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Title: Alcoholic Fermentation

Second Edition, 1914

Author: Arthur Harden

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Language: English

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ALCOHOLIC

FERMENTATION

2nd Edition, 1914

by

Arthur Harden

MONOGRAPHS ONBIOCHEMISTRY

EDITED BYR. H. A. PLIMMER, D.Sc.

ANDF. G. HOPKINS, M.A., M.B., D.Sc.,

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LONGMANS, GREEN AND CO.,LONDON, NEW YORK, BOMBAY, CALCUTTA, AND

MADRAS.

ALCOHOLICFERMENTATION

BY

ARTHUR HARDEN,

PH.D., D.SC., F.R.S.PROFESSOR OF BIOCHEMISTRY, LONDON

UNIVERSITYHEAD OF THE BIOCHEMICAL

DEPARTMENT, LISTER INSTITUTE,CHELSEA

SECOND EDITION

LONGMANS, GREENAND CO.

39 PATERNOSTER ROW,LONDON

FOURTH AVENUE & 30TH STREET,NEW YORK,

BOMBAY, CALCUTTA, AND MADRAS1914

PREFACE.

The following chapters are based oncourses of lectures delivered at theLondon University and the RoyalInstitution during 1909–1910. In theman account is given of the work done onalcoholic fermentation since Buchner'sepoch-making discovery of zymase,only in so far as it appears to throwlight on the nature of that phenomenon.Many interesting subjects, therefore,have perforce been left untouched,among them the problem of the

formation of zymase in the cell, andthe vexed question of the relation ofalcoholic fermentation to the metabolicprocesses of the higher plants andanimals.

My thanks are due to the Council ofthe Royal Society, and to thePublishers of the "Journal ofPhysiology" for permission to makeuse of blocks (Figs. 2, 4 and 7) whichhave appeared in their publications.

A. H.

PREFACE TO THESECOND EDITION.

In the New Edition no change has beenmade in the scope of the work. Therapid progress of the subject has,however, rendered necessary manyadditions to the text and a considerableincrease in the bibliography.

A. H.May,1914.

CONTENTS.

CHAPTER PAGE

I. HISTORICAL

INTRODUCTION

1

II. ZYMASE AND ITS

PROPERTIES

18

III. THE FUNCTION OF

PHOSPHATES IN

ALCOHOLIC

FERMENTATION

41

IV. THE CO-ENZYME OF59

YEAST-JUICE

V. ACTION OF SOME

INHIBITING AND

ACCELERATING

AGENTS ON THE

ENZYMES OF

YEAST-JUICE

70

VI. CARBOXYLASE81

VII. THE BY-PRODUCTS

OF ALCOHOLIC

FERMENTATION

85

VIII. THE CHEMICAL96

CHANGES

INVOLVED IN

FERMENTATION

IX. THE MECHANISM OF

FERMENTATION

119

BIBLIOGRAPHY136

INDEX155

CHAPTER I.HISTORICAL INTRODUCTION.

[p001]

The problem of alcoholic fermentation,of the origin and nature of thatmysterious and apparently spontaneouschange which converted the insipidjuice of the grape into stimulatingwine, seems to have exerted afascination over the minds of naturalphilosophers from the very earliesttimes. No date can be assigned to thefirst observation of the phenomena of

the process. History finds man in thepossession of alcoholic liquors, and inthe earliest chemical writings we findfermentation, as a familiar naturalprocess, invoked to explain andillustrate the changes with which thescience of those early days wasconcerned. Throughout the period ofalchemy fermentation plays animportant part; it is, in fact, scarcelytoo much to say that the language ofthe alchemists and many of their ideaswere founded on the phenomena offermentation. The subtle change inproperties permeating the whole mass

of material, the frothing of thefermenting liquid, rendering evidentthe vigour of the action, seemed tothem the very emblems of themysterious process by which the longsought for philosopher's stone was toconvert the baser metals into gold. Aschemical science emerged from themists of alchemy, definite ideas aboutthe nature of alcoholic fermentationand of putrefaction began to be formed.Fermentation was distinguished fromother chemical changes in which gaseswere evolved, such as the action ofacids on alkali carbonates (Sylvius de

le Boë, 1659); the gas evolved wasexamined and termed gas vinorum, andwas distinguished from the alcoholwith which it had at first been confused(van Helmont, 1648); afterwards it wasfound that like the gas from potashes itwas soluble in water (Wren, 1664). Thegaseous product of fermentation andputrefaction was identified byMacBride, in 1764, with the fixed airof Black, whilst Cavendish in 1766showed that fixed air alone wasevolved in alcoholic fermentation andthat a mixture of this with inflammableair was produced by putrefaction. In

the meantime it had been recognisedthat only sweet liquors could befermented ("Ubi notandum, nihilfermentare quod non sit dulce," Becher,1682), and finally Cavendish [p002]

[1776] determined the proportion offixed air obtainable from sugar byfermentation and found it to be 57 percent. It gradually became recognisedthat fermentation might yield eitherspirituous or acid liquors, whilstputrefaction was thought to be anaction of the same kind asfermentation, differing mainly in thecharacter of the products (Becher).

As regards the nature of the processvery confused ideas at first prevailed,but in the time of the phlogisticchemists a definite theory offermentation was proposed, first byWillis (1659) and afterwards by Stahl[1697], the fundamental idea of whichsurvived the overthrow of thephlogistic system by Lavoisier andformed the foundation of the views ofLiebig. To explain the spontaneousorigin of fermentation and itspropagation from one liquid to another,they supposed that the processconsisted in a violent internal motion

of the particles of the fermentingsubstance, set up by an aqueous liquid,whereby the combination of theessential constituents of this materialwas loosened and new particlesformed, some of which were thrust outof the liquid (the carbon dioxide) andothers retained in it (the alcohol).

Stahl specifically states that a bodyin such a state of internal disquietudecan very readily communicate thedisturbance to another, which is itselfat rest but is capable of undergoing asimilar change, so that a putrefying orfermenting liquid can set another liquid

in putrefaction or fermentation.Taking account of the gradual

accumulation of fact and theory wefind at the time of Lavoisier, fromwhich the modern aspect of theproblem dates, that Stahl's theoreticalviews were generally accepted.Alcoholic fermentation was known torequire the presence of sugar and wasthought to lead to the production ofcarbon dioxide, acetic acid, andalcohol.

The composition of organiccompounds was at that time notunderstood, and it was Lavoisier who

established the fact that they consistedof carbon, hydrogen, and oxygen, andwho made systematic analyses of thesubstances concerned in fermentation(1784–1789). Lavoisier [1789] appliedthe results of these analyses to thestudy of alcoholic fermentation, and byemploying the principle which heregarded as the foundation ofexperimental chemistry, "that there isthe same quantity of matter before andafter the operation," he drew up anequation between the quantities ofcarbon, hydrogen, and oxygen in theoriginal sugar and in the resulting

substances, alcohol, carbon dioxide,and acetic acid, showing that theproducts contained the whole matter ofthe sugar, and thus for the first timegiving a clear view of the chemical[p003] change which occurs infermentation. The conclusion to whichhe came was, we now know, verynearly accurate, but the research mustbe regarded as one of those remarkableinstances in which the genius of theinvestigator triumphs overexperimental deficiencies, for theanalytical numbers employedcontained grave errors, and it was only

by a fortunate compensation of thesethat a result so near the truth wasattained.

Lavoisier's equation or balance sheetwas as follows:—

Carbon. Hydrogen.95·9 poundsof sugar(canesugar)consist of 26·8 7·7

These yield:—57·7 poundsof alcoholcontaining 16·7 9·6

35·3 poundsof carbondioxidecontaining 9·9

2·5 poundsof aceticacidcontaining 0·6 0·2

——— ———Totalcontainedin products 27·2 9·8

The true composition of the sugarused was carbon 40·4, hydrogen 6·1,oxygen 49·4.

Lavoisier expressed no view as tothe agency by which fermentation wasbrought about, but came to a verydefinite and characteristic conclusionas to the chemical nature of the change.The sugar, which he regarded inharmony with his general views as anoxide, was split into two parts, one ofwhich was oxidised at the expense ofthe other to form carbonic acid, whilstthe other was deoxygenised in favourof the former to produce the

combustible substance alcohol, "so thatif it were possible to recombine thesetwo substances, alcohol and carbonicacid, sugar would result".

From this point commences themodern study of the problem. Providedby the genius of Lavoisier with theassurance that the hitherto mysteriousprocess of fermentation was to beranked along with familiar chemicalchanges, and that it proceeded inharmony with the same quantitativelaws as these simpler reactions,chemists were stimulated in theirdesire to penetrate further into the

mysteries of the phenomenon, and theimportance and interest of the problemattracted many workers.

So important indeed did the matterappear to Lavoisier's countrymen thatin the year 8 of the French Republic(1800) a prize—consisting of a goldmedal, the value of which, expressed interms of the newly introduced metricsystem, was that of one kilogram ofgold—was offered by the Institute forthe best answer to the question: "Whatare the characteristics by which animaland vegetable substances which act asferments can be distinguished from

those which they are capable offermenting?" [p004]

This valuable prize was againoffered in 1802 but was never awarded,as the fund from which it was to bedrawn was sequestrated from theInstitute in 1804. The first response tothis stimulating offer was an importantmemoir by citizen Thenard [1803],which provided many of the facts uponwhich Liebig subsequently based hisviews. Thenard combats the prevailingidea, first expressed by Fabroni (1787–1799), that fermentation is caused bythe action of gluten derived from grain

on starch and sugar, but is himselfuncertain as to the actual nature of theferment. He points out that allfermenting liquids deposit a materialresembling brewer's yeast, and heshows that this contains nitrogen, muchof which is evolved as ammonia ondistillation. His most important resultis, however, that when yeast is used toferment pure sugar, it undergoes agradual change and is finally left as awhite mass, much reduced in weight,which contains no nitrogen and iswithout action on sugar. Thenard,moreover, it is interesting to note,

differs from Lavoisier, inasmuch as heascribes the origin of some of thecarbonic acid to the carbon of theferment, an opinion which was stillheld in various degrees by manyinvestigators (see Seguin, quoted byThenard).

Thenard's memoir was followed by acommunication of fundamentalimportance from Gay-Lussac [1810]. Aprocess for preserving food had beenintroduced by Appert, which consistedin placing the material in bottles,closing these very carefully andexposing them to the temperature of

boiling water for some time. Gay-Lussac was struck by the fact that whensuch a bottle was opened fermentationor putrefaction set in rapidly. Analysisof the air left in such a sealed bottleshowed that all the oxygen had beenabsorbed, and these facts led to theview that fermentation was set up bythe action of oxygen on thefermentable material. Experimentappeared to confirm this in the moststriking way. A bottle of preservedgrape-juice was opened over mercuryand part of its contents passed throughthe mercury into a bell-jar containing

air, the remainder into a similar vesselfree from air. In the presence of airfermentation set in at once, in theabsence of air no fermentationwhatever occurred. This connectionbetween fermentation and the presenceof air was established by numerousexperiments and appearedincontestable. Fermentation, it wasfound, could be checked by boilingeven after the addition of oxygen, andhence food could be preserved in freecontact with the air, provided only thatit was raised to the temperature ofboiling water at short intervals of time.

Gay-Lussac's opinion was that theferment was formed by the action ofthe oxygen on the [p005] liquid, and thatthe product of this action was alteredby heat and rendered incapable ofproducing fermentation, as was alsobrewer's yeast, which, however, heregarded, on account of its insolubility,as different from the soluble fermentwhich initiated the change in thelimpid grape-juice. Colin, on the otherhand [1825], recognised that alcoholicfermentation by whatever substance itwas started, resulted in the formationof an insoluble deposit more active

than the original substance, and hesuggested that this deposit mightpossibly in every case be of the samenature.

So far no suspicion appears to havearisen in the minds of those who hadoccupied themselves with the study offermentation that this change differedin any essential manner from manyother reactions familiar to chemists.The origin and properties of theferment were indeed remarkable andinvolved in obscurity, but theuncertainty regarding this substancewas no greater than that surrounding

many, if not all, compounds of animaland vegetable origin. Although,however, the purely chemical view asto the nature of yeast was generallyrecognised and adopted, isolatedobservations were not wanting whichtended to show that yeast might besomething more than a mere chemicalreagent. As early as 1680 in letters tothe Royal Society Leeuwenhoekdescribed the microscopic appearanceof yeast of various origins as that ofsmall, round, or oval particles, but nofurther progress seems to have beenmade in this direction for nearly a

century and a half, when we find thatDesmazières [1826] examined the filmformed on beer, figured the elongatedcells of which it was composed, anddescribed it under the name ofMycoderma Cerevisiæ. He, however,regarded it rather as of animal than ofvegetable origin, and does not appearto have connected the presence of thesecells with the process of fermentation.

Upon this long period during whichyeast was regarded merely as achemical compound there followed, ashas so frequently occurred in similarcases, a sudden outburst of discovery.

No less than three observers hit almostsimultaneously upon the secret offermentation and declared that yeastwas a living organism.

First among these in strict order oftime was Cagniard-Latour [1838], whomade a number of communications tothe Academy and to the SociétéPhilomatique in 1835–6, the contentsof which were collected in a paperpresented to the Academy of Scienceson 12 June, 1837, and published in1838. The observations upon which thismemoir was based were almostexclusively microscopical. Yeast was

recognised as consisting of sphericalparticles, which were capable of [p006]

reproduction by budding but incapableof motion, and it was thereforeregarded as a living organism probablybelonging to the vegetable kingdom.Alcoholic fermentation was observedto depend on the presence of livingyeast cells, and was attributed to someeffect of their vegetative life (quelqueeffet de leur végétation). It was alsonoticed that yeast was not deprived ofits fermenting power by exposure tothe temperature of solid carbonic acid,a sample of which was supplied to

Cagniard-Latour by Thilorier, who hadonly recently prepared it for the firsttime.

Theodor Schwann [1837], whoseresearches were quite independent ofthose of Cagniard-Latour, approachedthe problem from an entirely differentpoint of view. During the year 1836Franz Schulze [1836] published aresearch on the subject of spontaneousgeneration, in which he proved thatwhen a solution containing animal orvegetable matter was boiled, noputrefaction set in provided that all airwhich was allowed to have access to

the liquid was previously passedthrough strong sulphuric acid. Schwannperformed a very similar experimentby which he showed that this sameresult, the absence of putrefaction, wasattained by heating all air which cameinto contact with the boiled liquid.Wishing to show that other processesin which air took part were not affectedby the air being heated, he madeexperiments with fermenting liquidsand found, contrary to his expectation,that a liquid capable of undergoingvinous fermentation and containingyeast did not undergo this change after

it had been boiled, provided that, as inthe case of his previous experiments,only air which had been heated wasallowed to come into contact with it.

Schwann's experiments on theprevention of putrefaction wereunexceptionable and quite decisive.The analogous experiments dealingwith alcoholic fermentation were notquite so satisfactory. Yeast was addedto a solution of cane sugar, the flaskcontaining the mixture placed inboiling water for ten minutes, and theninverted over mercury. About one-thirdof the liquid was then displaced by air

and the flasks corked and kept invertedat air temperature. In two flasks the airintroduced was ordinary atmosphericair, and in these flasks fermentation setin after about four to six weeks. Intothe other two flasks air which had beenheated was led, and in these nofermentation occurred. As described,the experiment is quite satisfactory, butSchwann found on repetition that theresults were irregular. Sometimes allthe flasks showed fermentation,sometimes none of them. This wascorrectly ascribed to the experimentaldifficulties, but none [p007] the less

served as a point of attack for hostileand damaging criticism at the hands ofBerzelius (p. 8).

The origin of putrefaction wasdefinitely attributed by Schwann to thepresence of living germs in the air, andthe similarity of the result obtainedwith yeast suggested the idea thatalcoholic fermentation was alsobrought about by a living organism, aconception which was at onceconfirmed by a microscopicalexamination of a fermenting liquid.The phenomena observed under themicroscope were similar to those noted

by Cagniard-Latour, and in accordancewith these observations alcoholicfermentation was attributed to thedevelopment of a living organism, thefermentative function of which wasfound to be destroyed by potassiumarsenite and not by extract of Nuxvomica, so that the organism wasregarded rather as of vegetable than ofanimal nature. This plant received thename of "Zuckerpilz" or sugar fungus(which has been perpetuated in thegeneric term Saccharomyces).Alcoholic fermentation was explainedas "the decomposition brought about by

this sugar fungus removing from thesugar and a nitrogenous substance thematerials necessary for its growth andnourishment, whilst the remainingelements of these compounds, whichwere not taken up by the plant,combined chiefly to form alcohol".

Kützing's memoir, the third of thetrio [1837], also dates from 1837, andhis opinions, like those of Cagniard-Latour, are founded on microscopicalobservations. He recognises yeast as avegetable organism and accuratelydescribes its appearance. Alcoholicfermentation depends on the formation

of yeast, which is produced when thenecessary elements and the properconditions are present and thenpropagates itself. The action on theliquid thus increases and theconstituents not required to form theorganism combine to form unorganisedsubstances, the carbonic acid andalcohol. "It is obvious," says Kützing,in a passage which roused the sarcasmof Berzelius, "that chemists must nowstrike yeast off the roll of chemicalcompounds, since it is not a compoundbut an organised body, an organism."

These three papers, which were

published almost simultaneously, werereceived at first with incredulity.Berzelius, at that time the arbiter anddictator of the chemical world,reviewed them all in his"Jahresbericht" for 1839 [1839] withimpartial scorn. The microscopicalevidence was denied all value, andyeast was no more to be regarded as anorganism than was a precipitate ofalumina. Schwann's experiment (p. 6)was criticised on the ground that thefermenting power of the added yeasthad been only partially destroyed in the[p008] flasks in which fermentation

ensued, completely in those whichremained unchanged, the admission ofheated or unheated air beingindifferent, a criticism to some extentjustified by Schwann's statement,already quoted, of the uncertain resultof the experiment.

Berzelius himself regardedfermentation as being brought about bythe yeast by virtue of that catalyticforce, which he had supposed tointervene in so many reactions, bothbetween substances of mineral and ofanimal and vegetable origin [1836],and which enabled "bodies, by their

mere presence, and not by theiraffinity, to arouse affinities ordinarilyquiescent at the temperature of theexperiment, so that the elements of acompound body arrange themselves insome different way, by which a greaterdegree of electro-chemicalneutralisation is attained".

To the scorn of Berzelius was soonadded the sarcasm of Wöhler andLiebig [1839, 1839]. Stimulated in partby the publications of the three authorsalready mentioned, and in part by thereport of Turpin [1838], who at therequest of the Academy of Sciences

had satisfied himself by observation ofthe accuracy of Cagniard-Latour'sconclusions, Wöhler prepared anelaborate skit on the subject, which hesent to Liebig, to whom it appealed sostrongly that he added some touches ofhis own and published it in the"Annalen," following immediatelyupon a translation of Turpin's paper.Yeast was here described with aconsiderable degree of anatomicalrealism as consisting of eggs whichdeveloped into minute animals, shapedlike a distilling apparatus, by which thesugar was taken in as food and digested

into carbonic acid and alcohol, whichwere separately excreted, the wholeprocess being easily followed under themiscroscope.

Close upon this pleasantry followeda serious and important communicationfrom Liebig [1839], in which the natureof fermentation, putrefaction, anddecay was exhaustively discussed.Liebig did not admit that thesephenomena were caused by livingorganisms, nor did he attribute themlike Berzelius to the catalytic action ofa substance which itself survived thereaction unchanged. As regards

alcoholic fermentation, Liebig's chiefarguments may be briefly summarised.As the result of alcoholic fermentation,the whole of the carbon of the sugarreappears in the alcohol and carbondioxide formed. This change is broughtabout by a body termed the ferment,which is formed as the result of achange set up by the access of air toplant juices containing sugar, andwhich contains all the nitrogen of thenitrogenous constituents of the juice.This ferment is a substance remarkablysusceptible of change, which undergoesan uninterrupted and progressive

metamorphosis, of [p009] the nature ofputrefaction or decay, and produces thefermentation of the sugar as aconsequence of the transformationwhich it is itself undergoing.

The decomposition of the sugar istherefore due to a condition ofinstability transferred to it from theunstable and changing ferment, andonly continues so long as thedecomposition of the ferment proceeds.This communication of instability fromone substance undergoing chemicalchange to another is the basis ofLiebig's conception, and is illustrated

by a number of chemical analogies, oneof which will suffice to explain hismeaning. Platinum is itself incapableof decomposing nitric acid anddissolving in it; silver, on the otherhand, possesses this power. Whenplatinum is alloyed with silver, thewhole mass dissolves in nitric acid, thepower possessed by the silver beingtransferred to the platinum. In likemanner the condition of activedecomposition of the ferment istransferred to the sugar, which by itselfis quite stable. The central idea is thatof Stahl (p. 2) which was thus

reintroduced into scientific thought.In a pure sugar solution the

decomposition of the ferment sooncomes to an end and fermentation thenceases. In beer wort or vegetablejuices, on the other hand, more fermentis continually formed in the manneralready described from the nitrogenousconstituents of the juice, and hence thesugar is completely fermented awayand unexhausted ferment left behind.Liebig's views were reiterated in hiscelebrated "Chemische Briefe," andbecame the generally accepted doctrineof chemists. There seems little doubt

that both Berzelius and Liebig in theirscornful rejection of the results ofCagniard-Latour, Schwann andKützing, were influenced, perhapsalmost unconsciously, by a desire toavoid seeing an important chemicalchange relegated to the domain of thatvital force from beneath the sway ofwhich a large part of organic chemistryhad just been rescued by Wöhler'sbrilliant synthetical production of ureaand by the less recognised synthesis ofalcohol by Hennell (see on this pointAhrens [1902]). A strong body ofevidence, however, gradually

accumulated in favour of the vegetablenature of yeast, so that it may be saidthat by 1848 a powerful minorityadhered to the views of Cagniard-Latour, Schwann, and Kützing [seeSchrohe, 1904, p. 218, and compareBuchner, 1904]. Among these must beincluded Berzelius [1848], who had soforcibly repudiated the idea only tenyears before, whereas Liebig in the1851 edition of his letters does notmention the fact that yeast is a livingorganism (Letter XV).

The recognition of the vegetablenature of yeast, however, by no [p010]

means disproved Liebig's view of thenature of the change by which sugarwas converted into carbon dioxide andalcohol, as was carefully pointed out bySchlossberger [1844] in a research onthe nature of yeast, carried out inLiebig's laboratory but withoutdecisive results.

Mitscherlich was also convinced ofthe vegetable character of yeast, andshowed [1841] that when yeast wasplaced in a glass tube closed byparchment and plunged into sugarsolution, the sugar entered the glasstube and was there fermented, but was

not fermented outside the tube. Heregarded this as a proof thatfermentation only occurred at thesurface of the yeast cells, andexplained the process by contact actionin the sense of the catalytic action ofBerzelius, rather than by Liebig'stransference of molecular instability.Similar results were obtained with ananimal membrane by Helmholtz[1843], who also expressed hisconviction that yeast was a vegetableorganism.

In 1854 Schröder and von Dusch[1854, 1859, 1861] strongly reinforced

the evidence in favour of this view bysucceeding in preventing theputrefaction and fermentation of manyboiled organic liquids by the simpleprocess of filtering all air which hadaccess to them through cotton-wool.These experiments, which werecontinued until 1861, led to theconclusion that the spontaneousalcoholic fermentation of liquids wasdue to living germs carried by the air,and that when the air was passedthrough the cotton-wool these germswere held back.

At the middle of the nineteenth

century opinions with regard toalcoholic fermentation,notwithstanding all that had been done,were still divided. On the one handLiebig's theory of fermentation waswidely held and taught. Gerhardt, forexample, as late as 1856 in the articleon fermentation in his treatise onorganic chemistry [1856], gives entiresupport to Liebig's views, and histreatment of the matter affords aninteresting glimpse of the argumentswhich were then held to be decisive.The grounds on which he rejects theconclusions of Schwann and the other

investigators who shared the belief inthe vegetable nature of yeast are that,although in some cases animal andvegetable matter and infusions can bepreserved from change by the methodsdescribed by these authors, in othersthey cannot, a striking case being thatof milk, which even after being boiledbecomes sour even in filtered air, andthis without showing any trace ofliving organisms. The action of heat,sulphuric acid, and filtration on the airis to remove, or destroy, not livingorganisms but particles ofdecomposing matter, that is to say,

ferments which would add theiractivity to that of the oxygen of the air.Moreover, many ferments, as forexample diastase, act without [p011]

producing any insoluble depositwhatever which can be regarded as anorganism.

"Evidemment," he concludes, "lathéorie de M. Liebig explique seuletous les phénomènes de la manière laplus complète et la plus logique; c'est àelle que tous les bons esprits nepeuvent manquer de se rallier."

On the other hand it was held bymany to have been shown that Liebig's

view of the origin of yeast by theaction of the air on a vegetable infusionwas erroneous, and that fermentationonly arose when the air transferred tothe liquid an active agent which couldbe removed from it by sulphuric acid(Schulze), by heat (Schwann), and bycotton-wool (Schröder and von Dusch).Accompanying alcoholic fermentationthere was a development of a livingorganism, the yeast, and fermentationwas believed, without any very strictproof, to be a phenomenon due to thelife and vegetation of this organism.This doctrine seems indeed [Schrohe,

1904] to have been widely taught inGermany from 1840–56, and to haveestablished itself in the practice of thefermentation industries.

In 1857 commenced the classicalresearches of Pasteur which finallydecided the question as to the originand functions of yeast and led him tothe conclusion that "alcoholicfermentation is an act correlated withthe life and organisation of the yeastcells, not with the death or putrefactionof the cells, any more than it is aphenomenon of contact, in which casethe transformation of sugar would be

accomplished in presence of theferment without yielding up to it ortaking from it anything" [1860]. It isimpossible here to enter in detail intoPasteur's experiments on this subject,or indeed to do more than indicate thegeneral lines of his investigation. Hisstarting-point was the lactic acidfermentation.

The organism to which this changewas due had hitherto escaped detection,and as we have seen the spontaneouslactic fermentation of milk was one ofthe phenomena adduced by Gerhardt(p. 10) in favour of Liebig's views.

Pasteur [1857] discovered the lacticacid producing organism andconvinced himself that it was in fact aliving organism and the active cause ofthe production of lactic acid. One ofthe chief buttresses of Liebig's theorywas thus removed, and Pasteur nextproceeded to apply the same methodand reasoning to alcoholicfermentation. Liebig's theory of theorigin of yeast by the action of theoxygen of the air on the nitrogenousmatter of the fermentable liquid wasconclusively and strikingly disprovedby the brilliant device of producing a

crop of yeast in a liquid mediumcontaining only comparatively [p012]

simple substances of knowncomposition—sugar, ammoniumtartrate and mineral phosphate. Herethere was obviously present in theoriginal medium no matter which couldbe put into a state of putrefaction bycontact with oxygen and extend itsinstability to the sugar. Any suchmaterial must first be formed by thevital processes of the yeast. In the nextplace Pasteur showed by carefulanalyses and estimations that,whenever fermentation occurred,

growth and multiplication of yeastaccompanied the phenomenon. Thesugar, he proved, was not completelydecomposed into carbon dioxide andalcohol, as had been assumed by Liebig(p. 8). A balance-sheet of materials andproducts was constructed whichshowed that the alcohol and carbondioxide formed amounted only to about95 per cent. of the invert sugarfermented, the difference being madeup by glycerol, succinic acid, cellulose,and other substances [1860, p. 347]. Inevery case of fermentation, even whena paste of yeast was added to a solution

of pure cane sugar in water, the yeastwas found by quantitativemeasurements to have taken somethingfrom the sugar. This "something" wasindeterminate in character, but,including the whole of the extractiveswhich had passed from the yeast cellsinto the surrounding liquid, itamounted to as much as 1·63 per cent.of the weight of the sugar fermented[1860, p. 344].

Pasteur was therefore led to considerfermentation as a physiological processaccompanying the life of the yeast. Hisconclusions were couched in

unmistakable words: "The chemical actof fermentation is essentially aphenomenon correlative with a vitalact, commencing and ceasing with thelatter. I am of opinion that alcoholicfermentation never occurs withoutsimultaneous organisation,development, multiplication of cells, orthe continued life of cells alreadyformed. The results expressed in thismemoir seem to me to be completelyopposed to the opinions of Liebig andBerzelius. If I am asked in whatconsists the chemical act whereby thesugar is decomposed and what is its

real cause, I reply that I am completelyignorant of it.

"Ought we to say that the yeast feedson sugar and excretes alcohol andcarbonic acid? Or should we rathermaintain that yeast in its developmentproduces some substance of the natureof a pepsin, which acts upon the sugarand then disappears, for no suchsubstance is found in fermentedliquids? I have nothing to reply to thesehypotheses. I neither admit them norreject them, and wish only to restrainmyself from going beyond the facts.And the facts tell me simply that all

true fermentations are correlative withphysiological phenomena."

Liebig felt to the full the weight ofPasteur's criticisms; his reply [p013] waslong delayed [1870], and, according tohis biographer, Volhard [1909], causedhim much anxiety. In it he admits thevegetable nature of yeast, but does notregard Pasteur's conclusion as in anyway a solution of the problem of thenature of alcoholic fermentation.Pasteur's "physiological act" is forLiebig the very phenomenon whichrequires explanation, and which he stillmaintains can be explained by his

original theory of communicatedinstability. On some of Pasteur'sresults, notably the very important oneof the cultivation of yeast in a syntheticmedium, he casts grave doubt, whilsthe explains the production of glyceroland succinic acid as due to independentreactions. The phenomenon offermentation is still for him one whichaccompanies the decomposition of theconstituents of the cell, rather thantheir building up by vegetative growth."When the fungus ceases to grow, thebond which holds together theconstituents of the cell contents is

relaxed, and it is the motion which isthus set up in them which is the meansby which the yeast cells are enabled tobring about a displacement ordecomposition of the elements of sugaror other organic molecules." Pasteurreplied in a brief and unanswerablenote [1872]. All his attention wasconcentrated on the one question of theproduction of yeast in a syntheticmedium, which he recognised asfundamental. The validity of thisexperiment he emphaticallyreaffirmed, and finally undertook, frommaterials supplied by Liebig himself,

to produce as much yeast as could bereasonably desired. This challenge wasnever taken up, and thiscommunication formed the last word ofthe controversy. Pasteur had at thistime firmly established his thesis, nofermentation without life, both foralcoholic fermentation and for thoseother fermentations which areproduced by bacteria, and had put upona sound and permanent basis theconclusions drawn by Schulze,Cagniard-Latour, Schwann, andKützing from their early experiments.It became generally recognised that

putrefaction and other fermentativechanges were due to specificorganisms, which produced them in theexercise of their vital functions.

Pasteur subsequently [1875] came tothe conclusion that fermentation wasthe result of life without oxygen, thecells being able, in the absence of freeoxygen, to avail themselves of theenergy liberated by the decompositionof substances containing combinedoxygen. This view, which did notinvolve any alteration of Pasteur'soriginal thesis but was an attempt toexplain the physiological origin and

function of fermentation, gave rise to aprolonged controversy, which cannotbe further discussed in these pages.[p014]

Nevertheless, Liebig's desire topenetrate more deeply into the natureof the process of fermentationremained in many minds, andnumerous endeavours were made toobtain further insight into the problem.In spite of an entire lack of directexperimental proof, the conception thatalcoholic fermentation was due to thechemical action of some substanceelaborated by the cell and not directly

to the vital processes of the cell as awhole found strenuous supporters evenamong those who were convinced ofthe vegetable character of yeast. Asearly as 1833 diastase, discovered stillearlier by Kirchhoff and Dubrunfaut,had been extracted by means of waterfrom germinating barley andprecipitated by alcohol as a whitepowder, the solution of which wascapable of converting starch into sugar,but lost this power when heated [Payenand Persoz, 1833]. Basing his ideas inpart upon the behaviour of thissubstance, Moritz Traube [1858]

enunciated in the clearest possiblemanner the theory that allfermentations produced by livingorganisms are caused by ferments,which are definite chemical substancesproduced in the cells of the organism.He regarded these substances as beingclosely related to the proteins andconsidered that their function was totransfer the oxygen and hydrogen ofwater to different parts of the moleculeof the fermentable substance and thusbring about that apparentintramolecular oxidation and reductionwhich is so characteristic of

fermentative change and had arrestedthe attention of Lavoisier and, longafter him, of Liebig.

Traube's main thesis, thatfermentation is caused by definiteferments or enzymes, attracted muchattention, and received fresh supportfrom the separation of invertase in1860 from an extract of yeast byBerthelot, and from the advocacy andauthority of this great countryman ofPasteur, who definitely expressed hisopinion that insoluble ferments existedwhich could not be separated from thetissues of the organism, and further,

that the organism could not itself beregarded as the ferment, but only as theproducer of the ferment [1857, 1860].Hoppe-Seyler [1876] also supportedthe enzyme theory of fermentation, butdiffered in some respects from Traubeas to the exact function of the ferment[see Traube, 1877; Hoppe-Seyler,1877].

Direct experimental evidence was,however, still wanting, and Pasteur'sreiterated assertion [1875] that allfermentation phenomena weremanifestations of the life of theorganism remained uncontroverted by

experience.Numerous and repeated direct

experimental attacks had been made[p015] from time to time upon theproblem of the existence of afermentation enzyme, but all hadyielded negative or unreliable results.

As early as 1846 a bold attempt hadbeen made by Lüdersdorff [1846] toascertain whether fermentation was orwas not bound up with the life of theyeast by grinding yeast and examiningthe ground mass. A single gram ofyeast was thoroughly ground, theprocess lasting for an hour, and the

product was tested with sugar solution.Not a single bubble of gas was evolved.A similar result was obtained in arepetition of the experiment bySchmidt in Liebig's laboratory [1847],the grinding being continued in thiscase for six hours, but the naturalconclusion that living yeast wasessential for fermentation was notaccepted, on the ground that during thelengthy process of trituration in contactwith air the yeast had become alteredand now no longer possessed the powerof producing alcoholic fermentation,but instead had acquired that of

changing sugar into lactic acid [seeGerhardt, 1856, p. 545].

Similar experiments made in 1871by Marie von Manasseïn [1872, 1897],in which yeast was ground for six tofifteen hours with powdered rockcrystal, yielded products whichfermented sugar, but they containedunbroken yeast cells, so that the resultsobtained could not be considereddecisive [Buchner and Rapp, 1898, 1],although Frau von Manasseïn herselfdrew from them and from others inwhich sugar solution was treated withheated yeast, but not under aseptic

conditions, the conclusion that livingyeast cells were not necessary forfermentation.

Quite unsuccessful were also theattempts made to accomplish theseparation of fermentation from theliving cell by Adolf Mayer [1879, p.66], and, as we learn from Roux, byPasteur himself, grinding, freezing, andplasmolysing the cells, having in hishands proved alike in vain. Extractionby glycerol or water, a method bywhich many enzymes can be obtainedin solution, gave no better results[Nägeli and Loew, 1878], and the

enzyme theory of alcoholicfermentation appeared quite unjustifiedby experiment.

