BOHLEY AND FRÖHLICH

Reprinted from New Beer in an Old Bottle: 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

Peter Bohley and Kai-Uwe Fröhlich

The discovery of cell-free fermentation by the Eduard Buchner (1860-1917) is now regarded as one of the great landmarks in the development of . 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 Fig. 1. Eduard Buchner’s letter of appointment as Professor of Analytical and had recently become vacant by granting it to one of the lecturers of Pharmaceutical at the University of Tübingen. (Source: Universitäts- philosophy. Fortunately even the rectorate confirmed that a professor- archiv Tübingen, Catalogue No. 126/76) ship of analytical and pharmaceutical chemistry was urgently needed, to assure a reliable and regular presentation of these subjects in the biological course of events, which would never be completely (gesicherte regelmäßige Vertretung dieser Fächer). After some argument the understood… This hypothesis of the vitalists might have dramatically factual necessity of chemical instruction (sachlichen Bedürfnisse des inhibited the progress of research into the processes of if it had not chemischen Unterrichts) in the Faculty of Natural Sciences prevailed, been possible to falsify it. The vitalists were totally convinced, for and Professor Eduard Buchner from Kiel was finally appointed (Fig. example, that only living cells could produce the much sought- 1) as a capable representative of these subjects (tüchtigen Vertreter für after from , together with the that escapes diese Fächer). as bubbles. Separation of this process of fermentation from the living As early as the summer semester of 1896, Buchner taught ana- cells seemed impossible, and had indeed never been achieved. On the lytical chemistry to ten students in Tübingen, and in the winter other hand the heretical idea existed that the “vital forces” were no semester of 1896–1897 he was teaching pharmaceutical chemistry to more than molecular forces, an idea that had been advocated by 14 students and the chemistry of fermentation to 27. He was later Buchner in his lectures on the chemistry of fermentation. His last appointed a professor in in 1898, but he had made his trail- lectures in Tübingen on this subject were given in the summer blazing discovery shortly after he had taken up his office in Tü- semester of 1898, and attracted students from far and wide: among the bingen. In view of the earlier anxieties of the Faculty of Philosophy, it 32 students (Fig. 2) were W. A. Osborne from Belfast and A. was fitting that it concerned a problem that was of the greatest interest Medwedev from Odessa, and we may also note the name of Otto to philosophers: the miracle of life could not be explained by the laws Dimroth, later famous as a chemist. of physics or chemistry, because a vis vitalis was at work in the 52 51 BOHLEY AND FRÖHLICH A PRIZE-WINNING DISCOVERY OF 1896

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 Fig. 2. The list of participants in Buchner’s last course at Tübingen, in the Fig. 1. Eduard Buchner’s letter of appointment as Professor of Analytical and had recently become vacant by granting it to one of the lecturers of summer semester of 1898. (Source: Universitätsarchiv Tübingen, Catalogue No. Pharmaceutical chemistry at the University of Tübingen. (Source: Universitäts- 51/66) philosophy. Fortunately even the rectorate confirmed that a professor- archiv Tübingen, Catalogue No. 126/76) ship of analytical and pharmaceutical chemistry was urgently needed, to assure a reliable and regular presentation of these subjects in the biological course of events, which would never be completely During a visit to his older brother Hans in in October (gesicherte regelmäßige Vertretung dieser Fächer). After some argument the understood… This hypothesis of the vitalists might have dramatically 1896, Eduard Buchner had managed to prove that it is possible to factual necessity of chemical instruction (sachlichen Bedürfnisse des inhibited the progress of research into the processes of life if it had not produce a cell-free extract capable of fermenting sugar. On 9th chemischen Unterrichts) in the Faculty of Natural Sciences prevailed, been possible to falsify it. The vitalists were totally convinced, for January 1897, he sent a preliminary message (Buchner, 1897) from and Professor Eduard Buchner from Kiel was finally appointed (Fig. example, that only living yeast cells could produce the much sought- Tübingen to Berlin, which arrived on 11th January; in it he describes 1) as a capable representative of these subjects (tüchtigen Vertreter für after alcohol from sugar, together with the carbon dioxide that escapes the experiments of October 1986 that had made the miracle possible: diese Fächer). as bubbles. Separation of this process of fermentation from the living As early as the summer semester of 1896, Buchner taught ana- cells seemed impossible, and had indeed never been achieved. On the It is first of all proved that the initiation of the process of lytical chemistry to ten students in Tübingen, and in the winter other hand the heretical idea existed that the “vital forces” were no fermentation does not require such a complicated apparatus as semester of 1896–1897 he was teaching pharmaceutical chemistry to more than molecular forces, an idea that had been advocated by the yeast cell. Rather a substance in solution is to be 14 students and the chemistry of fermentation to 27. He was later Buchner in his lectures on the chemistry of fermentation. His last considered as the carrier of the fermentative effect of the appointed a professor in Berlin in 1898, but he had made his trail- lectures in Tübingen on this subject were given in the summer extract, without doubt a ; it shall be named . blazing discovery shortly after he had taken up his office in Tü- semester of 1898, and attracted students from far and wide: among the The idea that a special protein derived from the yeast bingen. In view of the earlier anxieties of the Faculty of Philosophy, it 32 students (Fig. 2) were W. A. Osborne from Belfast and A. cells causes fermentation has previously been expressed by was fitting that it concerned a problem that was of the greatest interest Medwedev from Odessa, and we may also note the name of Otto M. Traube in 1858 as the or ferment theory, and this to philosophers: the miracle of life could not be explained by the laws Dimroth, later famous as a chemist. has later been defended especially by F. Hoppe-Seyler. The of physics or chemistry, because a vis vitalis was at work in the separation of such an enzyme from the yeast cells has not 52 51 53 BOHLEY AND FRÖHLICH A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH

1 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 , 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 Fig. 2. The list of participants in Buchner’s last course at Tübingen, in the separate the potential for fermentation from the living yeast Fig. 1. Eduard Buchner’s letter of appointment as Professor of Analytical and summer semester of 1898. (Source: Universitätsarchiv Tübingen, Catalogue No. cell. The purely vitalistic theory therefore remained the un- Pharmaceutical chemistry at the University of Tübingen. (Source: Universitäts- 51/66) conditional winner. archiv Tübingen, Catalogue No. 126/76) We have all been raised in the conceptions of Pasteur. When experimental facts came into my hands that in the biological course of events, which would never be completely During a visit to his older brother Hans in Munich in October appeared to argue in favour of cell-free fermentation, I understood… This hypothesis of the vitalists might have dramatically 1896, Eduard Buchner had managed to prove that it is possible to understandably confronted them with grave doubts. The inhibited the progress of research into the processes of life if it had not produce a cell-free extract capable of fermenting sugar. On 9th publication followed only after experiments repeated two been possible to falsify it. The vitalists were totally convinced, for January 1897, he sent a preliminary message (Buchner, 1897) from months later led to exactly the same results as before. example, that only living yeast cells could produce the much sought- Tübingen to Berlin, which arrived on 11th January; in it he describes Accordingly, it is possible to press cell-free juice from yeast that after alcohol from sugar, together with the carbon dioxide that escapes the experiments of October 1986 that had made the miracle possible: as bubbles. Separation of this process of fermentation from the living cells seemed impossible, and had indeed never been achieved. On the It is first of all proved that the initiation of the process of fermentation does not require such a complicated apparatus as other hand the heretical idea existed that the “vital forces” were no 1 the yeast cell. Rather a substance in solution is to be Zunächst ist bewiesen, dass es zur Einleitung des Gährungsvorganges more than molecular forces, an idea that had been advocated by keines so complicirten Apparates bedarf, wie ihn die Hefezelle vorstellt. Als Buchner in his lectures on the chemistry of fermentation. His last considered as the carrier of the fermentative effect of the Träger der Gährwirkung des Pressaftes ist vielmehr eine gelöste Substanz, lectures in Tübingen on this subject were given in the summer extract, without doubt a protein; it shall be named zymase. zweifelsohne ein Eiweisskörper zu betrachten; derselbe soll als Z y m a s e semester of 1898, and attracted students from far and wide: among the The idea that a special protein derived from the yeast bezeichnet werden. Die Anschauung, dass ein den Hefezellen entstammender, cells causes fermentation has previously been expressed by besonders gearteter Eiweisskörper die Gährung veranlasse, ist als Enzym - oder 32 students (Fig. 2) were W. A. Osborne from Belfast and A. Ferment-Theorie bereits 1858 von M. T r a u b e ausgesprochen und später Medwedev from Odessa, and we may also note the name of Otto M. Traube in 1858 as the enzyme or ferment theory, and this insbesondere von F. Hoppe-Seyler vertheidigt worden. Die Abtrennung eines Dimroth, later famous as a chemist. has later been defended especially by F. Hoppe-Seyler. The derartigen Enzyms von den Hefezellen war aber bisher nicht geglückt. separation of such an enzyme from the yeast cells has not 52 53 54 A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH PRIZE-WINNING DISCOVERY OF 1896 1 A succeeded up to now. 2 In a lecture before the Deutsche chemische Gesellschaft (the German brings sugar into fermentation. Chemical Society) in Berlin on 14th March 1898, Buchner (1898) This discovery was received immediately after its publication with proved his discovery in a series of well prepared experiments, with all both enthusiastic approval and furious contradiction. But soon Buch- details publicly demonstrated and elaborated: ner could state:

The alcoholic fermentation of sugar has frequently raised the The fact of cell-free fermentation is now probably generally interest of scientists. There were numerous theories about the accepted, especially since confirmation has come from achievement of this process until Pasteur’s pioneering works, reports from others. However, a controversy recently beginning in the middle of this century, led to the final emerged again about whether the juice pressed from and proposition that there is no fermentation without organisms. yeast capable of fermentation truly contained a substance Specifically, Pasteur viewed the process as a physiological act corresponding to an enzyme that is to be considered the closely and inseparably tied to the life processes of yeast cells. carrier of fermentation, or whether there were still living Other researchers, however, such as Moritz Traube, Berthelot, fragments of protoplasm (plasma hypothesis).3 Liebig and Hoppe-Seyler, were of the opinion that yeast, in the same manner as it produces a certain chemical substance, an This dispute also was finally decided in favour of Buchner’s enzyme unorganized ferment or enzyme, invertin, likewise produces theory, and now after all the way was cleared for modern a substance that brings about the effects of fermentation. Clear as the theory was, however, experimental proof was lacking, 2 because in spite of many attempts nobody had been able to Die alkoholische Gährung des Zuckers hat schon vielfach das Interesse der Naturforscher erregt. Zahlreiche Theorien über das Zustandekommen dieses Fig. 2. The list of participants in Buchner’s last course at Tübingen, in the separate the potential for fermentation from the living yeast Processes folgten aufeinander, bis die bahnbrechenden Arbeiten Pasteur’s, summer semester of 1898. (Source: Universitätsarchiv Tübingen, Catalogue No. cell. The purely vitalistic theory therefore remained the un- begonnen in der Mitte unseres Jahrhunderts, zur endgültigen Aufstellung des 51/66) conditional winner. Satzes führten: Keine Gährung ohne Organismen. Im Speciellen betrachtete We have all been raised in the conceptions of Pasteur. Pasteur den Vorgang als einen physiologischen Act, eng und untrennbar ver- bunden mit den Lebensvorgängen der Hefezellen. Andere Forscher dagegen, During a visit to his older brother Hans in Munich in October When experimental facts came into my hands that wie Moritz Traube, Berthelot, Liebig und Hoppe-Seyler waren der Ansicht, dass die 1896, Eduard Buchner had managed to prove that it is possible to appeared to argue in favour of cell-free fermentation, I Hefe, wie sie einen bestimmten chemischen Stoff, ein unorganisirtes Ferment produce a cell-free extract capable of fermenting sugar. On 9th understandably confronted them with grave doubts. The oder Enzym, das Invertin, producirt, welches Rohrzucker in Traubenzucker und publication followed only after experiments repeated two Fruchtzucker spaltet, dass sie wohl ähnlich auch eine Substanz erzeuge, der die January 1897, he sent a preliminary message (Buchner, 1897) from Gährwirkung zukommt. Aber, so anschaulich diese Theorie war, es fehlte jeder Tübingen to Berlin, which arrived on 11th January; in it he describes months later led to exactly the same results as before. experimentelle Beweis, denn niemals konnte trotz vieler Versuche eine Tren- the experiments of October 1986 that had made the miracle possible: Accordingly, it is possible to press cell-free juice from yeast that nung 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. It is first of all proved that the initiation of the process of Als mir im October 1896 experimentelle Thatsachen in die Hände fielen, fermentation does not require such a complicated apparatus as die für eine zellenfreie Gährung zu sprechen schienen, da stand ich denselben 1 the yeast cell. Rather a substance in solution is to be Zunächst ist bewiesen, dass es zur Einleitung des Gährungsvorganges daher begreiflicher Weise zunächst sehr zweifelnd gegenüber. Erst nachdem keines so complicirten Apparates bedarf, wie ihn die Hefezelle vorstellt. Als considered as the carrier of the fermentative effect of the zwei Monate später wiederholte Versuche genau zu den früheren Ergebnissen Träger der Gährwirkung des Pressaftes ist vielmehr eine gelöste Substanz, geführt hatten, erfolgte die Veröffentlichung. extract, without doubt a protein; it shall be named zymase. zweifelsohne ein Eiweisskörper zu betrachten; derselbe soll als Z y m a s e Danach ist es möglich, aus Hefe einen zellenfreien Saft auszupressen, welcher Zucker The idea that a special protein derived from the yeast bezeichnet werden. Die Anschauung, dass ein den Hefezellen entstammender, in Gährung versetzt. cells causes fermentation has previously been expressed by besonders gearteter Eiweisskörper die Gährung veranlasse, ist als Enzym - oder 3 Die Thatsache der zellenfreien Gährung wird jetzt, zumal wieder bestäti- Ferment-Theorie bereits 1858 von M. T r a u b e ausgesprochen und später M. Traube in 1858 as the enzyme or ferment theory, and this gende Mittheilungen von anderer Seite vorliegen, wohl allgemein anerkannt. insbesondere von F. Hoppe-Seyler vertheidigt worden. Die Abtrennung eines Dagegen ist von Neuem Streit entstanden, ob in dem gährkräftigen Hefepressaft has later been defended especially by F. Hoppe-Seyler. The derartigen Enzyms von den Hefezellen war aber bisher nicht geglückt. als Träger der Gährwirkung anzusprechen sind eine Enzym-ähnliche Substanz, separation of such an enzyme from the yeast cells has not die Zymase (Enzymtheorie), oder etwa noch lebende Protoplasmastückchen (Plasmahypothese). 53 54 55 BOHLEY AND FRÖHLICH A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH succeeded up to now.1 2 In a lecture before the Deutsche chemische Gesellschaft (the German brings sugar into fermentation. enzymology and with that for a molecular theory for an explanation Chemical Society) in Berlin on 14th March 1898, Buchner (1898) of the events of fermentation. This discovery was received immediately after its publication with proved his discovery in a series of well prepared experiments, with all There remained, however, the objection of Marie von Manassein both enthusiastic approval and furious contradiction. But soon Buch- details publicly demonstrated and elaborated: (1897), who saw Buchner’s work as little more than a confirmation of ner could state: her own:

The alcoholic fermentation of sugar has frequently raised the The fact of cell-free fermentation is now probably generally On 9th April 1871, I presented my work on alcoholic fermen- interest of scientists. There were numerous theories about the accepted, especially since confirmation has come from tation in German. I expressed the main result of my work achievement of this process until Pasteur’s pioneering works, reports from others. However, a controversy recently with the following words: “On the basis of all these experi- beginning in the middle of this century, led to the final emerged again about whether the juice pressed from and ments I consider myself entitled to state that living yeast cells proposition that there is no fermentation without organisms. yeast capable of fermentation truly contained a substance are not necessary for alcoholic fermentation… Consequently, Specifically, Pasteur viewed the process as a physiological act corresponding to an enzyme that is to be considered the I do not consider alcoholic fermentation as a physiological, closely and inseparably tied to the life processes of yeast cells. carrier of fermentation, or whether there were still living but as a chemical process…” Other researchers, however, such as Moritz Traube, Berthelot, fragments of protoplasm (plasma hypothesis).3 Nobody can admire the profound method of Mr. Buchner Liebig and Hoppe-Seyler, were of the opinion that yeast, in the more than I do, which enabled him to separate the alcoholic same manner as it produces a certain chemical substance, an This dispute also was finally decided in favour of Buchner’s enzyme ferment from yeast and to enable fermentation in a simple unorganized ferment or enzyme, invertin, likewise produces theory, and now after all the way was cleared for modern solution of ferment and cane sugar. However, the most a substance that brings about the effects of fermentation. Clear important aspect of the question, namely, that alcoholic as the theory was, however, experimental proof was lacking, fermentation is not, as Pasteur believed he had proved, a 2 because in spite of many attempts nobody had been able to Die alkoholische Gährung des Zuckers hat schon vielfach das Interesse physiological but merely a chemical process, has been found der Naturforscher erregt. Zahlreiche Theorien über das Zustandekommen dieses separate the potential for fermentation from the living yeast Processes folgten aufeinander, bis die bahnbrechenden Arbeiten Pasteur’s, by me more than a quarter of a century ago. cell. The purely vitalistic theory therefore remained the un- begonnen in der Mitte unseres Jahrhunderts, zur endgültigen Aufstellung des In conclusion, I have to remark that some extended conditional winner. Satzes führten: Keine Gährung ohne Organismen. Im Speciellen betrachtete travels, from which I have only recently returned, brought We have all been raised in the conceptions of Pasteur. Pasteur den Vorgang als einen physiologischen Act, eng und untrennbar ver- about the delay in the publication of this notice. bunden mit den Lebensvorgängen der Hefezellen. Andere Forscher dagegen, When experimental facts came into my hands that wie Moritz Traube, Berthelot, Liebig und Hoppe-Seyler waren der Ansicht, dass die St. Petersburg, end of November 1897 appeared to argue in favour of cell-free fermentation, I Hefe, wie sie einen bestimmten chemischen Stoff, ein unorganisirtes Ferment understandably confronted them with grave doubts. The oder Enzym, das Invertin, producirt, welches Rohrzucker in Traubenzucker und publication followed only after experiments repeated two Fruchtzucker spaltet, dass sie wohl ähnlich auch eine Substanz erzeuge, der die Gährwirkung zukommt. Aber, so anschaulich diese Theorie war, es fehlte jeder To this, of course, Buchner had to reply, and he did so quite bluntly months later led to exactly the same results as before. experimentelle Beweis, denn niemals konnte trotz vieler Versuche eine Tren- (Buchner and Rapp, 1898) : Accordingly, it is possible to press cell-free juice from yeast that nung des Gährvermögens von den lebenden Hefezellen erzielt werden. Die rein vitalistische Theorie blieb also unbedingt Siegerin. In the penultimate issue of these reports Mrs. von Wir alle sind in den Anschauungen Pasteur’s aufgewachsen. Als mir im October 1896 experimentelle Thatsachen in die Hände fielen, Manassein claims the discovery that living yeast cells are die für eine zellenfreie Gährung zu sprechen schienen, da stand ich denselben not necessary for alcoholic fermentation, based on an 1 Zunächst ist bewiesen, dass es zur Einleitung des Gährungsvorganges daher begreiflicher Weise zunächst sehr zweifelnd gegenüber. Erst nachdem examination carried out in 1871, which we have known about keines so complicirten Apparates bedarf, wie ihn die Hefezelle vorstellt. Als zwei Monate später wiederholte Versuche genau zu den früheren Ergebnissen for a long time. It is not our fault that under these Träger der Gährwirkung des Pressaftes ist vielmehr eine gelöste Substanz, geführt hatten, erfolgte die Veröffentlichung. zweifelsohne ein Eiweisskörper zu betrachten; derselbe soll als Z y m a s e Danach ist es möglich, aus Hefe einen zellenfreien Saft auszupressen, welcher Zucker circumstances it has to be stated explicitly: this work, bezeichnet werden. Die Anschauung, dass ein den Hefezellen entstammender, in Gährung versetzt. commendable as it was in its time, proves that the author was besonders gearteter Eiweisskörper die Gährung veranlasse, ist als Enzym - oder 3 Die Thatsache der zellenfreien Gährung wird jetzt, zumal wieder bestäti- subjectively convinced of the existence of a fermentative Ferment-Theorie bereits 1858 von M. T r a u b e ausgesprochen und später gende Mittheilungen von anderer Seite vorliegen, wohl allgemein anerkannt. enzyme, as M. Traube (1858) and M. Berthelot (1860) were insbesondere von F. Hoppe-Seyler vertheidigt worden. Die Abtrennung eines Dagegen ist von Neuem Streit entstanden, ob in dem gährkräftigen Hefepressaft derartigen Enzyms von den Hefezellen war aber bisher nicht geglückt. als Träger der Gährwirkung anzusprechen sind eine Enzym-ähnliche Substanz, before her; however, the objective proof of the truth of the die Zymase (Enzymtheorie), oder etwa noch lebende Protoplasmastückchen assumption was missing, as this could hardly have been (Plasmahypothese). provided with the state of knowledge and methodology that 54 55 56 A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH A PRIZE-WINNING DISCOVERY OF 1896

