Reprinted from New Beer in an Old Bottle: Eduard Buchner and the Growth of
Biochemical Knowledge, pp. 43–50, ed. A. Cornish-Bowden, Universitat de
València, Spain, 1997
A PRIZE-WINNING DISCOVERY OF
1896: BUCHNER PROVIDES EVIDENCE
OF CELL-FREE FERMENTATION
Peter Bohley and Kai-Uwe Fröhlich
The discovery of cell-free fermentation by the chemist Eduard
Buchner (1860-1917) is now regarded as one of the great landmarks in
the development of biochemistry. Nonetheless, his appointment to the
University of Tübingen in April 1896 almost failed. The Faculty of
Philosophy had proposed to fill an extraordinary professorship that
had recently become vacant by granting it to one of the lecturers of
philosophy. Fortunately even the rectorate confirmed that a professorship of analytical and pharmaceutical chemistry was urgently
needed, to assure a reliable and regular presentation of these subjects
(gesicherte regelmäßige Vertretung dieser Fächer). After some argument the
factual necessity of chemical instruction (sachlichen Bedürfnisse des
chemischen Unterrichts) in the Faculty of Natural Sciences prevailed,
and Professor Eduard Buchner from Kiel was finally appointed (Fig.
1) as a capable representative of these subjects (tüchtigen Vertreter für
diese Fächer).
As early as the summer semester of 1896, Buchner taught analytical chemistry to ten students in Tübingen, and in the winter
semester of 1896–1897 he was teaching pharmaceutical chemistry to
14 students and the chemistry of fermentation to 27. He was later
appointed a professor in Berlin in 1898, but he had made his trailblazing discovery shortly after he had taken up his office in Tübingen. In view of the earlier anxieties of the Faculty of Philosophy, it
was fitting that it concerned a problem that was of the greatest interest
to philosophers: the miracle of life could not be explained by the laws
of physics or chemistry, because a vis vitalis was at work in the
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B OHLEY AND FRÖHLICH
Fig. 1. Eduard Buchner’s letter of appointment as Professor of Analytical and
Pharmaceutical chemistry at the University of Tübingen. (Source: Universitätsarchiv Tübingen, Catalogue No. 126/76)
in the biological course of events, which would never be completely
understood… This hypothesis of the vitalists might have dramatically
inhibited the progress of research into the processes of life if it had not
been possible to falsify it. The vitalists were totally convinced, for
example, that only living yeast cells could produce the much soughtafter alcohol from sugar, together with the carbon dioxide that escapes
as bubbles. Separation of this process of fermentation from the living
cells seemed impossible, and had indeed never been achieved. On the
other hand the heretical idea existed that the “vital forces” were no
more than molecular forces, an idea that had been advocated by
Buchner in his lectures on the chemistry of fermentation. His last
lectures in Tübingen on this subject were given in the summer
semester of 1898, and attracted students from far and wide: among the
32 students (Fig. 2) were W. A. Osborne from Belfast and A.
Medwedev from Odessa, and we may also note the name of Otto
Dimroth, later famous as a chemist.
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A PRIZE-WINNING DISCOVERY OF 1896
Fig. 2. The list of participants in Buchner’s last course at Tübingen, in the
summer semester of 1898. (Source: Universitätsarchiv Tübingen, Catalogue No.
51/66)
During a visit to his older brother Hans in Munich in October
1896, Eduard Buchner had managed to prove that it is possible to
produce a cell-free extract capable of fermenting sugar. On 9th
January 1897, he sent a preliminary message (Buchner, 1897) from
Tübingen to Berlin, which arrived on 11th January; in it he describes
the experiments of October 1986 that had made the miracle possible:
It is first of all proved that the initiation of the process of
fermentation does not require such a complicated apparatus as
the yeast cell. Rather a substance in solution is to be
considered as the carrier of the fermentative effect of the
extract, without doubt a protein; it shall be named zymase.
