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Appl Microbiol Biotechnol (2013) 97:3747–3762 DOI 10.1007/s00253-013-4768-2

MINI-REVIEW

The roots—a short history of industrial and

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- mainly secondary metabolites, e.g. obtained by nating discoveries, such as as living being biotransformation. By the mid-twentieth century, biotech- responsible for the of and . Serious nology was becoming an accepted specialty with courses controversies arose between vitalists and chemists, resulting being established in the life sciences departments of several in the reversal of theories and paradigms, but prompting universities. Starting in the 1970s and 1980s, BT gained the continuing research and progress. Pasteur’s work led to the attention of governmental agencies in Germany, the UK, establishment of the science of microbiology by developing Japan, the USA, and others as a field of innovative potential pure monoculture in sterile medium, and together with the and economic growth, leading to expansion of the field. work of to the recognition that a single path- Basic research in and Molecular dra- ogenic organism is the causative agent for a particular matically widened the field of life sciences and at the same disease. Pasteur also achieved innovations for industrial time unified them considerably by the study of and processes of high economic relevance, including beer, wine their relatedness throughout the evolutionary process. The and . Several decades later Buchner, disproved the scope of accessible products and services expanded signif- hypothesis that processes in living cells required a meta- icantly. Economic input accelerated research and develop- physical ‘vis vitalis’ in addition to pure chemical laws. ment, by encouraging and financing the development of were shown to be the chemical basis of biocon- new methods, tools, machines and the foundation of new versions. Studies on the formation of products in microbial companies. The discipline of ‘New Biotechnology’ became , resulted in the manufacture of citric , and one of the lead sciences. Although biotechnology has histor- chemical components required for explosives particularly in ical roots, it continues to influence diverse industrial fields of war time, and butanol, and further products through activity, including food, feed and other commodities, for fermentation. The requirements for during the example polymer manufacture, biofuels and energy produc- Second World War lead to the industrial manufacture of tion, providing services such as environmental protection, and penicillin, and to the era of with further antibi- the development and production of many of the most effective otics, like streptomycin, becoming available. This was drugs. The understanding of biology down to the molecular followed by a new class of high value-added products, level opens the way to create novel products and efficient environmentally acceptable methods for their production. K. Buchholz (*) Institute for , Keywords Biotechnology . History . Fermentation theories . Technical University of Braunschweig, Hans-Sommer Str. 10, Industrial microbiology . Genetic techniques . Biotech 38106 Braunschweig, Germany companies e-mail: [email protected]

J. Collins Life Science Faculty, c/o Helmholtz Centre Introduction for InfectionResearch - HZI, AG Directed , Technical University of Braunschweig, Inhoffenstr. 7, 38124 Braunschweig, Germany Fermentation has been of great practical and economic e-mail: [email protected] relevance as a handicraft for thousands of years, notably 3748 Appl Microbiol Biotechnol (2013) 97:3747–3762 the production of beer, wine and . The written first with each other, others in opposition to each other, so that the document was by the Sumerians 6,000 years ago and de- first attract, the latter reject each other’. Kützing (1837, pp. 396, scribes the technique of brewing (Bud 1993). Beer and wine 397) believed that ‘… organic entities (living organisms) can manufacture was economically so important in ancient form themselves by spontaneous generation …’, and he as- Mesopotamia and Egypt that it became a major source of sumed two forces, the ‘organizing living force, and the chem- tax revenue. Soya fermentation was established in China ical affinity, fighting each other …’, and Quevenne (1838, around 3500 BP. Due to its great practical relevance alco- p.469) used the term ‘secret of life’. In contrast to the vitalist holic fermentation was of major technical as well as scien- school, Liebig, the head of the chemical school, vigorously tific interest. Controversies over basic concepts, e.g. argued against the concept of living bodies being active in vitalism versus materialism in and biology, fermentation processes and advanced his erroneous theory of resulted in the establishment, and reversal of theories and ferments that supposed a body undergoing decomposition paradigms, but finally lead to scientific rationalisation of which transfers its disturbed equilibrium onto other metastable causality, and continuous technical progress, resulting in substances (Liebig 1839). In his book on chemical technology, the emergence of BT. 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 The early period till 1850—fermentation mysteries with the large sections that were devoted to the topic in the books on technology and chemical engineering of the time Leeuwenhook, about 1680, had observed, with the aid of his (Otto 1838; Poppe 1842; Knapp 1847; Wagner 1857;Payen, microscope, tiny ‘animalcules’ in droplets of , which 1874). Knapp (1847, p.367) reported that brewing was he, however, did not associate with fermentation. Then, in performed in Germany at the level of handicraft, estimated the second half of the eighteenth century Spallanzani under- at a volume of about 22.7 million hectolitres (2,27 million m3) took microscopic investigations of many specimens, includ- in 1840, whereas in the UK it was carried out on an industrial ingspermandmicrobialgrowth.Bytheendofthe scale in large factories with fermenters of up to 240,000 L. eighteenth and beginning of the nineteenth centuries, re- Particularly beer, as well as wine, acetic and lactic acid pro- spectively, Lavoisier and Gay-Lussac had elaborated quan- duction contributed significantly to the national economies. A titative correlations for alcoholic fermentation, without ‘fast manufacture’ (‘Schnellessigfabrikation’)was giving explanations for the process underlying it. From the developed by Schützenbach in 1823. It worked, remarkably, mid-1830s evidence began to accumulate which pointed to with active acetic acid (of course not recognized at the biological of fermentation. Based on well- that time) immobilized on beechwood chips (Ost 1900). designed experiments, Schwann (1837) and Cagniard- Unformed, or unorganized ferments, obviously non-living Latour (1838) independently showed that yeast is a micro- matter, different from yeast, enzymes in today’sterms,were organism, an ‘organized’ body, and that alcoholic fermenta- recognized and further characterized. Notably diastase, of tion is linked to living yeast. Both observed the yeast of beer which small amounts were able to liquify large amounts of being little globular bodies able to reproduce themselves, was studied in detail (Payen and Persoz 1833). Further, excluding spontaneous generation, and presenting a theory enzymes described were, e.g. ‘emulsin’ and pepsin (Schwann on fermentation corresponding in essential parts to that 1836; see also Buchholz and Poulson 2000). The first indus- which Pasteur put forward about two decades later (for an trial processes that used enzymes (diastase) to produce dex- extended overview, see Buchholz and Collins 2010, part I). trins were established from the 1830s onwards in France, Many other scientists, including Kützing, Turpin and based on Payen’swork(Knapp1847). Quevenne, contributed significant advances in understanding The most relevant events of this period are summarized fermentation, confirming that living organisms were involved in Table 1. 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 fermenta- The period from 1850 to 1890—the emergence tion emerges from spontaneous generation, and is a conse- of microbiology as a science quence of a ‘secret living force’ (in contrast to chemical forces), a that view promoted, e.g. Gay-Lussac (Buchholz and It was only with Pasteur’s work that the scientific debate on the Collins 2010, chapter 2). The mysterious concepts are obvious nature of fermentation was settled in favor of the role of living from a textbook by Poppe (1842, p. 229): ‘Fermentation is seen , starting from hypotheses based on empirical as a—at a time and under circumstances spontaneous - occur- results provided by sophisticated experiments and ingenious ring mighty movement in a of different compounds …, theoretical conclusions. Pasteur’s outstanding accomplish- which is due to the fact that several compounds act in harmony ments have been documented in several biographies, e.g. Appl Microbiol Biotechnol (2013) 97:3747–3762 3749

