What is an alcoholic fermentation?

 

We have come a long way to understand alcoholic fermentation.

As early as the second millennium BC, the Paleo-Babylonians and perhaps others people (Egyptians, Indians) before them had identified and named a beige and greasy material collected in two ways. Either by recovering the beer dregs from the bottom of the jars, or by collecting the beige supernatant from the scum at the time of the tumultuous fermentation of beer. Once dried in the sun on a porous surface (wall, pottery shard, plant fibres, skins, etc.), the precious substance was used to revive the next brew or to make the bread leavening.

For thousands of years, man has been observing fermentation and making leaven. But before the year 1680, nobody had ever seen a yeast!

 

 

1st act

The first act of this adventure is the observation of living beings. In 1680, a Dutchman saw animalcules in beer with his own eyes and drew them. Antoni van Leeuwenhoek (1632-1723) lives in Delft, Holland. He writes: "Droplets floating in a clear substance; each was made up of six separate 'globules', which are the same size and shape as the globules of our blood" (letter to Thomas Gale, 14 June 1680).

Deux modèles de globules de levures de bière.Two "globules" of brewer's yeast observed by Van Leeuwenhoek. Drawing in sanguine pencil. Letter of 14 June 1680. After Ph. Boutibonnes, Antoni Van Leeuwenhoek 1632-1723. L'exercice du regard. Belin 1994.

 

Van Leeuwenhoek has just observed the multiplication of Saccharomyces by budding. He also notes the presence of bubbles which " come up in abundance from the lower part of the beer and settle on the surface "[1]. His mother Grietge Jacobstr van den Berch came from a family of brewers, as did his first wife Barbara de Mey. He was able to obtain samples of beer in the middle of fermentation, observed with one of his monocular microscopes he had built. With 2 or 3 lenses, it could magnify up to 300 times. A resolution of about 1.4 µm enabled him to draw precisely a few yeasts of 10 to 50 microns.

For more than 30 years, Leeuwenhoek's microscopy has been observing all this tiny world swarming around him. As a remarkable optician, preparer, observer and designer, he describes cross sections of seeds, certainly barley or malt with their germs :

« But the most remarkable point is this: just as the animal, in the womb, is fed by a cord which consists of many vessels, and allows it to grow, I believe that the grains that I have examined also possesses an identical cord; the cord is, in one case or another, either very short or very long - it exceeds then the size of the whole seed » (letter of 13th July 1685). This 'cord' is the plumule of malted grain (among other examples of germinating seeds described by Leeuvenhoek).

Jan Verkolje - Antonie van Leeuwenhoek

 

« We will see Him (the Creator) has not only placed in all grains a young plant in reduction, but also a material close to the flour, in which the plant is housed and which serves as a primary food; but that it is also various seeds in which there is no such nutrients and which are filled with the various parts of the plant itself » (letter of 13th june 1687. Boutibonnes 1994,134). Leeuwenhoek fuels a debate that will span the 18th-19th centuries between the defenders of "spontaneous generation" - the living is born only from the living -, and the partisans of animate or inanimate mechanics - matter follows only the laws of physics and chemistry [2].

In 1697, Georg Ernst Stahl, chemist and physicist at the Prussian court published his Zymotechnica Fundamentalis (Foundations of Fermentation Techniques). He advocates a study of fermentation by means of chemistry, an approach free of the alchemical conceptions of the time. Fermentation is a recombination of inanimate material elements that chemistry can analyse. However, Stahl only discusses the principles, without providing any quantitative analysis. The scientific approach to fermentation, made up of described and controlled experiments, with verifiable measurements, came into being a century later with the rationalists of the 18th century and the birth of chemistry.

 

 

 2rd act

Lavoisier 1774, Opuscules physiques et chimiques

 

Chemistry. Antoine Laurent de Lavoisier (1743-1794) explains how the fermentation of beer or wine transforms the elements but preserves the matter. It is based on Priestley's work published in 1771 on "l'air fixe"[3], i.e. carbon dioxide. Joseph Priestley (1733-1804) conducted his carbon dioxide experiments at the Meadow Lane Brewery in Leeds. The brewery is an inexhaustible source of CO2 collected in the fermentation vats and has long been known to brewers as a dangerous, asphyxiating gas. In 1789, Lavoisier generalised the results obtained with alcoholic fermentation in his Traité Elémentaire de Chimie (Elementary Treatise on Chemistry):

