, and presenting a theory
on fermentation corresponding in essential parts to that
which Pasteur put forward about two decades later (for an
extended overview, see Buchholz and Collins 2010, part I).
Many other scientists, including Ktzing, Turpin and
Quevenne, contributed significant advances in understanding
fermentation, confirming that living organisms were involved
in fermentation processes other than that leading to alcohol,
e.g., in acetic acid fermentation. However, their arguments were
often confused by mystic concepts, in particular that fermentation emerges from spontaneous generation, and is a consequence of a secret living force (in contrast to chemical
forces), a that view promoted, e.g. Gay-Lussac (Buchholz and
Collins 2010, chapter 2). The mysterious concepts are obvious
from a textbook by Poppe (1842, p. 229): Fermentation is seen
as aat 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. Ktzing (1837, pp. 396,
397) believed that organic entities (living organisms) can
form themselves by spontaneous generation , and he assumed two forces, the organizing living force, and the chemical affinity, fighting each other , and Quevenne (1838,
p.469) used the term secret of life. In contrast to the vitalist
school, Liebig, the head of the chemical school, vigorously
argued against the concept of living bodies being active in
fermentation processes and advanced his erroneous theory of
ferments that supposed a body undergoing decomposition
which transfers its disturbed equilibrium onto other metastable
substances (Liebig 1839). In his book on chemical technology,
Knapp (1847, p. 271) came to the conclusion that no one of the
hypotheses is up to now accepted as unequivocal truth.
The importance of fermentation processes corresponds
with the large sections that were devoted to the topic in the
books on technology and chemical engineering of the time
(Otto 1838; Poppe 1842; Knapp 1847; Wagner 1857; Payen,
1874). Knapp (1847, p.367) reported that brewing was
performed in Germany at the level of handicraft, estimated
at a volume of about 22.7 million hectolitres (2,27 million m3)
in 1840, whereas in the UK it was carried out on an industrial
scale in large factories with fermenters of up to 240,000 L.
Particularly beer, as well as wine, acetic and lactic acid production contributed significantly to the national economies. A
fast acetic acid manufacture (Schnellessigfabrikation) was
developed by Schtzenbach in 1823. It worked, remarkably,
with active acetic acid bacteria (of course not recognized at
that time) immobilized on beechwood chips (Ost 1900).
Unformed, or unorganized ferments, obviously non-living
matter, different from yeast, enzymes in todays terms, were
recognized and further characterized. Notably diastase, of
which small amounts were able to liquify large amounts of
starch was studied in detail (Payen and Persoz 1833). Further,
enzymes described were, e.g. emulsin and pepsin (Schwann
1836; see also Buchholz and Poulson 2000). The first industrial processes that used enzymes (diastase) to produce dextrins were established from the 1830s onwards in France,
based on Payens work (Knapp 1847).
The most relevant events of this period are summarized
in Table 1.
The period from 1850 to 1890the emergence

of microbiology as a science
It was only with Pasteurs work that the scientific debate on the
nature of fermentation was settled in favor of the role of living
microorganisms, starting from hypotheses based on empirical
results provided by sophisticated experiments and ingenious
theoretical conclusions. Pasteurs outstanding accomplishments have been documented in several biographies, e.g.
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Table 1 Dates and events in early biotechnology

Ancient handicraft
6000 BC
3500 BC
3500 BC
Cheese and bread fermentation
Fourteenth century
Beer fermentation
Wine fermentation
Soja fermentation
Industrial acetic acid fermentation
Early period up to 1850

Scientific events
Technical application
1680 Leeuwenhoek observes microorganisms

1783 Spallanzani observed protease action
1793 Lavoisier and
1810 Gay-Lussac: quantitative chemistry of alcoholic fermentation GayLussac: hypothesis of spontaneous generation
1833 Payen and Persoz: diastase (enzyme) characterization
1836 Berzelius: catalysis (including enzymes) a
1837, 1838 Schwann, Cagniard-Latour: living cells as fermentation agents
1834, 1838 Ktzing, Quevenne: hypotheses of spontaneous generation, (see
also before, Gay- Lussac); vital factor
1839 Liebig: chemical decay hypothesis
1830s Major controversy on fermentation theories
a
Early eighteenth century: technical beer and wine fermentation;

also industrial beer fermentation
1823 Immobilized bacteria used for acetic acid production
1840s industrial enzymatic dextrin production (Payen)
Berzelius (1836)
(Birch 1990) and Geison (1995). The first basic question which
Pasteur definitively answered was that of the origin and character of fermentation: Was it brought about by living microorganisms, or by pure chemical phenomena, as Liebig, Berzelius
and their school believed? In the 1850s, Pasteur had visited a
factory for alcohol production on a nearly daily basis and took
samples of the fermentation broth which he investigated in his
laboratory. Losses in alcoholic fermentation were an initial
stimulus to work on a scientific explanation and on finding
technical solutions. After numerous microscopical observations, he observed yeast buds in normal fermentation runs,
but rods that he soon identified as lactic acid yeast, when
the fermentation ran sour (due to the formation of acetic or
lactic acid) (Pasteur 1857b). He investigated lactic acid fermentation in detail. In his paper on the topic, Pasteur (1857a)
elaborated the essentials of fermentation processes. He
presented the means with which to isolate microorganisms in
a pure culture. In his discussion he introduced (1) the biological
conception of fermentation as the result of the activity of living
microorganisms; (2) he discussed the practice of inoculation for
starting a reliable fermentation, that was also common practice
in beer fermentation; (3) the notion of specificity, according to
which each fermentation could be traced to a specific microbe;
(4) the essential experimental factor that the fermentation medium must provide the nutrients for the microorganism; and (5)
specific chemical features characterized by the main fermentation products and by products (Pasteur 1857a, b).
One of the mysteries of fermentation had remained highly controversial, the hypothesis of a generatio spontanea,
spontaneous generation of living organisms. Pasteur (1862)
addressed this basic and controversial question efficiently.
He referred to Schwann and others whose serious work he
repeated and confirmed, with significant experimental modifications (see also Geison 1995 p. 115). In addition to
highly precise experiments using various methods, Pasteur
undertook something of a show in 1860 with expeditions to
high altitude mountains, most spectacularly to the Alps and
the glacier Mer de Glace, to demonstrate the existence of
germ free air, in contrast to air under normal conditions
carrying germs causing infection in sugar juices (and in
fermentation). The results of these experiments were
presented by Pasteur first in a lecture to the Socit
Chimique de Paris in 1861 and then in a famous lecture at
the Sorbonne in 1864, a demonstrative performance for tout
Paris. The finding of yeasts and their living nature, as well
as the knowledge of their origin, eliminates the mystery of
the spontaneous occurence of fermentations of natural sugar
juices (Pasteur 1876, pp. 229, 230). Pasteur made a
radical attack against the chemical school, with Liebig as
the head, this being the central arena of dispute on fermentation (Pasteur 1860; Geison 1995).
Pasteurs book Etudes sur la Bire (Pasteur 1876) gave a
thorough experimental, theoretical and scientific account of
his investigations, results, and conclusions. He developed
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technical solutions to a number of practical problems, and

