FERMENTATION. The process of fermentation in the preparation of wine, vinegar, beer and bread was known and practised in prehistoric times. The alchemists used the terms fermentation, digestion and putrefaction indiscriminately; any reaction in which chemical energy was displayed in some form or other - such, for instance, as the effervescence occasioned by the addition of an acid to an alkaline solution - was described as a fermentation (Lat. fervere, to boil); and the idea of the "Philosopher's Stone" setting up a fermentation in the common metals and developing the essence or germ, which should transmute them into silver or gold, further complicated the conception of fermentation. As an outcome of this alchemical doctrine the process of fermentation was supposed to have a purifying and elevating effect on the bodies which had been submitted to its influence. Basil Valentine wrote that when yeast was added to wort "an internal inflammation is communicated to the liquid, so that it raises in itself, and thus the segregation and separation of the feculent from the clear takes place." Johann Becher, in 1669, first found that alcohol was formed during the fermentation of solutions of sugar; he distinguished also between fermentation and putrefaction. In 1697 Georg Stahl admitted that fermentation and putrefaction were analogous processes, but that the former was a particular case of the latter.
The beginning of definite knowledge on the phenomenon of fermentation may be dated from the time of Antony Leeuwenhoek, who in 1680 designed a microscope sufficiently powerful to render yeast cells and bacteria visible; and a description of these organisms, accompanied by diagrams, was sent to the Royal Society of London. This investigator just missed a great discovery, for he did not consider the spherical forms to be living organisms but compared them with starch granules. It was not until 1803 that L. J. Thenard stated that yeast was the cause of fermentation, and held it to be of an animal nature, since it contained nitrogen and yielded ammonia on distillation, nor was it conclusively proved that the yeast cell was the originator of fermentation until the researches of C. Cagniard de la Tour, T. Schwann and F. Blitzing from 1836 to 1839 settled the point. These investigators regarded yeast as a plant, and Meyer gave to the germs the systematic name of "Saccharomyces" (sugar fungus). In 1839-1840 J. von Liebig attacked the doctrine that fermentation was caused by micro-organisms, and enunciated his theory of mechanical decomposition. He held that every fermentation consisted of molecular motion which is transmitted from a substance in a state of chemical motion - that is, of decomposition - to other substances, the elements of which are loosely held together. It is clear from Liebig's publications that he first regarded yeast as a lifeless, albuminoid mass; but, although later he considered they were living cells, he would never admit that fermentation was a physiological process, the chemical aspect being paramount in the mind of this distinguished investigator.
In 1857 Pasteur decisively proved that fermentation was a physiological process, for he showed that the yeast which produced fermentation was no dead mass, as assumed by Liebig, but consisted of living organisms capable of growth and multiplication. His own words are: "The chemical -action of fermentation is essentially a correlative phenomenon of a vital act, beginning and ending with it. I think that there is never any alcoholic fermentation without there being at the same time organization, development and multiplication of globules, or the continued consecutive life of globules already formed." Fermentation, according to Pasteur, was caused by the growth and multiplication of unicellular organisms out of contact with free oxygen, under which circumstance they acquire the power of taking oxygen from chemical compounds in the medium in which they are growing. In other words "fermentation is life without air, or life without oxygen." This theory of fermentation was materially modified in 1892 and 1894 by A. J. Brown, who described experiments which were in disagreement with Pasteur's dictum. A. J. Brown writes: "If for the theory ` life without air ' is substituted the consideration that yeast cells can use oxygen in the manner of ordinary aerobic fungi, and probably do require it for the full completion of their lifehistory, but that the exhibition of their fermentative functions is independent of their environment with regard to free oxygen, it will be found that there is nothing contradictory in Pasteur's experiments to such a hypothesis." Liebig and Pasteur were in agreement on the point that fermentation is intimately connected with the presence of yeast in the fermenting liquid, but their explanations concerning the mechanism of fermentation were quite opposed. According to M. Traube (1858), the active cause of fermentation is due to the action of different enzymes contained in yeast and not to the yeast cell itself. As will be seen later this theory was confirmed by subsequent researches of E. Fischer and E. Buchner.
