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ApplMicrobiolBiotechnol(2013)97:37473762
DOI10.1007/s00253-013-4768-2
MINI-REVIEW
Therootsashorthistoryofindustrialmicrobiology
andbiotechnology
KlausBuchholz & JohnCollins
Received:20December2012/Revised:8February2013/Accepted:9February2013/Publishedonline:17March2013
# Springer-VerlagBerlinHeidelberg2013
Abstract Earlybiotechnology(BT)haditsrootsinfascinatingdiscoveries,suchasyeastaslivingmatterbeing
responsibleforthefermentationofbeerandwine.Serious
controversiesarosebetweenvitalistsandchemists,resulting
inthereversaloftheoriesandparadigms,butprompting
continuingresearchandprogress.Pasteur
sworkledtothe
establishmentofthescienceofmicrobiologybydeveloping
puremonocultureinsterilemedium,andtogetherwiththe
workofRobertKochtotherecognitionthatasinglepathogenicorganismisthecausativeagentforaparticular
disease.Pasteuralsoachievedinnovationsforindustrial
processesofhigheconomicrelevance,includingbeer,wine
andalcohol.SeveraldecadeslaterBuchner,disprovedthe
hypothesisthatprocessesinlivingcellsrequiredametaphysical visvitalis inadditiontopurechemicallaws.
Enzymeswereshowntobethechemicalbasisofbioconversions.Studiesontheformationofproductsinmicrobial
fermentations,resultedinthemanufactureofcitricacid,and
chemicalcomponentsrequiredforexplosivesparticularlyin
wartime,acetoneandbutanol,andfurtherproductsthrough
fermentation.Therequirementsforpenicillinduringthe
SecondWorldWarleadtotheindustrialmanufactureof
penicillin,andtotheeraofantibioticswithfurtherantibiotics,likestreptomycin,becomingavailable.Thiswas
followedbyanewclassofhighvalue-addedproducts,
K.Buchholz( ) *
InstituteforChemicalEngineering,
TechnicalUniversityofBraunschweig,Hans-SommerStr.10,
38106Braunschweig,Germany
e-mail:k.buchholz@tu-bs.de
J.Collins
LifeScienceFaculty,c/oHelmholtzCentre
forInfectionResearch-HZI,AGDirectedEvolution,
TechnicalUniversityofBraunschweig,Inhoffenstr.7,
38124Braunschweig,Germany
e-mail:tojohncollins@gmail.com
mainlysecondarymetabolites,e.g.steroidsobtainedby
biotransformation.Bythemid-twentiethcentury,biotechnologywasbecominganacceptedspecialtywithcourses
beingestablishedinthelifesciencesdepartmentsofseveral
universities.Startinginthe1970sand1980s,BTgainedthe
attentionofgovernmentalagenciesinGermany,theUK,
Japan,theUSA,andothersasafieldofinnovativepotential
andeconomicgrowth,leadingtoexpansionofthefield.
BasicresearchinBiochemistryandMolecularBiologydramaticallywidenedthefieldoflifesciencesandatthesame
timeunifiedthemconsiderablybythestudyofgenesand
theirrelatednessthroughouttheevolutionaryprocess.The
scopeofaccessibleproductsandservicesexpandedsignificantly.Economicinputacceleratedresearchanddevelopment,byencouragingandfinancingthedevelopmentof
newmethods,tools,machinesandthefoundationofnew
companies.Thedisciplineof
NewBiotechnology became
oneoftheleadsciences.Althoughbiotechnologyhashistoricalroots,itcontinuestoinfluencediverseindustrialfieldsof
activity,includingfood,feedandothercommodities,for
examplepolymermanufacture,biofuelsandenergyproduction,providingservicessuchasenvironmentalprotection,and
thedevelopmentandproductionofmanyofthemosteffective
drugs.Theunderstandingofbiologydowntothemolecular
levelopensthewaytocreatenovelproductsandefficient
environmentallyacceptablemethodsfortheirproduction.
.
.
Keywords BiotechnologyHistoryFermentationtheories
.
.
IndustrialmicrobiologyGenetictechniquesBiotech
companies
Introduction
Fermentationhasbeenofgreatpracticalandeconomic
relevanceasahandicraftforthousandsofyears,notably
Appl Microbiol Biotechnol (2013) 97:37473762
3748
the production of beer, wine and bread. The written first

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

Leeuwenhook, about 1680, had observed, with the aid of his
microscope, tinyanimalculesin
droplets of liquids, which

he, however, did not associate with fermentation. Then, in
the second half of the eighteenth century Spallanzani undertook microscopic investigations of many specimens, including sperm and microbial growth. By the end of the
eighteenth and beginning of the nineteenth centuries, respectively, Lavoisier and Gay-Lussac had elaborated quantitative correlations for alcoholic fermentation, without
giving explanations for the process underlying it. From the
mid-1830s evidence began to accumulate which pointed to
the biological nature of fermentation. Based on welldesigned experiments, Schwann
1837)
(
and CagniardLatour 1( 838) independently showed that yeast is a microorganism, anorganizedbody,
and that alcoholic fermentation is linked to living yeast. Both observed the yeast of beer
being little globular bodies able to reproduce themselves,
excluding spontaneous generation, and presenting a theory
on fermentation corresponding in essential parts to that
which Pasteur put forward about two decades later (for an
extended overview, see Buchholz and Collins
2010, part I).
Many other scientists, including Ktzing, Turpin and
Quev enne, contributed significa ntadvanc esinun derstandin
g
ferm entat ion,confirm ingthatli vingorga nismsw ereinvolve
d
infe rment ationproces sesothert hanthatl eading toalcohol,
e.g. ,inac eticacidfer mentation .However ,their argumentsw
ere often confusedbym ysticconc epts,inp articu larthatfermen
forces), a that view promoted, e.g. Gay-Lussac (Buchholz and

Collins2010, chapter 2). The mysterious concepts are obvious
from a textbook by Poppe
1842,
( p. 229):Fermentation
is seen
as a
at a time and under circumstances spontaneous - occurring mighty movement in a liquid of different compounds
,
which is due to the fact that several compounds act in harmony
with each other, others in opposition to each other, so that the

first attract, the latter reject each other. Ktzing
1837,
( pp. 396,
397) believed that organic entities (living organisms) can
form themselves by spontaneous generation , and he assumed two forces, theorganizing
living force, and the chemical affinity, fighting each other , and Quevenne1838,
(
p.469) used the termsecret
of life. In contrast to the vitalist

iL ,lo hcs ht,g ibe e h fo da
c i m e h t o h c s l a v ,l r o g i
y lsuo
a g d e u r e h t s n i n oc t p e c
b g n i v l f o e b s i d o g ni i t c a
niev
t a n e m r f c o r p n i s e n a s e a d a v d e s i h d c n u o e n r t n e m r f o y e h t s u t a h s o p d o b a e s u y o g r e d n c e d g n i s o p m n o i t i s r e f n a t h c i w b r u t s i d e d i r b l u q om u m r e h t o n a t s e n a t s b u e l ( s e c g i b e L
hypotheses is up to now accepted as unequivocal truth.
The importance of fermentation processes corresponds

wit hthelarges ec tionsthatwer edevotedt othetopicinthe
boo ksontechno lo gyandchemica lengineer ingofthetime
(Ot to1838;Pop pe1842;Knapp18 47;Wagner1857;Payen,
187 4).Knapp(1 84 ,p.367)repo
7
rtedthatb rewingwas
per formedinGe rm anyattheleve lofhandic raft,estimated
ata volumeofab ou t22.7million hectolitr es(2,27millionm)
3
in 1840, whereas in the UK it was carried out on an industrial

sc alein largefa ct ori es wi thf ermente rso fu pt o240,000L.
Pa rticu larlybe er ,as we ll asw ine,ace tic an dl acticacidprodu ction contrib ut eds ig ni fic antlyto the na ti onaleconomies.A
fast acetic acid manufacture(Schnellessigfabrikation)wa

s
developed by Schtzenbach in 1823. It worked, remarkably,
with active acetic acid bacteria (of course not recognized at
that time) immobilized on beechwood chips (Ost
1900).
Unformed, or unorganized ferments, obviously non-living
m a t t e r , d i f f e r e n t f r o m y e a s t , e n z y m e s i n t o d a y s terms, were
recognizda dfu rthec
arcteizd. Not ablydi
stae,of
whicsmal oun tswera bletoiqufya rgeamou ntsofa tsrchwa dieu itaden Pal( naye nd ozrsPe
1836; see also Buchholz and Poulson

