Among the disaccharides, cane sugar and maltose are freely fermented, and the juice can be shown like living yeast to contain invertase and maltase. The extent of fermentation does not differ materially from that attained with glucose. Lactose is not fermented.

Of the higher sugars raffinose is fermented by juice from bottom yeast, but more slowly than cane sugar or maltose. No experiments seem to have been made with juice from top yeast. [p033]

The fermentation of glycogen by yeast-juice is of considerable interest, since it is known that the characteristic reserve carbohydrate of the yeast cell is glycogen [see Harden and Young, 1902, where the literature is cited], and moreover that in living yeast the intracellular fermentation of glycogen proceeds readily, whereas glycogen added to a solution in which yeast is suspended is not affected. Yeast-juice contains a diastatic enzyme which hydrolyses glycogen to a reducing and fermentable sugar, so that in a juice poor in zymase to which glycogen has been added, the amount of sugar is found to increase, the hydrolysis of the glycogen proceeding more quickly than the fermentation of the resulting sugar [Harden and Young, 1904], but the course of this enzymic hydrolysis of glycogen by yeast-juice has not yet been studied. As a rule, it is found both with juices from top and bottom yeast that the evolution of carbon dioxide from glycogen proceeds less rapidly and reaches a lower total than from an equivalent amount of glucose.

Since nearly all samples of yeast contain glycogen, yeast-juice and also zymin usually contain this substance as well as the products of its hydrolysis. These provide a source of sugar which enters into alcoholic fermentation, so that a slow spontaneous production of carbon dioxide and alcohol proceeds when yeast-juice is preserved without any addition of sugar. The extent of this autofermentation varies considerably, as might be expected, with the nature of the yeast employed or the preparation of the material, but is generally confined within the limits of 0·06 to 0·5 gram of carbon dioxide for 25 c.c. of juice.

In juice from bottom yeast it amounts to about 5 to 10 per cent. of the total fermentation obtainable with glucose [Buchner, 1900, 2], whereas in juice from top yeasts, which gives a smaller total fermentation with glucose, it may occasionally equal, or even exceed, the glucose fermentation, and frequently amounts to 30 to 50 per cent. of it. It is therefore generally advisable in studying the effect of yeast-juice on any particular substance to ascertain the extent of autofermentation by means of a parallel experiment.

The maceration extract of Lebedeff (p. 24) is usually, but not invariably [Oppenheimer, 1914, 2], free from glycogen, which is hydrolysed [p034] and fermented during the processes of drying and macerating, and therefore as a rule shows no appreciable autofermentation.

The kinetics of fermentation by zymase will be considered later on (p. 120), but the effect on the total fermentation of different concentrations of sugar, this substance being present throughout in considerable excess, may be advantageously discussed at this stage. The subject has been investigated by Buchner [Buchner, E. and H., and Hahn, 1903, pp. 150–8; Buchner and Rapp, 1897] using cane sugar, and he has found both for yeast-juice and for dried yeast-juice dissolved in water that (a) the total amount of fermentation increases with the concentration of the sugar; (b) the initial rate of fermentation decreases with the concentration of the sugar. The following are the results of a typical experiment, 20 c.c. of yeast-juice being employed in presence of toluene at 22°:—

The results as to the total fermentations in experiments of this kind are liable to be vitiated by the circumstance that when a low initial concentration of sugar is employed, the supply of sugar may be so greatly exhausted before the close of the experiment as to cause a marked diminution in the rate of fermentation and hence an unduly low total. Even allowing, however, for any effect of this kind, the foregoing table clearly shows the increase in total fermentation and the decrease in initial rate accompanying the increase of sugar concentration from 10 to 40 per cent. Working with a greater range of concentrations (3·3–53·3 grm. per 100 c.c.) Lebedeff has obtained similar results with maceration extract [1911, 4], but has found that the total amount fermented diminishes after a certain optimum concentration (about 33·3 grm. per 100 c.c.) is reached.

A practical conclusion from these experiments is that a high [p035] concentration of sugar tends to preserve the enzyme in an active state for a longer time. Simultaneously it prevents the development of bacteria and yeast cells.