explained his motives in doing so. His findings, and their
establishment, may be considered to be a new paradigm
guiding further research. Pasteur thus laid the foundations
of a new scientific discipline, microbiology, known as bacteriology at the time (Delaunay 1951, Avant-propos, p. 22).
Among others, Berthelot (1860, 1864) and Bchamp (1864)
published a range of relevant papers on fermentation, e.g. of
substrates other than sugar.
However, one final mystery in fermentation remained:
the vital force hypothesis linked all chemical transformations
in fermentations to a mysterious act depending on life,
stating that the chemical act of fermentation is essentially a phenomenon correspondent to a vital act, beginning and ending with the latter (Pasteur 1876, pp. 229,
230, 306).
Several new active substances (enzymes) from different
sources (e.g. flowers and fruits, pancreas) were discovered,
including invertase, lipase and fibrinolytic activities, and
emulsin (Buchholz and Poulson 2000; Buchholz and Collins
2010, chapter 3). By the 1870s, studies had established the
existence of two types of ferments. They became known as
unformed (unorganized) and formed (organized) ferments (the
latter referred to living bodies, such as yeast). The German
physiologist, Willy Khne (1877) referred to the pepsin type
of unformed ferments as enzymes.
A summary of important scientific discoveries and applications is given in Table 2.
Although the application of fermentation processes was

well established, there were still problems with the manufacture and quality of the most important products, alcohol,
beer and wine. Considerable losses occurred in factories
producing alcohol from beet, when the juice was turning
sour. Early in his investigations on fermentation, Pasteur
was engaged in several industrial problems. They were subjects of highly accurate and meticulous scientific investigations
by Bchamp and Pasteur and led to the solution of the most
urgent problemsan ingenious combination of scientific and
technical progress with mutual interaction (Geison 1995; Birch.
1990). Pasteur (1873; 1876, p. 328) patented his invention
of a closed vessel for brewing to protect the fermentation
process from air-borne infections (Fig. 1).
A range of fermentation products became an important
part of the overall economy in European, North American
and Asian countries. At the end of the nineteenth century,
the fermentation industry was growing fast. It encompassed
the manufacture of beer and wine, industrial alcohol, yeast,
acetic and lactic acid, cheese, soy sauce and sake. Beer
manufacture represented one of the most important economic
activities. Thus in Germany, it had grown to 50 mn (million)
hL (hectoliter, 100 L) in 1890 (Ullmann 1915, p. 533). The
production process was described in all technology textbooks of the nineteenth century. Wine was also an important fermentation product, having a major economic
impact. The production around 1890 was estimated at 120
mn hL world wide, 113 mn hL in Europe, in France alone
Table 2 The period from 1850 to 1890 (Scriban 1982, pp.13, 14; Buchholz and Collins 2010, chapters 3 and 4)
Time, scientists
1837/1838 Schwann
and Cagniard-Latour
1850 Rayer and
Davaine
18561877 Pasteur
1866 Mendel
1876 Koch
1877-86 Pasteur
1880 Winogradsky
1881 Pasteur
Scientific findings, events
Technical progress, industrial innovation
Experimental demonstration of living yeast as agent in

alcoholic fermentation
Detection of the origin of anthrax and the role of
microorganisms in diseases
Investigations on fermentation (from 1856 on):
Investigations on alcohol fermentation (1858)
Studies on spontaneous generation (18591862)
Growing importance of industrial fermentation of beer

(production 23 million hL in 1840, Germany) b
Technical-scale production of yeast, wine, soy sauce, sake.
Industrial-scale beer fermentation in GB
Detection of anaerobic fermentation (1861)

Studies on wine fermentation, invention of
Pasteurisation (1864)
Studies on beer fermentation (1871)
Theory of fermentation (1876)
Detection of facultative anaerobic fermentation of yeast
Heredity laws
Work on the bacterium leading to anthrax; agar plate method
Begin of investigations on anthrax (1877)
Soil microorganisms: the bacterial nature of nitrification
Vaccination against anthrax and rabies
1870s: Hansen breeding pure yeast for commercial application;

1874 Christian Hansens Laboratory (Denmark): production
of rennet (chymosin) for cheese manufacture
Beer production: 36 million hectolitres in 1873, Germany
New type of industrial beer fermenter (Pasteur; Fig. 1)

1895 Wehmer: Lactic acid production
There are, of course, more scientists and events which have been relevant; however, inevitably, a selection must be made
1 hL corresponds to 100 L, or 0.1 m3
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Fig. 1 Pasteurs technical

fermenter (Pasteur 1876, p. 328)
about 39 mn hL (Brockhaus 1895, vol. 16, pp. 591595).

Wine was attributed not only agreeable but also health
effects when administered properly, e.g. a remarkable means
for preserving the forces and improving the resistance to
infections. The physiology was described with stimulation
of the nervous system and blood circulation, improving
or enhancing the subjective feeling and performance
(Brockhaus 1895, vol. 16, pp. 591595). The alcohol production in Germany was estimated up to 3.7 million hL in
1893/1894. An advanced technology had been developed
and applied in large factories: the process using starch as the
raw material was operated at high pressure to ensure gelatinization (Henzedmpfer.); hydrolysis was then achieved by
adding diastase (malt) to stirred tank reactors, followed by
fermentation for 72 h, using yeast that had been produced
separately; distilleries were controlled automatically
(Brockhaus 1895, vol. 15, p. 172178). Yeast as a commercial product was mainly generated in high yield in distilleries (pressed yeast, Presshefe); it was then sold for use in
other industrial processes, for example bread manufacture
(Payen 1874, Vol. 2, p. 403). In Denmark, Hansen made
major progress in breeding pure yeast by working with solid
culture media (e.g. agar plates, as did Koch) isolating colonies from single cells which he could then propagate. This
became the basis for pure yeast fermentation and commercial applications which was adopted e.g. by the German
brewing industry, where the Berlin Institute and its first
director Max Delbrck played a major role. The work
of Pasteur and Koch placed emphasis on the particular
quality of individual pure cultures or clones. It was
realized that quality control and characterization of the
organisms used were important. This accompanied the
beginning of microbial diagnostics which involved specific
staining.
Ferments in terms of enzymes found application,

diastase on a major industrial scale, since the 1840s, a
few others in the second half of the nineteenth century.
The first company founded on an enzyme-based process
was Christian Hansens laboratory in Copenhagen
(Denmark), so named to this day. It pioneered the use
of rennet (lab ferment, chymosin), for cheese manufacture (Brockhaus 1894b, vol. 10, p. 863; Poulson and
Buchholz 2003). Further pancreas enzymes, trypsin or
pancreatin, and pepsin, isolated from pig or cow, were
used as drugs, for example, as digestive aids. Pepsin is
a rational drug insofar that a weekend function of the
stomach (dyspepsia) is enforced by little doses of pepsin, and, in fact, numerous positive reports by doctors
are available (Brockhaus 1894b).
A wave of foundation of research institutions, mainly
governmental institutes took place, devoted to research
on beer, wine and food manufacture, hygiene, medical
care, and water, as well as laboratories of the brewing
and bakery industries, notably in Europe, and several in
the USA. Institutions for brewing research and education were established in Weihenstephan near Munich
(18721876), Berlin (Institut fr Grungsgewerbe, 1874),
Hohenheim (1888) (all in Germany), in Copenhagen,
and in Paris the famous Institut Pasteur (1888). In
Britain, the British School of Malting and Brewing
was founded at the University of Birmingham in 1899.
In the USA, by states decrees, agricultural research institutions
were founded from 1863 on, that eventually became the
origins of big universities like MIT, Cornell, and Wisconsin
(Bud 1993).
Following Pasteur and Kochs success in identifying
causative agents of disease and establishing pure cultures,
pharmaceutical companies also established bacteriology
3752
departments which produced vaccines or tested substances