2000). The first industrial processes that used enzymes (diastase) to produce dextrins were established from the 1830s onwards in France,
based on Payenswo
rk(Knapp
1847).
The most relevant events of this period are summarized
in Table1.
The
periodrgesfromspo
from 1850 to
1890ge
theneration,andisaconseemergence
ta-tioneme
ntaneous
quenc eofasecr
of microbiology as a science
It was only with Pasteurs
work that the scientific debate on the
nature of fermentation was settled in favor of the role of living
microorganisms, starting from hypotheses based on empirical
results provided by sophisticated experiments and ingenious
theoretical conclusions. Pasteurs outstanding accomplishments have been documented in several biographies, e.g.
3749
Table 1 Dates and events in early biotechnology

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

Scientific events
1680 Leeuwenhoek observes microorganisms
1783 Spallanzani observed protease action
1793 Lavoisier and
1810 Gay-Lussac: quantitative chemistry of alcoholic fermentation GayLussac: hypothesis of spontaneous generation
1833 Payen and Persoz: diastase (enzyme) characterization
1836 Berzelius: catalysis (including enzymes) a
1837, 1838 Schwann, Cagniard-Latour: living cells as fermentation agents
1834, 1838 Ktzing, Quevenne: hypotheses of spontaneous generation, (see
also before, Gay- Lussac); vital factor
1839 Liebig: chemical decay hypothesis
1830s Major controversy on fermentation theories
a
Technical application
Early eighteenth century: technical beer and wine fermentation;

also industrial beer fermentation
1823 Immobilized bacteria used for acetic acid production
1840s industrial enzymatic dextrin production (Payen)
Berzelius (1836)
One of the mysteries of fermentation had remained high(Birch1990)andGeison(

1995).Thefirstbasicquestionwhich
Pasteur definitively answeredwas that of the origin and charly controversial, the hypothesis
of ageneratio spontanea,
spontaeug rationf
acter of fermentation: Was it brought about by living microorlivngoraddressed
asm .Pasteur(
)
ganisms, or by pure chemical phenomena, as Liebig, Berzelius
this basic and controversial question efficiently. 1 86 2
and their school believed? In the 1850s, Pasteur had visited a
He referred toSchwann and others whoseserious workhe
repatdnc onf irmed,w
factory for alcohol production on a nearly daily basis and took
thsignfcifications
a tex perimntal modsamples of the fermentation broth which he investigated in his
(see also Geison
1995p. 115). In addition to
highlyprec
isee xper imentsusingv ariou sm ethods,P aste
laboratory. Losses in alcoholic fermentation were an initial
ur
stimulus to work on a scientific explanation and on finding
undertooks omet hing ofashowin186 0with ex pedition st
technical solutions. After numerous microscopical observao
highaltitu demo unta ins,mostspec tacul ar lytotheA lpsan
tions, he observed yeast buds in normal fermentation runs,
d
b u t r o d s t h a t h e s o o n i d e n t i f i e d a s l a c t i c a c i d y e a stheglacier
t , when
Merd eGla ce,todemonst ratet he existenc eof
the fermentationran
sour(due
to the formation of acetic or
germfreeai r,in cont rasttoairund ernor ma lconditi ons
carryingge rmsc ausi nginfectioni nsuga rj uices(an din
lactic acid) (Pasteur1857b). He investigated lactic acid fermenfermentati on). Ther esultsofthes eexpe ri mentswer epresent edbyPa
tation in detail. In his paper on the topic, 1857a)
Pasteur (
elaborated the essentials offermentation processes. He
presented the means with whichto isolate microorganisms in
a pure culture. In his discussionhe introduced (1) the biological Paris.The

finding of yeasts and their living nature, as well
asth ekn owle dg f th eiro
conception of fermentation as the result of the activity of living
rig n,e lim nates th emys
microorganisms; (2) he discussed the practice of inoculation for
tjuices
ery of thespona ne ousc curen ofermnta
tio nsof at urals gar ( P steu r
starting a reliable fermentation, that was also common practice
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 metation (Pasteur1860; Geison1995).
dium must provide the nutrients for the microorganism; and (5)
Pasteurs book Etudes sur la Bire (Pasteur1876) gave a
thorough experimental, theoretical and scientific account of
specific chemical features characterized by the main fermentation products and by products (Pasteur
1857a,b).
his investigations, results, and conclusions. He developed
3750
technical solutions to a number of practical problems, and

explained his motives in doing so. His findings, and their
establishment, may be considered to be a new paradigm
guiding further research. Pasteur thus laid the foundations
of a new scientific discipline, microbiology, known as bacteriology at the time (Delaunay
1951, Avant-propos, p. 22).
Among others, Berthelot1860,1864)
(
and Bchamp1864)
(
published a range of relevant papers on fermentation, e.g. of
substrates other than sugar.
However, one final mystery in fermentation remained:
thevital
forcehypothesis
linked all chemical transformations

in fermentations to a mysterious act depending on life,
stating that thechemical
act of fermentation is essentially a phenomenon correspondent to a vital act, beginning and ending with the latter(Pasteur
1876, pp. 229,
230, 306).
Several new active substances (enzymes) from different
sources (e. g.flow ersandfrui ts,pancre as)w erediscovered,
includi ngi nverta se,lipasea ndfibrino lyti cactivities,and
emulsin (Bu chholz andPoulson
2000;Buch holz andCollin
s
2010,ch apt er3).B ythe1870s, studiesha dest ablishedthe
existen ceo ftwoty pesofferme nts.Theyb ecam eknownas
unforme d(u norgan ized)andfo rmed(orga nize d)ferments(
the
latterr efe rredto livingbodi es,suchas yeas t).TheGerman
physiol ogi st,Wil lyKhne(
18 77)referr edto thepepsintype
ofunfor med fermen tsasenzyme s.
A summary of important scientific discoveries and applications is given in Table

2.
Although the application of fermentation processes was

wellesta bli shed,therew er estillproble mswiththe manufacturea ndq ualityofthe mo stimportantp roducts,a lcohol,
beerandw ine .Considerab le lossesoccurr edinfacto ries
producin gal coholfrombe et ,whenthejuic ewasturni ng
sour.Ear lyi nhisinvesti ga tionsonferme ntation,P asteur
wasengag edi nseveralind us trialproblem s.Theywer esubjectsofh igh lyaccuratea nd meticuloussc ientifici nvestiga t
ions
byBcham pan dPasteurand le dtothesoluti onofthemo st
urgentpr obl ems
aningeni ous combination of scientifican d
t e c h n i a l p r o g re s w i t h m u t u a
l i n t e ra c o n ( Ge i s o
19 5
;Bi
rch.
19 0
) .P a s t e u r (
1873
; 1876
edvs ,p.328 lforb)peatn dhi aclos sinvewtgoprfe tc feht ermnta ion procesf m a ir-bo neif cti ons(Fig
A range of fermentation products became an important

p artof th eoverallecon omyinEur opean, No rthAmerican
a ndAsi an countries.At theendof thenin et eenthcentury,
t hefer me ntationindus trywasgr owingf as t.Itencompass
ed
t heman uf actureofbeer andwine, indust ri alalcohol,yeas
t,
a cetic an dlacticacid, cheese,s oysauc ea ndsake.Beer
m anufa ct urerepresent edoneoft hemost im portantecono
m ic
a ctivi ti es.ThusinGer many,ith adgrow nt o50mn(million
)
h L(hec to liter,100L)i n1890(Ul lmann
1 91 ,p.533).The
5
p roduc ti onprocesswas describe dinall te chnologytextb ookso ft henineteenth century. Winewa sa lsoanimp ortan tf ermentationp roduct,h avinga ma joreconomic
i mpact .T heproduction around18 90wase st imatedat120mn hL wor l
Table 2 The period from 1850 to 1890 (Scriban

1982, pp.13, 14; Buchholz and Collins
2010, chapters 3 and 4)
Time, scientists
1837/1838 Schwann
and Cagniard-Latour
1850 Rayer and
Davaine
18561877
Pasteur
1866 Mendel
1876 Koch
1877-86 Pasteur
1880 Winogradsky
1881 Pasteur
Scientific findings, events

