Chemistry of Alcoholic Fermentation ~-

These function on the .pray principle whereby streams of hot and cold water at ... combination ma\h and lauter tub, the brewhouqe yield from malt rnay...
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SEPTElZBEH, 1935

INDUSTRIAL AND E U C l Y E E R l N G CHEAIISTRY

three-arm rackerc: which nil1 deliver up to two hundred forty half-barrel< per hour n ithout foaming or other loss oi beer. The washing and cleaning of the kegs are an important part of the finiihing operatiom. Theae containers are &ject to all kind< of contamination, particularly those packages which hare been used for shipping trade. Recently great progress has been inade in developing more efficient and automatically operating keg-washing machine.. These function on the .pray principle whereby streams of hot and cold water a t preq-ures up to 60 pounds per square inch are injected into the package to dislodge and wash out dried beer remain.: and other accumulated foreign substanceb. A sound, clean trade package i i just as important as the maintenance of sanitary conditions in other parts of the brewery.

Economic lspects Unmalted cereals, and products derived therefrom, furnisli to the brewers' wort colids or extract in amount.: which are almost equal to the laboratory or theoretical yield. The brewery yield obtained from barley malt, the main ingredient of beer, varieq between 62 and 72 per cent for malt< produced from American six-rowed barley, depending upon the quality of this brewing material and the nature and condition of the brewhouse equipment. With the conventional combination ma\h and lauter tub, the brewhouqe yield from malt rnay be 4 to 8 per cent below the laboratory figure. With the separate mash and lauter tubs, laboratory yield may be approached more closcly, and with a mash filter i t is often exceeded. The iecond econoinic aspect is presented by process shrink-

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age during cellar operations. Beer is lost by being admixed with yeast crops in tank or cask iedinients, by entrainment, and by admixture with witer used to flush out conduits, such as pipe and hose lines, and in filters. Process shrinkage to rome extent depends upon the size of units coi1,qtituting the hrewery equipment, and therefore is smaller in large breweries and greater in the small plants. I n general, a wellconducted brewery will turn out about 97 barrels of beer per 100 barrels of wort brought into the starting tub, whereas in smaller plants a final yield of 88 to 90 barrels generally is obtained. d third economic Triewpoint involve,? the engineering services required for brewery operations-that is, production of steam, electric power, refrigeration, and the maintenance of wat'er and air supplies. Much has been done during the last few years to correlate and coordinate the development of t,hese services with breming procedure, but the so-called engineering departments of breweries still offer a splendid field for the mechanical and chemical engineer. As in any factory using process steam, the brewery offers many opportunities for steam to do considerable work before and after i t has been used in the actual brewing processes. Many mechanical improvements can be introduced into existing breweries to reduce operating costs without in any way reducing quality of finished product.

Acknowledgment Acknowledgment is made of the helpful assistance of S. Laufer and A. R. Erda in preparing material for this paper. RECEIVED .ipril 26, 1935.

Chemistry of Alcoholic Fermentation LEONOR MICHAELIS The Rockefeller Institute for Medical Research, New York, N. Y.

catalytic reaction* could easily he separated from the living LCOHOLIC fermentation has h e n known cells simply by extraction with water. and utilized from times immeniorial, but amroaches to the understanding of the chemical and biological processes underlying it are of rather ~i~~~~~~~of a Cell-Free Fermenting .iigent aecent date. I t is unnecessarv to dwell uDon the stages represented by such names as Schwann, Gay-Lussac, Liebig, Bucliner ( I ) dixovered in 1897 that by special methods t'he Wohler, Pasteur. The results of their works may be sumfermenting agent of the yeast cell can also be separated froin iiiarized as follows: Fermentation is caused by a living orthe living cell. This discovery made it clear that alcoholic ganism, the yeast cell, and confermentation, like ot'her enzysists mainly in the conversion matic reactions, is caused by a of sugar into a l c o h o l and carc h e m i c a l substance or rather bon dioxide although other end A brief reviewis given Of what is known at by a complicated s y s t e m of product,s may also be forined present about the chemical processes inchemical substances which can in smaller q u a n t i t i e s . T h e valved in alcoholic fermentation. The by no means be c a l l e d l i v i n g essential difficulty in a further large field ofa~coho~ic fermentation is With- m a t t e r . The achievement' of the living cell is to produce these approach consisted to the the out boundary and is interlinked with all the Hut once problem in theOf fact that no chemical agent endowed adjacent territories of chemistry and biproduced, these agents act in a ology. The alluring feature is the fact that v-ith the faculty of causing ferpurely chemical way, even in t,he any progress in its exploration has always absence of the living material by mentation could be isolated from the living yeast cell. This was meant a simultaneous progress in the field xhich they were produced. in c o n t r a s t t o t h e f a c t that Buchner's method cells consisted of general metabolism both of animals and grinding with many other agents, now called e n z y m e s , which bring about Plants, and vice versa. sand and Kieselguhr and, after o t h e r biologically i m p o r t a n t coniplet'e crushing of the cellh I

