historia de la biotecnologia-1

8/18/2019 Historia de La Biotecnologia-1

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MINI-REVIEW

The roots — a 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 fasci-nating discoveries, such as yeast as living matter being

responsible for the fermentation of beer and wine. Seriouscontroversies arose between vitalists and chemists, resultingin the reversal of theories and paradigms, but promptingcontinuing research and progress. Pasteur ’s work led to theestablishment of the science of microbiology by developing

pure monoculture in sterile medium, and together with thework of Robert Koch to the recognition that a single path-ogenic organism is the causative agent for a particular disease. Pasteur also achieved innovations for industrial

processes of high economic relevance, including beer, wineand alcohol. Several decades later Buchner, disproved thehypothesis that processes in living cells required a meta-

physical ‘vis vitalis’ in addition to pure chemical laws.Enzymes were shown to be the chemical basis of biocon-versions. Studies on the formation of products in microbialfermentations, resulted in the manufacture of citric acid, andchemical components required for explosives particularly inwar time, acetone and butanol, and further products throughfermentation. The requirements for penicillin during theSecond World War lead to the industrial manufacture of

penicillin, and to the era of antibiotics with further antibi-otics, like streptomycin, becoming available. This wasfollowed by a new class of high value-added products,

mainly secondary metabolites, e.g. steroids obtained by biotransformation. By the mid-twentieth century, biotech-

nology was becoming an accepted specialty with courses being established in the life sciences departments of severaluniversities. Starting in the 1970s and 1980s, BT gained theattention of governmental agencies in Germany, the UK,Japan, the USA, and others as a field of innovative potentialand economic growth, leading to expansion of the field.Basic research in Biochemistry and Molecular Biology dra-matically widened the field of life sciences and at the sametime unified them considerably by the study of genes andtheir relatedness throughout the evolutionary process. Thescope of accessible products and services expanded signif-icantly. Economic input accelerated research and develop-

ment, by encouraging and financing the development of new methods, tools, machines and the foundation of newcompanies. The discipline of ‘ New Biotechnology’ becameone of the lead sciences. Although biotechnology has histor-ical roots, it continues to influence diverse industrial fields of activity, including food, feed and other commodities, for example polymer manufacture, biofuels and energy produc-tion, providing services such as environmental protection, andthe development and production of many of the most effectivedrugs. 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 . Biotechcompanies

Introduction

Fermentation has been of great practical and economicrelevance as a handicraft for thousands of years, notably

K. Buchholz (*)Institute for Chemical Engineering,Technical University of Braunschweig, Hans-Sommer Str. 10,38106 Braunschweig, Germanye-mail: [email protected]

J. CollinsLife Science Faculty, c/o Helmholtz Centrefor InfectionResearch - HZI, AG Directed Evolution,Technical University of Braunschweig, Inhoffenstr. 7,38124 Braunschweig, Germanye-mail: [email protected]

Appl Microbiol Biotechnol (2013) 97:3747 – 3762

DOI 10.1007/s00253-013-4768-2


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the production of beer, wine and bread. The written first document was by the Sumerians 6,000 years ago and de-scribes the technique of brewing (Bud 1993). Beer and winemanufacture was economically so important in ancient Mesopotamia and Egypt that it became a major source of tax revenue. Soya fermentation was established in Chinaaround 3500 BP. Due to its great practical relevance alco-

holic fermentation was of major technical as well as scien-tific 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 inthe emergence of BT.

The early period till 1850 — fermentation mysteries

Leeuwenhook, about 1680, had observed, with the aid of his

microscope, tiny ‘animalcules’ in droplets of liquids, whichhe, however, did not associate with fermentation. Then, inthe second half of the eighteenth century Spallanzani under-took microscopic investigations of many specimens, includ-ing sperm and microbial growth. By the end of theeighteenth and beginning of the nineteenth centuries, re-spectively, Lavoisier and Gay-Lussac had elaborated quan-titative correlations for alcoholic fermentation, without giving explanations for the process underlying it. From themid-1830s evidence began to accumulate which pointed tothe biological nature of fermentation. Based on well-designed experiments, Schwann (1837) and Cagniard-

Latour (1838) independently showed that yeast is a micro-organism, an ‘organized’ body, and that alcoholic fermenta-tion 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 theoryon fermentation corresponding in essential parts to that which Pasteur put forward about two decades later (for anextended overview, see Buchholz and Collins 2010, part I).

Many other scientists, including Kützing, Turpin andQuevenne, contributed significant advances in understandingfermentation, confirming that living organisms were involvedin fermentation processes other than that leading to alcohol,

e.g., in acetic acidfermentation. However, their arguments wereoften confused by mystic concepts, in particular that fermenta-tion emerges from spontaneous generation, and is a conse-quence of a ‘secret living force’ (in contrast to chemicalforces), a that view promoted, e.g. Gay-Lussac (Buchholz andCollins 2010, chapter 2). The mysterious concepts are obviousfrom a textbook by Poppe (1842, p. 229): ‘Fermentation is seenas a — at a time and under circumstances spontaneous - occur-ring 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 thefirst attract, the latter reject each other ’. Kützing (1837, pp. 396,397) believed that ‘… organic entities (living organisms) canform themselves by spontaneous generation …’, and he as-sumed two forces, the ‘organizing living force, and the chem-ical 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, vigorouslyargued against the concept of living bodies being active infermentation processes and advanced his erroneous theory of ferments that supposed a body undergoing decompositionwhich transfers its disturbed equilibrium onto other metastablesubstances (Liebig 1839). In his book on chemical technology,Knapp (1847, p. 271) came to the conclusion that ‘nooneofthe… hypotheses is up to now accepted as unequivocal truth’.

The importance of fermentation processes correspondswith 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, estimatedat a volume of about 22.7 million hectolitres (2,27 million m3)in 1840, whereas in the UK it was carried out on an industrialscale in large factories with fermenters of up to 240,000 L.Particularly beer, as well as wine, acetic and lactic acid pro-duction contributed significantly to the national economies. A‘fast acetic acid manufacture’ (‘Schnellessigfabrikation’) wasdeveloped by Schützenbach 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 today’s terms, wererecognized 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 (Schwann1836; see also Buchholz and Poulson 2000). The first indus-trial processes that used enzymes (diastase) to produce dex-trins were established from the 1830s onwards in France,

based on Payen’s work (Knapp 1847).The most relevant events of this period are summarized

in Table 1.