Experimental demonstration of living yeast as agent in
alcoholic fermentation
Detection of the origin of anthrax and the role of
microorganisms in diseases
Investigations on fermentation (from 1856 on):
Investigations on alcohol fermentation (1858)
Studies on spontaneous generation (18591862)
Detection of anaerobic fermentation (1861)

Studies on wine fermentation, invention of
Pasteurisation (1864)
Studies on beer fermentation (1871)
Theory of fermentation (1876)
Detection of facultative anaerobic fermentation of yeast
Heredity laws
Work on the bacterium leading to anthrax; agar plate method
Begin of investigations on anthrax (1877)
Soil microorganisms: the bacterial nature of nitrification
Vaccination against anthrax and rabies
Technical progress, industrial innovation

Growing importance of industrial fermentation of beer
(production 23 million hL in 1840, Germany) b
Technical-scale production of yeast, wine, soy sauce, sake.
Industrial-scale beer fermentation in GB
1870s: Hansen breeding pure yeast for commercial application;

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

(Pasteur; Fig.
1895 Wehmer: Lactic acid production
There are, of course, more scientists and events which have been relevant; however, inevitably, a selection must be made
1 hL corresponds to 100 L, or 0.1 m3
3751
Fig. 1 Pasteurs technical

fermenter (Pasteur1876 ,p.328)
about 39 mn hL (Brockhaus
1895, vol. 16, pp. 591
595).
Wine was attributed not only agreeable but also health
effects when administered properly, e.g. a remarkable means
for preserving the forces and improving the resistance to
infections. The physiology was described withstimulation
of the nervous system and blood circulation, improving

or enhancing the subjective feeling and performance
(Brockhaus1895, vol. 16, pp. 591595).
The alcohol production in Germany was estimated up to 3.7 million hL in

1893/1894. An advanced technology had been developed
and applied in large factories: the process using starch as the
raw material was operated at high pressure to ensure gelatinization (Henzedmpfer.); hydrolysis was then achieved by
adding diastase (malt) to stirred tank reactors, followed by
fermentation for 72 h, using yeast that had been produced
separately; distilleries were controlled automatically
(Brockhaus1895, vol. 15, p. 172178).
Yeast as a commercial product was mainly generated in high yield in distilleries (pressed yeast, Presshefe); it was then sold for use in
other industrial processes, for example bread manufacture
(Payen1874, Vol. 2, p. 403). In Denmark, Hansen made
major progress in breeding pure yeast by working with solid
culture media (e.g. agar plates, as did Koch) isolating colonies from single cells which he could then propagate. This
became the basis for pure yeast fermentation and commercial applications which was adopted e.g. by the German
brewing industry, where the Berlin Institute and its first
director Max Delbrck played a major role. The work
of Pasteur and Koch placed emphasis on the particular
quality of individual pure cultures or clones. It was
realized that quality control and characterization of the
organisms used were important. This accompanied the
beginning of microbial diagnostics which involved specific
staining.
Ferments in terms of enzymes found application,

diastase on amajo ri ndustri alsca le,sincethe1840s,a
fewother si nthes ec ondhalf ofthe nineteenthcentury.
Thefirst co mpany fo undedon anenz yme-basedproces
s was Ch ri stian Ha nsensla borat oryinCopenhagen
( D e n m ar k ) , s o n a m e d t o t h
i s d a y .I t p o n e r e d t h e u
se
ofren t(lab fe rment ,c
h y m o s in ) , f r c h e s m a n
ufac-t re(B ro ckhau s
1 89 4
b l , v o . 1 0, p .8 6 3 ; P o u l so n a d B u c h o l z
a rational drug insofar that a weekend function of the

s t o m a c h ( d y sp e i a ) e n
f o r c e d b y l i t l e do s f p
ep-sin,a nd,ifact um erousp it iverp ortsb yd octrsa eav il able( Brockhau s
Awa
sea
inl
gov
sto
es
onb
ufa
v e of f o un d a t io f r e
r c hi n s ti u o n s , m a
y
e r nm
o k pl
a r ch
e r,
c t ur
e n ta l i n s u t e
a c e, d v ot o r
w i ne a d fo m a n
e , h y g i e n , m d i c a l r e , a n d w t e r s e l a s b o r t i e s f t h e b r w i n g a n d b a k e r y i n d u s t r i e s , n o t a b l y i E pu r o e , a n d s e v e r a l i n t h e U S A . I n s t i u o n s f r b e w i g r e s e a r c h n d e d u c a - t i o n w e r s a b l i s h e d i n W s t e p h a n e a r M u i c h ( 1 8 7 2 8 7 6 ) , B e r l i n ( I n s t i u t f r G r u n g s g e w r b , 1 8 7 4 ) ,
Hohenheim (1888) (all in Germany), in Copenhagen,

sem
st
uhdaonnP
iInstitut
afria
Pasteur
(1888).
In
Britain, the British
School of Malting and Brewing

was founded at the University of Birmingham in 1899.
In the USA, by states decrees, agricultural research institutions
were founded from 1863 on, that eventually became the
origins of big universities like MIT, Cornell, and Wisconsin
(Bud1993).
Following Pasteur and Kochs success in identifying
causative agents of disease and establishing pure cultures,
pharmaceutical companies also established bacteriology
3752
departments which produced vaccines or tested substances

for their antimicrobial properties (Metz
1997). J. E. Siebel in
Chicago issued a journalZymotechnic
Magazine: Zeitschrift
fr Grungsgewerbe and Food and Beverage Critic.In
German-speaking countries, 10 journals on brewing were

issued at that time (Brockhaus
1894a,b). Jrgensen, in
1885, founded the journal Zymotechnisk Tidende, and published a highly regarded Microorganisms
book on
and fer.Severalfurthe
rbo oksonm ycology and/orb acter ia
mentation
ormicroorganis msw ereiss ued.The termsBa cteri ol ogyor
Mycology,yZ mot ech nologi e,orMic robiolo gyden om inated
the new research field.

Thus theAge
of Bacteriologybegan
with a new paradigm, and a broadened industrial and economic base.
fermentation, during the period up to 1930, stimulated the

molecular approach to the study of the pathway of alcoholic
fermentation, mainly the research on the successive intermediates in metabolism. Buchner himself continued to
work on cell-free fermentation investigating intermediate
compounds and activities both in cell-free press juice as
well as in living yeast that would convert possible intermediates including trioses. By the end of the 1940s,
the scheme of glycolysis and alcohol formation was
finally complete (Florkin
1975; Kohler
1975; Buchholz
and Collins2010, chapter 4).
Of major impact on industrial microbiology were
Fernbachs systematic investigations at the Institut Pasteur
inPa rison me tabolicn ter
medi atesd ur ingalcoh lic
ferm en-t at ion(mal yofglcsi )byvar iousmcr organism,e. .yeast nd
Tyrothrix tenuis; this included the formation
of acids, notably acetic, succinic and pyruvic acids. He

identified corresponding enzymatic activities: the beginnings of what we now call biochemistry research. This
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 alcohols
(Fischer1909). With the work of Emil Fischer (18521919)
(Fernbach1910; Fernbach and Strange

1911).
Around 19071910,
there was a shortage of rubber on the

came the breakthrough in thedevelopment of structural
biochemistry; in the course of his scientific career he
world market. Perkin in the UK proposed an alliance, comcompletely 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
sugars, fats, and proteins (Fruton and Simmonds
1953).
butanol (butyl alcohol), which could be converted into buBuchner in the mid-1890s ended the hypothesis of
tadiene. This in turn could be polymerized to yield synthetic
visvita li s,t hatstillp ostul ate dhiddenmys teriou sforce rubber (Perkin Jr.1912). Shortly after this initiative, the First
s
World War created a demand that drove technical innovation
inferme nt ati on,whenhe publi she daseriesof papers
in the fermentation industries. Theacetone
butanolfer
(Buchne 1r 897
,1898;Buc hnera ndR 1898),w
app
hich
signale da bre akthrough infer men tationande nzymol ogy.
Buc hnerprocess
skeye became
xperimentw
epareapres
sjuicefro m
mentation
a keyastopr
technology
for explosives
production since acetone was required as a solvent, in short