« This operation is one of the most striking and extraordinary of all those that chemistry presents to us, and we have to examine where the carbonic acid gas that is released comes from, where the flammable spirit that is formed comes from, and how a soft body, a vegetable oxide, can be transformed into two so different substances, one of which is combustible, the other eminently incombustible. We see that, in order to arrive at the solution of these two questions, it was necessary first of all to have a good knowledge of the analysis and nature of the body likely to ferment, and of the products of fermentation; for nothing is created, neither in the operations of art nor in those of nature, and one can state as a principle that in every operation there is an equal quantity of matter before and after the operation; that the quality and quantity of the principles [ingredients] are the same, and that there are only changes, only modifications, and so on. » (Chapitre XIII: About the decomposition of plants oxides by the vinous fermentation, 101. Trans. Beer-Studies).

 

This major statement is preceded by a description of the main chemical transformations of alcoholic fermentation[4].

In 1810, Louis-Joseph Gay-Lussac (1778-1850) definitively established that ethanol and CO2 are the main products of the decomposition of sugar by alcoholic fermentation. He formalized the experimental techniques and proposed a method of quantitative analysis, pushing away definitively the magico-interpretative approaches of his contemporaries. The chemical equation glucose + water => ethanol + CO2 is now established.

The scientists of the beginning of the 19th century hold two cards in their hands: microscopic budding beings found in beer and the chemical equation of alcoholic fermentation. But neither the zoo-botanists (biology has not yet been born) nor the chemists are yet able to explain the secrets of the alcoholic fermentation. The brewing techniques will come to their rescue.

 

 

3rd act

Theodor Schwann

 

Three scientists published independently in the same year 1837 the description of the yeasts during alcoholic fermentation observed under the microscope: Cagniard-Latour in France, Theodor Schwann and Friedrich Traugot Kützing in Germany. Their scientific works have nothing in common with the righteous but not systematic observations made a century and a half earlier by Leeuwenhoek.

Unfortunately, they will be derided by the great figures of organic chemistry at the time: the Germans Friedrich Wöhler (1800-1882) and Justus von Liebig (1803-1873), and the Swedish Jöns Jacob Berzelius (1779-1848). The study of yeast activity is temporarily abandoned.

 

 

 4th act

Pasteur Brevet n° 91941 du 28 juin 1871Pasteur's patent n° 91941 of June 28th 1871. Pasteur's Patent n° 91941 dated June 28 1871. Application of an invention patent of fifteen years for a method of making beer. Filed June 28, 1871 at 3 o'clock. I hereby declare taking a patent of fifteen years for a new way of brewing beer which consists essentially in the fermentation sheltered from any contact with the air ... Louis Pasteur, member of the Institute, residing in Paris, 45 rue d'Ulm. June 26, 1871.

 

Louis Pasteur (1822-1895) offers a first synthesis of his work. With the help of Biot, he establishes the causal link between the yeast (organism) and the chemical transformation. To do this, he isolated Saccharomyces from all the microorganisms that can be found in sweet juices. Pasteur did not discover the yeasts, studied and observed long before him. He demonstrated that alcoholic fermentation and all other fermentations result from the activity of microscopic organisms[5].

 

With this scientific outcome, Pasteur rushes to rescue the large breweries[6] that are struggling to protect their brews from secondary fermentations (lactic, acetic, butyric), from contamination of bacteria (pungent beer, sulfur beer, stringy beer) or fungi (mycodermes). He proposes to heat (sterilization) anything that comes into contact with the wort after boiling, and remove it from the natural air (contamined) during the alcoholic fermentation (Etudes sur la bière 1876). Louis Pasteur submits to Paris 4 patents, 3 ot them related to the improved production of beer, and more directly to the protection of the wort during fermentation. Pasteur will also file his method and a patent to the U.S. Patent Office, an evidence of his large industrial views.

 

However, one question remains unanswered. How does the yeast work ?

Like a living organism or a chemical compound ?

It is still the brewery that will contribute to solving this crucial scientific problem. The alcoholic fermentation of beer provides the basis for laboratory experiments. Brewer's yeast becomes both an industrial and scientific issue. It is also because Hansen succeeded in isolating and cultivating pure strains of yeast, in the laboratories of the Danish Carlsberg brewery, that Buchner was able to demonstrate in Bavaria that these yeasts act by means of an enzyme.