The idea that a special protein derived from the yeast
cells causes fermentation has previously been expressed by
M. Traube in 1858 as the enzyme or ferment theory, and this
has later been defended especially by F. Hoppe-Seyler. The
separation of such an enzyme from the yeast cells has not
53
B OHLEY AND FRÖHLICH
succeeded up to now.1
In a lecture before the Deutsche chemische Gesellschaft (the German
Chemical Society) in Berlin on 14th March 1898, Buchner (1898)
proved his discovery in a series of well prepared experiments, with all
details publicly demonstrated and elaborated:
The alcoholic fermentation of sugar has frequently raised the
interest of scientists. There were numerous theories about the
achievement of this process until Pasteur’s pioneering works,
beginning in the middle of this century, led to the final
proposition that there is no fermentation without organisms.
Specifically, Pasteur viewed the process as a physiological act
closely and inseparably tied to the life processes of yeast cells.
Other researchers, however, such as Moritz Traube, Berthelot,
Liebig and Hoppe-Seyler, were of the opinion that yeast, in the
same manner as it produces a certain chemical substance, an
unorganized ferment or enzyme, invertin, likewise produces
a substance that brings about the effects of fermentation. Clear
as the theory was, however, experimental proof was lacking,
because in spite of many attempts nobody had been able to
separate the potential for fermentation from the living yeast
cell. The purely vitalistic theory therefore remained the unconditional winner.
We have all been raised in the conceptions of Pasteur.
When experimental facts came into my hands that
appeared to argue in favour of cell-free fermentation, I
understandably confronted them with grave doubts. The
publication followed only after experiments repeated two
months later led to exactly the same results as before.
Accordingly, it is possible to press cell-free juice from yeast that
Zunächst ist bewiesen, dass es zur Einleitung des Gährungsvorganges
keines so complicirten Apparates bedarf, wie ihn die Hefezelle vorstellt. Als
Träger der Gährwirkung des Pressaftes ist vielmehr eine gelöste Substanz,
zweifelsohne ein Eiweisskörper zu betrachten; derselbe soll als Z y m a s e
bezeichnet werden. Die Anschauung, dass ein den Hefezellen entstammender,
besonders gearteter Eiweisskörper die Gährung veranlasse, ist als Enzym - oder
F e r m e n t - T h e o r i e bereits 1858 von M. T r a u b e ausgesprochen und später
insbesondere von F. Hoppe-Seyler vertheidigt worden. Die Abtrennung eines
derartigen Enzyms von den Hefezellen war aber bisher nicht geglückt.
1
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A PRIZE-WINNING DISCOVERY OF 1896
brings sugar into fermentation.2
This discovery was received immediately after its publication with
both enthusiastic approval and furious contradiction. But soon Buchner could state:
The fact of cell-free fermentation is now probably generally
accepted, especially since confirmation has come from
reports from others. However, a controversy recently
emerged again about whether the juice pressed from and
yeast capable of fermentation truly contained a substance
corresponding to an enzyme that is to be considered the
carrier of fermentation, or whether there were still living
fragments of protoplasm (plasma hypothesis).3
This dispute also was finally decided in favour of Buchner’s enzyme
theory, and now after all the way was cleared for modern
Die alkoholische Gährung des Zuckers hat schon vielfach das Interesse
der Naturforscher erregt. Zahlreiche Theorien über das Zustandekommen dieses
Processes folgten aufeinander, bis die bahnbrechenden Arbeiten Pasteur’s,
begonnen in der Mitte unseres Jahrhunderts, zur endgültigen Aufstellung des
Satzes führten: Keine Gährung ohne Organismen. Im Speciellen betrachtete
Pasteur den Vorgang als einen physiologischen Act, eng und untrennbar verbunden mit den Lebensvorgängen der Hefezellen. Andere Forscher dagegen,
wie Moritz Traube, Berthelot, Liebig und Hoppe-Seyler waren der Ansicht, dass die
Hefe, wie sie einen bestimmten chemischen Stoff, ein unorganisirtes Ferment
oder Enzym, das Invertin, producirt, welches Rohrzucker in Traubenzucker und
Fruchtzucker spaltet, dass sie wohl ähnlich auch eine Substanz erzeuge, der die
Gährwirkung zukommt. Aber, so anschaulich diese Theorie war, es fehlte jeder
experimentelle Beweis, denn niemals konnte trotz vieler Versuche eine Trennung des Gährvermögens von den lebenden Hefezellen erzielt werden. Die rein
vitalistische Theorie blieb also unbedingt Siegerin.