for their antimicrobial properties (Metz 1997). J. E. Siebel in
Chicago issued a journal Zymotechnic Magazine: Zeitschrift
fr Grungsgewerbe and Food and Beverage Critic. In
German-speaking countries, 10 journals on brewing were
issued at that time (Brockhaus 1894a, b). Jrgensen, in
1885, founded the journal Zymotechnisk Tidende, and published a highly regarded book on Microorganisms and fermentation. Several further books on mycology and/or bacteria
or microorganisms were issued. The terms Bacteriology or
Mycology, Zymotechnologie, or Microbiology denominated
the new research field.
Thus the Age of Bacteriology began with a new paradigm, and a broadened industrial and economic base.
The period from 1890 to 1940The advent

of biochemistry, and new products
In 1891, Fischer established stereochemistry, illustrating his
theory on specificity with the famous picture of a lock and
key: To use a picture, I will say that enzyme and glucoside
must fit like lock and key in order to interact chemically. . .
(Fischer 1909). With the work of Emil Fischer (18521919)
came the breakthrough in the development of structural
biochemistry; in the course of his scientific career he
completely shifted the orientation of research in chemistry
towards the principal organic components of living matter:
sugars, fats, and proteins (Fruton and Simmonds 1953).
Buchner in the mid-1890s ended the hypothesis of
vis vitalis, that still postulated hidden mysterious forces
in fermentation, when he published a series of papers
(Buchner 1897, 1898; Buchner and Rapp 1898), which
signaled a breakthrough in fermentation and enzymology.
Buchners key experiment was to prepare a press juice from
yeast, which contained all the enzymes required for the
transformation of sugar into alcohol and carbon dioxide,
and to demonstrate that no living cells remained. He then
could show that this solution could perform the same reaction as did living yeast during fermentation, assuming one
enzyme, called zymase, being the catalyst. Buchner
presented the proof that (alcoholic) fermentation did not
require the presence of . . .such a complex apparatus as is
the yeast cell. The agent was in fact a soluble substance
without doubt a protein bodywhich he called zymase,
and what much later turned out to be the enzyme system
of the whole glycolytic pathway (Buchner 1897). With
Buchners work the dogma of the vis vitalis fell. It initiated
a new paradigm, the biochemical paradigm, which, in contrast to that of Pasteur, stated that enzyme catalysis, including complex phenomena like that of alcoholic fermentation,
was a chemical process not necessarily linked to the presence and action of living cells. The discovery of cell-free
fermentation, during the period up to 1930, stimulated the

molecular approach to the study of the pathway of alcoholic
fermentation, mainly the research on the successive intermediates in metabolism. Buchner himself continued to
work on cell-free fermentation investigating intermediate
compounds and activities both in cell-free press juice as
well as in living yeast that would convert possible intermediates including trioses. By the end of the 1940s,
the scheme of glycolysis and alcohol formation was
finally complete (Florkin 1975; Kohler 1975; Buchholz
and Collins 2010, chapter 4).
Of major impact on industrial microbiology were
Fernbachs systematic investigations at the Institut Pasteur
in Paris on metabolic intermediates during alcoholic fermentation (mainly of glycolysis) by various microorganisms,
e.g. yeast and Tyrothrix tenuis; this included the formation
of acids, notably acetic, succinic and pyruvic acids. He
identified corresponding enzymatic activities: the beginnings of what we now call biochemistry research. This
was not only important in elucidating the mechanism of
fermentation but was also of practical relevance for acid
production. Fernbach obtained patents on the fermentation
of starch for the production of acetone and higher alcohols
(Fernbach 1910; Fernbach and Strange 1911).
Around 19071910, there was a shortage of rubber on the
world market. Perkin in the UK proposed an alliance, comprising an extended list of chemists and bacteriologists,
including Fernbach and Weizmann, with the aim to produce
butanol (butyl alcohol), which could be converted into butadiene. This in turn could be polymerized to yield synthetic
rubber (Perkin Jr. 1912). Shortly after this initiative, the First
World War created a demand that drove technical innovation
in the fermentation industries. The acetone butanol fermentation process became a key technology for explosives
production since acetone was required as a solvent, in short
supply in Britain. Chaim Weizmann, who had worked in
Fernbachs laboratory, continued similar research in the
Biochemical Department of Manchester University, and
made a new contribution using a more abundant source of
raw material, viz. maize, in 1915 (Speakman 1919).
Weizmann brought his own laboratory experiments to the
notice of the Admiralty, in the spring of 1915. He asked
Winston Churchill, the first Lord of the Admiralty, to build a
plant, and in July, a pilot plant was erected in Nicholsons
London gin distillery. The process is usually referred to as
the Weizmann process (Weizmann 1917; Nathan 1919; for
more details and the political background refer to, Bud
1993). The manufacture of acetone by the Weizmann process attained the greatest success at the factory of British
Acetones, Toronto, Ltd., in Canada, on a large scale.
Fourteen new fermenters were constructed, about 18 ft
(5.5 m) in diameter and 20 ft (6.1 m) high, holding 24,000
gallons (91 m3) of mash (Nathan 1919; Speakman 1919). In
Germany, war requirements concerned glycerol (glycerin)

for the manufacture of explosives, when supplies of fat
became enormously curtailed as a result of the imposition
of the sea blockade. Investigations initiated by Ldecke with
the object of obtaining glycerol on an industrial scale by
means of fermentation became of supreme importance.
The process was developed by the Protol Company. The
monthly output of glycerol was about 1,000 tons
(Connstein and Ldecke 1919a, b).
The formation of oxalic and citric acids by Apergillus and
Penicillium species had been observed by Wehmer in the
1890s. Citric acid fermentation became an object of study
by several academic groups that were actively engaged in
optimizing the process and in elucidating the biochemical
mechanism leading from the sugar substrate to citric acid.
Currie undertook what came to be considered a classic
investigation of the factors controlling the production of
citric acid by a selected strain of Aspergillus niger; he
elaborated optimum conditions for the production of citric
acid. Currie joined Chas. Pfizer in New York, where a plant
was established which went into production in 1923. By
1933, this industry already contributed 85 % (in Europe
5,100 tons and in the USA 3,500 tons) of the worlds
citric acid production of 10,400 tons. Further products
manufactured by fermentation were gluconic acid and lactic
acid (May and Herrick 1930; Frey 1930; Roehr 1996, 1998).
Another important example of industrial research activity
was the development of the oxidation of sorbitol to sorbose
by Acetobacter suboxydans as an intermediate for vitamin
C. The corresponding so-called Reichstein process was used
from the 1930s on a large industrial scale (Buchholz and
Seibel 2008). Another process established in the 1930s was
the manufacture of L-ephedrine as a pharmaceutical using
the stereoselective condensation of benzaldehyde and
acetaldehyde by yeast (Vasic-Racki 2000). Ethanol continued to represent a major product of outstanding economic
importance.
A breakthrough event was in 1928 when Fleming observed that a culture of a Penicillium notatum inhibited the
growth of bacteria. He demonstrated the production of an
antibacterial substance in the culture broth and named it
penicillin. However, there was rather a long delay before
research and development aiming at production was undertaken, finally stimulated by Florey, Heatley, and Chain
who entered this field again toward the end of the 1930s
(Bud 2007; see below).
The nature of enzymes and the structure of proteins
required more than 40 years to be established, and it
remained controversial for decades (Sumner and Myrbck
1950). In the 1930s, Stanley successfully crystallized the
tobacco mosaic virus; it was the first time that any living form
had been crystallized, and it revolutionized thinking about
the chemical nature of life (VanDemark and Batzing 1987).
3753
Enzyme technology rapidly expanded. A range of enzymes,