This subject, which is of considerable importance with reference to the question of the protoplasmic or enzymic nature of the active agent in yeast-juice, has been examined in some detail by Buchner [Buchner, E. and H., and Hahn, 1903, pp. 158–65] and by Meisenheimer [1903] for juices from bottom yeast, by Harden and Young [1904] for those from top yeast, and by Lebedeff [1911, 4] for maceration extract, the results obtained being in substantial agreement.

Dilution of yeast-juice with sugar solution, so that the concentration of the sugar remains constant, produces a small progressive diminution in the total fermentation, which only becomes marked when more than 2 volumes are added, and this independently of the actual concentration of the sugar. Dilution with water produces a somewhat more decided diminution, which, however, does not exceed 50 per cent. of the total for the addition of 3 volumes of water. The effect on maceration extract is somewhat greater but of the same kind. The autofermentation of juice from top yeast is scarcely affected by dilution with 4 volumes of water.

On the whole, therefore, yeast-juice may be said to be only slightly affected by dilution even with pure water, and the effect of the latter can in no way be regarded as comparable with the poisonous effect which it exerts on living protoplasm, as suggested by Macfadyen, Morris, and Rowland [1900].

(g) The Effect of Antiseptics on the Fermentation of Sugars by Yeast-Juice.

Buchner has paid special attention to the effect of antiseptics on the course of fermentation by yeast-juice [Buchner and Rapp, 1897; 1898, 2, 3; 1899, 1; Buchner and Antoni, 1905, 1; Buchner and Hoffmann, 1907; Buchner, E. and H., and Hahn, 1903, pp. 169–205; see also Albert, 1899, 2; Gromoff and Grigorieff, 1904; Duchaček, 1909] in order (1) to obtain evidence as to the possibility of the active agent in yeast-juice consisting of fragments of protoplasm and not of a soluble enzyme, and (2) also to provide a safe method of avoiding contamination, by the growth of bacteria or yeasts, of the liquids used which were often kept at 25° for several days. The results of these experiments are briefly summarised in the following table, in which the effect of each substance on the total fermentation produced is noted:—

Hydrocyanic acid, even in dilute solution, completely suspends the fermenting power of the juice, without, however, producing any permanent change in the fermenting complex, as is shown by the fact that when the hydrocyanic acid is removed by a current of air, the juice regains its fermenting power. In this respect hydrocyanic acid behaves precisely as with many other enzymes and with colloidal platinum [Bredig, 1901]. Sodium arsenite is a pronounced protoplasmic poison, which rapidly destroys the power of growth and reproduction in living cells, and was therefore applied to yeast-juice to differentiate between protoplasmic and enzymic action. It was, however, found that the action of this substance was complicated by some unknown factor and very irregular results were obtained [Buchner, E. and H., and Hahn, 1903, pp. 193 ff.]. These phenomena appear to be of the same order as those produced by the addition of arsenates to yeast-juice [Harden and Young, 1906, 3], and will be discussed along with the latter (p. 77).

Permanent Preparations Containing Active Zymase.

A considerable number of preparations have been obtained in the dry state which retain some proportion of the fermenting power of yeast or yeast-juice.

Starting with yeast-juice, it is possible to arrive at this result either by evaporation or precipitation. When the juice is very rapidly evaporated to a syrup at 20° to 25° and then further dried at 35°, either in the air or in a vacuum and finally exposed over sulphuric acid in a vacuum desiccator, a dry brittle mass is obtained which is soluble in water and retains practically the whole of the fermenting power of the juice. The success of the preparation depends on the nature of the yeast from which the juice is derived, Berlin yeasts V and S yielding much less satisfactory results than Munich yeast. The powder when [p038] thoroughly dry is found to retain its properties almost unimpaired for at least a year, and can be heated to 85° for eight hours without undergoing any serious loss of fermenting power [Buchner and Rapp, 1898, 4; 1901; Buchner, E. and H., and Hahn, 1903, pp. 132–9].