y e a s t , w h i c h o nt a i e d l
supply in Britain. Chaim Weizmann, who had worked in
l t h e n z y m e s r qu i r e d f o
Fernbachs laboratory, continued similar research in the
the
Biochemical Department of Manchester University, and
t r a n s f o m a t i o no f s u g a r i
made a new contribution using a more abundant source of
n t o a l c h o l a n d ca r b o n d i
raw material, viz. maize, in 1915 (Speakman
1919).
xide,
Weizmann brought his own laboratory experiments to the
a n d t o e m o n s t r at e h n o
notice of the Admiralty, in the spring of 1915. He asked
l i v n g c e l s r e m a i n e d . H t h e n c o u l d s h w t h a i s o l u t i o n c d p e r f m t h e s a m r e a c - t i o n a s d i d l i v n g y e a s t d u r i n f e r m e n t a i o , a s u m i n g o e e n z y m e , c a l d z y m a s e , b i n g t h e c a t a l y s t . B u c h n e r p r e s n t e d h p r o f t h a t ( a l c o h i c ) f e r m e n t a i o n d d n o t r e q u i r t h p s e n c o f . . s u c h a o m p l e ax p r a t u s i s
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 Nicholsons
without doubt a protein bodywhich he calledzymase,
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; Nathan1919; for
of the whole glycolytic pathway (Buchner
1897). With
more details and the political background refer to, Bud
Buchners work the dogma of thevis
vitalisfell.
It initiated
1993). The manufacture of acetone by the Weizmann process attained the greatest success at the factory of British
a new paradigm, the biochemical paradigm, which, in contrast to that of Pasteur, stated that enzyme catalysis, includAcetones, Toronto, Ltd., in Canada, on a large scale.
ing complex phenomena like that of alcoholic fermentation,
Fourteen new fermenters were constructed, about 18 ft
(5.5 m) in diameter and 20 ft (6.1 m) high, holding 24,000
was a chemical process not necessarily linked to the presence and action of living cells. The discovery of cell-free
gallons (91 m)3 of mash (Nathan
1919; Speakman1919). In
The period from 1890 to 1940The advent
of biochemistry, and new products
Germany, war requirements concerned glycerol (glycerin)

for the manufacture of explosives, when supplies of fat
became enormously curtailed as a result of the imposition
of the sea blockade. Investigations initiated by Ldecke with
the object of obtaining glycerol on an industrial scale by
means of fermentation became of supreme importance.
The process was developed by the Protol Company. The
monthly output of glycerol was about 1,000 tons
(Connstein and Ldecke
1919a,b).
The formation of oxalic and citric acids by Apergillus and
Penicillium species had been observed by Wehmer in the
1890s. Citric acid fermentation became an object of study
by several academic groups that were actively engaged in
optimizing the process and in elucidating the biochemical
mechanism leading from the sugar substrate to citric acid.
Currie undertook what came to be considered a classic
investigation of the factors controlling the production of
citric acid by a selected strain of Aspergillus niger; he
elaborated optimum conditions for the production of citric
acid. Currie joined Chas. Pfizer in New York, where a plant
was established which went into production in 1923. By
1933, this industry already contributed 85 % (in Europe
5,100 tons and in the USA 3,500 tons) of the worlds
citric acid production of 10,400 tons. Further products
manufactured by fermentation were gluconic acid and lactic
acid (May and Herrick
1930; Frey1930; Roehr1996,1998).
Another important example of industrial research activity
was the development of the oxidation of sorbitol to sorbose
by Acetobacter suboxydans as an intermediate for vitamin
C. The corresponding so-called Reichstein process was used
from the 1930s on a large industrial scale (Buchholz and
Seibel2008). Another process established in the 1930s was
the manufacture of L-ephedrine as a pharmaceutical using
the stereoselective condensation of benzaldehyde and
acetaldehyde by yeast (Vasic-Racki
2000).
Ethanol continued to represent a major product of outstanding economic
importance.
A breakthrough event was in 1928 when Fleming observed that a culture of a Penicillium notatum inhibited the
growth of bacteria. He demonstrated the production of an
antibacterial substance in the culture broth and named it
penicillin. However, there was rather a long delay before
research and development aiming at production was undertaken, finally stimulated by Florey, Heatley, and Chain
who entered this field again toward the end of the 1930s
(Bud2007; see below).
The nature of enzymes and the structure of proteins
req uiredm or ethan40 yea rst obeestabl is hed,andit
rem ainedc on trovers ial for decades(S um nerandMyrb
ck
195 0).Int he 1930s,S tan ley successfu ll ycrystallizedthe
tob accomo sa icvirus ;it was thefirstt im ethatanylivingfo
rm hadbee nc rystall ize d,a nditrevol ut ionizedthinkingab
3753
Enzyme technology rapidly expanded. A range of enzymes,

including diastase (amylolytic enzymes), proteases and
pectinases, were isolated from different organisms for commercial use, mainly from Bacillus subtilis and other species
such as Aspergillus oryzae and A. niger ( Ta u b e r 1 9 4 9 , pp.
396494;
Buchholz and Poulson

2000). Takamine began
isolating bacterial amylases in the 1890s in Japan. In 1894,
he obtained a patent for the production of a diastatic enzyme
preparation from molds, which he calledTakadiastasefor
the production of amylases for the hydrolysis of starches in

food manufacture (Tauber
1949). Major applications of enzymes were proteases in the chill-proofing of beer, and the
addition of malt extract in dough-making by American
bakers in the USA. In 1922, they used 30 million pounds
(13,500 tons) of malt extract valued at $ 2.5 million. In
1907, Rhm patented the application of a mixture containing
pancreatic extract as a bating agent, replacing the unpleasant
use of dung, and he founded the Rhm and Haas Company
in the same year based on this application. From about 1930
onwards, the enzyme preparation was produced by fermentation (Tauber1949; Buchholz and Poulson
2000). For
education the Institute in Berlin offered various courses from
1888 (and later a curriculum for brewers), as did theInstitut
fr Grungsphysiologie und Bakteriologie that was

established at the Technical High School in Vienna in
1897, as well as other institutions. Courses on fermentation
were offered by Bernhauer at the German University in
Prague in the 1930s 1993/1995,
(Bud
pp. 60, 61, 104,
132, 202, 203; Clifton
1966).
Important scientific breakthroughs and applications are
summarized in Table
3.
The period from 1940 to 1970the era of antibiotics,

and the emergence of biotechnology
Florey, Heatley and Chain, towards the end of the 1930s,
began to investigate penicillin in the course of their systematic study of antibacterial substances at Oxford University.
The credit for resurrecting penicillin, described at the time
as unstable as an opera singer, certainly goes to the Oxford
group. They developed an assay, found a way of producing
penicillin in surface culture and demonstrated the marked
activity and therapeutic value of penicillin in a clinical trial
in 1940 (Bud2007; Coghill1970; Demain1981; Ohno et al.
2000). Early yields and recovery, however, were very
discouraging and the difficulties in wartime England led
them to visit authorities, laboratories, and industrial companies in the USA for help in July, 1941. They were advised
by research authorities to visit Peoria, Illinois, USA, to talk
with officials of the Northern Regional Research Laboratory
out thec he mical na tu reoflife(Van Dem 1987).
arkandBatzi ng
(NRRL) because this institution had just organized a fermentation division. The representatives offered Florey all
3754
Table 3 The period from 1890 to 1940 (Buchholz and Collins
2010, chapter 4; Roehr
1996)
Time, scientistsa
1894 E. Fischer
1897 Bu chnerFe
1900 sB uchnerR
1905 E. Fischer
comp an y(Germ
Specificity of enzymes
rmentationd ue toenzym
ersearchonf er mentati
andothersRe se archint
any)
Enzyme technology expanding (Takadiastase)

eactio nonlyFirst wastedisp osalbiogasreactor(B ombay)
oninte rmediates
henatu reofprotei ns1907Enz ymetechnology:Rhma ndHaas
1910f Fernbach
1911f Fernbach and Strange;
1912f Perkin
1915f Weizmann
1915f Connstein and Ldecke
1916 Thom and Currie
1920s
1920s and 1930s Embden,
Meyerhoff and others
1925, 1930s Sumner, Northrup
1928 Fleming
1933 Reichstein
End of 1930s Florey and Chain
1940
Rersearch on fermentation intermediates

Microbial formation of acetone and butanol
Finding of Clostridium acetobutylicum

Glycerol fermentation b
Citric acid fermentation b
Fermentation technology expanding: Production of butanol

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

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

Reichstein process for vitamin C production
Sterile enzyme fermentation for detergents etc.
Peak alcohol production
Selected scientists and events relevant for applied microbiology (see also first footnote2)in Table
Most intermediates mentioned here, butanol, acetone, citric acid, etc., have been observed before, but not developed further for industrial
production
the help they could give. Biosynthesis work began on July

