Other Theories.
Among other suggestions may be mentioned that of Kohl [[1909]] who asserts that sodium lactate is readily fermented, whilst Kusseroff [[1910]] holds the view that the glucose is first reduced to sorbitol and the latter fermented, in spite of the fact that sorbitol itself in the free state is not fermented by yeast.
The rapid appearance and disappearance of glycogen in the yeast cell at various stages of fermentation [see Pavy and Bywaters, [1907]; Wager and Peniston, [1910]] has led to the suggestion [Grüss, [1904]; Kohl, [1907]] that this substance is of great importance in fermentation, and represents a stage through which all the sugar must pass before being fermented. The fact that the formation of glycogen has been observed in yeast-juice by Cremer [[1899]], and that complex carbohydrates are also undoubtedly formed (p. [31]), are consistent with this theory. The low rate of autofermentation of living yeast, which is only a few per cent. of the rate of sugar fermentation, renders this supposition very improbable (Slator), as does the fact that the fermentation of glycogen by yeast-juice is usually slower than that of glucose [see also Euler, [1914]].
An entirely different explanation of the chemical changes attendant on alcoholic fermentation has been suggested by Löb [[1906]; [p117] [1908, 1], [2]; [1909, 1], [2,] [3,] [4;] [1910;] Löb and Pulvermacher, [1909]], founded on the idea that the various decompositions of the sugar molecule both by chemical and biological agents are to be explained by a reversal of the synthesis of sugar from formaldehyde. As the sugar molecule can be built up by the condensation of formaldehyde, so it tends to break down again into this substance, and the products observed in any particular case are formed either by partial depolymerisation in this sense or by partial re-synthesis following on depolymerisation.
Löb has adduced many striking facts in favour of this view, and has shown that very dilute alkalis produce no lactic acid but formaldehyde and a pentose as primary products. These substances represent the first stage of depolymerisation and are also formed by the electrolysis of glucose.
Löb has himself been unable to detect definite intermediate products of fermentation by adding reagents, such as aniline, ammonia, and phloroglucinol, which would combine with such substances and prevent their further decomposition [[1906]].
The occurrence of traces of formaldehyde as a product of alcoholic fermentation by yeast-juice [Lebedeff, [1908]] is at least consistent with this theory, but no decisive evidence has so far been obtained either for or against it.
In all the foregoing attempts to indicate the probable stages in the production of alcohol and carbon dioxide from sugar, a single molecule of the sugar forms the starting-point. The facts recounted in Chapter III as to the function of phosphates in alcoholic fermentation, which are summed up in the equation:—
2 C6H12O6 + 2 R2HPO4 = 2 CO2 + 2 C2H6O + 2 H2O + C6H10O4(PO4R2)2,
render it in the highest degree probable that two molecules of the sugar are concerned. The most reasonable interpretation of this equation appears to be that in the presence of phosphate and of the complicated machinery of enzyme and co-enzyme two molecules of the hexose, or possibly of the enolic form, are each decomposed primarily into two groups.
Of the four groups thus produced, two go to form alcohol and carbon dioxide and the other two are synthesised to a new chain of six carbon atoms, which forms the carbohydrate residue of the hexosephosphate. The introduction of the phosphoric acid groups may possibly occur before the rupture of the original molecules, and may even be the determining factor of this rupture, or again this introduction may take place during or after the formation of the new carbon [p118] chain. Sufficient information is not yet available for the exact formulation of a scheme for this reaction. Such a scheme, it may be noted, would not necessarily be inconsistent with the views of Wohl and of Buchner as to the way in which the carbon chain of a hexose is broken in the process of fermentation, but would interpret differently the subsequent changes which are undergone by the simpler groups which are the result of this rupture. The reaction might thus proceed without the formation of definite intermediate products, whilst opportunity would be afforded for the production of a small quantity of by-products such as formaldehyde, glycerol, lactic acid, acetic acid, etc., by secondary reactions.
A symmetrical scheme can readily be constructed for such a change, but much further information is required before any decisive conclusion can be drawn as to the precise course of the reaction which actually occurs in alcoholic fermentation.
CHAPTER IX. THE MECHANISM OF FERMENTATION.