2 brings sugar into fermentation. enzymology and with that for a molecular theory for an explanation existed in those times. The state of affairs results from the of the events of fermentation. following: This discovery was received immediately after its publication with There remained, however, the objection of Marie von Manassein both enthusiastic approval and furious contradiction. But soon Buch- (1897), who saw Buchner’s work as little more than a confirmation of ner could state: 1. The author’s experiments have almost all been her own: performed by incubating dry-heated or boiled pressed yeast The fact of cell-free fermentation is now probably generally On 9th April 1871, I presented my work on alcoholic fermen- with a boiled 10% solution of sugar for 2 to 56 days; it was then accepted, especially since confirmation has come from tation in German. I expressed the main result of my work distilled and the distillate tested for alcohol using the reports from others. However, a controversy recently with the following words: “On the basis of all these experi- extremely sensitive iodoform reaction. Today it has long been emerged again about whether the juice pressed from and ments I consider myself entitled to state that living yeast cells proved that boiling for 10 minutes is not sufficient to sterilize a yeast capable of fermentation truly contained a substance are not necessary for alcoholic fermentation… Consequently, solution of sugar. Indeed, microscopic investigation in corresponding to an enzyme that is to be considered the I do not consider alcoholic fermentation as a physiological, conclusion of the experiments showed the presence of “point- carrier of fermentation, or whether there were still living but as a chemical process…” like shapes and immeasurable grains” which at that time was fragments of protoplasm (plasma hypothesis).3 Nobody can admire the profound method of Mr. Buchner not considered suspicious. Micrococci were obviously un- more than I do, which enabled him to separate the alcoholic known at that time, and it was not yet known that certain This dispute also was finally decided in favour of Buchner’s enzyme ferment from yeast and to enable fermentation in a simple fission fungi ferment sugar and produce alcohol. theory, and now after all the way was cleared for modern solution of ferment and cane sugar. However, the most important aspect of the question, namely, that alcoholic 2. According to the author air-dried yeast, heated up to fermentation is not, as Pasteur believed he had proved, a 308°C for 3 hours 20 minutes, whereby the cells were charred 2 Die alkoholische Gährung des Zuckers hat schon vielfach das Interesse physiological but merely a chemical process, has been found beyond recognition, and likewise yeast boiled for 45 minutes, der Naturforscher erregt. Zahlreiche Theorien über das Zustandekommen dieses by me more than a quarter of a century ago. still had the ability to ferment. Therefore, methodical errors Processes folgten aufeinander, bis die bahnbrechenden Arbeiten Pasteur’s, must have occurred… begonnen in der Mitte unseres Jahrhunderts, zur endgültigen Aufstellung des In conclusion, I have to remark that some extended Satzes führten: Keine Gährung ohne Organismen. Im Speciellen betrachtete travels, from which I have only recently returned, brought At that time, Eduard Buchner could not know that his “zymase” con- Pasteur den Vorgang als einen physiologischen Act, eng und untrennbar ver- about the delay in the publication of this notice. bunden mit den Lebensvorgängen der Hefezellen. Andere Forscher dagegen, sists in fact of a multitude of , whose discovery became wie Moritz Traube, Berthelot, Liebig und Hoppe-Seyler waren der Ansicht, dass die St. Petersburg, end of November 1897 possible only after the beginning of the present century (e.g. alcohol Hefe, wie sie einen bestimmten chemischen Stoff, ein unorganisirtes Ferment dehydrogenase in 1909, pyruvate decarboxylase in 1911, hexokinase oder Enzym, das Invertin, producirt, welches Rohrzucker in Traubenzucker und Fruchtzucker spaltet, dass sie wohl ähnlich auch eine Substanz erzeuge, der die in 1927, glucosephosphate isomerase in 1933, pyruvate kinase in 1934, Gährwirkung zukommt. Aber, so anschaulich diese Theorie war, es fehlte jeder To this, of course, Buchner had to reply, and he did so quite bluntly enolase in 1935, phosphofructokinase in 1936, and aldolase in 1936). experimentelle Beweis, denn niemals konnte trotz vieler Versuche eine Tren- (Buchner and Rapp, 1898) : His lecture from 14th March 1898 was obviously very lively and nung des Gährvermögens von den lebenden Hefezellen erzielt werden. Die rein convincing as well (Buchner, 1898): vitalistische Theorie blieb also unbedingt Siegerin. In the penultimate issue of these reports Mrs. von Wir alle sind in den Anschauungen Pasteur’s aufgewachsen. Manassein claims the discovery that living yeast cells are The procedure to produce the pressed juice, developed with Als mir im October 1896 experimentelle Thatsachen in die Hände fielen, substantial help of Mr. Privatdozent Dr. Martin Hahn in the die für eine zellenfreie Gährung zu sprechen schienen, da stand ich denselben not necessary for alcoholic fermentation, based on an daher begreiflicher Weise zunächst sehr zweifelnd gegenüber. Erst nachdem examination carried out in 1871, which we have known about Institute of Hygiene in Munich, is in short as follows. Fresh zwei Monate später wiederholte Versuche genau zu den früheren Ergebnissen for a long time. It is not our fault that under these Munich bottom-fermenting pressed beer yeast is ground in a geführt hatten, erfolgte die Veröffentlichung. circumstances it has to be stated explicitly: this work, grinding machine; the latter, delivered by Hugershoff in Danach ist es möglich, aus Hefe einen zellenfreien Saft auszupressen, welcher Zucker Leipzig and set to work by a gas motor, consists… of a hori- in Gährung versetzt. commendable as it was in its time, proves that the author was 3 Die Thatsache der zellenfreien Gährung wird jetzt, zumal wieder bestäti- subjectively convinced of the existence of a fermentative zontally rotating porcelain mortar, in which a porcelain gende Mittheilungen von anderer Seite vorliegen, wohl allgemein anerkannt. enzyme, as M. Traube (1858) and M. Berthelot (1860) were piston, weighed down as one likes, constantly moves to and Dagegen ist von Neuem Streit entstanden, ob in dem gährkräftigen Hefepressaft before her; however, the objective proof of the truth of the fro in a straight line… The preparations that have been set up als Träger der Gährwirkung anzusprechen sind eine Enzym-ähnliche Substanz, clearly show the difference in the macroscopic appearance of die Zymase (Enzymtheorie), oder etwa noch lebende Protoplasmastückchen assumption was missing, as this could hardly have been (Plasmahypothese). provided with the state of knowledge and methodology that the yeast in the different states; initially you see fresh