The period from 1850 to 1890 — the emergence

of microbiology as a science

It was only with Pasteur ’s work that the scientific debate on thenature of fermentation was settled in favor of the role of livingmicroorganisms, starting from hypotheses based on empiricalresults provided by sophisticated experiments and ingenioustheoretical conclusions. Pasteur ’s outstanding accomplish-ments have been documented in several biographies, e.g.

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(Birch 1990) and Geison (1995). The first basic question whichPasteur definitively answered was that of the origin and char-acter of fermentation: Was it brought about by living microor-ganisms, or by pure chemical phenomena, as Liebig, Berzeliusand 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 hislaboratory. Losses in alcoholic fermentation were an initialstimulus to work on a scientific explanation and on findingtechnical solutions. After numerous microscopical observa-tions, he observed yeast buds in normal fermentation runs,

but rods that he soon identified as lactic acid ‘yeast ’, whenthe fermentation ‘ran sour ’ (due to the formation of acetic or lactic acid) (Pasteur 1857b). He investigated lactic acid fermen-tation 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 biologicalconception of fermentation as the result of the activity of livingmicroorganisms; (2) he discussed the practice of inoculation for starting a reliable fermentation, that was also common practicein beer fermentation; (3) the notion of specificity, according towhich each fermentation could be traced to a specific microbe;(4) the essential experimental factor that the fermentation me-dium must provide the nutrients for the microorganism; and (5)specific chemical features characterized by the main fermenta-tion products and by products (Pasteur 1857a, b).

One of the mysteries of fermentation had remained high-ly 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 mod-ifications (see also Geison 1995 p. 115). In addition tohighly precise experiments using various methods, Pasteur undertook something of a show in 1860 with expeditions tohigh altitude mountains, most spectacularly to the Alps andthe glacier Mer de Glace, to demonstrate the existence of germ free air, in contrast to air under normal conditionscarrying germs causing infection in sugar juices (and infermentation). The results of these experiments were

presented by Pasteur first in a lecture to the SociétéChimique 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 wellas 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 aradical attack against the chemical school, with Liebig asthe head, this being the central arena of dispute on fermen-tation (Pasteur 1860; Geison 1995).

Pasteur ’s book Etudes sur la Bière (Pasteur 1876) gave athorough experimental, theoretical and scientific account of his investigations, results, and conclusions. He developed

Table 1 Dates and events in early biotechnology

Ancient handicraft

6000 BC Beer fermentation

3500 BC Wine fermentation

3500 BC Soja fermentation

Cheese and bread fermentation

Fourteenth century 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 Gay-

Lussac: hypothesis of spontaneous generation

Early eighteenth century: technical beer and wine fermentation;

also industrial beer fermentation

1833 Payen and Persoz: diastase (enzyme) characterization 1823 Immobilized bacteria used for acetic acid production

1836 Berzelius: catalysis (including enzymes) a

1837, 1838 Schwann, Cagniard-Latour: living cells as fermentation agents

1834, 1838 Kützing, Quevenne: hypotheses of spontaneous generation, (see

also before, Gay- Lussac); vital factor

1839 Liebig: chemical decay hypothesis 1840s industrial enzymatic dextrin production (Payen)

1830s Major controversy on fermentation theories

a Berzelius (1836)

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technical solutions to a number of practical problems, andexplained his motives in doing so. His findings, and their establishment, may be considered to be a new paradigmguiding further research. Pasteur thus laid the foundationsof a new scientific discipline, microbiology, known as bac-teriology at the time (Delaunay 1951, Avant-propos, p. 22).Among others, Berthelot (1860, 1864) and Béchamp (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 linkedall chemical transformationsin fermentations to a mysterious act depending on life, …stating that the ‘chemical act of fermentation is essen-tially a phenomenon correspondent to a vital act, begin-ning 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 Collins2010, chapter 3). By the 1870s, studies had established theexistence of two types of ferments. They became known asunformed (unorganized) and formed (organized) ferments (thelatter referred to living bodies, such as yeast). The German

physiologist, Willy Kühne (1877) referred to the pepsin typeof unformed ferments as ‘enzymes’.

A summary of important scientific discoveries and appli-cations is given in Table 2.

Although the application of fermentation processes waswell established, there were still problems with the manu-facture and quality of the most important products, alcohol,

beer and wine. Considerable losses occurred in factories producing alcohol from beet, when the juice was turningsour. Early in his investigations on fermentation, Pasteur was engaged in several industrial problems. They were sub-

jects of highly accurate and meticulous scientific investigations by Béchamp and Pasteur and led to the solution of the most urgent problems — an ingenious combination of scientific andtechnical progresswith mutual interaction (Geison1995; Birch.1990). Pasteur (1873; 1876, p. 328) patented his inventionof 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 Americanand Asian countries. At the end of the nineteenth century,the fermentation industry was growing fast. It encompassedthe 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 economicactivities. 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 economicimpact. The production around 1890 was estimated at 120mn 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 a Scientific findings, events Technical progress, industrial innovation

1837/1838 Schwann

and Cagniard-Latour

Experimental demonstration of living yeast as agent in

alcoholic fermentation

Growing importance of industrial fermentation of beer

(production 23 million hL in 1840, Germany) b

1850 Rayer and

Davaine

Detection of the origin of anthrax and the role of

microorganisms in diseases

Technical-scale production of yeast, wine, soy sauce, sake.