The analysis of the process of alcoholic fermentation by yeast-juice and other preparations from yeast which has been carried out in the preceding chapters has shown that the phenomenon is one of a very complex character. The principal substances directly concerned in the change appear to be the enzyme and co-enzyme of the juice, a second enzyme, hexosephosphatase, and, in addition, sugar, phosphate, and the hexosephosphate formed from these. During autofermentation two other factors are involved, the complex carbohydrates of the juice, including glycogen and dextrins, and the diastatic ferment by which these are converted into fermentable sugars. It is also possible that the supply of free phosphate is partially provided by the action of proteoclastic ferments on phosphoproteins. Under special circumstances the rate at which fermentation proceeds may be controlled by the available amount of any one of these numerous substances.
When the juice from well-washed yeast is incubated, the phenomenon of autofermentation is observed. The juice contains an abundant supply of enzyme, co-enzyme, and phosphate or hexosephosphate, and in this case the controlling factor is usually the supply of sugar, which is conditioned by the concentration of the diastatic enzyme or of the complex carbohydrates as the case may be. When this is the case the measured rate of fermentation is the rate at which sugar is being produced in the juice, this being the slowest of the various reactions which are proceeding under these circumstances. If sugar be now added, an entirely different state of affairs is set up. As soon as any accumulated phosphate has been converted into hexosephosphate, the normal rate of fermentation which is usually higher than that of autofermentation is attained, and, provided that excess of sugar be present, fermentation continues for a considerable period at a slowly diminishing rate and finally ceases. During the first part of this fermentation the rate is controlled entirely by the supply of free phosphate, and this depends mainly on the concentration of the hexosephosphatase and of the hexosephosphate, and only in a secondary degree on the decomposition [p120] of other phosphorus compounds by other enzymes and on the concentration of the sugar. The amount of hexosephosphate in yeast-juice is usually such that an increase in its concentration does not greatly affect the rate of fermentation, and hence the measured rate during this period represents the rate at which hexosephosphate is being decomposed, and this in its turn depends on the concentration of hexosephosphatase, which is therefore the controlling factor. As fermentation proceeds, the concentration of both enzyme and co-enzyme steadily diminishes, as already explained, probably owing to the action of other enzymes, so that at an advanced stage of the fermentation, the controlling factor may be the concentration of either of these, or the product of the two concentrations (see p. [122]). The hexosephosphatase appears invariably to outlast the enzyme and co-enzyme. The condition at any moment could be determined experimentally if it were possible to add enzyme, co-enzyme and hexosephosphatase at will and so ascertain which of these produced an acceleration of the rate.
Unfortunately this can at present be only very imperfectly accomplished, owing to the impossibility of separating these substances from each other and from accompanying matter which interferes with the interpretation of the result.
A third condition can also be established by adding to the fermenting mixture of the juice and sugar a solution of phosphate. The supply of phosphate is now almost independent of the action of the hexosephosphatase, and the measured rate represents the rate at which reaction (1), p. [51], can occur between sugar and phosphate in the presence of the fermenting complex consisting of enzyme and co-enzyme. This change is controlled, so long as sugar and phosphate are present in the proper amounts, by the concentration of the fermenting complex or possibly of either the enzyme or the co-enzyme. If only a single addition of a small quantity of phosphate be made, the rate falls as soon as the whole of this has been converted into hexosephosphate and the reaction then passes into the stage just considered, in which the rate is controlled by the production of free phosphate.
Although these varying reactions have not yet been exhaustively studied from the kinetic point of view, owing to the experimental difficulties to which allusion has already been made, investigations have nevertheless been carried out on the effect of the variation of concentration of yeast-juice and zymin as a whole, as well as of the carbohydrate. Herzog [[1902], [1904]] has made experiments of this kind with zymin, and Euler [[1905]] with yeast-juice, whilst many of the results [p121] obtained by Buchner and by Harden and Young are also available.
The actual observations made by these authors show that the initial velocity of fermentation is almost independent of the concentration of sugar within certain limits, but decreases slowly as the concentration increases. When the velocity constant is calculated on the assumption that the reaction is monomolecular [see Bayliss, [1914], Chap. VI], approximate constancy is found for the first period of the fermentation. This method of dealing with the results is, however, as pointed out by Slator, misleading, the apparent agreement with the law of monomolecular reactions being probably due to the gradual destruction of the fermenting complex.
Experiments with low concentrations of sugar are difficult to interpret, the influence of the hydrolysis of glycogen and of dextrins on the one hand, and the synthesis of sugar to more complex carbohydrates on the other (p. [31]), having a relatively great effect on the concentration of the sugar. Unpublished experiments (Harden and Young) indicate, however, that the velocity of fermentation remains approximately constant, until a certain very low limit of sugar concentration is reached, and then falls rapidly. The fall in rate, however, only continues over a small interval of concentration, after which the velocity again becomes approximately constant and equal to the rate of autofermentation. During this last phase, as already indicated, the velocity is generally controlled by the rate of production of sugar and no longer by that of phosphate, this substance being now present in excess. In other words, the rate of fermentation of sugar by yeast-juice and zymin is not proportional to the concentration of the sugar present as required by the law of mass, but, after a certain low limit of sugar concentration, is independent of this and is actually slightly decreased by increase in the concentration of the sugar.