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pressing the mass with a hydraulic press. Thus a press juice is obtained which is a turbid but otherwise homogeneous liquid with the faculty of fermenting sugar. The press juice contains no living cell and exhibits fermentation even in the presence of drastic antiseptics, such as toluene. Rlethods other than Buchner's have been since described. Lebedew (11) prepared another active extract from yeast, which is now even more frequently used than Buchner's. H e dries the yeast a t 25" C. for several days and grinds the dry mass in a mill to a very fine powder. A suspension of this powder in water is kept for 2 hours a t 37" C. and then centrifuged. The supernatant fluid is a turbid solution with the faculty of fermenting. From this solution a durable dry preparation may be obtained by precipitation with acetone. The liquid Lebedew juice itself, very active in the beginning, gradually becomes inactive on standing, probably because of the proteolytic enzymes which are also present in such a juice and gradually decompose the fermenting agent. The discovery of a cell-free fermenting agent made possible a deeper insight both into the nature of the enzymes and the chemical processes underlying fermentation. On working with living yeast cells, all chemical processes take place &hin the celi. Sugar diffuses into the cell across the cell wall; the end products of fermentation diffuse out of the cell into the medium, and no insight can be gained into the series of intracellular chemical processes which finally lead to the end products. This is different when the fermentation is brought about by cell-free extracts. Here i t is possible to discover stages of the chemical processes intermediate between sugar and alcohol. On describing these various intermediate steps, it is advisable to follow their natural order and not the order in which they have been discovered. It is characteristic of the advancement of knowledge that the description of the processes now can be made according to their natural order instead of a historical one.

Rockefeller Institute laboratory this salt has been perfectly crystallized. The theory suggests itself that these phosphate esters are intermediate products in the fermentation. There is a criterion as to whether a substance is a n intermediate product. When instead of sugar the supposedly intermediate product is mixed with yeast juice, the fermentation should proceed at least a t the same rate as, or even a t a higher rate than, the fermentation of sugar. The most plausible theory would be that first a monophosphate is formed and then a diphosphate, and then that further steps of the development will follow. If this is true, diphosphate should be a t least as rapidly fermented as sugar. However, diphosphate is considerably more slowly fermented. Monophosphate i., in general, more rapidly fermented than diphosphate. The crystallized monophosphate is fermented a t a rate comparable to glucose but only in the initial stage of fermentation. The role of the phosphate esters may be better understood by the following experiment: When yeast juice is mixed with glucoqe, a certain time elapses before fermentation begins. This is called the induction time. This induction time is abolished when, besides glucose. a small amount of hexose phosphate ester'is added: I n this case fermentation starts immediately. Thus it appears that the phosphate esters are not only intermediate products, but that some interaction between the free sugar and the phosphate ester takes place. The monophosphate seems to be the more important, or better, the more active one, and the accumulation of diphosphate during fermentation seems to be something like a retarding reaction. Probably the diphosphate is a reserve from which the more active monophosphate is to be rebuilt during t h e further stages of fermentation. This problem, however, is not yet definitely settled.

Combination of Sugar and Phosphoric Acid

The next step is the breakdown of the 6-carbon chain of glucose or the hexose phosphate esters into two molecules, each containing a 3-carbon chain. This type of splitting had been surmised for a long time, but only recently has experimental evidence been given to support it. The scheme for the appearance and further fermentation of these 3-carbon phosphoric esters was the last achievement of Embden (4) before his premature death, and was not only confirmed but also supplemented in a rather surprising way by Meyerhof and Lohmann (18). As matters stand according to present developments this splitting process is as follows: Hexose diphosphate is split by an enzyme to give two molecules of a triose monophosphate. There are two trioses, dioxyacetone and glycerol aldehyde-the one a ketose, the other a n aldose. Meyerhof and Lohmann stated that the triose phosphate developed to be the ester of dioxyacetone. The splitting of the hexose into the two trioses does not go on to completion but leads to a n equilibrium. Hexose diphosphate is split by this enzyme to a certain extent. On the other hand, triose esters are synthesized by the same enzyme to form hexose diphosphate. The equilibrium reached is the same, whether we start from the one side or the other. The equilibrium is brought about in less than a minute. The situation of this equilibrium depends largely on temperature. Surprisingly, t h e synthesis is favored by increasing the temperature. Before proceeding farther, we may hint a t a weak point in our present knowledge. This breakdown of the 6-carbon chain has been demonstrated as yet only for hexose diphosphate, not for monophosphate. As stated above, the hexose monophosphate is more likely to be a faster reacting intermediate product than the diphosphate. Perhaps the discrepancy may be solved by a future discovery which would