In 1879 C. Nageli formulated his well-known molecularphysical theory, which supported Liebig's chemical theory on the one hand and Pasteur's physiological hypothesis on the other: "Fermentation is the transference of the condition of motion of the molecules, atomic groups and atoms of the various compounds constituting the living plasma, to the fermenting material, in consequence of which equilibrium in the molecules of the latter is destroyed, the result being their disintegration." He agreed with Pasteur that the presence of living cells is essential to the transformation of sugar into alcohol, but dissented from the view that the process occurs within the cell. This investigator held that the decomposition of the sugar molecules takes place outside the cell wall. In 1894 and 1895, Fischer, in a remarkable series of papers on the influence of molecular structure upon the action of the enzyme, showed that various species of yeast behave very differently towards solutions of sugars. For example, some species hydrolyse came sugar and maltose, and then carry on fermentation at the expense of the simple sugars (hexoses) so formed. Saccharomyces Marxianus will not hydrolyse maltose, but it does attack cane sugar and ferment the products of hydrolysis. Fischer next suggested that enzymes can only hydrolyse those sugars which possess a molecular structure in harmony with their own, or to use his ingenious analogy, "the one may be said to fit into the other as a key fits into a lock." The preference exhibited by yeast cells for sugar molecules is shared by mould fungi and soluble enzymes in their fermentative actions. Thus, Pasteur showed that Penicillium glaucum, when grown in an aqueous solution of ammonium racemate, decomposed the dextro-tartrate, leaving the laevotartrate, and the solution which was originally inactive to polarized light became dextro-rotatory. Fischer found that the enzyme "invertase," which is present in yeast, attacks methyl-d-glucoside but not methyl-l-glucoside.
In 1897 Buchner submitted yeast to great pressure, and isolated a nitrogenous substance, enzymic in character, which he termed "zymase." This body is being continually formed in the yeast cell, and decomposes the sugar which has diffused into the cell. The freshly-expressed yeast juice causes concentrated solutions of cane sugar, glucose, laevulose and maltose to ferment with the production of alcohol and carbon dioxide, but not milk-sugar and mannose. In this respect the plasma behaves in a similar manner towards the sugars as does the living yeast cell. Pasteur found that, when cane sugar was fermented by yeast, 49.4% of carbonic acid and 51.1% of alcohol were produced; with expressed yeast juice cane sugar yields 47% of carbonic acid and 47.7% of alcohol. According to Buchner the fermentative activity of yeast-cell juice is not due to the presence of living yeast cells, or to the action of living yeast protoplasm, but it is caused by a soluble enzyme. A. Macfadyen, G. H. Morris and S. Rowland, in repeating Buchner's experiments, found that zymase possessed properties differing from all other enzymes, thus: dilution with twice its volume of water practically destroys the fermentative power of the yeast juice. These investigators considered that differences of this nature cannot be explained by the theory that it is a soluble enzyme, which brings about the alcoholic fermentation of sugar. The remarkable discoveries of Fischer and Buchner to a great extent confirm Traube's views, and reconcile Liebig's and Pasteur's theories. Although the action of zymase may be regarded as mechanical, the enzyme cannot be produced by any other than living protoplasm.
Pasteur's important researches mark an epoch in the technical aspect of fermentation. His investigations on vinegar-making revolutionized that industry, and he showed how, instead of waiting two or three months for the elaboration of the process, the vinegar could be made in eight or ten days by exposing the vats containing the mixture of wine and vinegar to a temperature of 20 to 25° C., and sowing with a small quantity of the acetic organism. To the study of the life-history of the butyric and acetic organisms we owe the terms "anaerobic" and "aerobic." His researches from 1860 and onwards on the then vexed question of spontaneous generation proved that, in all cases where spontaneous generation appeared to have taken place, some defect or other was in the experiment. Although the direct object of Pasteur was to prove a negative, yet it was on these experiments that sterilization as known to us was developed. It is only necessary to bear in mind the great part played by sterilization in the laboratory, and pasteurization on the fermentation industries and in the preservation of food materials. Pasteur first formulated the idea that bacteria are responsible for the diseases of fermented liquids; the corollary of this was a demand for pure yeast. He recommended that yeast should be purified by cultivating it in a solution of sugar containing tartaric acid, or, in wort containing a small quantity of phenol. It was not recognized that many of the diseases of fermented liquids are occasioned by foreign yeasts; moreover, this process, as was shown later by Hansen, favours the development of foreign yeasts at the expense of the good yeast.