Having convinced himself of this,Nägeli [1879] suggested a newexplanation of the facts based onmolecular-physical grounds. Accordingto this view, which unites in itselfsome of the conceptions of Liebig,Pasteur, and Traube, fermentation isthe transference of a state of motionfrom the molecules, atomic groups, andatoms of the compounds constitutingthe living plasma of the cell to thefermentable material, whereby the

equilibrium existing in the moleculesof the latter is disturbed anddecomposition ensues [1879, p. 29].[p016]

This somewhat complex idea, whilstincluding, as did Liebig's theory,Stahl's fundamental conception of atransmission of a state of motion,satisfies Pasteur's contention thatfermentation cannot occur without life,and at the same time explains thespecific action of different organismsby differences in the constitution oftheir cell contents. The really essentialpart of Nägeli's theory consisted in the

limitation of the power of transferenceof molecular motion to the livingplasma, by which the failure of allattempts to separate the power offermentation from the living cell wasexplained. This was the specialphenomenon which requiredexplanation; to account for this thetheory was devised, and when this wasexperimentally disproved, the theorylost all significance.

For nearly twenty years no furtherprogress was made, and then in 1897the question which had aroused somuch discussion and conjecture, and

had given rise to so much experimentalwork, was finally answered by EduardBuchner, who succeeded in preparingfrom yeast a liquid which, in thecomplete absence of cells, was capableof effecting the resolution of sugar intocarbon dioxide and alcohol [1897, 1].

In the light of this discovery thecontribution to the truth made by eachof the great protagonists in theprolonged discussion on the problem ofalcoholic fermentation can bediscerned with some degree ofclearness. Liebig's main contention thatfermentation was essentially a

chemical act was correct, although hisexplanation of the nature of this actwas inaccurate. Pasteur, in so far as heconsidered the act of fermentation asindissolubly connected with the life ofthe organism, was shown to be in error,but the function of the organism hasonly been restricted by a single stage,the active enzyme of alcoholicfermentation has so far only beenobserved as the product of the livingcell. Nearest of all to the truth wasTraube, who in 1858 enunciated thetheorem, which was only proved foralcoholic fermentation in 1897, that all

fermentations produced by livingorganisms are due to ferments secretedby the cells.

Buchner's discovery of zymase hasintroduced a new experimental methodby means of which the problem ofalcoholic fermentation can be attacked,and the result has been that since 1897a considerable amount of informationhas been gained with regard to thenature and conditions of action of theenzymes of the yeast cell. It has beenfound that the machinery offermentation is much more complexthan had been surmised. The enzyme

zymase, which is essential forfermentation, cannot of itself bringabout the alcoholic fermentation ofsugar, but is dependent on the presenceof a second substance, termed, for[p017] want of a more reasonable name,the co-enzyme. The chemical natureand function of this mysteriouscoadjutor are still unknown, but as itwithstands the temperature of boilingwater and is dialysable, it is probablymore simple in constitution than theenzyme. This, however, is not all; forthe decomposition of sugar a phosphateis also indispensable. It appears that in

yeast-juice, and therefore also mostprobably in the yeast cell, thephosphorus present takes an active partin fermentation and goes through aremarkable cycle of changes. Thebreakdown of sugar into alcohol andcarbon dioxide is accompanied by theformation of a complexhexosephosphate, and the phosphate issplit off from this compound and thusagain rendered available for action bymeans of a special enzyme, termedhexosephosphatase. In addition to thiscomplex of ferments, the cell alsopossesses special enzymes by which

the zymase and the co-enzyme can bedestroyed, and, further, at least onesubstance, known as an anti-enzyme,which directly checks this destructiveaction. It seems probable, moreover,that the decomposition of the sugarmolecule takes place in stages,although much doubt yet exists as tothe nature of these.

At the present moment the subjectremains one of the most interesting inthe whole field of biological chemistry,the limited degree of insight which hasalready been gained into themarvellous complexity of the cell

lending additional zest to the attemptto penetrate the darkness whichshrouds the still hidden mysteries.

CHAPTER II.ZYMASE AND ITS PROPERTIES.

Discovery of Zymase.

[p018]

The history of Buchner's discovery isof great interest [Gruber, 1908; Hahn,1908]. As early as 1893 Hans andEduard Buchner found that the cells ofeven the smallest micro-organismcould be broken by being ground withsand [Buchner, E. and H., and Hahn,1903, p. 20], and in 1896 the same

process was applied by these twoinvestigators to yeast, with the objectof obtaining a preparation fortherapeutic purposes. Difficulties arosein the separation of the cell contentsfrom the ground-up mixture of cellmembranes, unbroken cells, and sand,but these were overcome by carryingout the suggestion of Martin Hahn (atthat time assistant to Hans Buchner)that kieselguhr should be added and theliquid squeezed out by means of ahydraulic press [Buchner, E. and H.,and Hahn, 1903, p. 58]. The yeast-juicethus obtained was, in the first instance,

employed for animal experiments, butunderwent change very rapidly. Theordinary antiseptics were found to beunsuitable, and hence sugar was addedas a preservative, and it was themarked action of the juice upon thisadded cane sugar that drew EduardBuchner's attention to the fact thatfermentation was proceeding in theabsence of yeast-cells.

As in the case of so manydiscoveries, the new phenomenon wasbrought to light, apparently by chance,as the result of an investigationdirected to quite other ends, but

fortunately fell under the eye of anobserver possessed of the genius whichenabled him to realise its importanceand give to it the true interpretation.

In his first papers [1897, 1, 2; 1898],Buchner established the followingfacts: (1) yeast-juice free from cells iscapable of producing the alcoholicfermentation of glucose, fructose, canesugar, and maltose; (2) the fermentingpower of the juice is neither destroyedby the addition of chloroform, benzene,or sodium arsenite [Hans Buchner,1897], by filtration through a Berkefeldfilter, by evaporation to dryness at 30°

to 35°, nor by precipitation withalcohol; (3) the fermenting power iscompletely destroyed when the liquidis heated to 50°. [p019]

From these facts he drew theconclusion "that the production ofalcoholic fermentation does not requireso complicated an apparatus as theyeast cell, and that the fermentativepower of yeast-juice is due to thepresence of a dissolved substance". Tothis active substance he gave the nameof zymase.

Buchner's discovery was notreceived without some hesitation. A

number of investigators preparedyeast-juice, but failed to obtain anactive product [Will, 1897; Delbrück,1897; Martin and Chapman, 1898;Reynolds Green, 1897; Lintner, 1899].A more accurate knowledge of thenecessary conditions and of theproperties of yeast-juice, however, ledto more successful results [Will, 1898;Reynolds Green, 1898; Lange, 1898],and it was soon established that, givensuitable yeast, an active preparationcould be readily procured by Buchner'smethod. Criticism was then directed tothe effect of the admitted presence of a

certain number of micro-organisms inyeast-juice [Stavenhagen, 1897], butBuchner [Buchner and Rapp, 1897] wasable to show by experiments in thepresence of antiseptics and with juicefiltered through a Chamberland candlethat the fermentation was not due toliving organisms of any kind.

The most weighty criticism ofBuchner's conclusion consisted in anattempt to show that the properties ofyeast-juice might be due to thepresence, suspended in it, of fragmentsof living protoplasm, which, althoughsevered from their original

surroundings in the cell, might retainfor some time the power of producingalcoholic fermentation. This, it will beseen, was an endeavour to extendNägeli's theory to include in it thenewly discovered fact.

In favour of this view were adducedthe similarity between the effects ofmany antiseptics on living yeast and onthe juice, the ephemeral nature of thefermenting agent present in the juice,the effect of dilution with water, andthe phenomenon of autofermentationwhich is exhibited by the juice in theabsence of added sugar [Abeles, 1898;

v. Kupffer, 1897; v. Voit, 1897;Wehmer, 1898; Neumeister, 1897;Macfadyen, Morris, and Rowland,1900; Bokorny, 1906; Fischer, 1903;Beijerinck, 1897, 1900; Wroblewski,1899, 1901].

A brief general description of theactual properties of yeast-juice and ofthe phenomena of fermentation by itsmeans is sufficient to show the greatimprobability of this view.

The juice prepared by Buchner'smethod forms a somewhat viscousopalescent brownish-yellow liquid,which is usually faintly acid in reaction

[compare Ahrens, 1900] and almostoptically inactive. It has a specificgravity of 1·03 to 1·06, contains 8·5 to14 per cent. [p020] of dissolved solids,and leaves an ash amounting to 1·4 to 2per cent. About 0·7 to 1·7 per cent. ofnitrogen is present, nearly all in theform of protein, which coagulates to athick white mass when the juice isheated.

A powerful digestive enzyme of thetype of trypsin is also present, so thatwhen the juice is preserved its albuminundergoes digestion at a rate whichdepends on the temperature [Hahn,

1898; Geret and Hahn, 1898, 1, 2;1900; Buchner, E. and H., and Hahn,1903, pp. 287–340], and is convertedinto a mixture of bases and amino-acids. After about six days at 37°, or 10to 14 days at the ordinary temperature,the digestion is so complete that nocoagulation occurs when the juice isboiled. As this proteoclastic enzyme,like the alcoholic enzyme, cannot beextracted from the living cells, it istermed yeast endotrypsin orendotryptase. Fresh yeast-juiceproduces a slow fermentation of sugar,which lasts for forty-eight to ninety-six

hours at 25° to 30°, about a week at theordinary temperature, and then ceases,owing, not to exhaustion of the sugar,but to the disappearance of thefermenting agent. When the juice ispreserved or incubated in the absenceof a fermentable sugar thisdisappearance occurs considerablysooner, so that even after standing for asingle day at room temperature, or twodays at 0°, no fermentation may occurwhen sugar is added. The reason forthis behaviour has not been definitelyascertained. As will be seen later on (p.64) the phenomenon is a complex one,

but the disappearance of the enzymewas originally ascribed by Buchner tothe digestive action upon it of theendotrypsin of the juice [1897, 2], andno better explanation has yet beenfound. Confirmation of this view isafforded by the fact that the addition ofa tryptic enzyme of animal origingreatly hastens the disappearance ofthe alcoholic enzyme [Buchner, E. andH., and Hahn, 1903, p. 126], and thatsome substances which hinder thetryptic action favour fermentation[Harden, 1903]. The amount offermentation produced is almost

unaffected by the presence of suchantiseptics as chloroform or toluene,although some others, such as arsenitesand fluorides, decrease it when addedin comparatively high concentrations,and it is only slightly diminished bydilution with three or four volumes ofsugar solution, somewhat moreconsiderably by dilution with water.When it is filtered through aChamberland filter the first portions ofthe filtrate are capable of bringingabout fermentation, but the fermentingpower diminishes in the succeedingportions and finally disappears. The

juice can be spun in a centrifugalmachine without being in any wayaltered, and no separation into more orless active layers takes place underthese conditions. [p021]

The amorphous powder obtained bydrying the precipitate produced whenthe juice is added to a mixture ofalcohol and ether is also capable ofproducing fermentation, and theprocess of precipitation may berepeated without seriously diminishingthe fermenting power of the product.

These facts clearly show that thevarious phenomena adduced by the

supporters of the theory ofprotoplasmic fragments are quiteconsistent with the presence of adissolved enzyme as the active agent ofthe juice, and at the same time that theproperties demanded of the livingfragments of protoplasm to whichfermentation is ascribed are such ascannot be reconciled with ourknowledge of living matter. If livingprotoplasm is the cause of alcoholicfermentation by yeast-juice, a newconception of life will be necessary;the properties of the postulatedfragments of protoplasm must be so

different from those which theprotoplasm of the living cell possessesas to deprive the theory of all realvalue [Buchner, 1900, 2; Buchner, E.and H., and Hahn, 1903, p. 33].

Further and very convincingevidence against the protoplasm theoryis afforded by the behaviour of yeasttowards various desiccating agents.When yeast is dried at the ordinarytemperature it retains its vitality for aconsiderable period. If, however, thedried yeast be heated for six hours at100° it loses the power of growth andreproduction but still retains that of

fermenting sugar, and when groundwith sand, kieselguhr and 10 per cent.glycerol solution yields an active juice[Buchner, 1897, 2; 1900, 1].Preparations (known as zymin)obtained by treating yeast with amixture of alcohol and ether [Albert,1900, 1901, 1], or with acetone andether [Albert, Buchner, and Rapp,1902], show precisely similarproperties (p. 38). The proof in thiscase has been carried a step further, forthe active juice obtained by grindingsuch acetone-yeast, when precipitatedwith alcohol and ether, yields an

amorphous powder, still capable offermenting sugar.

The Preparation of Yeast-Juice.

Buchner's process for the preparationof active yeast-juice is characterised byextreme simplicity. The yeastemployed, which should be freshbrewery yeast, is washed two or threetimes by being suspended in a largeamount of water and allowed to settlein deep vessels. It is then collected on afilter cloth, wrapped in a press cloth,

and submitted to a pressure of about 50kilos, per sq. cm. for five minutes. Theresulting friable mass contains about70 per cent. of water and is free fromadhering wort. The washed yeast isthen [p022] mixed with an equal weightof silver sand and 0·2 to 0·3 parts ofkieselguhr, care being taken that this isfree from acid. The correct amount ofkieselguhr to be added can only beascertained by experience, and varieswith different samples of yeast. Thedry powder thus obtained is brought inportions of 300 to 400 grams into alarge porcelain mortar and ground by

hand by means of a porcelain pestlefastened to a long iron rod whichpasses through a ring fixed in the wall(Fig. 1). The mortar used by Buchnerhas a diameter of 40 cm. and the pestleand rod together weigh 8 kilos.

FIG. 1.

As the grinding proceeds the light-coloured powder gradually darkens andbecomes brown, and the mass becomesmoist and adheres to the pestle, untilfinally, after two to three minutes'grinding, it takes the consistency ofdough, at which stage the process is

stopped. The mass is next enveloped ina press cloth and submitted to apressure of 90 kilos, per sq. cm. in ahydraulic hand press, the pressurebeing very gradually raised in order toavoid rupture of the cloth. The clothrequired for 1000 grams of yeastmeasures 60 by 75 cm. and ispreviously soaked in water and thensubmitted to a pressure of 50 kilos, persq. cm., retaining about 35 to 40 c.c. ofwater.

The juice runs from the press on to afolded filter paper, to removekieselguhr and yeast cells, and passes

into a vessel standing in ice water.The yield of juice obtained by

Buchner in an operation of this kindfrom 1 kilo. of yeast amounts to 320 to460 c.c. It may be increased by re-grinding the press cake and againsubmitting it to pressure, and thenamounts on the average to 450 to 500c.c.

Since the cell membranes constituteabout 20 per cent. of the weight of thedry yeast, this yield corresponds tomore than 60 per cent. of the total cellcontents of the yeast. It has beencomputed by Will [quoted by Buchner,

E. and H., and Hahn, 1903, p. 66] that[p023] only about 20 per cent. of thecells are left unaltered by one grindingand pressing, and only 4 per cent. aftera repetition of the process, at least 57per cent. of the cells being actuallyruptured by the double process, and theremainder to some extent altered. Itseems probable from these figures thata certain amount of the juice may bederived from the unbroken cells, andWill expressly states that manyunbroken cells have lost their vacuoles.

FIG. 2.

If the yeast be submitted to a processof regeneration, which consists inexposure to a well-aerated solution ofsugar and mineral salts untilfermentation is complete, the juicesubsequently obtained [p024] is moreactive than that yielded by the originalyeast [Albert, 1899, 1].

A modified method of grinding yeastwas introduced by Macfadyen, Morris,and Rowland [1900], who placed amixture of yeast and sand in a jacketedand cooled vessel, in which a spindlecarrying brass flanges was rapidlyrotated [Rowland, 1901]. One kilo. of

yeast ground in this way for 3·5 hoursyielded 350 c.c. of juice.

This grinding process was at firstadopted by Harden and Young in theirexperiments but was afterwardsabandoned in favour of Buchner'shand-grinding process, as it was foundliable to yield juices of low fermentingpower, probably on account ofinefficient cooling during the grindingprocess. A slight modification ofBuchner's process has, however, beenintroduced, the hand-ground massbeing mixed with a further quantity ofkieselguhr until a nearly dry powder is

formed, and the mass packed betweentwo layers of chain cloth in steel filterplates and pressed out in a hydraulicpress at about 2 tons to the square inch(300 kilos. per sq. cm.). The press andplates are shown in section in Fig. 2. Ithas also been found convenient toremove yeast cells and kieselguhr fromthe freshly pressed juice bycentrifugalisation instead of byfiltration through paper, and to washthe yeast before grinding by means of afilter-press.

Working with English top yeastsHarden and Young have found the yield

of juice extremely variable, the generalrule being that the amount of juiceobtainable from freshly skimmed yeastis smaller than that yielded by the sameyeast after standing for a day or twoafter being skimmed. The yield for1000 grams of pressed brewer's yeastvaries from 150 to 375 c.c., and is onthe average about 250 c.c.

Very fresh yeast occasionallypresents the peculiar phenomenon thatscarcely any juice can be expressedfrom the ground mass, although thelatter does not differ in appearance orconsistency from a mass which gives a

good yield.

Extraction of Zymase fromUnground Yeast.

1. Maceration of Dried Yeast.A valuable addition to the methods

of obtaining an active solution ofzymase was made in 1911 by Lebedeff[1911, 2; 1912, 2; see also 1911, 3, 7,and 1912, 1]. This investigator hadbeen in the habit of grinding driedyeast with water for preparing samplesof yeast-juice of uniform character and

observed that when the dried yeast wasdigested with sugar solution and themixture heated, coagulation [p025] tookplace throughout the whole liquid, theproteins of the yeast having passed outof the cells. Further examinationrevealed the interesting fact that driedyeast readily yielded an active extractwhen macerated in water for sometime. The quality of the resulting"maceration extract" depends on aconsiderable number of factors, thechief of which are: (1) the temperatureof drying of the yeast; (2) thetemperature of maceration; (3) the

duration of maceration; and (4) thenature of the yeast, as well as, ofcourse, the amount of water added inmaceration.

In general the yeast should be driedat 25°–30° and then macerated with 3parts of water for 2 hours at 35°.

The temperature of maceration mayas a rule be varied, without detrimentto the product provided that the time ofmaceration is also suitably altered;thus with dried Munich yeast,maceration for 4·5 hours at 25° is aboutas effective as 2 hours at 35°, whereastreatment for a shorter time at 25° or a

longer time at 35° produces in generala less efficacious extract. Yeast driedat a lower temperature than 25° tendsto yield an extract poor in co-enzyme(p. 59) and hence of low fermentingpower, this being especially marked atair temperature.

The subsequent treatment of theyeast during maceration may, however,be of great influence in such cases.Thus a yeast dried at 15° gave bymaceration at 25° for 4·5 hours a weakextract (yielding with excess of sugar0·33g. CO2), whereas when maceratedat 35° for 2 hours it yielded a normal

extract (1·36g. CO2).The nature of the yeast is of

paramount importance. Thus whileMunich (bottom) yeast usually gives agood result, a top yeast from a Parisbrewery was found to yield extractscontaining neither zymase nor its co-enzyme in whatever way thepreparation was conducted. Theexistence of such yeasts is of greatinterest, and it was probably due to theunfortunate selection of such a yeastfor his experiments that Pasteur wasunable to prepare active fermentingextracts and therefore failed to

anticipate Buchner by more than 30years (see p. 15). The English topyeasts as a rule give poor results [seeDixon and Atkins, 1913] andsometimes yield totally inactivemaceration extract. It is not understoodwhy the enzyme passes out of the cellduring the process of maceration andthe whole method gives rise to anumber of extremely interestingproblems.

Method.—A suitable yeast is washedby decantation, filtered through a cloth,lightly pressed by means of a handpress, and then passed through a sieve

of 5mm. mesh, spread out in a layer 1–1·5cm. thick and left at 25°–35° for twodays. Fifty grams of the dried yeast is[p026] thoroughly and carefully mixedwith 150 c.c. of water in a basin bymeans of a spatula and the wholedigested for two hours at 35°. The massoften froths considerably. It is thenfiltered through ordinary folded filterpaper, preferably in two portions, andcollected in a vessel cooled by ice. Theseparation may also be effected bycentrifuging or pressing out the mass,and the maceration may beconveniently conducted in a flask

immersed in the water of a thermostat.It is not advisable to macerate morethan 50 grams in one operation. Underthese conditions 25–30 c.c. of extractare obtained after 20 minutes'filtration, 70–80 c.c. in twelve hours.Dried Munich yeast can be boughtfrom Messrs. Schroder of Munich andserves as a convenient source of the

extract.[1]

[1] The material supplied isoccasionally found to

yield an inactive extractand every sample should

be tested.

This extract closely resembles inproperties the juice obtained bygrinding the same yeast, but it isusually more active and contains moreinorganic phosphate (see p. 46).

2. Other Methods.Attempts to prepare active extracts

from undried yeast in an analogousmanner have so far not been verysuccessful. Thus Rinckleben [1911]found that plasmolysis by glycerol (8per cent.) or sodium phosphate (5 percent.) sometimes yielded an activejuice and sometimes a juice which

contained enzyme but no co-enzyme,but more often an inactive juiceincapable of activation (p. 64) [see alsoKayser, 1911].

Giglioli [1911] by the addition ofchloroform also obtained an activeliquid. It appears in fact as thoughalmost any method of plasmolysing theyeast cell may yield a certainproportion of zymase in the exudate.

An ingenious process has beendevised by Dixon and Atkins [1913]who applied the method of freezing inliquid air which they had foundefficacious for obtaining the sap from

various plant organs. They thussucceeded in obtaining from yeast,derived from Guinness' brewery inDublin, liquids capable of fermentingsugar and of about the same efficacy asthe maceration extracts prepared byLebedeff's method from the sameyeast. The results were, however, inboth cases very low, the maximumtotal production of CO2 by 25 c.c. ofliquid from excess of sugar being 32·5c.c. (air temperature) or about 0·06g.Munich yeast on the other hand yields,either by maceration or grinding, aliquid giving as much as 1·5–2g. of

CO2 per 25 c.c., whilst [p027] Englishyeast-juice prepared by grinding oftengives as much as 0·5–0·7g. of CO2.

No direct comparison with the juiceprepared by grinding was made byDixon and Atkins, but it may beconcluded from their results that thebest method of obtaining an activepreparation from the top yeasts used inthis country is that of grinding.Maceration, freezing and plasmolysisalike yield poor results. With Munichyeast on the other hand the macerationprocess yields excellent results, whilstthe liquid air process has not so far

been tried.

Practical Methods for theEstimation of the

Fermenting Power of Yeast-Juice.

In order to estimate the amount ofcarbon dioxide evolved in a given timeand the total amount evolved by theaction of yeast-juice on sugar, Buchneradopted an extremely simple method,which consisted in carrying out thefermentation in an Erlenmeyer flaskprovided with a small wash-bottle,

which contained sulphuric acid and wasclosed by a Bunsen valve, andascertaining the loss of weight duringthe experiment. Corrections arenecessary for the carbon dioxidepresent in the original juice andretained in the liquid at the close of theexperiment and for that present in theair space of the apparatus, but it wasfound that for most purposes thesecould be neglected. In cases in whichgreater accuracy was desired, thecarbon dioxide was displaced by airbefore the weighings were made. Atypical experiment of this kind, without

displacement of carbon dioxide, is thefollowing:—

March 22, 1899, Berlin bottom yeastV. 20 c.c. juice + 8 grams canesugar + 0·2 c.c. toluene as

antiseptic at 16°. Grams of carbondioxide after

24 48 72 96 hours.0·40 0·64 0·99 1·11

The total weight of carbon dioxideevolved under these conditions istermed the fermenting power of thejuice (Buchner).

A more accurate method[Macfadyen, Morris, and Rowland,1900] consists in passing the carbon

dioxide into caustic soda solution andestimating it by titration. The yeast-juice, sugar, and antiseptic are placedin an Erlenmeyer flask provided with astraight glass tube, through which aircan be passed over the surface of theliquid, and a conducting tube leadinginto a second flask which contains 50c.c. of 10 per cent. caustic sodasolution and is connected with the airby a guard tube containing soda lime.The juice can be freed from carbondioxide by agitation in a current of airbefore the flask is connected to [p028]

that containing the caustic soda

solution, and at the end of the period ofincubation air is passed through theapparatus, the liquid being boiled out ifgreat accuracy is required. Theabsorption flask is then disconnectedand the amount of absorbed carbondioxide estimated by titration. This iscarried out by making up the contentsof the flask to 200 c.c., taking out analiquot portion, rendering this exactlyneutral to phenophthalein by theaddition first of normal and finally ofdecinormal acid, adding methyl orangeand titrating with decinormal acid toexact neutrality. Each c.c. of

decinormal acid used in this lasttitration represents 0·0044 gram ofcarbon dioxide in the quantity ofsolution titrated.

FIG. 3.

These methods are only suitable for

observations at considerable intervalsof time. For the continuous observationof the course of fermentation Harden,Thompson and Young [1910] connectthe fermentation flask with a Schiff'sazotometer filled with mercury andmeasure the volume of gas evolved, theliquid having been previously saturatedwith carbon dioxide (Fig. 3). The levelof the mercury in the reservoir is keptconstant by a syphon overflow, asshown in the figure, or, according to amodification introduced by S. G. Paine,by a specially constructed bottleprovided with two tubulures near the

bottom. This ensures that no change inthe pressure in the flask occurs, and thevolume of gas observed is reduced tonormal pressure by means of a table.Before making a reading it is necessaryto shake the fermenting mixturethoroughly, as the albuminous liquidvery readily becomes greatlysupersaturated with carbon dioxide, somuch so in fact that very little gas isevolved in the intervals between theshakings. The exact procedure inmaking an observation consists inshaking the flask [p029] thoroughly,replacing in the thermostat, allowing to

remain for one minute, and thenreading the level of the mercury in theazotometer. After the required time,say five minutes, has elapsed from thetime at which the flask was firstshaken, it is again removed from thebath, shaken as before, replaced,allowed to remain for one minute andthe reading then taken. In this wayreadings can be conveniently made atintervals of three or five minutes oreven less, and much more detailedinformation obtained about the courseof the reaction than is possible bymeans of observations made at

intervals of several hours.Another form of volumetric

apparatus, designed by Walton [1904],has been used by Lebedeff [1909].

An apparatus on a different principlehas been designed by Slator [1906] foruse with living yeast, but is equallyapplicable to yeast-juice, and a verysimilar form has been more recentlyemployed by Iwanoff [1909, 2]. In thisapparatus the change of pressureproduced by the evolution of carbondioxide is measured at constantvolume, and comparative rates ofevolution can be obtained with

considerable accuracy, although themethod has the disadvantage that theabsolute volume of gas evolved is notmeasured. The apparatus consists of abottle or flask connected with amercury manometer. The fermentingmixture is placed in the bottle alongwith glass beads to facilitate agitation,the pressure is reduced to a smallamount by the water-pump, and the riseof pressure is then observed atintervals, this being proportional to thevolume of gas produced. As in thepreceding case, the liquid must be wellshaken before a reading is made.

The Alcoholic Fermentationof the Sugars by Yeast-

Juice.

Yeast-juice brings about a slowfermentation of those sugars which arefermented by the yeast from which it isprepared as well as of dextrin, and ofstarch and glycogen, which are notfermented by living yeast.

(a) Relation to Fermentationby living Yeast.

Both in rate of fermentation and inthe total fermentation produced, yeast-

juice stands far behind the equivalentamount of living yeast. Taking 25 c.c.of yeast-juice to be equivalent to atleast 36 grams of pressed yeastcontaining 70 per cent. of moisture, itis found that whereas the yeast-juice(from English top yeast) gives withglucose a maximum rate offermentation of about 3 c.c. in fiveminutes, the living yeast ferments thesugar at the rate of about 126 c.c. in thesame time, or [p030] about forty timesas quickly. The total carbon dioxideobtainable from the yeast-juice,moreover, corresponds to the

fermentation of only 2 to 3 grams ofsugar, whilst the living yeast willreadily ferment a much larger quantity,although the exact limit in this respecthas not been accurately determined.The reasons for this great difference inbehaviour will be discussed later on,after the various factors concerned infermentation have been considered (p.123).

(b) Relation of Alcohol toCarbon Dioxide.

In all cases of fermentation by yeast-juice and zymin, the relative amounts

of carbon dioxide and alcohol producedare substantially in the ratio of themolecular weights of the compounds,that is as 44: 46, so that for 1 part ofcarbon dioxide 1·04 of alcohol areformed. This has been shown for thejuice and zymin from bottom yeasts byBuchner [Buchner, E. and H., andHahn, 1903, pp. 210, 211], whoobtained the ratios 1·01, 0·98, 1·01, and0·99 from experiments in which from 8to 15 grams of alcohol were produced.Similar numbers, 0·90, 1·12, 0·95, 0·91and 0·92, have been obtained for thejuice from top yeasts by Harden and

Young [1904], who worked with muchsmaller quantities. The variable resultsobtained with juice from top yeast byMacfadyen, Morris and Rowland[1900], have not been confirmed.

(c) Relation of CarbonDioxide and Alcohol

Produced to the Amount ofSugar Fermented.

The construction of a balance-sheetbetween the sugar fermented and theproducts formed is of special interestin the case of alcoholic fermentation byyeast-juice, because, there being no

cell growth as in the case of livingyeast, an opportunity appears to beafforded of ascertaining whether thewhole of the sugar is converted intoalcohol and carbon dioxide, or whethersome fraction of the sugar passes intoany of the well-known subsidiaryproducts of alcoholic fermentation byyeast, such as glycerol, fusel oil, orsuccinic acid. Unfortunately thequestion cannot be settled in this way.When the loss of sugar during thefermentation is estimated directly, it isusually found to be considerablygreater than the sum of the alcohol and

carbon dioxide produced from it. Thisfact was first observed by Macfadyen,Morris and Rowland [1900], and wasthen confirmed by Buchner [Buchner,E. and H., and Hahn, 1903, p. 212], inone instance, the excess of sugar lostover products being in this case about15 per cent. of the total sugar whichhad disappeared. The matter was thenmore thoroughly investigated byHarden and Young [1904]. [p031]

The conditions under which theexperiment must be carried out are notvery favourable to the attainment ofextreme accuracy. Yeast-juice contains

glycogen and a diastatic enzyme whichconverts this into dextrins and finallyinto sugar. This process goes onthroughout fermentation, tending toincrease the sugar present and to makethe apparent loss of sugar less than thesum of the products. In spite of this itwas found that a certain amount ofsugar invariably disappeared withoutbeing accounted for as alcohol orcarbon dioxide, and this whether thefermentation lasted sixty or a hundredand eight hours, and independently ofthe dilution of the juice. Thisdisappearing sugar amounted in some

cases to 44 per cent. of the total loss ofsugar, and on the average of twenty-five experiments was 38 per cent.Further information was sought byconverting all the sugar-yieldingconstituents of the juice into sugar byhydrolysis before and after thefermentation. This process revealed thefact that when the glucose equivalentof the juice before and afterfermentation was determined afterhydrolysis with three times normalacid for three hours (and a correctionmade for the loss of reducing powerexperienced by glucose itself when

submitted to this treatment), thedifference was almost exactly equal tothe alcohol and carbon dioxideproduced. In other words,accompanying fermentation, a changeproceeds by which sugar is convertedinto a less reducing substance,reconvertible into sugar by hydrolysiswith acids. Similar results weresubsequently obtained by Buchner andMeisenheimer [1906], who employed1·5 normal acid and observed a smallnett loss of sugar. Still more recentlyLebedeff [1909, 1910, see also 1913, 2]has carried out similar estimations with

the same result. It is doubtful whetherthe experiments which have so far beenmade on this point are sufficientlyaccurate to decide with certaintywhether or not the loss of sugar isexactly equal to the sum of the carbondioxide and alcohol produced. It hasbeen shown by Buchner andMeisenheimer [1906] that glycerol is aconstant product of alcoholicfermentation by yeast-juice (p. 95), andno other source for this than the sugarhas yet been found, so that it is notimprobable that a small amount ofsugar is converted into non-

carbohydrate substances other thancarbon dioxide and alcohol.

It has also been shown [Harden andYoung, 1913] that the deficit of sugaris not due to the formation ofhexosephosphate (p. 47), which has alower reduction than glucose, and thatthe solution from which the sugar(either glucose or fructose) hasdisappeared actually contains somesubstance of relatively highdextrorotation and of low reducingpower. [p032]

However this may be, it may beconsidered as established that during

alcoholic fermentation sugar isconverted by an enzyme into somecompound of less reducing power,which again yields sugar on hydrolysiswith acids. The exact nature of thissubstance has not been ascertained, butit appears likely that the process is asynthetical one resulting in theformation of some polysaccharide,possibly intermediate between thehexoses and glycogen.

A similar phenomenon has beenobserved with living yeast by Euler andJohansson [1912, 1], and Euler andBerggren [1912], whose interpretation

of the observation is discussed later on(p. 57).

(d) Fermentation of DifferentCarbohydrates.

Autofermentation.Yeast-juice and zymin ferment all

the sugars which are fermented by theyeast from which they are prepared,and, in addition, a number of colloidalsubstances which cannot pass throughthe membrane of the living yeast cell,but which are hydrolysed by enzymesin the juice and thus converted intosimpler sugars capable of fermentation

[Buchner and Rapp, 1898, 3; 1899, 2].Of the simple sugars which have beenexamined, glucose, fructose, andmannose are freely fermented, l-arabinose not at all, whilst the case ofgalactose is doubtful. Galactose is,however, fermented by juice preparedfrom a yeast which has been "trained"to ferment galactose [Harden andNorris, 1910]. As regards both the rateof fermentation and the total amount ofcarbon dioxide evolved from glucoseand fructose by the action of a definiteamount of yeast-juice, Buchner andRapp obtained practically identical

numbers. Harden and Young [1909],using juice from top yeast, found thatfructose was slightly more rapidlyfermented and gave a somewhat largertotal than glucose, whilst mannose wasinitially fermented at almost the samerate as glucose, but gave a decidedlylower total, the following being theaverage result:—

Sugar. RelativeRates.

RelativeTotals.

Glucose 1 1 Fructose 1·29 1·15Mannose 1·04 0·67

Among the disaccharides, cane sugarand maltose are freely fermented, andthe juice can be shown like living yeastto contain invertase and maltase. Theextent of fermentation does not differmaterially from that attained withglucose. Lactose is not fermented.

Of the higher sugars raffinose isfermented by juice from bottom yeast,but more slowly than cane sugar ormaltose. No experiments seem to have

been made with juice from top yeast.[p033]

As regards the fermentation of thehigher carbohydrates, very littleexperimental work has been carriedout. Buchner and Rapp found that thefermentation of starch paste wasdoubtful, but that soluble starch andcommercial dextrin were fermentedwith some freedom. No special studyhas been made of the diastatic enzymeswhich bring about the hydrolysis ofthese substances.

The fermentation of glycogen byyeast-juice is of considerable interest,

since it is known that the characteristicreserve carbohydrate of the yeast cell isglycogen [see Harden and Young,1902, where the literature is cited], andmoreover that in living yeast theintracellular fermentation of glycogenproceeds readily, whereas glycogenadded to a solution in which yeast issuspended is not affected. Yeast-juicecontains a diastatic enzyme whichhydrolyses glycogen to a reducing andfermentable sugar, so that in a juicepoor in zymase to which glycogen hasbeen added, the amount of sugar isfound to increase, the hydrolysis of the

glycogen proceeding more quickly thanthe fermentation of the resulting sugar[Harden and Young, 1904], but thecourse of this enzymic hydrolysis ofglycogen by yeast-juice has not yetbeen studied. As a rule, it is found bothwith juices from top and bottom yeastthat the evolution of carbon dioxidefrom glycogen proceeds less rapidlyand reaches a lower total than from anequivalent amount of glucose.

