Fermentation

FERMENTATION, a rapidly occurring decomposition of organic matter caused by microorganisms, for which this process serves as a source of kinetic energy. True fermentations, in the strict sense of the word, are not associated with oxidative processes at the expense of atmospheric O₂ and are divided into the following three main types: alcoholic, lactic acid, and butyric acid fermentation. In alcoholic fermentation, simple sugars decompose into ethyl alcohol and CO₂: C₆H₁₂O₆ = 2CH₃.CH₂OH + 2CO₂ + 28 calories. In lactic acid fermentation, simple sugars decompose with the formation of only lactic acid: C₆H₁₂O₆ = 2CH₃.CH(OH).COOH + 18 calories. In butyric acid fermentation, simple sugars decompose with the formation of butyric acid, carbon dioxide, and hydrogen: C₆H₁₂O₆ = CH₃.CH₂.CH₂.COOH + 2CO₂ + 2H₂ + 15 calories. All three types of fermentation were discovered by Louis Pasteur. He also established their chemical balance and elucidated the most important conditions influencing them. Being processes associated with the release of energy, true fermentations replace respiration for the microbes that cause them and, thus, make possible anaerobiosis, i.e., life without access to air. In addition to the above pure types of fermentation, there are also mixed ones, in which products of two and even all three main types of fermentation are formed simultaneously. In addition to the above-listed main fermentation products, by-products also frequently accumulate, of which volatile acids, glycerin, succinic acid, and certain alcohols are particularly widespread. Oxidative fermentations are processes that occupy an intermediate position between true fermentations and normal respiration. The main type of oxidative fermentation is acetic acid fermentation, also discovered and studied by Pasteur. In acetic acid fermentation, ethyl alcohol is oxidized to acetic acid: CH₃.CH₂OH + O₂ = CH₃.COOH + H₂O. In addition, microbiological oxidations of certain polyhydric alcohols are known. A characteristic feature of fermentation is, firstly, the release of free energy, and secondly, the mass processing of the fermentable material. Since during the fermentation of a gram-molecule of simple sugar, even in alcoholic fermentation, only 28 calories are released (in other true fermentations, even less energy is released), whereas during the combustion of the same gram-molecule of sugar to CO₂ and water in the respiration process, 674 calories are released, it is necessary to decompose at least 25 times more sugar by means of alcoholic fermentation than through normal oxygen respiration to cover the energy needs of the microorganism. In acetic acid fermentation, the release of energy calculated per unit of alcohol is greater than in true fermentations, because the hydrogen of the alcohol is partially burned to water, but the product of fermentation is acetic acid, a substance that still possesses a large reserve of latent energy. Consequently, the release of energy during acetic acid fermentation is still much less than during the complete combustion of alcohol to carbon dioxide and water. For this reason, acetic acid fermentation also bears the character of a rapidly proceeding process. - Each fermentation is caused by microorganisms specially adapted to it. The geochemical role of fermentation is very important: organic matter is decomposed by means of fermentation in the absence of atmospheric oxygen, thus ensuring the possibility of a complete carbon cycle on our planet. Under natural conditions, butyric acid fermentation has the largest scale. Alcoholic fermentation has been studied in more detail than the others because it has great technical importance, being so far the sole method for preparing alcohol and alcoholic beverages. The typical organism causing alcoholic fermentation in industry is yeast, i.e., single-celled ascomycetous fungi of the genus Saccharomyces. In addition, alcoholic fermentation is also caused by certain Mucor fungi and many bacteria. All higher and lower plants that normally respire in the presence of air produce a certain amount of alcohol and CO₂ in the absence of oxygen. During the alcoholic fermentation of yeast, in addition to the normal fermentation products, ethyl alcohol and CO₂, a small amount of by-products is formed, namely: acetaldehyde, acetic acid, glycerin, succinic acid, malic acid, and so-called fusel oils. Acetic acid, acetaldehyde, and glycerin represent products of the accidental perversion of the normal course of fermentation; non-volatile acids and fusel oils originate from the amino acids of proteins present in the liquid or in the yeast itself. Succinic and malic acids are formed from dibasic amino acids, and fusel oils—i.e., monohydric primary alcohols whose structure is determined by the structure of the corresponding amino acids after the hydrolytic cleavage of CO₂ and NH₃—are formed from monobasic amino acids. Thus, for example, from the amino acid leucine, isoamyl alcohol is obtained: CH₃/CH-CH₂-CH(NH₂)-COOH + H₂O = ... [fragment OCR note]