including diastase (amylolytic enzymes), proteases and
pectinases, were isolated from different organisms for commercial use, mainly from Bacillus subtilis and other species
such as Aspergillus oryzae and A. niger (Tauber 1949, pp.
396494; Buchholz and Poulson 2000). Takamine began
isolating bacterial amylases in the 1890s in Japan. In 1894,
he obtained a patent for the production of a diastatic enzyme
preparation from molds, which he called Takadiastase for
the production of amylases for the hydrolysis of starches in
food manufacture (Tauber 1949). Major applications of enzymes were proteases in the chill-proofing of beer, and the
addition of malt extract in dough-making by American
bakers in the USA. In 1922, they used 30 million pounds
(13,500 tons) of malt extract valued at $ 2.5 million. In
1907, Rhm patented the application of a mixture containing
pancreatic extract as a bating agent, replacing the unpleasant
use of dung, and he founded the Rhm and Haas Company
in the same year based on this application. From about 1930
onwards, the enzyme preparation was produced by fermentation (Tauber 1949; Buchholz and Poulson 2000). For
education the Institute in Berlin offered various courses from
1888 (and later a curriculum for brewers), as did the Institut
fr Grungsphysiologie und Bakteriologie that was
established at the Technical High School in Vienna in
1897, as well as other institutions. Courses on fermentation
were offered by Bernhauer at the German University in
Prague in the 1930s (Bud 1993/1995, pp. 60, 61, 104,
132, 202, 203; Clifton 1966).
Important scientific breakthroughs and applications are
summarized in Table 3.
The period from 1940 to 1970the era of antibiotics,

and the emergence of biotechnology
Florey, Heatley and Chain, towards the end of the 1930s,
began to investigate penicillin in the course of their systematic study of antibacterial substances at Oxford University.
The credit for resurrecting penicillin, described at the time
as unstable as an opera singer, certainly goes to the Oxford
group. They developed an assay, found a way of producing
penicillin in surface culture and demonstrated the marked
activity and therapeutic value of penicillin in a clinical trial
in 1940 (Bud 2007; Coghill 1970; Demain 1981; Ohno et al.
2000). Early yields and recovery, however, were very
discouraging and the difficulties in wartime England led
them to visit authorities, laboratories, and industrial companies in the USA for help in July, 1941. They were advised
by research authorities to visit Peoria, Illinois, USA, to talk
with officials of the Northern Regional Research Laboratory
(NRRL) because this institution had just organized a fermentation division. The representatives offered Florey all
3754
Table 3 The period from 1890 to 1940 (Buchholz and Collins 2010, chapter 4; Roehr 1996)
Time, scientistsa
1894 E. Fischer
1897 Buchner
1900s Buchner
1905 E. Fischer and others
Specificity of enzymes
Fermentation due to enzyme action only
Rersearch on fermentation intermediates
Research in the nature of proteins
Enzyme technology expanding (Takadiastase)

First waste disposal biogas reactor (Bombay)
1910f Fernbach
1911f Fernbach and Strange;
1912f Perkin
1915f Weizmann
1915f Connstein and Ldecke
1916 Thom and Currie
1920s
1920s and 1930s Embden,
Meyerhoff and others
1925, 1930s Sumner, Northrup
1928 Fleming
1933 Reichstein
End of 1930s Florey and Chain
1940
Rersearch on fermentation intermediates

Microbial formation of acetone and butanol
Finding of Clostridium acetobutylicum

Glycerol fermentation b
Citric acid fermentation b
1907 Enzyme technology: Rhm and Haas company

(Germany)
b
Fermentation technology expanding: Production of butanol

for rubber manufacture b
War requirements: acetone and butanol production
Glycerol production for explosives
Research on glycolysis
Pfizer: Industrial production of citric acid

Large-scale industrial yeast production for bakeries
Enzyme crystallization
Finding of penicillin action
Sorbitol transformation into L-sorbose
Resumed research on penicillin
Protein structure solved
Large-scale waste water treatment (1928, Essen, Germany)

Reichstein process for vitamin C production
Sterile enzyme fermentation for detergents etc.
Peak alcohol production
Selected scientists and events relevant for applied microbiology (see also first footnote in Table 2)
Most intermediates mentioned here, butanol, acetone, citric acid, etc., have been observed before, but not developed further for industrial
production
the help they could give. Biosynthesis work began on July

15, 1941, at the NRRL under the general direction of Dr.
Coghill (Greene and Schmitz Jr. 1970; see also AIChE 1970)
. Research studies were also initiated at the Universities of
Minnesota (on microbial strains), Wisconsin (on fermentation), Penn State (on recovery), the Carnegie Institute,
Wisconsin and Stanford (on mutation) and at MIT (on drying
and packaging) (Coghill 1970). By the fall of 1941, yields of
penicillin began to climb to 6, 10, and to 24 Oxford units per
mL, using an improved mould strain, as compared with
about 3 units/mL obtained by the Oxford group.
In December 1941, the US Government became interested.
The US Department of Agriculture (USDA) called a meeting
in New York which included representatives from the
National Research Council, and four companies, Merck,
Squibb, Pfizer and Lederle, an event that was considered to
represent the real turning point for penicillin production
(Coghill 1970; Greene and Schmitz Jr. 1970) (some 18 more
companies became involved subsequently; Elder 1970).
Industry representatives agreed to make research teams available to work on the problem of supplying adequate quantities
of penicillin. By 1943, the amazing curative properties of
penicillin were becoming pretty well-known, and there was
a huge demand for the drug. The prime goal established by the
government representatives was to have ample stocks on hand
for the US armys invasion of Europe in the spring of 1944.
That goal finally was metby a huge effort, and a hitherto
unknown approach to interdisciplinary cooperation and project organization (Coghill 1970).

