A prize-winning discovery of 1896:   Buchner provides evidence of cell-free fermentation (From: Peter Bohley and Kai-Uwe Fröhlich (1997) "A prize-winning discovery of 1896," pp. 51–60 in New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge (ed. A. Cornish-Bowden), Universitat de València, Valencia, Spain.

 

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. After some argument the factual necessity of chemical instruction 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.

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 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 sought-after 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. Medwedew from Odessa, and we may also note the name of Otto Dimroth, later famous as a chemist.

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 succeeded up to now.

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 vitalist 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 brings sugar into fermentation.

 

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 yeast and 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).

 

This dispute also was finally decided in favour of Buchner’s enzyme theory, and now after all the way was cleared for modern 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 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 "point-like 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, glucose phosphate 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 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.

 

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 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 far-reaching 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 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

BUCHNER, E. (1897) "Alkoholische Gährung ohne Hefezellen" Ber. Dt. Chem. Ges. 30, 117–124; 1110–1113

BUCHNER, E. (1898) "Ueber zellenfreie Gährung" Ber. Dt. Chem. Ges. 31, 568–574

BUCHNER, 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

VON MANNASEÏN, M. (1897) "Zur Frage von der alkoholischen Gährung ohne lebende Hefezellen" Ber. Dt. Chem. Ges. 30, 3061–3062

 

 

 

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Last update: 26 September 2002

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