Table 1 Dates and events in early biotechnology

Ancient handicraft

6000 BC Beer fermentation 3500 BC Wine fermentation 3500 BC Soja fermentation 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 action 1793 Lavoisier and 1810 Gay-Lussac: quantitative chemistry of alcoholic fermentation Gay- Early eighteenth century: technical beer and wine fermentation; Lussac: hypothesis of spontaneous generation also industrial beer fermentation 1833 Payen and Persoz: diastase () characterization 1823 Immobilized bacteria used for acetic acid production 1836 Berzelius: (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)

(Birch 1990)andGeison(1995).Thefirstbasicquestionwhich One of the mysteries of fermentation had remained high- Pasteur definitively answered was that of the origin and char- ly controversial, the hypothesis of a ‘generatio spontanea’, acter of fermentation: Was it brought about by living microor- spontaneous generation of living organisms. Pasteur (1862) ganisms, or by pure chemical phenomena, as Liebig, Berzelius addressed this basic and controversial question efficiently. and their school believed? In the 1850s, Pasteur had visited a He referred to Schwann and others whose ‘serious work’ he factory for alcohol production on a nearly daily basis and took repeated and confirmed, with significant experimental mod- samples of the fermentation broth which he investigated in his ifications (see also Geison 1995 p. 115). In addition to laboratory. Losses in alcoholic fermentation were an initial highly precise experiments using various methods, Pasteur stimulus to work on a scientific explanation and on finding undertook something of a show in 1860 with expeditions to technical . After numerous microscopical observa- high altitude mountains, most spectacularly to the Alps and tions, he observed yeast buds in normal fermentation runs, the glacier Mer de Glace, to demonstrate the existence of but rods that he soon identified as lactic acid ‘yeast’,when germ free air, in contrast to air under normal conditions the fermentation ‘ran sour’ (due to the formation of acetic or carrying germs causing in juices (and in lactic acid) (Pasteur 1857b). He investigated lactic acid fermen- fermentation). The results of these experiments were tation in detail. In his paper on the topic, Pasteur (1857a) presented by Pasteur first in a lecture to the Société elaborated the essentials of fermentation processes. He Chimique de Paris in 1861 and then in a famous lecture at presented the means with which to isolate microorganisms in the Sorbonne in 1864, a demonstrative performance for ‘tout a pure culture. In his discussion he introduced (1) the biological Paris’. ‘The finding of and their living nature, as well conception of fermentation as the result of the activity of living as the knowledge of their origin, eliminates the mystery of microorganisms; (2) he discussed the practice of inoculation for the spontaneous occurence of fermentations of natural sugar starting a reliable fermentation, that was also common practice juices…’ (Pasteur 1876, pp. 229, 230). Pasteur made a in beer fermentation; (3) the notion of specificity, according to radical attack against the chemical school, with Liebig as which each fermentation could be traced to a specific microbe; the head, this being the central arena of dispute on fermen- (4) the essential experimental factor that the fermentation me- tation (Pasteur 1860; Geison 1995). dium must provide the nutrients for the ; and (5) Pasteur’s book Etudes sur la Bière (Pasteur 1876) gave a specific chemical features characterized by the main fermenta- thorough experimental, theoretical and scientific account of tion products and by products (Pasteur 1857a, b). his investigations, results, and conclusions. He developed 3750 Appl Microbiol Biotechnol (2013) 97:3747–3762 technical solutions to a number of practical problems, and Although the application of fermentation processes was explained his motives in doing so. His findings, and their well established, there were still problems with the manu- establishment, may be considered to be a new paradigm facture and quality of the most important products, alcohol, guiding further research. Pasteur thus laid the foundations beer and wine. Considerable losses occurred in factories of a new scientific discipline, microbiology, known as bac- producing alcohol from beet, when the juice was turning teriology at the time (Delaunay 1951, Avant-propos, p. 22). sour. Early in his investigations on fermentation, Pasteur Among others, Berthelot (1860, 1864) and Béchamp (1864) was engaged in several industrial problems. They were sub- published a range of relevant papers on fermentation, e.g. of jects of highly accurate and meticulous scientific investigations substrates other than sugar. by Béchamp and Pasteur and led to the of the most However, one final mystery in fermentation remained: urgent problems—an ingenious combination of scientific and the ‘vital force’ hypothesis linked all chemical transformations technical progress with mutual interaction (Geison 1995;Birch. in fermentations to a mysterious act depending on life, … 1990). Pasteur (1873; 1876, p. 328) patented his invention stating that the ‘chemical act of fermentation is essen- of a closed vessel for brewing to protect the fermentation tially a phenomenon correspondent to a vital act, begin- process from air-borne (Fig. 1). ning and ending with the latter’ (Pasteur 1876, pp. 229, A range of fermentation products became an important 230, 306). part of the overall economy in European, North American Several new active substances (enzymes) from different and Asian countries. At the end of the nineteenth century, sources (e.g. flowers and fruits, pancreas) were discovered, the fermentation industry was growing fast. It encompassed including , and fibrinolytic activities, and the manufacture of beer and wine, industrial alcohol, yeast, emulsin (Buchholz and Poulson 2000; Buchholz and Collins acetic and lactic acid, cheese, soy sauce and sake. Beer 2010, chapter 3). By the 1870s, studies had established the manufacture represented one of the most important economic existence of two types of ferments. They became known as activities. Thus in Germany, it had grown to 50 mn (million) unformed (unorganized) and formed (organized) ferments (the hL (hectoliter, 100 L) in 1890 (Ullmann 1915, p. 533). The latter referred to living bodies, such as yeast). The German production process was described in all technology text- physiologist, Willy Kühne (1877) referred to the pepsin type books of the nineteenth century. Wine was also an im- of unformed ferments as ‘enzymes’. portant fermentation product, having a major economic A summary of important scientific discoveries and appli- impact. The production around 1890 was estimated at 120 cations is given in Table 2. 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 a Scientific findings, events Technical progress, industrial innovation