Strain screening and development, including mutation
procedures, proved to be a key factor for success. From
many sources, including soil samples from around the
world, collected by the US Army, many hundreds of strains
of penicillin-producers were isolated. The best producer of
all (labeled NRRL 1951), ironically, came from a moldy
cantaloupe melon from a Peoria fruit market. Genetic
changes were undertaken at the Carnegie Institute and at
the University of Wisconsin. Moyers (NRRL) medium
improvement and use of a better NRRL strain raised the
concentration of penicillin to 100 units/mL. Subsequent
improvements raised this by another order of magnitude to
about 1,500 units/mL with the Wisconsin strain (Coghill
1970). As a result, we began to get a trickle of a supply
of penicillin during the early months of 1942, as Richards
reported (Greene and Schmitz Jr. 1970; Silcox 1970). By
June 1942, enough penicillin to treat 10 patients had been
produced, and by February 1943 there was sufficient material to treat approximately 100 patients. Production was by
surface culture flasks, the most reliable method at the time.
In 1942, 2-years intensive development had resulted in
increasing the level of output of penicillin by some
140,000-fold. The most efficient approach was submerged
or deep-tank fermentation, but there were a number of
severe practical problems, the solutions of which were not
obvious, but which were finally achieved (Fig. 2) (Greene

and Schmitz Jr. 1970; Silcox 1970). Downstream operations, the isolation of penicillin, also represented a huge
challenge. They included new methods for biological processes, such as liquidliquid extraction, centrifugation,
freeze drying, crystallization and others (Silcox 1970;
Perlman 1970).
Thus began a wartime collaboration which was to involve the efforts of literally hundreds of biochemists, chemists, bacteriologists, biologists, chemical engineers,
physicians, toxicologists, pharmacologists, and pathologists
on both sides of the Atlantic, managed and coordinated by
industrial executives, academic administrators, and government leaders. (Greene and Schmitz Jr. 1970). At the political level, the injection of funds, people, companies, and
government interest meant a transformation in the ways of
doing science. A range of smaller projects on penicillin
production were undertaken in several other countries, including Germany, the Netherlands, France and by Czech scientist
(Bud 2007, pp. 7596). Penicillin became a public property
and big business (Bud 2007, pp. 2353, 5474). The prospects, and later the success of penicillin, prompted further
research on antibiotics. Waksman isolated actinomycyin in
1940, streptotricin in 1942, and streptomycin in 1944 from
cultures of actinomycetes (Ohno et al. 2000). (The patent on
Streptomycin and for starter cultures for yoghurt largely financed the establishment of the Life Sciences Faculty at the
University of Wisconsin in Madison for the next 50 years and
provided many stipends for students.) However, the therapeutic potential has been threatened by the emergence of increasingly resistant bacterial strains as a natural consequence of
their use, first observed by Abraham and Chain (1940). In
clinical settings, more than 50 % of Escherichia coli isolates
and more than 90 % of S. aureus isolates are ampicillin
Fig. 2 Penicillin fermenters in operation at E.R. Squibb & Sons, 1946

(Langlykke 1970)
3755
resistant. Factors that exacerbate this phenomenon are misuse

and overuse, and the widespread use of antibiotics in
aquariums, in agriculture and animal husbandry (Bud 2007,
pp.116-139; Hubschwerlen 2007).
By the 1950s, large-scale production not only of traditional goods, for example, beer, alcohol, cheese, but also
new products, including citric acid and pharmaceuticals and
other products of particularly high social and economic
relevance, had become well established. Growing economic
relevance followed notably the success of penicillin manufacture, and further antibiotics, like streptomycin, became
available, followed by a new class of high value-added
products, mainly secondary metabolites, e.g. steroids
obtained by biotransformation. Other major products of
growing market relevance included amino acids, organic
acids, carbohydrates, and derivatives (hydrolysates, isomers), vitamins, solvents, and enzymes for new applications
(Demain 1981; Demain 2001).
A new generation of biocatalysts, based on immobilization techniques developed in the academic field, led to a
breakthrough in processing of food and pharmaceutical
compounds. Large-scale processes were established using
biocatalysts for penicillin hydrolysis (for the synthesis of
semisynthetic -lactam antibiotics) and glucose isomerization (Poulson and Buchholz 2003; Buchholz et al. 2012,
chapters 7 and 8). Waste water treatment became more
wide-spread, due to legislation, and gained great attention.
This resulted in new developments, and capital investment,
both in the public and industrial sectors (Jrdening and
Winter 2005).
Significant events are summarized in Table 4
Starting in the 1970s and 1980s, BT gained the attention
of governmental agencies in Germany, the UK, Japan, the
USA and others as a field of innovative potential and economic growth. This was also in response to the first oil price
crisis in the beginning 1970s, and the realization that renewable material resources would become more important in the
future. These approaches led to expansion of the field. The
first enthusiastic report by the German chemical technology
organization Dechema was issued in 1974 for the German
Ministry for Education and Science (Bundesministerium fr
Bildung und Wissenschaft, BMBW). It was the first systematic approach for BT research funding, emphasizing classical BT, and developing a research and development strategy,
which finally aimed at encouraging innovations in industry
(Dechema 1974; Buchholz 1979; Bud 1994, pp. 192198).
This study has been an intriguing example of interaction
between policy makers, industry and science, and was
termed a corporatist approach by Jasanoff (1985). Further
studies on BT were issued in the UK, Japan and France (Bud
1993, pp. 189210). Essential topics and aims of the
Dechema study reflected the main established scientific
and applied fields of BT at that time. The basic disciplines
3756
Table 4 The period from 1940 to 1975 (Buchholz and Poulson 2000; Bud 2007; Buchholz and Collins 2010, chapters 4 and 5)
Time, scientists
End of 1930s Florey and Chain

1940
1940s Waksman
Resume research on penicillin

Protein structure solved
Extended research on antibiotics: actinomycin,
streptomycin
1941
1944
USA: penicillin project, due to war requirements

Large-scale industrial penicillin production; Pfizer:
deep tank penicillin fermentation
1948 Brotzu and Oxford team

1949
1952/1953
Cephalosporin, broad spectrum antibiotic

First biochemical engineering symposium
1953 Watson, Crick, Franklin

1950s
1958 Gaden (Ed.)
Structure of DNA
Development of immobilized enzymes
First biotech journal a
Production of further antibiotics: Pfizer, Lederle:

tetracycline; Eli Lilly: erythromycin
Industrial steroid biotransformation (prednisolone)
Expanding waste water treatment due to government
requirements
1959 Chain et al. with Beecham Begin of research on 6-APA

End of 1960s
1971
1972
1973 Cohen and Boyer
Gene cloning
1974
Political level: Germany: DECHEMA-report,
followed by other studies on biotechnology
in UK, Japan, France
Large-scale enzyme processes: detergents, starch processing;