15, 1941, at the NRRL under the general direction of Dr.
Coghill (Greene and Schmitz Jr.
1970; see also AIChE
1970)
. Research studies were also initiated at the Universities of
Minnesota (on microbial strains), Wisconsin (on fermentation), Penn State (on recovery), the Carnegie Institute,
Wisconsin and Stanford (on mutation) and at MIT (on drying
and packaging) (Coghill1970). By the fall of 1941, yields of
penicillin began to climb to 6, 10, and to 24 Oxford units per
mL, using an improved mould strain, as compared with
about 3 units/mL obtained by the Oxford group.
In December 1941, the US Government became interested.
Th eUSDepar tment ofA gr iculture(U SDA)ca lledameeting
in NewYorkw hichi ncl ud edrepresen tative sfromthe
Na tionalRe searc hCo un cil,andfou rcompa nies,Merck,
Sq uibb,Pfi zeran dLe de rle,aneven tthatw asconsideredto
re presentt herea ltu rn ingpointfo rpenic illinproduction
(C oghill19 70;Gr een ea ndSchmitzJ 1970)(some18more
r.
co mpaniesb ecame inv ol vedsubsequ ently; Elder
1970).
In dustryre prese nta ti vesagreedt omaker esearchteamsava il ab letowork onthe pro bl emofsupply ingade quatequantities
of penicill in.By 194 3, theamazing curati vepropertiesof
pe nicillin wereb eco mi ngprettywe ll-kno wn,andtherewas
ah ugedeman dfort hed ru g.Theprime goales tablishedbythe
go vernment repre sen ta tiveswasto haveam plestocksonhand
fo rtheUSar mysin vas io nofEuropei nthesp ringof1944.
That goal finally was met

by a huge effort, and a hitherto
unknown approach to interdisciplinary cooperation and project organization (Coghill1970).

Strain screening and development, including mutation
proced ures,prov edt obeakeyfacto rforsucce ss.From
manyso urces,inc lud ingsoilsampl esfromaro undthe
world, collected byt heUSArmy,man yhundreds ofstrain
s
ofpeni cillin-pr odu cerswereisol ated.Theb estproducero
f
all(la beledNRRL 195 1),ironicall y,camefro mamoldy
cantal oupemelon fro maPeoriafrui tmarket.G enetic changeswereu
improve n ta dus eof ab e

t erNR Ls ai nra ise dt h
e
con e tra i ofp eni ci l
l i n t o 1 0 u s / m L . S u b s e q u e n t i m p r o v e m n t s r a i e d t h yi as nb o r d e r o f m a g n i t u e d t o a b o u t 1 , 5 0 0 u n i t Ls w/ m h e W c o i n s t r a i n ( C o g h i l
of penicillin during the early months of 1942, as Richards

repo t d( G re n a dS c hm i
tzJr.
1970
; Sil cox
1 97 0
). B
y
June1942,
into rea 1
en
produce , a
943ther w a
terialto e
nou ghpe ni c il
0pa tien ts h ad b
ndb yFe ru a ry 1
s u f ic ie n tm a
t a p r o x i m a t e l y 1 0 t i e n sp a . P r o d u c t i n w a s b y u r f a c e c u l t r e f l a s k , t h e m o s t r e l i a b l e m t h o d a t h e i m . I n 1 9 4 2 , 2 - y e a r s i n t e n s i v e d e l o p m e n t h a d r s u l t e d i n n c r e a s i n g t h e l v o f u t p u t o f p e n i c i l n b y s o m e 1 4 0 , - f o l d . T h e m o s t e f i c i e n t a p r o a c h w s s u b m e r g e d o r d e p - t a n k f e r m e n t a i o n , b u t h e r e w a n u m b e r o f s e v r p r a c t i l o b l e m s , t h e s o l u t i o n s o f w h i c e r n o t
3755
obvious, but which were finally achieved 2)

(Fig.
(Greene
resistant. Factors that exacerbate this phenomenon are misuse
a nd Sc hm itz J1970;S
r.
colix
1970). Dow n st re am o pe ra -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 propp.116-139; Hubschwerlen2007).
cesses, such as liquidliquid
extraction, centrifugation,
By the 1950s, large-scale production not only of traditi ona lgoods ,forexample ,beer,alco hol ,che es e,butalso
freeze drying, crystallization and others (Silcox
1970;
ne wpr oducts ,includingc itricacida ndp harm ac euticalsa
Perlman1970).
nd
Thus began a wartime collaboration which was to inot her produc tsofparticu larlyhighs oci alan de conomic
re lev ance,h adbecomewel lestablish ed. Grow in gecono
volve the efforts of literally hundreds of biochemists, chemmi c
ists, bacteriologists, biologists, chemical engineers,
re lev ancefo llowednotab lythesucce sso fpen ic illinmanu
physi cians,toxicologi sts,pharmac ologists ,andpathologist onb
s
othsidesoftheAtl antic,manag edandcoo rdinatedby
fa ctu re,and furtheranti biotics,li kes trep to mycin,became
indus trialexecutives, academicadm inistrat ors,andgovern- av ail able,f ollowedbyan ewclassofh igh valu e- added
mentl eaders.(Greenean dSchmitzJr.
1970).At thepolitpr odu cts,ma inlyseconda rymetaboli tes ,e.g .s teroids
ob tai nedbyb iotransform ation.Othe rma jorp ro ductsof

gr owi ngmark etrelevance includedam ino acid s, organicacids ,carbo
ical level,the
injection of funds, people, companies, and
governmti tersm ant
atrnsfom ionthe wayso f doingsce. Ar ang eofs malerp ojectsn p enic l in
p r o d u c t i n w e r e u n d re t a k n
i n s e v r la o t h e r c o u tn r i e s ,
A new generation of biocatalysts, based on immobilizai n c l u d - i n g G e r m a n ,y t h e N e r l a n sd , F r a n ec a dn b y C z e c h s c i e n t ( B u d
t ion techniques de velopedinthea cadem ic field,ledtoa
b rea kthroughin pr ocessingoffoo dandp ha rmaceutical

and big business (Bud
2007,pp.2353, 5474).
The prosc omp ounds.Larg e- scaleprocesse swere es tablishedusing
pects, and later the success of penicillin, prompted further
b ioc atalystsfo rp enicillinhydr olysi s( forthesynthesiso f
s emi synthetic -l actamantibiot ics)a nd glucoseisomeriza research on antibiotics. Waksman isolated actinomycyin in
1940,str ep totricinin19 42,ands treptomy cinin1944fro m
cultures of actinomycete s(Ohnoe 2000).(Thepatent

tal.
on
t
i
o
n
(
P
o
u
l
s
n
a
d
B
u
c
h
o
l
Streptom yc inandforstar tercult uresfory oghurtlargel yfiz 20 3
; Buch o lz eta l.
2 01
nancedth ee stablishment oftheLi feScienc esFacultyatt he
2 ,
Universi ty ofWisconsini nMadiso nforthen ext50yearsan d
provided ma nystipendsfo rstuden ts.)Howe ver,thethera peu- chapte rs7and8) .Wa stewa
ticpoten ti alhasbeenthr eatened bytheeme rgenceofincr eas- ter atmenb cam emor
inglyres is tantbacteria lstrain sasanatu ralconsequen ceof
w i d e - s p r e a d , u e t o l e g i s a t o n , n d g a i e g r e a t n i o . T h s r e s u l t d i n e e w d v le o p m n t s , a n d c p i t a l n v e s m , b o t h i n t h e p u b l i c a d i n d u s t r i a l s e c t o r s ( J d e n i g a n d W i t e r
theiruse ,f irstobserved byAbrah amandCha1940).In
in(
clinical se ttings,moret han50%o fandmor ethan90%of
Significant events are summarized in Table
4
Starting in the 1970s and 1980s, BT gained the attention
ofgovern me nta lagen cie sinGer ma ny,the UK, Japan,the
Escherichia coli isolates
USAandot he rsa safie ldo finnov at ivepot ent ialandecoS. aureus isolates are ampicillin
nomicgro wt h.T hiswa sal soinre sp onseto the firstoilpric e
crisisin th ebe ginni ng1 970s,a nd therea liz ationthatren ewablemate ri alr esour ces wouldb ec omemor eim portantinth
e
future.T he sea pproa che sledto ex pansio nof thefield.The
firstent hu sia sticr epo rtbyth eG ermanc hem icaltechnolo g
y
organiza ti onD echem awa sissue di n1974f ort heGerman
Ministry fo rEd ucati ona ndScie nc e(Bund esm inisteriumf
r
Bildungu nd Wis sensc haf t,BMBW ). Itwast hef irstsystematicappr oa chf orBTr ese archfu nd ing,em pha sizingclassi -calBT,andd
This t
igu n
tion
betw
dustry
Fig. 2 Penicillin fermenters in operation at E.R. Squibb & Sons, 1946
(Langlykke1970)
udyha sbe ena in tr

examp leo fint er ac
npoli cym aker s, in
andsc ien ce,a nd was term d acorp at ista p ro chbyJ n f (
Dech mast udyre flect d

h e m a i n s t a b l i s h e d s c i n t i f c a n d a p l i e f l d s o f BT a t h i m e . T h b a s i c d i s c i p l i n e s
3756
Table 4 The period from 1940 to 1975 (Buchholz and Poulson