Since nearly all samples of yeastcontain glycogen, yeast-juice and alsozymin usually contain this substance aswell as the products of its hydrolysis.

These provide a source of sugar whichenters into alcoholic fermentation, sothat a slow spontaneous production ofcarbon dioxide and alcohol proceedswhen yeast-juice is preserved withoutany addition of sugar. The extent ofthis autofermentation variesconsiderably, as might be expected,with the nature of the yeast employedor the preparation of the material, butis generally confined within the limitsof 0·06 to 0·5 gram of carbon dioxidefor 25 c.c. of juice.

In juice from bottom yeast itamounts to about 5 to 10 per cent. of

the total fermentation obtainable withglucose [Buchner, 1900, 2], whereas injuice from top yeasts, which gives asmaller total fermentation withglucose, it may occasionally equal, oreven exceed, the glucose fermentation,and frequently amounts to 30 to 50 percent. of it. It is therefore generallyadvisable in studying the effect ofyeast-juice on any particular substanceto ascertain the extent ofautofermentation by means of aparallel experiment.

The maceration extract of Lebedeff(p. 24) is usually, but not invariably

[Oppenheimer, 1914, 2], free fromglycogen, which is hydrolysed [p034]

and fermented during the processes ofdrying and macerating, and therefore asa rule shows no appreciableautofermentation.

(e) Effect of Concentration ofSugar on the Total Amount of

Fermentation.The kinetics of fermentation by

zymase will be considered later on (p.120), but the effect on the totalfermentation of differentconcentrations of sugar, this substance

being present throughout inconsiderable excess, may beadvantageously discussed at this stage.The subject has been investigated byBuchner [Buchner, E. and H., andHahn, 1903, pp. 150–8; Buchner andRapp, 1897] using cane sugar, and hehas found both for yeast-juice and fordried yeast-juice dissolved in waterthat (a) the total amount offermentation increases with theconcentration of the sugar; (b) theinitial rate of fermentation decreaseswith the concentration of the sugar.The following are the results of a

typical experiment, 20 c.c. of yeast-juice being employed in presence oftoluene at 22°:—

Cane Sugar. CO2 in grams afterWeight. Per cent. 6 hours. 24 hours.

2·2 10 0·173·52 15 0·145 20 0·136·66 25 0·138·56 30 0·1210·76 35 0·1213·33 40 0·11

The results as to the totalfermentations in experiments of thiskind are liable to be vitiated by thecircumstance that when a low initialconcentration of sugar is employed, the

supply of sugar may be so greatlyexhausted before the close of theexperiment as to cause a markeddiminution in the rate of fermentationand hence an unduly low total. Evenallowing, however, for any effect ofthis kind, the foregoing table clearlyshows the increase in totalfermentation and the decrease in initialrate accompanying the increase ofsugar concentration from 10 to 40 percent. Working with a greater range ofconcentrations (3·3–53·3 grm. per 100c.c.) Lebedeff has obtained similarresults with maceration extract [1911,

4], but has found that the total amountfermented diminishes after a certainoptimum concentration (about 33·3grm. per 100 c.c.) is reached.

A practical conclusion from theseexperiments is that a high [p035]

concentration of sugar tends topreserve the enzyme in an active statefor a longer time. Simultaneously itprevents the development of bacteriaand yeast cells.

(f) Effect of VaryingConcentration of Yeast-Juice.

This subject, which is of

considerable importance with referenceto the question of the protoplasmic orenzymic nature of the active agent inyeast-juice, has been examined in somedetail by Buchner [Buchner, E. and H.,and Hahn, 1903, pp. 158–65] and byMeisenheimer [1903] for juices frombottom yeast, by Harden and Young[1904] for those from top yeast, and byLebedeff [1911, 4] for macerationextract, the results obtained being insubstantial agreement.

Dilution of yeast-juice with sugarsolution, so that the concentration ofthe sugar remains constant, produces a

small progressive diminution in thetotal fermentation, which only becomesmarked when more than 2 volumes areadded, and this independently of theactual concentration of the sugar.Dilution with water produces asomewhat more decided diminution,which, however, does not exceed 50 percent. of the total for the addition of 3volumes of water. The effect onmaceration extract is somewhat greaterbut of the same kind. Theautofermentation of juice from topyeast is scarcely affected by dilutionwith 4 volumes of water.

Natureof Juice.

Per cent.of SugarEmployed

byWeight.

Volumesof

SugarSolutionAdded.

BottomYeast

1 29

0124

2 9

0124

3 9

————

1

0(Auto-ferment-ation)

——

—0

TopYeast 2 29

1246

3 7·4

————

[p036]

On the whole, therefore, yeast-juicemay be said to be only slightly affectedby dilution even with pure water, andthe effect of the latter can in no way beregarded as comparable with thepoisonous effect which it exerts onliving protoplasm, as suggested byMacfadyen, Morris, and Rowland

[1900].

(g) The Effect of Antisepticson the Fermentation of Sugars

by Yeast-Juice.Buchner has paid special attention to

the effect of antiseptics on the courseof 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 theactive agent in yeast-juice consisting offragments of protoplasm and not of asoluble enzyme, and (2) also to providea safe method of avoidingcontamination, by the growth ofbacteria or yeasts, of the liquids usedwhich were often kept at 25° forseveral days. The results of theseexperiments are briefly summarised inthe following table, in which the effectof each substance on the totalfermentation produced is noted:—

Substance.

Concentrated solution ofglycerolConcentrated solution ofsugarToluene (to saturation orexcess)

Chloroform 0·5 percent.0·8 percent.(saturation)Large excess(17 percent.)

Chloralhydrate

0·7 percent.

3·5–5·4 per

cent.Phenol 0·1 per

cent.0·5 "

1·2 "

Thymol 1 "

5 "Benzoic acid 0·1 "

0·25 "Salicylicacid

0·1 "0·27 "

Formaldehyde 0·12 "0·24 "

Acetone 6 "14 "

Alcohol 6 "14 "

Sodium 0·5 "

fluoride 2 "

Ammoniumfluoride

0·55 percent.

Sodiumazoimide,NaN3,

0·36 percent.0·71 "

Quininehydrochloride

1 "

Ozone 10·4–34·8mgs. per 20c.c.

Hydrocyanicacid

1·2 percent.

[p037]

The general result of theseexperiments is to show that quantitiesof antiseptics which are sufficient toinhibit the characteristic action of

living cells have only a slight effect onthe fermentative activity of yeast-juice.A large excess of the antiseptic inmany cases produces a very decideddiminution or total destruction of thefermenting power, and accompanyingthis a precipitation of the constituentsof the juice. The decided increase ofactivity produced by small quantitiesof chloral hydrate, and to a less markedextent by chloroform and a few othersubstances, is of considerable interest.It is ascribed by Duchaček to aselective action on the proteoclasticenzyme, but without satisfactory

evidence.Hydrocyanic acid, even in dilute

solution, completely suspends thefermenting power of the juice, without,however, producing any permanentchange in the fermenting complex, asis shown by the fact that when thehydrocyanic acid is removed by acurrent of air, the juice regains itsfermenting power. In this respecthydrocyanic acid behaves precisely aswith many other enzymes and withcolloidal platinum [Bredig, 1901].Sodium arsenite is a pronouncedprotoplasmic poison, which rapidly

destroys the power of growth andreproduction in living cells, and wastherefore applied to yeast-juice todifferentiate between protoplasmic andenzymic action. It was, however, foundthat the action of this substance wascomplicated by some unknown factorand very irregular results wereobtained [Buchner, E. and H., andHahn, 1903, pp. 193 ff.]. Thesephenomena appear to be of the sameorder as those produced by the additionof arsenates to yeast-juice [Harden andYoung, 1906, 3], and will be discussedalong with the latter (p. 77).

Permanent PreparationsContaining Active Zymase.

A considerable number ofpreparations have been obtained in thedry state which retain some proportionof the fermenting power of yeast oryeast-juice.

Starting with yeast-juice, it ispossible to arrive at this result eitherby evaporation or precipitation. Whenthe juice is very rapidly evaporated to asyrup at 20° to 25° and then furtherdried at 35°, either in the air or in avacuum and finally exposed over

sulphuric acid in a vacuum desiccator,a dry brittle mass is obtained which issoluble in water and retains practicallythe whole of the fermenting power ofthe juice. The success of thepreparation depends on the nature ofthe yeast from which the juice isderived, Berlin yeasts V and S yieldingmuch less satisfactory results thanMunich yeast. The powder when [p038]

thoroughly dry is found to retain itsproperties almost unimpaired for atleast a year, and can be heated to 85°for eight hours without undergoing anyserious 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 obtainedby precipitating yeast-juice withalcohol, alcohol and ether, or acetone.The preparation is best effected bybringing the juice into 10 volumes ofacetone, centrifuging at once and asrapidly as possible, washing, first withacetone and then with ether, and finallydrying over sulphuric acid. The whitepowder thus obtained is not completelysoluble in water but is almost entirelydissolved by aqueous glycerol (2·5 to

20 per cent.), forming a solution whichhas practically the same fermentingpower as the original juice. Theprecipitation can be repeated withoutany serious loss of fermenting power.Prolonged contact of the precipitatewith the supernatant liquid, especiallywhen alcohol or alcohol and ether areused, causes a rapid loss of thecharacteristic property [Albert andBuchner, 1900, 1, 2; Buchner, E. andH., and Hahn, 1903, pp. 228–246;Buchner and Duchaček, 1909].

Dry preparations capable offermenting sugar can also be readily

obtained from yeast without anypreliminary rupture of the cells. Heatalone (yielding a product known ashefanol) or treatment with dehydratingagents may be used for this purpose,and a brief allusion has already beenmade (p. 21) to the different varietiesof permanent yeast (Dauerhefe)obtainable in these ways. The mostimportant of these products are thedried Munich yeast (Lebedeff, see p.25), and the material known as zymin,which is now made under patent rightsfor medicinal purposes by Schroder ofMunich. The latter has proved of value

in the investigation of the productionof zymase in the yeast cell [Buchnerand Spitta, 1902], and of many otherproblems concerned with alcoholicfermentation. In order to prepare it 500grams of finely divided pressedbrewer's yeast, containing about 70 percent. of water, are brought into 3 litresof acetone, stirred for ten minutes, andfiltered and drained at the pump. Themass is then well mixed with 1 litre ofacetone for two minutes and againfiltered and drained. The residue isroughly powdered, well kneaded with250 c.c. of ether for three minutes,

filtered, drained, and spread on filterpaper or porous plates. After standingfor an hour in the air it is dried at 45°for twenty-four hours. About 150grams of an almost white powdercontaining only 5·5 to 6·5 per cent. ofwater are obtained. This is quiteincapable of growth or reproductionbut produces a very considerableamount of alcoholic fermentation, fargreater indeed than a corresponding[p039] quantity of yeast-juice. Twograms of the powder corresponding to6 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 wouldon the average only ferment from one-quarter to one-sixth of this amount ofsugar. The rate produced by thisamount of zymin is about one-eighth ofthat given by the corresponding amountof living yeast [Albert, 1900; Albert,Buchner, and Rapp, 1902]. Theproteoclastic ferment is still present inzymin, which undergoes autolysis inpresence of water in a similar mannerto yeast-juice [Albert, 1901, 2].

As already mentioned an active juicecan be prepared by grinding acetone-

yeast with water, sand, and kieselguhr,and this process presents the advantagethat samples of yeast-juice ofapproximately constant compositioncan be prepared at intervals fromsuccessive portions of a uniformsupply of acetone-yeast.

Preparations of acetone-yeast, madefrom yeast freed from glycogen byexposure in a thin layer to the air forthree or four hours at 35° to 45°, oreight hours at the ordinary temperature[Buchner and Mitscherlich, 1904],show practically no autofermentationand may be used analytically for the

estimation of fermentable sugars.All the foregoing preparations

exhibit the same general properties asyeast-juice, as regards their behaviourtowards the various sugars, antiseptics,etc.

When zymin is mixed with sugarsolution without being previouslyground, it exhibits a peculiarity whichis of some practical interest. The timewhich elapses before the normal rate offermentation is attained and the totalfermentation obtainable vary with theamount of sugar solution added, thetime increasing and the total

diminishing as the quantity of thisincreases. This phenomenon appears tohave been noticed by Trommsdorff[1902], and a single experiment ofBuchner shows the influence of thesame conditions [Buchner, E. and H.,and Hahn, 1903, p. 265, Nos. 700–1].Harden and Young have found thatwhen 2 grams of zymin are mixed withvarying quantities of 10 per cent. sugarsolution the following results areobtained:—

Volumesof

SugarSolution

Total Gas Evolved in

1 2 3

5c.c. 15·7 31·6 44·8

10 2·2 10·5 23 20 0·9 2·4 13·640 1·4 1·7 2·3

[p040]

This behaviour appears to be due tothe removal of soluble matter essentialfor fermentation from the cell, which isdiscussed later on. It follows that whenzymin is being tested for fermentingpower, a uniform method should beadopted, and all comparative testsshould be made with the same volumes

of added sugar solution. Ground zyminappears to begin to ferment somewhatmore slowly than unground (2 grm. to12·4 c.c. of sugar solution in eachcase), but eventually produces the sametotal volume of gas [Buchner andAntoni, 1905, 1].

CHAPTER III.THE FUNCTION OF

PHOSPHATES IN ALCOHOLIC

FERMENTATION.

[p041]

In the course of some preliminaryexperiments (commenced by the lateAllan Macfadyen, but subsequentlyabandoned) on the production of anti-ferments by the injection of yeast-juiceinto animals, the serum of the treatedanimals was tested for the presence of

such antibodies both for the alcoholicand proteoclastic enzymes of yeast-juice, and it was then observed that theserum of normal and of treated animalsalike greatly diminished the autolysisof yeast-juice.

As the explanation of thecomparatively rapid disappearance ofthe fermenting power from yeast-juicehad been sought, as already mentioned(p. 20), in the hydrolytic action of thetryptic enzyme which alwaysaccompanies zymase, the experimentwas made of carrying out thefermentation in the presence of serum,

with the result that about 60 to 80 percent. more sugar was fermented than inthe absence of the serum [Harden,1903].

This fact was the starting-point of aseries of attempts to obtain a similareffect by different means, in the courseof which a boiled and filtered solutionof autolysed yeast-juice was used, inthe hope that the products formed bythe action of the tryptic enzyme on theproteins of the juice would, inaccordance with the general rule, proveto be an effective inhibitant of thatenzyme. This solution was, in fact,

found to produce a very markedincrease in the total fermentationeffected by yeast-juice, the addition ofa volume of boiled juice equal to thatof the yeast-juice doubling the amountof carbon dioxide evolved [Harden andYoung, 1905, 1]. This effect was foundto be common to the filtrates fromboiled fresh yeast-juice and fromboiled autolysed yeast-juice, and wasultimately traced in the main, not to theantitryptic effect which had beensurmised, but to two independentfactors, either of which was capable insome degree of bringing about the

observed result.Boiled yeast-juice was indeed found

to possess a decided anti-autolyticeffect, as determined by a comparisonof the amounts of nitrogen renderednon-precipitable by tannic acid inyeast-juice alone [p042] and in a mixtureof yeast-juice and boiled juice onpreservation [Harden, 1905]. The anti-autolytic effect, however, appeared tovary independently of the effect on thefermentation, and the conclusion wasdrawn, as stated above, that theincrease in the alcoholic fermentationwas not directly dependent on the

decrease in the action of theproteoclastic enzyme but was due tosome independent cause. The propertypossessed by boiled yeast-juice ofdiminishing the autolysis of yeast-juicehas now been carefully examined byBuchner and Haehn [1910, 2] andascribed by them to a solubleantiprotease (p. 65).

The two factors to which theincrease in fermentation produced bythe addition of boiled juice wereultimately traced were (1) the presenceof phosphates in the liquid, and (2) theexistence in boiled fresh yeast-juice of

a co-ferment or co-enzyme, thepresence of which is indispensable forfermentation [Harden and Young,1905, 1, 2].

The former of these factors will behere discussed and the co-enzyme willform the subject of the followingchapter.

The general fact that sodiumphosphate increases the totalfermentation produced by a givenvolume of yeast juice was observed onseveral 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 toits alkalinity, comparing it in thisrespect with potassium carbonate andremarking that the increase in bothcases took place chiefly in the firsttwenty hours of fermentation. Theincreased amount of fermentationfollowing the addition of boiled yeast-juice was also noted by Buchner andRapp [1899, 2, No. 265, p. 2093] in asingle experiment.

Observations made at intervals of afew minutes instead of twenty hourshave, however, revealed the fact thatphosphates play a part of fundamental

importance in alcoholic fermentationand that their presence is absolutelyessential for the production of thephenomenon.

Effect of the Addition ofPhosphate to a FermentingMixture of Yeast-Juice and

Sugar.

When a suitable quantity[2] of asoluble phosphate is added to afermenting mixture of glucose,fructose, or mannose with yeast-juice,

the rate of fermentation rapidly rises,sometimes increasing as much astwenty-fold, continues at this highvalue for a certain period and then fallsagain to a value approximately equalto, but generally [p043] somewhathigher than, that which it originallyhad. Careful experiments have shownthat during this period of enhancedfermentation the amounts of carbondioxide and alcohol produced exceedthose which would have been formed inthe absence of added phosphate by aquantity exactly equivalent to thephosphate added in the ratio CO2 or

C2H6O:R′2HPO4 [Harden and Young,1906, 1].

[2] The effect of an excess ofphosphate is discussed

later on, p. 71.

This result is of fundamentalimportance, and the evidence on whichit rests deserves some consideration.Quantitative experiments on thissubject require certain preliminaryprecautions. The acid phosphates aretoo acid to permit of any extendedfermentation and the phosphates of theformula R′2HPO4 absorb a

considerable volume of carbon dioxidewith production of a bicarbonate,according to the reaction:—

R2HPO4 + H2CO3 ⇌ RHCO3 + RH2PO4.

The method which has been adopted,therefore, is to employ either asecondary phosphate saturated withcarbon dioxide at the temperature ofthe experiment, or a mixture of fivemolecular proportions of the secondaryphosphate with one molecularproportion of a primary phosphate, inwhich the amount of bicarbonate

formed is negligible. In the former caseit is necessary to ascertain whether anyof the carbon dioxide evolved isderived from the bicarbonate by theaction of acid originally present orproduced in the yeast-juice or by adisturbance of the original equilibriumowing to the chemical change whichoccurs. This is done by acidifyingduplicate samples with hydrochloricacid before and after the fermentationand measuring the gas evolved in eachcase. Any necessary correction canthen be made. The calculation of theextra amount of carbon dioxide

evolved from yeast-juice containingsugar when a phosphate is addedinvolves an estimation of the amountwhich would have been evolved in theabsence of added phosphate, and this isa matter of some difficulty. Since thefinal steady rate of fermentationattained is often slightly different fromthe initial rate, the practice has beenadopted of ascertaining this final rateand then calculating the total evolutioncorresponding to it for the wholeperiod from the time of the addition ofthe phosphate to the end of theobservations. This amount deducted

from the observed total leaves the extraamount of carbon dioxide formed, andit is this quantity which is equivalent tothe phosphate added. Alcohol issimultaneously produced in the normalratio. The justification for this methodof calculation will be found later (p.54).

The following table, containing theresults of experiments with [p044]

glucose, fructose, and mannose,indicates very clearly the nature of themethod of calculation and also of theagreement between observation andtheory.

Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution containing 1gram of the sugar to be examined (alarge excess) were incubated withtoluene at 25° for one hour, in order toremove all free phosphate, and to eachwere then added 5 c.c. of a solution ofsodium phosphate corresponding to0·1632 gram of Mg2P2O7 andequivalent to 32·6 c.c. of carbondioxide at N.T.P. The rates offermentation were then observed untilthey had passed through the period ofacceleration and had fallen and attaineda steady value, the gases being

measured moist at 19·3° and 760·15mm.

Glucose. Mannose.Maximum rateattained,c.cs. perfive minutes 9·6 7

Final rate offermentation 1·1 0·96

Total carbondioxideproduced byfermentationin fifty-five minutesafteraddition ofphosphate 49·7 47·8

Correctionforevolution in

absence ofphosphate infifty-fiveminutes 12·1 10·6

Extra carbondioxideequivalentto phosphate 37·6 37·2

Extra carbondioxideequivalentto phosphateat N.T.P. 34·4 34

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

Another experiment is illustratedgraphically in Fig. 4, in which the

volume of carbon dioxide evolved isplotted against time. The determinationwas in this case made by adding 25 c.c.of an aqueous solution containing 5grams of glucose to one quantity of 25c.c. of yeast-juice (curve A) and 5 c.c.of 0·3 molar solution of the mixedprimary and secondary sodiumphosphates, and 20 c.c. of a solutioncontaining 5 grams of glucose to asecond equal quantity of yeast-juice(curve B). Curve A shows the normalcourse of fermentation of yeast-juicewith glucose. There is a slightpreliminary acceleration during the

first twenty minutes, due to freephosphate in the juice, and the rate thenbecomes steady at about 1·4 c.c. in fiveminutes. During this preliminaryacceleration 10 c.c. of extra carbondioxide are evolved, this number beingobtained graphically by continuing theline of steady rate back to the axis ofzero time. Curve B shows the effect ofthe added phosphate. The rate rises toabout 9·5 c.c. in five minutes, i.e. tomore than six times the normal rate,and then gradually falls until after anhour it is again steady and almostexactly equal to 1·4 c.c. per five

minutes. Continuing the line of steadyrate back to the axis of zero [p045] timeit is found that the extra amount ofcarbon dioxide is 48 c.c. Subtractingfrom this the 10 c.c. shown in curve Aas due to the juice alone, a differenceof 38 c.c. is obtained due to the addedphosphate. The amount calculated fromthe phosphate added in this case is, atatmospheric temperature and pressure,38·9 c.c.

FIG. 4.

After the expiration of seventyminutes from the commencement ofthe experiment, a second addition ismade of an equal amount of phosphate.

The whole phenomenon then recurs, asshown in curve C, the maximum ratebeing slightly lower than before, about6 c.c. per five minutes, and the rateagain becoming finally steady at 1·4c.c. as before. The extra amount ofcarbon dioxide evolved in this secondperiod obtained graphically as in theformer case, is 107–68 = 39 c.c.

It may be noted that in this case theobservations after each addition lastfifty to seventy minutes, so that anerror of 0·1 c.c. per five minutes in theestimated final rate would make anerror of 1 to 1·4 c.c. in the extra

amount of carbon dioxide, i.e. about 3to 4 per cent. of the total, and this isapproximately the limit of accuracy ofthe method. [p046] The results are moreprecise when the yeast-juice employedis an active one, since, when thefermenting power of the juice is low,the initial period of acceleratedfermentation is unduly prolonged andthe calculation of the extra amount ofcarbon dioxide is rendered uncertain.

Zymin (p. 38) yields preciselysimilar results to yeast-juice, but inthis case the rate of fermentation is notso largely increased. This has the effect

that the extra amount of carbon dioxidecannot be quite so accurately estimatedfor zymin, because a slight error in thedetermination of the final rate offermentation has a greater influence onthe result. The equivalence between theextra amount of carbon dioxideevolved and the phosphate added is,however, unmistakable, as is shown bythe following results of an experimentwith zymin, in which 6 grams of zymin(Schroder) + 3 grams of fructose(Schering) + 25 c.c. of water wereincubated at 25° in presence of tolueneuntil a steady rate had been attained.

Five c.c. of a solution of sodiumphosphate equivalent to 32·2 c.c.carbon dioxide at N.T.P. were thenadded.

Maximum rateattained, c.c. perfive minutes 14·1

Final rate offermentation 6·2

Total evolved byfermentation ineighty minutesafter addition ofphosphate 131

Correction forevolution inabsence ofphosphate ineighty minutes 99·2

Extra carbon dioxideat 16° and 767·1mm 31·8

Extra carbon dioxideat N.T.P 29·8

Considering the small proportionalrise in rate and the long period of

accelerated fermentation, theagreement between the volumeobserved, 29·8 c.c., and that calculatedfrom the phosphate, 32·2, is quitesatisfactory [Harden and Young, 1910,1.] Precisely the same relations holdfor maceration extract, but in this caseit must be remembered that a largeamount of free phosphate is present inthe extract, as much as 0·3129 grm.Mg2P2O7 being obtained from 20 c.c.in one preparation, so that the originalextract had the concentration of a 0·14molar solution of sodium phosphate. Itis in fact not improbable that the delay

in the onset of fermentation sometimesobserved with maceration extract (seeLebedeff, 1912, 2; Neuberg andRosenthal, 1913) may be due to thepresence of phosphate in so great anexcess of the amount which can berapidly esterified by the enzymes thatthe rate of fermentation is at firstgreatly lowered (see p. 71). When thisphosphate is removed by incubationwith glucose or fructose, thesubsequent addition of phosphateproduces the characteristic action andthe extra carbon dioxide evolved is, aswith other yeast preparations,

equivalent to the phosphate added. Anactual estimation carried out in thisway gave 35 c.c. of CO2 for an additionof phosphate equivalent to 32·9 c.c.[Harden and Young, 1912]. [p047]

Within the limits imposed by theexperimental conditions, then, the factis well established that the addition ofa soluble phosphate to a fermentingmixture of a hexose with yeast-juice,maceration extract, dried yeast, orzymin causes the production of anequivalent amount of carbon dioxideand alcohol.

This fact indicates that a definite

chemical reaction occurs in whichsugar and phosphate are concerned, andthis conclusion is confirmed when thefate of the added phosphate isinvestigated. If an experiment, such asone of those described above, beinterrupted as soon as the rate offermentation has again become normal,and the liquid be boiled and filtered, itis found that nearly the whole of thephosphorus present passes into thefiltrate, but that only a smallproportion of this exists as mineralphosphate, whilst the remainder,including that added in the form of a

soluble phosphate, is no longerprecipitable by magnesium citratemixture [Harden and Young, 1905, 2].

A similar observation was made at alater date by Iwanoff [1907], who hadpreviously observed [1905] that livingyeast, like many other vegetableorganisms, converted mineralphosphates into organic derivatives.Iwanoff employed zymin and hefanol(p. 38) instead of yeast-juice, andfound that phosphates were therebyrendered non-precipitable by uraniumacetate solution, but did not observethe accelerated fermentation caused by

their addition.The foregoing conclusions have been

strikingly confirmed by experimentswith maceration extract carried out byEuler and Johansson [1913], in whichboth the carbon dioxide evolved andthe phosphate rendered non-precipitable by magnesia weredetermined at intervals. When driedyeast is employed as the fermentingagent, the amount of phosphateesterified in the earlier stages is greaterthan would be expected, but ultimatelybecomes exactly equivalent to thecarbon dioxide evolved.

Nature of the Phospho-organic Compound formedby Yeast-Juice and Zymin

from the Hexoses andPhosphate.

The formation and properties of thecompound produced from phosphatesin the manner just described have beeninvestigated 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; Eulerand Kullberg, 1911, 3; Euler andOhlsén, 1911; 1912; Euler andJohansson, 1912, 4; Euler andBäckström, 1912], but its exactconstitution cannot as yet be regardedas definitely known. [p048]

Phosphates undergo thischaracteristic change when the sugarundergoing fermentation is glucose,mannose, or fructose, and it may besaid at once that no distinction can beestablished between the productsformed from these various hexoses;they all appear to be identical. The

compound produced is, as alreadymentioned, not precipitated byammoniacal magnesium citratemixture, nor by uranium acetatesolution. It can, however, beprecipitated by copper acetate(Iwanoff) and by lead acetate (Young).The preparation of the pure lead saltfrom the liquid obtained by fermentinga sugar with yeast-juice or zymin inpresence of phosphate is commencedby boiling and filtering the liquid.Magnesium nitrate solution and a smallquantity of caustic soda solution arethen added to precipitate any free

phosphate, and the liquid well stirredand allowed to stand over night. To theneutralised filtrate lead acetate is thenadded together with sufficient causticsoda solution to maintain the reactionneutral to litmus, until no furtherprecipitate is formed. The liquid is thenfiltered or, better, centrifugalised, andthe precipitate repeatedly washed withwater until a portion of the clearfiltrate gives no reduction when boiledwith Fehling's solution. It is essentialthat this washing should be thorough asevidence has recently been obtained ofthe formation under certain conditions

of a hexosephosphate, the lead salt ofwhich is not so sparingly soluble asthat of the hexosediphosphate [Hardenand Robison, 1914]. The leadprecipitate is then suspended in water,decomposed by a current ofsulphuretted hydrogen, the clear filtratefreed from sulphuretted hydrogen by acurrent of air, and finally neutralisedwith caustic soda. The removal ofphosphate and conversion into lead saltare repeated twice, and the resultinglead salt is then found to be free fromnitrogen and to have a compositionrepresented by the formula

C6H10O4(PO4Pb)2. Lebedeff carriesout the preparation in a somewhatdifferent manner. The fermentation iseffected by means of air-dried yeast(150 grams to 1 litre of water, 210grams cane-sugar and 105 grams of amixture of 2 parts Na2HPO4 and 1 partNaH2PO4) and the liquid (about 700c.c.) after boiling and filtering, istreated with an equal volume ofacetone. About 300 c.c. of a thickliquid is precipitated and this isredissolved in water and precipitatedby an equal volume of acetone two orthree times. The final liquid is then

precipitated with warm lead acetatesolution and filtered and washed withdilute lead acetate solution until thefiltrate is clear and no longer reducesFehling's solution after removal of thelead [1910]. Euler and Fodor [1911] onthe other hand precipitate the freephosphate with magnesia mixture andthen add acetone, dissolve the syrupthus precipitated in water and addcopper [p049] acetate solution. A bluecopper salt is precipitated which isthoroughly washed with water and usedfor the preparation of solutions of theacid. A solution of the free acid can

readily be prepared by the action ofsulphuretted hydrogen on the lead saltsuspended in water. It forms a stronglyacid liquid, which requires exactly twoequivalents of base for each atom ofphosphorus present to render it neutralto phenolphthalein. It decomposeswhen evaporated, leaving a charredmass containing free phosphoric acid.The acid is slightly optically active,and has [aD] = + 3·4°. A number ofamorphous salts have been prepared byprecipitation from a solution of thesodium salt, and of these the silver,barium, and calcium salts have been

analysed with results agreeing with thegeneral formula C6H10O4(PO4R′2)2.The magnesium, calcium, barium, andmanganese salts, which are onlysparingly soluble, are all precipitatedwhen their solutions are boiled but re-dissolve on cooling, and this propertycan be utilised for their purification.The alkali salts have only beenobtained as viscid residues.

A difference of opinion exists as tothe molecular weight and constitutionof this substance. Iwanoff [1909, 1]regards it as a triosephosphoric acid, C3H5O2(PO4H2), basing this view on

the preparation of an osazone whichmelted at 142°, but when recrystallisedfrom benzene gave a product melting at127°–8°, which had the sameappearance, melting-point, andnitrogen content as the triosazoneformed by the action ofphenylhydrazine on the oxidationproducts of glycerol. Neither Lebedeff[1909] nor Young could obtainIwanoff's osazone, and all attempts toreduce the acid with formation ofglycerol either by sodium amalgam orhydriodic acid were unsuccessful(Young). There is therefore practically

no serious experimental evidence infavour of Iwanoff's view.

On the other hand, Harden andYoung regard the acid as adiphosphoric ester of a hexose. Thisview is based on the fact that when theacid is boiled with water, or an acid,free phosphoric acid is produced alongwith a levo-rotatory solutioncontaining fructose and possibly asmall proportion of some other sugaror sugars. (Euler and Fodor howeverdid not obtain a hexose in this way[1911].) The acid itself only reducesFehling's solution after some hours in

the cold, rapidly when boiled, whereaswhen its solution is first boiled, andthen treated with Fehling's solution inthe cold, the products of decompositionbring about reduction in a few minutes.The reduction brought about when theacid is boiled with Fehling's solution isconsiderably less (33 per cent.) thanthat produced by an equivalent amountof glucose. The behaviour of thecompound towards phenylhydrazine isalso in complete agreement [p050] withthis view. Lebedeff found [1909, 1910]that the acid or its salts heated withphenylhydrazine in presence of acetic

acid gave an insoluble compoundwhich was ultimately found to be thephenylhydrazine salt ofhexosemonophosphoric acid osazone

C6H5NH·NH2·H2PO4·C4H5(OH)3·C(N

[Lebedeff, 1910; 1911, 6; Young,1911]. After recrystallisation fromalcohol this compound forms yellowneedles, melting at 151°–152°. It isdecomposed by caustic soda yielding asodium salt

Na2PO4·C4H5(OH)3·(CN2HC6H5)·CH(N

and on boiling with caustic soda

decomposes giving a hexosazone (freefrom phosphorus) which is probablyglucosazone, and in additionglyoxalosazone, probably as the resultof a secondary decomposition. Towardsacids it is remarkably stable yieldingwith hydrochloric acid ahexosonephosphoric ester from whichthe original osazone can be regenerated(Lebedeff). Lebedeff at first [1910]argued from the formation of thisosazone that the originalhexosephosphate contained only onephosphoric acid group per molecule ofhexose. It was however shown by

Young [1911] and subsequentlyconfirmed by Lebedeff [1911, 6] thatone molecule of phosphoric acid issplit off during the formation of theosazone, even in neutral solution.Moreover it has been found that in thecold hexosediphosphoric acid reactswith 3 molecules of phenylhydrazineforming the diphenylhydrazine salt ofhexosediphosphoric acidphenylhydrazone

(C6H5NH·NH2·H2PO4)2·C6H7(OH)3·N

This compound crystallises out when 1volume of alcohol is added to a

solution of 3 molecules ofphenylhydrazine in one of the acid andforms colourless needles melting at115°–117°. p-Bromophenylhydrazineyields an analogous compound meltingat 127°–128°.

Precisely the same products aregiven with phenylhydrazine by thehexosephosphoric acid prepared fromglucose, mannose, and fructose,proving that all these sugars yield thesame hexosediphosphoric acid, a pointof fundamental importance.

Direct measurements of themolecular weight of the acid by the

freezing-point method, combined withthe determination of the degree ofdissociation by the rate of cane-sugarinversion, are indecisive, but indicatethat the acid has a molecular weightconsiderably higher than that requiredfor a triosephosphoric acid.

A similar uncertainty attaches to thedetermination of the molecular weightfrom the freezing-point depression andconductivity of the acid potassium salt[Euler and Fodor, 1911]. Euler howeverconcludes [p051] that both ahexosediphosphoric acid and atriosemonophosphoric acid are formed,

but has not prepared any derivatives ofthe latter.