 

 

5th act

Emil Christian Hansen (1842-1909) knew how to isolate pure strains of yeast, cultivate them and preserve their technical characteristics as they multiplied. He worked in Denmark and between 1876 and 1909 he headed the physiology department of the Carlsberg Brewery laboratories, working closely with some Bavarian and Viennese brewers to promote the so-called bottom-fermentation beers. The pure strains isolated by Hansen are bottom-fermenting yeasts.

Emil Christian Hansen 

In 1883 he described a strain of bottom-fermenting yeast called Saccharomyces carlsbergensis. As a young man, he worked in Paris with Pasteur who never stopped supporting this work. A renowned mycologist, he published in 1896 "Practical studies in fermentation; being contributions to the life history of micro-organisms".

Emil Christian Hansen in the laboratory of the Carlsberg brewery. In the background, the device he has developed to grow pure strains of yeast, in the hands of his assistant..

 

The note adressed by Louis Pasteur in 1886 is worth quoting almost verbatim, because it sums up the situation of the European brewery in the late 19th century. The technological mutations are coming about against a backdrop of fierce commercial competition between the German, Austro-Hungarian, Danish, English, Belgian, Dutch and French brewing industries.

« The wort used to make beer, being carried at the start of production to its boiling point is deprived thereby of any microbe disease. These are introduced soon by the vessels, and especially by the yeast that makes the wort fermenting after cooling. Making a yeast deprived of any ferment of disease germs is therefore required for a brewer concerned about the quality of its products and their conservation. But these are not the only enemies that leaven brings in beer.
Everyone knows that there are two kinds of commercial beers, the said bottom-fermented beer and said top-fermented beer; the first specific to German manufacturing; the second to the British manufacturing. The qualities of these two types of beers correspond to two different yeasts, which are referred to as the bottom yeast and the top yeast. However, if these ferments are only used in breweries, they are not the only ones able to ferment sugars into alcohol, and with these yeasts one could brew beers different from the two commercial beers spoken above. Their tastes would be peculiar, and their consumption will probably be rejected. In my "Etudes sur la bière", I pointed out the existence of some of these yeasts.

Here, we touch upon the noticeable progress brought by Mr. Hansen into the brewery in recent years. The alcoholic yeast in question, which we call wild in order to distinguish them from the top and bottom yeasts proper to good beers, are often mixed in the leavens of the breweries with these latter yeasts whose quality is sought. Hansen was the first to understood very well that the yeast of the consumed beers should be pure, not only in relation to the microbes, ferments of actual diseases, but also that they should be deprived of the wild yeast cells.

Today, in all microbiology laboratories, we have safe and various methods to isolate in pure state a particular microbe, such and such a cell. Hansen had only to put into practice these methods, and it is an honor for my lab that he has addressed first those that are applied here. These preparations of pure yeasts have already render many services to the brewery in various countries, because it is easily understood that given a pure cell of yeast, its propagation in the worts is easy and plentiful. I should add that Hansen's long and patient studies were made under the patronage and inspiration of one of the most prominent brewers in Europe, Mr. Jacobsen, in Copenhagen. Mr. Jacobsen is among those great industrialists who best understood the necessary alliance of science and industry. Many universities would envy the beautiful lab for chemistry he has founded. » (December 1886 [7])

 

We read between the lines the expelling the "wild yeasts" out of the brewery given as a industrial purpose. Laboratories and industrial breweries want to let enter only top or bottom yeasts inside the brewing vats. This anathema will sign in the 20th century the death sentence in Europe for numerous local beers tradional, renamed "beers from uncontrolled or spontaneous fermentation". In industry jargon, this means unwanted beers.

Hansen's research and techniques do not allow us to understand how yeast works during alcoholic fermentation. Nevertheless, they are part of the overall solution that will come from Germany soon.

 

6th act

6th and last act. In 1896, Eduard Buchner (1860-1917) succeeded in starting the fermentation of a beer wort with an extract of crushed brewer's yeast. He demonstrates that the integrity of the yeast cells is not necessary for alcoholic fermentation to take place. The zymase - as the chemical compound extracted from the yeast is then called - acts outside the living cell. This discovery was the birth of biochemistry. It will lead to the identification of enzymes, their separation and purification throughout the 20th century. The loop closes on the malt diastases identified by Payen and Persoz fifty years earlier. Pasteur's "vitalist" positions are sharply refuted.

Eduard Buchner

 

Buchner's procedures for grinding yeast are described in his memoir "Alcoholic fermentation without Yeast Cells" published in 1897[8]. He succeeded where others have failed before him (Moritz Traube, Berthelot, Liebig, Hoppe-Seyler, and perhaps Pasteur). Eduard Buchner's chance, on an experimental level, was Munich, where a few German breweries adopted the bottom fermentation and its specific yeasts.

The Munich brewers adopt in 1886 the pure bottom-fermenting yeast proposed by Hansen. This evolution is driven by the WSB [9] :

  • ­1876 : foundation of the Wissenschaftiche Station für Brauerei (WSB) in Munich, headed by Aubry.
  • ­1884 : Aubry learns in Copenhagen, with Hansen personally, the techniques to select and propagate the bottom-fermenting yeast, called pure (a single strain).
  • ­1885 : beginning of the "pure bottom yeast" culture at the WSB in Munich.
  • ­1885 : providing and testing of the WSB yeasts in Munich, at the Spaten brewery led by Gabriel Sedlmayr.
  • ­1886 : generalisation to other Munich breweries, and final adoption of the culture method of brewing yeasts.
  • ­ The neighbouring brewers in Bohemia (today's Czech Republic) are following the same path.

 

In contrast to top-fermenting yeasts with a thick cell wall, a "bottom yeast" has a thin wall which allows also the coenzyme to be extracted from the zymase, without which the zymase is inactive. Without bottom-fermenting yeasts, Buchner's laboratory experiment would probably have failed in 1896.

The Munich brewers used at that time the kieselguhr to filter their beers very finely, where other breweries in Europe were using sieves, cotton, fibres, filters, etc. Kieselguhr cold-pressed filtration was the trademark of Bavarian and Bohemian breweries, which export a pale, fresh, water-crystalline beer crowned with an immaculate white froth. Kieselguhr is a fine, chemically neutral powder of pulverised diatoms (fossil shells). It has enabled Buchner to crush yeast cells without changing the pH value. Finally, he used a hydraulic press developed by his brother Hans to extract the contents of the bacteria cells, a tool that Eduard Buchner used with his bottom-fermenting yeasts. His experimental success depended on advanced brewing techniques developed by his contemporaries in Munich and Copenhagen.

The industrial brewery is the biggest beneficiary of these scientific advances, and with it the entire food industry still in gestation. On the other hand, the brewery has helped and inspired many research. From malt enzymes to pure yeasts, brewing illustrates the fruitful coupling between science and applied techniques.

The old building of the Carlsberg Laboratory in Valby, Copenhagen, Denmark in 2005

 

The Carlsberg Brewery's laboratories also funded the research of Johan Kjeldahl and of Søren Sørensen. The Danish chemist Kjedal has developed a method for measuring the nitrogen content in organic matter. It makes it possible to determine the amount of protein present in the malt used to make beer, which must contain little protein compared to starch. Between 1876 and 1900, Kjedal was head of the chemistry department of the Carlsberg laboratory, which was founded in Copenhagen in 1875, and worked with his colleague Emil Hansen in the physiology department. The chemist Søren Sørensen introduced the concept of Hydrogen Potential, the pH that measures the acidity of a solution. The concentration of hydrogen ions conditions the activity of proteins, especially that of enzymes, which is so important in all brewing operations. Sørensen headed the Carlsberg Laboratories in Copenhagen between 1901 and 1938, succeeding Kjedal who died in 1900. At the dawn of the 20th century, European industrial brewers have an unprecedented arsenal of scientific knowledge and technology at their disposal to master and soon direct all the biochemical transformations that contribute to the brewing of a certain type of standard beer. Lager beer will soon flood the entire planet.

The industrial brewery is the biggest beneficiary of these scientific advances, and with it the entire food industry still in gestation. On the other hand, the brewery has helped and inspired many research. From malt enzymes to pure yeasts, brewing illustrates the fruitful coupling between science and applied techniques.

 

*  *  *  *  *

In conclusion, without the new technical arsenal and the economic context of the Munich brewery, based on Hansen's methods for selecting bottom-fermenting yeasts, Buchner would have missed his experiments. One might wonder whether Pasteur himself had not tried them, without success, a few years earlier. In Germany, Moritz Traube (1826-1894) had suggested as early as 1858 (Theorie der Fermentwirkungen), again in 1879, such experiments[10]. In France, the Pasteur-Berthelot controversy of 1878 had pushed Pasteur into his entrenchment, shaken by Claude Bernard's scientific position in favour of "soluble ferments", i.e. the purely chemical explanation of alcoholic fermentation.