Active powders can also be obtained by precipitating yeast-juice with alcohol, alcohol and ether, or acetone. The preparation is best effected by bringing the juice into 10 volumes of acetone, centrifuging at once and as rapidly as possible, washing, first with acetone and then with ether, and finally drying over sulphuric acid. The white powder thus obtained is not completely soluble in water but is almost entirely dissolved by aqueous glycerol (2·5 to 20 per cent.), forming a solution which has practically the same fermenting power as the original juice. The precipitation can be repeated without any serious loss of fermenting power. Prolonged contact of the precipitate with the supernatant liquid, especially when alcohol or alcohol and ether are used, causes a rapid loss of the characteristic property [Albert and Buchner, 1900, 1, 2; Buchner, E. and H., and Hahn, 1903, pp. 228–246; Buchner and Duchaček, 1909].

Dry preparations capable of fermenting sugar can also be readily obtained from yeast without any preliminary rupture of the cells. Heat alone (yielding a product known as hefanol) or treatment with dehydrating agents may be used for this purpose, and a brief allusion has already been made (p. 21) to the different varieties of permanent yeast (Dauerhefe) obtainable in these ways. The most important of these products are the dried Munich yeast (Lebedeff, see p. 25), and the material known as zymin, which is now made under patent rights for medicinal purposes by Schroder of Munich. The latter has proved of value in the investigation of the production of zymase in the yeast cell [Buchner and Spitta, 1902], and of many other problems concerned with alcoholic fermentation. In order to prepare it 500 grams of finely divided pressed brewer's yeast, containing about 70 per cent. of water, are brought into 3 litres of acetone, stirred for ten minutes, and filtered and drained at the pump. The mass is then well mixed with 1 litre of acetone for two minutes and again filtered and drained. The residue is roughly powdered, well kneaded with 250 c.c. of ether for three minutes, filtered, drained, and spread on filter paper or porous plates. After standing for an hour in the air it is dried at 45° for twenty-four hours. About 150 grams of an almost white powder containing only 5·5 to 6·5 per cent. of water are obtained. This is quite incapable of growth or reproduction but produces a very considerable amount of alcoholic fermentation, far greater indeed than a corresponding [p039] quantity of yeast-juice. Two grams of the powder corresponding to 6 grams of yeast and about 3·5 to 4 c.c. of yeast-juice, are capable of fermenting about 2 grams of sugar, whereas the 4 c.c. of yeast-juice would on the average only ferment from one-quarter to one-sixth of this amount of sugar. The rate produced by this amount of zymin is about one-eighth of that given by the corresponding amount of living yeast [Albert, 1900; Albert, Buchner, and Rapp, 1902]. The proteoclastic ferment is still present in zymin, which undergoes autolysis in presence of water in a similar manner to yeast-juice [Albert, 1901, 2].

As already mentioned an active juice can be prepared by grinding acetone-yeast with water, sand, and kieselguhr, and this process presents the advantage that samples of yeast-juice of approximately constant composition can be prepared at intervals from successive portions of a uniform supply of acetone-yeast.

Preparations of acetone-yeast, made from yeast freed from glycogen by exposure in a thin layer to the air for three or four hours at 35° to 45°, or eight hours at the ordinary temperature [Buchner and Mitscherlich, 1904], show practically no autofermentation and may be used analytically for the estimation of fermentable sugars.

All the foregoing preparations exhibit the same general properties as yeast-juice, as regards their behaviour towards the various sugars, antiseptics, etc.

When zymin is mixed with sugar solution without being previously ground, it exhibits a peculiarity which is of some practical interest. The time which elapses before the normal rate of fermentation is attained and the total fermentation obtainable vary with the amount of sugar solution added, the time increasing and the total diminishing as the quantity of this increases. This phenomenon appears to have been noticed by Trommsdorff [1902], and a single experiment of Buchner shows the influence of the same conditions [Buchner, E. and H., and Hahn, 1903, p. 265, Nos. 700–1]. Harden and Young have found that when 2 grams of zymin are mixed with varying quantities of 10 per cent. sugar solution the following results are obtained:—

This behaviour appears to be due to the removal of soluble matter essential for fermentation from the cell, which is discussed later on. It follows that when zymin is being tested for fermenting power, a uniform method should be adopted, and all comparative tests should be made with the same volumes of added sugar solution. Ground zymin appears to begin to ferment somewhat more slowly than unground (2 grm. to 12·4 c.c. of sugar solution in each case), but eventually produces the same total volume of gas [Buchner and Antoni, 1905, 1].