2000; Bud2007; Buchholz and Collins
2010, chapters 4 and 5)
Time, scientists
End of 1930s Florey and Chain

1940
1940s Waksman
Resume research on penicillin

Protein structure solved
Extended research on antibiotics: actinomycin,
streptomycin
1941USA:penicilli
1944Large-scalein
deeptankpenicilli
npro ject,dueto warrequi rem en ts

dust rialpenici llinprod uct io n;P fizer:
nfer mentation
1948 Brotzu and Oxford team

1949
1952/1953
Cephalosporin, broad spectrum antibiotic

First biochemical engineering symposium
1953 Watson, Crick, Franklin

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

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

End of 1960s
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,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
There were of course other failures which would be worth investigation
An exception are glucose sensors
involved in BT research and development work were midiscipline and there were no books, rather no journals, curcrobiology, cell biology, biochemistry, and
to a limited
ricula or scientific conferences devoted to the subject. A few
extent molecular biology and genetics 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 (197274)
(Buchholz
1979; Buchholz and
and another BT curriculum was established in the 1970s at
the Technical University of Berlin (Buchholz
1979, pp. 69,
Collins2010, chapter 5).
Research work in the field of BT proceeded as subtopic
71). The first BT journal of high reputation was established in
withinam otle yc oll ectio no fs cientific an dengineeringdi
1958 by Elmer Gaden as the Journal of Microbiological and
sBiochemical
Engineering
. It later
Biotechnology
ciplines with al owl evelo fc oh erenceand li ttleintegration u ptill
the1960san
d1 970s.Durin
gthbecame
e1940s,Step
hensons and
Bioengineering and is still a leading journal in the field. A

few other journals appeared in the 1950s and 1960s, for
Bacterial Metabolism and of Kluyvers Chemical Activities
of Micro-Organisms appeared, the Grungschemische
example Applied Microbiology, renamed Environmental and
Praktikum, by Bernhauer was published in 1936 (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
th ef ie ld:onappli edmicro biology(Re1967,Pi
hm
rt1975
The period from 1975 onthe new biotechnology
), as
we ll as onbiochemi calengi neering(Ai baetal.
19 65;Bail
ey
The turning point in genetics ensued from the establishment
an dO ll 1977).Th
is
efirste ncyclopedi asandseri esonBTwe reissu edbyRe hm andReed
1981)andFli
(
ckingerandDrew(
1999).Thus, bi o
of a model for the molecular structure of DNA by Jim
Watson and Francis Crick, based on the crystallography data

of Rosalind Franklin, who was working in Morris Wilkins
lab in 1953 (Watson and Crick
1953). This was the culmination of work initiated by Sir William Henry Bragg and his
son William Lawrence on X-ray diffraction by crystals, to
study molecular structure, initially of minerals but later of
more complex organic structures, including the first 3-D
structure of a protein, myoglobin (Max Perutz and John
Kendrew, see Kendrew et1958),
al. further of penicillin,
vitamin B 12, and insulin (Hodgkin
1979). The significance
of DNA structure, as the material of which genes are made,
was immediately recognized due to the ground-breaking
work, during the preceding 50 years, of a great number of
scientists in chemistry and biology, mostly microbiology
including Gregor Mendel, Friedrich Miescher, Phoebus
Levene, William Astbury, Erwin Chargaff, Oswald Avery,
Francois Jacob, Jacques Monod, Ole Maaloe, Max
Delbrck, Sydney Brenner and others (Judson
1979;
Winnacker1987; Buchholz and Collins
2010, Chapter 7).
But theDNA
Revolutionas Hotchkiss termed it,

progressed or penetrated slowly into technology, initially
having little effect on traditional processes and products
(Hotchkiss1979). The Asilomar conference 1975 initiated
a public discussion on the possible hazards of recombinant
DNA research (for details, see Buchholz and Collins
2010,
section 8.1.2). The following two decades saw many years
of discussion of possible risks and containment requirements associated with recombinant technologies which
eventually formed the basis of the guidelines for recombinant DNA work and finally culminated in international
legislation (see for example Cartagena Protocol on Biosafety,
http://bch.cbd.int/protocol/t ext/ .)
Subsequent to Watson and Cricks publication in 1953 of
the DNA structure, a large number of significant scientific
breakthrough events as well as technological progress provided a new basis for BT. Selected events are summarized in
Table5. Berg, Cohen, and Boyer in 1972 introduced recombinant DNA (rDNA) technology when they constructed the
first recombinant plasmids and viruses, which were introduced into bacteria, or animal cells respectively, where they
were autonomously propagated. A patent granted to Cohen
and Boyer, and the University of California was critically
commented by Berg (Cohen et1972;
al. Cohen and Boyer
1979/1980; Berg and Mertz
2010).Entrepreneurwas
still
a dirty word in molecular biology, leading one to reflect on

the situation in engineering a century earlier with the slandering of George Stephenson (later inventor of the steam
engine) by Sir Humphrey Davy at the time of hisinvention
of the miners lamp (not patented), already produced as

Stephensons prototype (patented).
Based on the new genetic techniques, a significant
change occurred during the 1980s and 1990s with common
approaches in different disciplines underlying BT, and the
3757
merging of molecular biology and biochemical engineering.

Industrial interest and the range of products expanded significantly, and many new companies, mainly in the USA,
were founded. New methods and tools played a key role in
the rapid expansion of recombinant technologies. These
include: gel electrophoresis, centrifugation, restriction endonucleases, plasmid cloning, a range of further cloning
methods extending to most known species of microorganisms and eukaryotes, in particular in plants, cloning of larger
(gene-sized) DNA fragments via virus cosmid, fosmid,
BAC and YAC (this latter in yeast) cloning, oligonucleotide
synthesis, DNA sequencing, gene mining, metagenomics,
and recently synthetic biology; protein design has become a
rational tool for biopharmaceuticals and enzyme development (Winnacker1987; Demain2001; Bornscheuer and
Buchholz 2005; Buchholz and Collins
2010,chapetrs 7,9).
Once the tools for gene cloning in the Gram-negative E. coli
had been established it became easy to develop gene cloning
vectors which could be transferred to other species. This
involved the identification of plasmids that replicated in
other hosts and genes (promoters) that could be expressed
and used for selection in the new host, including bacteria,
yeast, insect cell lines and plant cells (Collins
1977). Thus
all the elements for the new recombinant DNA technology,
at least for bacterial and animal cells are available: Methods
to prepare DNA, which, following restriction cleavage
(i.e. treatment with restriction endonucleases) could be covalently joined to avectorwith
a DNA ligase; avector
(plasmid or virus) to ensure maintenance in the cell; a

method to preparecleanvector
DNA; an efficient method

to incorporate DNA into the cell; culture techniques to
isolate single clones carrying a single recombinant hybrid
molecule, including selective techniques to enrich for the
cellstransformedwith
the vectors, for example selection

for antibiotic-resistance genes (Buchholz and Collins
2010,
chapter 7). More recently, since the 1990s, the so called
omicsapproaches:
genomics, proteomics, metabolomics,

bioinformatics, and their integration into systems biology
and biotechnology aimed at understanding, quantitative description and rational modification of whole organisms.
Biosystems engineering or systems biotechnology aims at
the integration of biology, mathematics, bioinformatics, and
systems engineering to gaina holistic view of complex
biological and biotechnological systems, including quantitative description and improvement of whole organisms and
the rational development of novel production processes
(Reuss2001; Deckwer et 2006;
al.
Klein-Marcuschamer et
al.2010; Papini et 2010;
al. Buchholz and Collins
2010,
sections 13.6 and 15.6).
As a consequence of this development, in the USA, also
on t hepolitical le vel, theperceptio no fBT dive rgedgre atl y
by t he1980sasco mp ared tothatinEuro pe par ticu larlyin
Ge r manyinthe19 70 s.Th isisperceive df rom arep ortofth e
3758
Table 5 The new biotechnology
Scientific events
1944
1950
1953
1953
Technical application
Avery et al.: chemical nature of chromosomes: DNA