Industrial production of 6-APA (Bayer, Germany; Beecham GB))
Large-scale enzymatic glucose isomerisation
Expanding production of amino and organic acids, vitamins,
enzymes in food manufacture
Failures: SCP production; cellulosics utilization; biosensorsb,c
This and the following table overlap in time scale due to events that are part of the two different periods
6-APA 6-aminopenicillanic acid, intermediate for the production of ampicillin and other semisynthetic penicillin derivatives
a
Journal of Microbiological and Biochemical Engineering; it later became Biotechnology and Bioengineering
There were of course other failures which would be worth investigation
An exception are glucose sensors
involved in BT research and development work were microbiology, cell biology, biochemistry, andto a limited
extentmolecular biology and genetics in addition to
chemical engineering. Recombinant DNA methods were
not mentioned since they were not available at the time of
writing the study (197274) (Buchholz 1979; Buchholz and
Collins 2010, chapter 5).
Research work in the field of BT proceeded as subtopic
within a motley collection of scientific and engineering disciplines with a low level of coherence and little integration up
till the 1960s and 1970s. During the 1940s, Stephensons
Bacterial Metabolism and of Kluyvers Chemical Activities
of Micro-Organisms appeared, the Grungschemische
Praktikum, by Bernhauer was published in 1936 (Bud
1993). Later, textbooks dealt with specific topics (not on
BT as an integrated field), signifying increased attention to
the field: on applied microbiology (Rehm 1967, Pirt 1975), as
well as on biochemical engineering (Aiba et al. 1965; Bailey
and Ollis 1977). The first encyclopedias and series on BT
were issued by Rehm and Reed (1981) and Flickinger and
Drew (1999). Thus, biotechnology did not exist as a scientific
discipline and there were no books, rather no journals, curricula or scientific conferences devoted to the subject. A few
UK and American universities offered special courses;
University College London established a curriculum granting
a Master of Science in Biochemical Engineering in the 1960s,
and another BT curriculum was established in the 1970s at
the Technical University of Berlin (Buchholz 1979, pp. 69,
71). The first BT journal of high reputation was established in
1958 by Elmer Gaden as the Journal of Microbiological and
Biochemical Engineering. It later became Biotechnology and
Bioengineering and is still a leading journal in the field. A
few other journals appeared in the 1950s and 1960s, for
example Applied Microbiology, renamed Environmental and
Applied Microbiology and Applied Microbiology and
Biotechnology.
The period from 1975 onthe new biotechnology

The turning point in genetics ensued from the establishment
of a model for the molecular structure of DNA by Jim
Watson and Francis Crick, based on the crystallography data

of Rosalind Franklin, who was working in Morris Wilkins
lab in 1953 (Watson and Crick 1953). This was the culmination of work initiated by Sir William Henry Bragg and his
son William Lawrence on X-ray diffraction by crystals, to
study molecular structure, initially of minerals but later of
more complex organic structures, including the first 3-D
structure of a protein, myoglobin (Max Perutz and John
Kendrew, see Kendrew et al. 1958), further of penicillin,
vitamin B 12, and insulin (Hodgkin 1979). The significance
of DNA structure, as the material of which genes are made,
was immediately recognized due to the ground-breaking
work, during the preceding 50 years, of a great number of
scientists in chemistry and biology, mostly microbiology
including Gregor Mendel, Friedrich Miescher, Phoebus
Levene, William Astbury, Erwin Chargaff, Oswald Avery,
Francois Jacob, Jacques Monod, Ole Maaloe, Max
Delbrck, Sydney Brenner and others (Judson 1979;
Winnacker 1987; Buchholz and Collins 2010, Chapter 7).
But the DNA Revolution as Hotchkiss termed it,
progressed or penetrated slowly into technology, initially
having little effect on traditional processes and products
(Hotchkiss 1979). The Asilomar conference 1975 initiated
a public discussion on the possible hazards of recombinant
DNA research (for details, see Buchholz and Collins 2010,
section 8.1.2). The following two decades saw many years
of discussion of possible risks and containment requirements associated with recombinant technologies which
eventually formed the basis of the guidelines for recombinant DNA work and finally culminated in international
legislation (see for example Cartagena Protocol on Biosafety,
http://bch.cbd.int/protocol/text/. )
Subsequent to Watson and Cricks publication in 1953 of
the DNA structure, a large number of significant scientific
breakthrough events as well as technological progress provided a new basis for BT. Selected events are summarized in
Table 5. Berg, Cohen, and Boyer in 1972 introduced recombinant DNA (rDNA) technology when they constructed the
first recombinant plasmids and viruses, which were introduced into bacteria, or animal cells respectively, where they
were autonomously propagated. A patent granted to Cohen
and Boyer, and the University of California was critically
commented by Berg (Cohen et al. 1972; Cohen and Boyer
1979/1980; Berg and Mertz 2010). Entrepreneur was still
a dirty word in molecular biology, leading one to reflect on
the situation in engineering a century earlier with the slandering of George Stephenson (later inventor of the steam
engine) by Sir Humphrey Davy at the time of his invention
of the miners lamp (not patented), already produced as
Stephensons prototype (patented).
Based on the new genetic techniques, a significant
change occurred during the 1980s and 1990s with common
approaches in different disciplines underlying BT, and the
3757
merging of molecular biology and biochemical engineering.

Industrial interest and the range of products expanded significantly, and many new companies, mainly in the USA,
were founded. New methods and tools played a key role in
the rapid expansion of recombinant technologies. These
include: gel electrophoresis, centrifugation, restriction endonucleases, plasmid cloning, a range of further cloning
methods extending to most known species of microorganisms and eukaryotes, in particular in plants, cloning of larger
(gene-sized) DNA fragments via virus cosmid, fosmid,
BAC and YAC (this latter in yeast) cloning, oligonucleotide
synthesis, DNA sequencing, gene mining, metagenomics,
and recently synthetic biology; protein design has become a
rational tool for biopharmaceuticals and enzyme development (Winnacker 1987; Demain 2001; Bornscheuer and
Buchholz 2005; Buchholz and Collins 2010, chapters 7, 9).
Once the tools for gene cloning in the Gram-negative E. coli
had been established it became easy to develop gene cloning
vectors which could be transferred to other species. This
involved the identification of plasmids that replicated in
other hosts and genes (promoters) that could be expressed
and used for selection in the new host, including bacteria,
yeast, insect cell lines and plant cells (Collins 1977). Thus
all the elements for the new recombinant DNA technology,
at least for bacterial and animal cells are available: Methods
to prepare DNA, which, following restriction cleavage
(i.e. treatment with restriction endonucleases) could be covalently joined to a vector with a DNA ligase; a vector
(plasmid or virus) to ensure maintenance in the cell; a
method to prepare clean vector DNA; an efficient method
to incorporate DNA into the cell; culture techniques to
isolate single clones carrying a single recombinant hybrid
molecule, including selective techniques to enrich for the
cells transformed with the vectors, for example selection
for antibiotic-resistance genes (Buchholz and Collins 2010,
chapter 7). More recently, since the 1990s, the so called
omics approaches: genomics, proteomics, metabolomics,
bioinformatics, and their integration into systems biology
and biotechnology aimed at understanding, quantitative description and rational modification of whole organisms.
Biosystems engineering or systems biotechnology aims at
the integration of biology, mathematics, bioinformatics, and
systems engineering to gain a holistic view of complex
biological and biotechnological systems, including quantitative description and improvement of whole organisms and
the rational development of novel production processes
(Reuss 2001; Deckwer et al. 2006; Klein-Marcuschamer et
al. 2010; Papini et al. 2010; Buchholz and Collins 2010,
sections 13.6 and 15.6).
As a consequence of this development, in the USA, also
on the political level, the perception of BT diverged greatly
by the 1980s as compared to that in Europe particularly in
Germany in the 1970s. This is perceived from a report of the
3758
Table 5 The new biotechnology

Scientific events
1944
1950
1953
1953
Technical application
Avery et al.: chemical nature of chromosomes: DNA

Chargaff: rule of nucleotide ratios
Sanger: sequence of insulin
Watson and Crick: structure of DNA
(For technical application up to the 1960s, see Table 4)
1955f Kornberg et al.: enzymatic DNA replication