As regards the constitution of thehexosephosphoric ester severalsuggestions have been made by Young,but no decisive evidence at presentexists. The identity of the productsfrom glucose, mannose, and fructosemay be explained by regarding the acidas a derivative of the enolic formcommon to these three sugars (p. 97),or by supposing that portions of twosugar molecules may be concerned inits production. The formation andcomposition of the hydrazone and

osazone are of great importance as theyindicate that in all probability one ofthe phosphoric acid residues is unitedwith the carbon atom adjacent to thecarbonyl group of the hexose. Theymoreover render it certain that theoriginal phosphoric ester is ahexosediphosphoric ester and not atriosemonophosphoric ester.

Hexosediphosphoric acid has not asyet been discovered in the animal body.The action of a number of enzymesupon it has been examined [Euler,1912, 2; Euler and Funke, 1912;Harding, 1912; Plimmer, 1913] with

the following results.The lipase of castor oil seeds, a

glycerol extract of the intestinalmucous membrane of the rabbit andpig, and an aqueous extract of branhave a slow hydrolytic action, whereaspepsin and trypsin are without effect.Feeding experiments with rabbits anddogs indicate that the ester is capableof hydrolysis in the animal body, alarge proportion of the phosphorusbeing excreted as inorganic phosphate.The ester is also decomposed byBacillus coli communis.

It is remarkable that the

hexosephosphate is not fermented norhydrolysed by living yeast, a factobserved by Iwanoff, Harden andYoung, and Euler, although, accordingto the experiments of Paine [1911], theyeast cell is at all events partiallypermeable to the sodium salt.

The Equation of AlcoholicFermentation.

An equation can readily beconstructed for the reaction in whichhexosephosphate is formed, the dataavailable being the formula of the

product and the relation between thephosphate added and the carbondioxide and alcohol produced:—

(1) 2 C6H2O6 + 2 PO4HR3  = 2 CO2 + 2 C2H6O + 2 H2O + C6H10O4(PO4R2)2.

According to this, two molecules ofsugar are concerned in the change, thecarbon dioxide and alcohol being equalin weight to one [p052] half of the sugarused, and the hexosephosphate andwater representing the other half.

Additional confirmation of thisequation is afforded by the

determination of the ratio betweensugar used and carbon dioxide evolvedwhen a known weight of sugar togetherwith an excess of phosphate is added toyeast-juice at 25°. The phenomena thenobserved are precisely similar to thosewhich occur when a phosphate is addedto a fermenting mixture of yeast-juiceand excess of sugar as described above.The rate of fermentation rapidly risesand then gradually falls until a rate isattained approximately equal to that ofthe autofermentation of the juice inpresence of phosphate. At this point itis found that the extra amount of

carbon dioxide evolved, beyond thatwhich would have been given off in theabsence of added sugar, bears the ratioexpressed in equation (1) to the sugaradded [Harden and Young, 1910, 2].The results of four estimations made inthis way were (a) 0·2 grams of glucosegave 26·5 and 27·9 c.c. of carbondioxide at N.T.P.; (b) 0·2 grams offructose gave 27·9 and 28·9 c.c. Thecarbon dioxide calculated from thesugar added in each of the four cases is26·96 c.c.

It has also been shown by Euler andJohansson [1913] that in the

fermentation of a mixture of equivalentamounts of phosphate and glucose, thewhole of the glucose had disappearedwhen the whole of the phosphate hadbecome esterified.

Cycle of ChangesUndergone by Phosphate in

Alcoholic Fermentation.

According to equation (1) the freephosphate present is used up in thereaction, and the question at oncearises whether there is any source fromwhich a constant supply of free

phosphate can be elaborated in thejuice, or whether some other changeoccurs which results in the formationof carbon dioxide and alcohol in theabsence of free phosphate. Theexperimental evidence points in thedirection of the former of thesealternatives, but the question is a verydifficult one to decide with absolutecertainty.

When a mixture of a phosphate withyeast-juice and sugar is examined atintervals and the amount of freephosphate estimated, the followingstages are observed:—

1. During the initial period ofaccelerated fermentation following theaddition of the phosphate, theconcentration of free phosphate rapidlydiminishes.

2. At the close of this period, theamount of free phosphate [p053] presentis very low, and, as long as activefermentation continues, no markedincrease in it occurs.

3. As alcoholic fermentationslackens and finally ceases, a markedand rapid rise in the amount of freephosphate occurs at the expense of thehexosephosphate, which steadily

diminishes in amount, and this changeis brought about by an enzyme in thejuice and ceases if the liquid be boiled.

This last reaction may berepresented by the equation

(2) C6H10O4(PO4R2)2 + 2 H2O = C6H12O6 + 2 PO4HR2.

In the light of this equation, togetherwith equation No. 1, given above, allthese facts can be simply and easilyunderstood.

The rapid diminution in the amountof free phosphate during stage 1corresponds with the occurrence of

reaction (1). During the whole periodof fermentation the enzymic hydrolysisof the hexosephosphate is proceedingaccording to equation (2). Up to theend of stage 2 the phosphate thusproduced enters into reaction,according to equation (1), with thesugar which is present in excess and isthus reconverted into hexosephosphate,so that as long as alcoholicfermentation is proceeding freely, noaccumulation of free phosphate canoccur.

As soon as alcoholic fermentationceases, however, it is no longer

possible for the phosphate to pass backinto hexosephosphate, and hence itaccumulates in the free state.

A similar hydrolysis ofhexosephosphate and accumulation ofphosphate occur when a solution ofhexosephosphate is treated with yeast-juice which has been deprived of thepower of fermentation by dialysis, orwith zymin freed from co-enzyme bywashing (p. 63).

The actual rate of fermentationobserved in any particular case inpresence of excess of sugar, enzyme,and co-enzyme must on this view

depend on the supply of phosphatewhich is available.

In presence of an adequate amountof phosphate, as well as of sugar, thehighest rate attained represents themaximum velocity at which reaction(1) can proceed in that sample of yeast-juice or zymin, and this high rate ischaracteristic of the initial period ofaccelerated fermentation which followsthe addition of a suitable quantity ofphosphate. By the simple expedient ofrenewing the supply of phosphate asrapidly as it is converted intohexosephosphate, this high rate can be

maintained for a considerable time[Harden and Young, 1908, 1]. In thisway, for example, an average rate ofevolution of carbon dioxide of 15 c.c.in five minutes was maintained for anhour and a [p054] quarter, whereas thenormal rate in the absence of addedphosphate was 3 c.c.

As soon as all the free phosphate hasentered into the reaction, however, thesupply of phosphate depends in themain on the rate at which the resultinghexosephosphate is decomposed, andthe rate of fermentation now attained isconditioned by the rate at which

reaction (2) proceeds, and thisevidently depends on the existingconcentration of the hydrolyticenzyme, which may be provisionallytermed hexosephosphatase.

The rates attained during the initialperiod of rapid fermentation and thesubsequent period of slow fermentationare thus seen to represent the velocitiesof two entirely different chemicalreactions.

These considerations also explainwhy it is the extra carbon dioxideevolved during the initial period, andnot the total carbon dioxide, which is

equivalent to the added phosphate. Asthe production of phosphate isproceeding throughout the wholeperiod at a rate which is equivalent tothe normal rate of fermentation, it isobviously necessary to deduct thecorresponding amount of carbondioxide from the total evolved in orderto ascertain the amount equivalent tothe added phosphate.

An explanation is also afforded ofthe fact that a considerable increase inthe concentration of hexosephosphatedoes not materially increase the normalrate of fermentation. This is probably

due to the circumstance that, inaccordance with the general behaviourof enzymes in presence of excess of thefermentable substance, thehexosephosphatase hydrolysesapproximately equal amounts ofhexosephosphate in equal timeswhatever the concentration of the lattermay be, above a certain limit.

According to the experiments ofEuler and Johansson [1913] thehydrolytic activity of thehexosephosphatase is greatlydiminished by the presence of toluene.

Effect of Phosphate on theTotal Fermentation

Produced by Yeast-Juice.

The addition of a phosphate to yeast-juice not only produces the effectalready described, but also enables agiven volume of yeast-juice to effect alarger total fermentation, even afterallowance is made for the carbondioxide equivalent to the quantity ofphosphate added. The increase in thecase of ordinary yeast-juice has beenfound to amount to from 10 to 150 percent. of the original total fermentation

[p055] produced by the juice in theabsence of added phosphate. Thenumbers contained in columns 1 and 2of the table on p. 56 illustrate thiseffect, the ratio of the total in thepresence of phosphate to that obtainedin its absence being given, as well asthat of the total in presence ofphosphate less the equivalent of thephosphate added, to the originalfermentation. The cause of thisincrease in the total fermentation isprobably to be sought mainly in aprotective action of the excess ofhexosephosphate on the various

enzymes, for, as has been stated above,the rate of fermentation after thetermination of the initial period, ispractically the same as in the absenceof added phosphate (see p. 43).

Now it follows from equation (1) (p.51) that in the total absence ofphosphate no fermentation shouldoccur, and the experimental realisationof this result would afford very strongevidence in favour of thisinterpretation of the phenomenon.

Hitherto, however, it has not beenfound possible to free the materialsemployed completely from phosphorus

compounds which yield phosphates byenzymic hydrolysis during theexperiment, but it has been found thatwhen the phosphate contents arereduced to as low a limit as possible,the amount of sugar fermentedbecomes correspondingly small, and,further, that in these circumstances theaddition of a small amount ofphosphate or hexosephosphateproduces a relatively large increase inthe fermenting power of the enzyme.

When the total phosphorus present isthus largely reduced, the increaseproduced by the addition of a small

amount of phosphate may amount to asmuch as eighty-eight times theoriginal, in addition to the quantityequivalent to the phosphate, whilst theactual total evolved, including thisequivalent, may be as much as twentytimes the original fermentation. Thisresult must be regarded as strongevidence in favour of the view thatphosphates are indispensable foralcoholic fermentation.

The results indicated above wereexperimentally obtained in threedifferent ways and are exhibited in thefollowing table. In the first place (cols.

3 and 4), advantage was taken of thefact that the residues obtained byfiltering yeast-juice through a Martingelatin filter (p. 59) are sometimesfound to be almost free from mineralphosphates, whilst they still contain asmall amount of co-enzyme. Theexperiment then consists in comparingthe fermentation produced by such aresidue poor in phosphate with thatobserved when a small amount ofphosphate is added. The second method(col. 5) consisted in carrying out twoparallel fermentations by means of aresidue rendered inactive by filtration

[p056] and a solution of co-enzyme freefrom phosphate and hexosephosphate(p. 67) [Harden and Young, 1910, 2].

The third method (col. 6) consistedin washing zymin with water, toremove soluble phosphates, and thenadding to it a solution of co-enzymecontaining only a small amount ofphosphate, and ascertaining the effectof the addition of a small knownamount of hexosephosphate upon thefermentation produced by this mixture[Harden and Young, 1911, 1].

1c.c.

2c.c.

Gas evolvedin absenceof addedphosphate 369 220

In thepresence ofaddedphosphate 629 629

Increase dueto phosphate 260 409

Carbonic acidequivalentto phosphate 63 61

Increaseafterinitialperiod 197 348

Ratio oftotals 1·7 2·9

Ratio ofincreaseafterinitial

period tooriginalfermentation 0·5 1·6

Production of a FermentableSugar from

Hexosephosphate by theAction of an Enzyme

Contained in Yeast-Juice.

The sugar which, according toequation (2) accompanies thephosphate formed by the enzymichydrolysis of hexosephosphate is underordinary circumstances fermented bythe alcoholic enzyme of the juice and

thus escapes detection.When, however, a solution of a

hexosephosphate is exposed to theaction of either yeast-juice or zymin,entirely or partially freed from co-enzyme, this sugar, being no longerfermented, accumulates and can beexamined. It has thus been found[Harden and Young, 1910, 2] that asugar is in fact produced in this waywhich can be fermented by living yeastand exhibits the reactions of fructose,although the presence of other hexosesis not excluded. The products of theenzymic hydrolysis of the

hexosephosphates therefore appear tobe the same as, or similar to, thoseformed by the action of acids [Young,1909].

A further consequence of these factsis that a hexosephosphate will yieldcarbon dioxide and alcohol when it isadded to yeast-juice or zymin, and thishas also been found to be the case[Harden and Young, 1910, 2; Iwanoff,1909, 1]. [p057]

Mechanism of theFormation of

Hexosediphosphoric Acid.

On this subject little is yet known,but a number of extremely interestingresults, the interpretation of which isstill doubtful, have been obtained byEuler and his colleagues. Euler hasobtained a yeast [Yeast H of the St.Erik's brewery in Stockholm] whichdiffers from Munich yeast in severalrespects. A maceration extract preparedfrom the yeast dried at 40° in a vacuumproduces no effect on a glucosesolution containing phosphate. If,however, the glucose solution bepreviously partially fermented withliving yeast and then boiled and

filtered, the addition of the extractprepared from Yeast H brings about theesterification of phosphoric acidwithout any accompanying evolution ofcarbon dioxide [Euler and Ohlsén,1911, 1912].

Euler interprets this as follows: (a)Glucose itself is not directly esterified,but must first undergo somepreliminary change, which is broughtabout by the action of living yeast. Noproof of the existence of a newmodification of glucose in this solutionhas however been advanced, other thanits behaviour to extract of Yeast H, so

that Euler's conclusion cannot beunreservedly accepted. It is moreoverpossible and even more probable thatsome thermostable catalytic substance(perhaps a co-enzyme) passes from theyeast into the glucose solution andenables the yeast extract to attack theglucose and phosphoric acid. A verysmall degree of esterification was alsoproduced when an extract having noaction on glucose and phosphate wasadded to glucose which had beentreated with 2 per cent. caustic soda forforty hours, but the nature of thecompound formed was not ascertained

[Euler and Johansson, 1912, 4]. (b) Theesterification of phosphoric acidwithout the evolution of carbon dioxideimplies that the enzyme by which thisprocess is effected is distinct from thatwhich causes the actual decompositionof the sugar. Euler goes further thanthis and regards the enzyme as a purelysynthetic one, giving it the name ofhexosephosphatese to distinguish itfrom the hexosephosphatase whichhydrolyses the hexosephosphate.

The evidence on which thisconclusion is based cannot be regardedas satisfactory, inasmuch as it consists

in the observation that in presence ofsugar yeast extract does not hydrolysethe phosphoric ester. This, however,could not be expected since hydrolysisand synthesis under these conditionswould ultimately proceed at equalrates.

In any case the adoption of thisnomenclature is inconsistent with theconception of an enzyme as a catalystand is therefore inadvisable until thereaction has been much morethoroughly studied. [p058]

It may further be pointed out that noproof has yet been advanced that the

phosphoric ester produced withoutevolution of carbon dioxide is identicalwith hexosediphosphoric acid producedwith evolution of carbon dioxide. It isby no means improbable that itrepresents some intermediate stage inthe production of the latter (see p. 117).

Euler's other results on this subjectmay be briefly summarised as follows:—

(1) In presence of excess of sugar theesterification of the phosphoric acidproceeds by a monomolecular reactionand is most rapid in faintly alkalinereaction [Euler and Kullberg, 1911, 3].

(2) When yeast extract has beenheated for 30 minutes to 40° it effectsthe esterification of phosphoric acid ata much greater rate than the unheatedextract (2–10 times). Heating at 50° for30 minutes however completelyinactivates the extract. The cause of theactivation is as yet unknown. Thetemperature coefficient for theunheated extract (17·5°–30°) is 1·4–1·5for 10° rise of temperature [Euler andOhlsén, 1911].

(3) Yeasts which in the dried stateall produce rapid esterification ofphosphoric acid, yield extracts of very

unequal powers in this respect [Euler,1912, 1].

CHAPTER IV.THE CO-ENZYME OF YEAST-

JUICE.

[p059]

In the previous chapter reference wasmade to the fact that the addition ofboiled yeast-juice greatly increases theamounts of carbon dioxide and alcoholformed from sugar by the action of agiven volume of yeast-juice.

FIG. 5.

When the boiled juice is dialysed thesubstance or substances to which thiseffect is due pass into the dialysate, theresidue being quite inactive. In order toascertain the effect on the process ofalcoholic fermentation of the complete

removal of these unknown substancesfrom yeast-juice itself, dialysisexperiments were instituted with freshyeast-juice, capable of bringing aboutan active production of carbon dioxideand alcohol from sugar. It was alreadyknown from the experiments ofBuchner and Rapp [1898, 1] thatdialysis in parchment paper forseventeen hours at 0° against water orphysiological salt solution onlyproduced a diminution of about 20 percent. in the total amount offermentation obtainable, and in view ofthe less permanent character of the

juice from top yeasts a more rapidmethod of dialysis was sought. Thiswas found in the process of filtrationunder pressure through a film ofgelatin, supported in the pores of aChamberland filter candle, which hadbeen introduced by Martin [1896].

In this way it was found possible todivide the juice into a residue and afiltrate, each of which was itselfincapable of setting up the alcoholicfermentation of glucose, whereas, whenthey were reunited, the mixtureproduced almost as active afermentation as the original juice

[Harden and Young, 1905, 1; 1906, 2].The apparatus employed for this

purpose consists of a brass tubeprovided with a flange in which thegelatinised candle is held by acompressed india-rubber ring, and isshown in section in Fig. 5. Two suchapparatus are used, each capable ofholding about 70 c.c. of the liquid to befiltered. The tubes, after being filledwith the yeast-juice, are connected bymeans of a screw joint with a cylinderof compressed air and the filtrationcarried out under a pressure of 50atmospheres, [p060] the arrangement

employed being shown in Fig. 6. In theearlier experiments 25 to 50 c.c. ofyeast-juice were placed in each tubeand the filtration continued until nomore liquid passed through. Theresidue was then washed several timesin situ by adding water and forcing itthrough the candle. The time occupiedin this process varied from six totwelve hours with differentpreparations of yeast-juice. The candlewas then removed from the brasscasing and the thick, brown-colouredresidue scraped off, dissolved in water,and at once examined. It was

subsequently found to be possible todry this residue in vacuo over sulphuricacid without seriously altering thefermenting power, and this led to aslight modification of the method,which is now conducted as follows.Two quantities of 50 c.c. each of yeast-juice are filtered, without washing, andthe residues spread on watch-glassesand dried in vacuo. Two freshquantities of 50 c.c. are then filteredthrough the same candles and theresidues also dried. The 200 c.c. ofjuice treated in this way give a dryresidue of 17 to 24 grams. The residue

is then dissolved in 100 c.c. of waterand filtered in quantities of 50 c.c.through two fresh gelatinised candlesand the residue again dried. Aconsiderable diminution in weightoccurs, partly owing to incompleteremoval from the candle and brasscasing, and the final residue onlyamounts to about 9 to 12 grams.Occasionally it is necessary to repeatthe processes of dissolving in water,filtering, and drying, but a considerableloss both of material and fermentingpower attends each such operation.

The sticky residue dries up very

rapidly in vacuo to a brittle, scalymass, which is converted by grindinginto a light yellow powder.

The filtrate was invariably found tobe quite devoid of fermenting power,none of the enzyme passing through thegelatin.

FIG. 6.

Properties of the Filtered andWashed Residue.—The residueprepared as described above consists

mainly of the protein, glycogen, anddextrins of the yeast-juice, and isalmost free from mineral phosphates,but contains a certain amount ofcombined phosphorus. It also containsthe enzymes of the juice, including theproteoclastic enzyme, and thehexosephosphatase (p. 54). Its solutionin water is usually quite inactive toglucose or fructose, but in some casesproduces a small and evanescentfermentation. When the original filtrateor a corresponding [p061] quantity of thefiltrate from boiled fresh yeast-juice isadded, the mixture ferments glucose or

fructose quite readily. The followingtable shows the quantitative relationsobserved, the sugar being in all casespresent in excess:—

No. Material. Volume.Fil-trateadded.c.c.

1 Undriedandunwashedresidue

15 c.c. 015 " 15

2 15 " 015 " 0

3 Undriedandwashedresidue

25 " 025 " 0

4 20 " 020 " 20

5 Washedanddriedresidue

1 gramin 15c.c.

0

0

61 gramin 25c.c.

0

0

[p062]

These experiments lead to theconclusion that the fermentation of

glucose and fructose by yeast-juice isdependent upon the presence, not onlyof the enzyme, but also of anothersubstance which is dialysable andthermostable.

Precisely similar results weresubsequently obtained by Buchner andAntoni [1905, 2] by the dialysis ofyeast-juice. One hundred c.c. of juicewere dialysed for twenty-four hours at0° against 1300 c.c. of distilled water,and the dialysate was then evaporatedat 40° to 50° to 20 c.c. The fermentingpower of 20 c.c. of the dialysed juicewas then determined with the following

additions:—

(1) 20 c.c. of dialysed juice + 10 c.c.of water gave 0·02 gram CO2.

(2) 20 c.c. of dialysed juice + 10 c.c.of evaporated dialysate gave 0·52gram CO2.

(3) 20 c.c. of dialysed juice + 10 c.c.of boiled juice gave 0·89 gramCO2.

It was shown in the previous chapterthat phosphates are essential tofermentation, and hence it becomesnecessary to inquire whether the effectof dialysis is simply to remove these.Experiment shows that this is not the

case. Soluble phosphates do not conferthe power of producing fermentationon the inactive residue obtained byfiltration. Moreover, when yeast-juiceis digested for some time before beingboiled, it is found, as will besubsequently described, that the boiledautolysed juice is quite incapable ofsetting up fermentation in the inactiveresidue, although free phosphates areabundantly present [Harden and Young,1906, 2].

The filtration residue is neverobtained quite free from combinedphosphorus, but the production from

this of the phosphate necessary forfermentation to proceed, may be soslow as to render the test for co-enzyme uncertain, owing to theabsence of sufficient phosphate. Whena filtration residue is being tested it istherefore necessary to secure thepresence of sufficient phosphate toenable the characteristic reaction toproceed, and at the same time to avoidadding phosphate in too greatconcentration, as this may, in thepresence of only small amounts ofenzyme or co-enzyme, inhibit thefermentation (p. 71). The proof that a

filtration residue or dialysed juice isquite free from co-enzyme is thereforea somewhat complicated matter, andnot only involves the experimentaldemonstration that the material willnot ferment sugar, but also that thispower is not imparted to it by theaddition of a small concentration ofphosphate. As it has been found (p. 73)that the fermentation of fructose is lessaffected than that of glucose by thepresence of excess of phosphate, thepractical method of examining afiltration residue for co-enzyme is totest its action on a solution of fructose

(1) alone and (2) in presence of a smallconcentration of phosphate. If theresidue produces no action [p063] ineither case, but produces fermentationwhen a solution of co-enzyme is addedin the presence of the sameconcentration of phosphate as waspreviously employed, it may beconcluded that this sample was freefrom co-enzyme but contained enzyme;such an experiment also affords adefinite proof that the co-enzyme doesnot consist of phosphate.

This dialysable, thermostablesubstance, without which alcoholic

fermentation cannot proceed, has beenprovisionally termed the co-ferment orco-enzyme of alcoholic fermentation.This expression was first introduced byBertrand [1897], to denote substancesof this kind, and he applied it in twoinstances—to the calcium salt which heconsidered was necessary for the actionof pectase on pecten substances, and tothe manganese which he supposed to beessential for the activity of laccase.Without inquiring whether thesesubstances are precisely comparable infunction with that contained in yeast-juice, the term may be very well

applied to signify the substance ofunknown constitution without the co-operation of which the thermolabileenzyme of yeast-juice is unable to setup the process of alcoholicfermentation. The active agent ofyeast-juice consisting of both enzymeand co-enzyme may be convenientlyspoken of as the fermenting complex,and this term will occasionally beemployed in the sequel.

The co-enzyme is present alike inthe filtrates from fresh yeast-juice andfrom boiled yeast-juice, and is alsocontained in the liquids obtained by

boiling yeast with water and bywashing zymin or dried yeast withwater.

Practically the only chemicalproperty of the co-enzyme, other thanthat of rendering possible the processof alcoholic fermentation, which has sofar been observed, is that it is capableof being decomposed, probably byhydrolysis, by a variety of reagents,prominent among which is yeast-juice.This was observed by Harden andYoung in the course of their attemptsto prepare a completely inactiveresidue by filtration. In many cases a

residue was obtained which stillpossessed a very limited power offermentation, only a small amount ofcarbon dioxide being formed and theaction ceasing entirely after theexpiration of a short period; on thesubsequent addition of boiled juice,however, a very considerable evolutionof carbon dioxide was produced. Thiswas interpreted to mean that theresidue in question contained an amplesupply of enzyme but only a smallproportion of co-enzyme, and that thelatter was rapidly destroyed, so that thefermentation soon ceased. The boiled

juice then added provided a furtherproportion of co-enzyme by the aid ofwhich the surplus enzyme was [p064]

enabled to carry on the fermentation.This view was confirmed by adding toa solution of a completely inactivefiltration residue and glucosesuccessive small quantities of boiledjuice and observing the volumes ofcarbon dioxide evolved after each suchaddition. Thus in one case successiveadditions of volumes of 3 c.c. of boiledjuice produced evolutions of 8·2, 6, and6 c.c. of carbon dioxide. In anothercase two successive additions of 15 c.c.

of boiled juice produced evolutions of54 and 41·2 c.c. On the other hand, theenzyme itself also gradually disappearsfrom yeast-juice when the latter isincubated either alone or with sugar (p.20).

The cessation of fermentation in anyparticular mixture of enzyme and co-enzyme may, therefore, be due to thedisappearance of either of these factorsfrom the liquid. If the amount of co-enzyme present be relatively small it isthe first to disappear, and fermentationcan then only be renewed by theaddition of a further quantity, whilst

the addition of more enzyme producesno effect. If, on the other hand, theamount of co-enzyme be relativelylarge, the inverse is true; the enzyme isthe first to disappear, and fermentationcan only be renewed by the addition ofmore enzyme, a further quantity of co-enzyme producing no effect. It has,moreover, been found that the co-enzyme, like the enzyme, disappearsmore rapidly in the absence of glucosethan in its presence, incubation at 25°for two days being as a rule sufficientto remove all the co-enzyme fromyeast-juice from top yeasts in the

absence of sugar, whilst in the presenceof fermentable sugar co-enzyme maystill be detected at the end of four days.

In all the experiments carried out byHarden and Young with juice fromEnglish top yeast it was found thatwhen a mixture of the juice withglucose was incubated untilfermentation had ceased, the furtheraddition of co-enzyme in the form ofboiled juice did not cause any renewalof the action; in other words, the wholeof the enzyme had disappeared.

On the other hand, Buchner andKlatte [1908], working with juice and

zymin prepared from bottom yeast,observed the extremely interesting factthat after the cessation of fermentationthe addition of an equal volume ofboiled juice caused a reneweddecomposition of sugar, and that theprocesses of incubation until no furtherevolution of gas occurred and re-excitation of fermentation by theboiled juice could be repeated as manyas six times. Thus in one experimentthe duration of the fermentation wasextended from three to a total oftwenty-four days, and the total gasevolved from 0·73 gram to 2·19 grams.

The phenomenon has been found to becommon to yeast from Munich and[p065] from Berlin as well as to zyminand maceration extract, and it wasfurther observed that the boiled juicefrom one yeast could regenerate thejuice from another, although thequantitative relations were different.

In these samples of yeast-juice,therefore, there is present a naturalcondition of affairs precisely similar tothat obtaining in the artificial mixturesof inactive filtration residue and co-enzyme solution made by Harden andYoung. The balance of quantities is

such that the co-enzyme disappearsbefore the enzyme, leaving a certainamount of enzyme capable ofexercising its usual function as soon assufficient co-enzyme is added. Thisestablishes an interesting point ofcontrast with the juice prepared fromtop yeast in England, in which theenzyme does not outlast the co-enzyme[Harden and Young, 1907]. Thedifference may be due to somevariation in the relative proportions ofenzyme and co-enzyme or of theenzymes to which the disappearance ofeach of these is presumptively due, or

to a combination of these two causes. Itwas, however, found, even in the juicefrom bottom yeast, that incubation forthree days at 22° without the additionof sugar caused the disappearance ofthe enzyme as well as of the co-enzyme, and left a residue alikeincapable of being regenerated by theaddition of co-enzyme or of restoringthe power of producing fermentation toan inactive mixture containing enzymeand sugar.

If the fermenting power of the juiceis to be preserved by repeatedregeneration for a long period, it is

absolutely necessary to add the co-enzyme solution each time as soon asfermentation has ceased, since theenzyme in the absence of this additionrapidly disappears, even in thepresence of sugar.

This result is probably to beexplained, at all events in the main, bythe presence in the co-enzyme solutionof the antiprotease to which referencehas already been made [Buchner andHaehn, 1910, 2]. This agent, theconstitution of which is still unknown,protects proteins in general from theaction of digestive enzymes, and on the

assumption that the alcoholic enzymeof yeast-juice belongs to the class ofproteins, may be supposed to lessen therate at which this enzyme is destroyedby the endotryptase of the juice. Thisantiprotease is, like the co-enzyme (p.68), destroyed by lipase but is morestable than the co-enzyme towardshydrolytic agents, and can be obtainedfree from co-enzyme by boiling thesolution for some hours alone or byheating with dilute sulphuric acid. Sucha solution possesses no regenerativepower, but still retains its power ofprotecting proteins against digestion

and of preserving the fermenting powerof yeast-juice. [p066]

It must, however, be rememberedthat the addition of a phosphate alonemay greatly prolong the period offermentation of yeast-juice (p. 55), andsugar is well known to exert a similaraction. It appears, therefore, that theexistence of the enzyme is prolongednot only by the presence of theantiprotease but also by that of sugarand hexosephosphate, into whichphosphate passes in presence of sugar.Similar effects are exerted on the co-enzyme by sugar and probably also by

hexosephosphate.The fermenting complex, therefore,

in the presence of these substances,either separately or together, falls offmore slowly in activity and is presentfor a longer time, and for both of thesereasons produces an increased amountof fermentation. It seems probable alsothat the hexosephosphatase is similarlyaffected, so that the supply of freephosphate is at the same time bettermaintained, and the rate offermentation for this reason decreasesmore slowly than would otherwise bethe case.

It is in this way that an explanationmay be found of the remarkableincrease in total fermentation, which isproduced by the addition to yeast-juiceand sugar of boiled yeast-juice,containing free phosphate (whichpasses into hexosephosphate) as well asco-enzyme, of boiled autolysed yeast-juice, containing free phosphate but noco-enzyme, or of phosphate solutionalone.

In no case is the original rate offermentation greatly increased after theinitial acceleration has disappeared, butin every case the total fermentation is

considerably augmented, and this is nodoubt mainly to be attributed, as justexplained, to the diminished rate ofdecomposition of the fermentingcomplex and probably of thehexosephosphatase.

Although both enzyme and co-enzyme are completely precipitatedfrom yeast-juice, as already described(p. 38), by 10 volumes of acetone, theco-enzyme is less easily precipitatedthan the enzyme, and a certain degreeof separation can therefore be attainedby fractional precipitation [Buchnerand Duchaček, 1909]. The enzyme

cannot, however, be completely freedfrom co-enzyme in this manner, andthe process is attended by a veryconsiderable loss of enzyme. This isprobably due to the fact that only smallquantities of acetone can be added (1·5to 3 volumes), in order to avoidprecipitation of co-enzyme, and thatthe precipitates thus formed contain alarge proportion of water, a conditionwhich appears to be fatal to thepreservation of the enzyme.

It is, however, not quite certainwhether it is the zymase or thehexosephosphatase which is destroyed

in these cases, as no attempt [p067] wasmade to distinguish between them. Inany case the precipitates obtained byfractional treatment with acetone, evenwhen reunited, produce a much smallerfermentation than the original juice orthe powder prepared by bringing it into10 volumes of acetone.

Attempts to isolate the co-enzymefrom boiled yeast-juice have also beenhitherto unsuccessful. It has, however,been found possible to remove aconsiderable amount of material fromthe solution without affecting the co-enzyme. When 1 volume of alcohol is

added to boiled yeast-juice, a bulkyprecipitate, consisting largely ofcarbohydrates, is produced, and thefiltrate from this is found to contain theco-enzyme and can be freed fromalcohol by evaporation. Furtherprecipitation with alcohol has not ledto useful results.

When a solution which has beentreated in this way is precipitated withlead acetate and kept neutral to litmus,the free phosphate andhexosephosphate are thrown down andthe co-enzyme remains in solution. Thefiltrate can be freed from lead by

means of sulphuretted hydrogen andneutralised, and then forms a solutionof co-enzyme free from phosphate andhexosephosphate but still containingcombined phosphorus. More completepurification than this has not yet beenaccomplished. Occasionally theprecipitate of lead salts retains some ofthe co-enzyme, apparently byadsorption, but usually the greater partremains in the solution (Harden andYoung).

The co-enzyme is partially removedfrom yeast-juice by means of acolloidal solution of ferric hydroxide

(Resenscheck). A precipitate is thusobtained which contains phosphorusand resembles boiled yeast-juice in itsregenerative action on yeast-juicerendered inactive by fermentation. Ithas not, however, so far been foundpossible to isolate any definitecompound from this precipitate. Thereare also indications that when yeast-juice, either fresh or boiled, iselectrolysed, the co-enzyme tends toaccumulate at the cathode[Resenscheck, 1908, 1, 2].

Buchner and Klatte [1908] made useof yeast-juice rendered free from co-

enzyme by incubation with sugarsolution to examine the nature of theagent by which the co-enzyme isdestroyed. This agent is certainly anenzyme, since boiled yeast-juice can bepreserved with unimpaired powers fora considerable length of time, andsuspicion fell naturally, in the firstinstance, on the endotryptase of theyeast cell. Direct experiment showed,however, that yeast-juice, which, whenfresh, rapidly destroyed the co-enzymeof boiled juice, lost this power onpreservation, but retained itsproteoclastic properties without

diminution, so that the tryptic enzymecould not be the one concerned. Thedirect action of commercial trypsin onboiled yeast-juice also yielded [p068] anegative result, although this cannotstrictly be regarded as an indication ofthe effect of the specific proteoclasticenzymes of yeast-juice. On the otherhand, it was found that when boiledjuice was treated for some time with anemulsion containing the lipase ofcastor oil seeds, the co-enzyme wascompletely destroyed. This is a resultof great importance, inasmuch as itprobably indicates that the co-enzyme

is chemically allied to the class ofsubstances hydrolysable by lipase, i.e.to the fats and other esters.

Further, observations by Buchnerand Haehn [1909] have shown thatdigestion with potassium carbonatesolution containing 2·5 grams per 100c.c. also brings about the destruction ofthe co-enzyme, and that this is alsoslowly accomplished by the repeatedboiling of the juice. The co-enzyme isalso destroyed both by acid andalkaline hydrolysis, and when thesolution is evaporated to dryness andthe residue ignited.

Beyond this general indicationnothing is known of the chemicalnature of the co-enzyme. The intimaterelation of phosphoric acid to theprocess of fermentation renders it notimpossible that the co-enzyme maycontain this group, but there is nodefinite evidence for such a belief.Purely negative results have beenobtained with all the substances ofknown composition which have yetbeen tested, among these being solublephosphates, hexosephosphates and anumber of oxidisable and reduciblesubstances, such as quinol, p-

phenylenediamine, methylene blue,peptone beef broth, etc. (Harden andYoung; Harden and Norris [1914]; seealso Euler and Bäckström [1912]),glycero-phosphates (Buchner andKlatte).