The first chemical process is, astonishingly, a synthetic one and not a breakdown. I t consists in the combination of sugar with phosphoric acid to form various hexose phosphate esters. S o fermentation takes place in the absence of phosphates. Harden and Young (9) discovered that during the firbt stage of fermentation the inorganic phosphate is diminished and organic phosphate compounds are formed. They have been recognized by Harden and Young (7) as phosphate esters of hexoses. A series of such phosphate esters has been discovered. The first one described by Harden and Young (IO) is a hexose diphosphate which, according to our prebent knowledge, is combined with one phosphoric acid molecule a t carbon atom 1, and another a t 6. The hexose of this eater appears to be fructose even when the sugar from which it generated is pure glucose. Furthermore, hexose monophosphate esters have been isolated. I n the first place, there is the so-called Robison ester (8), which contains only one phosphoric acid molecule a t carbon atom 6. This preparation appears to be a mixture of a glucose ester and a fructose ester. There is, besides, a trehalose monophosphate (24). Another hexose monophosphate ester, the Neuberg ester (ZO), has been prepared also by partially hydrolyzing Harden's diphosphate ester. This ester seems to be identical with the fructose part of Robison's ester. We have not yet reached a complete insight into the possible varieties of these esters. It is difficult to obtain them in crystallized form. Two hexose monophosphate esters have also been synthesized by Levene and Raymond (12); one is probably identical with Robison's but has not yet been crystallized. Recently Warburg (27) succeeded in obtaining the Robison ester in the form of its calcium salt in a microcrystalline form. I n the

Breakdown of the 6-Carbon Chain

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not be quite unexpected-namely, that a comparable splitting process is also possible for hexose monophosphate.

Dismutations The dioxyacetone phosphate formed from hexose diphosphate immediately undergoes further changes. If we assume that dioxyacetone phosphate is in equilibrium with its isomer, glycerol aldehyde phosphate, we may expect that a reaction common to aldehydes occurs; that is, two molecules react upon each other in such a way that one is reduced by the other which, in turn, is oxidized. Such a process is called a Cannizzaro reaction or a dismutation, and other examples will be encountered later on in the further stages I-If fermentation. This prqcess converts two molecules of dioxyacetone phosphate into one molecule of glycerophosphoric acid and one molecule of what is called phosphoglyceric acid. Phosphoglyceric acid undergoes a remarkable further enzymatic alteration discovered by Embden. Free phosphoric acid is split out, not hydrolytically by adding a molecule of water in the way an ester is usually split by water, but in such a way that the hydrogen atom which is necessary to supplement the phosphoric acid residue to phosphoric acid is withdrawn from the organic part of the molecule. What is left is then pyruvic acid. Quite recently, Lohmann and hIeyerhof (16), studying this process in the metabolism of frog muscle, discovered that this reaction is not direct but goes through the intermediate state of a phosphate ester of pyruvic acid. The process is as follows: Phosphoglyceric acid is converted by an enzyme to phosphopyruvic acid, and the latter is hydrolysed in the presence of a coenzyme to pyruvic acid and phosphoric acid. The fate of pyruvic acid ha3 been known for many years by Neuberg's discovery (22) that, because of a sDecial enzvme called carboxvlase., Iilvruvic " acid is split inio carbon dioxide and acetaldkhyde. Acetaldehyde as an intermediate product of fermentation had been surmised for a long time and has been definitely proved by Neuburg (23) to arise during fermentation. When fermentation takes plgce in the presence of sodium sulfite or calcium sulfite, the aldehyde combines with this reagent to form the well-known aldehyde bisulfite compound. Because of this reaction, acetaldehyde escapes further alterations by yeast and can be recovered quantitatively. Under ordinary conditions, without sulfite, acetaldehyde undergoes a further change. We recall now the above-mentioned glycerophosphoric acid which is the other moiety of the splitting process of the 6-carbon chain. This substance is oxidized by acetaldehyde to form a phosphate ester of glycerol aldehyde, and the acetaldehyde in its turn is reduced to ethyl alcohol. Here we come t o the desired end product of fermentation. The other end product, carbon dioxide, has been accounted for already; it is generated from the splitting of pyruvic acid. We now come to the phosphate ester of glycerol aldehyde. By another process of dismutation this ester is again converted into equal parts of glycerophosphoric acid and phosphoglyceric acid. A fresh portion of phosphoglyceric acid is thus produced which undergoes the changes already described. It furnishes a new portion of alcohol and the remainder undergoes the cycle described above. Thus the cycle is started over again until all sugar is finally converted into alcohol and carbon dioxide.