leition CH, \ CH - CH) - CH₂OH + CO* + NH,. pi isoamyl alcohol. In reality, this reaction represents not hydrolysis, but oxidation followed by reduction, and consists of at least 4 steps (simple reactions that together make up the total process of fusel oil formation). Thus, the formation of fusel oils has nothing to do with the process of sugar fermentation. Of the simple sugars, d-glucose, d-fructose, d-mannose, and d-galactose undergo fermentation, the last one weaker than the other three hexoses. In addition, those carbohydrates and glycosides are fermented from which yeast can split off simple sugars capable of fermentation. Thus, cane sugar and maltose are fermented by yeast, because the named disaccharides are split by yeast enzymes; on the contrary, milk sugar is not fermented (although upon splitting it yields glucose and galactose capable of fermentation), because the yeast does not contain an enzyme splitting milk sugar. With an abundant supply of O, yeast exhibits normal oxygen respiration and uses it as a source of energy, but, nevertheless, fermentation does not stop, although the yields of alcohol in relation to the amount of decomposed sugar decrease. Normal respiration strongly promotes the reproduction of yeast, as a result of which passing air through the fermenting wort is used in industry. Fermentation stops when the alcohol concentration reaches 10-14%; therefore, there is no point in subjecting solutions to fermentation in which the sugar concentration exceeds 25%. In fact, yeast is capable of fermenting even in stronger sugar solutions, as can be seen in the example of the fermentation of honey. However, complete fermentation of the sugar in such cases is impossible: upon reaching the limiting alcohol concentration, the process stops. Fermentation caused by mucor fungi stops already at 5% alcohol. In the absence of sugar, yeast nevertheless develops weak fermentation at the expense of reserve carbohydrates contained in their body (so-called self-fermentation of yeast). Among these carbohydrates, glycogen takes first place. At the same time, powerful proteolytic enzymes of yeast come into action, and self-digestion begins. In the presence of sugar and in the presence of vigorous fermentation, self-digestion does not occur, and the action of yeast tryptase on proteins present in the surrounding liquid is also significantly weakened. 30 years ago, the discovery of so-called extracellular fermentation, caused by killed yeast and juice extracted from it, made a great impression. The putative enzyme of alcoholic fermentation was named zymase. Later on, however, it turned out that if fermentation is actually caused by a special enzyme, the latter possesses completely special properties that sharply distinguish it from other enzymes known to us; the final solution to the question of the enzymatic nature of fermentation can only be given by further in-depth studies on the kinetics of fermentation. In industry, alcoholic fermentation is used for the preparation of wine, beer, pure alcohol, fruit wines, kefir, kumys, and other alcoholic beverages. For the preparation of wine, juice is squeezed from wine berries and subjected to fermentation in large vats. Yeast is not added, and fermentation is carried out by wine yeast (Saccharomyces ellipsoideus), which is always found in abundance on wine berries. Sweet wines are prepared from dried berries, in which the sugar content is increased, and fermentation is not allowed to go to completion. Wines with an alcohol content of over 16% are obtained by means of artificial addition of wine alcohol after fermentation. Sparkling wines undergo secondary fermentation in corked bottles, during which the wine is supersaturated with carbon dioxide. The acidity of wines depends mainly on tartaric acid, contained in abundance in grapes, and on acetic acid formed during fermentation. Beer is prepared from malt, i.e., germinated barley seeds killed by careful drying. Malt is infused with water at 50-70°, during which the enzyme amylase (diastase) contained in the malt converts the starch of this material into maltose. The liquid turns brown and becomes sweet (beer wort). It is boiled with hops to impart aroma and bitterness to the product, then fermented with brewer's yeast (Saccharomyces cerevisiae). The alcohol content in beer is 2.5-5%. There are many races of cultivated brewer's yeast, differing from each other by certain features of fermentation. In brewing practice, top and bottom fermentation are distinguished. The first goes turbidly, yeast floats on the surface in the form of mucous islands, the liquid heats up. Bottom fermentation is caused by other