The original French article appears in the Appendix, pages 265-267

1857,  Louis Pasteur
 

MY WORK ON THE PROPERTIES OF THE amyl alcohols and on the very remarkable crystallographic properties of their derivatives has led me to a study of the process of fermentation. Later I will have the honour to present to the Academy observations which show an unexpected relationship between fermentation and the optically active properties of organic molecules found m nature.
The necessary materials for the preparation and production of lactic acid are well known to chemists. It is known that it is only necessary to take a solution of sugar and add chalk, which keeps the medium neutral, a nitrogenous material, such as casein, gluten, animal membranes, etc., in order to have the sugar transformed into lactic acid. But the explanation of this phenomenon is quite obscure, since the way in which the decomposable nitrogenous material acts is completely ignored. Its weight does not change significantly. It does not putrefy. Although it becomes modified and is continually in a marked state of change, it would be difficult to speak of what its composition is.
Careful studies up to the present have not revealed the development of organised beings during the fermentation process. Those observers who have recognised such beings have always established at the same time that they were accidental and harmful to the fermentation process.
The facts appear therefore to be very favourable for the ideas of M. Liebig. In his eyes, the ferment is a substance which is highly alterable which decomposes and in so doing induces the fermentation because of the alteration which it experiences itself, communicating this agitation to the molecular group of the fermentable material and in this way bringing about its decomposition. According to M. Liebig, this is the principal cause of all fermentations and the origin of the majority of contagious diseases. Each day his opinion receives more favour....
I propose to establish in the first part of this work that, in the same way that there exists an alcoholic ferment, the yeast of beer, which is always found wherever sugar is decomposed into alcohol and carbon dioxide, there also exists a particular ferment, the lactic yeast, which is always present when sugar becomes converted into lactic acid. Furthermore, the decomposable nitrogenous material which is able to bring about the conversion of sugar into this acid is used as a convenient nutrient for the development of this ferment.
It is possible to observe in ordinary lactic acid fermentations, on top of the sediment of chalk and nitrogenous material, a grey substance which occurs at the surface of this deposit. Under microscopic examination it can be barely distinguished from the casein, disintegrating gluten, etc. so that nothing indicates that it may be a special material, nor that it has arisen during the fermentation. Nevertheless, it is this substance which plays the principal role in the fermentation. I will shortly reveal the method for its isolation and preparation in a state of purity.
I have extracted from beer yeast its soluble material by boiling it several times with l 5 to 20 times its weight of water. This extract is filtered carefully. In this are dissolved 50 grams of sugar per litre, and some chalk is added. Then it is seeded with a trace of the grey material which I mentioned above, and a good lactic acid fermentation is obtained of the usual sort. On the next day the fermentation is vigorous and regular. The liquid which had been perfectly clear at the beginning becomes turbid, the chalk gradually disappears, and at the same time a deposit is produced which increases continually and progressively at the same rate that the chalk disappears. In addition, all of the characteristics and symptoms of the well known lactic acid fermentation are observed. In this experiment it is possible to replace the yeast water with a decoction of any decomposable nitrogenous material, whether fresh or decomposed. Let us now see the nature of this substance which is correlated with all of the phenomena that are included under the words lactic acid fermentations. Its appearance is similar to that of the beer yeast when it is studied en masse and squeezed or pressed. Under the microscope it is seen to form tiny globules or small objects which are very short, isolated or in groups of irregular masses. These globules are much smaller than those of beer yeast and move actively by Brownian movement. If washed with a large amount of water by decantation, then diluted into a solution of pure sugar, they immediately begin to make acid, but quite slowly, since acid inhibits significantly their action on sugar. If chalk is added so that the medium is maintained at neutrality, the conversion of sugar is considerably accelerated, and even though only a small amount of material is acting, in less than an hour the production of gas is observed and the liquid contains large amounts of calcium lactate and butyrate. Only a small amount of this yeast is needed to convert a large amount of sugar. This fermentation is preferably carried out in the absence of air, since it is inhibited by plants or by parasitic infusoria.
Therefore the lactic fermentation, like the ordinary alcoholic fermentation, is always correlated with the production of a nitrogenous material which has all the properties of an organised body of the mycodermal type, and is probably closely related to the yeast of beer. But the difficulties of the subject are only half solved. There are many complications. Lactic acid is indeed the principal product of the fermentation which has been given its names but it is far from the only product Butyric acid, alcohol, mannitol and a viscous material are always found accompanying the lactic acid. The proportions of these materials are highly variable. The circumstances with mannitol are especially mysterious. The proportions which are formed of this substance are subject to large variations. In addition, M. Berthelot has shown that if sugar is replaced by mannitol in the lactic fermentation, mannitol is fermented to alcohol, lactic acid, and butyric acid. How is one to explain the formation of mannitol in the fermentation from sugar, when it should be destroyed at the same rate that it is produced? Let us examine carefully the chemical properties of this new yeast. I have stated that if it is washed well and placed in a pure sugar solution, it acidifies the liquid over a period of time. The conversion of sugar becomes gradually slower under these conditions, since the liquid becomes quite acidic. The liquid can be analysed successfully only after the acid is neutralised with chalk and the excess sugar is destroyed by beer yeast. One then finds, if the liquid is evaporated, variable proportions of mannitol and a viscous material. However washed lactic yeast brings about the transformation of sugar into various products including always mannitol, only if the liquid is allowed to become quickly acid. If the same experiment is repeated with the precaution that an amount of chalk is added which is sufficient to continually neutralise the liquid, then neither the gum nor the mannitol are formed, or, perhaps more exactly, are quickly transformed further.
I reported above that M. Berthelot had shown that when mannitol is substituted for sugar in the lactic fermentation, this material is fermented. But it is simple to show that in this case, the fermentation of mannitol occurs when the lactic yeast develops and brings about its production. If a solution of pure mannitol is mixed with powdered chalk and fresh washed lactic yeast, within an hour gas appears and the chemical transformation of mannitol begins. Carbon dioxide and hydrogen are released, and the liquid contains alcohol, lactic acid and butyric acid, all productions of the fermentation of mannitol.
Concerning butyric acid, experiments have shown that the lactic yeast acts directly upon calcium lactate and converts it into calcium carbonate and calcium butyrate. But sugar is acted upon first, and if it is present in the liquid, the yeast prefers to ferment it to lactic acid.
In a later communication I will have the honour of presenting to the Academy some general ideas and new methods of experimentation from work on other fermentations.
 

Comments by Thomas Brock
This paper announces the entry of Pasteur into the field of fermentation, and represents the beginning of the science of microbiology. Previously Pasteur had worked in crystallography, and had shown that optical isomers of an organic compound differed in crystal structure. He became interested in optical isomers and made many studies. Through these he formulated the idea that optical isomerism existed through the agency of living organisms. His studies on the optical isomers of amyl alcohol led him to the study of fermentation. His first reported studies were on the lactic acid fermentation, as seen above. As he mentions, the doctrine of Liebig was well established by this time, the earlier works of Schwann and Cagniard-Latour (see pages 16 and 20) having been forgotten. Pasteur entered into a battle with Liebig which lasted for a number of years, but which was essentially ended when Pasteur published his paper on alcoholic fermentation (see page 31).
However in this, his earlier work, he discusses lactic acid fermentation, announcing that it is brought about by a living organism. He describes crude microscopical observations of this organism, and manages to separate it from the fermentation broth by washing. He is able to show that this organism can grow in a medium containing yeast extract, sugar, and chalk, and that in this medium it produces lactic acid. These results, only described briefly here, were sufficient to convince him that the organism caused the fermentation.
As he himself comments, there are a number of complications in the natural lactic fermentation. He was not dealing with a pure culture. In fact, the very concept of a pure culture was foreign to him, an d it was only later, through more detailed studies on fermentation that it became obvious that many of the complex changes occurring were not due to the action of a single organism. This is obvious in the present work and there would be no point in attempting to explain the variable appearance of mannitol, butyric acid, and gum. Pasteur himself later solved this problem well enough (see page 39).
In his publications, Pasteur followed the French habit, still prevalent today, of writing many short papers, with very few pieces of actual data in each paper. In this way the essential features of a process were revealed to the readers only gradually over a period of months or years, leading to many misconceptions and misinterpretations. The number of papers Pasteur published in total runs to over six volumes, but many of these are repetitions of earlier papers, with a few additions. The papers of Pasteur that are included in the present volume will give the reader a brief idea of some of the areas that he worked in, although whole areas of his work are not included because they did not seem to lend themselves well to the purpose of the present volume.