Sources of Knowledge of Chemical Processes The methods by which the present knowledge of the chemical processes during fermentation has been reached can be only briefly mentioned here. The foundation of all experimentation is, as stated above, the cell-free press juice or

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maceration juice. On working with living yeast, the appearance of the phosphate esters and most of the other intermediate products escapes detection because everything occurs inside the cell. Hexose phosphates, added to living yeast, are not fermented, or, at best, are very sluggishly fermented. These esters cannot penetrate the cell wall and are not accessible to the enzymes within the cell. But even on working with the cell-free extract, obtaining the intermediate products is not an easy matter. The hexose phosphates can be prepared by interrupting the fermentation a t a suitable stage. The system is fixed with trichloroacetic acid, and the filtrate is treated with lead acetate; from this precipitate the phosphate esters can be isolated by complicated processes in the form of their barium or calcium salts. Certain poisons were especially useful to obtain the other intermediate products. Sodium fluoride has a specific inhibiting action on the enzyme which splits phosphoglyceric acid. When a triose phosphoric acid ib added to the enzyme containing also sodium fluoride, the dismutation into glycerophosphoric acid and phosphoglyceric acid takes place, but further processes are inhibited. In this way Meyerhof succeeded in accumulating these two otherwise transitory substances. Addition of sodium sulfite prevents the acetaldehyde from further chemical alteration as stated above and so permits its detection. Pyruvic acid cannot be obtained in the same way, although pyruvic acid also forms a bisulfite compound. Unfortunately this compound is just as easily fermented as pyruvic acid itself, whereas the acetaldehydebisulfite compound is not attacked by yeast. The best evidence that pyruvic acid is an intermediate product of fermentation is given by Seuberg's discovery that pyruvic acid is fermented by yeast a t least as fast as glycose itself. The splitting of the 6-carbon chain into two 3-carbon chains has been moved s t r i c t l y e x p e r i m e n t a l l y by Embden and by *Meyerhof and -Lohmann f o r m u s c Ik extract. From analogy, we transfer this idea also to yeast fermentation. Later on we shall d e m o n s t r a t e the justification of drawing a n a 1o gi e4 f r o m muwle metabolism to yeast metabolism.

Enzymes All of the chemical processes discusbed to this point are caused by enzymes. More than enough enzymes in yeast are known to account for the many chemical reactions discussed. h'one of these enzymes has been prepared in a pure condition. But a great many of them can be separated and isolated from each other in such a may as to ascertain the hypothesis that we have to deal with a great number of different enzyme-. It is certain that there are many enzymes each with a special function and not a single enzyme that would exhibit different functions under different conditions. There are even more enzymes than those concerned with the chemical reactionalready mentioned. Some of these enzymes are not concerned a t all with fermentation, such as the enzymes capable of splitting polypeptides. We shall pass over the complicated enzyme system involved in respiration. Other enzymes are linked with fermentation only in a preparatory way. Thus, invertase hydrolyzes sucrose, and maltase hydrolyzes maltose to a simple, fermentable hexose, although it is almost certain that these disaccharides are accessible to fermentation to a certain extent, even without this preparatory hydrolysis. There is also a phosphatase which is generally able to hydrolyze phosphate esters, and probably the same enzyme can also, according to conditions, bring about the synthesis of phosphate ester. Furthermore, there is the enzyme that splits phosphoglyceric acid into phosphoric acid and pyruvic acid, and another enzyme (Neuberg's carboxylase) that splits carbon dioxide from pyruvic acid. There is also an enzyme