1837/1838 Schwann Experimental demonstration of living yeast as agent in Growing importance of of beer and Cagniard-Latour alcoholic fermentation (production 23 million hL in 1840, Germany) b 1850 Rayer and Detection of the origin of and the role of Technical-scale production of yeast, wine, soy sauce, sake. Davaine microorganisms in diseases 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: Hansen breeding pure yeast for commercial application; 1874 Christian Hansen’s Laboratory (Denmark): production of (chymosin) for cheese manufacture Detection of anaerobic fermentation (1861) Studies on wine fermentation, invention of Pasteurisation (1864) Beer production: 36 million hectolitres in 1873, Germany Studies 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 Appl Microbiol Biotechnol (2013) 97:3747–3762 3751

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

about 39 mn hL (Brockhaus 1895, vol. 16, pp. 591–595). Ferments in terms of enzymes found application, Wine was attributed not only agreeable but also health diastase on a major industrial scale, since the 1840s, a effects when administered properly, e.g. a remarkable means few others in the second half of the nineteenth century. for preserving the forces and improving the resistance to The first company founded on an enzyme-based process infections. The was described with ‘stimulation was ‘Christian Hansen’s laboratory in Copenhagen of the nervous system and blood circulation, improving (Denmark), so named to this day. It pioneered the use or enhancing the subjective feeling and performance’ of rennet (lab ferment, chymosin), for cheese manufac- (Brockhaus 1895, vol. 16, pp. 591–595). The alcohol pro- ture (Brockhaus 1894b, vol. 10, p. 863; Poulson and duction in Germany was estimated up to 3.7 million hL in Buchholz 2003). Further pancreas enzymes, trypsin or 1893/1894. An advanced technology had been developed pancreatin, and pepsin, isolated from pig or cow, were and applied in large factories: the process using starch as the used as drugs, for example, as digestive aids. ‘Pepsin is raw material was operated at high to ensure gelati- a rational drug insofar… that a weekend function of the nization (Henzedämpfer.); hydrolysis was then achieved by stomach (dyspepsia) is enforced by little doses of pep- adding diastase (malt) to stirred tank reactors, followed by sin, and, in fact, numerous positive reports by doctors fermentation for 72 h, using yeast that had been produced are available’ (Brockhaus 1894b). separately; distilleries were controlled automatically A wave of foundation of research institutions, mainly (Brockhaus 1895, vol. 15, p. 172–178). Yeast as a commer- governmental institutes took place, devoted to research cial product was mainly generated in high yield in distiller- on beer, wine and food manufacture, hygiene, medical ies (pressed yeast, Presshefe); it was then sold for use in care, and water, as well as laboratories of the brewing other industrial processes, for example bread manufacture and bakery industries, notably in Europe, and several in (Payen 1874, Vol. 2, p. 403). In Denmark, Hansen made the USA. Institutions for brewing research and educa- major progress in breeding pure yeast by working with tion were established in Weihenstephan near Munich culture media (e.g. agar plates, as did Koch) isolating colo- (1872–1876), Berlin (‘Institut für Gärungsgewerbe,’ 1874), nies from single cells which he could then propagate. This Hohenheim (1888) (all in Germany), in Copenhagen, became the basis for pure yeast fermentation and commer- andinParisthefamous‘Institut Pasteur’ (1888). In cial applications which was adopted e.g. by the German Britain, the ‘British School of Malting and Brewing’ brewing industry, where the Berlin Institute and its first was founded at the University of Birmingham in 1899. director Max Delbrück played a major role. The work In the USA, by states decrees, agricultural research institutions of Pasteur and Koch placed emphasis on the particular were founded from 1863 on, that eventually became the quality of individual pure cultures or clones. It was origins of big universities like MIT, Cornell, and Wisconsin realized that quality control and characterization of the (Bud 1993). organisms used were important. This accompanied the Following Pasteur and Koch’s success in identifying beginning of microbial diagnostics which involved specific causative agents of disease and establishing pure cultures, staining. pharmaceutical companies also established bacteriology 3752 Appl Microbiol Biotechnol (2013) 97:3747–3762 departments which produced vaccines or tested substances fermentation, during the period up to 1930, stimulated the for their antimicrobial properties (Metz 1997). J. E. Siebel in molecular approach to the study of the pathway of alcoholic Chicago issued a journal ‘Zymotechnic Magazine: Zeitschrift fermentation, mainly the research on the successive inter- für Gärungsgewerbe and Food and Beverage Critic.’ In mediates in metabolism. Buchner himself continued to German-speaking countries, 10 journals on brewing were work on -free fermentation investigating intermediate issued at that time (Brockhaus 1894a, b). Jörgensen, in compounds and activities both in cell-free press juice as 1885, founded the journal Zymotechnisk Tidende, and pub- well as in living yeast that would convert possible in- lished a highly regarded book on Microorganisms and fer- termediates including trioses. By the end of the 1940s, mentation. Several further books on and/or bacteria the scheme of and alcohol formation was or microorganisms were issued. The terms Bacteriology or finally complete (Florkin 1975; Kohler 1975; Buchholz Mycology, ‘Zymotechnologie’, or Microbiology denominated and Collins 2010, chapter 4). the new research field. Of major impact on industrial microbiology were Thus the ‘Age of Bacteriology’ began with a new para- Fernbach’s systematic investigations at the Institut Pasteur digm, and a broadened industrial and economic base. in Paris on metabolic intermediates during alcoholic fermen- tation (mainly of glycolysis) by various microorganisms, e.g. yeast and Tyrothrix tenuis; this included the formation The period from 1890 to 1940—The advent of , notably acetic, succinic and pyruvic acids. He of biochemistry, and new products identified corresponding enzymatic activities: the begin- nings of what we now call biochemistry research. This In 1891, Fischer established stereochemistry, illustrating his was not only important in elucidating the mechanism of theory on specificity with the famous picture of a lock and fermentation but was also of practical relevance for acid key: ‘To use a picture, I will say that enzyme and glucoside production. Fernbach obtained patents on the fermentation must fit like lock and key in order to interact chemically. . .’ of starch for the production of acetone and higher (Fischer 1909). With the work of Emil Fischer (1852–1919) (Fernbach 1910; Fernbach and Strange 1911). came the breakthrough in the development of structural Around 1907–1910, there was a shortage of rubber on the biochemistry; in the course of his scientific career he world market. Perkin in the UK proposed an alliance, com- completely shifted the orientation of research in chemistry prising an extended list of chemists and bacteriologists, towards the principal organic components of living matter: including Fernbach and Weizmann, with the aim to produce , fats, and (Fruton and Simmonds 1953). butanol (butyl alcohol), which could be converted into bu- Buchner in the mid-1890s ended the hypothesis of tadiene. This in turn could be polymerized to yield synthetic vis vitalis, that still postulated hidden mysterious forces rubber (Perkin Jr. 1912). Shortly after this initiative, the First in fermentation, when he published a series of papers World War created a demand that drove technical innovation (Buchner 1897, 1898; Buchner and Rapp 1898), which in the fermentation industries. The ‘acetone butanol’ fer- signaled a breakthrough in fermentation and enzymology. mentation process became a key technology for explosives Buchner’s key experiment was to prepare a press juice from production since acetone was required as a , in short yeast, which contained all the enzymes required for the supply in Britain. Chaim Weizmann, who had worked in transformation of sugar into alcohol and , Fernbach’s laboratory, continued similar research in the and to demonstrate that no living cells remained. He then Biochemical Department of Manchester University, and could show that this solution could perform the same reac- made a new contribution using a more abundant source of tion as did living yeast during fermentation, assuming one raw material, viz. maize, in 1915 (Speakman 1919). enzyme, called zymase, being the catalyst. Buchner Weizmann brought his own laboratory experiments to the presented the proof that (alcoholic) fermentation did not notice of the Admiralty, in the spring of 1915. He asked require the presence of ‘. . .such a complex apparatus as is Winston Churchill, the first Lord of the Admiralty, to build a the yeast cell’. The agent was in fact a soluble substance— plant, and in July, a pilot plant was erected in Nicholson’s without doubt a body—which he called ‘zymase,’ London gin distillery. The process is usually referred to as and what much later turned out to be the enzyme system the Weizmann process (Weizmann 1917; Nathan 1919; for of the whole glycolytic pathway (Buchner 1897). With more details and the political background refer to, Bud Buchner’s work the dogma of the ‘vis vitalis’ fell. It initiated 1993). The manufacture of acetone by the Weizmann pro- a new paradigm, the biochemical paradigm, which, in con- cess attained the greatest success at the factory of British trast to that of Pasteur, stated that enzyme catalysis, includ- , Toronto, Ltd., in Canada, on a large scale. ing complex phenomena like that of alcoholic fermentation, Fourteen new fermenters were constructed, about 18 ft was a not necessarily linked to the pres- (5.5 m) in diameter and 20 ft (6.1 m) high, holding 24,000 ence and action of living cells. The discovery of cell-free gallons (91 m3) of mash (Nathan 1919; Speakman 1919). In Appl Microbiol Biotechnol (2013) 97:3747–3762 3753