Charg af f:ru leofnucl eotide ra tios
Sange r: sequ enceofin sulin
Watso na ndCr ick:stru ctureo fD NA
(For technical application up to the 1960s, see 4)
Table
1955f Kornberg et al.: enzymatic DNA replication

1957f Zamecnik and Hoagland: amino acid activation, translation in
protein synthesis1959 Kendrew: first X-ray enzyme structure
1960196 1JacobandMonod :operonm odelo fgene regula tion;
concept of mRNA
19611966
Nirenberg, Khorana et al.: genetic code

1963 Merrifield: solid-phase protein synthesis
1968 Arber and Linn: restriction endonucleases
1971 fNath ans ;Sout hern:DNAsep aration1971Farley,C ape,Glas er:establ ish mentofCetu
1972 Mertz ,Da vies: recombinant DNA1972Industrialpr oduction of6-amino -pe nicillanic
Berg :firs tre combi nantvirus
s,th efirstB iotec hCompa ny

acid
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 Khler and Millstein: monoclonal antibodies
1976 Swanson, Boyer: foundation of second biotech company: Genentec
1975 Asilomar conference (moratorium on recombinant DNA research) 1977f Further New Biotech companies founded
1978 Heffron et al.: directed mutagenesis
1978 Recombinant human insulin (Genentec)
1979 Mayer, Collins and Wagner: recombinant penicillin acylase
1980 Chakrabarty: first patent for recombinant bacterium
1983f Frank and Blcker; Carruthers: mechanized DNA synthesis
1980f Work on recombinant-amylase
(Novo)
1982 FDA approval of human insulin (Eli Lilly)
1982 Large-scale production of recombinant-galactosidase
(Boehringer Mannheim, D)
1983 Schell and Montagu: first transgenic plant (tobacco)

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

1999 Start of CELERA
industrial genome sequencing
1999 Vitamin C via microbial pathway
These topics are difficult to assign, a range of arguments being raised in terms of their classification as technical application, not fundamental
research
b
Bailey (1991,1996)
OTA of 1984 (OTA

1984). It refers to methods that arose
with knowledge on DNA and that revolutionized what
was thinkable. In contrast to the reports mentioned
before, emphasis in the OTA study was on genetic engineering and rDNA technology, resulting in commercial
opportunity and support of fast commercial exploitation
of scientific results, closely associated with the business

world.
The industrial breakthrough came with recombinant
hum aninsulin, developedbyG enen tech incooperationwit
hE lyLillyin1 978,andappro vedb ythe USFoodandDrug
Adm inistratio nin1982(Bud
1 993/ 1995 ,pp.232,237;
Walsh2007, pp. 297, 298); this was at a time when some

heads of European pharmaceutical companies did not believe that a recombinant DNA product would ever be approved for clinical use. This precedent , notably the
approval human insulin as the first recombinant DNA
product on the market, was followed by a series of further
recombinant products, mostly drugs, which in general could
not be produced by other technical means, and which are of
great medical interest. Some of these products previously
isolated in small amounts from human blood or tissue were
in danger of being contaminated with human pathogenic
viruses (not all known at that time, e.g. AIDS virus, HCV).
In this respect, this alternative production route provided products not only in sufficient quantity for general use but also with
an improved and reproducible quality. The products included
human growth hormone in 1983,-interferon, and a hepatitis
B vaccine in 1986, tissue plasminogen activator (tPA) in 1987,
and erythropoietin in 1989 (product approval). Actually, recombinant proteins, including hormones and growth factors,
blood clotting factors, cytokines, monoclonal antibodies and
vaccines are most important biopharmaceuticals, with a market size estimated of some $50 billion per year around 2010
(Walsh 2007, Aggarwal2007). Antibiotics remained an important sector of biopharmaceuticals, with many different
specialties and sales estimated at more than $50 billion per
year (Hubschwerlen2007).
Large investment by multinational companies, the foundatio nofmanysma ll newcompanies, afewofwhic hha ve
grown remarkably ,a ndstatefunded bigresearc hme rgedin
agold rushintoth eN ewBiotechnolo gy,asrecom bin ant
technology was termed in the USA. Key steps toward the

transfer of science into the economic sphere resulted in the
foundation of new BT companies, the first being Cetus,
started in 1971, later the originator of thepolymerase
chain
reaction(PCR;
Kary Mullis) which gave birth to the era of

gene diagnostics and personalized medicine. Herbert Boyer
and Robert Swanson founded Genentech in 1976; amongst
the most important companies founded were Biogen (1978),
Amgen (1980) and Chiron (1981), later bought by Cetus
(Demain2001, 2003, personal communication; Buchholz
and Collins2010, chapters 5, 6, 17; for a recent survey,
see Table 17.5).
Industrial products, other than pharmaceuticals, expanded
aswell,bas edbothont radit iona landrecombinantm ethods,
withsalesw orldwidee stima tedo ver50billion.The most
i m p o r ta n b u l k p r od u c t s a
r e t h an o l , m i n o an d o r g
nic
a c i d s ,p r o u c e d i nl a r g e
m o u n t s, v i a m i n s ,a n d b i o
polym e r s . M t a b o l i c e ng i e r
i n g h a sb e u s e d uc e s f l y f o r t h e p t i m z a t i n o f y e l d s , . g . f o r t h e p r o d u c t i o n f a m n o a c i d s ( f o r a s u r v e y , B c h o l z a n d C o l i n s
3759
both aerobic and anaerobic, being applied in numerous