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that converts methylglyoxal into lactic acid, discovered by Dakin ( 8 ) and by Neuberg (19). The presence of such an enzyme conveyed the idea that methylglyoxal is an internediate product of fermentation, although lactic acid is not an end product, or a t least not a conspicuous end product of fermentation by yeast. Methylglyoxal was thought of as that 3-carbon compound which originates from the breaking of the hexose in two halves. This idea has a t present been abandoned, and the process described above adopted instead. It is hard to imagine that the occurrence of methylglyoxalase is quite fortuitous and of no purpoqe, and it is possible that some day methylglyoxal will be again given a part to play in the drama of fermentation. Some of these enzymes are in a certain sense simple, since they need no coenzyme for their action. Among the enzymes concerned with the processes proper of fermentation, carboxylase may be mentioned. Other enzymes need home supplementary agent to develop their faculties. Rlethylglyoxalase requires the presence of glutathione, as Lohmann (13) . , has shown. The enzymes concerned with the first stages of fermentation are of a still more complicated nature. When yeast juice is dialyzed, neither the remaining juice nor the dialyzate is capable of fermenting. A mixture of both is again active. Thus the enzyme consists of two constituents, one of a more colloidal nature, not dialyzable, the other of obviously smaller molecular weight and easily diffusible. They are distinguished as zymase and cozymase. Zymase is very sensitive to heat, whereas cozymase is rather resistant to heat, almost up to the boiling point. I t has been further shown by Lohmann (14) that, besides these two constituents of the enzyme system, a small amount of a magnesium salt is indispensable. S o t much purification of the zymase has been attained as yet. The cozymase has been prepared by Euler (6)in a much purer state, but not yet nearly pure enough to be designated a definable chemical compound. Carboxylase can easily be separated from most of the other enzymes. Another auxiliary substance has been found by Euler (6)which he called the a-factor. It is very heat-resistant (much more than cozymasej and increases considerably the ferment’ation with living yeast cells, but it has no effect on the fermentation with yeast juice. 4 great number of other substances of a more or less enzymatic nature can find no place in this paper because they are not involved in the fermentation of carbohydrates-namely, the vitamins of yeast.

Origin of Secondary End Products Returning to the chemical processes in fermentation, we have accounted for what may be called the normal end products of fermentation-ethyl alcohol and carbon dioxide. The origin of the other end products, which may occur sometimes only in small amounts, sometimes to such an extent as to exceed the normal end products in quantity, will now be discussed. Glycerol in small quantities is usually found among the end products of fermentation. The normally developed small amount of glycerol is probably formed from phosphoglyceric acid, which in part escapes the cyclic process described above and is hydrolyzed instead by an enzyme called phosphatase into glycerol and phosphoric acid. Under special conditions, however, the course of fermentation can be artificially modified in such a may as to furnish glycerol as a main product of fermentation. When fermentation occurs in the presence of sodium sulfite, acetaldehyde is thrown out of the reaction and can no longer be reduced to alcohol. Instead, other compounds are reduced. The trioses, when subjected to this reduction which normally takes place with acetaldehyde, will give glycerol even in the absence of sodium sulfite when the fermentation takes place in a n alkaline medium. Sormally

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yeast becomes acid on fermentation. When artificially the fermentation is forced on an alkaline medium, glycerol and also acetic acid are formed instead of alcohol and carbon dioxide, as Seuberg (91) discovered. Other important by-products of fermentation are the fusel oils and the higher alcohols, especially isoamyl and d-amyl. The origin of fusel oil has been discovered by Ehrlich (3). The parent substance is not a carbohydrate but consists of amino acids, although the presence of a fermentable sugar is requisite for the action of yeast on amino acids. The source of these acids is the protein of the fermenting mixture. T h e amino acid is first converted into a keto acid. The latter, like pyruvic acid, loses carbon dioxide, and the remaining material is an aldehyde. Just as acetaldehyde is reduced to ethyl alcohol, these aldehydes are reduced to their corresponding alcohols. From isoleucin d-amyl alcohol is formed, and from leucin isoamyl alcohol is formed. If the amino arid happens to be dicarboxylic, such as aspartic acid, it is first converted into the keto acid. The latter loses only one molecule of carbon dioxide, and the resulting aldehyde-carbonic acid is, instead of being reduced to hydroxy acid, oxidized to a dicarboxylic acid with one carbon atom less than the original amino acid. From aspartic acid succinic acid is developed, which has been known for a long time to be one of the minor end products of alcoholic fermentation. Other amino acids furnish in the same way a part of those substances responsible for the various flavors of fermented beverages. The mechanism by which a carbohydrate breaks down by the metabolism of yeast appears a t first glance to be very different from the may a carbohydrate is utilized by the cell\ of higher living organisms. Here the end products of the normal aerobic metabolism are carbon dioxide and water. Under anaerobic conditions however, the main end product i5 lactic acid. This production of lactic acid is comparable to that of alcohol by yeast and is often also referred to as fermentation, in distinction to respiration which takes place in the presence of oxygen. The eqergy evolved from the respiration of one gram of sugar is about twenty times the energy evolved by fermentation. Here two important problems ariqe: I n which way is the lactic acid production by higher cells correlated with the alcohol production by yeast; and what explanation can be given for the difference of metabolism in the presence and in the absence of oxygen in higher cells, and is there such a difference also in the metabolism of yeast?

