What is an alcoholic fermentation?

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!

1^st 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).

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).

« 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 18^th-19^th 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 18^th century and the birth of chemistry.

 2^rd act

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 19^th 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.

3^rd act

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. "The yeast of beer is a mass of small globular bodies  liable to reproduce, consequently organised, and not an inert or purely chemical substance, as was supposed; these bodies seem to belong to the vegetable kingdom and regenerate in two different ways. The yeast of beer can be born and develop in certain circumstances, with great promptitude, even in carbonic acid, as in the brewers' vat." (Cagniard-Latour  1837)

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.

 4^th act

Pasteur'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.

In 1854, Pasteur was appointed Dean of the newly created Lille Faculty of Sciences. His stay there marked a turning point in his career. He began his work on fermentations in relation to the industrial sector, particularly Louis Emmanuel Bigo-Tilloy, a sugar and alcohol manufacturer. He studied the flaws observed during alcoholic fermentation and demonstrated that fermentation was due to the presence of a micro-organism, the birth of microbiology. After his publication at the Lille Society of Sciences, Agriculture and Arts on lactic fermentation (3 August 1857), Pasteur was appointed Administrator and Director of Scientific Studies at the Ecole Normale de Paris. He left Lille in October 1857.

5^th 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.

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 19^th 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 20^th 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.

6^th act

6^th 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 20^th century. The loop closes on the malt diastases identified by Payen and Persoz fifty years earlier. Pasteur's "vitalist" positions are sharply refuted.

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 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 20^th 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 20^th 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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