Fermentation is the result of chemical activity. The mysteries of the alcoholic fermentation seem to be solved. Not completely! While scientists are analysing these mechanisms, a discreet monk is working with his peas. During the 20th century, genetics discovered DNA, the biochemistry of amino acids and proteins. Enzymes are proteins produced by living organisms. No living, no proteins, no fermentation. Back to the initial question.

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[1] Boutibonnes Philippe 1994, Van Leeuwenhoek, l'exercice du regard. Belin, p. 70.
A synthetic article about the yeast and the brewery in 18è-19è centuries : Lietz Peter 2003, Die Bedeutung der Bierhefe in Verlauf der Entwicklung der Bierherstellung. Gesellschaft für die Geschichte und Bibliographie des Brauwesens, Jahrbuch 2003, 79:127.

[2] Ruestow Edward 1984, Leeuwenhoek and the campaign against spontaneous generation, Journal of the History of Biology 17 (2) : 225-248, p. 226.

[3] " Mr. Priestley first examines fixed air itself, the one produced by the spirituous fermentation, or any effervescence. The breweries have offered him a simple and easy way to get a large amount of this air in almost a perfect state of purity; there is constantly a layer nine inches thick above the tanks where the beer is fermenting, and as it is continually renewed by the one provided by the beer, it is not mixed in this thick with the air from the vicinity. This air, according to the experiments of Mr. Black, is heavier than air, and it is probably for this reason it remains in some way attached to the surface of the beer, without being separated; it is also because of its excess of weight that someone can carry it from one room to another in an open jar, provided that the opening is facing up. In the first moments, the fixed air does meddle but little with the air from the atmosphere." (Lavoisier 1774, Opuscules physiques et chimiques, p. 112-113. Translation Beer-Studies)
Oeuvres de Lavoisier. Tome premier. Opuscules physiques et chimiques

[4] "Everyone knows how wine, cider, mead are make, and in general all fermented drinks. Juices are pressed from grapes and apples; the latter is diluted with water, and the liquor is put in large vats, and stored in a place where the temperature is at least 10 degrees of Reaumur's thermometer. Soon, it is excited by a rapid movement of fermentation, many air bubbles are coming up on the surface, and when the fermentation is at its highest peak, the amount of these bubbles is so large, the amount of emerging gas is so large, that we believe the liquor is on a fiery inferno that excites a violent boil. The gas that emerges is of carbonic acid, and when it is collected with care, it is perfectly pure and free of the mixture of any other species of air or gas. The juice of grapes, soft and sweet before, has been changed during this operation into a vinous liquor, which contains no more sugar when the fermentation is complete, and can be converted by distillation into a liquor flammable, which is known in the trade and the arts under the name spirit-of-wine. Being a result of the fermentation of any sweet material enough diluted with water, we feel that it would be against the principles of our nomenclature to name the liquor spirit-of-wine, or spirit-of-cider, or rather spirit-of-fermented-sugar. So, we were forced to adopt a more general name, and 'alcohol' that comes from the Arabs seemed to us to fulfill our own purpose." (Lavoisier 1789, TRAITÉ ÉLÉMENTAIRE DE CHIMIE, Chapitre XIII). Translation Beer-Studies.
Oeuvres de Lavoisier. Tome premier. Traité élémentaire de chimie.

[5] In 1787, Adamo Fabbroni had been the first to argue that the fermentation of the wine is produced by a living substance in the wort (Ragionamento sull'arte di far vino, Florence).

[6] Breweries Champigneulles in Lorraine and Brewery Velten in Marseille.

[7] Bulletin de la Société d'Encouragement pour l'Industrie nationale, 4ème série, Déc. 1886, pp. 601-603. Between 1878 and 1888, Hansen published his research reports in the Danish Meddelelser fra Carlsberg-Laboratoriet, udgivne ved Laboratoriets Bestyrelse, with summaries in French who seem intended to Pasteur's teams.

[8] Cornish-Bowden Athel 1997, New Beer in an Old Bottle: Eduard Buchner and the Growth of Biochemical Knowledge. http://bip.cnrs-mrs.fr/bip10/buchner.htm

[9] Ceccatti John Simmons 2001, Science in the Brewery: pure yeast culture and the transformation of brewing practices in Germany at the end of the 19th century. PhD University of Chicago, 91-95.

[10] Long before Buchner, Traube has isolated an enzyme from potato able to bluing the guaiacum, an experimental evidence for the effective chemical action of enzymes after their extraction from the cells of potato.

30/11/2020  Christian Berger