a

Correlation of Lactic Acid and Alcohol As regards the correlation between lactic acid and alcohol, it bas been shown that relatively slight changes of the enzyme system are able to bring about this difference in the end product. On the whole, the intermediate products of fermentation in higher organisms and yeast are rather similar. In either case, sugar is subjected to phosphorylation. The two cases are not comparable in all details, however. I n the metabolism of muscle there is a chain of phosphorylations in which not only carbohydrates but also creatinine and adenylic acid are involved. Otherwise, the similarity is striking (17) until we arrive a t the stage where pyruvic acid is formed. We have seen how this acid leads to formation of alcohol in yeast, mainly because of the action of carboxylase, which splits out carbon dioxide from pyruvic acid. I n higher organisms this process does not take place, but pyruvic acid i; transformed into lactic acid by a simple reduction. In the presence of oxygen, according to Neyerhof’s discovery, only about one-fifth of the lactic acid is oxidized to carbon dioxide plus water, and the remainder is resynthesized to form a carbohydrate, glycogen, which now can serve as a further sub-

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d r a t e for fermentation and respiration. The energy evolved hy the combustion of that small fraction of lactic iicid is sufficient not only for the energy required for the life of the cell but also for t'he resynthesis of the remainder of lactic acid to glycogen. The problem arises as to whether a comparable change of metabolisni according to the presence or absence of oxygen occurs also in yeast. Such a problem has heen clearly stated already by Pasteur. He advanced the idea that ferinentation is & saris air; that is, the energy evolved in the absence of air by fermentation is the .source of energy for the cell in the same way that the energy evolved from respiration is the source of energy for respiring cell..

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Thus it i i obvious that the ~netabolibinof yeaht ia intere.ting on it. own account, either from the purely scientific or from the industrial point of view. It is no exaggeration to assert that any progre+ in the knowledge of yea\t fernientation immediately brings about an advancement in the underitanding of the metaboliTm of all other living organi5nii, and \?ice versa.

Some Compounds Involved in Fermentation

Effect of Air on Fermentation This ingenious idea led Pasteur to the postulate that a cell capable of fermenting sugar should do so only in the absence of air and that the same cell in the presence of air should burn up the carbohydrate to carbon dioxide and water. However, he did not succeed in proving this postulate satisfactorily by experiment. Yeast ferments sugar both in the absence and in the presence of oxygen. But in the presence of oxygen, besides fermentation there is also combustion t o carbon dioxide and water-in other words, respiration. When this respiration takes place, the extent of the fermentation is somewhat diminished. This is a certain approach to his postulate, but it does not justify t'he claim that in the presence of oxygen respiration entirely replace,? the fermentation which occurs without oxygen. The experimental test for the soundness of Pasteur's idea was demonstrated niuch later by lleyerhof (16). Instead of working with cultivated strains of yeast, such as are used in manufacturing beer or mine, he studied naturally occurring wild yeast varieties and found the variety Torula utilis especially suitable. This variety complies with Pasteur's hypothesis. It ferments in the absence of oxygen, hut fails to ferment a1moi;t entirely in the presence of oxygen and re.spires instead. =llso this behavior is quit'e analogous to what happens in inost higher organisms; they produce lactic acid in the absence of oxygen but none or very little lactic acid in the presence of oxygen, in which case they respire instead. This phenomenon that an anaerobic fermentation is quantit'atively shifted to respiration by the presence of oxygen has been termed by Warburg ( 2 5 , 26) the Pasteur-Meyerhof reaction to show that t'he occurrence of this phenomenon was predicted by Pasteur and experimentally confirmed by Meyerhof. This Pasteur-lleyerhof reaction is not necessarily cornplete in all types of cells. Sometimes the various enzyme systems of a cell may not be entirely adapted to the purpose of shifting anaerobic fermentation quantitatively to aerobic respiration. This may be thought of as a kind of insufficiency of the chemical structure or coordination of the organism. I n such a case a smaller or greater percentage of the fermentation may persist even during the aerobic respiration. The industrially used cultivated yeasts show this phenomenon to a particularly high extent. They are cultivated for t,he purpose of fermentation and possess this faculty in so high a degree that aerobic respiration cannot cope with the enormous amount of fermentation products. Thus Pasteur happened to meet in his cultivated yeast a somewhat unnatural, domesticated organism. Later, Warburg (25) demonstrated that certain tissues in higher animals also show what we may call an incomplete Pasteur reaction. They produce lactic acid even when respiring in oxygen, although to a smaller extent than in absence of oxygen. He showed this to be true especially for embryonic cells and, what is most, interesting, for the cells of malignant tumors. These cells, like domesticated yeast, cause such a high fermentation that respiration is incapable of burning u p all the products of fermentation.