55 56 57 A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH

Munich bottom-fermenting pressed beer yeast, then the same existed in those times. The state of affairs results from the after pressing at 50 atmospheres and mixing with quartz sand following: and kieselguhr as a dry powder, then as a plastic mass after grinding and finally as a rather firm pressed cake at the end 1. The author’s experiments have almost all been of the total procedure. The fresh yeast press juice, as you see it performed by incubating dry-heated or boiled pressed yeast in front of you — this preparation was transferred here from with a boiled 10% solution of sugar for 2 to 56 days; it was then Munich in a large ice bucket at 0°C — is a yellowish liquid, distilled and the distillate tested for alcohol using the almost clear when the light passes through it, otherwise extremely sensitive iodoform reaction. Today it has long been opalizing, with a pleasant smell of yeast; it contains a lot of proved that boiling for 10 minutes is not sufficient to sterilize a dissolved carbon dioxide, which begins to escape on warming solution of sugar. Indeed, microscopic investigation in to 40°C… conclusion of the experiments showed the presence of “point- On pouring a volume (10 ccm) of a 50% solution of cane like shapes and immeasurable grains” which at that time was sugar at 30°C into 10 ccm of fresh press juice in a test tube, a not considered suspicious. Micrococci were obviously un- distinct production of gas starts off about 10 minutes after known at that time, and it was not yet known that certain mixing, which continues for several days at room fission fungi ferment sugar and produce alcohol. temperature (experiment). The gas formed is carbonic acid, as is easy to prove. 4 2. According to the author air-dried yeast, heated up to 308°C for 3 hours 20 minutes, whereby the cells were charred These experiments were the beginning of the discovery of many beyond recognition, and likewise yeast boiled for 45 minutes, thousands of enzymes that act in the cellular as biocata- still had the ability to ferment. Therefore, methodical errors lysts in a way to allow cells, and therefore organisms, to live. It is the must have occurred… multitude of molecular interactions which make the miracle of life

At that time, Eduard Buchner could not know that his “zymase” con- sists in fact of a multitude of enzymes, whose discovery became 4 Das Verfahren zur Herstellung des Pressaftes, unter wesentlicher Beihülfe possible only after the beginning of the present century (e.g. alcohol des Hrn. Privatdocenten Dr. Martin Hahn im Münchener hygienischen Institut dehydrogenase in 1909, pyruvate decarboxylase in 1911, hexokinase ausgebildet, ist kurz folgendes. Frische Münchener untergährige Bierpress- in 1927, glucosephosphate isomerase in 1933, pyruvate kinase in 1934, hefe,... wird... in einer Zerreibungsmaschine zerrieben; die letztere, von Hugershoff in Leipzig geliefert und durch einen Gasmotor in Betrieb gesetzt, enolase in 1935, phosphofructokinase in 1936, and aldolase in 1936). besteht... aus einer horizontal rotirenden Porzellanreibschale, in welcher sich His lecture from 14th March 1898 was obviously very lively and ein beliebig beschwertes Porzellanpistill fortwährend geradlinig hin und her convincing as well (Buchner, 1898): bewegt... Die aufgestellten Präparate zeigen deutlich den Unterschied im makroskopischen Aussehen der Hefe in den verschiedenen Stadien, zunächst The procedure to produce the pressed juice, developed with haben Sie hier frische Münchener untergährige Bierpreßhefe, dann solche substantial help of Mr. Privatdozent Dr. Martin Hahn in the nach Auspressen bei 50 Atmosphären und Mischen mit Quarzsand und Institute of Hygiene in Munich, is in short as follows. Fresh Kieselguhr als trocknes Pulver, hierauf als plastische Masse nach dem Zerreiben und endlich als ziemlich festen Presskuchen am Ende der Gesammtoperation. Munich bottom-fermenting pressed beer yeast is ground in a Der frische Hefepressaft, wie Sie ihn hier vor sich sehen — dieses Präparat grinding machine; the latter, delivered by Hugershoff in wurde in einem grossen Eiskübel bei 0°C von München hierher überführt — Leipzig and set to work by a gas motor, consists… of a hori- stellt eine gelbliche, im durchfallenden Licht fast klare, sonst opalisierende zontally rotating porcelain mortar, in which a porcelain Flüssigkeit von angenehmem Hefegeruch vor; er enthält viel Kohlendioxyd gelöst, welches beim Erwärmen auf 40°C zu entweichen beginnt.... piston, weighed down as one likes, constantly moves to and Beim Eingiessen eines Volumens (10ccm) einer 30°C warmen, 50-pro- fro in a straight line… The preparations that have been set up centigen Rohrzuckerlösung in 10 ccm frischen Pressaft im Reagenzrohr tritt clearly show the difference in the macroscopic appearance of etwa 10 Minuten nach dem Mischen deutliche Gasentwickelung ein, welche bei the yeast in the different states; initially you see fresh Zimmertemperatur einige Tage ... andauert (Versuch).… Das Gas, welches dabei entsteht, ist Kohlensäure, wie leicht zu beweisen.