races of yeast. It is carried out at a low t° (4-6°), using cooling of the wort with ice. Beer of bottom fermentation is less stable, but has a significantly better taste and therefore has supplanted top fermentation beer in almost all countries. Ropiness of beer is caused by wild yeast, which can be avoided by using pure races of cultivated yeast for factory fermentation. For the preparation of pure alcohol, a significant amount of starch in the form of a crushed mass of potato or rye seeds is added to the malt. As a result, a wort with a high sugar concentration is obtained. After fermentation, the alcohol concentration reaches 12-14%. Alcohol is distilled off and its strength is increased by repeated distillations, during which the alcohol is simultaneously purified from extraneous impurities (rectification of alcohol). Baker's yeast, used for raising dough with CO₂, is obtained by pressing top fermentation yeast, with the admixture of starch for the viscosity of the pressed mass. Kefir and kumys are products of alcoholic and lactic acid fermentation of milk occurring simultaneously. Kefir yeast belongs to a special genus Torula. Lactic acid fermentation is caused not by fungi, but by bacteria, and is divided into two types: pure and impure fermentation. In the first (typical representative Bacterium lactis acidi Leichm.), the only product is lactic acid (see above); in the second (typical representative Bacterium lactis aerogenes), in addition to lactic acid, volatile acids, ethyl alcohol, CO₂, H, and sometimes other products are formed. Bacteria of pure fermentation prefer a complete absence of O₂ and, unlike yeast, are completely devoid of normal respiration; bacteria of impure fermentation, on the other hand, prefer access to air and can breathe normally. Fermentation proceeds especially vigorously in milk. The formation of clots depends on the precipitation of milk proteins due to the acidic reaction. Upon the accumulation of 0.8-1.5% acid, fermentation stops, but upon neutralization of the acid it goes to the end, even with large quantities of sugar. Those same simple sugars that undergo alcoholic fermentation can ferment, and those carbohydrates that are converted into fermentable hexoses by microbes of lactic acid fermentation. Unlike yeast, lactic acid bacteria are capable of splitting and then fermenting milk sugar, but most of them are not able to split and, consequently, ferment cane sugar. Fermentation proceeds best at a high temperature (30-40°). Butyric acid fermentation is caused by anaerobes. Some of them assimilate molecular nitrogen of the atmosphere. Pure butyric acid fermentation (see above) is rare (representative - Clostridium Pasteurianum). Usually, in addition to butyric acid, H and CO₂, volatile acids, ethyl alcohol, and other products are also formed. Between butyric acid and lactic acid fermentation, there is a number of transitions, such that it is sometimes difficult to determine whether a given fermentation should be considered essentially lactic acid or butyric acid. Many butyric acid bacteria can split and ferment complex carbohydrates, such as starch, dextrins, cellulose, and pectin substances. Therefore, butyric acid fermentation is extremely widespread in nature. Fermentation of cellulose and pectins is especially interesting. Cellulose is fermented with the formation of CO₂, volatile acids, and hydrogen or methane. Pectic fermentation lies at the basis of flax retting. It has been little studied yet. Acetic acid fermentation is caused by specific bacteria belonging to different species. These microbes always develop on the surface of an alcohol solution in the form of a film. For the industrial preparation of wine vinegar, fermentation is carried out in wide vats at 20°-30°. Acetic acid fermentation stops at 6-14% free acetic acid, depending on the species of bacteria. The reproduction of acetic acid bacteria occurs with extraordinary speed. The chemical side of fermentation is of outstanding interest. To clarify the unknown reactions underlying actual fermentation, attempts are made to capture intermediate products of these processes. Yeast and other fermentative organisms produce powerful reductions of various substances and are able to displace the hydrogen of alcohol groups, transferring it to an active state. This property, according to modern views, lies at the basis of fermentation. In addition, it turned out that phosphates are necessary for alcoholic and lactic acid fermentations. The first stage of fermentation consists in the formation of mono- and diphosphoric ester of fermenting hexoses: C₆H₁₀O₄(Me₂PO₃)₂ and C₆H₁₀O₄(Me₂PO₄)₂. Diphosphoric ester is perhaps a by-product. Then the carbon chain of hexose is broken in half. It is assumed that glycerine aldehyde is first obtained in this case, but it has not been detected up to the present time.