CHqOH CHOH CHiOH CHiOH CHOH CHO CHzOH CO CH20H CHzOH CHOH COOH

G13cerol GI>cerolaldehyde Dioxyatetone a-Glycerlc acid

1 2 3 4 5 6 CHOH CHOH CHOH CHOH CH CHiOH 0

L-

Aldose (glucose)

_1

5 6 CHzOH CH CHOH CHOH CHOH CHIOH 1

2

4

3

0 2 4

L -

1

2

3

5

Ketose

( h i

tose)

6

CHOH CHOH CHOH CH CHOH CH O(POaH9)

Hexoee monophos-~ 0 phoric acid CH>O(POBHI) CH CHOH CHOH COH CH?O(PO?Hg) Hexose diphos-0-phorrc acid C H:

hTPak?I I

CHO'_H_- -

__

1 I

+

JOOH Phosphoglycerir acid CH2 O H
CH3 or

COOH (enolic form)

Pyruvic acid

CO I COOH

'

1

+ HsPOi

(ketonic form)

Literature Cited Buchner, E., Ber., 30,117, 1110 (1897). Dakin, H . D . , and Dudley, H. \T., J . B i d . Chern., 14, 155, 423; 15, 127 (1913). Ehrlich, F., Jahr. I'ersuchs- I A m n s t a l t BraiLerie Berlin, 10,515 (1907); Ehrlich, F., and Jarohsen, K. A,, Ber.. 44,888 (1911); Ehrlich, F., and Piotschimuka, P., Ibid.,45,1006 (1912). Embden, G. H . , Deuticke, J., and Graff, G., Klin. Woehschr., 12, 213 (1933). Euler, H . von, and Myrbkck, K., 2. physiol. Chem., 131, 179 (1923); 133,260; 136,107; 139,15, 287 (1924). Ihid., 141,297 (1924). Harden, A., ".llcoholic Fermentation," hlonographs in Biochemistry, 4th ed., London, 1932. Harden, d., and Robison, R., Proc. C'hern. Soc., 30,10 (1914). Harden, -1.. and Young, W.,J., Ihid., 21,189 (1905). Harden, A,, and Young, IT. ,J., Proc. Rou. Soc. ( L o ~ ~ d o i i ) , B80,299 (1908). Lebedew, A. Ton, Conapt. rend., 152,49, 1129 (1911). Lerene, P. A., and Raymond, A. L., J . B i d . Chert&.,89, 479 (1930); 92,765 (1931). Lohmann, K.,Biochem.Z.,254,332 (193%). Lohmann, K., Saturwi'ssei~schaften,19,180 (1931). Lohmann, K., and Meyerhof, O., Biochem. Z., 272,60 (1934). Meyerhof, O., Ihid., 162,43 (1925) ; .\'uturwissensc/iuften, 13, 980 (1925). Meyerhof, O., "Die cheniischcn Vorginge in1 Muskel," Berlin, 1930. Meyerhof, O., and Kiessling, H., Biochem. Z., 264,40; 267,318 (1933). Neuberg, C., Ibid., 49,6 0 2 ; 51,484 (1913). Ibid., 88,432 (1818). Neuberg, C., and F i r b e r , E., Biochem. Z., 78,238 (1917); Neuberg, C., and Hirsch, J., Ibid., 98,141 (1919). Neuberg, O., and Hildesheimer, A , , I h i d . , 31,170 (1911).