Wir alle sind in den Anschauungen Pasteur’s aufgewachsen.
Als mir im October 1896 experimentelle Thatsachen in die Hände fielen,
die für eine zellenfreie Gährung zu sprechen schienen, da stand ich denselben
daher begreiflicher Weise zunächst sehr zweifelnd gegenüber. Erst nachdem
zwei Monate später wiederholte Versuche genau zu den früheren Ergebnissen
geführt hatten, erfolgte die Veröffentlichung.
Danach ist es möglich, aus Hefe einen zellenfreien Saft auszupressen, welcher Zucker
in Gährung versetzt.
3 Die Thatsache der zellenfreien Gährung wird jetzt, zumal wieder bestätigende Mittheilungen von anderer Seite vorliegen, wohl allgemein anerkannt.
Dagegen ist von Neuem Streit entstanden, ob in dem gährkräftigen Hefepressaft
als Träger der Gährwirkung anzusprechen sind eine Enzym-ähnliche Substanz,
die Zymase (Enzymtheorie), oder etwa noch lebende Protoplasmastückchen
(Plasmahypothese).
2
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B OHLEY AND FRÖHLICH
enzymology and with that for a molecular theory for an explanation
of the events of fermentation.
There remained, however, the objection of Marie von Manassein
(1897), who saw Buchner’s work as little more than a confirmation of
her own:
On 9th April 1871, I presented my work on alcoholic fermentation in German. I expressed the main result of my work
with the following words: “On the basis of all these experiments I consider myself entitled to state that living yeast cells
are not necessary for alcoholic fermentation… Consequently,
I do not consider alcoholic fermentation as a physiological,
but as a chemical process…”
Nobody can admire the profound method of Mr. Buchner
more than I do, which enabled him to separate the alcoholic
ferment from yeast and to enable fermentation in a simple
solution of ferment and cane sugar. However, the most
important aspect of the question, namely, that alcoholic
fermentation is not, as Pasteur believed he had proved, a
physiological but merely a chemical process, has been found
by me more than a quarter of a century ago.
In conclusion, I have to remark that some extended
travels, from which I have only recently returned, brought
about the delay in the publication of this notice.
St. Petersburg, end of November 1897
To this, of course, Buchner had to reply, and he did so quite bluntly
(Buchner and Rapp, 1898) :
In the penultimate issue of these reports Mrs. von
Manassein claims the discovery that living yeast cells are
not necessary for alcoholic fermentation, based on an
examination carried out in 1871, which we have known about
for a long time. It is not our fault that under these
circumstances it has to be stated explicitly: this work,
commendable as it was in its time, proves that the author was
subjectively convinced of the existence of a fermentative
enzyme, as M. Traube (1858) and M. Berthelot (1860) were
before her; however, the objective proof of the truth of the
assumption was missing, as this could hardly have been
provided with the state of knowledge and methodology that
56
A PRIZE-WINNING DISCOVERY OF 1896
existed in those times. The state of affairs results from the
following:
1. The author’s experiments have almost all been
performed by incubating dry-heated or boiled pressed yeast
with a boiled 10% solution of sugar for 2 to 56 days; it was then
distilled and the distillate tested for alcohol using the
extremely sensitive iodoform reaction. Today it has long been
proved that boiling for 10 minutes is not sufficient to sterilize a
solution of sugar. Indeed, microscopic investigation in
conclusion of the experiments showed the presence of “pointlike shapes and immeasurable grains” which at that time was
not considered suspicious. Micrococci were obviously unknown at that time, and it was not yet known that certain
fission fungi ferment sugar and produce alcohol.