From subsequent intermediate products, it was possible to isolate methylglyoxal CH₃.CO.CHO and acetaldehyde CH₃.CHO. Furthermore, it turned out that yeast vigorously decomposes pyruvic acid with the formation of acetaldehyde and carbon dioxide: CH₃.CO.COOH = CH₃.CHO + CO₂. Finally, it has been proven that yeast reduces acetaldehyde to ethyl alcohol. With pure lactic acid fermentation, it was possible to isolate the following intermediate products: hexose phosphate, methylglyoxal, and pyruvic acid. Since bacteria of pure lactic acid fermentation lack the ability to decompose pyruvic acid, it is probable that the chemistry of alcoholic and lactic acid fermentation is identical up to the stage of pyruvic acid formation: 1) C₆H₁₂O₆ = 2CH₂OH.CHOH.CHO (glyceraldehyde); 2) 2CH₂OH.CHOH.CHO = 2CH₃.CO.CHO + 2H₂O + 4H (methylglyoxal); 3) 2CH₃.CO.CHO + 2O = 2CH₃.CO.COOH (pyruvic acid); 4) 2CH₃.CO.COOH + 4H = 2CH₃.CHOH.COOH (lactic acid, for lactic acid fermentation) or 4) 2CH₃.CO.COOH = 2CH₃.CHO + 2CO₂ (acetaldehyde) and 5) 2CH₃.CHO + 4H = 2CH₃.CH₂OH (ethyl alcohol, for alcoholic fermentation). The formation of hexose phosphate probably facilitates the transfer of hydrogen and oxygen. Butyric acid fermentation has been little studied because, owing to the layering of many heterogeneous reactions, its dissection encounters great difficulties. In butyric acid fermentation, pyruvic acid and acetaldehyde probably also form, but since hydrogen is released as a gas, reduction of the carbonyl group does not occur, and acetaldehyde is converted into butyric acid via intramolecular rearrangements through aldol.

S. Kostychev. Ammoniacal (alkaline) fermentation of urine is observed during more or less prolonged storage of urine discharged from the bladder. Urine acquires a sharply alkaline reaction, and a significant amount of NH₃ appears in it. Under pathological conditions, ammoniacal fermentation of urine can also occur in the bladder. This process is associated with the multiplication of bacteria (Micrococcus ureae, Bacterium ureae, etc.) containing the enzyme urease (see). Under the influence of the latter, urea undergoes hydrolytic cleavage with the formation of NH₃ and CO₂: CO(NH₂)₂ + H₂O = CO₂ + 2NH₃. Calling this process fermentation is, in essence, incorrect, because fermentation is understood to mean processes that provide the cell with the energy necessary for its vital activity, whereas the cleavage of urea is not accompanied by the release of any significant amount of energy. Apparently, this is one of the intermediate metabolic processes of bacteria. During ammoniacal fermentation, urine pales, becomes covered with a film, and turns turbid due to the proliferation of microbes and the precipitation of substances insoluble in an alkaline medium: calcium phosphate of the composition Ca₃(PO₄)₂, the double phosphate salt of ammonium and magnesium (triple phosphate), acid ammonium urate, and calcium carbonate.