The precise function of the co-enzyme is even more obscure than itschemical nature. The system ofreacting substances consisting offermentable material, enzyme and co-enzyme, bears, however, an obvioussuperficial resemblance to many of thesystems required for theaccomplishment of chemical changes

in the animal or vegetable organism.Such a triad of substances is, forexample, requisite for the process bywhich the red blood corpuscles of ananimal are broken up by the serum of adifferent animal into the blood ofwhich the red corpuscles of the firstanimal have been injected. This effectis only produced when two substancesare present, the amboceptor or immunebody and the complement. The analogymay be carried to a further stage sincethe amboceptor is, like the co-enzyme,more thermostable than thecomplement, which therefore

corresponds with the enzyme. Immuneserum can, in fact, be freed fromcomplement by being heated at 57–60°for half an hour, whereas theamboceptor is unaffected by thistreatment. On the other hand, thecomplement and amboceptor do not[p069] appear to act like enzymes butrather like ordinary chemical reagents,remaining in combination even afterthe blood corpuscle has been brokenup, whereas the enzyme and co-enzymeof yeast-juice are again liberated whenthe reaction between sugar andphosphate has been completed.

CHAPTER V.ACTION OF SOME INHIBITING

AND ACCELERATING AGENTS

ON THE ENZYMES OF YEAST-

JUICE.

[p070]

One of the most interesting and at thesame time most difficult problemsconcerning enzyme action in general isthe nature of the inhibiting oraccelerating effect produced by manysubstances upon the rate or total result

of the chemical process set up inpresence of the enzyme. Inhibition, it isusually supposed, involves either thedecomposition of the enzyme, in whichcase it is irreversible, its removal fromthe sphere of action by some change inits mode of solution, or the formationof an inactive or less active compoundbetween the enzyme and the inhibitingagent. This compound it maysometimes be possible to decompose,with the result that the activity of theenzyme is restored. A striking exampleof this, to which allusion has alreadybeen made, is the effect of hydrocyanic

acid on alcoholic fermentation (p. 37).Acceleration of enzyme action can in

some cases be ascribed to the fact thatthe accelerating substance possesses anassignable chemical function in thereaction, so that an increase in theconcentration of this substance causesan increase in the rate of the reaction.As we have seen in Chapter III, this isthe explanation of the acceleratingeffect of phosphates on fermentationby yeast-juice. In many other cases,however, no such chemical functioncan be traced, as, for example, in theeffect of neutral salts on the hydrolytic

action of invertase, or the effect of theaddition of the co-enzyme to zymase,and it is necessary to fall back on someassumption, such as that theaccelerating agent acts by increasingthe effective concentration of theenzyme or by combining either withthe enzyme or the substrate, forming acompound which undergoes thereaction more readily.

The interest in the followingexamples of inhibition and accelerationof fermentation by yeast-juice lies notonly in their relation to these generalproblems but also, and perhaps chiefly,

in their bearing on the specific problemof the nature and mode of action of thevarious agents concerned in theproduction of alcohol and carbondioxide from sugar in the yeast-cell.[p071]

I. Influence ofConcentration of Phosphate

on the Course ofFermentation.

Prominent among these instances ofinhibition and acceleration are thephenomena attendant on the addition of

excess of phosphate to yeast-juice.When a phosphate is added to a

fermenting mixture of a sugar andyeast-juice, the effect varies with theconcentration of the phosphate and thesugar and with the particular specimenof yeast-juice employed. With lowconcentrations of phosphate inpresence of excess of glucose theacceleration produced is so transientthat no accurate measurements of ratecan be made. As soon as the amount ofphosphate added is sufficiently large, itis found that the rate of evolution ofcarbon dioxide very rapidly increases

from five to ten times, and then quicklyfalls approximately to its originalvalue.

As the concentration of phosphate isstill further increased, it is firstobserved that the maximum velocity,which is still attained almostimmediately after the addition of thephosphate, is maintained for a certainperiod before the fall commences, andthen, as the increase in concentration ofphosphate proceeds, that the maximumis only gradually attained after theaddition, the period required for thisincreasing with the concentration of the

phosphate. Moreover, with still higherconcentrations, the maximum rateattained is less than that reached withlower concentrations, and further, therate falls off more slowly. Theconcentration of phosphate whichproduces the highest rate, which maybe termed the optimum concentration,varies very considerably with differentspecimens of yeast-juice [Harden andYoung, 1908, 1].

All these points are illustrated by theaccompanying curves (Fig. 7) whichshow the rate of evolution per fiveminutes plotted against the time for

four solutions in which the initialconcentrations of phosphate were (A)0·033, (B) 0·067, (C) 0·1, and (D) 0·133molar, the volumes of 0·3 molarphosphate being 5, 10, 15, and 20 c.c.in each case added to 25 c.c. of yeast-juice, and made up to 45 c.c, eachsolution containing 4·5 grams ofglucose. The time of addition is takenas zero, the rate before addition beingconstant, as shown in the curves.

FIG. 7.

It will be observed that 5 and 10 c.c.(A and B) give the same maximum,whilst 15 c.c. (C) produce a muchlower maximum, and 20 c.c. (D) a stilllower one, the rate at which the

velocity diminishes after theattainment of the maximum beingcorrespondingly slow in these last twocases. By calculating the amount ofphosphate which has disappeared assuch from the amount of carbondioxide evolved, [p072] it is found thatthe maximum does not occur at thesame concentration of free phosphatein each case.

These results suggest that thephosphate is capable of forming two ormore different unstable associationswith the fermenting complex. One ofthese, formed with low concentrations

of the phosphate, has the compositionmost favourable for the decompositionof sugar, whilst the others, formed withhigher concentrations of phosphate,contain more of the latter, probablyassociated in such a way with thefermenting complex as to render thelatter partially or wholly incapable ofeffecting the decomposition of thesugar molecule. As the fermentationproceeds slowly in the presence ofexcess of phosphate, the concentrationof the latter is reduced by conversioninto hexosephosphate, and a re-distribution of phosphate occurs,

resulting in the gradual change of theless active into the more activeassociation of phosphate withfermenting complex, and a consequentrise in the rate of fermentation.

In those cases in which themaximum rate corresponding to theoptimum concentration of phosphate isnever attained, some secondary causemay be supposed to intervene, such asa permanent change in a portion of thefermenting complex, accumulation ofthe products of the reaction, etc.

It is also possible as suggested byBuchner for the analogous case of

arsenite (p. 78) that the addition ofincreasing amounts of phosphatecauses a progressive but reversiblechange in the mode of dispersion [p073]

of the colloidal enzyme, and that thishas the secondary effect of altering therate of fermentation. No decisiveevidence is as yet available upon thesubject.

The results obtained by Euler andJohansson [1913] to which referencehas already been made indicate that inpresence of a moderate excess ofphosphate esterification is more rapidthan production of carbon dioxide. No

explanation of this phenomenon has yetbeen given, but it might obviously bedue either to the production of somephosphorus compound whichsubsequently takes part in theproduction both of hexosediphosphateand of carbon dioxide, or, lessprobably, to the entire independence ofthe two changes—esterification ofphosphate and production of carbondioxide—which might then bedifferently affected by the presence ofexcess of phosphate and therefore takeplace at different rates.

II. Reaction of Fructose withPhosphates in Presence of

Yeast-Juice.

Although, as has been pointed out (p.42), glucose, mannose, and fructose allreact with phosphate in a similarmanner in presence of yeast-juice,there are nevertheless certainquantitative differences between thebehaviour of glucose and mannose onthe one hand, and fructose on the other,which appear to be of considerableimportance. Fructose differs from theother two fermentable hexoses in two

particulars: (1) the optimumconcentration of phosphate is muchgreater; (2) the maximum rate offermentation attainable is much higher[Harden and Young, 1908, 2; 1909].

These points are clearly illustratedby the following results, which all referto 10 c.c. of yeast-juice, and show thatthe optimum concentration ofphosphate for the fermentation offructose is from 1·5 to 10 times that ofglucose, and that the maximum rate offermentation for fructose in presenceof phosphate is 2 to 6 times that ofglucose.

Sugarin

Grams.TotalVolume.

Optimum Volume of0·6 Molar

Phosphate in c.c.

Glucose. Fructose.2 35 2 4 50 1 10 1·6 23 2 1 25 1·752 25 5 2 20 2 2 22·5 0·75

[p074]

It is interesting to note that the twohigh rates, 32·2 and 31·2 c.c. per fiveminutes, are equal to about half the rateobtainable with an amount of livingyeast corresponding to 10 c.c. of yeast-

juice, assuming that about 16 to 20grams of yeast are required to yieldthis volume of juice, and that thisamount of yeast would give about 56 to70 c.c. of carbon dioxide per fiveminutes at 25°, which has been foundexperimentally to be about the rateobtainable with the top yeast employedfor these experiments.

III. Effect of the Addition ofFructose on the

Fermentation of Glucose orMannose in Presence of a

Large Excess of Phosphate.

When the maximum rate offermentation of glucose or mannose byyeast-juice in presence of phosphate isgreatly lowered by the addition of alarge excess of phosphate, the additionof a relatively small amount of fructose(as little as 2·5 per cent. of the weightof the glucose) causes rapidfermentation to occur. This inducedactivity is not due solely to theselective fermentation of the addedfructose, since the amount of gasevolved may be greatly in excess of

that obtainable from the quantityadded.

Another way of expressing the samething is to say that the optimumconcentration of phosphate (p. 71) isgreatly raised when 2·5 per cent. offructose is added to glucose, and thatconsequently the rate of fermentationrises. The effect is extremely striking,since a mixture of glucose and yeast-juice fermenting in the presence of alarge excess of phosphate at the rate ofless than 1 c.c. of carbon dioxide infive minutes may be made to fermentat six to eight times this rate by the

addition of only 0·05 gram of fructose(2·5 per cent. of the glucose present),and to continue until the total gasevolved is at least five to six times asgreat as that obtainable from the addedfructose, the concentration of thephosphate being the whole time at sucha height as would limit thefermentation of glucose alone to itsoriginal value.

The effect is not produced when theconcentration of the phosphate is sohigh that the rate of fermentation offructose is itself greatly lowered.

This remarkable inductive effect is

specific to fructose and is not producedwhen glucose is added to mannose orfructose, or by mannose when added toglucose or fructose, under the properconditions of concentration ofphosphate in each case.

This interesting property of fructose,taken in connection with the [p075] factsthat this sugar in presence of phosphateis much more rapidly fermented thanglucose or mannose, and that theoptimum concentration of phosphatefor fructose is much higher than forglucose or mannose, appears toindicate that fructose when added to

yeast-juice does not merely act as asubstance to be fermented, but inaddition, bears some specific relationto the fermenting complex.

All the phenomena observed are,indeed, consistent with the suppositionthat fructose actually forms apermanent part of the fermentingcomplex, and that, when theconcentration of this sugar in the yeast-juice is increased, a greater quantity ofthe complex is formed. As the result ofthis increase in the concentration of theactive catalytic agent, the yeast-juicewould be capable of bringing about the

reaction with sugar in presence ofphosphate at a higher rate, and at thesame time the optimum concentrationof phosphate would become greater,exactly as is observed. The questionwhether, as suggested above, fructoseactually forms part of the fermentingcomplex, and the further questions,whether, if so, it is an essentialconstituent, or whether it can bereplaced by glucose or mannose withformation of a less active complex,remain at present undecided, andcannot profitably be more fullydiscussed until further information is

available.It must, moreover, be remembered

that different samples of yeast-juicevary to a considerable extent in theirrelative behaviour to glucose andfructose, so that the phenomena underdiscussion may be expected to varywith the nature and past history of theyeast employed.

IV. Effect of Arsenates onthe Fermentation of Sugarsby Yeast-Juice and Zymin.

The close analogy which exists

between the chemical functions ofphosphorus and arsenic lends someinterest to the examination of theaction of sodium arsenate upon amixture of yeast-juice and sugar, andexperiments reveal the fact thatarsenates produce a very considerableacceleration in the rate of fermentationof such a mixture [Harden and Young,1906, 3; 1911, 1]. The phenomenaobserved, however, differ markedlyfrom those which accompany theaction of phosphate.

The acceleration produced is of thesame order of magnitude as that

obtained with phosphate, but it ismaintained without alteration for aconsiderable period, so that there is noequivalence between the amount ofarsenate added and the extra amount offermentation effected. Further, noorganic arsenic compoundcorresponding in composition with thehexosephosphates appears to beformed.

Increase of concentration of arsenateproduces a rapid inhibition of [p076]

fermentation, probably due to somesecondary effect on the fermentingcomplex, possibly to be interpreted as

the formation of compounds incapableof combining with sugar and henceunable to carry on the process offermentation. An optimumconcentration of arsenate thereforeexists just as of phosphate, at which themaximum rate is observed, and thisoptimum concentration and thecorresponding rate vary with differentsamples of juice and are less forglucose than for fructose. The rate offermentation by zymin is relativelyless increased than that by yeast-juice.

Owing to the fact that the rate ispermanently maintained the addition of

a suitable amount of arsenate increasesthe total fermentation produced to amuch greater extent than phosphate.

The nature of these effects may begathered from the result of a fewtypical experiments. In one case therate of fermentation of glucose byyeast-juice was raised by the presenceof 0·03 molar arsenate from 2 to 23 c.c.per five minutes, and the total evolvedin ninety-five minutes from 51 to 459c.c. The accelerating effect on 20 c.c.of juice, of as little as 0·005 c.c. of 0·3molar arsenate, containing 0·11 mgrm.of arsenic, can be distinctly observed,

but the maximum effect is usuallyproduced by about 1 to 3 c.c., theconcentration being therefore 0·015 to0·045 molar. Greater concentrationsthan this produce a less degree ofacceleration accompanied by a shorterduration of fermentation, as shown bythe following numbers which refer to20 c.c. of yeast-juice in a total volumeof 40 c.c. containing 10 per cent. ofglucose:—

C.cs. of0·3 MolarArsenate in40 c.c.

MolarConcentrationof Arsenate.

Maximum Rate

Fermentation.

0 0 0·005 0·0000375 0·01 0·000075 0·02 0·00015 0·04 0·0003 0·1 0·00075 0·2 0·0015 0·5 0·00375 1·0 0·0075 2·0 0·015 5·0 0·037510·0 0·07515·0 0·112520 0·15

The contrast between glucose andfructose in their relations to [p077]

arsenate are well exhibited in thefollowing table, in which the rates offermentation produced by arsenate inpresence of excess of glucose andfructose respectively are given:—

Concentrationof Arsenate.

Rate.Glucose. Fructose.

0·0075molar 12·1 26·6

0·0225(opt. forglucose) 13·4 —

0·0525(opt. forfructose) — 45·8

0·1125 5·1 39

Here the optimum concentration forfructose is more than twice that for

glucose, whilst the maximum rate offermentation obtainable with fructoseis between three and four times themaximum given by glucose.

V. Effect of Arsenites on theFermentation Produced by

Yeast-Juice.

Effects somewhat similar to thoseproduced by arsenates were observedby Buchner [Buchner and Rapp, 1897;1898, 1, 2, 3; 1899, 2; Buchner, E. andH., and Hahn, 1903, pp. 184–205] whenpotassium arsenite was added to yeast-

juice. This substance, the action ofwhich on yeast had been adduced bySchwann as a proof of the vegetablenature of this organism, was employedby Buchner on account of its poisonouseffect on vegetable cells as anantiseptic and as a means of testing forthe protoplasmic nature of the agentpresent in yeast-juice. Its effect on thefermentation was, however, found to beirregular, and at the same time it didnot act as an efficient antiseptic in theconcentrations which could beemployed. Even 2 per cent. ofarsenious oxide, added as the

potassium salt, had in many cases adecided effect in diminishing the totalfermentation obtained with cane sugar,and this effect increased with theconcentration. A number ofirregularities were also observed whichcannot here be discussed. It was furtherfound that in some cases 2 per cent. ofarsenious oxide inhibited thefermentation of glucose but not ofsaccharose, or of a mixture of glucoseand fructose, whilst its effect onfructose alone was of an intermediatecharacter.

The important observation was also

made by Buchner that the addition of asuitable quantity of arsenite as a rulecaused a greatly increased fermentationduring the first sixteen hours even inexperiments in which the totalfermentation was diminished. Byexamining the effect of arsenite onfermentation in a similar manner tothat of arsenate, Harden and Young[1911, 1] have found that a closeanalogy exists [p078] between theeffects and modes of action of thesesubstances, but that arsenite produces amuch smaller acceleration thanarsenate. An optimum concentration of

arsenite exists, just as in the case ofarsenate, which produces a maximumrate of fermentation. Further increasein concentration leads to inhibition,and in no case is there any indication ofthe production of an exactly equivalentamount of fermentation as in the caseof phosphate. In various experimentswith dialysed, evaporated, and dilutedyeast-juice in which 2 per cent. ofarsenious oxide was found by Buchnerto inhibit fermentation, it is probablethat, owing to the small amount offermenting complex left, this amountof arsenious oxide was considerably in

excess of the optimum concentration,although Buchner ascribes the effect tothe removal of some of the protectivecolloids of the juice, owing to theprolonged treatment to which it hadbeen subjected.

The extent of the action of arseniteappears from the following results. Inone case a rate of 1·7 c.c. was increasedto 7 c.c. by 0·06 molar arsenite. Inanother experiment it was found thatthe optimum concentration was 0·04molar arsenite, the addition of whichincreased the rate three-fold. As in thecase of arsenate the optimum

concentration and the correspondingmaximum rate of fermentation areconsiderably greater for fructose thanfor glucose. The relative rates producedby the addition of equivalent amountsof arsenate and arsenite (1 c.c. of 0·3molar solution in each case to 20 c.c. ofyeast-juice) were 27·5 and 3·1, theoriginal rate of the juice being 1·7. Ingeneral the optimum concentration ofarsenite is considerably greater thanthat of arsenate.

The inhibiting effects of higherconcentrations of arsenite and arsenatealso present close analogies, but this

most interesting aspect of the questionhas not yet been sufficiently examinedto repay detailed discussion. Buchner[Buchner, E. and H., and Hahn, 1903,pp. 199–205] has suggested that theinhibition is due primarily to somechange in the colloidal condition of theenzyme and has shown that certaincolloidal substances appear to protectit, as does also sugar. The possibility isalso present that inactive combinationsof some sort are formed between thefermenting complex and the inhibitingagent, in the manner suggested toaccount for the inhibiting effect of

excess of phosphate (p. 72). It seemsmost probable that the effect is acomplex one, in which many factorsparticipate.

Nature of the AccelerationProduced by Arsenate and

Arsenite.

In explanation of the remarkableaccelerating action of arsenates andarsenites two obvious possibilitiespresent themselves. In the [p079] firstplace the arsenic compound mayactually replace phosphate in the

reaction characteristic of alcoholicfermentation, the resulting arsenicanalogue of the hexosephosphate beingso unstable that it undergoesimmediate hydrolysis, and is thereforeonly present in extremely smallconcentration at any period of thefermentation and cannot be isolated. Inthe second place it is possible that thearsenic compound may accelerate theaction of the hexosephosphatase of thejuice, and thus by increasing the rate ofcirculation of the phosphate producethe permanent rise of rate. With thiseffect may possibly be associated a

direct acceleration of the action of thefermenting complex.

The experimental decision betweenthese alternative explanations isrendered possible by the use of amixture of enzyme and co-enzyme freefrom phosphate and hexosephosphate.As has already been described (p. 55) amixture of boiled yeast-juice, whichhas been treated with lead acetate,glucose or fructose, and washed zymincan be prepared which scarcelyundergoes any fermentation unlessphosphate be added. If now arsenatesor arsenites can replace phosphate, they

should be capable of setting upfermentation in such a mixture.Experiment shows that they do notpossess this power. For fermentation toproceed phosphate must be present andit cannot be replaced either by arsenateor arsenite [Harden and Young, 1911,1].

The effect of these salts on theaction of the hexosephosphatase canalso be ascertained by a modificationof the foregoing experiment. If ahexosephosphate be made the solesource of phosphate in such a mixtureas that described above, in which it

must be remembered abundance ofsugar is present, the rate at whichfermentation can proceed will becontrolled by the rate at which thehexosephosphate is decomposed withformation of phosphate. Experimentshows that in the presence of addedarsenate or arsenite the rate offermentation is largely increased, sothat the effect of these salts must be toincrease the rate of liberation ofphosphate, or in other words, toaccelerate the hydrolytic action of thehexosephosphatase.

This conclusion is even more

strikingly confirmed by a comparisonof the direct action of yeast-juice onhexosephosphate in presence and inabsence of arsenate, as measured by theactual production of free phosphate. Ina particular experiment this gave riseto 0·0707 gram of Mg2P2O7 in theabsence of arsenate and 0·6136 gram ofMg2P2O7 in the presence of arsenate.

The results obtained with arseniteare throughout very similar to thosegiven by arsenate, but are not quite sostriking. It may therefore be affirmedwith some confidence that the chiefaction of arsenates [p080] and arsenites

in accelerating the rate of fermentationof sugars by yeast-juice or zymin,consists in an acceleration of the rate atwhich phosphate is produced from thehexosephosphate by the action of thehexosephosphatase.

It has further been found thatarsenates, and to a less degreearsenites, also produce an accelerationof the rate of autofermentation ofyeast-juice and of the rate at whichglycogen is fermented. This turns outto be due in all probability to anincrease in the activity of theglycogenase by the action of which the

sugar is supplied which is the directsubject of fermentation. Thus in onecase an initial rate of fermentation ofglycogen of 1·9 c.c. per five minuteswas increased by 0·05 molar arsenateto 9·7 and the amount of carbondioxide evolved in two hours from 38to 158 c.c. Even this enhancedproduction of glucose from glycogen,however, is not nearly sufficient for thecomplete utilisation of the phosphatealso being liberated by the action onthe hexosephosphatase, for the additionof an excess of sugar produces a muchhigher rate, in this case 36 c.c. per five

minutes. The effect of arsenate on therate of action of the glycogenase seemstherefore to be much smaller than onthat of the hexosephosphatase.

No other substances have yet beenfound which share these interestingproperties with arsenates and arsenites,and no advance has been made towardsan understanding of the mechanism ofthe accelerating action of these salts onthe specific enzymes which areaffected by them.

CHAPTER VI.CARBOXYLASE.

[p081]

An observation of remarkable interest,which promises to throw light onseveral important features of thebiochemistry of yeast, was made in1911, and has since then formed thesubject of detailed investigation byNeuberg and a number of co-workers.

It was found that yeast had the powerof rapidly decomposing a large numberof hydroxy-and keto-acids [Neuberg

and Hildesheimer, 1911; Neuberg andTir, 1911; see also Karczag, 1912, 1,2]. The most important among theseare pyruvic acid, CH3·CO·COOH, and aconsiderable number of other aliphatica-keto-acids which are decomposedwith evolution of carbon dioxide andformation of the correspondingaldehyde:—

R·CO·COOH = R·CHO + CO2.

The reaction is produced by all racesof brewer's yeast which have beentried, as well as by active yeastpreparations and extracts and by wine

yeasts [Neuberg and Karczag, 1911, 4;Neuberg and Kerb, 1912, 2]. Thephenomenon can readily be exhibitedas a lecture experiment by shaking up 2g. of pressed yeast with 12 c.c. of 1 percent. pyruvic acid, placing the mixturein a Schrötter's fermentation tube,closing the open limb by means of arubber stopper carrying a long glasstube and plunging the whole in water of38–40°. Comparison tubes of yeast andwater and yeast and 1 per cent. glucosemay be started at the same time, and itis then seen that glucose and pyruvicacid are fermented at approximately

the same rate [Neuberg and Karczag,1911, 1]. If English top yeast be used itis well to take 0·5 per cent. pyruvicacid solution and to saturate the liquidswith carbon dioxide beforecommencing the experiment. Theproduction of acetaldehyde can bereadily demonstrated by distilling themixture at the close of fermentationand testing for the aldehyde either byRimini's reaction (a blue colorationwith diethylamine and sodiumnitroprusside) or by means of p-nitrophenylhydrazine whichprecipitates the hydrazone, melting at

128·5° [Neuberg and Karczag, 1911, 2,3]. [p082]

As the result of quantitativeexperiments it has been shown that 80per cent. of the theoretical amount ofacetaldehyde can be recovered. Thesalts of the acids are also attacked, thecarbonate of the metal, which may bestrongly alkaline, being formed. Thustaking the case of pyruvic acid, thesalts are decomposed according to thefollowing equation:—

2 CH3·CO·COOK + H2O = 2 CH3·CHO + K2CO3 + CO2.

Under these conditions aconsiderable portion of the aldehydeundergoes condensation to aldol[Neuberg, 1912]:—

2 CH3·CHO = CH3·CH(OH)·CH2·CHO.

This change appears to be dueentirely to the alkali and not to anenzyme since the aldol obtained yieldsinactive β-hydroxybutyric acid onoxidation [Neuberg and Karczag, 1911,3; Neuberg, 1912]. The variouspreparations derived from yeast whichare capable of producing alcoholic

fermentation also effect thedecomposition of pyruvic acid in thesame manner as living yeast. They are,however, more sensitive to the acidityof the pyruvic acid, and it is thereforeadvisable to employ a salt of the acidin presence of excess of a weak acid,such as boric or arsenious acid, whichdecomposes the carbonate formed buthas no inhibiting action on the enzyme[Harden, 1913; Neuberg and Rosenthal,1913].

As already mentioned the action isexerted on α-ketonic acids as a classand proceeds with great readiness with

oxalacetic acid, COOH·CH2·CO·COOH, all the threeforms of which are decomposed, withα-ketoglutaric acid, and with α-ketobutyric acid. Hydroxypyruvic acid CH2(OH)·CO·COOH is slowlydecomposed yielding glycolaldehyde, CH2(OH)·CHO, and this condenses to asugar [Neuberg and Kerb, 1912, 3;1913, 1]. Positive results have alsobeen obtained with diketobutyric,phenylpyruvic, p-hydroxyphenylpyruvic,phenylglyoxylic andacetonedicarboxylic acids [Neuberg

and Karczag, 1911, 5].

Relation of Carboxylase toAlcoholic Fermentation.

With regard to the relation ofcarboxylase to the process of alcoholicfermentation, nothing definite is yetknown. As Neuberg points out [seeNeuberg and Kerb, 1913, 1] theuniversal presence of the enzyme inyeasts capable of producing alcoholicfermentation, and the extremereadiness with which the fermentationof pyruvic acid takes place create a

[p083] strong presumption that thedecomposition of pyruvic acid actuallyforms a stage in the process of thealcoholic fermentation of the sugars.On the other hand Ehrlich's alcoholicfermentation of the amino-acids (p. 87)provides another function forcarboxylase—that of decomposing theα-ketonic acids produced by thedeaminisation of the amino-acids. Itmust be remembered in this connectionthat carboxylase is not specific in itsaction, but catalyses the decompositionnot only of pyruvic acid but also of alarge number of other α-ketonic acids,

including many of those whichcorrespond to the amino-acids ofproteins and are doubtless formed inthe characteristic decomposition ofthese amino-acids by yeast.Carboxylase undoubtedly effects onestage in the production of alcoholsfrom amino-acids, whether it is alsothe agent by which one stage in thealcoholic fermentation of sugar isbrought about still remains to beproved.

A comparison of the conditions ofaction of carboxylase and zymase hasrevealed several interesting points of

difference. Neuberg and Rosenthal[1913] have observed that thefermentation of pyruvic acid bymaceration extract commences muchmore rapidly than that of glucose andinterpret this to mean that in thefermentation of glucose a longpreliminary process occurs beforesufficient pyruvic acid has beenproduced to yield a perceptible amountof carbon dioxide. The long delay (3hours) which they sometimes observedin the action of maceration juice onglucose is however by no meansinvariable (see p. 46), but in any case

indicates that the sugar fermentationcan be affected by conditions which arewithout influence on the pyruvicfermentation. A similar conclusion isto be drawn from the fact that thepyruvic acid fermentation is lessaffected by antiseptics than the glucosefermentation [Neuberg and Karczag,1911, 4; Neuberg and Rosenthal, 1913],chloroform sufficient to stop theglucose fermentation brought about byyeast or dried yeast being usuallywithout effect on the fermentation ofthe pyruvates either alone or inpresence of boric or arsenious acid. A

more important difference is thatcarboxylase decomposes pyruvic acidin the absence of the co-enzyme whichis necessary for the fermentation ofglucose [Harden, 1913; Neuberg andRosenthal, 1913]. This can bedemonstrated experimentally bywashing dried yeast or zymin withwater (see p. 63) until it is no longercapable of decomposing glucose(Harden), or by allowing macerationextract to autolyse or dialyse until it isfree from co-enzyme (Neuberg andRosenthal). The zymase of macerationextract is moreover inactivated in 10

minutes at 50–51°, whereas after thistreatment the carboxylase is stillactive. [p084]

The only conclusion that can belegitimately drawn from these highlyinteresting facts is that if thedecomposition of pyruvic acid actuallybe a stage in the alcoholic fermentationof glucose the soluble co-enzyme isrequired for some change precedent tothis, so that in its absence theproduction of pyruvic acid cannot beeffected.

CHAPTER VII.THE BY-PRODUCTS OF

ALCOHOLIC FERMENTATION.

[p085]

When pure yeast is allowed to developin a solution of sugar containing asuitable nitrogenous diet and the propermineral salts, the liquid at the close ofthe fermentation contains not onlyalcohol and some carbon dioxide butalso a considerable number of othersubstances, some arising from the

carbonaceous and others from thenitrogenous metabolism of the cell.Prominent among the non-nitrogenoussubstances which are thus found infermented sugar solutions are fusel oil,succinic acid, glycerol, acetic acid,aldehyde, formic acid, esters, andtraces of many other aldehydes andacids. In addition to these substanceswhich are found in the liquid, there arealso the carbonaceous constituents ofthe newly formed cells of theorganism, comprising the material ofthe cell walls, yeast gum, glycogen,complex organic phosphates, as well as

other substances.The attention of chemists has been

directed to these compounds sincePasteur first emphasised theirimportance as essential products of thealcoholic fermentation of sugar, andhis example was generally followed inattributing their origin to the sugar.

The study of cell-free fermentationby means of yeast-juice or zymin has,however, revealed the facts that certainof these substances are not formed inthe absence of living cells, and thattheir origin is to be sought in themetabolic processes which accompany

the life of the cell. Their source,moreover, has been traced not to thesugar but to the amino-acids, formedby the hydrolysis of the proteins, whichoccur in all such liquids as beer wort,grape juice, etc., which are usuallysubmitted to alcoholic fermentation.This has so far been proved withcertainty for the fusel oil and succinicacid, and rendered highly probable forall the various aldehydes and acids ofwhich traces have been detected.

Fusel Oil.

All forms of alcohol prepared byfermentation contain a fraction of highboiling-point, which is termed fuseloil, and amounts to about [p086] 0·1 to0·7 per cent. of the crude spiritobtained by distillation. This materialis not an individual substance, butconsists of a mixture of very variedcompounds, all occurring in smallamount relatively to the ethyl alcoholfrom which they have been separated.The chief constituents of the mixtureare the two amyl alcohols, isoamylalcohol,

(CH3)2·CH·CH2·CH2·OH,

and d-amyl alcohol,

CH3·CH(C2H5)·CH2·OH,

which contains an asymmetric carbonatom and is optically active. Inaddition to these, much smalleramounts of propyl alcohol and isobutylalcohol are present, together withtraces of fatty acids, aldehydes, andother substances.

The origin of these purely non-nitrogenous compounds was usuallysought in the sugar of the liquidfermented, from which they were

thought to be formed by the yeast itselfor by the agency of bacteria[Emmerling, 1904, 1905; Pringsheim,1905, 1907, 1908, 1909], whilst otherstraced their formation to the directreduction of fatty acids. Felix Ehrlichhas, however, conclusively shown in aseries of masterly researches that thealcohols, and probably also thealdehydes, contained in fusel oil are inreality derived from the amino-acidswhich are formed by the hydrolysis ofthe proteins.

The close relationship between thecomposition of leucine,

(CH3)2·CH·CH2·CH(NH2)·COOH,

and isoamyl alcohol,

(CH3)2·CH·CH2·CH2·OH,

had previously led to the surmise that agenetic relation might exist betweenthese substances, but the idea had notbeen experimentally confirmed. In1903 Ehrlich discovered [1903; 1904,1, 2; 1907, 2; 1908; Ehrlich andWendel, 1908, 2] that proteins alsoyield on hydrolysis an isomeride ofleucine known as isoleucine, which hasthe constitution

CH3·CH(C2H5)·CH(NH2)·COOH,

and therefore stands to d-amyl alcohol,

CH3·CH(C2H5)·CH2·OH,

in precisely the same relation asleucine to isoamyl alcohol. Thissuggestive fact at once directed hisattention to the problem of the originof the amyl alcohols in alcoholicfermentation. Using a pure culture ofyeast, and thus excluding theparticipation of bacteria in the change,he found that leucine readily yieldedisoamyl alcohol, and isoleucine d-amylalcohol when these amino-acids were

added in the pure state [p087] to asolution of sugar and treated with aconsiderable proportion of yeast [1905;1906, 2, 3; 1907, 1, 3]. The chemicalreactions involved are simple ones andare represented by the followingequations:—

(1) (CH3)2·CH·CH2·CH(NH2)·COOH

Leucine

+ H2O =

(CH3)2CH·CH2CH2·OHIsoamyl alcohol

+ CO2 + NH3

(2) CH3·CH(C2H5)·CH(NH2)·COOH

Isoleucine

+ H2O =

CH3·CH(C2H5)·CH2·OHd-Amyl alcohol

+ CO2 + NH3

The experiments by which theseimportant changes were demonstratedwere of a very simple and convincingcharacter [Ehrlich, 1907, 1]. Twohundred grams of sugar and 3 to 10grams of the nitrogenous substance tobe examined were dissolved in 2 to 2·5

litres of tap water in a 3 to 4 litre flask,the liquid was sterilised by beingboiled for several hours, and aftercooling 40 to 60 grams of fresh yeastwere added and the flask allowed tostand at room temperature until thewhole of the sugar had beendecomposed by fermentation. In theearlier experiments the amyl alcoholswere isolated and identified byconversion into the correspondingvalerianic acids, but as a rule the fuseloil as a whole was quantitativelyestimated in the filtrate by the Röse-Herzfeld method [Lunge, 1905, p. 571].

The following are typical results. (1)An experiment carried out as abovewithout any addition of leucine gave97·32 grams of alcohol containing 0·40per cent. of fusel oil. (2) When 6 gramsof synthetic, optically inactive leucinewere added, 97·26 grams of alcoholwere obtained, containing 2·11 percent. of fusel oil, which was alsooptically inactive; 2·5 grams of leucinewere recovered, so that 87 per cent. ofthe theoretical yield of isoamyl alcoholwas obtained from the 3·5 grams ofleucine decomposed. (3) In thepresence of 2·5 grams of d-isoleucine

(prepared from molasses residues), 200grams of sugar gave 93·99 grams ofalcohol, containing 1·44 per cent. offusel oil, which was lævo-rotatory.This corresponds with 80 per cent. ofthe theoretical yield of d-amyl alcoholfrom the isoleucine added.