2. According to the author air-dried yeast, heated up to
308°C for 3 hours 20 minutes, whereby the cells were charred
beyond recognition, and likewise yeast boiled for 45 minutes,
still had the ability to ferment. Therefore, methodical errors
must have occurred…
At that time, Eduard Buchner could not know that his “zymase” consists in fact of a multitude of enzymes, whose discovery became
possible only after the beginning of the present century (e.g. alcohol
dehydrogenase in 1909, pyruvate decarboxylase in 1911, hexokinase
in 1927, glucosephosphate isomerase in 1933, pyruvate kinase in 1934,
enolase in 1935, phosphofructokinase in 1936, and aldolase in 1936).
His lecture from 14th March 1898 was obviously very lively and
convincing as well (Buchner, 1898):
The procedure to produce the pressed juice, developed with
substantial help of Mr. Privatdozent Dr. Martin Hahn in the
Institute of Hygiene in Munich, is in short as follows. Fresh
Munich bottom-fermenting pressed beer yeast is ground in a
grinding machine; the latter, delivered by Hugershoff in
Leipzig and set to work by a gas motor, consists… of a horizontally rotating porcelain mortar, in which a porcelain
piston, weighed down as one likes, constantly moves to and
fro in a straight line… The preparations that have been set up
clearly show the difference in the macroscopic appearance of
the yeast in the different states; initially you see fresh
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B OHLEY AND FRÖHLICH
Munich bottom-fermenting pressed beer yeast, then the same
after pressing at 50 atmospheres and mixing with quartz sand
and kieselguhr as a dry powder, then as a plastic mass after
grinding and finally as a rather firm pressed cake at the end
of the total procedure. The fresh yeast press juice, as you see it
in front of you — this preparation was transferred here from
Munich in a large ice bucket at 0°C — is a yellowish liquid,
almost clear when the light passes through it, otherwise
opalizing, with a pleasant smell of yeast; it contains a lot of
dissolved carbon dioxide, which begins to escape on warming
to 40°C…
On pouring a volume (10 ccm) of a 50% solution of cane
sugar at 30°C into 10 ccm of fresh press juice in a test tube, a
distinct production of gas starts off about 10 minutes after
mixing, which continues for several days at room
temperature (experiment). The gas formed is carbonic acid, as
is easy to prove. 4
These experiments were the beginning of the discovery of many
thousands of enzymes that act in the cellular metabolism as biocatalysts in a way to allow cells, and therefore organisms, to live. It is the
multitude of molecular interactions which make the miracle of life
Das Verfahren zur Herstellung des Pressaftes, unter wesentlicher Beihülfe
des Hrn. Privatdocenten Dr. Martin Hahn im Münchener hygienischen Institut
ausgebildet, ist kurz folgendes. Frische Münchener untergährige Bierpresshefe,... wird... in einer Zerreibungsmaschine zerrieben; die letztere, von
Hugershoff in Leipzig geliefert und durch einen Gasmotor in Betrieb gesetzt,
besteht... aus einer horizontal rotirenden Porzellanreibschale, in welcher sich
ein beliebig beschwertes Porzellanpistill fortwährend geradlinig hin und her
bewegt... Die aufgestellten Präparate zeigen deutlich den Unterschied im
makroskopischen Aussehen der Hefe in den verschiedenen Stadien, zunächst
haben Sie hier frische Münchener untergährige Bierpreßhefe, dann solche
nach Auspressen bei 50 Atmosphären und Mischen mit Quarzsand und
Kieselguhr als trocknes Pulver, hierauf als plastische Masse nach dem Zerreiben
und endlich als ziemlich festen Presskuchen am Ende der Gesammtoperation.
Der frische Hefepressaft, wie Sie ihn hier vor sich sehen — dieses Präparat
wurde in einem grossen Eiskübel bei 0°C von München hierher überführt —
stellt eine gelbliche, im durchfallenden Licht fast klare, sonst opalisierende
Flüssigkeit von angenehmem Hefegeruch vor; er enthält viel Kohlendioxyd
gelöst, welches beim Erwärmen auf 40°C zu entweichen beginnt....