The relations here are very similar to those shown to exist by Duclaux [[1899]] and Adrian Brown [[1902]] for the action of invertase on cane sugar and are probably to be explained in the manner suggested by the latter. According to this investigator, the enzyme unites with the fermentable material, or as it is now termed, the substrate or zymolyte, forming a compound which only slowly decomposes so that it remains in existence for a perceptible interval of time. The rate of fermentation depends on the rate of decomposition of this compound and hence varies with its concentration. This conception leads to the result that the rate of fermentation will increase with the concentration of the substrate up to a certain limit and will then remain [p122] constant, unless interfered with by secondary actions. This limit of concentration is that at which there is just sufficient of the material in question present to combine with practically the whole of the enzyme, so that no further increase in its amount can cause a corresponding increase in the quantity of its compound with the enzyme or in the rate of fermentation which depends on the concentration of that compound.
The curve relating the rate of action of such an enzyme with the concentration of the zymolyte therefore consists of two portions, one in which the rate at any moment is proportional to the concentration of the zymolyte, according to the well-known law of the action of mass, and a second in which the rate at any moment is almost independent of that concentration, approximately equal amounts being decomposed in equal times whatever the concentration of the substrate.
The results of the experiments with yeast-juice therefore indicate that what is being measured is a typical enzyme action, but afford no information as to which of the many possible actions is the controlling one, a fact which must be ascertained for each particular case in the manner indicated above.
Clowes [[1909]], using washed zymin free from fermenting power and adding various volumes of boiled yeast extract, found that the velocity of reaction was proportional to the product of the concentrations of zymin and yeast extract up to a certain optimum concentration. He interprets these concentrations as representing the concentrations of zymase and co-enzyme, but they also represent the concentrations of hexosephosphatase (present in the zymin) and phosphate (present in the yeast extract), so that at least four factors were being altered instead of only two.
It has already been mentioned that Euler and Kullberg [[1911, 3]] found the conversion of phosphate into hexosephosphate in presence of excess of glucose to proceed according to a monomolecular reaction (p. [58]).
The rate of fermentation is diminished by dilution of the yeast-juice, but less rapidly than the concentration of the juice. Herzog found that when the relation between concentration of enzyme and the velocity constant of the reaction is expressed by the formula K1/K2 = (C1/C2)n where K1 and K2 are the velocity constants corresponding with the enzyme concentrations C1 and C2, the value for n is 2 for zymin, whilst Euler working with yeast-juice obtained values varying from 1·29 to 1·67 and decreasing as K increased.
The temperature coefficient of fermentation by zymin was found [p123] by Herzog to be K24·5°/K14·5° = 2·88, which agrees well with the value found by Slator for yeast-cells (p. [129]).
When we endeavour to apply the results of the investigations of the fermentation of sugar by yeast-juice, zymin, etc., to the process which goes on in the living cell, considerable difficulties present themselves. A scheme of fermentation in the living cell can, however, easily be imagined, which is in harmony with these results. According to the most simple form of this ideal scheme, the sugar which has diffused into the cell unites with the fermenting complex and undergoes the characteristic reaction with phosphate, already present in the cell, yielding carbon dioxide, alcohol, and hexosephosphate. The latter is then decomposed, just as it is in yeast-juice, but more rapidly, and the liberated phosphate again enters into reaction, partly with the sugar formed from the hexosephosphate and partly with fresh sugar supplied from outside the cell. The main difference between fermentation by yeast-juice and by the living cell would then consist in the rate of decomposition of the hexosephosphate, for it has been shown that yeast-juice in presence of sufficient phosphate can ferment sugar at a rate of the same order of magnitude (from 30 to 50 per cent.) as that attained by living yeast.
The difference between the two therefore would appear to lie not so much in their content of fermenting complex as in their very different capacity for liberating phosphate from hexosephosphate and thus supplying the necessary conditions for fermentation.
A simple calculation based on the phosphorus content of living yeast [Buchner and Haehn, [1910, 2]] shows that the whole of this phosphate must pass through the stage of hexosephosphate every five or six minutes in order to maintain the normal rate of fermentation, whereas in an average sample of yeast-juice the cycle, calculated in the same way, would last nearly two hours.