1957f Zamecnik and Hoagland: amino acid activation, translation in
protein synthesis
1959 Kendrew: first X-ray enzyme structure
19601961 Jacob and Monod: operon model of gene regulation;
concept of mRNA
19611966 Nirenberg, Khorana et al.: genetic code
1963 Merrifield: solid-phase protein synthesis
1968 Arber and Linn: restriction endonucleases
1971f Nathans; Southern: DNA separation
1972 Mertz, Davies: recombinant DNA
Berg: first recombinant virus
Khorana et al.: first chemically synthesized gene
1973 Cohen, Boyer: recombinant plasmid/microorganism
1971 Farley, Cape, Glaser: establishment of Cetus, the first Biotech

Company
1972 Industrial production of 6-amino-penicillanic acid
1974 Large-scale production of glucose/fructose syrup

1975f Maxam and Gilbert; Sanger: methods for DNA sequencing
1975 Khler and Millstein: monoclonal antibodies
1976 Swanson, Boyer: foundation of second biotech company: Genentec
1975 Asilomar conference (moratorium on recombinant DNA research) 1977f Further New Biotech companies founded
1978 Heffron et al.: directed mutagenesis
1978 Recombinant human insulin (Genentec)
1979 Mayer, Collins and Wagner: recombinant penicillin acylase
1980 Chakrabarty: first patent for recombinant bacterium
1983f Frank and Blcker; Carruthers: mechanized DNA synthesis
1980f Work on recombinant -amylase (Novo)

1982 FDA approval of human insulin (Eli Lilly)
1982 Large-scale production of recombinant -galactosidase
(Boehringer Mannheim, D)
1983 Schell and Montagu: first transgenic plant (tobacco)

1984 Political level: OTA study; mechanized DNA sequencing
1988 Mullis: polymerase chain reaction (PCR)
1990 Start of human genome project a
1994 Stemmer: DNA shuffling
1995 First complete bacterial genome sequence
1995f Metabolic engineering b
1997 First cloned animal: Dolly
1998 Argonne Structural Genomics Meeting: human chromosome 22
2000 First approximate version of the human Genome a
1988 Leder, Stewart: patent for transgenic mouse
1996 Mass cultivation of recombinant seeds (commercial corn seeds)

1999 Start of CELERAindustrial genome sequencing
1999 Vitamin C via microbial pathway
These topics are difficult to assign, a range of arguments being raised in terms of their classification as technical application, not fundamental
research
Bailey (1991, 1996)
OTA of 1984 (OTA 1984). It refers to methods that arose

with knowledge on DNA and that revolutionized what
was thinkable. In contrast to the reports mentioned
before, emphasis in the OTA study was on genetic engineering and rDNA technology, resulting in commercial
opportunity and support of fast commercial exploitation
of scientific results, closely associated with the business

world.
The industrial breakthrough came with recombinant
human insulin, developed by Genentech in cooperation with
Ely Lilly in 1978, and approved by the US Food and Drug
Administration in 1982 (Bud 1993/1995, pp. 232, 237;
Walsh 2007, pp. 297, 298); this was at a time when some
heads of European pharmaceutical companies did not believe that a recombinant DNA product would ever be approved for clinical use. This precedent , notably the
approval human insulin as the first recombinant DNA
product on the market, was followed by a series of further
recombinant products, mostly drugs, which in general could
not be produced by other technical means, and which are of
great medical interest. Some of these products previously
isolated in small amounts from human blood or tissue were
in danger of being contaminated with human pathogenic
viruses (not all known at that time, e.g. AIDS virus, HCV).
In this respect, this alternative production route provided products not only in sufficient quantity for general use but also with
an improved and reproducible quality. The products included
human growth hormone in 1983, -interferon, and a hepatitis
B vaccine in 1986, tissue plasminogen activator (tPA) in 1987,
and erythropoietin in 1989 (product approval). Actually, recombinant proteins, including hormones and growth factors,
blood clotting factors, cytokines, monoclonal antibodies and
vaccines are most important biopharmaceuticals, with a market size estimated of some $50 billion per year around 2010
(Walsh 2007, Aggarwal 2007). Antibiotics remained an important sector of biopharmaceuticals, with many different
specialties and sales estimated at more than $50 billion per
year (Hubschwerlen 2007).
Large investment by multinational companies, the foundation of many small new companies, a few of which have
grown remarkably, and state funded big research merged in
a gold rush into the New Biotechnology, as recombinant
technology was termed in the USA. Key steps toward the
transfer of science into the economic sphere resulted in the
foundation of new BT companies, the first being Cetus,
started in 1971, later the originator of the polymerase chain
reaction (PCR; Kary Mullis) which gave birth to the era of
gene diagnostics and personalized medicine. Herbert Boyer
and Robert Swanson founded Genentech in 1976; amongst
the most important companies founded were Biogen (1978),
Amgen (1980) and Chiron (1981), later bought by Cetus
(Demain 2001, 2003, personal communication; Buchholz
and Collins 2010, chapters 5, 6, 17; for a recent survey,
see Table 17.5).
Industrial products, other than pharmaceuticals, expanded
as well, based both on traditional and recombinant methods,
with sales worldwide estimated over 50 billion . The most
important bulk products are ethanol, amino and organic
acids, produced in large amounts, vitamins, and biopolymers. Metabolic engineering has been used successfully for
the optimization of yields, e.g. for the production of amino
acids (for a survey, see Buchholz and Collins 2010, chapter
16). A very large sector for application of biotechnology is
in fact environmental technology which has become an
important industry. This includes waste water treatment,
3759
both aerobic and anaerobic, being applied in numerous