Germany, war requirements concerned (glycerin) Enzyme technology rapidly expanded. A range of enzymes, for the manufacture of explosives, when supplies of fat including diastase (amylolytic enzymes), and became enormously curtailed as a result of the imposition pectinases, were isolated from different organisms for com- of the sea blockade. Investigations initiated by Lüdecke with mercial use, mainly from Bacillus subtilis and other species the object of obtaining glycerol on an industrial scale by such as Aspergillus oryzae and A. niger (Tauber 1949,pp. means of fermentation became of supreme importance. 396–494; Buchholz and Poulson 2000). Takamine began The process was developed by the Protol Company. The isolating bacterial in the 1890s in Japan. In 1894, monthly output of glycerol was about 1,000 tons he obtained a patent for the production of a diastatic enzyme (Connstein and Lüdecke 1919a, b). preparation from , which he called ‘Takadiastase’ for The formation of oxalic and citric acids by Apergillus and the production of amylases for the hydrolysis of in species had been observed by Wehmer in the food manufacture (Tauber 1949). Major applications of en- 1890s. fermentation became an object of study zymes were proteases in the chill-proofing of beer, and the by several academic groups that were actively engaged in addition of malt extract in dough-making by American optimizing the process and in elucidating the biochemical bakers in the USA. In 1922, they used 30 million pounds mechanism leading from the sugar substrate to citric acid. (13,500 tons) of malt extract valued at $ 2.5 million. In Currie undertook what came to be considered a classic 1907, Röhm patented the application of a mixture containing investigation of the factors controlling the production of pancreatic extract as a bating agent, replacing the unpleasant citric acid by a selected of ;he use of dung, and he founded the Röhm and Haas Company elaborated optimum conditions for the production of citric in the same year based on this application. From about 1930 acid. Currie joined Chas. in New York, where a plant onwards, the enzyme preparation was produced by fermen- was established which went into production in 1923. By tation (Tauber 1949; Buchholz and Poulson 2000). For 1933, this industry already contributed 85 % (in Europe education the Institute in Berlin offered various courses from 5,100 tons and in the USA 3,500 tons) of the world’s 1888 (and later a curriculum for brewers), as did the ‘Institut citric acid production of 10,400 tons. Further products für Gärungsphysiologie und Bakteriologie’ that was manufactured by fermentation were gluconic acid and lactic established at the Technical High School in Vienna in acid (May and Herrick 1930; Frey 1930; Roehr 1996, 1998). 1897, as well as other institutions. Courses on fermentation Another important example of industrial research activity were offered by Bernhauer at the German University in was the development of the oxidation of sorbitol to sorbose Prague in the 1930s (Bud 1993/1995, pp. 60, 61, 104, by Acetobacter suboxydans as an intermediate for vitamin 132, 202, 203; Clifton 1966). C. The corresponding so-called Reichstein process was used Important scientific breakthroughs and applications are from the 1930s on a large industrial scale (Buchholz and summarized in Table 3. Seibel 2008). Another process established in the 1930s was the manufacture of L-ephedrine as a pharmaceutical using the stereoselective of benzaldehyde and The period from 1940 to 1970—the era of antibiotics, acetaldehyde by yeast (Vasic-Racki 2000). con- and the emergence of biotechnology tinued to represent a major product of outstanding economic importance. Florey, Heatley and Chain, towards the end of the 1930s, A breakthrough event was in 1928 when Fleming ob- began to investigate penicillin in the course of their system- served that a culture of a Penicillium notatum inhibited the atic study of antibacterial substances at Oxford University. growth of bacteria. He demonstrated the production of an The credit for resurrecting penicillin, described at the time antibacterial substance in the culture broth and named it ‘as unstable as an opera singer’, certainly goes to the Oxford penicillin. However, there was rather a long delay before group. They developed an assay, found a way of producing research and development aiming at production was un- penicillin in surface culture and demonstrated the marked dertaken, finally stimulated by Florey, Heatley, and Chain activity and therapeutic value of penicillin in a clinical trial who entered this field again toward the end of the 1930s in 1940 (Bud 2007; Coghill 1970; Demain 1981; Ohno et al. (Bud 2007; see below). 2000). Early yields and recovery, however, were very The nature of enzymes and the structure of proteins discouraging and the difficulties in wartime led required more than 40 years to be established, and it them to visit authorities, laboratories, and industrial compa- remained controversial for decades (Sumner and Myrbäck nies in the USA for help in July, 1941. They were advised 1950). In the 1930s, Stanley successfully crystallized the by research authorities to visit Peoria, Illinois, USA, to talk tobacco mosaic ; it was the first time that any living form with officials of the Northern Regional Research Laboratory had been crystallized, and it revolutionized thinking about (NRRL) because this institution had just organized a fer- the chemical nature of life (VanDemark and Batzing 1987). mentation division. The representatives offered Florey all 3754 Appl Microbiol Biotechnol (2013) 97:3747–3762