57 58 A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH

Munich bottom-fermenting pressed beer yeast, then the same possible. These processes of life take place absolutely according to the after pressing at 50 atmospheres and mixing with quartz sand laws of physics and of chemistry, and we all are endebted to Eduard and kieselguhr as a dry powder, then as a plastic mass after Buchner, who was honoured in 1907 with one of the first Nobel prizes grinding and finally as a rather firm pressed cake at the end to be awarded, for a substantial milestone on the way to their en- of the total procedure. The fresh yeast press juice, as you see it lightenment, experimental falsification of the hypothesis that fermen- in front of you — this preparation was transferred here from tation without living cells would not be possible. Munich in a large ice bucket at 0°C — is a yellowish liquid, Eduard Buchner’s friend Carl Harries (1917) wrote as follows in almost clear when the light passes through it, otherwise an obituary: opalizing, with a pleasant smell of yeast; it contains a lot of dissolved carbon dioxide, which begins to escape on warming Buchner had a brilliant intelligence, which could be enjoyed to 40°C… again and again; besides, his line of thought was clear and On pouring a volume (10 ccm) of a 50% solution of cane simple. Usually deliberate in understanding in order to avoid sugar at 30°C into 10 ccm of fresh press juice in a test tube, a jumping to conclusions, he would sometimes draw far- distinct production of gas starts off about 10 minutes after reaching but certain conclusions in a flash. He followed all mixing, which continues for several days at room communications about other scientific subjects, even if they temperature (experiment). The gas formed is carbonic acid, as 4 were far from him, with lively, I might say charming, is easy to prove. sympathy. He did not exhibit the one-sidedness of some university teachers who are only interested in their narrow These experiments were the beginning of the discovery of many subject. In his scientific essays this quality became obvious in thousands of enzymes that act in the cellular metabolism as biocata- the extensive inclusion of outside publications for the lysts in a way to allow cells, and therefore organisms, to live. It is the discussion of his results. However, what especially attracted multitude of molecular interactions which make the miracle of life 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 4 Das Verfahren zur Herstellung des Pressaftes, unter wesentlicher Beihülfe des Hrn. Privatdocenten Dr. Martin Hahn im Münchener hygienischen Institut characteristically Upper-Bavarian reserve with foreigners. ausgebildet, ist kurz folgendes. Frische Münchener untergährige Bierpress- Indeed, he could be quite rude with people who were not his hefe,... wird... in einer Zerreibungsmaschine zerrieben; die letztere, von type. His highest effort was to be reliable, and I have indeed Hugershoff in Leipzig geliefert und durch einen Gasmotor in Betrieb gesetzt, known barely a man of higher reliability and punctuality. 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 An enthusiastic mountaineer, Buchner knew well how import- makroskopischen Aussehen der Hefe in den verschiedenen Stadien, zunächst ant a sovereign circumspection is for the success of plans for the haben Sie hier frische Münchener untergährige Bierpreßhefe, dann solche nach Auspressen bei 50 Atmosphären und Mischen mit Quarzsand und future. So his respect of the exposed administrators of state authority, at Kieselguhr als trocknes Pulver, hierauf als plastische Masse nach dem Zerreiben that time still officially addressed as “Excellency” was quite limited. und endlich als ziemlich festen Presskuchen am Ende der Gesammtoperation. An example for this is the following little story reported by his friend Der frische Hefepressaft, wie Sie ihn hier vor sich sehen — dieses Präparat Carl Harries (1917): 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 From 1904 onwards, Buchner’s name is found on very many gelöst, welches beim Erwärmen auf 40°C zu entweichen beginnt.... lists of appointment, especially after Lossen’s retirement from Beim Eingiessen eines Volumens (10ccm) einer 30°C warmen, 50-pro- centigen Rohrzuckerlösung in 10 ccm frischen Pressaft im Reagenzrohr tritt Königsberg in 1904. etwa 10 Minuten nach dem Mischen deutliche Gasentwickelung ein, welche bei Buchner was invited by phone for a conference about his Zimmertemperatur einige Tage ... andauert (Versuch).… Das Gas, welches dabei appointment to Königsberg by Excellency Althoff, then entsteht, ist Kohlensäure, wie leicht zu beweisen.

59 58 A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH BOHLEY AND FRÖHLICH

Munich bottom-fermenting pressed beer yeast, then the same possible. These processes of life take place absolutely according to the holder of power in the Ministry of Culture. Buchner had after pressing at 50 atmospheres and mixing with quartz sand laws of physics and of chemistry, and we all are endebted to Eduard urgent commitments, and stated that he could only come if and kieselguhr as a dry powder, then as a plastic mass after Buchner, who was honoured in 1907 with one of the first Nobel prizes he would be dealt with immediately. Althoff then promised grinding and finally as a rather firm pressed cake at the end to be awarded, for a substantial milestone on the way to their en- to meet him in no more than ten minutes. He went there, was of the total procedure. The fresh yeast press juice, as you see it lightenment, experimental falsification of the hypothesis that fermen- directed to the well-known anteroom, and waited. An hour in front of you — this preparation was transferred here from tation without living cells would not be possible. went by, then an hour and a half, and Althoff did not appear. Munich in a large ice bucket at 0°C — is a yellowish liquid, Eduard Buchner’s friend Carl Harries (1917) wrote as follows in Finally the door opened, Buchner was sitting with the watch almost clear when the light passes through it, otherwise an obituary: in hand, pointed to the watch face and asked: “Are these ten opalizing, with a pleasant smell of yeast; it contains a lot of minutes?”, to which Excellency Althoff crossly replied: “So dissolved carbon dioxide, which begins to escape on warming Buchner had a brilliant intelligence, which could be enjoyed you do not want to be appointed to Königsberg”, slammed the to 40°C… again and again; besides, his line of thought was clear and door shut and walked out on Buchner. This experience he On pouring a volume (10 ccm) of a 50% solution of cane simple. Usually deliberate in understanding in order to avoid told me freshly few hours afterwards. sugar at 30°C into 10 ccm of fresh press juice in a test tube, a jumping to conclusions, he would sometimes draw far- distinct production of gas starts off about 10 minutes after reaching but certain conclusions in a flash. He followed all We can well suppose that today also Eduard Buchner would not retreat mixing, which continues for several days at room communications about other scientific subjects, even if they into resigned or even servile silence when it becomes necessary to temperature (experiment). The gas formed is carbonic acid, as were far from him, with lively, I might say charming, protest openly against serious mistakes of the administrators of state is easy to prove. 4 sympathy. He did not exhibit the one-sidedness of some authority. university teachers who are only interested in their narrow These experiments were the beginning of the discovery of many subject. In his scientific essays this quality became obvious in REFERENCES thousands of enzymes that act in the cellular metabolism as biocata- the extensive inclusion of outside publications for the lysts in a way to allow cells, and therefore organisms, to live. It is the discussion of his results. However, what especially attracted BUCHNER, E. (1897) “Alkoholische Gährung ohne Hefezellen” Ber. multitude of molecular interactions which make the miracle of life me to him was his honest noble-mindedness, combined with Dt. Chem. Ges. 30, 117–124; 1110–1113 great courage and his true kindness. This last was more BUCHNER, E. (1898) “Ueber zellenfreie Gährung” Ber. Dt. Chem. Ges. difficult for strangers to notice, because he had the 4 Das Verfahren zur Herstellung des Pressaftes, unter wesentlicher Beihülfe 31, 568–574 des Hrn. Privatdocenten Dr. Martin Hahn im Münchener hygienischen Institut characteristically Upper-Bavarian reserve with foreigners. BUCHNER, E. and RAPP, R. (1898) “Alkoholische Gährung ohne ausgebildet, ist kurz folgendes. Frische Münchener untergährige Bierpress- Indeed, he could be quite rude with people who were not his Hefezellen” Ber. Dt. Chem. Ges. 31, 209–217 hefe,... wird... in einer Zerreibungsmaschine zerrieben; die letztere, von type. His highest effort was to be reliable, and I have indeed HARRIES, C. (1917) “Eduard Buchner” Ber. Dt. Chem. Ges. 50, 1842–1876 Hugershoff in Leipzig geliefert und durch einen Gasmotor in Betrieb gesetzt, known barely a man of higher reliability and punctuality. besteht... aus einer horizontal rotirenden Porzellanreibschale, in welcher sich V O N MANNASEIN, M. (1897) “Zur Frage von der alkoholischen ein beliebig beschwertes Porzellanpistill fortwährend geradlinig hin und her Gährung ohne lebende Hefezellen” Ber. Dt. Chem. Ges. 30, bewegt... Die aufgestellten Präparate zeigen deutlich den Unterschied im An enthusiastic mountaineer, Buchner knew well how import- 3061–3062 makroskopischen Aussehen der Hefe in den verschiedenen Stadien, zunächst ant a sovereign circumspection is for the success of plans for the haben Sie hier frische Münchener untergährige Bierpreßhefe, dann solche nach Auspressen bei 50 Atmosphären und Mischen mit Quarzsand und future. So his respect of the exposed administrators of state authority, at Kieselguhr als trocknes Pulver, hierauf als plastische Masse nach dem Zerreiben that time still officially addressed as “Excellency” was quite limited. und endlich als ziemlich festen Presskuchen am Ende der Gesammtoperation. An example for this is the following little story reported by his friend Der frische Hefepressaft, wie Sie ihn hier vor sich sehen — dieses Präparat Carl Harries (1917): 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 From 1904 onwards, Buchner’s name is found on very many gelöst, welches beim Erwärmen auf 40°C zu entweichen beginnt.... lists of appointment, especially after Lossen’s retirement from Beim Eingiessen eines Volumens (10ccm) einer 30°C warmen, 50-pro- centigen Rohrzuckerlösung in 10 ccm frischen Pressaft im Reagenzrohr tritt Königsberg in 1904. etwa 10 Minuten nach dem Mischen deutliche Gasentwickelung ein, welche bei Buchner was invited by phone for a conference about his Zimmertemperatur einige Tage ... andauert (Versuch).… Das Gas, welches dabei appointment to Königsberg by Excellency Althoff, then entsteht, ist Kohlensäure, wie leicht zu beweisen.