This change, which Ehrlich hastermed the alcoholic fermentation ofthe amino-acids, although broughtabout by living yeast, does not appearto occur at all when zymin [Ehrlich,1906, 4; Pringsheim, 1906] or yeast-juice [Buchner and Meisenheimer,1906] is substituted for the intact

organism, nor is it effected even byliving yeast in the absence of afermentable sugar [Ehrlich, 1907, 1].The reaction appears indeed to beintimately connected with thenitrogenous metabolism of the cell, andthe whole of the ammonia produced isat once assimilated and does not appearin the fermented liquid. Other amino-acids [p088] undergo a correspondingchange, and the reaction appears to be ageneral one. Thus tyrosine, OH·C6H4·CH2·CH(NH2)·COOH, yieldsp-hydroxyphenylethyl alcohol, ortyrosol [Ehrlich, 1911, 1; Ehrlich and

Pistschimucka, 1912, 2], OH·C6H4·CH2·CH2OH, a substance ofintensely bitter taste, which was firstprepared in this way and is probablyone of the most important factors indetermining the flavour of beers, etc.Phenylalanine, C6H5·CH2·CH(NH2)·COOH, in asimilar way yields phenylethyl alcohol,C6H5·CH2·CH2OH, one of theconstituents of oil of roses, whilsttryptophane,

C6H4╱ │

HN │╲ │HC═══C·CH2·CH(NH2)·COOH

yields tryptophol,

C6H4╱ │

HN │╲ │HC═══C·CH2·CH2OH

which was also first prepared in thisway [Ehrlich, 1912] and has a veryfaintly bitter, somewhat biting taste.

The extent to which the amino-acidsof a medium in which yeast isproducing fermentation aredecomposed in this sense depends onthe amount of the available nitrogenand on the form in which it is present.Thus the addition of ammoniumcarbonate to a mixture of yeast andsugar was found to lower theproduction of fusel oil from 0·7 to 0·33per cent. of the alcohol produced. Theaddition of leucine alone raised thepercentage from 0·7 to 2·78, but theaddition of both leucine andammonium carbonate resulted in the

formation of only 0·78 per cent. offusel oil, The production of fusel oiltherefore and the character of theconstituents of the fusel oil alikedepend on the composition of themedium in which fermentation occurs.This affords a ready explanation of thefact that molasses, which containsalmost equal amounts of leucine andisoleucine, yields a fusel oil alsocontaining approximately equalamounts of isoamyl alcohol and d-amylalcohol [Marckwald, 1902], whilst cornand potatoes, in which leucinepreponderates over isoleucine, yield

fusel oils containing a relatively largeamount of the inactive alcohol. Thesubject is, in fact, one of great interestto the technologist, for as Ehrlichpoints out "the great variety of thebouquets of wine and aromas ofbrandy, cognac, arrak, rum, etc., maybe very simply referred to the manifoldvariety of the proteins of the rawmaterials (grapes, corn, rice, sugarcane, etc.) from which they arederived".

Yeast can also form fusel oil at theexpense of its own protein, but thisonly occurs to any considerable extent

when the external [p089] supply ofnitrogen is insufficient. Under thesecircumstances the amino-acids formedby autolysis may be decomposed andtheir nitrogen employed over again forthe construction of the protein of thecell.

The yield is also influenced by thecondition of the yeast employed withregard to nitrogen, a yeast poor innitrogen being more efficacious indecomposing amino-acids than onewhich is already well supplied withnitrogenous materials. The nature ofthe carbonaceous nutriment and finally

the species of yeast are also of greatimportance [see Ehrlich, 1911, 2;Ehrlich and Jacobsen, 1911].

A very important characteristic ofthe action of yeast on the amino-acidsis that the two stereo-isomerides ofthese optically active compounds arefermented at different rates. Wheninactive, racemic leucine is treatedwith yeast and sugar, the naturallyoccurring component, the l-leucine, ismore rapidly attacked, so that if theexperiment be interrupted at the propermoment the other component, the d-leucine, alone is present and may be

isolated in the pure state. In an actualexperiment 3·8 grams of thiscomponent were obtained in the purestate from 10 grams of dl-leucine[Ehrlich, 1906, 1], so that the whole ofthe l-leucine (5 grams) had beendecomposed but only 1·2 grams of thed-leucine. This mode of action hasbeen found to be characteristic of thealcoholic fermentation of the amino-acids by yeast. In all the instances sofar observed, both components of theinactive amino-acid are attacked, butusually the naturally occurringisomeride is the more rapidly

decomposed, although in the case of β-aminobutyric acid both componentsdisappear at the same rate [Ehrlich andWendel, 1908, 1]. This reactiontherefore must be classed along withthe action of moulds on hydroxy-acids[McKenzie and Harden, 1903], and theaction of lipase on inactive esters[Dakin, 1903, 1905], in which bothisomerides are attacked but at unequalrates, and differs sharply from theaction of yeast itself on sugars [Fischerand Thierfelder, 1894], and of emulsin,maltase, etc., which only act on oneisomeride and leave the other entirely

untouched [see Bayliss, 1914, pp. 55,77, 117].

Succinic Acid.

The origin of the succinic acidformed in fermentation has also beentraced by Ehrlich [1909] to thealcoholic fermentation of the amino-acids. It was shown by Buchner and byKunz [1906] that succinic acid likefusel oil is not formed duringfermentation by yeast-juice or zymin,and, in the light of Ehrlich's work onfusel oil, several [p090] modes of

formation appeared possible for thissubstance [Ehrlich, 1906, 3]. Thedibasic amino-acids might, forexample, undergo simple reduction, theNH2 group being removed as ammoniaand replaced by hydrogen. Asparticacid would thus pass into succinic acid:—

COOH·CH2·CH(NH2)·COOH + 2 H = COOH·CH2·CH2·COOH + NH3.

This change can be effected in thelaboratory only by heating withhydriodic acid. Biologically it has beenobserved [E. and H. Salkowski, 1879]when aspartic acid is submitted to theaction of putrefactive bacteria, andalmost quantitatively when Bacilluscoli communis is cultivated in amixture of aspartic acid and glucose[Harden, 1901]. In this case a well-defined source of hydrogen exists inthe glucose, which when acted on by

this bacillus yields a large volume ofgaseous hydrogen, which is not evolvedin the presence of aspartic acid. Somesuch source is also available in the caseof yeast, although it cannot bechemically defined, for this organismis known to produce many reducingactions, which are usually ascribed tothe presence of reducing ferments orreductases in the cell.

A similar action would convertglutamic acid,

COOH·CH2·CH2·CH(NH)2·COOH,

into glutaric acid,

COOH·CH2·CH2·CH2·COOH,

which also is found among the productsof fermentation, whilst the monamino-acids would pass into the simple fattyacids.

On submitting these ideas to the testof experiment, however, Erhlich foundthat the addition of aspartic acid didnot in any way increase the yield ofsuccinic acid, and that of all the amino-acids which were tried only glutamicacid, COOH·CH2·CH2·CH(NH2)·COOH,produced a definite increase in the

amount of this substance. Furtherexperiments showed that glutamic acidwas actually the source of the succinicacid, the relations being quite similarto those which exist for the productionof fusel oil.

Succinic acid is formed wheneversugar is fermented by yeast, even in theabsence of added nitrogenous matter,and amounts to 0·2 to 0·6 per cent. ofthe weight of the sugar decomposed, itsorigin in this case being the glutamicacid formed by the autolysis of theyeast protein. When some other sourceof nitrogen is present, such as

asparagine or an ammonium salt, theamount falls to 0·05 to 0·1. If glutamicacid be added it rises to about 1 to 1·5per cent. but falls again to about 0·05 to0·1 when other sources of nitrogen,such as asparagine or ammonium salts,are simultaneously available, either inthe presence or [p091] absence of addedglutamic acid. As in the case of fuseloil, the production does not occur inthe absence of sugar, and is noteffected by yeast-juice or zymin.

The chemical reaction involved inthe production of succinic acid differsto some extent from that by which

fusel oil is formed, inasmuch as anoxidation is involved:—

COOH·CH2·CH·CH(NH2)·COOH + 2 O = COOH·CH2·CH2·COOH + NH3 + CO2.

From analogy with the production ofamyl alcohol from leucine, glutamicacid would be expected to yield γ-hydroxybutyric acid:—

COOH·CH2·CH2·CH(NH2)·COOH + H2O = NH3 + CO2 + COOH·CH2·CH2·CH2·OH.

As a matter of fact this substance

cannot be detected among the productsof fermentation, but succinic acid asalready explained is formed. This acidmight, however, possibly be formed bythe oxidation of the γ-hydroxybutyricacid:—

COOH·CH2·CH2·CH2·OH + 2 O = COOH·CH2·CH2·COOH + H2O,

although this change is on biologicalgrounds improbable.

The conversion of the group —CH(NH2)— into the terminal CH2·OHin fusel oil, or COOH in succinic acid,may possibly be effected in several

different ways, the most probable ofwhich are the following:—

I. Direct elimination of carbondioxide, followed by hydrolysis of theresulting amine:—

(1) R·CH(NH2)·COOH = R·CH2·NH2 + CO2.(2) R·CH2·NH2 + H2O = R·CH2·OH + NH3.

The reaction (1) is actually effectedby many bacteria and has beenemployed for the preparation of basesfrom amino-acids [cf. Barger, 1914, p.7], although there is no direct evidence

that it can be brought about by yeast.On the other hand reaction (2) hasactually been observed with someyeasts. Thus it has been found [Ehrlichand Pistschimuka, 1912, 1] that many"wild" yeasts produce this change withgreat readiness in presence of sugar,glycerol or ethyl alcohol as sources ofcarbon and grow well in media inwhich amines, such as p-hydroxyphenylethylamine or iso-amylamine, form the only source ofnitrogen. Willia anomala (Hansen), ayeast which forms surface growths,succeeds admirably under these

conditions, whereas culture yeasts aremuch less active in this way, althoughthey produce a certain amount ofchange. It is therefore possible that thismode of decomposition plays somepart in the production of fusel oil, butin the case of culture yeasts it isentirely subordinated to the mode nextto be discussed. [p092]

II. Oxidative removal of the –NH2group with formation of an α-ketonicacid:—

(1) R·CH(NH2)·COOH + O = R·CO·COOH + NH3

followed by the decomposition of theketonic acid into carbon dioxide and analdehyde and the subsequent reductionor oxidation of the aldehyde:—

(2) R·CO·COOH = R·CHO + CO2.(3) (a) R·CHO + 2 H = R·CH2OH. (b) R·CHO + O = R·COOH.

The evidence for the occurrence ofreaction (1) is supplied by theexperiments of Neubauer and Fromherz[1911]. Having previously found thatamino-acids undergo a change of thiskind in the animal body, Neubauerinvestigated their behaviour towards

yeast. Taking dl-phenylaminoaceticacid, C6H5·CH(NH2)·COOH, it wasfound that the changes produced wereessentially the same as in the animalbody. The l-component of the acid waspartly acetylated and partly unchanged,whereas the d-component of the acidyielded benzyl alcohol, C6H5·CH2·OH,phenylglyoxylic acid, C6H5·CO·COOH,and the hydroxy-acid C6H5·CH(OH)·COOH. Since howeverthis hydroxy-acid was produced in thel-form it probably arose by theasymmetric reduction ofphenylglyoxylic acid, a reaction which

can be effected by yeast as was alsofound to be the case in the animal body[see Dakin, 1912, pp. 52, 78].Moreover it was shown that when theeffects of yeast on a ketonic acid andthe corresponding hydroxy-acid werecompared, the alcohol was formed inmuch better yield from the ketonic acid(70 per cent.) than from the hydroxy-acid (3–4 per cent.), the actual examplebeing the production of tyrosol (p-hydroxyphenylethyl alcohol), OH·C6H4·CH2·CH2OH, from p-hydroxyphenylpyruvic acid, OH·C6H4·CH2·CO·COOH, and p-

hydroxyphenyl-lactic acid, OH·C6H4·CH2·CH(OH)·COOHrespectively.

Neubauer by these experimentsestablished two extremely importantpoints. 1. That the amino-acids actuallyyield the corresponding α-ketonic acidswhen treated with yeast and sugarsolution. 2. That the a-ketonic acidsunder similar conditions give thealcohol containing one carbon atomless in good yield, whereas thecorresponding hydroxy-acids only givean extremely small amount of thesealcohols.

It is therefore probable that at anearly stage in the decomposition of theamino-acids by yeast a ketonic acid isproduced, which then undergoes furtherchange.The source of the oxygen required for

this reaction and the mechanism ofoxidation have not yet been definitelyascertained. It is possible [p093] thathydrated imino-acids of the type OH│

R·C—COOH│NH2

are first formed [Knoop,

1910], but these have not as yet beenisolated.

The spontaneous production ofketonic aldehydes from amino-acidsand from hydroxy-acids in aqueoussolution, which has been demonstratedby Dakin and Dudley [1913], pointshowever to the possibility that theketonic acid may be a secondaryproduct derived from thecorresponding ketonic aldehyde [seealso Dakin, 1908; Neuberg, 1908,1909]. This itself may either arisedirectly from the amino-acid or from apreviously formed hydroxy-acid, the

latter alternative being, however,improbable in view of the small yieldof alcohol obtained from hydroxy-acidsby the action of yeast in theexperiments of Neubauer andFromherz.

R·CH(NH2)·COOH → R·CH(OH)·COOH⇅ ⇅R·CO·CHO

↓ + OxygenR·CO·COOH

(2) Whatever be the exact mode bywhich the ketonic acid is formed, itappears most probable that a compound

of this nature forms the starting-pointfor the next stage in the production ofthe alcohols. The researches ofNeuberg, which have already beendiscussed on p. 81, have revealed amechanism in yeast—the enzymecarboxylase—by which these α-ketonicacids are rapidly broken up into analdehyde and carbon dioxide:

R·CO·COOH = R·CHO + CO2

and it can scarcely be doubted that thisis the actual course of the reaction.

(3) The final conversion of thealdehyde into the corresponding

alcohol is also a change which it hasbeen proved can be effected by yeast[Neuberg and Rosenthal, 1913]probably by the aid of the reductasewhich is one of the weapons in itsarmoury of enzymes.

Yeast is capable of producing manyvigorous reducing actions and rapidlyreduces methylene blue and sodiumselenite. It is in all probability due to areaction of this kind that the iso-amylaldehyde and isovaleraldehydewere reduced to the alcohols inNeuberg and Steenbock's experiments[1913, 1914], and that considerable

quantities of ethyl alcohol are formedin the sugar free fermentation ofpyruvic acid [Neuberg and Kerb, 1913,1] (see later p. 110 for a discussion ofthis question).

A further possibility exists that insome cases the aldehyde may [p094] besimultaneously oxidised and reduced orthe molecule of one aldehyde reducedand that of another oxidised withproduction of the corresponding acidand alcohol by an "aldehydo-mutase,"similar to that which has been observedby Parnas [1910] in many animaltissues. Finally the aldehyde may

simply be converted into thecorresponding acid by oxidation asappears to take place in the formationof succinic acid.

The intermediate production of analdehyde would thus be consistent bothwith the production of alcohols andacids from amino-acids.

Fusel oil would be formed by thereduction of the aldehydes arising fromthe simple monobasic amino-acids,succinic acid would be produced byoxidation of the aldehyde derived fromthe dibasic glutamic acid.

In favour of this view is to be

adduced the fact that aldehydes such asisobutyraldehyde and valeraldehydehave been found in crude spirit, whilstacetaldehyde is a regular product ofalcoholic fermentation [see Ashdownand Hewitt, 1910]. Benzaldehyde,moreover, has been actually detected asa product of the alcoholic fermentationof phenylaminoacetic acid, C6H5·CH(NH2)·COOH [Ehrlich, 1907,1]. Further, the aldehydes so producedwould readily pass by oxidation intothe corresponding fatty acids, smallquantities of which are invariablyproduced in fermentation.

This view of the nature of thealcoholic fermentation of the amino-acids is undoubtedly to be preferred tothat previously suggested by Ehrlich[1906, 3] according to which ahydroxy-acid is first formed and theneither directly decomposed into analcohol and carbon dioxide or into analdehyde and formic acid, the aldehydebeing reduced and the formic aciddestroyed (see p. 115).

R·CH(NH2)·COOH → R·CH(OH)·COOH↓ or

R·CH2OH + CO2

The most probable course of thedecomposition by which isoamylalcohol and succinic acid are producedfrom leucine and glutamic acidrespectively is therefore the following:—

(a) Isoamyl Alcohol.(1)

(1) (CH3)2·CH·CH2·CH(NH2)·COOH

Leucine

(2) (CH3)2·CH·CH2·CO·COOH

α-Ketoisovalerianic acid

(3) (CH3)2CHCH2·CHO

Isovaleraldehyde  + CO2

(4) (CH3)2·CH·CH2·CH2OH

Isoamyl alcohol(b) Succinic Acid.

(1) COOH·CH2·CH2·CH(NH2)·COOH

Glutamic acid

(2) COOH·CH2·CH2·CO·COOH

α-Keto-glutaric acid

(3) COOH·CH2CH2·CHO  + CO2

(3) COOH·CH2CH2·CHOSuccinic semialdehyde

 + CO2

(4) COOH·CH2·CH2·COOH

Succinic acid

Glycerol.

[p095]

Of the three chief by-products ofalcoholic fermentation, only glycerolremains at present referable directly tothe sugar. This substance, as shown bythe careful experiments of Buchner andMeisenheimer [1906], is formed by theaction both of yeast-juice and zymin tothe extent of 3·8 per cent. of the sugar

decomposed, and no other source forits production has so far beenexperimentally demonstrated. If it betrue that during the decomposition ofsugar into alcohol and carbon dioxide,substances containing three carbonatoms are formed as intermediatecompounds (see p. 100), it is obviousthat these might by reduction beconverted into glycerol which wouldthus be a true by-product of thealcoholic fermentation of sugar. [SeeOppenheimer, 1914, 2.] It has,however, been suggested that it may inreality be a product of decomposition

of lipoid substances or of the nuclein ofthe cell (Ehrlich).

The effect of Ehrlich's work has beenclearly to distinguish the chemicalchanges involved in the production offusel oil and succinic acid from thoseconcerned in the decomposition ofsugar into alcohol and carbon dioxide,and to bring to light a most importantseries of reactions by means of whichthe yeast-cell is able to supply itselfwith nitrogen, one of the indispensableconditions of life.

CHAPTER VIII.THE CHEMICAL CHANGES

INVOLVED IN FERMENTATION.

[p096]

It has long been the opinion ofchemists that the remarkable andalmost quantitative conversion of sugarinto alcohol and carbon dioxide duringthe process of fermentation is mostprobably the result of a series ofreactions, during which variousintermediate products are momentarily

formed and then used up in thesucceeding stage of the process. Novery good ground can be adduced forthis belief except the contrast betweenthe chemical complexity of the sugarmolecule and the comparativesimplicity of the constitution of theproducts. Many attempts have,however, been made to obtain evidenceof such a series of reactions, andnumerous suggestions have been madeof probable directions in which suchchanges might proceed. In makingthese suggestions, investigators havebeen guided mainly by the changes

which are produced in the hexoses byreagents of known composition. Thefermentable hexoses, glucose, fructose,mannose, and galactose, appear to berelatively stable in the presence ofdilute acids at the ordinarytemperature, and are only slowlydecomposed at 100°, more rapidly byconcentrated acids, with formation ofketonic acids, such as levulinic acid,and of coloured substances of complexand unknown constitution.

In the presence of alkalis, on theother hand, the sugar molecule isextremely susceptible of change. In the

first place, as was discovered by Lobryde Bruyn [1895; Bruyn and Ekenstein,1895; 1896; 1897, 1, 2, 3, 4], each ofthe three hexoses, glucose, fructose,and mannose is converted by dilutealkalis into an optically almost inactivemixture containing all three, andprobably ultimately of the samecomposition whichever hexose isemployed as the starting-point.

This interesting phenomenon is mostsimply explained on the assumptionthat in the aqueous solution of any oneof these hexoses, along with themolecules of the hexose itself, there

exists a small proportion of those of anenolic form which is common to all thethree hexoses, as illustrated by thefollowing formulæ, the aldehydeformulæ [p097] being employed insteadof the γ-oxide formulæ for the sake ofsimplicity:—

CHO CHO CH2│ │ │HCOH HOCH CO│ │ │

HOCH HOCH HOCH│ │ │HCOH HCOH HCOH│ │ │HCOH HCOH HCOH│ │ │CH2(OH) CH2(OH) CH2Glucose Mannose Fructose

This enolic form is capable of givingrise to all three hexoses, and the changeby which the enolic form is produced

and converted into an equilibriummixture of the three correspondinghexoses is catalytically accelerated byalkalis, or rather by hydroxyl ions. Inneutral solution the change is so slowthat it has never been experimentallyobserved; in the presence ofdecinormal caustic soda solution at 70°the conversion is complete in threehours. Precisely similar effects areproduced with galactose, which yieldsan equilibrium mixture containingtalose and tagatose, sugars whichappear not to be fermentable.

The continued action even of dilute

alkaline solutions carries the changemuch further and brings about acomplex decomposition which is muchmore rapidly effected by moreconcentrated alkalis and at highertemperatures. This change has been thesubject of very numerousinvestigations [for an account of thesesee E. v. Lippmann, 1904, pp. 328, 713,835], but for the present purpose theresults recently obtained byMeisenheimer [1908] may be quoted astypical. Using normal solutions ofcaustic soda and concentrations offrom 2 to 5 grams of hexose per 100

c.c., it was found that at airtemperature in 27 to 139 days from 30to 54 per cent. of the hexose wasconverted into inactive lactic acid,C3H6O3, from 0·5 to 2 per cent. intoformic acid, CH2O2, and about 40 percent. into a complex mixture ofhydroxy-acids, containing six and fourcarbon atoms in the molecule. Usuallyonly about 74 to 90 per cent. of thesugar which had disappeared wasaccounted for, but in one case theproducts amounted to 97 per cent. ofthe sugar. About 1 per cent. of thesugar was probably converted into

alcohol and carbon dioxide. Noglycollic acid, oxalic acid, glycol, orglycerol was produced.

The fact that alcohol is actuallyformed by the action of alkalis onsugar was established by Buchner andMeisenheimer [1905], who obtainedsmall quantities of alcohol (1·8 to 2·8grams from 3 kilos. of cane sugar) byacting on cane sugar with boilingconcentrated caustic soda [p098]

solution. It is evident that under theseconditions an extremely complex seriesof reactions occurs, but the formationof alcohol and carbon dioxide and of a

large proportion of lactic acid deservesmore particular attention.

The direct formation of alcohol fromsugar by the action of alkalis appearsfirst to have been observed by Duclaux[1886], who exposed a solution ofglucose and caustic potash to sunlightand obtained both alcohol and carbondioxide. As much as 2·6 per cent. of thesugar was converted into alcohol in asimilar experiment made by Buchnerand Meisenheimer [1904]. When theweaker alkalis, lime water or barytawater, were employed instead ofcaustic potash, however, no alcohol

was formed, but 50 per cent. of thesugar was converted into inactive lacticacid [Duclaux, 1893, 1896]. Duclauxtherefore regarded the alcohol andcarbon dioxide as secondary productsof the action of a comparatively strongalkali on preformed lactic acid. Ethylalcohol can, in fact, be produced fromlactic acid both by the action ofbacteria [Fitz, 1880] and of moulds[Mazé, 1902], and also by chemicalmeans. Thus Duclaux [1886] found thatcalcium lactate solution exposed tosunlight underwent decomposition,yielding alcohol and calcium carbonate

and acetate, whilst Hanriot [1885,1886], by heating calcium lactate withslaked lime obtained a considerablequantity of a liquid which he regardedas ethyl alcohol, but which was shownby Buchner and Meisenheimer [1905]to be a mixture of ethyl alcohol withisopropyl alcohol.

It appears, therefore, that inactivelactic acid can be quite readilyobtained in large proportion from thesugars by the action of alkalis, whilstalcohol can only be prepared incomparatively small amount andprobably only as a secondary product

of the decomposition of lactic acid.The study of the action of alkalis on

sugar has, however, yielded still furtherinformation as regards the mechanismof the reaction by which lactic acid isformed. A considerable body ofevidence has accumulated, tending toshow that some intermediate product ofthe nature of an aldehyde or ketonecontaining three carbon atoms is firstformed.

Thus Pinkus [1898] andsubsequently Nef [1904, 1907], byacting on glucose with alkali inpresence of phenylhydrazine obtained

the osazone of methylglyoxal, CH3·CO·CHO. This osazone may beformed either from methylglyoxalitself, from acetol, CH3·CO·CH2·OH,or from lactic aldehyde, CH3·CH(OH)·CHO [Wohl, 1908].Methylglyoxal itself may also beregarded as a secondary [p099] productderived from glyceraldehyde, CH2(OH)·CH(OH)·CHO, ordihydroxyacetone, CH2(OH)·CO·CH2(OH), by a processof intramolecular dehydration, so thatthe osazone might also be derivedindirectly from either of these

compounds [see also Neuberg andOertel, 1913]. Methylglyoxal itselfreadily passes into lactic acid when itis treated with alkalis, a molecule ofwater being taken up:—

CH3·CO·CHO + H2O = CH3·CH(OH)·COOH.

Further evidence in the same directionis afforded by the interesting discoveryof Windaus and Knoop [1905], thatglucose is converted by ammonia inpresence of zinc hydroxide intomethyliminoazole,

CH3·C·NH·CH║ ║ ,HC────N

a substance which is a derivative ofmethylglyoxal.

The idea suggested by Pinkus thatacetol is the first product of the actionof alkalis on sugar has been renderedvery improbable by the experiments ofNef, and the prevailing view (Nef,Windaus and Knoop, Buchner andMeisenheimer) is that the first productis glyceraldehyde, which then passesinto methylglyoxal, and finally intolactic acid:—

(1) C6H12O6 = 2 CH2(OH)·CH(OH)·CHO.(2) CH2(OH)·CH(OH)·CHO = CH3·CO·CHO + H2O.(3) CH3·CO·CHO + H2O = CH3·CH(OH)·COOH.

All these changes may occur atordinary temperatures in the presenceof a catalyst, and in so far resemble theprocesses of fermentation by yeastsand bacteria.

The first attempt to suggest ascheme of chemical reactions by whichthe changes brought about by living

organisms might be effected was madein 1870 by Baeyer [1870], who pointedout that these decompositions might beproduced by the successive removaland re-addition of the elements ofwater. The result of this would be tocause an accumulation of oxygenatoms towards the centre of the chainof six carbon atoms, which, inaccordance with general experience,would render the chain more easilybroken. Baeyer formulated the changescharacteristic of the alcoholic andlactic fermentations as follows, theintermediate stages being derived from

the hydrated aldehyde formula ofglucose by the successive removal andaddition of the elements of water: [p100]

I.CH2·OH│CH·OH│CH·OH│CH·OH│CH·OH│CH(OH)2

II.CH2 . . . OH│COH . . H│C . . OH . . H│COH . . . H│COH . . . H│CH . . . (OH)2

III. IV. V.

III.CH3│CH . OH│C(OH)2│C(OH)2│C(OH)2│CH3

IV.CH3│CH(OH)│CO╱O╲CO│CH(OH)│CH3

V.CH3│CH2╱O╲CO╱O╲CO╱O╲CH2│CH3

H3

The immediate precursor of alcoholand carbon dioxide is here seen to bethe anhydride of ethoxycarboxylic acid(V), whilst that of lactic acid is lacticanhydride (IV). (Baeyer does notappear, as recently stated byMeisenheimer [1907, p. 8], Wohl[1907, 2], and Buchner andMeisenheimer [1909] to havesuggested that lactic acid was anintermediate product in alcoholicfermentation, but rather to haverepresented independently the courseof the two different kinds of

fermentation, the alcoholic and thelactic.)

It was subsequently pointed out byBuchner and Meisenheimer [1904] thatBaeyer's principle of oxygenaccumulation might be applied in adifferent way, so that a ketonic acidwould be produced, the decompositionof which, in a manner analogous to thatof acetoacetic acid, would lead to theformation of two molecules of lacticacid, from which the final productsalcohol and carbon dioxide might bedirectly derived, as shown in thefollowing formulæ:—

CHO·CH(OH)·CH(OH)·CH(OH)·CH(OH)·CH2(OH)

COOH·CH(OH)·CH2·CO·CH(OH)·CH3

COOH·CH(OH)·CH3────COOH·CH(OH)·CH3

CO2────CH2·OH·CH3────

────CO2────CH2·OH·CH3

A scheme based on somewhatdifferent principles has beenpropounded by Wohl [Lippmann, 1904,p. 1891], and has been accepted byBuchner and Meisenheimer [1905] asmore probable than that quoted above.Wohl and Oesterlin [1901] were able totrace experimentally the various stagesof the conversion of tartaric acid (I)

into oxalacetic acid (III), which can becarried out by reactions taking place atthe ordinary temperature, and theyfound that the first stage consisted inthe removal of the elements of waterleaving an unsaturated hydroxyderivative (II) which in the secondstage underwent intramolecular changeinto the corresponding keto-compound(III): [p101]

COOH COOH COOH· · ·CH(OH) H C(OH) CO· − · = ║ ⇌ ·CH(OH) OH CH CH· · ·COOH COOH COOH

I.Tartaricacid

II.Oxalacetic

This change differs in principle fromthat assumed by Baeyer, inasmuch asthe second stage is not effected by there-addition of water, but by the keto-enol transformation, which is nowusually ascribed to the migration of the

hydrogen atom, although the sameresult can theoretically be arrived at bythe addition and removal of theelements of water. The analogy of thisprocess to what might be supposed tooccur in the conversion of sugar intocarbon dioxide and alcohol was pointedout by Wohl and Oesterlin, andsubsequently Wohl developed atheoretical scheme of reactions bywhich the process of alcoholicfermentation could be represented. Inthe first place the elements of water areremoved from the α and β carbonatoms of glucose (I) and the resulting

enol (II) undergoes conversion into thecorresponding ketone (III), which hasthe constitution of a condensationproduct of methylglyoxal andglyceraldehyde, and hence is readilyresolved by hydrolysis into thesecompounds (IV). The glyceraldehydepasses by a similar series of changes(V, VI) into methylglyoxal, and this isthen converted by addition of waterinto lactic acid (VII), a reaction whichis common to all ketoaldehydes of thiskind. Finally, the lactic acid is split upinto alcohol and carbon dioxide (VIII):—

CHO CHO│ │CH(OH) C(OH)│ H ║CH(OH) − OH CH│ │CH(OH) CH(OH)│ │CH(OH) CH(OH)│ │CH2(OH) CH2(OH)

I.Glucose.

II.

Methyl-glyoxalCHO │

│CO +H2O│CH3CHO CHO CHO│ │ │CH(OH) H C(OH) ⇌ CO + 

H2O │ · ║ │CH2(OH) − HO CH2 CH3

IV.Glyceral-dehyde.

V. VI.Methyl-glyoxal.

[p102]

This scheme agrees well with the

current ideas as to the formation oflactic acid from glucose under theinfluence of alkalis (p. 99). Itpostulates the formation asintermediate products of no less thanthree compounds containing a chain ofthree carbon atoms—glyceraldehyde,methylglyoxal, and lactic acid.

The Lactic Acid Theory ofAlcoholic Fermentation.

A practical interest was given tothese various schemes by the fact thatBuchner and Meisenheimer adduced

experimental evidence in favour of theview that lactic acid is an intermediateproduct in the formation of alcohol andcarbon dioxide from sugar byfermentation [1904, 1905, 1906, 1909].

These observers proved by a seriesof very careful analyses that yeast-juice frequently, but not invariably,contains small quantities of lactic acid,not exceeding 0·2 per cent. Whenyeast-juice is incubated alone or withsugar the amount of lactic acid mayeither increase or decrease. Moreover,lactic acid added to the juice issometimes diminished and sometimes

increased in quantity. On the whole itappears that the addition of aconsiderable quantity of sugar or ofsome lactic acid favours thedisappearance of lactic acid. Juices oflow fermenting power produce adiminution in the lactic acid present,those of high fermenting power anincrease.

In all cases the amounts of lacticacid either produced or destroyed arevery small in relation to the volume ofthe yeast-juice employed.

Throughout the whole series ofexperiments the greatest increase

amounted to 0·47 per cent. on the juiceemployed, and the greatest decrease to0·3 per cent. [See also Oppenheimer,1914, 1.] Buchner and Meisenheimer atone time regarded these facts as strongevidence that lactic acid is anintermediate product of alcoholicfermentation. It was thought probablethat the production of alcohol andcarbon dioxide from sugar occurred inat least two stages and under theinfluence of two distinct enzymes. Thefirst stage consisted in the conversionof sugar into lactic acid, and for theenzyme which brought about this

decomposition was reserved the namezymase or yeast-zymase. The lacticacid was then broken down into alcoholand carbon dioxide by the secondenzyme, lactacidase.

This theory, which is quite inharmony with the current ideas as tothe mode of decomposition of sugarsby alkalis, and is also consistent withWohl's scheme of reactions, is open toadverse criticism from several pointsof view. In the first place, it isnoticeable that the total amount oflactic acid used up by the juice isextremely small, even [p103] in the most

favourable cases, relatively to theamount of the juice [Harden, 1905],and it may be added to the sugar-fermenting power of the juice.Moreover, as pointed out by Buchnerand Meisenheimer themselves [1909],no proof is afforded that the lactic acidwhich disappears is converted intoalcohol and carbon dioxide. It is noteven certain, although doubtlessprobable, that the lactic acid whichoccurs or is produced in the juice isreally derived from sugar.

The most weighty criticism of thetheory is that of Slator [1906, 1907;

1908, 1, 2], which is based on theconsideration that if lactic acid be anintermediate product of alcoholicfermentation the reaction by which it isfermented must proceed at least asrapidly as that by which it is formed, inorder to prevent accumulation of lacticacid. The fermentation of lactic acid byyeast should therefore proceed at leastas rapidly as that of glucose. So far isthat from being the case that it hasbeen experimentally demonstrated thatlactic acid is not fermented at all byliving yeast. This conclusion wasrendered extremely probable by Slator,

who showed that lactic acid, even inconcentrations insufficient to preventthe fermentation of glucose, is notfermented to any considerable extent.The final proof that lactic acid isneither formed nor fermented by pureyeast has been brought by Buchner andMeisenheimer in a series of verycareful quantitative experimentscarried out with a pure yeast and withstrict precautions against bacterialcontamination [1909, 1910].

At first sight this fact appearsdecisive against the validity of thelactic acid theory, and it is recognised

as such by Buchner and Meisenheimer.Wohl has, however, suggested that thenon-fermentability of lactic acid byyeast is not really conclusive [1907, 1;see also Franzen and Steppuhn, 1912,1]. The production of lactic acid fromglucose is attended by the evolution ofa considerable amount of heat (22 cal.),and it is possible that at the moment ofproduction the molecule of the acid isin a condition of activity correspondingwith a much higher temperature thanthe average temperature of thefermenting liquid. Under thesecircumstances the molecule would be

much more susceptible of chemicalchange than at a later period whentemperature equilibrium had beenattained. It has, however, been pointedout by Tafel [1907], that such adecomposition of the lactic acid wouldoccur at the very instant of formationof the molecule, so that no groundremains even on this view for assumingthe actual existence of lactic acid as adefinite intermediate product. It hasalso been suggested by Luther [1907]that an unknown isomeride of lacticacid is formed as an intermediateproduct and fermented, and that traces

of lactic [p104] acid are formed by asecondary reaction from this, but nosatisfactory evidence for this view isforthcoming. There still remains adoubt as to whether the living yeast-cell is permeable to lactic acid, a factwhich would of course afford a verysimple explanation of the non-fermentability of the acid. Apart fromthis, however, it is difficult, in face ofthe evidence just quoted, to believe thatlactic acid is in reality an intermediateproduct in alcoholic fermentation.