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 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; Microbial formation of acetone and butanol b Fermentation technology expanding: Production of butanol 1912f Perkin 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, Research on glycolysis Large-scale industrial yeast production for bakeries Meyerhoff and others 1925, 1930s Sumner, Northrup Enzyme 1928 Fleming Finding of penicillin action Large-scale waste water treatment (1928, Essen, Germany) 1933 Reichstein Sorbitol transformation into L-sorbose Reichstein process for production End of 1930s Florey and Chain Resumed research on penicillin Sterile enzyme fermentation for detergents etc. 1940 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 the help they could give. work began on July unknown approach to interdisciplinary cooperation and pro- 15, 1941, at the NRRL under the general direction of Dr. ject organization (Coghill 1970). Coghill (Greene and Schmitz Jr. 1970; see also AIChE 1970) Strain screening and development, including . Research studies were also initiated at the Universities of procedures, proved to be a key factor for success. From Minnesota (on microbial strains), Wisconsin (on fermenta- many sources, including soil samples from around the tion), Penn State (on recovery), the Carnegie Institute, world, collected by the US Army, many hundreds of strains Wisconsin and Stanford (on mutation) and at MIT (on of penicillin-producers were isolated. The best producer of and packaging) (Coghill 1970). By the fall of 1941, yields of all (labeled NRRL 1951), ironically, came from a moldy penicillin began to climb to 6, 10, and to 24 Oxford units per cantaloupe melon from a Peoria fruit market. Genetic mL, using an improved mould strain, as compared with changes were undertaken at the Carnegie Institute and at about 3 units/mL obtained by the Oxford group. the University of Wisconsin. Moyer’s(NRRL)medium In December 1941, the US Government became interested. improvement and use of a better NRRL strain raised the The US Department of Agriculture (USDA) called a meeting of penicillin to 100 units/mL. Subsequent in New York which included representatives from the improvements raised this by another order of magnitude to National Research Council, and four companies, Merck, about 1,500 units/mL with the Wisconsin strain (Coghill Squibb, Pfizer and Lederle, an event that was considered to 1970). ‘As a result, we began to get a trickle of a supply represent the real turning point for penicillin production of penicillin during the early months of 1942’, as Richards (Coghill 1970; Greene and Schmitz Jr. 1970) (some 18 more reported (Greene and Schmitz Jr. 1970; Silcox 1970). By companies became involved subsequently; Elder 1970). June 1942, enough penicillin to treat 10 patients had been Industry representatives agreed to make research teams avail- produced, and by February 1943 there was sufficient mate- able to work on the problem of supplying adequate quantities rial to treat approximately 100 patients. Production was by of penicillin. By 1943, the amazing curative properties of surface culture flasks, the most reliable method at the time. penicillin were becoming pretty well-known, and there was In 1942, 2-years intensive development had resulted in a huge demand for the drug. The prime goal established by the increasing the level of output of penicillin by some government representatives was to have ample stocks on hand 140,000-fold. The most efficient approach was submerged for the US army’s invasion of Europe in the spring of 1944. or deep-tank fermentation, but there were a number of That goal finally was met—by a huge effort, and a hitherto severe practical problems, the solutions of which were not Appl Microbiol Biotechnol (2013) 97:3747–3762 3755 obvious, but which were finally achieved (Fig. 2) (Greene resistant. Factors that exacerbate this phenomenon are misuse and Schmitz Jr. 1970;Silcox1970). Downstream opera- and overuse, and the widespread use of antibiotics in tions, the isolation of penicillin, also represented a huge aquariums, in agriculture and animal husbandry (Bud 2007, challenge. They included new methods for biological pro- pp.116-139; Hubschwerlen 2007). cesses, such as liquid–liquid , , By the 1950s, large-scale production not only of tradi- freeze drying, crystallization and others (Silcox 1970; tional goods, for example, beer, alcohol, cheese, but also Perlman 1970). new products, including citric acid and pharmaceuticals and ‘Thus began a wartime collaboration which was to in- other products of particularly high social and economic volve the efforts of literally hundreds of biochemists, chem- relevance, had become well established. Growing economic ists, bacteriologists, biologists, chemical engineers, relevance followed notably the success of penicillin manu- physicians, toxicologists, pharmacologists, and pathologists facture, and further antibiotics, like streptomycin, became on both sides of the Atlantic, managed and coordinated by available, followed by a new class of high value-added industrial executives, academic administrators, and govern- products, mainly secondary metabolites, e.g. steroids ment leaders’. (Greene and Schmitz Jr. 1970). At the polit- obtained by biotransformation. Other major products of ical level, ‘the injection of funds, people, companies, and growing market relevance included amino acids, organic government interest meant a transformation in the ways of acids, carbohydrates, and derivatives (hydrolysates, iso- doing science’. A range of smaller projects on penicillin mers), vitamins, , and enzymes for new applications production were undertaken in several other countries, includ- (Demain 1981; Demain 2001). ing Germany, the Netherlands, France and by Czech scientist A new generation of biocatalysts, based on immobiliza- (Bud 2007,pp.75–96). Penicillin became a public property tion techniques developed in the academic field, led to a and big business (Bud 2007,pp.23–53, 54–74). The pros- breakthrough in processing of food and pharmaceutical pects, and later the success of penicillin, prompted further compounds. Large-scale processes were established using research on antibiotics. Waksman isolated actinomycyin in biocatalysts for penicillin hydrolysis (for the synthesis of 1940, streptotricin in 1942, and streptomycin in 1944 from semisynthetic β-lactam antibiotics) and isomeriza- cultures of actinomycetes (Ohno et al. 2000). (The patent on tion (Poulson and Buchholz 2003; Buchholz et al. 2012, Streptomycin and for starter cultures for yoghurt largely fi- chapters 7 and 8). Waste water treatment became more nanced the establishment of the Life Sciences Faculty at the wide-spread, due to legislation, and gained great attention. University of Wisconsin in Madison for the next 50 years and This resulted in new developments, and capital investment, provided many stipends for students.) However, the therapeu- both in the public and industrial sectors (Jördening and tic potential has been threatened by the emergence of increas- Winter 2005). ingly resistant bacterial strains as a natural consequence of Significant events are summarized in Table 4 their use, first observed by Abraham and Chain (1940). In Starting in the 1970s and 1980s, BT gained the attention clinical settings, more than 50 % of isolates of governmental agencies in Germany, the UK, Japan, the and more than 90 % of S. aureus isolates are USA and others as a field of innovative potential and eco- nomic growth. This was also in response to the first oil price crisis in the beginning 1970s, and the realization that renew- able 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 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 was termed a corporatist approach by Jasanoff (1985). Further studies on BT were issued in the UK, Japan and France (Bud 1993, pp. 189–210). Essential topics and aims of the Fig. 2 Penicillin fermenters in operation at E.R. Squibb & Sons, 1946 Dechema study reflected the main established scientific (Langlykke 1970) and applied fields of BT at that time. The basic disciplines 3756 Appl Microbiol Biotechnol (2013) 97:3747–3762