About this time Hansen, who had long been engaged in researches on the biology of the fungi of fermentation, demonstrated that yeast free from bacteria could nevertheless occasion diseases in beer. This discovery was of great importance to the zymo-technical industries, for it showed that bacteria are not the only undesirable organisms which may occur in yeast. Hansen set himself the task of studying the properties of the varieties of yeast, and to do this he had to cultivate each variety in a pure state. Having found that some of the commonest diseases of beer, such as yeast turbidity and the objectionable changes in flavour, were caused not by bacteria but by certain species of yeast, and, further, that different species of good brewery yeast would produce beers of different character, Hansen argued that the pitching yeast should consist only of a single species - namely, that best suited to the brewery in question. These views met with considerable opposition, but in 1890 Professor E. Duclaux stated that the yeast question as regards low fermentation has been solved by Hansen's investigations. He emphasized the opinion that yeast derived from one cell was of no good for top fermentation, and advocated Pasteur's method of purification. But in the course of time, notwithstanding many criticisms and objections, the reform spread from bottom fermentation to top fermentation breweries on the continent and in America. In the United Kingdom the employment of brewery yeasts selected from a single cell has not come into general use; it may probably be accounted for in a great measure by conservatism and the wrong application of Hansen's theories.
Pure Cultivation of Yeasts.-The methods which were first adopted by Hansen for obtaining pure cultures of yeast were similar in principle to one devised by J. Lister for isolating a pure culture of lactic acid bacterium. Lister determined the number of bacteria present in a drop of the liquid under examination by counting, and then diluted this with a sufficient quantity of sterilized water so that each drop of the mixture should contain, on an average, less than one bacterium. A number of flasks containing a nutrient medium were each inoculated with one drop of this mixture; it was found that some remained sterile, and Lister assumed that the remaining flasks each contained a pure culture. This method did not give very certain results, for it could not be guaranteed that the growth in the inoculated flask was necessarily derived from a single bacterium. Hansen counted the number of yeast cells suspended in a drop of liquid diluted with sterilized water. A volume of the diluted yeast was introduced into flasks containing sterilized wort, the degree of dilution being such that only a small proportion of the flasks became infected. The flasks were then well shaken, and the yeast cell or cells settled to the bottom, and gave rise to a separate yeast speck. Only those cultures which contained a single yeast speck were assumed to be pure cultivations. By this method several races of Saccharomycetes and brewery yeasts were isolated and described.
The next important advance was the substitution of solid for liquid media; due originally to Schroter. R. Koch subsequently improved the method. He introduced bacteria into liquid sterile nutrient gelatin. After being well shaken, the liquid was poured into a sterile glass Petrie dish and covered with a moist and sterile bell-jar. It was assumed that each separate speck contained a pure culture. Hansen pointed out that this was by no means the case, for it is more difficult to separate the cells from each other in the gelatin than in the liquid. To obtain an absolutely pure culture with certainty it is necessary, even when the gelatin method is employed, to start from a single cell. To effect this some of the nutrient gelatin containing yeast cells is placed on the under-surface of the cover-glass of the moist chamber. Those cells are accurately marked, the position of which is such that the colonies, to which they give rise, can grow to their full size without coming into contact with other colonies. The growth of the marked cells is kept under observation for three or four days, by which time the colonies will be large enough to be taken out of the chamber and placed in flasks. The contents of the flasks can then be introduced into larger flasks, and finally into an apparatus suitable for making enough yeast for technical purposes. Such, in brief, are the methods devised by that brilliant investigator Hansen; and these methods have not only been the basis on which our modern knowledge of the Saccharomycetes is founded, but are the only means of attack which the present-day observer has at his disposal.
From the foregoing it will be seen that the term fermentation has now a much wider significance than when it was applied to such changes as the decomposition of must or wort with the production of carbon dioxide and alcohol. Fermentation now includes all changes in organic compounds brought about by ferments elaborated in the living animal or vegetable cell. There are two distinct types of fermentation: (1) those brought about by living organisms (organized ferments), and (2) those brought about by non-living or unorganized ferments (enzymes). The first class include such changes as the alcoholic fermentation of sugar solutions, the acetic acid fermentation of alcohol, the lactic acid fermentation of milk sugar, and the putrefaction of animal and vegetable nitrogenous matter. The second class include all changes brought about by the agency of enzymes, such as the action of diastase on starch, invertase on cane sugar, glucase on maltose, &c. The actions are essentially hydrolytic.