59 58 60 A PRIZE-WINNING DISCOVERY OF 1896 BOHLEY AND FRÖHLICH possible. These processes of life take place absolutely according to the holder of power in the Ministry of Culture. Buchner had laws of physics and of chemistry, and we all are endebted to Eduard urgent commitments, and stated that he could only come if Buchner, who was honoured in 1907 with one of the first Nobel prizes he would be dealt with immediately. Althoff then promised to be awarded, for a substantial milestone on the way to their en- to meet him in no more than ten minutes. He went there, was lightenment, experimental falsification of the hypothesis that fermen- directed to the well-known anteroom, and waited. An hour tation without living cells would not be possible. went by, then an hour and a half, and Althoff did not appear. Eduard Buchner’s friend Carl Harries (1917) wrote as follows in Finally the door opened, Buchner was sitting with the watch an obituary: in hand, pointed to the watch face and asked: “Are these ten minutes?”, to which Excellency Althoff crossly replied: “So Buchner had a brilliant intelligence, which could be enjoyed you do not want to be appointed to Königsberg”, slammed the again and again; besides, his line of thought was clear and door shut and walked out on Buchner. This experience he simple. Usually deliberate in understanding in order to avoid told me freshly few hours afterwards. jumping to conclusions, he would sometimes draw far- reaching but certain conclusions in a flash. He followed all We can well suppose that today also Eduard Buchner would not retreat communications about other scientific subjects, even if they into resigned or even servile silence when it becomes necessary to were far from him, with lively, I might say charming, protest openly against serious mistakes of the administrators of state sympathy. He did not exhibit the one-sidedness of some authority. university teachers who are only interested in their narrow subject. In his scientific essays this quality became obvious in REFERENCES the extensive inclusion of outside publications for the discussion of his results. However, what especially attracted BUCHNER, E. (1897) “Alkoholische Gährung ohne Hefezellen” Ber. me to him was his honest noble-mindedness, combined with Dt. Chem. Ges. 30, 117–124; 1110–1113 great courage and his true kindness. This last was more BUCHNER, E. (1898) “Ueber zellenfreie Gährung” Ber. Dt. Chem. Ges. difficult for strangers to notice, because he had the 31, 568–574 characteristically Upper-Bavarian reserve with foreigners. BUCHNER, E. and RAPP, R. (1898) “Alkoholische Gährung ohne Indeed, he could be quite rude with people who were not his Hefezellen” Ber. Dt. Chem. Ges. 31, 209–217 type. His highest effort was to be reliable, and I have indeed HARRIES, C. (1917) “Eduard Buchner” Ber. Dt. Chem. Ges. 50, 1842–1876 known barely a man of higher reliability and punctuality. V O N MANNASEIN, M. (1897) “Zur Frage von der alkoholischen Gährung ohne lebende Hefezellen” Ber. Dt. Chem. Ges. 30, An enthusiastic mountaineer, Buchner knew well how import- 3061–3062 ant 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

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