Beim Eingiessen eines Volumens (10ccm) einer 30°C warmen, 50-procentigen Rohrzuckerlösung in 10 ccm frischen Pressaft im Reagenzrohr tritt
etwa 10 Minuten nach dem Mischen deutliche Gasentwickelung ein, welche bei
Zimmertemperatur einige Tage ... andauert (Versuch).… Das Gas, welches dabei
entsteht, ist Kohlensäure, wie leicht zu beweisen.
4
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A PRIZE-WINNING DISCOVERY OF 1896
possible. These processes of life take place absolutely according to the
laws of physics and of chemistry, and we all are endebted to Eduard
Buchner, who was honoured in 1907 with one of the first Nobel prizes
to be awarded, for a substantial milestone on the way to their enlightenment, experimental falsification of the hypothesis that fermentation without living cells would not be possible.
Eduard Buchner’s friend Carl Harries (1917) wrote as follows in
an obituary:
Buchner had a brilliant intelligence, which could be enjoyed
again and again; besides, his line of thought was clear and
simple. Usually deliberate in understanding in order to avoid
jumping to conclusions, he would sometimes draw farreaching but certain conclusions in a flash. He followed all
communications about other scientific subjects, even if they
were far from him, with lively, I might say charming,
sympathy. He did not exhibit the one-sidedness of some
university teachers who are only interested in their narrow
subject. In his scientific essays this quality became obvious in
the extensive inclusion of outside publications for the
discussion of his results. However, what especially attracted
me to him was his honest noble-mindedness, combined with
great courage and his true kindness. This last was more
difficult for strangers to notice, because he had the
characteristically Upper-Bavarian reserve with foreigners.
Indeed, he could be quite rude with people who were not his
type. His highest effort was to be reliable, and I have indeed
known barely a man of higher reliability and punctuality.
An enthusiastic mountaineer, Buchner knew well how important a sovereign circumspection is for the success of plans for the
future. So his respect of the exposed administrators of state authority, at
that time still officially addressed as “Excellency” was quite limited.
An example for this is the following little story reported by his friend
Carl Harries (1917):
From 1904 onwards, Buchner’s name is found on very many
lists of appointment, especially after Lossen’s retirement from
Königsberg in 1904.
Buchner was invited by phone for a conference about his
appointment to Königsberg by Excellency Althoff, then
59
B OHLEY AND FRÖHLICH
holder of power in the Ministry of Culture. Buchner had
urgent commitments, and stated that he could only come if
he would be dealt with immediately. Althoff then promised
to meet him in no more than ten minutes. He went there, was
directed to the well-known anteroom, and waited. An hour
went by, then an hour and a half, and Althoff did not appear.
Finally the door opened, Buchner was sitting with the watch
in hand, pointed to the watch face and asked: “Are these ten
minutes?”, to which Excellency Althoff crossly replied: “So
you do not want to be appointed to Königsberg”, slammed the
door shut and walked out on Buchner. This experience he
told me freshly few hours afterwards.
We can well suppose that today also Eduard Buchner would not retreat
into resigned or even servile silence when it becomes necessary to
protest openly against serious mistakes of the administrators of state
authority.
REFERENCES
B UCHNER , E. (1897) “Alkoholische Gährung ohne Hefezellen” Ber.
Dt. Chem. Ges. 30, 117–124; 1110–1113
B UCHNER , E. (1898) “Ueber zellenfreie Gährung” Ber. Dt. Chem. Ges.
31, 568–574
B U C H N E R , E. and RAPP , R. (1898) “Alkoholische Gährung ohne
Hefezellen” Ber. Dt. Chem. Ges. 31, 209–217
HARRIES, C. (1917) “Eduard Buchner” Ber. Dt. Chem. Ges. 50, 1842–1876
V O N MA N N A S E I N , M. (1897) “Zur Frage von der alkoholischen
Gährung ohne lebende Hefezellen” Ber. Dt. Chem. Ges. 3 0 ,
3061–3062
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