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 requirements 1944 Large-scale industrial penicillin production; Pfizer: deep tank penicillin fermentation 1948 Brotzu and Oxford team , broad spectrum 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 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 Large-scale enzymatic glucose isomerisation 1974 Political level: Germany: DECHEMA-report, Expanding production of amino and organic acids, vitamins, followed by other studies on biotechnology enzymes in food manufacture in UK, Japan, France 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 b There were of course other failures which would be worth investigation c An exception are glucose sensors involved in BT research and development work were mi- discipline and there were no books, rather no journals, cur- crobiology, , biochemistry, and—to a limited ricula or scientific conferences devoted to the subject. A few extent— and in addition to UK and American universities offered special courses; chemical engineering. Recombinant DNA methods were University College London established a curriculum granting not mentioned since they were not available at the time of a Master of Science in Biochemical Engineering in the 1960s, writing the study (1972–74) (Buchholz 1979; Buchholz and and another BT curriculum was established in the 1970s at Collins 2010, chapter 5). the Technical University of Berlin (Buchholz 1979, pp. 69, Research work in the field of BT proceeded as subtopic 71). The first BT journal of high reputation was established in within a motley collection of scientific and engineering dis- 1958 by Elmer Gaden as the Journal of Microbiological and ciplines with a low level of coherence and little integration up Biochemical Engineering. It later became Biotechnology and till the 1960s and 1970s. During the 1940s, Stephenson’s Bioengineering and is still a leading journal in the field. A Bacterial Metabolism and of Kluyver’s Chemical Activities few other journals appeared in the 1950s and 1960s, for of Micro-Organisms appeared, the Gärungschemische example Applied Microbiology,renamedEnvironmental and Praktikum,byBernhauerwaspublishedin1936(Bud Applied Microbiology and Applied Microbiology and 1993). Later, textbooks dealt with specific topics (not on Biotechnology. BT as an integrated field), signifying increased attention to the field: on applied microbiology (Rehm 1967,Pirt1975), as well as on biochemical engineering (Aiba et al. 1965;Bailey The period from 1975 on—the new biotechnology and Ollis 1977). The first encyclopedias and series on BT were issued by Rehm and Reed (1981) and Flickinger and The turning point in genetics ensued from the establishment Drew (1999). Thus, biotechnology did not exist as a scientific of a model for the molecular structure of DNA by Jim Appl Microbiol Biotechnol (2013) 97:3747–3762 3757