Industrial-scale beer fermentation in GB

1856 – 1877 Pasteur Investigations on fermentation (from 1856 on):

Investigations on alcohol fermentation (1858)

Studies on spontaneous generation (1859 – 1862) 1870s:Hansenbreedingpure yeast for commercialapplication;

1874 Christian Hansen’s Laboratory (Denmark): production

of rennet (chymosin) for cheese manufacture

Detection of anaerobic fermentation (1861)

Studies on wine fermentation, invention of

Pasteurisation (1864) Beer production: 36 million hectolitres in 1873, GermanyStudies on beer fermentation (1871)

Theory of fermentation (1876)Detection of facultative anaerobic fermentation of yeast New type of industrial beer fermenter (Pasteur; Fig. 1)

1866 Mendel Heredity laws

1876 Koch Work on the bacterium leading to anthrax; agar plate method 1895 Wehmer: Lactic acid production

1877-86 Pasteur Begin of investigations on anthrax (1877)

1880 Winogradsky Soil microorganisms: the bacterial nature of nitrification

1881 Pasteur Vaccination against anthrax and rabies

a There are, of course, more scientists and events which have been relevant; however, inevitably, a selection must be made b 1 hL corresponds to 100 L, or 0.1 m3



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about 39 mn hL (Brockhaus 1895, vol. 16, pp. 591 – 595).Wine was attributed not only agreeable but also healtheffects when administered properly, e.g. a remarkable meansfor preserving the forces and improving the resistance toinfections. The physiology was described with ‘stimulationof the nervous system and blood circulation, improvingor enhancing the subjective feeling and performance’(Brockhaus 1895, vol. 16, pp. 591 – 595). The alcohol pro-duction in Germany was estimated up to 3.7 million hL in1893/1894. An advanced technology had been developedand applied in large factories: the process using starch as theraw material was operated at high pressure to ensure gelati-

nization (Henzedämpfer.); hydrolysis was then achieved byadding diastase (malt) to stirred tank reactors, followed byfermentation for 72 h, using yeast that had been producedseparately; distilleries were controlled automatically(Brockhaus 1895, vol. 15, p. 172 – 178). Yeast as a commer-cial product was mainly generated in high yield in distiller-ies (pressed yeast, Presshefe); it was then sold for use inother industrial processes, for example bread manufacture(Payen 1874, Vol. 2, p. 403). In Denmark, Hansen mademajor progress in breeding pure yeast by working with solidculture media (e.g. agar plates, as did Koch) isolating colo-nies from single cells which he could then propagate. This

became the basis for pure yeast fermentation and commer-cial applications which was adopted e.g. by the German

brewing industry, where the Berlin Institute and its first director Max Delbrück played a major role. The work of Pasteur and Koch placed emphasis on the particular quality of individual pure cultures or clones. It wasrealized that quality control and characterization of theorganisms used were important. This accompanied the

beginning of microbial diagnostics which involved specificstaining.

Ferments in terms of enzymes found application,diastase on a major industrial scale, since the 1840s, afew others in the second half of the nineteenth century.The first company founded on an enzyme-based processwas ‘Christian Hansen’s laboratory in Copenhagen(Denmark), so named to this day. It pioneered the useof rennet (lab ferment, chymosin), for cheese manufac-ture (Brockhaus 1894b, vol. 10, p. 863; Poulson andBuchholz 2003). Further pancreas enzymes, trypsin or

pancreatin, and pepsin, isolated from pig or cow, wereused as drugs, for example, as digestive aids. ‘Pepsin isa rational drug insofar … that a weekend function of the

stomach (dyspepsia) is enforced by little doses of pep-sin, and, in fact, numerous positive reports by doctorsare available’ (Brockhaus 1894b).

A wave of foundation of research institutions, mainlygovernmental institutes took place, devoted to researchon beer, wine and food manufacture, hygiene, medicalcare, and water, as well as laboratories of the brewingand bakery industries, notably in Europe, and several inthe USA. Institutions for brewing research and educa-tion were established in Weihenstephan near Munich(1872 – 1876), Berlin (‘Institut für Gärungsgewerbe,’ 1874),Hohenheim (1888) (all in Germany), in Copenhagen,

and in Paris the famous ‘Institut Pasteur ’ (1888). InBritain, the ‘British School of Malting and Brewing’was founded at the University of Birmingham in 1899.In the USA, by states decrees, agricultural researchinstitutionswere founded from 1863 on, that eventually became theorigins of big universities like MIT, Cornell, and Wisconsin(Bud 1993).

Following Pasteur and Koch’s success in identifyingcausative agents of disease and establishing pure cultures,

pharmaceutical companies also established bacteriology

Fig. 1 Pasteur ’s technicalfermenter (Pasteur 1876, p. 328)



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departments which produced vaccines or tested substancesfor their antimicrobial properties (Metz 1997). J. E. Siebel inChicago issued a journal ‘Zymotechnic Magazine: Zeitschrift für Gärungsgewerbe and Food and Beverage Critic.’ InGerman-speaking countries, 10 journals on brewing wereissued at that time (Brockhaus 1894a, b). Jörgensen, in1885, founded the journal Zymotechnisk Tidende, and pub-

lished a highly regarded book on Microorganisms and fer-mentation. Several further books on mycology and/or bacteriaor microorganisms were issued. The terms Bacteriology or Mycology, ‘Zymotechnologie’, or Microbiology denominatedthe new research field.

Thus the ‘Age of Bacteriology’ began with a new para-digm, and a broadened industrial and economic base.

The period from 1890 to 1940 — The advent

of biochemistry, and new products

In 1891, Fischer established stereochemistry, illustrating histheory on specificity with the famous picture of a lock andkey: ‘To use a picture, I will say that enzyme and glucosidemust fit like lock and key in order to interact chemically. . .’(Fischer 1909). With the work of Emil Fischer (1852 – 1919)came the breakthrough in the development of structural

biochemistry; in the course of his scientific career hecompletely shifted the orientation of research in chemistrytowards 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), whichsignaled a breakthrough in fermentation and enzymology.Buchner ’s key experiment was to prepare a press juice fromyeast, which contained all the enzymes required for thetransformation of sugar into alcohol and carbon dioxide,and to demonstrate that no living cells remained. He thencould show that this solution could perform the same reac-tion as did living yeast during fermentation, assuming oneenzyme, 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 body — which he called ‘zymase,’and what much later turned out to be the enzyme systemof the whole glycolytic pathway (Buchner 1897). WithBuchner ’s work the dogma of the ‘vis vitalis’ fell. It initiateda new paradigm, the biochemical paradigm, which, in con-trast to that of Pasteur, stated that enzyme catalysis, includ-ing complex phenomena like that of alcoholic fermentation,was a chemical process not necessarily linked to the pres-ence and action of living cells. The discovery of cell-free

fermentation, during the period up to 1930, stimulated themolecular approach to the study of the pathway of alcoholicfermentation, mainly the research on the successive inter-mediates in metabolism. Buchner himself continued towork on cell-free fermentation investigating intermediatecompounds and activities both in cell-free press juice aswell as in living yeast that would convert possible in-

termediates including trioses. By the end of the 1940s,the scheme of glycolysis and alcohol formation wasfinally complete (Florkin 1975; Kohler 1975; Buchholzand Collins 2010, chapter 4).