CHAPTER III. THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION.

As the explanation of the comparatively rapid disappearance of the fermenting power from yeast-juice had been sought, as already mentioned (p. 20), in the hydrolytic action of the tryptic enzyme which always accompanies zymase, the experiment was made of carrying out the fermentation in the presence of serum, with the result that about 60 to 80 per cent. more sugar was fermented than in the absence of the serum [Harden, 1903].

This fact was the starting-point of a series of attempts to obtain a similar effect by different means, in the course of which a boiled and filtered solution of autolysed yeast-juice was used, in the hope that the products formed by the action of the tryptic enzyme on the proteins of the juice would, in accordance with the general rule, prove to be an effective inhibitant of that enzyme. This solution was, in fact, found to produce a very marked increase in the total fermentation effected by yeast-juice, the addition of a volume of boiled juice equal to that of the yeast-juice doubling the amount of carbon dioxide evolved [Harden and Young, 1905, 1]. This effect was found to be common to the filtrates from boiled fresh yeast-juice and from boiled autolysed yeast-juice, and was ultimately traced in the main, not to the antitryptic effect which had been surmised, but to two independent factors, either of which was capable in some degree of bringing about the observed result.

Boiled yeast-juice was indeed found to possess a decided anti-autolytic effect, as determined by a comparison of the amounts of nitrogen rendered non-precipitable by tannic acid in yeast-juice alone [p042] and in a mixture of yeast-juice and boiled juice on preservation [Harden, 1905]. The anti-autolytic effect, however, appeared to vary independently of the effect on the fermentation, and the conclusion was drawn, as stated above, that the increase in the alcoholic fermentation was not directly dependent on the decrease in the action of the proteoclastic enzyme but was due to some independent cause. The property possessed by boiled yeast-juice of diminishing the autolysis of yeast-juice has now been carefully examined by Buchner and Haehn [1910, 2] and ascribed by them to a soluble antiprotease (p. 65).

The two factors to which the increase in fermentation produced by the addition of boiled juice were ultimately traced were (1) the presence of phosphates in the liquid, and (2) the existence in boiled fresh yeast-juice of a co-ferment or co-enzyme, the presence of which is indispensable for fermentation [Harden and Young, 1905, 1, 2].

The former of these factors will be here discussed and the co-enzyme will form the subject of the following chapter.

The general fact that sodium phosphate increases the total fermentation produced by a given volume of yeast juice was observed on several occasions by Wroblewski [1901] and also by Buchner [Buchner, E. and H., and Hahn, 1903, pp. 141–2], who ascribed the action of this salt to its alkalinity, comparing it in this respect with potassium carbonate and remarking that the increase in both cases took place chiefly in the first twenty hours of fermentation. The increased amount of fermentation following the addition of boiled yeast-juice was also noted by Buchner and Rapp [1899, 2, No. 265, p. 2093] in a single experiment.

Observations made at intervals of a few minutes instead of twenty hours have, however, revealed the fact that phosphates play a part of fundamental importance in alcoholic fermentation and that their presence is absolutely essential for the production of the phenomenon.

Effect of the Addition of Phosphate to a Fermenting Mixture of Yeast-Juice and Sugar.

When a suitable quantity[2] of a soluble phosphate is added to a fermenting mixture of glucose, fructose, or mannose with yeast-juice, the rate of fermentation rapidly rises, sometimes increasing as much as twenty-fold, continues at this high value for a certain period and then falls again to a value approximately equal to, but generally [p043] somewhat higher than, that which it originally had. Careful experiments have shown that during this period of enhanced fermentation the amounts of carbon dioxide and alcohol produced exceed those which would have been formed in the absence of added phosphate by a quantity exactly equivalent to the phosphate added in the ratio CO₂ or C₂H₆O:R′₂HPO₄ [Harden and Young, 1906, 1].