Methylglyoxal,

Dihydroxyacetone andGlyceraldehyde.

As regards the fermentability byyeast of compounds containing threecarbon atoms, which may possiblyappear as intermediate products in thetransformation of sugar into carbondioxide and alcohol, many experimentshave been carried out, with somewhatuncertain results. Care has to be takenthat the substance to be tested is notadded in such quantity as to inhibit thefermenting power of the yeast or yeast-juice, and further that the conditions

are such that the substance in question,often of a very unstable nature, is notconverted by some chemical changeinto a different fermentable compound.It is also possible that the substance tobe tested may accelerate the rate ofautofermentation in a similar mannerto arsenates (pp. 80, 126) and manyother substances. These are all pointswhich have not up to the presentreceived sufficient attention. In thecase of living yeast the further questionarises of the permeability of the cell.

Methylglyoxal, CH3·CO·CHO, hasbeen tested by Mayer [1907] and Wohl

[1907, 2] with yeast, and by Buchnerand Meisenheimer both with acetone-yeast [1906] and yeast-juice [1910], inevery case with negative results, but itmay be noted that the concentrationemployed in the last mentioned ofthese experiments was such asconsiderably to diminish theautofermentation of the juice.

Glyceraldehyde, CH2(OH)·CH(OH)·CHO, was alsotested with yeast with negative resultsby Wohl [1898] and by Emmerling[1899], who employed a number ofdifferent yeasts. The same negative

result attended the experiments ofPiloty [1897] and Emmerling [1899]with pure dihydroxyacetone. Fischerand Tafel [1888, 1889], however, hadpreviously found that glycerose, amixture of glyceraldehyde anddihydroxyacetone prepared by theoxidation of glycerol, was readilyfermented by yeast, agreeing in thisrespect with the still older observationsof Van Deen and of Grimaux. Thereason for this diversity of result hasnot been definitely ascertained, but ithas been supposed by Emmerling to liein the formation of some fermentable

sugar from [p105] glycerose when thelatter is subjected to too high atemperature during its preparation.

On the other hand, Bertrand [1904]succeeded in fermenting puredihydroxyacetone by treating a solutionof 1 gram in 30 c.c. of liquid with asmall quantity of yeast for ten days at30°, the best result being afermentation of 25 per cent. of thesubstance taken. Moreover, Boysen-Jensen [1908, 1910, 1914] states thathe has also observed both theformation from glucose and thefermentation of this substance by

living yeast, but the amounts of alcoholand carbon dioxide produced were sominute and the evidence for theproduction of dihydroxyacetone soinconclusive that the experimentscannot be regarded as in any waydecisive [see Chick, 1912; Euler andFodor, 1911; Karauschanoff, 1911;Buchner and Meisenheimer, 1912]. Acareful investigation by Buchner[1910] and Buchner and Meisenheimer[1910] has led them to the conclusionthat both glyceraldehyde anddihydroxyacetone are fermentable.Glyceraldehyde exerts a powerful

inhibiting action both on yeast andyeast-juice, and was only found to giverise to a very limited amount of carbondioxide, quantities of 0·15 to 0·025gram being treated with 1 gram ofyeast or 5 c.c. of yeast-juice and aproduction of 4 to 12 c.c. of carbondioxide being attained.

When 0·1 gram of dihydroxyacetonein 5 c.c. of water was brought incontact with 1 gram of living yeast,about half was fermented, 17 c.c. ofcarbon dioxide (at 20° and 600 mm.)being evolved in excess of theautofermentation of the yeast (13 c.c.).

A much greater effect was obtained bythe aid of yeast-juice, and theremarkable observation was made thatwhilst yeast-juice alone producedcomparatively little action a mixture ofyeast-juice and boiled yeast-juice wasmuch more effective, quantities of 20to 50 c.c. of yeast-juice mixed with anequal volume of boiled juice, which insome experiments was concentrated,yielding with 0·4, 1, and 2 grams ofdihydroxyacetone almost thetheoretical amount of carbon dioxideand alcohol in excess of that evolved inthe absence of this substance. It was

further observed that the fermentationof this substance commenced muchmore slowly than that of glucose. Noexplanation of either of these facts hasat present been offered. The conclusiondrawn from their experiments byBuchner and Meisenheimer thatdihydroxyacetone is readilyfermentable, was confirmed byLebedeff [1911, 1], who further madethe important observation that duringthe fermentation of dihydroxyacetonethe same hexosephosphoric acid isproduced as is formed during thefermentation of the hexoses. Lebedeff

accordingly propounded a scheme ofalcoholic fermentation according towhich the hexose [p106] was firstconverted into two molecules of triose,the latter being first esterified totriosephosphoric acid and thencondensed to hexosediphosphoric acid,which then underwent fermentation,after being hydrolysed to phosphoricacid, and some unidentified substance,probably an unstable modification of ahexose, much more readily attacked byan appropriate enzyme than theoriginal glucose or fructose [1911, 1,pp. 2941–2].

The idea that the sugar is firstconverted into triose and this intotriosemonophosphoric acid had beenpreviously suggested by Iwanoff whopostulated the agency of a specialenzyme termed synthease [1909, 1],and supposed that thistriosemonophosphoric acid was thendirectly fermented to alcohol, carbondioxide and phosphoric acid.According both to Iwanoff andLebedeff the phosphoric ester is anintermediate product and itsdecomposition provides this solesource of carbon dioxide and alcohol.

This is quite inconsistent with the factsrecounted above (Chap. III), whichprove that the formation of thehexosephosphate is accompanied by anamount of alcoholic fermentationexactly equivalent to the quantity ofhexosephosphate produced, and that therate of fermentation rapidly falls assoon as the free phosphate hasdisappeared, in spite of the fact that atthat moment the concentration of thehexosephosphate is at its highest,whereas according to Iwanoff's theoryit is precisely under these conditionsthat the maximum rate of fermentation

should be maintained.It has also been shown that the

arguments adduced by Iwanoff infavour of the existence of his syntheaseare not valid [Harden and Young, 1910,1].

The fermentation ofdihydroxyacetone was moreoverproved by Harden and Young [1912] tobe effected by yeast-juice andmaceration extract at a much slowerrate than that of the sugars, in spite ofthe fact that the addition ofdihydroxyacetone did not inhibit thesugar fermentation. The same thing has

been shown for living yeast by Slator[1912] in agreement with the earlierresults of Buchner [1910] and Buchnerand Meisenheimer [1910].

The logical conclusion fromLebedeff's experiments would appearrather to be that dihydroxyacetone isslowly condensed to a hexose and thatthis is then fermented in the normalmanner [Harden and Young, 1912;Buchner and Meisenheimer, 1912;Kostytscheff, 1912, 2]. Buchner andMeisenheimer, however, regard this asimprobable on the ground thatdihydroxyacetone, being symmetric in

constitution, would yield an inactivehexose of which only at most 50 percent. would be fermentable. Againstthis it may be urged, however, [p107]

that enzymic condensation ofdihydroxyacetone might very probablyoccur asymmetrically yielding anactive and completely fermentablehexose. Buchner and Meisenheimer,however, still support the view thatdihydroxyacetone forms anintermediate stage in the fermentationof glucose and adduce as confirmatoryevidence of the probability of such achange the observation of Fernbach

[1910] that this compound is producedfrom glucose by a bacillus, Tyrothrixtenuis, which effects the change bothwhen living and after treatment withacetone.

The balance of evidence, however,appears to be in favour of the opinionthat dihydroxyacetone does not fulfilthe conditions laid down by Slator (seep. 103) as essential for an intermediateproduct in the process of fermentation[see also Löb, 1910].

Lebedeff subsequently [1912, 4;Lebedeff and Griaznoff, 1912]extended his experiments to

glyceraldehyde and modified his theoryvery considerably. Using macerationextract it was found in generalagreement with the results of Buchnerand Meisenheimer (p. 105) that 20 c.c.of juice were capable of producingabout half the theoretical amount ofcarbon dioxide from 0·2 gram ofglyceraldehyde, whereas 0·4 gramcaused coagulation of the extract and adiminished evolution of carbondioxide. The addition of phosphatediminished rather than increased thefermentation. Even in the mostfavourable concentration however (0·2

gram per 20 c.c.) the glyceraldehyde isfermented much more slowly thandihydroxyacetone or saccharose, as isshown by the following figures:—

20 c.c.Extract +0·2 gram.

CO2 in grams insuccessive periods of6

hours.18

hours. hours.Cane sugar 0·050 0·000 0·000Dihydroxy-acetone 0·042 0·000 0·000

Glycer-aldehyde 0·008 0·022 0·005

Further, during an experiment in which0·129 gram of CO2 was evolved in 22·5hours from 0·9 gram of glyceraldehydein presence of phosphate, no change in

free phosphate was observed, whereasin a similar experiment with glucose aloss of about 0·2 gram of P2O5 wouldhave occurred. Hence the fermentationtakes place without formation ofhexosediphosphate. This wasconfirmed by the fact that the osazoneof hexosephosphoric acid was readilyisolated from the products offermentation of dihydroxyacetone(0·259 gram of CO2 having beenevolved in twenty hours) but could notbe obtained from those ofglyceraldehyde (0·138 gram CO2 intwenty hours). [p108]

This result is extremely interesting,although it is not impossible that therate of fermentation of theglyceraldehyde is so slow that anyphosphoric ester produced ishydrolysed as rapidly as it is formed.

Lebedeff regards the experiments asproof that phosphate takes no part inthe fermentation of glyceraldehyde andbases on this conclusion and his otherwork the following theory of alcoholicfermentation.

1. The sugar is split up intoequimolecular proportions ofglyceraldehyde and dihydroxyacetone:

—

(a) C6H12O6 = C3H6O3 + C3H6O3.

2. The dihydroxyacetone then passesthrough the stages previouslypostulated (p. 106).

(b) 4 C3H6O3 + 4 R2HPO4 = 4 C3H5O2PO4R2 + 4 H2O.(c) 4 C3H5O2PO4R2 = 2 C6H10O4(R2PO4)2.(d) 2 C6H10O4(R2PO4)2 + 4 H2O = 2 C6H12O6 + 4 R2HPO4.

After which the hexose, C6H12O6 re-enters the cycle at (a).

3. The fermentation of theglyceraldehyde occurs according to thescheme developed by Kostytscheff (p.109), pyruvic acid being formed alongwith hydrogen and then decomposedinto carbon dioxide and acetaldehyde,which is reduced by the hydrogen.Lebedeff, however, suggests [1914, 1,2] that glyceric acid is first formed (1)and then converted by an enzyme,which he terms dehydratase intopyruvic acid (2):—

(1) CH2(OH)·CH(OH)·CHO + H2O → CH2(OH)·CH(OH)·CH(OH)2 → CH2(OH)·CH(OH)·COOH + 2 H(2) CH2(OH)·CH(OH)·COOH = CH3·CO·COOH + H2O.

The experimental basis for this idea isthe fact that glyceric acid is fermentedby dried yeast and maceration juice[compare Neuberg and Tir, 1911].

This scheme has the merit ofrecognising the fact that the carbondioxide does not wholly arise from theproducts of decomposition ofhexosephosphate, nor from its direct

fermentation. The function assigned tothe phosphate is that of removingdihydroxyacetone and thus preventingit from inhibiting further conversion ofhexose into triose, according to thereversible reaction

C6H12O6 ⇌ 2 C3H6O3.

This however appears to be quiteinadequate, since, on the one hand, thefermentation of glucose proceeds quitefreely in presence of as much as 5grams per 100 c.c. of dihydroxyacetone[Harden and Young, 1912], and on theother hand alcoholic fermentation

appears not to proceed at all in theabsence of phosphate (see p. 55). Thisforms the chief objection to the theoryin its present form. The slow rate atwhich [p109] glyceraldehyde isfermented also affords an argumentagainst the validity of Lebedeff's view,but this may possibly be accounted forto some extent by the fact thatglyceraldehyde is a strong inhibitingagent so that it might be more rapidlyfermented if added in a more dilutecondition.

The unfermented glyceraldehydecannot be recovered from the solution

and nothing is known as to its fateexcept that it readily gives rise both tolactic acid and glycerol [Oppenheimer,1914, 1, 2]. Evidently the reactionbetween glyceraldehyde and yeast-juice is by no means a simple one.

The Pyruvic Acid Theory.

The third stage of Lebedeff's theorypostulates the intermediate formationof pyruvic acid. This idea immediatelysuggested itself when it became knownthat yeast was capable of rapidlydecomposing a-ketonic acids with

evolution of carbon dioxide [seeNeubauer and Fromherz, 1911, p. 350;Neuberg and Kerb, 1912, 4;Kostytscheff, 1912, 2].

This scheme has been differentlyelaborated by different workers.According to Kostytscheff it involves(1) the production of pyruvic acid fromthe hexoses, a process accompanied byloss of hydrogen; (2) thedecomposition of pyruvic acid intoacetaldehyde and carbon dioxide; and(3) the reduction of the acetaldehyde toethyl alcohol.

(1) C6H12O6 = 2 CH3·CO·COOH + 4[H].(2) 2 CH3·CO·COOH = 2 CH3·CHO + 2 CO2.(3) 2 CH3·CHO + 4 H = 2 CH3·CH2·OH.

1. As regards the production ofpyruvic acid from the hexoses by yeast,the only direct evidence is afforded bythe experiments of Fernbach andSchoen [1913] who have obtained acalcium salt having the qualitativeproperties of a pyruvate by carrying outalcoholic fermentation by yeast in

presence of calcium carbonate, buthave not yet definitely settled eitherthe identity of the acid or its originfrom sugar. Pyruvic acid is, however,very closely related to severalsubstances which are intimatelyconnected both chemically andbiochemically with the hexoses. Thuslactic acid is its reduction product,

CH3·CO·COOH + 2 H → CH3·CH(OH)·COOH,

glyceraldehyde can readily beconverted into it by oxidation toglyceric acid followed by abstraction

of water (Erlenmeyer), [p110]

CH2(OH)·CH(OH)·CHO + O → CH2(OH)·CH(OH)·COOHCH2(OH)·CH(OH)·COOH − H2O → CH3·CO·COOH,

and finally methylglyoxalCH3·CO·CHO is its aldehyde.

2. The decomposition of pyruvicacid into acetaldehyde and carbondioxide has already been fullydiscussed (Chapter VI). Theuniversality of the enzyme carboxylasein yeasts and the rapidity of its actionon pyruvic acid form the strongest

evidence at present available in favourof the pyruvic acid theory. Given thepyruvic acid, there is no doubt thatyeast is provided with a mechanismcapable of decomposing it at the samerate as an equivalent amount of sugar.

3. The final step postulated by thepyruvic acid theory is the quantitativereduction to ethyl alcohol of theacetaldehyde formed from the pyruvicacid.

The idea that acetaldehyde is anintermediate product in the variousfermentations of sugar has frequentlybeen entertained [Magnus Levy, 1902;

Leathes, 1906; Buchner andMeisenheimer, 1908; Harden andNorris, D., 1912] although no verydefinite experimental foundation existsfor the belief. It is, however, a well-known fact that traces of acetaldehydeare invariably formed during alcoholicfermentation [see Ashdown and Hewitt,1910], and this is of course consistentwith the occurrence of acetaldehyde asan intermediate product. Importantevidence as to the specific capability ofyeast to reduce acetaldehyde to alcoholhas been obtained by several workers.Thus Kostytscheff [1912, 3;

Kostytscheff and Hübbenet, 1913]found that pressed yeast, dried yeastand zymin all reduced acetaldehyde toalcohol, 50 grams of yeast in 10 hoursproducing from 660 mg. of aldehyde265 mg. of alcohol in excess of theamount produced by autofermentationin absence of added aldehyde.Maceration extract was found to reduceboth in absence and in presence ofsugar, whereas Lebedeff and Griaznoff[1912] obtained no reduction inpresence of sugar, and observed thatthe power of reduction was lost by theextract on digestion, a circumstance

which suggests the co-operation of aco-enzyme in the process. Neuberg andKerb [1912, 4; 1913, 1] have also beenable to show by large scaleexperiments that alcohol is produced inconsiderable quantity by thefermentation of pyruvic acid by livingyeast in absence of sugar and that theyield is increased by the presence ofglycerol. When treated with 22 kilos,of yeast, 1 kilo, of pyruvic acid yielded241 grams of alcohol in excess of thatgiven by the yeast alone, whilst inpresence of glycerol the amount was360 grams, the amount theoretically

obtainable being 523 grams. Thefunction of the glycerol is notunderstood but is probably that oflessening the rate of destruction of theyeast enzymes. [p111]

That yeast possesses powerfulreducing properties has long beenknown and many investigations havebeen made as to the relation of theseproperties to the process of alcoholicfermentation. Thus Hahn (Buchner, E.and H., and Hahn, 1903, p. 343) foundthat the power of reducing methyleneblue was possessed both by yeast andzymin and on the whole ran parallel to

the fermenting power in the process ofalcoholic fermentation. Theintervention of a reducing enzyme wassuggested by Grüss [1904, 1908, 1, 2]and was supported by Palladin [1908].The latter observed that zymin whichreduces sodium selenite and methyleneblue in absence of sugar almost ceasesto do so in presence of a fermentablesugar, and concluded that the greatdiminution of reduction duringfermentation was due to the fact thatthe reducing enzyme was largelycombined with a different substratearising from the sugar, the reduction of

which was necessary for alcoholicfermentation. Grüss, however, foundthat with living yeast the reduction isgreatly increased in presence of afermentable sugar, while Harden andNorris, R. V. [1914] confirmed theobservation of Grüss, but found that thereducing power of zymin is notseriously affected by the presence of afermentable sugar in concentration lessthen 20 grams per 100 c.c., whilst itsfermenting power for glucose isinhibited by 1 per cent. sodiumselenite. Hence Palladin's conclusioncannot be regarded as proved.

Interesting attempts have been madeby Kostytscheff and later by Lvoff toobtain evidence of the participation ofa reductase in alcoholic fermentationby adding some substance which wouldbe capable either of taking up hydrogenand thus preventing the reduction ofthe acetaldehyde or of converting thealdehyde into some compound lessliable to reduction.

Kostytscheff [1912, 1; 1913, 1, 2;1914; Kostytscheff and Hübbenet,1913; Kostytscheff and Scheloumoff,1913; Kostytscheff and Brilliant, 1913]has examined the effect of the addition

of zinc chloride, chosen with the ideathat it might polymerise the aldehydeand thus remove it from the sphere ofaction. As pointed out by Neuberg andKerb [1912, 1] this action is not veryprobable, and it was subsequentlyfound [Kostytscheff and Scheloumoff,1913] that the effect of added zinc saltswas more probably specifically due tothe zinc ion. Fermentation of sugar bydried yeast still proceeds when 0·6gram of ZnCl2 is added to 10 grams ofthe yeast and 50 c.c. of water, whereasit ceases in the presence of 1·2 gram ofZnCl2. Even the addition of 0·075 gram

however greatly diminishes the rate offermentation and the total amount ofsugar decomposed. The mostnoteworthy effect is that the productionof acetaldehyde is increased both inautofermentation and [p112] in sugarfermentation. The course of thereaction is further modified in thesense that the percentage of sugar usedup which can be accounted for in theproducts decreases, in other words the"disappearing sugar" (p. 31) increases.In long continued fermentationsmoreover and particularly with highconcentrations of zinc chloride less

alcohol is produced than is equivalentto the carbon dioxide evolved. Theinterpretation of these results isdifficult. Kostytscheff takes them tomean (1) that the zinc salt modifies onestage of the reaction so that a higherconcentration of intermediate productsis obtained, and (2) that the carbondioxide and alcohol must be producedat different stages or their ratio, in theabsence of secondary changes, wouldbe unalterable.

Alternative interpretations are,however, by no means excluded. ThusNeuberg and Kerb [1912, 1; 1913, 2] do

not regard it as conclusively provedthat the aldehyde really arises from thesugar since they have observed itsproduction in maceration extract freefrom autofermentation. The methodused by Kostytscheff for the separationof alcohol and aldehyde (treatmentwith bisulphite) has also provedunsatisfactory in their hands and theresults obtained as to the reduction ofacetaldehyde by yeast, etc., are notaccepted. They also consider that inany case the small amounts produced(less than 0·2 per cent. of the sugarused) would not afford convincing

evidence that the aldehyde is anintermediate product, although it mustbe admitted that no large accumulationof an intermediate product could bereasonably expected. It may also bepointed out that the increase in"disappearing sugar" may be simplydue to the fact that in the controls thewhole of the sugar was fermented, sothat any polysaccharide formed at anearlier stage would have beenhydrolysed and fermented, whereas inthe presence of zinc chloride excess ofsugar was present throughout the wholeexperiment.

Lvoff [1913, 1, 2, 3] has madequantitative experiments on the effectof methylene blue both on the sugarfermentation and autofermentation ofdried yeast and maceration extract. Inpresence of sugar the methylene bluecauses a decrease in the extent offermentation, the difference during thetime required for reduction of themethylene blue being represented by anamount of glucose equimolecular to thelatter. In the absence of sugar on theother hand an excess of carbon dioxideequimolecular to the methylene blue isevolved but no corresponding increase

in the alcohol production occurs. Theeffect of methylene blue is evidentlycomplex and it is impossible at presentto say whether Lvoff's contention iscorrect that the methylene blue actually[p113] interferes with the fermentationby taking up hydrogen (2 atoms permolecule of glucose) destined for thesubsequent reduction of someintermediate product or whether theeffect is one of general depression ofthe fermenting power which would bepresumably proportional to theconcentration of methylene blue andinversely proportional to that of the

fermenting complex [see Harden andNorris, R. V., 1914]. In any case it willbe noticed that Lvoff s interpretation ofthe results is at variance with therequirements of Kostytscheff's theory(p. 109) according to which 4 atoms ofhydrogen should be given off by amolecule of glucose.

Kostytscheff [1913, 2; Kostytscheffand Scheloumoff, 1913] has alsoobserved a depression of the extent offermentation by methylene bluewithout any serious alteration in theratio of CO2 to alcohol, although anincrease occurs in the production of

acetaldehyde.On the whole it cannot be said that

the evidence gathered fromexperiments on the reduction ofacetaldehyde and methylene blue isvery convincing. All that is establishedbeyond doubt seems to be that yeastpossesses a reducing mechanism formany aldehydes [see also in thisconnection Lintner and Luers, 1913;Lintner and von Liebig, 1911; as wellas Neuberg and Steenbock, 1913, 1914]and colouring matters. This mechanismappears to be capable of activity in theabsence of sugar and it is to be

supposed that in accordance with theviews of Bach [1913] the necessaryhydrogen is derived from water andthat some acceptor for the oxygensimultaneously liberated is alsopresent. There seems however at themoment to be no sufficient reason tosuppose that this mode of reduction isin any way altered by the presence ofsugar and until the production ofintermediate products equivalent to theamount of substance reduced isactually demonstrated, the conclusionsof these workers may be regarded asnot fully justified.

Neuberg and Kerb [1913, 2]themselves tentatively propose acomplicated scheme possessing somenovel features according to whichmethylglyoxal is the starting-point forthe later stages of the change.

(a) A small portion of this isconverted by a reaction which may bevariously interpreted as a Cannizzarotransformation or a reductase reactioninto glycerol and pyruvic acid.

CH2:C(OH)·CHO + H2O

H2+ │ = O

CH2(OH)·CHOH·CH(glycerol

CH2:C(OH)·CHO CH2:C(OH)·COOH(Pyruvic acid

(b) The pyruvic acid is thendecomposed by carboxylase yieldingaldehyde and carbon dioxide (equation2, p. 109). [p114]

(c) The aldehyde and a molecule ofglyoxal then undergo a Cannizzaroreaction and yield alcohol and pyruvicacid,

CH3·CO·CHO O CH3·CO·COOH+ │ = +

CH3·CHO H2 CH3CH2(OH)

and the latter then undergoes reaction(b).

A small amount of glycerol is thusnecessarily formed, as is actually foundto be the case.

The experimental foundation forstages (a) and (c) will be awaited withgreat interest, as well as the proof thatmethylglyoxal is readily fermentable(see p. 104).

The Formic Acid Theory.

An interesting interpretation of thephenomena of fermentation wasattempted by Schade [1906] basedupon the conception that glucose underthe influence of catalytic agents readilydecomposes into acetaldehyde andformic acid. It was subsequently foundthat the experimental evidence uponwhich this conclusion was founded hadbeen wrongly interpreted [Buchner,Meisenheimer, and Schade, 1906;Schade, 1907], but Schade hassucceeded in devising an interesting

series of reactions by means of whichalcohol and carbon dioxide can beobtained from sugar by the successiveaction of various catalysts. Thefollowing are the stages of this series:(1) Glucose, fructose, and mannose areconverted by alkalis into lactic acidalong with other products. (2) Lacticacid when heated with dilute sulphuricacid yields a mixture of acetaldehydeand formic acid:—

CH3·CH(OH)·COOH = CH3·CHO + H·COOH.

(3) It has long been known that formic

acid is catalysed by metallic rhodiumat the ordinary temperature intohydrogen and carbon dioxide, andSchade has found that when a mixtureof acetaldehyde and formic acid issubmitted to the action of rhodium theacetaldehyde is reduced to alcohol atthe expense of the hydrogen and thecarbon dioxide is evolved:—

CH3·CHO + H·COOH = CH3·CH2(OH) + CO2.

Schade suggests [1908] that thefermentation of sugar may proceed bya similar series of reactions catalysed

by enzymes, the acetaldehyde andformic acid being derived not from therelatively stable lactic acid but moreprobably from a labile substancecapable of undergoing change eitherinto lactic acid or into aldehyde andformic acid.

It will be noticed that this theoryresembles the pyruvic acid [p115] theoryin postulating the immediate formationof acetaldehyde but differs from it bysupposing that the reduction is effectedat the expense of formic acid producedat the same time.

The acetaldehyde question has

already been discussed. In view of thefact that formic acid is a regularproduct of the action of many bacteriaon glucose [see Harden, 1901],Schade's theory of alcoholicfermentation may be said to be apossible interpretation of the facts.Formic acid is known to be present insmall amounts in fermented sugarsolutions and the actual behaviour ofyeast towards this substance has beeninvestigated in some detail by Franzenand Steppuhn [1911; 1912, 1, 2], whohave obtained results stronglyreminiscent of those obtained with

lactic acid by Buchner andMeisenheimer (p. 102). Many yeastswhen grown in presence of sodiumformate decompose a certainproportion of it, whereas in absence offormate they actually produce a smallamount of formic acid—the absolutequantities being usually of the order of0·0005 gram molecule (0·023 gram)per 100 c.c. of medium in 4 to 5 days.Only in the case of S. validus did theconsumption of formic acid in 5 daysreach 0·0017 gram molecule (0·08gram). Somewhat similar but rathersmaller results were given by yeast-

juice, a small consumption of formicacid being usually observed. Thepossibility thus exists that formic acidmay be an intermediate product ofalcoholic fermentation and Franzenargues strongly in favour of this view.

Direct experiment, on the otherhand, shows that yeast-juice cannotferment a mixture of acetaldehyde andformic acid, even when these aregradually produced in molecularproportions in the liquid by the slowhydrolysis of a compound of the two,ethylideneoxyformate, OHC·O·CH(CH3)·O·CH(CH3)·O·CHO,

this method being adopted to avoid theinhibiting effect of free acetaldehydeand formic acid [Buchner andMeisenheimer, 1910]. Nor is thereduction of acetaldehyde assisted bythe presence of formate [Neuberg andKerb, 1912, 4; Kostytscheff andHübbenet, 1912].

A modified form of Schade's theoryhas been suggested by Ashdown andHewitt [1910], who have found thatwhen brewer's yeast is cultivated inpresence of sodium formate the yieldof aldehyde, as a rule, becomes less.They regard the aldehyde as derived

from alanine, CH3·CH(NH2)·COOH,one of the amino-acids formed fromthe proteins by hydrolysis, which isknown to be attacked by yeast in thecharacteristic manner (p. 87), formingalcohol, carbon dioxide, and ammonia.Fermentation is supposed to proceed insuch a way that the sugar is firstdecomposed into two smallermolecules, C3H6O3 [p116] (equation i),and that these react with formamide toproduce alanine and formic acid (ii).The alanine then enters into reactionwith formic acid, producing alcohol,carbon dioxide, and formamide (iii):—

(i) C6H12O6 = 2 C3H6O3.(ii) C3H6O3 + H·CO·NH2 = CH3·CH(NH2)·COOH + H·COOH.(iii) CH3·CH(NH2)·COOH + H·COOH = CH3·CH2·OH + CO2 + H·CO·NH2.

According to this scheme all the sugarfermented passes through the form ofalanine, and the formic acid acts alongwith the enzyme as catalyst, passinginto formamide in reaction (iii) andbeing regenerated in (ii). The alanine isin the first place derived from thehydrolysis of proteins, or possibly by

the reaction of the C3H6O3 group withone of the higher amino-acids:—

C3H6O3 + CnH(2n+1)·CH(NH2)·COOH =CnH(2n+1)·CH2·OH + CO2 + CH(NH2)·COOH.

There is as little positive evidencefor this course of events as for thatpostulated by Schade, and the theorysuffers from the additional disabilitythat the chemical reactions involvedhave not been realised in thelaboratory. Direct experiments withyeast-juice, moreover, show that a

mixture of alanine with formic acid ora formate is not fermented, whilstneither the added mixture norformamide seriously effects the actionof the juice on glucose.

Other Theories.

Among other suggestions may bementioned that of Kohl [1909] whoasserts that sodium lactate is readilyfermented, whilst Kusseroff [1910]holds the view that the glucose is firstreduced to sorbitol and the latterfermented, in spite of the fact that

sorbitol itself in the free state is notfermented by yeast.

The rapid appearance anddisappearance of glycogen in the yeastcell at various stages of fermentation[see Pavy and Bywaters, 1907; Wagerand Peniston, 1910] has led to thesuggestion [Grüss, 1904; Kohl, 1907]that this substance is of greatimportance in fermentation, andrepresents a stage through which all thesugar must pass before beingfermented. The fact that the formationof glycogen has been observed inyeast-juice by Cremer [1899], and that

complex carbohydrates are alsoundoubtedly formed (p. 31), areconsistent with this theory. The lowrate of autofermentation of livingyeast, which is only a few per cent. ofthe rate of sugar fermentation, rendersthis supposition very improbable(Slator), as does the fact that thefermentation of glycogen by yeast-juice is usually slower than that ofglucose [see also Euler, 1914].

An entirely different explanation ofthe chemical changes attendant onalcoholic fermentation has beensuggested by Löb [1906; [p117] 1908, 1,

2; 1909, 1, 2, 3, 4; 1910; Löb andPulvermacher, 1909], founded on theidea that the various decompositions ofthe sugar molecule both by chemicaland biological agents are to beexplained by a reversal of the synthesisof sugar from formaldehyde. As thesugar molecule can be built up by thecondensation of formaldehyde, so ittends to break down again into thissubstance, and the products observed inany particular case are formed eitherby partial depolymerisation in thissense or by partial re-synthesisfollowing on depolymerisation.

Löb has adduced many striking factsin favour of this view, and has shownthat very dilute alkalis produce nolactic acid but formaldehyde and apentose as primary products. Thesesubstances represent the first stage ofdepolymerisation and are also formedby the electrolysis of glucose.

Löb has himself been unable todetect definite intermediate products offermentation by adding reagents, suchas aniline, ammonia, andphloroglucinol, which would combinewith such substances and prevent theirfurther decomposition [1906].

The occurrence of traces offormaldehyde as a product of alcoholicfermentation by yeast-juice [Lebedeff,1908] is at least consistent with thistheory, but no decisive evidence has sofar been obtained either for or againstit.

In all the foregoing attempts toindicate the probable stages in theproduction of alcohol and carbondioxide from sugar, a single moleculeof the sugar forms the starting-point.The facts recounted in Chapter III as tothe function of phosphates in alcoholicfermentation, 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 probablethat two molecules of the sugar areconcerned. The most reasonableinterpretation of this equation appearsto be that in the presence of phosphateand of the complicated machinery ofenzyme and co-enzyme two moleculesof the hexose, or possibly of the enolicform, are each decomposed primarilyinto two groups.

Of the four groups thus produced,two go to form alcohol and carbondioxide and the other two aresynthesised to a new chain of sixcarbon atoms, which forms thecarbohydrate residue of thehexosephosphate. The introduction ofthe phosphoric acid groups maypossibly occur before the rupture of theoriginal molecules, and may even bethe determining factor of this rupture,or again this introduction may takeplace during or after the formation ofthe new carbon [p118] chain. Sufficientinformation is not yet available for the

exact formulation of a scheme for thisreaction. Such a scheme, it may benoted, would not necessarily beinconsistent with the views of Wohland of Buchner as to the way in whichthe carbon chain of a hexose is brokenin the process of fermentation, butwould interpret differently thesubsequent changes which areundergone by the simpler groups whichare the result of this rupture. Thereaction might thus proceed withoutthe formation of definite intermediateproducts, whilst opportunity would beafforded for the production of a small

quantity of by-products such asformaldehyde, glycerol, lactic acid,acetic acid, etc., by secondaryreactions.

A symmetrical scheme can readilybe constructed for such a change, butmuch further information is requiredbefore any decisive conclusion can bedrawn as to the precise course of thereaction which actually occurs inalcoholic fermentation.

CHAPTER IX.THE MECHANISM OF

FERMENTATION.

[p119]

The analysis of the process of alcoholicfermentation by yeast-juice and otherpreparations from yeast which has beencarried out in the preceding chaptershas shown that the phenomenon is oneof a very complex character. Theprincipal substances directly concernedin the change appear to be the enzyme

and co-enzyme of the juice, a secondenzyme, hexosephosphatase, and, inaddition, sugar, phosphate, and thehexosephosphate formed from these.During autofermentation two otherfactors are involved, the complexcarbohydrates of the juice, includingglycogen and dextrins, and the diastaticferment by which these are convertedinto fermentable sugars. It is alsopossible that the supply of freephosphate is partially provided by theaction of proteoclastic ferments onphosphoproteins. Under specialcircumstances the rate at which

fermentation proceeds may becontrolled by the available amount ofany one of these numerous substances.

When the juice from well-washedyeast is incubated, the phenomenon ofautofermentation is observed. The juicecontains an abundant supply ofenzyme, co-enzyme, and phosphate orhexosephosphate, and in this case thecontrolling factor is usually the supplyof sugar, which is conditioned by theconcentration of the diastatic enzymeor of the complex carbohydrates as thecase may be. When this is the case themeasured rate of fermentation is the

rate at which sugar is being producedin the juice, this being the slowest ofthe various reactions which areproceeding under these circumstances.If sugar be now added, an entirelydifferent state of affairs is set up. Assoon as any accumulated phosphate hasbeen converted into hexosephosphate,the normal rate of fermentation whichis usually higher than that ofautofermentation is attained, and,provided that excess of sugar bepresent, fermentation continues for aconsiderable period at a slowlydiminishing rate and finally ceases.