Of major impact on industrial microbiology wereFernbach’s systematic investigations at the Institut Pasteur in Paris on metabolic intermediates during alcoholic fermen-tation (mainly of glycolysis) by various microorganisms,e.g. yeast and Tyrothrix tenuis; this included the formationof acids, notably acetic, succinic and pyruvic acids. Heidentified corresponding enzymatic activities: the begin-nings 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 fermentationof starch for the production of acetone and higher alcohols(Fernbach 1910; Fernbach and Strange 1911).

Around 1907 – 1910, there was a shortage of rubber on theworld market. Perkin in the UK proposed an alliance, com-

prising an extended list of chemists and bacteriologists,including Fernbach and Weizmann, with the aim to produce

butanol (butyl alcohol), which could be converted into bu-tadiene. This in turn could be polymerized to yield syntheticrubber (Perkin Jr. 1912). Shortly after this initiative, the First

World War created a demand that drove technical innovationin the fermentation industries. The ‘acetone butanol’ fer-mentation 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 inFernbach’s laboratory, continued similar research in theBiochemical Department of Manchester University, andmade a new contribution using a more abundant source of raw material, viz. maize, in 1915 (Speakman 1919).Weizmann brought his own laboratory experiments to thenotice of the Admiralty, in the spring of 1915. He askedWinston Churchill, the first Lord of the Admiralty, to build a

plant, and in July, a pilot plant was erected in Nicholson’sLondon gin distillery. The process is usually referred to asthe Weizmann process (Weizmann 1917; Nathan 1919; for more details and the political background refer to, Bud1993). The manufacture of acetone by the Weizmann pro-cess attained the greatest success at the factory of BritishAcetones, 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 24,000gallons (91 m3) of mash (Nathan 1919; Speakman 1919). In



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Germany, war requirements concerned glycerol (glycerin)for the manufacture of explosives, when supplies of fat

became enormously curtailed as a result of the impositionof the sea blockade. Investigations initiated by Lüdecke withthe object of obtaining glycerol on an industrial scale bymeans 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 Lüdecke 1919a, b).

The formation of oxalic and citric acids by Apergillus and Penicillium species had been observed by Wehmer in the1890s. Citric acid fermentation became an object of study

by several academic groups that were actively engaged inoptimizing the process and in elucidating the biochemicalmechanism leading from the sugar substrate to citric acid.Currie undertook what came to be considered a classicinvestigation of the factors controlling the production of citric acid by a selected strain of Aspergillus niger ; heelaborated 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. By1933, this industry already contributed 85 % (in Europe5,100 tons and in the USA 3,500 tons) of the world’scitric acid production of 10,400 tons. Further productsmanufactured by fermentation were gluconic acid and lacticacid (May and Herrick 1930; Frey 1930; Roehr 1996, 1998).Another important example of industrial research activitywas the development of the oxidation of sorbitol to sorbose

by Acetobacter suboxydans as an intermediate for vitaminC. The corresponding so-called Reichstein process was usedfrom the 1930s on a large industrial scale (Buchholz and

Seibel 2008). Another process established in the 1930s wasthe manufacture of L-ephedrine as a pharmaceutical usingthe stereoselective condensation of benzaldehyde andacetaldehyde by yeast (Vasic-Racki 2000). Ethanol con-tinued to represent a major product of outstanding economicimportance.

A breakthrough event was in 1928 when Fleming ob-served that a culture of a Penicillium notatum inhibited thegrowth of bacteria. He demonstrated the production of anantibacterial substance in the culture broth and named it

penicillin. However, there was rather a long delay beforeresearch and development aiming at production was un-

dertaken, finally stimulated by Florey, Heatley, and Chainwho entered this field again toward the end of the 1930s(Bud 2007; see below).

The nature of enzymes and the structure of proteinsrequired more than 40 years to be established, and it remained controversial for decades (Sumner and Myrbäck 1950). In the 1930s, Stanley successfully crystallized thetobacco mosaic virus; it was the first time that any living formhad been crystallized, and it revolutionized thinking about the chemical nature of life (VanDemark and Batzing 1987).

Enzyme technology rapidly expanded. A range of enzymes,including diastase (amylolytic enzymes), proteases and

pectinases, were isolated from different organisms for com-mercial use, mainly from Bacillus subtilis and other speciessuch as Aspergillus oryzae and A. niger (Tauber 1949, pp.396 – 494; Buchholz and Poulson 2000). Takamine beganisolating 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 infood manufacture (Tauber 1949). Major applications of en-zymes were proteases in the chill-proofing of beer, and theaddition 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. In1907, Röhm patented the application of a mixture containing

pancreatic extract as a bating agent, replacing the unpleasant use of dung, and he founded the Röhm and Haas Companyin the same year based on this application. From about 1930

onwards, the enzyme preparation was produced by fermen-tation (Tauber 1949; Buchholz and Poulson 2000). For education the Institute in Berlin offered various courses from1888 (and later a curriculum for brewers), as did the ‘Institut für Gärungsphysiologie und Bakteriologie’ that wasestablished at the Technical High School in Vienna in1897, as well as other institutions. Courses on fermentationwere offered by Bernhauer at the German University inPrague in the 1930s (Bud 1993/1995, pp. 60, 61, 104,132, 202, 203; Clifton 1966).

Important scientific breakthroughs and applications aresummarized in Table 3.

The period from 1940 to 1970 — the 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 system-atic 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 Oxfordgroup. 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 trialin 1940 (Bud 2007; Coghill 1970; Demain 1981; Ohno et al.2000). Early yields and recovery, however, were verydiscouraging and the difficulties in wartime England ledthem to visit authorities, laboratories, and industrial compa-nies 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 fer-mentation division. The representatives offered Florey all



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the help they could give. Biosynthesis work began on July15, 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 fermenta-

tion), Penn State (on recovery), the Carnegie Institute,Wisconsin and Stanford (on mutation) and at MIT (on dryingand 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 withabout 3 units/mL obtained by the Oxford group.