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(23) Neuberg, O., and Reinfurth, E., Ibid., 89,365 (1918) (24) Robison, R., and Morgan, W. T. J., B i o c h a . J., 22, 1277 (1928). (25) Warburg, O., “Uber den Stoffwechsel der Tumoren,” Berlin, 1926. I

VOL. 27, NO. 9

(26) Warburg, O., “Uber die katalytischen Wirkungen der lebendigen Substanz,” Berlin, 1928. (27) Warburg, O., and Christian, W., Biochem. Z.,254, 438 (1932).

RECEIVED April 27, 1935.

Application of Oxidation-Reduction Potential to Brewing Control

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The theory of the oxidation-reduction systems and their potentials has recently been applied to brewing control work and the preliminary results appear to justify a more intense study. Certain factors influencing the flavor and keeping quality of beer, which have been little understood, may thereby find an explanation. Outstanding are the influence of aeration during the various stages of the brewing process, the propagation of microorganisms, and the effect of oxidizing and reducing reactions on beer flavor. The oxidation-reduction potential is

generally expressed by the symbol, rH (the negative logarithm of the pressure of the reducing hydrogen present in the solution). The oxidizing power of a solution is the greater the higher its potential, and the reducing power is greater the lower its potential. An electrometric and a colorimetric method are available for the determination of rH, providing means for a methodical study of the oxidation factor wherever it occurs during the entire brewing process. Its application is described in the study of aeration, of the so-called light-taste, and of yeast turbidities.

F. P. SIEBEL, JR., ARD E. SINGRUENJ’ French wineries recognize the effect of the oxygen in the air on the wine five decades the Siebel Institute of Technology, aroma and are now investigating the brewing industry Chicago, Ill. oxidation-reduction potential as a has more and more turned to science possible means of controlling aerafor assistance in the solution of its tion. It also finds application in sewage treatment and in the practical problems. The advancements in physical, colloidal, study of the biological condition of soils. and biochemistry, in particular, have contributed a great De Clerck in France ( 1 ) and iMendlik in Holland (2) were deal to the better understanding of purely empirical experithe first to study the merits of the oxidation-reduction potenence and rule-of-thumb methods. I n many cases brewing tial for brewing control work. Although their investigations scientists have traced the causes of frequent disturbances are still in the experimental stage, the preliminary results a p and, based on this knowledge, have been successful in devising pear to be deserving of attention. means to remedy faulty conditions and eliminate them for the future. Theory of Oxidation-Reduction Systems The application of the theory of hydrogen-ion concentration to malting and brewing has become a well-known, comUnder oxidation, originally all those reactions were classiparatively simple, and effective method for controlling procfied in which oxygen combined with another substance to essing methods. Sumerous other problems of physicoform a new compound. I n the meantime, however, a better chemical character, however, have remained unsolved. It understanding of intermolecular arrangement has taught us is only during the past year that European brewing scientists that atoms consist of an inner, positively charged nucleus have put another modern theory-that of the oxidation-re(protons) around which particles with negative charges duction systems-to work, to study into some of the hereto(electrons) revolve. Atoms which lose a n electron in the fore least understood factors influencing the flavor and the course of a chemical reaction gain a positive charge. keeping quality of the beer. These include all oxidation and For instance, an increase in valence constitutes a gain of a reduction reactions which take place during beer production. positive charge-in other words, an oxidation. Since no Outstanding in this respect are the influence of aeration, the electron can exist by itself, it will be bound by another subpropagation of microorganisms, and the effect of oxidation stance which is thereby reduced. It is evident, therefore, and reduction on the beer flavor. that neither oxidation nor reduction can take place alone but Other industries are already making practical use of the oxithat both reactions always are concurrent and consist in the dation-reduction potential. I n the dairy industry the detransfer of electrons from the oxidized to the reduced subgree and, to a certain extent, the nature of milk infections are stance, If we consider the gain of a positive electric charge determined by means of suitable oxidation-reduction indicaas oxidation, although no oxygen may be involved at all in the tors; it is also claimed that the suitability of milk for certain reaction, and the gain of a negative electric charge as reductypes of cheese can be predicted by this method. tion, the modern definition of oxidation would be “loss of electrons.” 1 Present address, 542 Arlington Place, Chicago, Ill.

URING the past