Watson and Francis Crick, based on the data merging of molecular biology and biochemical engineering. of Rosalind Franklin, who was working in Morris Wilkins Industrial interest and the range of products expanded sig- lab in 1953 (Watson and Crick 1953). This was the culmi- nificantly, and many new companies, mainly in the USA, nation of work initiated by Sir William Henry Bragg and his were founded. New methods and tools played a key role in son William Lawrence on X-ray diffraction by , to the rapid expansion of recombinant technologies. These study molecular structure, initially of but later of include: gel , centrifugation, restriction endo- more complex organic structures, including the first 3-D nucleases, cloning, a range of further cloning structure of a protein, myoglobin (Max Perutz and John methods extending to most known species of microorgan- Kendrew, see Kendrew et al. 1958), further of penicillin, isms and eukaryotes, in particular in plants, cloning of larger vitamin B 12, and (Hodgkin 1979). The significance (gene-sized) DNA fragments via virus cosmid, fosmid, of DNA structure, as the material of which genes are made, BAC and YAC (this latter in yeast) cloning, oligonucleotide was immediately recognized due to the ground-breaking synthesis, DNA sequencing, gene , metagenomics, work, during the preceding 50 years, of a great number of and recently synthetic biology; protein design has become a scientists in chemistry and biology, mostly microbiology rational tool for and enzyme develop- including Gregor Mendel, Friedrich Miescher, Phoebus ment (Winnacker 1987; Demain 2001; Bornscheuer and Levene, William Astbury, Erwin Chargaff, Oswald Avery, Buchholz 2005; Buchholz and Collins 2010,chapters7,9). Francois Jacob, Jacques Monod, Ole Maaloe, Max Once the tools for gene cloning in the Gram-negative E. coli Delbrück, Sydney Brenner and others (Judson 1979; had been established it became easy to develop gene cloning Winnacker 1987; Buchholz and Collins 2010, Chapter 7). vectors which could be transferred to other species. This But the ‘DNA Revolution’ as Hotchkiss termed it, involved the identification of that replicated in progressed or penetrated slowly into technology, initially other hosts and genes (promoters) that could be expressed having little effect on traditional processes and products and used for selection in the new host, including bacteria, (Hotchkiss 1979). The Asilomar conference 1975 initiated yeast, insect cell lines and plant cells (Collins 1977). Thus a public discussion on the possible hazards of recombinant all the elements for the new recombinant DNA technology, DNA research (for details, see Buchholz and Collins 2010, at least for bacterial and animal cells are available: Methods section 8.1.2). The following two decades saw many years to prepare DNA, which, following restriction cleavage of discussion of possible risks and containment require- (i.e. treatment with restriction endonucleases) could be co- ments associated with recombinant technologies which valently joined to a ‘vector’ with a DNA ligase; a ‘vector’ eventually formed the basis of the guidelines for recombi- (plasmid or virus) to ensure maintenance in the cell; a nant DNA work and finally culminated in international method to prepare ‘clean’ vector DNA; an efficient method legislation (see for example Cartagena Protocol on Biosafety, to incorporate DNA into the cell; culture techniques to http://bch.cbd.int/protocol/text/.) isolate single clones carrying a single recombinant hybrid Subsequent to Watson and Crick’s publication in 1953 of , including selective techniques to enrich for the the DNA structure, a large number of significant scientific cells ‘transformed’ with the vectors, for example selection breakthrough events as well as technological progress pro- for antibiotic-resistance genes (Buchholz and Collins 2010, vided a new basis for BT. Selected events are summarized in chapter 7). More recently, since the 1990s, the so called Table 5. Berg, Cohen, and Boyer in 1972 introduced recom- ‘’ approaches: genomics, proteomics, metabolomics, binant DNA (rDNA) technology when they constructed the , and their integration into systems biology first recombinant plasmids and , which were intro- and biotechnology aimed at understanding, quantitative de- duced into bacteria, or animal cells respectively, where they scription and rational modification of whole organisms. were autonomously propagated. A patent granted to Cohen Biosystems engineering or systems biotechnology aims at and Boyer, and the University of California was critically the integration of biology, mathematics, bioinformatics, and commented by Berg (Cohen et al. 1972; Cohen and Boyer systems engineering to gain a holistic view of complex 1979/1980; Berg and Mertz 2010). ‘Entrepreneur’ was still biological and biotechnological systems, including quanti- a dirty word in molecular biology, leading one to reflect on tative description and improvement of whole organisms and the situation in engineering a century earlier with the slan- the rational development of novel production processes dering of George Stephenson (later inventor of the steam (Reuss 2001; Deckwer et al. 2006; Klein-Marcuschamer et engine) by Sir Humphrey Davy at the time of his ‘invention’ al. 2010; Papini et al. 2010; Buchholz and Collins 2010, of the miner’s lamp (not patented), already produced as sections 13.6 and 15.6). Stephenson’s prototype (patented). As a consequence of this development, in the USA, also Based on the new genetic techniques, a significant on the political level, the perception of BT diverged greatly change occurred during the 1980s and 1990s with common by the 1980s as compared to that in Europe particularly in approaches in different disciplines underlying BT, and the Germany in the 1970s. This is perceived from a report of the 3758 Appl Microbiol Biotechnol (2013) 97:3747–3762

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- 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/ 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- (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 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 fundamental research b Bailey (1991, 1996)

OTA of 1984 (OTA 1984). It refers to methods that arose of scientific results, closely associated with the business with knowledge on DNA and that revolutionized what world. was ‘thinkable’. In contrast to the reports mentioned The industrial breakthrough came with recombinant before, emphasis in the OTA study was on genetic engi- human insulin, developed by Genentech in cooperation with neering and rDNA technology, resulting in commercial Ely Lilly in 1978, and approved by the US Food and Drug opportunity and support of fast commercial exploitation Administration in 1982 (Bud 1993/1995, pp. 232, 237; Appl Microbiol Biotechnol (2013) 97:3747–3762 3759