In December 1941, the US Government became interested.The US Department of Agriculture (USDA) called a meetingin New York which included representatives from the

National Research Council, and four companies, Merck,Squibb, Pfizer and Lederle, an event that was considered torepresent the real turning point for penicillin production

(Coghill 1970; Greene and Schmitz Jr. 1970) (some 18 morecompanies became involved subsequently; Elder 1970).Industry representatives agreed to make research teams avail-able to work on the problem of supplying adequate quantitiesof penicillin. By 1943, the amazing curative properties of

penicillin were becoming pretty well-known, and there wasa huge demand for the drug. The prime goal established by thegovernment representatives was to have ample stocks on handfor the US army’s invasion of Europe in the spring of 1944.That goal finally was met — by 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. Frommany sources, including soil samples from around the

world, collected by the US Army, many hundreds of strainsof penicillin-producers were isolated. The best producer of all (labeled NRRL 1951), ironically, came from a moldycantaloupe melon from a Peoria fruit market. Geneticchanges were undertaken at the Carnegie Institute and at the University of Wisconsin. Moyer ’s (NRRL) mediumimprovement and use of a better NRRL strain raised theconcentration of penicillin to 100 units/mL. Subsequent improvements raised this by another order of magnitude toabout 1,500 units/mL with the Wisconsin strain (Coghill1970). ‘As a result, we began to get a trickle of a supplyof penicillin during the early months of 1942’, as Richards

reported (Greene and Schmitz Jr. 1970; Silcox 1970). ByJune 1942, enough penicillin to treat 10 patients had been

produced, and by February 1943 there was sufficient mate-rial to treat approximately 100 patients. Production was bysurface culture flasks, the most reliable method at the time.In 1942, 2-years intensive development had resulted inincreasing the level of output of penicillin by some140,000-fold. The most efficient approach was submergedor deep-tank fermentation, but there were a number of severe practical problems, the solutions of which were not

Table 3 The period from 1890 to 1940 (Buchholz and Collins 2010, chapter 4; Roehr 1996)

Time, scientistsa Scientific findings, events Technical progress, industrial innovation

1894 E. Fischer Specificity of enzymes Enzyme technology expanding (Takadiastase)

1897 Buchner Fermentation due to enzyme action only First waste disposal biogas reactor (Bombay)

1900s Buchner Rersearch on fermentation intermediates

1905 E. Fischer and others Research in the nature of proteins 1907 Enzyme technology: Röhm and Haas company

(Germany)

1910f Fernbach Rersearch on fermentation intermediates

1911f Fernbach and Strange;

1912f Perkin

Microbial formation of acetone and butanol b Fermentation technology expanding: Production of butanol

for rubber manufacture b

1915f Weizmann Finding of Clostridium acetobutylicum War requirements: acetone and butanol production

1915f Connstein and Lüdecke Glycerol fermentation b Glycerol production for explosives

1916 Thom and Currie Citric acid fermentation b

1920s Pfizer: Industrial production of citric acid

1920s and 1930s Embden,

Meyerhoff and others

Research on glycolysis Large-scale industrial yeast production for bakeries

1925, 1930s Sumner, Northrup Enzyme crystallization

1928 Fleming Finding of penicillin action Large-scale waste water treatment (1928, Essen, Germany)

1933 Reichstein Sorbitol transformation into L

-sorbose Reichstein process for vitamin C productionEnd of 1930s Florey and Chain Resumed research on penicillin Sterile enzyme fermentation for detergents etc.

1940 Protein structure solved Peak alcohol production

a Selected scientists and events relevant for applied microbiology (see also first footnote in Table 2) b Most intermediates mentioned here, butanol, acetone, citric acid, etc., have been observed before, but not developed further for industrial production



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obvious, but which were finally achieved (Fig. 2) (Greeneand Schmitz Jr. 1970; Silcox 1970). Downstream opera-tions, the isolation of penicillin, also represented a hugechallenge. They included new methods for biological pro-cesses, such as liquid – liquid extraction, centrifugation,freeze drying, crystallization and others (Silcox 1970;Perlman 1970).

‘Thus began a wartime collaboration which was to in-volve the efforts of literally hundreds of biochemists, chem-ists, bacteriologists, biologists, chemical engineers,

physicians, toxicologists, pharmacologists, and pathologistson both sides of the Atlantic, managed and coordinated byindustrial executives, academic administrators, and govern-ment leaders’. (Greene and Schmitz Jr. 1970). At the polit-ical level, ‘the injection of funds, people, companies, andgovernment interest meant a transformation in the ways of doing science’. A range of smaller projects on penicillin

production were undertaken in several other countries, includ-ing Germany, the Netherlands, France and by Czech scientist

(Bud 2007, pp. 75 – 96). Penicillin became a public propertyand big business (Bud 2007, pp. 23 – 53, 54 – 74). The pros-

pects, and later the success of penicillin, prompted further research on antibiotics. Waksman isolated actinomycyin in1940, streptotricin in 1942, and streptomycin in 1944 fromcultures of actinomycetes (Ohno et al. 2000). (The patent onStreptomycin and for starter cultures for yoghurt largely fi-nanced the establishment of the Life Sciences Faculty at theUniversity of Wisconsin in Madison for the next 50 years and

provided many stipends for students.) However, the therapeu-tic potential has been threatened by the emergence of increas-ingly resistant bacterial strains as a natural consequence of

their use, first observed by Abraham and Chain (1940). Inclinical settings, more than 50 % of Escherichia coli isolatesand more than 90 % of S. aureus isolates are ampicillin

resistant. Factors that exacerbate this phenomenon are misuseand overuse, and the widespread use of antibiotics inaquariums, in agriculture and animal husbandry (Bud 2007,

pp.116-139; Hubschwerlen 2007).By the 1950s, large-scale production not only of tradi-

tional goods, for example, beer, alcohol, cheese, but alsonew products, including citric acid and pharmaceuticals and

other products of particularly high social and economicrelevance, had become well established. Growing economicrelevance followed notably the success of penicillin manu-facture, and further antibiotics, like streptomycin, becameavailable, followed by a new class of high value-added

products, mainly secondary metabolites, e.g. steroidsobtained by biotransformation. Other major products of growing market relevance included amino acids, organicacids, carbohydrates, and derivatives (hydrolysates, iso-mers), vitamins, solvents, and enzymes for new applications(Demain 1981; Demain 2001).