The Saccharomycetes belong to that division of the Thallophyta called the Hyphomycetes or Fungi (q.v.). Two great divisions are recognized in the Fungi: (i.) the Pycomycetes or Algal Fungi, which retain a definitely sexual method of reproduction as well as asexual (vegetative) methods, and (ii.) the Mycomycetes, characterized by extremely reduced or very doubtful sexual reproduction. The Mycomycetes may be divided as follows: (A) forms bearing both sporangia and conidia (see Fungi), (B) forms bearing conidia only, e.g. the common mushroom. Division A comprises (a) the true Ascomycetes, of which the moulds Eurotium and Penicillium are examples, and (b) the Hemiasci, which includes the yeasts. The gradual disappearance of the sexual method of reproduction, as we pass upwards in the fungi from the points of their departure from the Algae, is an important fact, the last traces of sexuality apparently disappearing in the ascomycetes.
With certain rare exceptions the Saccharomycetes have three methods of asexual reproduction: I. The most common. - The formation of buds which separate to form new cells. A portion of the nucleus of the parent cell makes its way through the extremely narrow neck into the daughter cell. This method obtains when yeast is vigorously fermenting a saccharine solution.
2. A division by fission followed by Endogenous spore formation, characteristic of the Schizosaccharomycetes. Some species show fermentative power.
3. Endospore formation, the conditions for which are as follows: (1) suitable temperature, (2) presence of air, (3) presence of moisture, (4) young and vigorous cells, (5) a food supply in the case of one species at least is necessary, and is in no case prejudicial. In some cases a sexual act would appear to precede spore formation. In most cases four spores are formed within the cell by free formation. These may readily be seen after appropriate staining.
In some of the true Ascomycetes, such as Penicillium glaucum, the conidia if grown in saccharine solutions, which they have the power of fermenting, develop single cell yeast-like forms, and do not - at any rate for a time - produce again the characteristic branching mycelium. This is known as the T orula condition. It is supposed by some that Saccharomyces is a very degraded Ascomycete, in which the Torula condition has become fixed.
The yeast plant and its allies are saprophytes and form no chlorophyll. Their extreme reduction in form and loss of sexuality may be correlated with the saprophytic habit, the proteids and other organic material required for the growth and reproduction being appropriated ready synthesized, the plant having entirely lost the power of forming them for itself, as evidenced by the absence of chlorophyll. The beer yeast S. cerevisiae, is never found wild, but the wine yeasts occur abundantly in the soil of vineyards, and so are always present on the fruit, ready to ferment the expressed juice.
Lavoisier was the first investigator to study fermentation from a quantitative standpoint. He determined the percentages of carbon, hydrogen and oxygen in the sugar and in the products of fermentation, and concluded that sugar in fermenting breaks up into alcohol, carbonic acid and acetic acid. The elementary composition of sugar and alcohol was fixed in 1815 by analyses made by GayLussac, Thenard and de Saussure. The first-mentioned chemist proposed the following formula to represent the change which takes place when sugar is fermented: - Ca-11206 = 2CO 2 2C2H60.
Sugar. Carbon dioxide. Alcohol.
This formula substantially holds good to the present day, although a number of definite bodies other than carbon dioxide and alcohol occur in small and varying quantities, according to the conditions of the fermentation and the medium fermented. Prominent among these are glycerin and succinic acid. In this connexion Pasteur showed that ioo parts of cane sugar on inversion gave 105.4 parts of invert sugar, which, when fermented, yielded 51.1 parts alcohol, 49.4 carbonic acid, o. 7 succinic acid, 3.2 glycerin and 1. o unestimated. A. Bechamp and E. Duclaux found that acetic acid is formed in small quantities during fermentation; aldehyde has also been detected. The higher alcohols such as propyl, isobutyl, amyl, capryl, oenanthyl and caproyl, have been identified; and the amount of these vary according to the different conditions of the fermentation. A number of esters are also produced. The characteristic flavour and odour of wines and spirits is dependent on the proportion of higher alcohols, aldehydes and esters which may be produced.