This result is of fundamental importance, and the evidence on which it rests deserves some consideration. Quantitative experiments on this subject require certain preliminary precautions. The acid phosphates are too acid to permit of any extended fermentation and the phosphates of the formula R′₂HPO₄ absorb a considerable volume of carbon dioxide with production of a bicarbonate, according to the reaction:—

The method which has been adopted, therefore, is to employ either a secondary phosphate saturated with carbon dioxide at the temperature of the experiment, or a mixture of five molecular proportions of the secondary phosphate with one molecular proportion of a primary phosphate, in which the amount of bicarbonate formed is negligible. In the former case it is necessary to ascertain whether any of the carbon dioxide evolved is derived from the bicarbonate by the action of acid originally present or produced in the yeast-juice or by a disturbance of the original equilibrium owing to the chemical change which occurs. This is done by acidifying duplicate samples with hydrochloric acid before and after the fermentation and measuring the gas evolved in each case. Any necessary correction can then be made. The calculation of the extra amount of carbon dioxide evolved from yeast-juice containing sugar when a phosphate is added involves an estimation of the amount which would have been evolved in the absence of added phosphate, and this is a matter of some difficulty. Since the final steady rate of fermentation attained is often slightly different from the initial rate, the practice has been adopted of ascertaining this final rate and then calculating the total evolution corresponding to it for the whole period from the time of the addition of the phosphate to the end of the observations. This amount deducted from the observed total leaves the extra amount of carbon dioxide formed, and it is this quantity which is equivalent to the phosphate added. Alcohol is simultaneously produced in the normal ratio. The justification for this method of calculation will be found later (p. 54).

The following table, containing the results of experiments with [p044] glucose, fructose, and mannose, indicates very clearly the nature of the method of calculation and also of the agreement between observation and theory.

These numbers agree well with the value calculated from the phosphate added, viz. 32·6 [Harden and Young, 1909].

Another experiment is illustrated graphically in Fig. 4, in which the volume of carbon dioxide evolved is plotted against time. The determination was in this case made by adding 25 c.c. of an aqueous solution containing 5 grams of glucose to one quantity of 25 c.c. of yeast-juice (curve A) and 5 c.c. of 0·3 molar solution of the mixed primary and secondary sodium phosphates, and 20 c.c. of a solution containing 5 grams of glucose to a second equal quantity of yeast-juice (curve B). Curve A shows the normal course of fermentation of yeast-juice with glucose. There is a slight preliminary acceleration during the first twenty minutes, due to free phosphate in the juice, and the rate then becomes steady at about 1·4 c.c. in five minutes. During this preliminary acceleration 10 c.c. of extra carbon dioxide are evolved, this number being obtained graphically by continuing the line of steady rate back to the axis of zero time. Curve B shows the effect of the added phosphate. The rate rises to about 9·5 c.c. in five minutes, i.e. to more than six times the normal rate, and then gradually falls until after an hour it is again steady and almost exactly equal to 1·4 c.c. per five minutes. Continuing the line of steady rate back to the axis of zero [p045] time it is found that the extra amount of carbon dioxide is 48 c.c. Subtracting from this the 10 c.c. shown in curve A as due to the juice alone, a difference of 38 c.c. is obtained due to the added phosphate. The amount calculated from the phosphate added in this case is, at atmospheric temperature and pressure, 38·9 c.c.

It may be noted that in this case the observations after each addition last fifty to seventy minutes, so that an error of 0·1 c.c. per five minutes in the estimated final rate would make an error of 1 to 1·4 c.c. in the extra amount of carbon dioxide, i.e. about 3 to 4 per cent. of the total, and this is approximately the limit of accuracy of the method. [p046] The results are more precise when the yeast-juice employed is an active one, since, when the fermenting power of the juice is low, the initial period of accelerated fermentation is unduly prolonged and the calculation of the extra amount of carbon dioxide is rendered uncertain.

Zymin (p. 38) yields precisely similar results to yeast-juice, but in this case the rate of fermentation is not so largely increased. This has the effect that the extra amount of carbon dioxide cannot be quite so accurately estimated for zymin, because a slight error in the determination of the final rate of fermentation has a greater influence on the result. The equivalence between the extra amount of carbon dioxide evolved and the phosphate added is, however, unmistakable, as is shown by the following results of an experiment with zymin, in which 6 grams of zymin (Schroder) + 3 grams of fructose (Schering) + 25 c.c. of water were incubated at 25° in presence of toluene until a steady rate had been attained. Five c.c. of a solution of sodium phosphate equivalent to 32·2 c.c. carbon dioxide at N.T.P. were then added.