A new generation of biocatalysts, based on immobiliza-

tion techniques developed in the academic field, led to a breakthrough in processing of food and pharmaceuticalcompounds. Large-scale processes were established using

biocatalysts for penicillin hydrolysis (for the synthesis of semisynthetic β-lactam antibiotics) and glucose isomeriza-tion (Poulson and Buchholz 2003; Buchholz et al. 2012,chapters 7 and 8). Waste water treatment became morewide-spread, due to legislation, and gained great attention.This resulted in new developments, and capital investment,

both in the public and industrial sectors (Jördening andWinter 2005).

Significant events are summarized in Table 4

Starting in the 1970s and 1980s, BT gained the attentionof governmental agencies in Germany, the UK, Japan, theUSA and others as a field of innovative potential and eco-nomic growth. This was also in response to the first oil pricecrisis in the beginning 1970s, and the realization that renew-able material resources would become more important in thefuture. These approaches led to expansion of the field. Thefirst enthusiastic report by the German chemical technologyorganization Dechema was issued in 1974 for the GermanMinistry for Education and Science (Bundesministerium für Bildung und Wissenschaft, BMBW). It was the first system-atic approach for BT research funding, emphasizing classi-

cal BT, and developing a research and development strategy,which finally aimed at encouraging innovations in industry(Dechema 1974; Buchholz 1979; Bud 1994, pp. 192 – 198).This study has been an intriguing example of interaction

between policy makers, industry and science, and wastermed a corporatist approach by Jasanoff (1985). Further studies on BT were issued in the UK, Japan and France (Bud1993, pp. 189 – 210). Essential topics and aims of theDechema study reflected the main established scientificand applied fields of BT at that time. The basic disciplines

Fig. 2 Penicillin fermenters in operation at E.R. Squibb & Sons, 1946(Langlykke 1970)



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involved in BT research and development work were mi-crobiology, cell biology, biochemistry, and — to a limitedextent — molecular biology and genetics in addition tochemical engineering. Recombinant DNA methods werenot mentioned since they were not available at the time of writing the study (1972 – 74) (Buchholz 1979; Buchholz andCollins 2010, chapter 5).

Research work in the field of BT proceeded as subtopicwithin a motley collection of scientific and engineering dis-ciplines with a low level of coherence and little integration uptill the 1960s and 1970s. During the 1940s, Stephenson’s

Bacterial Metabolism and of Kluyver ’s Chemical Activitiesof Micro-Organisms appeared, the Gärungschemische

Praktiku m, by Bernhauer was published in 1936 (Bud1993). Later, textbooks dealt with specific topics (not onBT as an integrated field), signifying increased attention tothe field: on applied microbiology (Rehm 1967, Pirt 1975), aswell as on biochemical engineering (Aiba et al. 1965; Baileyand Ollis 1977). The first encyclopedias and series on BTwere issued by Rehm and Reed (1981) and Flickinger andDrew (1999). Thus, biotechnology did not exist as a scientific

discipline and there were no books, rather no journals, cur-ricula or scientific conferences devoted to the subject. A fewUK and American universities offered special courses;University College London established a curriculum grantinga 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 in1958 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

App lied Microb iolog y and Applied Microbiology and Biotechnology.

The period from 1975 on — the new biotechnology

The turning point in genetics ensued from the establishment of a model for the molecular structure of DNA by Jim

Table 4 The period from 1940 to 1975 (Buchholz and Poulson 2000; Bud 2007; Buchholz and Collins 2010, chapters 4 and 5)

Time, scientists Scientific findings, events Technical progress, industrial innovation

End of 1930s Florey and Chain Resume research on penicillin

1940 Protein structure solved

1940s Waksman Extended research on antibiotics: actinomycin,

streptomycin

1941 USA: penicillin project, due to war requirements1944 Large-scale industrial penicillin production; Pfizer:

deep tank penicillin fermentation

1948 Brotzu and Oxford team Cephalosporin, broad spectrum antibiotic

1949 First biochemical engineering symposium

1952/1953 Production of further antibiotics: Pfizer, Lederle:

tetracycline; Eli Lilly: erythromycin

1953 Watson, Crick, Franklin Structure of DNA

1950s Development of immobilized enzymes Industrial steroid biotransformation (prednisolone)

1958 Gaden (Ed.) First biotech journal a Expanding waste water treatment due to government

requirements

1959 Chain et al. with Beecham Begin of research on 6-APA

End of 1960s Large-scale enzyme processes: detergents, starch processing;

1971

1972 Industrial production of 6-APA (Bayer, Germany; Beecham GB))

1973 Cohen and Boyer Gene cloning Large-scale enzymatic glucose isomerisation

1974 Political level: Germany: DECHEMA-report,

followed by other studies on biotechnology

in UK, Japan, France

Expanding production of amino and organic acids, vitamins,

enzymes in food manufacture

Failures: SCP production; cellulosics utilization; biosensors b,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 derivativesa Journal of Microbiological and Biochemical Engineering ; it later became Biotechnology and Bioengineering b There were of course other failures which would be worth investigationc An exception are glucose sensors



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Watson and Francis Crick, based on the crystallography dataof Rosalind Franklin, who was working in Morris Wilkinslab in 1953 (Watson and Crick 1953). This was the culmi-nation of work initiated by Sir William Henry Bragg and hisson William Lawrence on X-ray diffraction by crystals, tostudy 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 JohnKendrew, see Kendrew et al. 1958), further of penicillin,vitamin B 12, and insulin (Hodgkin 1979). The significanceof DNA structure, as the material of which genes are made,was immediately recognized due to the ground-breakingwork, during the preceding 50 years, of a great number of scientists in chemistry and biology, mostly microbiologyincluding Gregor Mendel, Friedrich Miescher, PhoebusLevene, William Astbury, Erwin Chargaff, Oswald Avery,Francois Jacob, Jacques Monod, Ole Maaloe, MaxDelbrück, 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, initiallyhaving little effect on traditional processes and products(Hotchkiss 1979). The Asilomar conference 1975 initiateda 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 yearsof discussion of possible risks and containment require-ments associated with recombinant technologies whicheventually formed the basis of the guidelines for recombi-nant DNA work and finally culminated in internationallegislation (see for example Cartagena Protocol on Biosafety,

http://bch.cbd.int/protocol/text/ . )Subsequent to Watson and Crick ’s publication in 1953 of

the DNA structure, a large number of significant scientific breakthrough events as well as technological progress pro-vided a new basis for BT. Selected events are summarized inTable 5. Berg, Cohen, and Boyer in 1972 introduced recom-

binant DNA (rDNA) technology when they constructed thefirst recombinant plasmids and viruses, which were intro-duced into bacteria, or animal cells respectively, where theywere autonomously propagated. A patent granted to Cohenand Boyer, and the University of California was criticallycommented by Berg (Cohen et al. 1972; Cohen and Boyer