Certain yeasts exercise a reducing action, forming sulphuretted hydrogen, when sulphur is present. The "stinking fermentations" occasionally experienced in breweries probably arise from this, the free sulphur being derived from the hops. Other yeasts are stated to form sulphurous acid in must and wort. Another fact of considerable technical importance is, that the various races of yeast show considerable differences in the amount and proportion of fermentation products other than ethyl alcohol and carbonic acid which they produce. From these remarks it will be clear that to employ the most suitable kind of yeast for a given alcoholic fermentation is of fundamental importance in certain industries. It is beyond the scope of the present article to attempt to describe the different forms of budding fungi (Saccharomyces), mould fungi and bacteria which are capable of fermenting sugar solutions. Thus, six species isolated by Hansen, Saccharomyces cerevisiae, S. Pasteurianus I., 1 II., III., and S. ellipsoideus, contained invertase and maltase, and can invert and subsequently ferment cane sugar and maltose. S. exiguus and S. Ludwigii contain only invertase and not maltase, and therefore ferment cane sugar but not maltose. S. apiculatus (a common wine yeast) contains neither of these enzymes, and only ferments solutions of glucose or laevulose.
Previously to Hansen's work the only way of differentiating I Hansen found there were three species of spore-bearing Saccharomycetes and that these could be subdivided into varieties. Thus, S. cerevisiae I., S. cerevisiae II., S. Pasteurianus I., &c.
yeasts was by studying morphological differences with the aid of the microscope. Max Reess distinguished the species according to the appearance of the cells thus, the ellipsoidal cells were designated Saccharomyces ellipsoideus, the sausage-shaped Saccharomyces Pasteurianus, and so on. It was found by Hansen that the same species of yeast can assume different shapes; and it therefore became necessary to determine how the different varieties of yeast could be distinguished with certainty. The formation of spores in yeast (first discovered by T. Schwann in 1839) was studied by Hansen, who found that each species only developed spores between certain definite temperatures. The time taken for spore formation varies greatly; thus, at 52° F., S. cerevisiae takes io, S. Pasteurianus I. and II. about 4, S. Pasteurianus III. about 7, and S. ellipsoideus about 4z days. The formation of spores is used as an analytical method for determining whether a yeast is contaminated with another species, - for example: a sample of yeast is placed on a gypsum or porcelain block saturated with water; if in ten days at a temperature of 52° F. no spores make their appearance, the yeast in question may be regarded as S. cerevisiae, and not associated with S. Pasteurianus or S. ellipsoideus. The formation of films on fermented. liquids is a well-known phenomenon and common to all micro-organisms. A free still surface with a direct access of air are the necessary conditions. Hansen showed that the microscopic appearance of film cells of the same species of Saccharomycetes varies according to the temperature of growth; the limiting temperatures of film formation, as well as the time of its appearance for the different species, also vary.
In the zymo-technical industries the various species of yeast exhibit different actions during fermentations. A well-known instance of this is the "top" and "bottom" brewery fermentations (see Brewing). In a top fermentation - typical of English breweries - the yeast rises, in a bottom fermentation, as the phrase implies, it settles in the vessel. Sometimes a bottom yeast may for a time exhibit signs of a top fermentation. It has not, however, been possible to transform a typical top yeast into a permanent typical bottom yeast. There appear to be no true distinctive characteristics for these two types. Their selection for a particular purpose depends upon some special quality which they possess; thus for brewing certain essentials are demanded as regards stability, clarification, taste and smell; whereas, in distilleries, the production of alcohol and a high multiplying power in the yeast are required. Culture yeasts have also been successfully employed in the manufacture of wine and cider. By the judicious selection of a type of yeast it is possible to improve the bouquet, and from an inferior must obtain a better wine or cider than would otherwise be produced.
Certain acid fermentations are of common occurrence. The Bacterium acidi lacti described by Pasteur decomposes milk sugar into lactic acid. Bacillus amylobacter usually accompanies the lactic acid organism, and decomposes lactic and other higher acids with formation of butyric acid. Moulds have been isolated which occasion the formation of citric acid from glucose. The production of acetic acid from alcohol has received much attention at the hands of investigators, and it has an important technical aspect in the manufacture of vinegar. The phenomenon of nitrification (see BACTERIOLOGY, AGRICULTURE and MANURE), i.e. the formation of nitrites and nitrates from ammonia and its compounds in the soil, was formerly held to be a purely chemical process, until Schloesing and Mintz suggested in 1877 that it was biological. It is now known that the action takes place in two stages; the ammonium salt is first oxidized to the nitrite stage and subsequently to the nitrate. (J. L. B.)
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