Wherein this difference resides is a difficult question, which cannot at present be answered with certainty.
In the first place it must be remembered that a very great acceleration of the action of the hexosephosphatase is produced by arsenates (p. [79]), and this suggests the possibility that some substance possessing a similar accelerating power is present in the yeast-cell and is lost or destroyed in the various processes involved in rendering the yeast susceptible to phosphate. The great variety of these processes—extraction of yeast-juice by grinding and pressing, drying and macerating, heating, treating with acetone and with toluene—renders this somewhat improbable, and so far no such substance has been detected. [p124]
A comparison of living yeast, zymin, and yeast-juice shows that these are situated on an ascending scale with respect to their response to phosphate. Taking fructose as the substrate in each case, yeast does not respond to phosphate at all (Slator), the rate of fermentation by zymin is approximately doubled (p. [46]), and that by yeast-juice increased ten to forty times, whilst the maximum rates are in each case of the same order of magnitude. Euler and Kullberg, however, have observed an acceleration of about 25 per cent. in the rate of fermentation of yeast in presence of a 2 per cent. solution of monosodium phosphate, NaH2PO4 [[1911, 1], [2]].
The high rate of fermentation by living yeast and its lack of response to phosphate may possibly be explained by supposing that the balance of enzymes in the living cell is such that the supply of phosphate is maintained at the optimum, and the rate of fermentation cannot therefore be increased by a further supply.
A further difference lies in the fact that yeast-juice and zymin respond to phosphate more strongly in presence of fructose than of glucose, whereas yeast ferments both sugars at the same rate (p. [131]), and this property has been shown to be connected with the specific relations of fructose to the fermenting complex. It seems possible that these differences are associated with the gradual passage from the complete living cell of yeast, through the dead and partially disorganised cell of zymin to yeast-juice in which the last trace of cellular organisation has disappeared and the contents of the cell are uniformly diffused throughout the liquid. Living yeast is, moreover, not only unaffected by phosphate but only decomposes hexosephosphate extremely slowly (Iwanoff).
Some light is thrown on these interesting problems by the effect of antiseptics on fermentation by yeast-cells and by yeast-juice. The action of toluene has hitherto been most completely studied, and this substance is an extremely suitable one for the purpose since it has practically no action whatever on fermentation by yeast-juice. The experiments of Buchner have, in fact, shown that the normal rate of fermentation and the total fermentation produced, are almost unaffected by the presence of toluene even in the proportion of 1 c.c. to 20 c.c. of yeast-juice. What then is the effect of toluene on the living yeast-cell? When toluene in large excess is agitated with a fermenting mixture of yeast and sugar, the rate of fermentation falls rapidly at first and then more slowly until a relatively constant rate is attained which gradually decreases in a similar manner to the rate of fermentation by yeast-juice. Thus at air temperature (16°) 10 grams of [p125] yeast suspended in 50 c.c. of 6 per cent. glucose solution gave the following results when agitated with toluene:—
| Time after Addition of Toluene, Minutes | C.c. of CO2 per Minute. | Time. | C.c. per Minute. |
|---|---|---|---|
| 0 | 4·6 | 6 | 1·6 |
| 1 | 4 | 8 | 1·2 |
| 2 | 3·3 | 12 | 0·85 |
| 3 | 2·6 | 24 | 0·8 |
| 4 | 2 | 32 | 0·5 |
| 5 | 1·8 | constant |
Simultaneously with this, the yeast acquires the property of decomposing and fermenting hexosephosphate and of responding to the addition of phosphate. This last property is only acquired to a small degree in this way but it becomes much more strongly developed if the pressed yeast be washed with toluene on the filter pump. Thus 10 grams of yeast after this treatment fermented fructose at 1·2 c.c. per three minutes; after the addition of phosphate (5 c.c. of 0·6 molar phosphate) the rate rose to 6·9 and then gradually fell in the typical manner [Harden, [1910]; see also Euler and Johansson, [1912, 3]].
The current explanation of the great decrease in rate of fermentation which attends the action of toluene and other antiseptics on living yeast, and also follows upon the disintegration of the cell, appears to be that in living yeast the high rate of fermentation is maintained by the continued production of relatively large fresh supplies of fermenting complex, and that when the power of producing this catalytic agent is destroyed by the poison, the rate of fermentation falls to a low value, corresponding to the store of zymase still present in the cell (cf. Buchner, E. and H., and Hahn, [1903], pp. 176, 180).