1979/1980; Berg and Mertz 2010). ‘Entrepreneur ’ was stilla dirty word in molecular biology, leading one to reflect onthe situation in engineering a century earlier with the slan-dering of George Stephenson (later inventor of the steamengine) by Sir Humphrey Davy at the time of his ‘invention’of the miner ’s lamp (not patented), already produced asStephenson’s prototype (patented).

Based on the new genetic techniques, a significant change occurred during the 1980s and 1990s with commonapproaches in different disciplines underlying BT, and the

merging of molecular biology and biochemical engineering.Industrial interest and the range of products expanded sig-nificantly, and many new companies, mainly in the USA,were founded. New methods and tools played a key role inthe rapid expansion of recombinant technologies. Theseinclude: gel electrophoresis, centrifugation, restriction endo-nucleases, plasmid cloning, a range of further cloning

methods extending to most known species of microorgan-isms 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, oligonucleotidesynthesis, DNA sequencing, gene mining, metagenomics,and recently synthetic biology; protein design has become arational tool for biopharmaceuticals and enzyme develop-ment (Winnacker 1987; Demain 2001; Bornscheuer andBuchholz 2005; Buchholz and Collins 2010, chapters 7, 9).Once the tools for gene cloning in the Gram-negative E. colihad been established it became easy to develop gene cloningvectors which could be transferred to other species. This

involved the identification of plasmids that replicated inother hosts and genes (promoters) that could be expressedand used for selection in the new host, including bacteria,yeast, insect cell lines and plant cells (Collins 1977). Thusall the elements for the new recombinant DNA technology,at least for bacterial and animal cells are available: Methodsto prepare DNA, which, following restriction cleavage(i.e. treatment with restriction endonucleases) could be co-valently joined to a ‘vector ’ with a DNA ligase; a ‘vector ’(plasmid or virus) to ensure maintenance in the cell; amethod to prepare ‘clean’ vector DNA; an efficient methodto incorporate DNA into the cell; culture techniques to

isolate single clones carrying a single recombinant hybridmolecule, including selective techniques to enrich for thecells ‘transformed’ with the vectors, for example selectionfor 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 biologyand biotechnology aimed at understanding, quantitative de-scription and rational modification of whole organisms.Biosystems engineering or systems biotechnology aims at the integration of biology, mathematics, bioinformatics, andsystems engineering to gain a holistic view of complex

biological and biotechnological systems, including quanti-tative description and improvement of whole organisms andthe 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, alsoon the political level, the perception of BT diverged greatly

by the 1980s as compared to that in Europe particularly inGermany in the 1970s. This is perceived from a report of the


http://bch.cbd.int/protocol/text/http://bch.cbd.int/protocol/text/


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OTA of 1984 (OTA 1984). It refers to methods that arosewith knowledge on DNA and that revolutionized what was ‘thinkable’. In contrast to the reports mentioned

before, emphasis in the OTA study was on genetic engi-neering and rDNA technology, resulting in commercialopportunity and support of fast commercial exploitation

of scientific results, closely associated with the businessworld.

The industrial breakthrough came with recombinant human insulin, developed by Genentech in cooperation withEly Lilly in 1978, and approved by the US Food and DrugAdministration in 1982 (Bud 1993/1995, pp. 232, 237;

Table 5 The new biotechnology

Scientific events Technical application

1944 Avery et al.: chemical nature of chromosomes: DNA

1950 Chargaff: rule of nucleotide ratios

1953 Sanger: sequence of insulin

1953 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

1960 – 1961 Jacob and Monod: operon model of gene regulation;

concept of mRNA

1961 – 1966 Nirenberg, Khorana et al.: genetic code

1963 Merrifield: solid-phase protein synthesis

1968 Arber and Linn: restriction endonucleases

1971f Nathans; Southern: DNA separation 1971 Farley, Cape, Glaser: establishment of Cetus, the first Biotech

Company

1972 Mertz, Davies: recombinant DNA 1972 Industrial production of 6-amino-penicillanic acid

Berg: first recombinant virus

Khorana et al.: first chemically synthesized gene

1973 Cohen, Boyer: recombinant plasmid/microorganism

1974 Large-scale production of glucose/fructose syrup

1975f Maxam and Gilbert; Sanger: methods for DNA sequencing

1975 Köhler 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 1980f Work on recombinant α-amylase (Novo)

1982 FDA approval of human insulin (Eli Lilly)

1983f Frank and Blöcker; Carruthers: mechanized DNA synthesis 1982 Large-scale production of recombinant α-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) 1988 Leder, Stewart: patent for transgenic mouse

1990 Start of human genome project a

1994 Stemmer: DNA shuffling

1995 First complete bacterial genome sequence

1995f Metabolic engineering b 1996 Mass cultivation of recombinant seeds (commercial corn seeds)

1997 First cloned animal: Dolly

1998 Argonne Structural Genomics Meeting: human chromosome 22 1999 Start of CELERA — industrial genome sequencing

2000 First approximate version of the human Genome a 1999 Vitamin C via microbial pathway

a

These topics are difficult to assign, a range of arguments being raised in terms of their classification as technical application, not fundamentalresearch b Bailey (1991, 1996)



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Walsh 2007, pp. 297, 298); this was at a time when someheads of European pharmaceutical companies did not be-lieve that a recombinant DNA product would ever be ap-

proved for clinical use . This precedent , notably theapproval 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 previouslyisolated in small amounts from human blood or tissue werein danger of being contaminated with human pathogenicviruses (not all known at that time, e.g. AIDS virus, HCV).In this respect, this alternative production route provided prod-ucts not only in sufficient quantity for general use but also withan improved and reproducible quality. The products includedhuman growth hormone in 1983, β-interferon, and a hepatitisB vaccine in 1986, tissue plasminogen activator (tPA) in 1987,and erythropoietin in 1989 (product approval). Actually, re-combinant proteins, including hormones and growth factors,

blood clotting factors, cytokines, monoclonal antibodies andvaccines are most important biopharmaceuticals, with a mar-ket size estimated of some $50 billion per year around 2010(Walsh 2007, Aggarwal 2007). Antibiotics remained an im-

portant 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 foun-dation of many small new companies, a few of which havegrown remarkably, and state funded big research merged ina ‘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 thefoundation of new BT companies, the first being Cetus,started in 1971, later the originator of the ‘ polymerase chainreaction’ (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; Buchholzand 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 organicacids, produced in large amounts, vitamins, and biopoly-mers. Metabolic engineering has been used successfully for the optimization of yields, e.g. for the production of aminoacids (for a survey, see Buchholz and Collins 2010, chapter 16). A very large sector for application of biotechnology isin fact environmental technology which has become animportant industry. This includes waste water treatment,

both aerobic and anaerobic, being applied in numeroussmall up to very large-scale installations, as well as a great number of exhaust air treatment units (Jördening and Winter 2005). Ethanol, traditionally based on starch and sugar to

produce it as gasoline additive on a very large scale, pro-voked heavy criticism, with respect to using traditionalagriculture 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 growinguse of cereals for ethanol was thought to be in part respon-sible for this price increase. Recently a trend emerged for using cellulosic biomass as a source of biofuels (Buchholzet al. 2012, section 12.2). Production of biogas and electric-ity generated by microbial fuel cells gained much attentionand impetus (Buchholz and Collins 2010, chapter 16).

Recombinant DNA methods also greatly affected enzymetechnology since the late 1970s. Over expression in fast-growing host organisms with high protein productivityallowed many enzymes, which were not readily accessible,

to be produced cheaply on an industrial scale. This technol-ogy allowed design of enzymes with modified specificitythrough iterative rapid cycles of gene mutation, screening or selection and testing in addition to crystallography andmolecular modelling. Such products are used on a largescale for starch products (used in food preparations with a

production volume of >10 million t/a, and ethanol with >37million t/a), enzymes in detergents, for pharmaceuticalsmanufacture, and many other fields (Buchholz et al. 2012,chapters 7, 8, sections 12.1, 12.2). Plant biotechnology hassuccessfully been established, aiming at improved yields,disease and herbicide resistance, etc. of crops. However,

controversies are ongoing with respect to political, ethicaland biosafety aspects. Transgenic crops are cultivated on avery large scale notably in the USA, Argentina, Brazil,Canada, and other countries (Slater et al. 2008; Buchholzand Collins 2010, Chapter 18).

Two achievements since 2000 gained major public reso-nance: First, the major goal of the Human Genome Project was achieved in 2000 with international cooperation and atotal expenditure of some $ 3 billion. The task which wascarried out by a major international consortium and largelyindependently by Craig Venters group was recognized asessentially complete in 2000 and commemorated by a com-

munication in the presence of Francis Collins, Craig Venter and the President of the USA. The result of the HumanGenome Project may possibly allow the discovery and pro-duction of hundreds of novel pharmaceuticals, many of which are natural human gene products previously not available in significant amounts or as virus-free prepara-tions, significantly improving diagnosis and eventually rev-olutionizing medicine. However, a number of argumentshave been raised in terms of their classification as technicalapplication, not fundamental research. After 10 years of



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expectation, e.g. with respect to drug targeting, the follow-ing comment was put forward ‘… a transformational tech-nology will always have its immediate consequencesoverestimated and its long-term consequences underestimated,and ....you may just start to imagine all the projects that willspin-off …’ (C&EN 2010). Much progress took placelargely through the involvement of flexible biotech compa-

nies such as Genentech, Cetus, Amgen and Biogen whichconcentrated on innovative development in parallel with alethargy and bad management in large (particularlyEuropean) pharmaceutical companies which lost their dominance in this new field.

The second major event may be considered the under-standing of the factors which control pluripotent and toti-

potent stem-cells and the controlled reprogramming of manydifferentiated cells to such stem cells. This opens a new areaof medical research, production of models for geneticdiseases (for personalized medicine), and a radical newapproach to understanding cancer, developments which will

give potential to a new area of biotechnological develop-ment. This found its origin in the work of those studying themolecular 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 andShinya Yamanaka.

A further event that received inordinate publicity was thechemical synthesis of the entire genome of Mycoplasmagenitalium by the group of Craig Venter; transferring thisDNA 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 experi-ment, however, has been extensively debated, but subsequent steps in synthetic biology may become a key technology(Bornscheuer 2010). Although it is definitely not ‘creationof 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 novelcells with new synthetic potential.

Conclusions

The history of biotechnology comprises exciting develop-ments over more than 200 years, from mysterious conceptsto rational science and technology, with great social andmedical achievements, and commercial impact. A reviewof this history suggests that basic research and the solutionto open problems and unknown phenomena, have provideda rational basis for a range of major technical innovations,with which new industries emerged. Thus might be

interpreted the development from early fermentation re-search to Pasteurs concept of microbiology and technicalinnovations, from Buchner and Fernbach towards Perkin’sand Weizmann’s processes, from Fleming towards Florey’sand Chain’s work, and the penicillin project, and Watson’sand Crick ’s solution of the DNA structure towards thecloning 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 biotechnologyas a new scientific discipline in its own right, sharing acommon paradigm at the molecular level with all the other life sciences (Buchholz 2007). Biotechnology continues, aswell, 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 subdisci-

plines, such as genomics, transcriptomics, proteomics, met-abolic flux analysis with quantitative analysis of complexmetabolites, and finally biochemical engineering, which

merged into biosystems engineering.Finally, we note that critical events during the historic

development of Biotechnology are associated with excep-tional 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 inven-tion finally leading to innovation. Public and private invest-ment programs often came slowly on advice or practicalvalidation of radical advances by a few pioneers (This latter aspect is treated in more detail throughout Buchholz andCollins 2010).

Acknowledgment The authors gratefully acknowledge valuableinformation by Arnold Demain.

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