Considering the small proportional rise in rate and the long period of accelerated fermentation, the agreement between the volume observed, 29·8 c.c., and that calculated from the phosphate, 32·2, is quite satisfactory [Harden and Young, 1910, 1.] Precisely the same relations hold for maceration extract, but in this case it must be remembered that a large amount of free phosphate is present in the extract, as much as 0·3129 grm. Mg₂P₂O₇ being obtained from 20 c.c. in one preparation, so that the original extract had the concentration of a 0·14 molar solution of sodium phosphate. It is in fact not improbable that the delay in the onset of fermentation sometimes observed with maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal, 1913) may be due to the presence of phosphate in so great an excess of the amount which can be rapidly esterified by the enzymes that the rate of fermentation is at first greatly lowered (see p. 71). When this phosphate is removed by incubation with glucose or fructose, the subsequent addition of phosphate produces the characteristic action and the extra carbon dioxide evolved is, as with other yeast preparations, equivalent to the phosphate added. An actual estimation carried out in this way gave 35 c.c. of CO₂ for an addition of phosphate equivalent to 32·9 c.c. [Harden and Young, 1912]. [p047]

This fact indicates that a definite chemical reaction occurs in which sugar and phosphate are concerned, and this conclusion is confirmed when the fate of the added phosphate is investigated. If an experiment, such as one of those described above, be interrupted as soon as the rate of fermentation has again become normal, and the liquid be boiled and filtered, it is found that nearly the whole of the phosphorus present passes into the filtrate, but that only a small proportion of this exists as mineral phosphate, whilst the remainder, including that added in the form of a soluble phosphate, is no longer precipitable by magnesium citrate mixture [Harden and Young, 1905, 2].

A similar observation was made at a later date by Iwanoff [1907], who had previously observed [1905] that living yeast, like many other vegetable organisms, converted mineral phosphates into organic derivatives. Iwanoff employed zymin and hefanol (p. 38) instead of yeast-juice, and found that phosphates were thereby rendered non-precipitable by uranium acetate solution, but did not observe the accelerated fermentation caused by their addition.

The foregoing conclusions have been strikingly confirmed by experiments with maceration extract carried out by Euler and Johansson [1913], in which both the carbon dioxide evolved and the phosphate rendered non-precipitable by magnesia were determined at intervals. When dried yeast is employed as the fermenting agent, the amount of phosphate esterified in the earlier stages is greater than would be expected, but ultimately becomes exactly equivalent to the carbon dioxide evolved.

Nature of the Phospho-organic Compound formed by Yeast-Juice and Zymin from the Hexoses and Phosphate.

The formation and properties of the compound produced from phosphates in the manner just described have been investigated by Harden and Young [1905, 2; 1908, 1; 1909; 1911, 2], Young [1909; 1911], Iwanoff [1907; 1909, 1], Lebedeff [1909; 1910; 1911, 5, 6; 1912, 3; 1913, 1]; and Euler [1912, 1; Euler and Fodor, 1911; Euler and Kullberg, 1911, 3; Euler and Ohlsén, 1911; 1912; Euler and Johansson, 1912, 4; Euler and Bäckström, 1912], but its exact constitution cannot as yet be regarded as definitely known. [p048]