small up to very large-scale installations, as well as a great
number of exhaust air treatment units (Jrdening and Winter
2005). Ethanol, traditionally based on starch and sugar to
produce it as gasoline additive on a very large scale, provoked heavy criticism, with respect to using traditional
agriculture crops for biofuels rather than food. A major
crisis occurred in 2007 and most notably in mid-2008,
causing a dramatic increase in food prices. The growing
use of cereals for ethanol was thought to be in part responsible for this price increase. Recently a trend emerged for
using cellulosic biomass as a source of biofuels (Buchholz
et al. 2012, section 12.2). Production of biogas and electricity generated by microbial fuel cells gained much attention
and impetus (Buchholz and Collins 2010, chapter 16).
Recombinant DNA methods also greatly affected enzyme
technology since the late 1970s. Over expression in fastgrowing host organisms with high protein productivity
allowed many enzymes, which were not readily accessible,
to be produced cheaply on an industrial scale. This technology allowed design of enzymes with modified specificity
through iterative rapid cycles of gene mutation, screening or
selection and testing in addition to crystallography and
molecular modelling. Such products are used on a large
scale for starch products (used in food preparations with a
production volume of >10 million t/a, and ethanol with >37
million t/a), enzymes in detergents, for pharmaceuticals
manufacture, and many other fields (Buchholz et al. 2012,
chapters 7, 8, sections 12.1, 12.2). Plant biotechnology has
successfully been established, aiming at improved yields,
disease and herbicide resistance, etc. of crops. However,
controversies are ongoing with respect to political, ethical
and biosafety aspects. Transgenic crops are cultivated on a
very large scale notably in the USA, Argentina, Brazil,
Canada, and other countries (Slater et al. 2008; Buchholz
and Collins 2010, Chapter 18).
Two achievements since 2000 gained major public resonance: First, the major goal of the Human Genome Project
was achieved in 2000 with international cooperation and a
total expenditure of some $ 3 billion. The task which was
carried out by a major international consortium and largely
independently by Craig Venters group was recognized as
essentially complete in 2000 and commemorated by a communication in the presence of Francis Collins, Craig Venter
and the President of the USA. The result of the Human
Genome Project may possibly allow the discovery and production of hundreds of novel pharmaceuticals, many of
which are natural human gene products previously not
available in significant amounts or as virus-free preparations, significantly improving diagnosis and eventually revolutionizing medicine. However, a number of arguments
have been raised in terms of their classification as technical
application, not fundamental research. After 10 years of
3760
expectation, e.g. with respect to drug targeting, the following comment was put forward a transformational technology will always have its immediate consequences
overestimated and its long-term consequences underestimated,
and ....you may just start to imagine all the projects that will
spin-off (C&EN 2010). Much progress took place
largely through the involvement of flexible biotech companies such as Genentech, Cetus, Amgen and Biogen which
concentrated on innovative development in parallel with a
lethargy and bad management in large (particularly
European) pharmaceutical companies which lost their
dominance in this new field.
The second major event may be considered the understanding of the factors which control pluripotent and totipotent stem-cells and the controlled reprogramming of many
differentiated cells to such stem cells. This opens a new area
of medical research, production of models for genetic
diseases (for personalized medicine), and a radical new
approach to understanding cancer, developments which will
give potential to a new area of biotechnological development. This found its origin in the work of those studying the
molecular biology of cell differentiation and embryogenesis,
originally in insect, worm or animal models, as did for
example the Nobel laureate Christiane Nusslein-Volhardt
and as those most recently recognized with a Nobel Prize
(2012 Physiology or Medicine) for Sir John B. Gurdon and
Shinya Yamanaka.
A further event that received inordinate publicity was the
chemical synthesis of the entire genome of Mycoplasma
genitalium by the group of Craig Venter; transferring this
DNA into a foreign Mycoplasma caused replacement of the
resident genome by the completely synthetic genome,
forming a novel strain capable of continuous self-replication
(Gibson et al. 2010). The scientific relevance of this experiment, however, has been extensively debated, but subsequent
steps in synthetic biology may become a key technology
(Bornscheuer 2010). Although it is definitely not creation
of life, as many journalists sensationalized this milestone, it
may still be considered as a further step in the tradition of
Pasteur making use of living organisms, e.g. creating novel
cells with new synthetic potential.
Conclusions
The history of biotechnology comprises exciting developments over more than 200 years, from mysterious concepts
to rational science and technology, with great social and
medical achievements, and commercial impact. A review
of this history suggests that basic research and the solution
to open problems and unknown phenomena, have provided
a rational basis for a range of major technical innovations,
with which new industries emerged. Thus might be
interpreted the development from early fermentation research to Pasteurs concept of microbiology and technical
innovations, from Buchner and Fernbach towards Perkins
and Weizmanns processes, from Fleming towards Floreys
and Chains work, and the penicillin project, and Watsons
and Cricks solution of the DNA structure towards the
cloning concept by Berg, Cohen, Boyer, and towards the
establishment of new companies and New Biotechnology.
Recently, applied microbiology, biochemical engineering
and molecular biology have merged to form biotechnology
as a new scientific discipline in its own right, sharing a
common paradigm at the molecular level with all the other
life sciences (Buchholz 2007). Biotechnology continues, as
well, as a field of technology, to develop new technical
processes and products based on a rational scientific basis.
A diversification arose through the formation of subdisciplines, such as genomics, transcriptomics, proteomics, metabolic flux analysis with quantitative analysis of complex
metabolites, and finally biochemical engineering, which
merged into biosystems engineering.
Finally, we note that critical events during the historic
development of Biotechnology are associated with exceptional personalities who often had the vision and insight of
how their findings could be developed for the benefit of
science and humanity, translating them into practical invention finally leading to innovation. Public and private investment programs often came slowly on advice or practical
validation of radical advances by a few pioneers (This latter
aspect is treated in more detail throughout Buchholz and
Collins 2010).
Acknowledgment The authors gratefully acknowledge valuable

information by Arnold Demain.
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Appl Microbiol Biotechnol (2013) 97:37473762

The rootsa short history of industrial microbiology

mainly secondary metabolites, e.g. steroids obtained by

the production of beer, wine and bread. The written first

The early period till 1850fermentation mysteries

Appl Microbiol Biotechnol (2013) 97:37473762

with each other, others in opposition to each other, so that the

The period from 1850 to 1890the emergence

Appl Microbiol Biotechnol (2013) 97:37473762

Table 1 Dates and events in early biotechnology

Early period up to 1850

1680 Leeuwenhoek observes microorganisms

Early eighteenth century: technical beer and wine fermentation;

1840s industrial enzymatic dextrin production (Payen)

Appl Microbiol Biotechnol (2013) 97:37473762

technical solutions to a number of practical problems, and

Although the application of fermentation processes was

Scientific findings, events

Technical progress, industrial innovation

Experimental demonstration of living yeast as agent in

Growing importance of industrial fermentation of beer

Detection of anaerobic fermentation (1861)

1870s: Hansen breeding pure yeast for commercial application;

Beer production: 36 million hectolitres in 1873, Germany

New type of industrial beer fermenter (Pasteur; Fig. 1)

1 hL corresponds to 100 L, or 0.1 m3

Appl Microbiol Biotechnol (2013) 97:37473762

Fig. 1 Pasteurs technical

about 39 mn hL (Brockhaus 1895, vol. 16, pp. 591595).

Ferments in terms of enzymes found application,

departments which produced vaccines or tested substances

The period from 1890 to 1940The advent

Appl Microbiol Biotechnol (2013) 97:37473762

fermentation, during the period up to 1930, stimulated the

Appl Microbiol Biotechnol (2013) 97:37473762

Germany, war requirements concerned glycerol (glycerin)

Enzyme technology rapidly expanded. A range of enzymes,

The period from 1940 to 1970the era of antibiotics,

Appl Microbiol Biotechnol (2013) 97:37473762

Scientific findings, events

Technical progress, industrial innovation

Enzyme technology expanding (Takadiastase)

Rersearch on fermentation intermediates

Finding of Clostridium acetobutylicum

1907 Enzyme technology: Rhm and Haas company

Fermentation technology expanding: Production of butanol

Pfizer: Industrial production of citric acid

Large-scale waste water treatment (1928, Essen, Germany)

the help they could give. Biosynthesis work began on July

unknown approach to interdisciplinary cooperation and project organization (Coghill 1970).

Appl Microbiol Biotechnol (2013) 97:37473762

obvious, but which were finally achieved (Fig. 2) (Greene

Fig. 2 Penicillin fermenters in operation at E.R. Squibb & Sons, 1946

resistant. Factors that exacerbate this phenomenon are misuse

Appl Microbiol Biotechnol (2013) 97:37473762

Scientific findings, events

Technical progress, industrial innovation

End of 1930s Florey and Chain

Resume research on penicillin

USA: penicillin project, due to war requirements

1948 Brotzu and Oxford team

Cephalosporin, broad spectrum antibiotic

1953 Watson, Crick, Franklin

Production of further antibiotics: Pfizer, Lederle: