FERMENTATION. Fermentation seen in the making of bread, in wine making and brewing is the classical example of the change brought about in materials containing starch or sugar, by the action of yeast. In the case of wine making, brewing and the making of bread, the fundamental chemical changes are the same, for starch must first be transformed into sugar before it can be fermented in the accepted sense of the word, that is to say, before it can undergo the decomposition into alcohol and car bon dioxide which is the essence of alcoholic fermentation. The yeast decomposing the sugar gives rise to carbon dioxide gas which first saturates the fluid or fills the interstices of the solid. The excess of gas escapes, causing a frothing and bubbling of the liquor, but in most fermented materials some of the carbonic acid gas remains and is partly responsible for the exhilarating and pleasing properties of the fluid. The rising of dough is due to the pressure generated by carbon dioxide gas, and this action is ob served in an accelerated manner when the bread is first put into the oven. The rate at which the dough rises or is fermented de pends upon several factors such as the temperature, and the amount of salt present, so that by varying such factors the baker is able to hold the process under control.
Yeast is now known to be a living material, and the spores or germs of yeast are to be found everywhere adherent to the par ticles of dust in the air. Consequently any sugary material exposed to the air quickly becomes fermented. Prior to the discovery of such germs in the atmosphere the ferment or leaven was supposed either to have been produced spontaneously, generated as it were, within the material itself by some occult force, or else to have been derived from pre-existing yeast from time immemorial. This latter view was nearer the truth, for it conceived of yeast as something akin to life. Indeed when questioned as to the origin of the Kephir ferment, which is analogous to yeast, Mohammedans in the Cau casus to this day will declare that the first grains of Kephir were put there by Allah. And is not, in the New Testament, the com parison of the Kingdom of Heaven, made at one time to a seed, and at another time to leaven? It would be fallacious, therefore, to imagine because of the materialistic tendencies of the alchemists, that humanity had never had any conception of yeast as a living thing, or of fer mentation as a process akin to life.
The alchemists, however, mixed up all sorts of phenomena, on the strength of superficial analogies, and applied the name fermen tation to any change accompanied by an evolution of gas, as, for example, the action of acids on chalk or soda, or the formation of a froth or scum on metals when calcined. The philosopher's stone was supposed to set up a fermentation in the base metals and give rise to the noble metals. In short, fermentation was any sort of boiling or bubbling set up in a cauldron or crucible (Latin Fer vere, to boil).
Pliny tells us that wine was liable to become vinegar during a voyage, but that such wines as withstood the voyage were actually more mature than they would have been in the same time on land. He points out the necessity of leaving a space between jars of wine in a cellar, to prevent "contagion." He gives also an interest ing description of the preparation of a medicinal wine called (3ios meaning life. Pliny praises this wine, as justly reputed to cure many diseases. It should be prepared, he says, by gathering the grapes in the sunshine which follows rain, turning the grapes daily, twice or thrice before crushing, and subsequently maturing the wine in the sun. In the absence of rain, the indications were to expose the grapes still longer to the sun, presumably to destroy the impurities which the rain would have washed away.
Belon writes that wines of Crete were always brought to the boil prior to exportation.
Columelle, apparently the most reliable of the early writers on this subject, indicates how wine may be prevented from alteration by a preliminary heating of the grape juice (must), and the importance of adding water from a well in the same district as that of the vineyard to replace the water lost by evaporation. This writer recognizes also that the best wines are such as do not require any treatment, for, as he says, that which can please without any resort to artifice is superior to all.
Ideas of Fermentation at the Beginning of the 19th Cen tury.—Such ancient practices, however, hardly found any echo in the ideas of the 18th and early 19th century. Vitalism was in disrepute, the microscope had not been perfected, and its revela tions were but little credited; chemistry had just made rapid strides to the front, and organic chemistry, through the masterful work especially of Liebig and Wohler, had become established as the sure way to a study of physiology, medicine and agriculture. Any attempt to introduce vague and unknown causes, as for ex ample a possible influence of microscopic forms of life in the process of fermentation, was considered retrograde and branded as vitalistic.
It was thought that the classical way to treat the subject of fermentation was as Lavoisier had treated it, namely to study the chemical changes, but to leave the question of the nature of the ferment strictly alone. A theory due to Georg Stahl (169 7) was advocated by Berzelius (1843) and Liebig about the same time. Liebig's idea was much the same as that of Stahl, namely that a ferment was an albuminoid substance in the act of decomposing, and hence in a condition of internal or molecular vibration, and that this vibration could be communicated to other materials, caus ing them thus to ferment. Berzelius' idea was less committal. For him it sufficed that the presence of the ferment caused the de composition to be set up in the material and he did not specify anything as to the internal condition of the ferment itself. Ber zelius introduced the term catalytic action, to express the idea that the ferment did not really take a part in the changes in any chemical sense, but acted by contact.
Liebig had certainly strong evidence that certain changes analo gous to fermentation could occur in the absence of life. Bitter almonds crushed with a little water underwent a change, which led to the decomposition of a sugar compound (glucoside), with the liberation of sugar, prussic acid, and an aromatic principle which gives to the almonds a most characteristic odour. This de composition did not occur if the almonds were boiled with water for a few minutes. The change was therefore brought about ap parently by some agency very analogous to a ferment which was also destroyed by heat. Liebig and Wohler showed, however, that destroying the life in the almonds did not prevent the action of this ferment-like principle, for they could precipitate the active substance by means of concentrated alcohol, and isolate it as a white amorphous powder still possessing the ability to decompose the glucoside.
And another very remarkable fact tended to strengthen the then current views that fermentation could be explained without invok ing the idea of the intervention of life. This was the discovery made by Edmund Davy (182o) that finely divided platinum, sometimes called spongy platinum or platinum black, could by its mere contact with alcohol or alcohol vapour produce acetic acid, which was known to be the acid produced from wine in the ordinary process of vinegar fermentation. The platinum black, in contact with alcohol, became spontaneously red hot, and remained glowing as long as alcohol vapour was present.
The various above-mentioned considerations, however, repre sented rather isolated pieces of information than any definite knowledge as to the nature of fermentation, and it was by no means sure that there was any real connection, for example, be tween the soluble ferment which Liebig obtained from almonds, and the classical process of alcoholic fermentation, which, after all, in view of its vast practical importance in industry, was really the type of fermentation by which all others were to be judged.
When the facts of the biologist were denied by the chemist, there was clearly no prospect of agreement or decision unless someone could come forward who might be authorized to speak in the name of both sciences. Only the rarest genius could at such a time have occupied such a position. The genius was found in Louis Pasteur. Pasteur opened the eyes of mankind to a new world of living things, the world of microscopic life. Such micro scopic beings, he proved, could live upon mineral matter, using for body building and as a source of energy the simplest forms of carbon compounds such as alcohol, acetic acid or sugar, while ammonia sufficed as a source of nitrogen. Each type of fermenta tion was shown to be correlated with the life of some special microscopic organism, multiplying with incredible velocity. Fer mentation, putrefaction and the slow process of combustion by which dead organic matter is resolved into mineral matter, at that time designated spontaneous combustion, were all shown to be caused by the agency of microbes, and to cease when the life was destroyed. Pasteur foresaw disease as the outcome of a failure of plant or animal to resist the attacks of inimical forms of micro scopic life ever present in the dust of the atmosphere, in soil, water or as a contamination of food.
It would be impossible to enter here into the great variety of fermentations which Pasteur studied, and it will be better to con fine our attention to one case, namely alcoholic fermentation set up by yeast, for this will serve as an example for all other types.
The contribution made by Pasteur to our knowledge of alcoholic fermentation, can, however, be better appraised if we first resume what was known of the matter before he began. This can be done briefly, for it does not amount to much.
Facts Known About Alcoholic Fermentation Prior to the Work of Pasteur.—Of facts definitely established before Pasteur came upon the scene, we have the following. Lavoisier (I 793) regarded alcoholic fermentation as a chemical change, in which the yeast was a mere agent which, in a quantitative study of the changes, could be ignored. He showed that the amount of alcohol and carbon dioxide gas produced in the course of the fermentation weighed the same as the sugar used at the beginning of the experiment, and he used this result to announce his far reaching generalization, that nothing is ever lost or destroyed, but that matter only undergoes change of form. Actually there were errors in Lavoisier's experimental work, but the deductions were correct, and were soon supported on theoretical grounds by Gay Lussac. This investigator also showed that oxygen was necessary for the commencement of the fermentation. He placed grapes in a closed tube over mercury, and introduced various gases before crushing the grape. No fermentation followed unless air or oxy gen was allowed to enter the tube. The elementary composition of yeast had also been determined by Dumas (1828), and the gen eral analysis of main constituents had been made by Payen (1839), and Mitscherlich (1835), who gives the composition of the ash of beer yeast, and also by the careful work of Schloss berger (1844) who distinguishes the composition of top and bot tom yeast.
Colin (1825) had found that the best medium for the "growth" of yeast was an extract by hot water of the yeast itself, and the albuminous nature of yeast was generally recognized, though the origin of the albuminous matter was not understood, the general view being that it must have arisen by the decomposition of pre existing animal or vegetable matter.
Thenard (1803) emphasized the importance of not confusing alcoholic fermentation with other processes, and taught that yeast was the cause of the fermentation. He likewise took up an independent position with regard to the fate of the yeast during fermentation. Liebig taught that during fermentation the yeast underwent decomposition (this was, of course, essential to his theory) , and that the evidence of the decomposition was the for mation of ammonia in the solution, for all albuminous substances were known to yield ammonia on decomposition. Thenard, how ever, was in doubts as to whether ammonia was formed, as he himself did not find it in appreciable amount. The yeast likewise did not appear always to lose weight during the fermentation, though sometimes it appeared to do so.
The fact should also be mentioned that traces of products other than alcohol and carbon dioxide had been 'detected in the fer mented fluid and were supposed to be derived from sugar, in par ticular, acetic acid and lactic acid. The amounts produced were so small, however, that it could not be considered proved that these materials truly had been derived from the sugar. They might have ariseli from the nitrogenous matter present in the solution.
Pasteur's Studies in Alcoholic Fermentation.—Pasteur admitted that the idea of the living cause of fermentation was a preconceived one with him, but it was strengthened towards a conviction by the consideration that the amyl alcohol produced as a by-product in fermentation was different from amyl alcohol produced synthetically in the laboratory, the latter was optically, and hence structurally symmetric, the former asymmetric. He then discovered the fact that racemic ammonium tartrate which is itself optically inactive, is decomposed by a mould such as Penicil ium glaucvm, in such a way that the solution develops optical properties. The observation instrument or polarimeter lets through light vibrating in one plane only, and may be set by cross ing the prisms, so that no light vibrating in this plane can come through. The solution examined at the beginning of the experi ment under these conditions appears in complete darkness, but as the fermentation progresses light appears, for it is now trans mitted through the solution in another plane, owing to the develop ment in the solution of an optically asymmetric substance. What actually has happened in this case is that one of the two optically opposite tartrates which are constituents of the racemic tartrate has been consumed or fermented by the fungus, leaving the other therefore exhibiting its optically asymmetric property. The re markable fact, therefore, that in the process of fermentation one type of structure was selected and the other rejected, indicated to Pasteur that the relation between the ferment and the sub stance which it fermented was of the most intimate kind, and con vinced him that nothing but life could be responsible for such a preferential treatment of molecules. This led to the view that each type of fermentation must be correlated with a specific organism, and Pasteur set to work to find the organisms.
In rapid succession he discovered the organisms responsible for the formation of lactic acid, butyric acid and acetic acid, as well as certain other organisms which, like the butyric acid organism, he found could act in the complete absence of air, which led to the distinction of aerobic and anaerobic forms of life. The main issue, however, was that of the part played by yeast in the alco holic fermentation. Few people took the attitude of Liebig of denying absolutely that yeast was alive, but many denied that the life of the yeast and the process of fermentation were connected in any essential way, while others stated that the yeast acted only after it was dead, in common with any other albuminous matter, as was postulated by the theory of Stahl, modified by Liebig.
Pasteur showed that Liebig's theory of fermentation set up by decomposing albuminous matter was utterly untenable. Liebig cited the alleged evidence that ammonia was produced by the de composition of the yeast. Pasteur demonstrated that no ammonia was formed during alcoholic fermentation, but on the contrary a large amount was absorbed by the yeast and synthesized into its protoplasm. Pasteur found he could actually grow yeast on a purely artificial medium made of various salts, with ammonia as a source of nitrogen, and with sugar as a source of carbon, and he offered to show Liebig a kilogram of pure white yeast produced exclusively out of these materials from an amount of yeast origi nally as large as a pin's head. The Academy offered to defray the cost of Liebig's visit to Paris. But Liebig was obstinate and re fused the offer. The production of a kilogram of pure yeast out of sugar and salts and ammonia was masterful evidence and left very little of Liebig's theory of fermentation set up by decom position.
Pasteur next concerned himself with the origin of yeasts and traced them to the atmospheric dust. He showed that all the germs or spores of these microscopic organisms causing fermentation, putrefaction and analogous changes, existed in the air, in amount varying with the place, the altitude, the temperature and the sea son, and by ingeniously filtering the air through guncotton, and dissolving the guncotton in ether, he isolated the germs from the air, and was able to examine them under the microscope and prove by experiment their respective properties.