small up to very large-scale installations, as well as a great
number of exhaust air treatment units (Jrdening and Winter
2005). Ethanol, traditionally based on starch and sugar to
produce it as gasoline additive on a very large scale, provoked heavy criticism, with respect to using traditional
agriculture crops for biofuels rather than food. A major
crisis occurred in 2007 and most notably in mid-2008,
causing a dramatic increase in food prices. The growing
use of cereals for ethanol was thought to be in part responsible for this price increase. Recently a trend emerged for
using cellulosic biomass as a source of biofuels (Buchholz
et al.2012, section 12.2). Production of biogas and electricity generated by microbial fuel cells gained much attention
and impetus (Buchholz and Collins
2010, chapter 16).
Recombinant DNA methods also greatly affected enzyme
technologys inc ethelat e197 0s.Over expressi oninfastgrowinghost org anismsw ithh ighprot einprodu ctivity
allowedmany enz ymes,wh ichw erenotr eadilyac cessible,
tobeproduce dch eaplyon anin dustria lscale.T histechnologyallowedd esi gnofenz ymes withmod ifiedspe cificity
throughiter ati verapid cycl esofgen emutatio n,screeningor
selectionan dte stingin addi tiontoc rystallo graphyand
molecularmo del ling.Su chpr oductsa reusedon alarge
scaleforsta rch product s(us edinfoo dprepara tionswitha
productionv olu meof>10 mill iont/a, andethan olwith>37
milliont/a) ,en zymesin dete rgents, forpharm aceuticals
manufacture ,an dmanyot herf ields(B uchholze 2012,
tal.
chapters7,8 ,se ctions1 2.1, 12.2).P lantbiot echnologyhas
successfull ybe enestab lish ed,aimi ngatimpr ovedyields,
diseaseandh erb icidere sist ance,et c.ofcrop s.However,
controversi esa reongoi ngwi threspe cttopoli tical,ethical
andbiosafet yas pects.T rans geniccr opsarecu ltivatedona
verylargesc ale notably inth eUSA,Ar gentina, Brazil,
Canada,ando the rcountr ies( Slatere tal.
2008;Buchholz
andCollins2 010,Chapte r18) .
Two achievements since 2000 gained major public resonan ce:First,the major goal oftheH umanG enomeP roject
was achievedin20 00wit hint ernati onalc oopera tionanda
tot alexpenditur eofso me$3 billio n.The taskwh ichwas
car riedoutbyama jorin tern ationa lcons ortium andlargely
ind ependentlyby Craig Vent ersgro upwas recogn izedas
ess entiallycomp letei n200 0andco mmemo ratedb yacommun icationinthe prese nceo fFranc isCol lins,C raigVenter
and thePresident ofthe USA. Theres ultof theHum an
Gen omeProjectma yposs ibly allowt hedis covery andproduc tionofhundre dsofn ovel pharma ceuti cals,m anyof
whi charenatural human gene produc tspre viousl ynot
ava ilableinsign ifica ntam ountso rasvi rus-fr eepreparatio ns,significa ntlyi mpro vingdi agnos isande ventuallyrev olu tionizingmed icine .How ever,a numbe rofarg uments
hav ebeenraisedi nterm soft heircl assif icatio nastechnical
app lication,not funda ment alrese arch. After1 0yearsof
3760
expectation, e.g. with respect to drug targeting, the following comment was put forward a transformational technology will always have its immediate consequences
overestimated and its long-term consequences underestimated,
and ....you may just start to imagine all the projects that will
spin-off (C&EN2010). Much progress took place
largely through the involvement of flexible biotech companies such as Genentech, Cetus, Amgen and Biogen which
conc entr at edoninnova tivede velop men tinpar allelwith
ale thar gy andbadmana gement inlar ge( partic ularly
Euro pean )p harmaceuti calcom panie swh ichlos ttheir
domi nanc ei nthisnewfi eld.
interpreted the development from early fermentation research to Pasteurs concept of microbiology and technical
innovations, from Buchner and Fernbach towards Perkins
and Weizmanns processes, from Fleming towards Floreys
and Chains work, and the penicillin project, and Watsons
and Cricks solution of the DNA structure towards the
cloning concept by Berg, Cohen, Boyer, and towards the
establishment of new companies and New Biotechnology.
Recently, applied microbiology, biochemical engineering
andmolecu larbiol ogyhavemerged toformbiote chnology
asanewsci entific disciplineini tsownright, sharinga
commonpar adigmat themolecularl evelwithall theother
lifescien ces(Buc hholz2007).Bi otechnology continues,as
well,asaf ieldoft echnology,tod evelopnewte chnical
The second major event may be considered the understa ndingo fthef actor swh ic hcontrolpl uri potentandtoti- processes andprod uctsbasedonar ationalscie ntificbasis.
pot entste m-cel lsand the co ntrolledre pro grammingofma Adiversif ication arosethrought heformation ofsubdisciplines,su chasgen omics,transcr iptomics,pr oteomics,metny
dif ferent iated cells tos uc hstemcells .Th isopensaneware abolicflu xanalys iswithquantit ativeanalys isofcomplex
metabolit es,andf inallybiochem icalenginee ring,which
ao fmedic alres earch ,pr od uctionofmo del sforgenetic
mergedint obiosys temsengineeri ng.
dis eases( forpe rsona liz ed medicine), and aradicalnew
app roacht ounde rstan din gc ancer,deve lop mentswhich
wil l
giv epoten tialt oanew are ao fbiotechno log icaldevelopmen t.This found itsor igi ni ntheworkof tho sestudyingthe
Finally, we note that critical events during the historic
mol ecular biolo gyofc ell di fferentiat ion andembryogene developm
en tofB iote chnology areass ociate dwi thexcepsis ,
tionalpe rs onal itie swhoofte nhadth evisio nan dinsightof
ori ginall yinin sect, wor mo ranimalmod els ,asdidfor
fi ndin gsco ntlyrec
uldbedev eloped forthe
ben
efitof(20 12Physiolo
exa mpleth eNobe llaur eat eC hristianeN uss lein-Volhardta howtheir
nd ast hosemos
aNopra
belPrize
sciencea
nd humatrece
nity ,translaognizedwith
tingth eminto
cticalinve
ntionfina ll ylea ding toinnova tion.P ublica ndp rivateinvest
mentprog ra msof tenc ameslowl yonadv iceorp rac tical
validati on ofra dica ladvance sbyafe wpione ers (Thislatteraspectis tr
A further event that received inordinate publicity was the
c hemical synth esis oftheent iregenomeo fMycoplas ma
g enitali umbyt hegr oupofCra igVenter;t ransferri ngt his
D NAintoa forei gnMy coplasma causedrepl acementof the
r esident genom ebyt hecomple telysynthe ticgenome ,
f orminga novel stra incapabl eofcontinu ousself-r epl ication
( Gibsone tal.
2 010) .Thescie ntificrele vanceofth ise xperim ent,how ever, hasb eenexten sivelydeba ted,butsu bse quent Acknowledgment The authors gratefully acknowledge valuable
s tepsins ynthe ticb iologyma ybecomeake ytechnolo gy
information by Arnold Demain.
( Bornsch euer
2 010) .Althoug hitisdefin itelynotc rea tion
of life, as many journalists sensationalized this milestone, it

tmays elbi co asedrin
tafur trshe ep diraethn
otin sPaf te usngkimar eo f ongvil anrg is ,ms teacrg. celoving wilse nth ew hentsy tienocp .al
Conclusions
The history of biotechnology comprises exciting developmen tsoverm or ethan200years ,frommyst eriousco ncepts
tor ational sc ienceandtechn ology,wit hgreatso cialand
med icalach ie vements,andco mmerciali mpact.Ar eview
oft hishist or ysuggeststhat basicrese archandt hesolution
too penprob le msandunknownp henomena, haveprov ided
ara tionalb as isforarangeof majortech nicalinn ovations,
wit hwhichn ew industrieseme rged.Thus mightbe
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the production of beer, wine and bread. The written first

The early period till 1850fermentation mysteries

droplets of liquids, which

forces), a that view promoted, e.g. Gay-Lussac (Buchholz and

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

of life. In contrast to the vitalist

hypotheses is up to now accepted as unequivocal truth.

The importance of fermentation processes corresponds

in 1840, whereas in the UK it was carried out on an industrial

1836; see also Buchholz and Poulson

Table 1 Dates and events in early biotechnology

Early period up to 1850

Early eighteenth century: technical beer and wine fermentation;

1840s industrial enzymatic dextrin production (Payen)

One of the mysteries of fermentation had remained high(Birch1990)andGeison(

a pure culture. In his discussionhe introduced (1) the biological Paris.The

Appl Microbiol Biotechnol (2013) 97:37473762

technical solutions to a number of practical problems, and

linked all chemical transformations

A summary of important scientific discoveries and applications is given in Table

Although the application of fermentation processes was

A range of fermentation products became an important

Table 2 The period from 1850 to 1890 (Scriban

Scientific findings, events

Detection of anaerobic fermentation (1861)

Technical progress, industrial innovation

1870s: Hansen breeding pure yeast for commercial application;

Beer production: 36 million hectolitres in 1873, Germany

New type of industrial beer fermenter 1)

1 hL corresponds to 100 L, or 0.1 m3

Appl Microbiol Biotechnol (2013) 97:37473762

Fig. 1 Pasteurs technical

of the nervous system and blood circulation, improving

The alcohol production in Germany was estimated up to 3.7 million hL in

Ferments in terms of enzymes found application,

a rational drug insofar that a weekend function of the

Hohenheim (1888) (all in Germany), in Copenhagen,

School of Malting and Brewing

departments which produced vaccines or tested substances

German-speaking countries, 10 journals on brewing were

the new research field.

with a new paradigm, and a broadened industrial and economic base.

Appl Microbiol Biotechnol (2013) 97:37473762

fermentation, during the period up to 1930, stimulated the

of acids, notably acetic, succinic and pyruvic acids. He

(Fernbach1910; Fernbach and Strange

there was a shortage of rubber on the

production since acetone was required as a solvent, in short

Winston Churchill, the first Lord of the Admiralty, to build a

London gin distillery. The process is usually referred to as

Appl Microbiol Biotechnol (2013) 97:37473762

Germany, war requirements concerned glycerol (glycerin)

Enzyme technology rapidly expanded. A range of enzymes,

Buchholz and Poulson

the production of amylases for the hydrolysis of starches in

fr Grungsphysiologie und Bakteriologie that was

The period from 1940 to 1970the era of antibiotics,

Appl Microbiol Biotechnol (2013) 97:37473762

Scientific findings, events

Technical progress, industrial innovation

Enzyme technology expanding (Takadiastase)

Rersearch on fermentation intermediates

Finding of Clostridium acetobutylicum