Walsh 2007, pp. 297, 298); this was at a time when some both aerobic and anaerobic, being applied in numerous heads of European pharmaceutical companies did not be- small up to very large-scale installations, as well as a great lieve that a recombinant DNA product would ever be ap- number of exhaust air treatment units (Jördening and Winter proved for clinical use. This precedent , notably the 2005). Ethanol, traditionally based on starch and sugar to approval human insulin as the first recombinant DNA produce it as gasoline additive on a very large scale, pro- product on the market, was followed by a series of further voked heavy criticism, with respect to using traditional recombinant products, mostly drugs, which in general could agriculture crops for biofuels rather than food. A major not be produced by other technical means, and which are of crisis occurred in 2007 and most notably in mid-2008, great medical interest. Some of these products previously causing a dramatic increase in food prices. The growing isolated in small amounts from human blood or tissue were use of cereals for ethanol was thought to be in part respon- in danger of being contaminated with human pathogenic sible for this price increase. Recently a trend emerged for viruses (not all known at that time, e.g. AIDS virus, HCV). using cellulosic as a source of biofuels (Buchholz In this respect, this alternative production route provided prod- et al. 2012, section 12.2). Production of biogas and electric- ucts not only in sufficient quantity for general use but also with ity generated by microbial fuel cells gained much attention an improved and reproducible quality. The products included and impetus (Buchholz and Collins 2010, chapter 16). human growth hormone in 1983, β-, and a hepatitis Recombinant DNA methods also greatly affected enzyme B vaccine in 1986, tissue plasminogen activator (tPA) in 1987, technology since the late 1970s. Over expression in fast- and erythropoietin in 1989 (product approval). Actually, re- growing host organisms with high protein productivity combinant proteins, including hormones and growth factors, allowed many enzymes, which were not readily accessible, blood clotting factors, cytokines, monoclonal antibodies and to be produced cheaply on an industrial scale. This technol- vaccines are most important biopharmaceuticals, with a mar- ogy allowed design of enzymes with modified specificity ket size estimated of some $50 billion per year around 2010 through iterative rapid cycles of gene mutation, screening or (Walsh 2007, Aggarwal 2007). Antibiotics remained an im- selection and testing in addition to crystallography and portant sector of biopharmaceuticals, with many different molecular modelling. Such products are used on a large specialties and sales estimated at more than $50 billion per scale for starch products (used in food preparations with a year (Hubschwerlen 2007). production volume of >10 million t/a, and ethanol with >37 Large investment by multinational companies, the foun- million t/a), enzymes in detergents, for pharmaceuticals dation of many small new companies, a few of which have manufacture, and many other fields (Buchholz et al. 2012, grown remarkably, and state funded big research merged in chapters 7, 8, sections 12.1, 12.2). Plant biotechnology has a ‘gold rush’ into the ‘New Biotechnology,’ as recombinant successfully been established, aiming at improved yields, technology was termed in the USA. Key steps toward the disease and herbicide resistance, etc. of crops. However, transfer of science into the economic sphere resulted in the controversies are ongoing with respect to political, ethical foundation of new BT companies, the first being Cetus, and biosafety aspects. Transgenic crops are cultivated on a started in 1971, later the originator of the ‘polymerase chain very large scale notably in the USA, Argentina, Brazil, reaction’ (PCR; Kary Mullis) which gave birth to the era of Canada, and other countries (Slater et al. 2008; Buchholz gene diagnostics and personalized medicine. Herbert Boyer and Collins 2010, Chapter 18). and Robert Swanson founded Genentech in 1976; amongst Two achievements since 2000 gained major public reso- the most important companies founded were Biogen (1978), nance: First, the major goal of the Human Genome Project Amgen (1980) and Chiron (1981), later bought by Cetus was achieved in 2000 with international cooperation and a (Demain 2001, 2003, personal communication; Buchholz total expenditure of some $ 3 billion. The task which was and Collins 2010, chapters 5, 6, 17; for a recent survey, carried out by a major international consortium and largely see Table 17.5). independently by Craig Venters group was recognized as Industrial products, other than pharmaceuticals, expanded essentially complete in 2000 and commemorated by a com- as well, based both on traditional and recombinant methods, munication in the presence of Francis Collins, Craig Venter with sales worldwide estimated over 50 billion €. The most and the President of the USA. The result of the Human important bulk products are ethanol, amino and organic Genome Project may possibly allow the discovery and pro- acids, produced in large amounts, vitamins, and biopoly- duction of hundreds of novel pharmaceuticals, many of mers. Metabolic engineering has been used successfully for which are natural human gene products previously not the optimization of yields, e.g. for the production of amino available in significant amounts or as virus-free prepara- acids (for a survey, see Buchholz and Collins 2010, chapter tions, significantly improving diagnosis and eventually rev- 16). A very large sector for application of biotechnology is olutionizing medicine. However, a number of arguments in fact environmental technology which has become an have been raised in terms of their classification as technical important industry. This includes waste water treatment, application, not fundamental research. After 10 years of 3760 Appl Microbiol Biotechnol (2013) 97:3747–3762 expectation, e.g. with respect to drug targeting, the follow- interpreted the development from early fermentation re- ing comment was put forward ‘… a transformational tech- search to Pasteurs concept of microbiology and technical nology will always have its immediate consequences innovations, from Buchner and Fernbach towards Perkin’s overestimated and its long-term consequences underestimated, and Weizmann’s processes, from Fleming towards Florey’s and ....you may just start to imagine all the projects that will and Chain’s work, and the penicillin project, and Watson’s spin-off…’ (C&EN 2010). Much progress took place and Crick’s solution of the DNA structure towards the largely through the involvement of flexible biotech compa- cloning concept by Berg, Cohen, Boyer, and towards the nies such as Genentech, Cetus, Amgen and Biogen which establishment of new companies and New Biotechnology. concentrated on innovative development in parallel with a Recently, applied microbiology, biochemical engineering lethargy and bad management in large (particularly and molecular biology have merged to form biotechnology European) pharmaceutical companies which lost their as a new scientific discipline in its own right, sharing a dominance in this new field. common paradigm at the molecular level with all the other The second major event may be considered the under- life sciences (Buchholz 2007). Biotechnology continues, as standing of the factors which control pluripotent and toti- well, as a field of technology, to develop new technical potent stem-cells and the controlled reprogramming of many processes and products based on a rational scientific basis. differentiated cells to such stem cells. This opens a new area A diversification arose through the formation of subdisci- of medical research, production of models for genetic plines, such as genomics, transcriptomics, proteomics, met- diseases (for personalized medicine), and a radical new abolic flux analysis with quantitative analysis of complex approach to understanding cancer, developments which will metabolites, and finally biochemical engineering, which give potential to a new area of biotechnological develop- merged into biosystems engineering. ment. This found its origin in the work of those studying the Finally, we note that critical events during the historic molecular biology of cell differentiation and embryogenesis, development of Biotechnology are associated with excep- originally in insect, worm or animal models, as did for tional personalities who often had the vision and insight of example the Nobel laureate Christiane Nusslein-Volhardt how their findings could be developed for the benefit of and as those most recently recognized with a Nobel Prize science and humanity, translating them into practical inven- (2012 Physiology or Medicine) for Sir John B. Gurdon and tion finally leading to innovation. Public and private invest- Shinya Yamanaka. ment programs often came slowly on advice or practical A further event that received inordinate publicity was the validation of radical advances by a few pioneers (This latter chemical synthesis of the entire genome of Mycoplasma aspect is treated in more detail throughout Buchholz and genitalium by the group of Craig Venter; transferring this Collins 2010). 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 Acknowledgment The authors gratefully acknowledge valuable (Gibson et al. 2010). The scientific relevance of this experi- information by Arnold Demain. ment, however, has been extensively debated, but subsequent steps in synthetic biology may become a key technology (Bornscheuer 2010). 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