Following up the classical experiment of Gay-Lussac which appeared to demonstrate that fermentation could not take place without air, Pasteur was led to recognize that oxygen was needed for the germination of the yeast spores, but that for the process of reproduction by budding it was not essential, and a close study of the behaviour of yeast under varying conditions of aeration re vealed the remarkable fact that yeast could at one time behave as an aerobe and at another time as an anaerobe. In the presence of air the growth was very vigorous, much more so than in the absence of air, but on the other hand fermentation as measured by the production of alcohol was much in abeyance, while con versely, when the yeast was deprived of air, growth was not so abundant, but fermentation very much more vigorous. Here, thought Pasteur, is the long-sought secret of fermentation, the yeast, and in fact, any organism deprived of the free oxygen of the atmosphere, will react with substances such as sugar dissolved in the medium, in the effort to obtain from them their oxygen of combination, and in proportion as an organism can succeed in obtaining oxygen in this way it will be capable of setting up fer mentation. All tissues of plants or animals might thus produce alcoholic fermentation for a brief fraction of time, and some by a gradual development of this faculty would become ferments, either facultative or permanent anaerobes.
It may be thought that Pasteur went too far in thus denoting fermentation Vie sans air, yet as A. J. Brown (1892) pointed out, exception could not be taken to the view if into the words with which Pasteur summarises his theory, we read the meaning that that part of the life of the yeast which is most closely corre lated with the phenomenon of alcoholic fermentation can take place in the absence of air, or to be precise, oxygen.
One final point in regard to the chemistry of alcoholic fermenta tion should be mentioned by way of definitely recording the fact that Pasteur regarded the change set up in sugar by yeast as more complicated than had been stated by Lavoisier. Pasteur was at pains repeatedly to point out that accompanying the main change of sugar to alcohol and carbon dioxide there was always a forma tion in definite proportion, amounting to about 2% of the sugar fermented, of two substances, glycerine and succinic acid. These were formed even when sugar was the only source of carbon, and must therefore have been derived from the sugar. The constant production of these substances, albeit in small amount, revealed to Pasteur the essential complexity of the fermentative act, and later progress has substantiated fully his view.
Their logical sequence is such an instructive feature of Pasteur's researches, that it would be wrong not to point out how the re searches on fermentation led to the investigation of disease in wine and vinegar, and how from the conception of epidemics of yeast and other benign organisms, Pasteur was led to the concep tion of the cause of epidemic disease in plants, in animals and man.
Fermentation has, in common parlance, come to have two mean ings ; on the one hand it refers to alcoholic fermentation, which on account of its great industrial importance, overshadows all other types of fermentation; and, on the other hand, it refers to a large variety of changes which occur in connection with animal and plant life, and which are especially of interest to the physiologist, the microbiologist, the pathologist and the physician. Academi cally speaking, however, these phenomena all come under one head. Ambiguity will be avoided if we use the word fermentation to describe the whole process set up by the living cells, the growth and multiplication of the cells, accompanied by fermentation, and the formation of the enzymes within the cells, as well as the de composition set up in the medium by their agency either in, around or outside the cells, while we reserve the word enzyme action to describe the individual fermentative acts, when dis associated from the living organism, or considered, even for purposes of discussion, as acting independently of the life of the cell. Thus, fermentation implies co-ordinated enzyme action, though any one or more enzyme actions may not amount to fermentation.
The chief advance in the chemistry of alcoholic fermentation, in recent years, has been made by Harden (1905) who showed that prior to its decomposition into alcohol and carbon dioxide, the molecule of sugar combines with phosphoric acid. In other words, that there is a synthesis prior to the decomposition. The work of C. Neuberg (1918) should also be mentioned, for he has succeeded in diverting the normal course of fermentation by yeast, into other channels, with the production, for example, of glycerine and acetaldehyde, the latter having been already de tected and recognized as of importance in connection with the theory of fermentation.
Yeasts, bacteria and moulds are now being utilized on a com mercial scale for various types of fermentation leading to the production of useful substances, as, for example, hydrogen, ace tone, glycerine, as well as to obviate the use of acids in the digestion of cellulose or the "saccharification" of starch.
The technical biological side of fermentation, especially as re gards its application in brewing and wine making, owes a very great deal to the work of Hansen, for it was he who applied first to the industry the methods of J. Lister and R. Koch, in order to obtain pure yeasts. For Hansen found that not only did bac teria lead to contamination in the brewery, but certain varieties of yeasts (wild yeasts) were also harmful, and it was therefore necessary to isolate pure yeasts, and begin the fermentation with these. The method has had wide application, and has revolu tionized both brewing and wine making. In wine making, for example, it is now possible by a proper choice of yeast, to modify at will the bouquet of the wine.