Phosphates undergo this characteristic change when the sugar undergoing fermentation is glucose, mannose, or fructose, and it may be said at once that no distinction can be established between the products formed from these various hexoses; they all appear to be identical. The compound produced is, as already mentioned, not precipitated by ammoniacal magnesium citrate mixture, nor by uranium acetate solution. It can, however, be precipitated by copper acetate (Iwanoff) and by lead acetate (Young). The preparation of the pure lead salt from the liquid obtained by fermenting a sugar with yeast-juice or zymin in presence of phosphate is commenced by boiling and filtering the liquid. Magnesium nitrate solution and a small quantity of caustic soda solution are then added to precipitate any free phosphate, and the liquid well stirred and allowed to stand over night. To the neutralised filtrate lead acetate is then added together with sufficient caustic soda solution to maintain the reaction neutral to litmus, until no further precipitate is formed. The liquid is then filtered or, better, centrifugalised, and the precipitate repeatedly washed with water until a portion of the clear filtrate gives no reduction when boiled with Fehling's solution. It is essential that this washing should be thorough as evidence has recently been obtained of the formation under certain conditions of a hexosephosphate, the lead salt of which is not so sparingly soluble as that of the hexosediphosphate [Harden and Robison, 1914]. The lead precipitate is then suspended in water, decomposed by a current of sulphuretted hydrogen, the clear filtrate freed from sulphuretted hydrogen by a current of air, and finally neutralised with caustic soda. The removal of phosphate and conversion into lead salt are repeated twice, and the resulting lead salt is then found to be free from nitrogen and to have a composition represented by the formula C₆H₁₀O₄(PO₄Pb)₂. Lebedeff carries out the preparation in a somewhat different manner. The fermentation is effected by means of air-dried yeast (150 grams to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture of 2 parts Na₂HPO₄ and 1 part NaH₂PO₄) and the liquid (about 700 c.c.) after boiling and filtering, is treated with an equal volume of acetone. About 300 c.c. of a thick liquid is precipitated and this is redissolved in water and precipitated by an equal volume of acetone two or three times. The final liquid is then precipitated with warm lead acetate solution and filtered and washed with dilute lead acetate solution until the filtrate is clear and no longer reduces Fehling's solution after removal of the lead [1910]. Euler and Fodor [1911] on the other hand precipitate the free phosphate with magnesia mixture and then add acetone, dissolve the syrup thus precipitated in water and add copper [p049] acetate solution. A blue copper salt is precipitated which is thoroughly washed with water and used for the preparation of solutions of the acid. A solution of the free acid can readily be prepared by the action of sulphuretted hydrogen on the lead salt suspended in water. It forms a strongly acid liquid, which requires exactly two equivalents of base for each atom of phosphorus present to render it neutral to phenolphthalein. It decomposes when evaporated, leaving a charred mass containing free phosphoric acid. The acid is slightly optically active, and has [a_D] = + 3·4°. A number of amorphous salts have been prepared by precipitation from a solution of the sodium salt, and of these the silver, barium, and calcium salts have been analysed with results agreeing with the general formula C₆H₁₀O₄(PO₄R′₂)₂. The magnesium, calcium, barium, and manganese salts, which are only sparingly soluble, are all precipitated when their solutions are boiled but re-dissolve on cooling, and this property can be utilised for their purification. The alkali salts have only been obtained as viscid residues.

A difference of opinion exists as to the molecular weight and constitution of this substance. Iwanoff [1909, 1] regards it as a triosephosphoric acid, C₃H₅O₂(PO₄H₂), basing this view on the preparation of an osazone which melted at 142°, but when recrystallised from benzene gave a product melting at 127°–8°, which had the same appearance, melting-point, and nitrogen content as the triosazone formed by the action of phenylhydrazine on the oxidation products of glycerol. Neither Lebedeff [1909] nor Young could obtain Iwanoff's osazone, and all attempts to reduce the acid with formation of glycerol either by sodium amalgam or hydriodic acid were unsuccessful (Young). There is therefore practically no serious experimental evidence in favour of Iwanoff's view.

On the other hand, Harden and Young regard the acid as a diphosphoric ester of a hexose. This view is based on the fact that when the acid is boiled with water, or an acid, free phosphoric acid is produced along with a levo-rotatory solution containing fructose and possibly a small proportion of some other sugar or sugars. (Euler and Fodor however did not obtain a hexose in this way [1911].) The acid itself only reduces Fehling's solution after some hours in the cold, rapidly when boiled, whereas when its solution is first boiled, and then treated with Fehling's solution in the cold, the products of decomposition bring about reduction in a few minutes. The reduction brought about when the acid is boiled with Fehling's solution is considerably less (33 per cent.) than that produced by an equivalent amount of glucose. The behaviour of the compound towards phenylhydrazine is also in complete agreement [p050] with this view. Lebedeff found [1909, 1910] that the acid or its salts heated with phenylhydrazine in presence of acetic acid gave an insoluble compound which was ultimately found to be the phenylhydrazine salt of hexosemonophosphoric acid osazone