This explanation implies that the rate of fermentation after the action of the toluene represents the amount of fermenting complex present, a supposition which has been shown (p. [53]) to be highly improbable. It further necessitates, as also pointed out independently by Euler and Ugglas [[1911]], a rapid destruction of the fermenting complex both in the process of fermentation and by the action of the antiseptic, as otherwise the store of zymase remaining in the dead cell would be practically the same as that contained in the living cell at the moment when it was subjected to the antiseptic, and this store would therefore suffice to carry out fermentation at the same rate in the dead as in the living cell. No such rapid destruction, however, occurs in yeast-juice, as judged by the rate of fermentation, which falls off [p126] slowly and to about the same extent in the presence or absence of toluene. Moreover, as shown above, it is highly probable that the actual amount of fermenting complex in yeast-juice is a large fraction of that present at any moment in the cell, and is capable under suitable conditions of producing fermentation at a rate comparable with that of the living cell.
This last criticism also applies to the view expressed by Euler [Euler and Ugglas, [1911]; Euler and Kullberg, [1911, 1], [2]] that in the living cell the zymase is partly free and partly combined with the protoplasm; when the vital activity of the cell is interfered with, the combined portion of the zymase is thrown out of action and only that which was free remains active.
The suggestion made by Rubner [[1913]] that the action of yeast on sugar is in reality chiefly a vital act, but that a small proportion of the change is due to enzyme action, is similar in its consequences to that of Euler and may be met by the same arguments. Buchner and Skraup [[1914]] have moreover shown that the effects of sodium chloride and toluene on the fermenting power of yeast which were observed by Rubner, can be explained in other ways.
Some other explanation must therefore be sought for this phenomenon. Great significance must be attached in this connection to the relation noted above between the degree of disintegration and disorganisation of the cell and the fall in the normal rate of fermentation. It seems not impossible that fermentation may be associated in the living cell with some special structure, or carried on in some special portion of the cell, perhaps the nuclear vacuole described by Janssens and Leblanc [[1898]], Wager [[1898], [1911]; Wager and Peniston, [1910]] and others which undergoes remarkable changes both during fermentation and autofermentation [Harden and Rowland, [1901]]. The disorganisation of the cell might lead to many modifications of the conditions, among others to the dilution of the various catalytic agents by diffusion throughout the whole volume of the cell. As a matter of observation the dilution of yeast-juice leads to a considerable diminution of the rate of fermentation of sugar, and it is possible that this is one of the chief factors concerned. That phenomena of this kind may be involved is shown by the remarkable effect of toluene on the autofermentation of yeast. Whereas the fermentation of sugar is greatly diminished by the action of toluene, the rate of autofermentation, which is carried on at the expense of the glycogen of the cell, is greatly increased. In a typical case, for example, the autofermentation of 10 grams of yeast suspended in 20 c.c. of water amounted to 28 c.c. in 4·8 hours [p127] at 25°, whereas the same amount of yeast in presence of 2 c.c. of toluene gave 97·6 c.c. in the same time.
Many salts produce a similar effect on English top yeasts (in which the autofermentation is large) [Harden and Paine, [1912]], whereas Neuberg and Karczag in Berlin [[1911, 2]] were unable to observe this phenomenon.
A necessary preliminary of the fermentation of glycogen is its conversion by a diastatic enzyme into a fermentable sugar, and it is probable that the effect of the disorganisation of the cell by toluene is that this enzyme finds more ready access to the glycogen, which is stored in the plasma of the cell. No such acceleration of autofermentation is effected by the addition of toluene to yeast-juice, and hence the result is not due to an acceleration of the action of the diastatic enzyme on the glycogen.
This effect of toluene is similar in character to the action of anæsthetics on the leaves of many plants containing glucosides and enzymes, whereby an immediate decomposition of the glucoside is initiated [see H. E. and E. F. Armstrong, [1910]].
Although as indicated above Euler's theory cannot apply to zymase itself, if applied to the hexosephosphatase it would afford a consistent explanation of the facts. According to this modified view it would be the hexosephosphatase of yeast which existed largely in the combined form, so that in extracts, in dried yeast and in presence of toluene only the small fraction which was free would remain active. The zymase on the other hand would have to be regarded as existing to a large extent in the free state so that it would pass into extracts comparatively unimpaired in amount and capable under proper conditions (i.e. when supplied with sufficient phosphate) of bringing about a very vigorous fermentation. The theory of combined and free enzymes is undoubtedly of considerable value, although it cannot be considered as fully established.