During the first part of thisfermentation the rate is controlledentirely by the supply of freephosphate, and this depends mainly onthe concentration of thehexosephosphatase and of thehexosephosphate, and only in asecondary degree on the decomposition[p120] of other phosphorus compoundsby other enzymes and on theconcentration of the sugar. The amountof hexosephosphate in yeast-juice isusually such that an increase in itsconcentration does not greatly affectthe rate of fermentation, and hence the

measured rate during this periodrepresents the rate at whichhexosephosphate is being decomposed,and this in its turn depends on theconcentration of hexosephosphatase,which is therefore the controllingfactor. As fermentation proceeds, theconcentration of both enzyme and co-enzyme steadily diminishes, as alreadyexplained, probably owing to the actionof other enzymes, so that at anadvanced stage of the fermentation, thecontrolling factor may be theconcentration of either of these, or theproduct of the two concentrations (see

p. 122). The hexosephosphataseappears invariably to outlast theenzyme and co-enzyme. The conditionat any moment could be determinedexperimentally if it were possible toadd enzyme, co-enzyme andhexosephosphatase at will and soascertain which of these produced anacceleration of the rate.

Unfortunately this can at present beonly very imperfectly accomplished,owing to the impossibility ofseparating these substances from eachother and from accompanying matterwhich interferes with the interpretation

of the result.A third condition can also be

established by adding to the fermentingmixture of the juice and sugar asolution of phosphate. The supply ofphosphate is now almost independentof the action of the hexosephosphatase,and the measured rate represents therate at which reaction (1), p. 51, canoccur between sugar and phosphate inthe presence of the fermenting complexconsisting of enzyme and co-enzyme.This change is controlled, so long assugar and phosphate are present in theproper amounts, by the concentration

of the fermenting complex or possiblyof either the enzyme or the co-enzyme.If only a single addition of a smallquantity of phosphate be made, the ratefalls as soon as the whole of this hasbeen converted into hexosephosphateand the reaction then passes into thestage just considered, in which the rateis controlled by the production of freephosphate.

Although these varying reactionshave not yet been exhaustively studiedfrom the kinetic point of view, owingto the experimental difficulties towhich allusion has already been made,

investigations have nevertheless beencarried out on the effect of thevariation of concentration of yeast-juice and zymin as a whole, as well asof the carbohydrate. Herzog [1902,1904] has made experiments of thiskind with zymin, and Euler [1905] withyeast-juice, whilst many of the results[p121] obtained by Buchner and byHarden and Young are also available.

The actual observations made bythese authors show that the initialvelocity of fermentation is almostindependent of the concentration ofsugar within certain limits, but

decreases slowly as the concentrationincreases. When the velocity constantis calculated on the assumption that thereaction is monomolecular [seeBayliss, 1914, Chap. VI], approximateconstancy is found for the first periodof the fermentation. This method ofdealing with the results is, however, aspointed out by Slator, misleading, theapparent agreement with the law ofmonomolecular reactions beingprobably due to the gradual destructionof the fermenting complex.

Experiments with low concentrationsof sugar are difficult to interpret, the

influence of the hydrolysis of glycogenand of dextrins on the one hand, andthe synthesis of sugar to more complexcarbohydrates on the other (p. 31),having a relatively great effect on theconcentration of the sugar.Unpublished experiments (Harden andYoung) indicate, however, that thevelocity of fermentation remainsapproximately constant, until a certainvery low limit of sugar concentration isreached, and then falls rapidly. The fallin rate, however, only continues over asmall interval of concentration, afterwhich the velocity again becomes

approximately constant and equal tothe rate of autofermentation. Duringthis last phase, as already indicated, thevelocity is generally controlled by therate of production of sugar and nolonger by that of phosphate, thissubstance being now present in excess.In other words, the rate of fermentationof sugar by yeast-juice and zymin isnot proportional to the concentration ofthe sugar present as required by the lawof mass, but, after a certain low limitof sugar concentration, is independentof this and is actually slightlydecreased 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 actionof invertase on cane sugar and areprobably to be explained in the mannersuggested by the latter. According tothis investigator, the enzyme uniteswith the fermentable material, or as itis now termed, the substrate orzymolyte, forming a compound whichonly slowly decomposes so that itremains in existence for a perceptibleinterval of time. The rate offermentation depends on the rate of

decomposition of this compound andhence varies with its concentration.This conception leads to the result thatthe rate of fermentation will increasewith the concentration of the substrateup to a certain limit and will thenremain [p122] constant, unless interferedwith by secondary actions. This limitof concentration is that at which thereis just sufficient of the material inquestion present to combine withpractically the whole of the enzyme, sothat no further increase in its amountcan cause a corresponding increase inthe quantity of its compound with the

enzyme or in the rate of fermentationwhich depends on the concentration ofthat compound.

The curve relating the rate of actionof such an enzyme with theconcentration of the zymolyte thereforeconsists of two portions, one in whichthe rate at any moment is proportionalto the concentration of the zymolyte,according to the well-known law of theaction of mass, and a second in whichthe rate at any moment is almostindependent of that concentration,approximately equal amounts beingdecomposed in equal times whatever

the concentration of the substrate.The results of the experiments with

yeast-juice therefore indicate that whatis being measured is a typical enzymeaction, but afford no information as towhich of the many possible actions isthe controlling one, a fact which mustbe ascertained for each particular casein the manner indicated above.

Clowes [1909], using washed zyminfree from fermenting power and addingvarious volumes of boiled yeastextract, found that the velocity ofreaction was proportional to theproduct of the concentrations of zymin

and yeast extract up to a certainoptimum concentration. He interpretsthese concentrations as representingthe concentrations of zymase and co-enzyme, but they also represent theconcentrations of hexosephosphatase(present in the zymin) and phosphate(present in the yeast extract), so that atleast four factors were being alteredinstead of only two.

It has already been mentioned thatEuler and Kullberg [1911, 3] found theconversion of phosphate intohexosephosphate in presence of excessof 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 theconcentration of the juice. Herzogfound that when the relation betweenconcentration of enzyme and thevelocity constant of the reaction isexpressed by the formula K1/K2 = (C1/C2)n where K1 and K2 arethe velocity constants correspondingwith the enzyme concentrations C1 andC2, the value for n is 2 for zymin,whilst Euler working with yeast-juiceobtained 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 foundby Slator for yeast-cells (p. 129).

When we endeavour to apply theresults of the investigations of thefermentation of sugar by yeast-juice,zymin, etc., to the process which goeson in the living cell, considerabledifficulties present themselves. Ascheme of fermentation in the livingcell can, however, easily be imagined,which is in harmony with these results.

According to the most simple form ofthis ideal scheme, the sugar which hasdiffused into the cell unites with thefermenting complex and undergoes thecharacteristic reaction with phosphate,already present in the cell, yieldingcarbon dioxide, alcohol, andhexosephosphate. The latter is thendecomposed, just as it is in yeast-juice,but more rapidly, and the liberatedphosphate again enters into reaction,partly with the sugar formed from thehexosephosphate and partly with freshsugar supplied from outside the cell.The main difference between

fermentation by yeast-juice and by theliving cell would then consist in therate of decomposition of thehexosephosphate, for it has been shownthat yeast-juice in presence ofsufficient phosphate can ferment sugarat a rate of the same order ofmagnitude (from 30 to 50 per cent.) asthat attained by living yeast.

The difference between the twotherefore would appear to lie not somuch in their content of fermentingcomplex as in their very differentcapacity for liberating phosphate fromhexosephosphate and thus supplying

the necessary conditions forfermentation.

A simple calculation based on thephosphorus content of living yeast[Buchner and Haehn, 1910, 2] showsthat the whole of this phosphate mustpass through the stage ofhexosephosphate every five or sixminutes in order to maintain thenormal rate of fermentation, whereas inan average sample of yeast-juice thecycle, calculated in the same way,would last nearly two hours.

Wherein this difference resides is adifficult question, which cannot at

present be answered with certainty.In the first place it must be

remembered that a very greatacceleration of the action of thehexosephosphatase is produced byarsenates (p. 79), and this suggests thepossibility that some substancepossessing a similar accelerating poweris present in the yeast-cell and is lost ordestroyed in the various processesinvolved in rendering the yeastsusceptible to phosphate. The greatvariety of these processes—extractionof 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 beendetected. [p124]

A comparison of living yeast, zymin,and yeast-juice shows that these aresituated on an ascending scale withrespect to their response to phosphate.Taking fructose as the substrate in eachcase, yeast does not respond tophosphate at all (Slator), the rate offermentation by zymin isapproximately doubled (p. 46), and thatby yeast-juice increased ten to fortytimes, whilst the maximum rates are in

each case of the same order ofmagnitude. Euler and Kullberg,however, have observed an accelerationof about 25 per cent. in the rate offermentation of yeast in presence of a 2per cent. solution of monosodiumphosphate, NaH2PO4 [1911, 1, 2].

The high rate of fermentation byliving yeast and its lack of response tophosphate may possibly be explainedby supposing that the balance ofenzymes in the living cell is such thatthe supply of phosphate is maintainedat the optimum, and the rate offermentation cannot therefore be

increased by a further supply.A further difference lies in the fact

that yeast-juice and zymin respond tophosphate more strongly in presence offructose than of glucose, whereas yeastferments both sugars at the same rate(p. 131), and this property has beenshown to be connected with thespecific relations of fructose to thefermenting complex. It seems possiblethat these differences are associatedwith the gradual passage from thecomplete living cell of yeast, throughthe dead and partially disorganised cellof zymin to yeast-juice in which the

last trace of cellular organisation hasdisappeared and the contents of the cellare uniformly diffused throughout theliquid. Living yeast is, moreover, notonly unaffected by phosphate but onlydecomposes hexosephosphateextremely slowly (Iwanoff).

Some light is thrown on theseinteresting problems by the effect ofantiseptics on fermentation by yeast-cells and by yeast-juice. The action oftoluene has hitherto been mostcompletely studied, and this substanceis an extremely suitable one for thepurpose since it has practically no

action whatever on fermentation byyeast-juice. The experiments ofBuchner have, in fact, shown that thenormal rate of fermentation and thetotal fermentation produced, are almostunaffected by the presence of tolueneeven in the proportion of 1 c.c. to 20c.c. of yeast-juice. What then is theeffect of toluene on the living yeast-cell? When toluene in large excess isagitated with a fermenting mixture ofyeast and sugar, the rate offermentation falls rapidly at first andthen more slowly until a relativelyconstant rate is attained which

gradually decreases in a similarmanner to the rate of fermentation byyeast-juice. Thus at air temperature(16°) 10 grams of [p125] yeastsuspended in 50 c.c. of 6 per cent.glucose solution gave the followingresults when agitated with toluene:—

Timeafter

Additionof

Toluene,Minutes

C.c. ofCO2 perMinute.

Time. C.c. perMinute.

0 4·6 6 1·6 1 4 8 1·2 2 3·3 12 0·853 2·6 24 0·8 4 2 32 0·5 5 1·8 constant

Simultaneously with this, the yeastacquires the property of decomposingand fermenting hexosephosphate and ofresponding to the addition ofphosphate. This last property is onlyacquired to a small degree in this waybut it becomes much more strongly

developed if the pressed yeast bewashed with toluene on the filter pump.Thus 10 grams of yeast after thistreatment fermented fructose at 1·2 c.c.per three minutes; after the addition ofphosphate (5 c.c. of 0·6 molarphosphate) the rate rose to 6·9 and thengradually fell in the typical manner[Harden, 1910; see also Euler andJohansson, 1912, 3].

The current explanation of the greatdecrease in rate of fermentation whichattends the action of toluene and otherantiseptics on living yeast, and alsofollows upon the disintegration of the

cell, appears to be that in living yeastthe high rate of fermentation ismaintained by the continuedproduction of relatively large freshsupplies of fermenting complex, andthat when the power of producing thiscatalytic agent is destroyed by thepoison, the rate of fermentation falls toa low value, corresponding to the storeof zymase still present in the cell (cf.Buchner, E. and H., and Hahn, 1903,pp. 176, 180).

This explanation implies that therate of fermentation after the action ofthe toluene represents the amount of

fermenting complex present, asupposition which has been shown (p.53) to be highly improbable. It furthernecessitates, as also pointed outindependently by Euler and Ugglas[1911], a rapid destruction of thefermenting complex both in the processof fermentation and by the action of theantiseptic, as otherwise the store ofzymase remaining in the dead cellwould be practically the same as thatcontained in the living cell at themoment when it was subjected to theantiseptic, and this store wouldtherefore suffice to carry out

fermentation at the same rate in thedead as in the living cell. No such rapiddestruction, however, occurs in yeast-juice, as judged by the rate offermentation, which falls off [p126]

slowly and to about the same extent inthe presence or absence of toluene.Moreover, as shown above, it is highlyprobable that the actual amount offermenting complex in yeast-juice is alarge fraction of that present at anymoment in the cell, and is capableunder suitable conditions of producingfermentation at a rate comparable withthat of the living cell.

This last criticism also applies to theview expressed by Euler [Euler andUgglas, 1911; Euler and Kullberg,1911, 1, 2] that in the living cell thezymase is partly free and partlycombined with the protoplasm; whenthe vital activity of the cell isinterfered with, the combined portionof the zymase is thrown out of actionand only that which was free remainsactive.

The suggestion made by Rubner[1913] that the action of yeast on sugaris in reality chiefly a vital act, but thata small proportion of the change is due

to enzyme action, is similar in itsconsequences to that of Euler and maybe met by the same arguments.Buchner and Skraup [1914] havemoreover shown that the effects ofsodium chloride and toluene on thefermenting power of yeast which wereobserved by Rubner, can be explainedin other ways.

Some other explanation musttherefore be sought for thisphenomenon. Great significance mustbe attached in this connection to therelation noted above between thedegree of disintegration and

disorganisation of the cell and the fallin the normal rate of fermentation. Itseems not impossible that fermentationmay be associated in the living cellwith some special structure, or carriedon in some special portion of the cell,perhaps the nuclear vacuole describedby Janssens and Leblanc [1898], Wager[1898, 1911; Wager and Peniston,1910] and others which undergoesremarkable changes both duringfermentation and autofermentation[Harden and Rowland, 1901]. Thedisorganisation of the cell might leadto many modifications of the

conditions, among others to thedilution of the various catalytic agentsby diffusion throughout the wholevolume of the cell. As a matter ofobservation the dilution of yeast-juiceleads to a considerable diminution ofthe rate of fermentation of sugar, and itis possible that this is one of the chieffactors concerned. That phenomena ofthis kind may be involved is shown bythe remarkable effect of toluene on theautofermentation of yeast. Whereas thefermentation of sugar is greatlydiminished by the action of toluene, therate of autofermentation, which is

carried on at the expense of theglycogen of the cell, is greatlyincreased. In a typical case, forexample, the autofermentation of 10grams of yeast suspended in 20 c.c. ofwater amounted to 28 c.c. in 4·8 hours[p127] at 25°, whereas the same amountof yeast in presence of 2 c.c. of toluenegave 97·6 c.c. in the same time.

Many salts produce a similar effecton English top yeasts (in which theautofermentation is large) [Harden andPaine, 1912], whereas Neuberg andKarczag in Berlin [1911, 2] wereunable to observe this phenomenon.

A necessary preliminary of thefermentation of glycogen is itsconversion by a diastatic enzyme into afermentable sugar, and it is probablethat the effect of the disorganisation ofthe cell by toluene is that this enzymefinds more ready access to theglycogen, which is stored in the plasmaof the cell. No such acceleration ofautofermentation is effected by theaddition of toluene to yeast-juice, andhence the result is not due to anacceleration of the action of thediastatic enzyme on the glycogen.

This effect of toluene is similar in

character to the action of anæstheticson the leaves of many plants containingglucosides and enzymes, whereby animmediate decomposition of theglucoside is initiated [see H. E. and E.F. Armstrong, 1910].

Although as indicated above Euler'stheory cannot apply to zymase itself, ifapplied to the hexosephosphatase itwould afford a consistent explanationof the facts. According to this modifiedview it would be thehexosephosphatase of yeast whichexisted largely in the combined form,so that in extracts, in dried yeast and in

presence of toluene only the smallfraction which was free would remainactive. The zymase on the other handwould have to be regarded as existingto a large extent in the free state so thatit would pass into extractscomparatively unimpaired in amountand capable under proper conditions(i.e. when supplied with sufficientphosphate) of bringing about a veryvigorous fermentation. The theory ofcombined and free enzymes isundoubtedly of considerable value,although it cannot be considered asfully established.

Fermentation by LivingYeast.

Much important information as tothe nature of the processes involved infermentation has been acquired by thedirect experimental study of the actionof living yeast on different sugars.

This phenomenon has formed thesubject of several investigations fromthe kinetic point of view, and itsgeneral features may now be regardedas well established.

The difficulty, which must as far aspossible be avoided in quantitative

experiments of this sort with livingyeast, is the alteration [p128] in theamount or properties of the yeast, dueto growth or to some change in thecells. This has been obviated in thework of Slator [1906] by determiningin every case the initial rate offermentation, so that the process onlycontinues for a very short period,during which any change in the amountor constitution of the yeast isnegligible. The method has the furtheradvantage that interference of theproducts of the reaction is to a largeextent avoided. The pressure apparatus

already described (p. 29) was employedby Slator, the rate of production ofcarbon dioxide being measured by theincrease of pressure in theexperimental vessel.

Influence of Concentrationof Dextrose on the Rate of

Fermentation.

With regard to this important factorit is found that the action of livingyeast follows the same law as that ofmost enzymes (p. 121); within certainwide limits the rate of fermentation is

almost independent of theconcentration of the sugar. Thisconclusion has been drawn by manyprevious investigators from theirexperiments [Dumas, 1874; Tammann,1889; Adrian Brown, 1892; O'Sullivan,1898, 1899] and is implicitly containedin the results of Aberson [1903],although he himself regarded thereaction as monomolecular.

FIG. 8.

Slator, working with a suspension often to twelve yeast-cells per 1/4000cubic millimetre at 30°, obtained theresults which are embodied in thecurve (Fig. 8).

This shows that, for the amount ofyeast in question, the rate of

fermentation is almost constant forconcentrations of glucose between[p129] 1 and 10 grams per 100 c.c., butgradually decreases as theconcentration increases. Below 1 gramper 100 c.c. the rate decreases veryrapidly with the concentration.

It follows from this, in the light ofwhat has already been said (p. 121),that the action of living yeast on sugarfollows the same course as a typicalenzyme reaction, although in this case,as in that of yeast-juice, no informationis given as to the exact nature of thisreaction.

Influence of theConcentration of Yeast.

It appears to be well established that,when changes in the quantity andconstitution of the yeast employed areeliminated, the rate of fermentation isexactly proportional to the number ofthe yeast-cells present (Aberson,Slator). This result might beanticipated, as pointed out by Slator,from the fact that the fermentationtakes place within the cell, each cellacting as an independent individual.

The diffusion of sugar into the yeast-

cell which necessarily precedes the actof fermentation has been shown bySlator and Sand [1910] to occur at sucha rate that the supply of sugar is alwaysin excess of the amount which can befermented by the cell.

Temperature Coefficient ofAlcoholic Fermentation by

Yeast.

The temperature coefficient offermentation by living yeast has beencarefully determined by Slator bymeasurements of the initial rates at a

series of temperatures from 5° to 40°C. The coefficient is found to be of thesame order as that for many chemicalreactions, but to vary considerably withthe temperature, a rise in temperaturecorresponding with a diminution in thecoefficient. The following values wereobtained for glucose; they areindependent of the concentration ofyeast and glucose, the class of yeast,and presence or absence of nutrientsalts, and remain the same wheninhibiting agents are present. Almostprecisely the same ratios are obtainedfor fructose and mannose:—

t. V(t+5)/Vt. V(t+10)/Vt.5 2·65 5·6 10 2·11 3·8 15 1·80 2·8 20 1·57 2·2525 1·43 1·9530 1·35 1·6 35 1·20

Aberson's result, K(t+10)/Kt = 2·72,which represents the mean coefficientfor 10° between 12° and 33°, agreeswell with this. [p130]

Action of AcceleratingAgents on Living Yeast.

Slator [1908, 1] was unable to find

any agent which greatly accelerated therate of fermentation of living yeast.Small concentrations of variousinhibiting agents which are oftensupposed to act in this way were quiteineffective, and phosphates, whichproduce such a striking change inyeast-juice, were almost without action(cp. p. 124).

Euler and Bäckström [1912],however, have made the importantobservation that sodiumhexosephosphate causes a considerableacceleration although it is itself neitherfermented nor hydrolysed under these

conditions. The extent of this is evidentfrom the following numbers:—

20 c.c. of 20 per cent.glucose solution.

0·25 g. yeast [Yeast H of St.Erik's brewery].

Withoutaddition.

+ 0·5 g. Nahexosephosphate.

Time.Min. CO2.

Time.Min. CO2.

46 10·5 37 8 76 17·5 73 19 197 45 188 52·5347 74·5 321 123 488 95 450 193·5

The observation has been confirmedwith English top yeast (Harden andYoung, unpublished experiments), but

no explanation of the phenomenon is atpresent forthcoming.

Euler has also found [Euler andCassel, 1913; Euler and Berggren,1912] that yeast extract, sodiumnucleinate and ammonium formate alsoincrease the rate of fermentation ofglucose by yeast, but these results havebeen criticised by Harden and Young[1913] on the ground that thepossibility of growth of the yeastduring the experiment has not beenexcluded.

Fermentation of Different

Sugars by Yeast.

Many valuable ideas as to the natureof fermentation have been obtained bya consideration of the phenomenapresented by the action of yeast on thedifferent hexoses. Of these onlyglucose, fructose, mannose, andgalactose are susceptible of alcoholicfermentation by yeast, thestereoisomeric hexoses prepared in thelaboratory being unfermentable, as arealso the pentoses, tetroses, and thealcohols corresponding to all thesugars. The yeast-cell is therefore

much more limited in its power ofproducing fermentation than such anorganism as, for example, Bacillus colicommunis, which attacks substances as[p131] diverse as arabinose, glucose,glycerol and mannitol, and yields withall of them products of the samechemical character, although in varyingproportions.

A careful examination of a numberof different genera and species of theSaccharomycetaceæ and alliedorganisms by E. F. Armstrong [1905]has shown that all yeasts whichferment glucose also ferment fructose

and mannose. Armstrong grew hisyeasts in a nutrient solution containingthe sugar to be investigated, and hisexperiments are open to the criticismthat the organisms were herebyafforded an opportunity for becomingacclimatised to the sugar. His results,therefore, only demonstrate the factthat the organisms in question whencultivated in presence of the sugarsexamined brought about theirfermentation, and do not exclude thepossibility that the same organismwhen grown in presence of a differentsugar might not be capable of

fermenting the one to which it had inthe other type of experiment becomeacclimatised.

This has actually been shown to bethe case for galactose by Slator [1908,1], and it is possible that thiscircumstance explains the negativeresults obtained by Lindner [1905] withS. exiguus and SchizosaccharomycesPombe upon mannose, a sugar which,according to Armstrong, is fermentedby both these organisms.

The same problem has been attackedquantitatively by Slator, who hasshown that living yeast of various

species and genera ferments glucoseand fructose at approximately the samerate. Moreover, when the yeast is actedupon by various inhibiting agents, suchas heat, iodine, alcohol, or alkalis, thecrippled yeast also ferments glucoseand fructose at the same rate.

With mannose the relations aresomewhat different. The relative rateof fermentation of mannose andglucose by yeast is dependent on thevariety of the yeast and the treatmentwhich it has received. Fresh samples ofyeast ferment mannose more quicklythan glucose, but by older samples the

glucose is the more rapidlydecomposed. This is especially the casewith yeast, the activity of which hasbeen partly destroyed by heat, therelative fermenting power to mannosebeing sometimes reduced by thistreatment from 120 per cent. of that ofglucose to only 12 per cent. (Slator).

A further difference consists in thefact that with certain yeasts the rate offermentation of glucose is somewhatincreased by monosodium phosphatewhilst that of mannose is unaffected[Euler and Lundeqvist, 1911].

Mixtures of glucose and fructose are

fermented by yeast at the [p132] samerate as either the glucose or thefructose contained in the mixturewould be alone. When, however,mannose and glucose are fermentedsimultaneously interference betweenthe reactions takes place, and this isespecially evident when the yeast hascomparatively little action on mannose.The following are the results obtainedby Slator:—

Yeast.

Relative Rates.

2·5 percent.

Glucose.

2·5 percent.

Mannose.

S.Thermantitonum 100

Brewery yeast,53 per cent.activitydestroyed byheat 100

Brewery yeast,60 per cent.activitydestroyed byheat 100

The case of galactose merits specialattention. Previous investigations [see

Lippmann, 1904, p. 734] have shownthat the fermentation of galactose byyeast differs greatly from that of theother hexoses. The subject has been re-investigated by E. F. Armstrong[1905], and by Slator [1908, 1].Armstrong carried out his experimentsin the manner already described (p.131), and found that some yeasts had,and others had not, the power offermenting galactose, although all werecapable of fermenting glucose,fructose, and mannose.

Slator made quantitativeexperiments on the same subject. He

was able to confirm the statementwhich had previously been made, thatcertain yeasts which have the propertyof fermenting galactose possess it onlyafter the yeast has become acclimatisedby culture in presence of the sugar.This was shown for brewery yeast andfor the species mentioned below. Thisphenomenon is one of great interestand is strictly analogous to theadaptation of bacteria which has nowbeen quite conclusively established[Neisser, 1906].

Yeast.Mode ofCulture.Grown in:

S. Carlsbergensis wort

"hydrolysedlactose

S. Cerevisiæ wort

"hydrolysedlactose

S. Thermantitonum. wort

"hydrolysedlactose

S. Ludwigii wort

"hydrolysedlactose

[p133]

It will be seen that in one case therate of fermentation of galactose was

considerably greater than that ofglucose. S. Ludwigii did not respond tothe cultivation in hydrolysed lactose,but, as Slator points out, it is quitepossible that repeated cultivation inthis medium might effect the change,and this would be strictly analogous tothe results obtained with bacteria.Slator's results have been confirmed byHarden and Norris, R. V. [1910], andby Euler and Johansson [1912, 2] whohave made an exceedingly interestingstudy of the progress of the adaptation.As in the case of mannose the rates offermentation of glucose and galactose

are differently affected by agents suchas heat and alcohol; moreover, the rateof fermentation of mixtures of dextroseand galactose is in no case either thesum or the mean of the rates obtainedwith the separate sugars. Thetemperature coefficient of thefermentation of galactose also differsslightly from that of the other hexoses.

Yeast.Relative Rates.

Glucose. Galactose.

S. Cerevisiæ 100" 100

S. Carlsbergensis 100S. Thermantitonum 100

Assuming that his conclusion that allyeasts which ferment glucose alsoferment fructose and mannose iscorrect, Armstrong has drawn attentionto the fact that these three hexoses arealso related by the possession of acommon enolic form (p. 97) and hassuggested that this enolic form is thesubstance actually fermented to carbondioxide and alcohol [1904].

The idea that such an intermediateform is the direct subject offermentation has much to recommendit. In the first place it is almost certain,as already pointed out, that the sugars

in aqueous solution do exist, althoughto a very small extent, in this enolicform. The slow rate at whichequilibrium is established in aqueoussolution, however, must be taken asdefinite evidence that under thesecircumstances the enolic form is onlyproduced very slowly [compare Lowry,1903]. This has been used by Slator[1908, 1] as an argument against theprobability of the preliminaryconversion of the sugars into the enolicform before fermentation. It appears,however, quite possible that under theinfluence of the fermenting complex of

the yeast-cell, or of special enzymes,this change might occur much morerapidly, [p134] and at different rateswith the different sugars. This reactionmight in fact control the observed rateof fermentation. This conceptionaffords a simple explanation of thedifferent rates of fermentation ofmannose and glucose, and also ofgalactose, the enolic form of which isquite different, by yeast under differentcircumstances, but does not explain theuniformity of rate observed by Slatorfor glucose and fructose nor the resultswith mixtures of sugars. The direct

fermentation of a common enolic formis also consistent with the fact that thesame hexosephosphate is producedfrom all three hexoses.

Slator himself prefers the view thatthe first stage of fermentation consistsin the rapid combination of the sugarwith the enzyme, producing acompound, which then breaks up at arate which determines the observedrate of fermentation. This rate will ofcourse vary with the nature of thecompound, so that if two sugars formthe same compound they will befermented at the same rate; if they

form different compounds, differentrates may result. Slator supposes thatglucose and fructose form the samecompound with the enzyme. This,however, appears to involve anintramolecular change of the sameorder as the production of the enolicform, and moreover is not absolutelyessential, as it is probably sufficient tosuppose that the two compoundsderived from glucose and fructose arevery similar, although possibly notabsolutely identical. Mannose andgalactose, on the other hand, formstereoisomeric compounds, and the

capacity of the fermenting complex toform these compounds may be affectedby various agents to a different extentfrom its capacity for combining withglucose or fructose.

A third theory has also beensuggested to explain these phenomena,according to which the various sugarsare fermented by different enzymes[see Slator, 1908, 1]. The uniformity ofthe result obtained with glucose andfructose suggests that these two sugarsare fermented by the same enzyme(glucozymase), mannose and galactoseby different ones (mannozymase and

galactozymase). This would afford asimple explanation of the differentrates of fermentation for differentsugars and of different degrees ofsensitiveness towards reagents.

If, however, a separate andindependent mechanism were presentfor each sugar, the rate of fermentationof mixtures should be the sum of therates for the constituents. This, asshown above, is not found to be thecase, and it is therefore necessary tosuppose, either that one sugarinfluences the fermentation of anotherin some unknown way, or that only a

part of the mechanism of fermentationis specific for the particular sugar.Thus the enzyme may be specific and,the co-enzyme [p135] non-specific, sothat only a certain maximum rate isattainable, or again, the supply of freephosphate may be the controllingfactor.

In the prevailing state of ignoranceas to the exact function of the co-enzyme and of the conditions uponwhich the velocity of fermentation inthe cell depends, it is at presentimpossible to decide between thesevarious theories, but they all offer

points of attack which justify the hopethat much further information can beobtained by experimental inquiry.

It will be seen from the foregoingthat Buchner's discovery of zymase hasopened a chapter in the history ofalcoholic fermentation which is yet farfrom being completed. In everydirection fresh problems presentthemselves, and it cannot be doubtedthat as in the past, the investigation ofthe action of the yeast-cell will stillprove to be of fundamental importancefor our knowledge of the mode inwhich chemical change is brought

about by living organisms. [p136]

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Ehrlich, Felix (1904, 2), Ueberden neuen optisch-aktivenNichtzucker, das Isoleucin

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Slator, Arthur, and H. J. S. SAND

(1910), Studies in fermentation.Part III. The rôle of diffusion infermentation by yeast-cells

J. Chem. Soc, 922–927.

129

Stahl, Georg Ernst (1697), 2

Zymotechnia fundamentalis(Franckfurth, 1734), 304.

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19

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128

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19

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47,50

INDEX.

ACETALDEHYDE, as an intermediateproduct of alcoholic fermentation,110.

— reduction of by yeast, 110.Acetone-yeast, 38.Alanine, as an intermediate product of

alcoholic fermentation, 115.Alcohol, formation of, from sugar by

alkalis, 97.Alcoholic fermentation, attempts to

separate enzymes of, from yeast- cell,15.

— — by-products of, 85.— — equation of, 51.

— — Gay-Lussac's theory of, 4.— — Iwanoff's theory of, 106.— — kinetics of, 120, 128.— — Lavoisier's views on, 3.— — Liebig's theory of, 8.— — Nägeli's theory of, 15.— — of the amino-acids, 87.— — — — theory of, 91.— — Pasteur's researches on, 11.— — Traube's enzyme theory of,

14.Alkalis, effect of, on hexoses, 96.Amino-acids, alcoholic fermentation of,

87.— stereoisomerides of, fermented at

different rates by yeast, 89.d-Amyl alcohol, formation of from

isoleucine, 86.

Antiprotease in yeast-juice, 42, 65.Antiseptics, action of, on yeast-juice, 19,

36.Arsenate, effect of, on fermentation by

yeast-juice and zymin, 73.— — on autofermentation of yeast-

juice, 80.— nature of acceleration produced

by, 78.Arsenite, effect of, on fermentation by

yeast-juice, 77.— — on autofermentation of yeast-

juice, 80.— nature of acceleration produced

by, 78.Autofermentation of yeast-juice, 33,

119.— — effect of arsenates and

arsenites on, 80.

BAEYER'S theory of fermentation, 99.Boiled yeast-juice, effect of, on

fermentation by yeast-juice, 41.

CARBOXYLASE, 81, 93.— relation of to alcoholic

fermentation, 83.Co-enzyme, effect of electric current on,

67.— enzymic destruction of, 63.— of yeast-juice, 59.— precipitation of, by ferric

hydroxide, 67.— properties of, 63.— removal of, from yeast-juice, 59.— separation from phosphate and

hexosephosphate, 67.

Concentration of sugar, effect of, onfermentation by yeast-juice, 34.

DAUERHEFE, 38.Diastatic enzyme of yeast-juice, 33.Dihydroxyacetone, fermentability of,

104.— formation of, in fermentation,

105.Dried yeast (Lebedeff), 24, 38.

ENDOTRYPTASE, 20.Enzyme action, laws of, 121.Enzymes, combined with protoplasm,

126.Equation of alcoholic fermentation, 51.

FERMENTATION by yeast-juice, causes ofcessation of, 64.

Fermenting complex, 63.— power of yeast-juice, estimation

of, 27.Formaldehyde, production of in

alcoholic fermentation, 117.Formic acid theory of fermentation, 114.Fructose, fermentation of, by yeast-juice,

32.— — in presence of phosphate, 73.— relation of, to fermenting

complex, 74.Fusel oil, formation of, from amino-

acids, 85.

GALACTOSE, fermentation of, by yeast,131.

— fermentation of, by yeast-juice,32.

Glucose, fermentation of, by yeast-juice,32.

Glyceraldehyde, fermentability of, 104.Glyceric acid, fermentation of, 108.Glycerol, formation in fermentation, 95.Glycogen as an intermediate product of

alcoholic fermentation, 116.— fermentation of, by yeast-juice,

33.— removal of, from yeast, 39.

Grinding of yeast by hand, 22.— — — mechanical, 23.

Glutamic acid, decomposition of, byyeast, 90.

HEFANOL, 38.Hexosediphosphoric acid

phenylhydrazone, hydrazine salt of,

50.Hexosemonophosphoric acid osazone,

hydrazine salt of, 50.Hexosephosphatase, 54.— effect of arsenate and arsenite on

action of, 79.Hexosephosphate, constitution of, 51.

— enzymic decomposition of, inyeast-juice, 56.

— — hydrolysis of, 51.— formation of, 48.— hydrolysis of, by acids, 49.— preparation of, 48.— properties of, 49.— theory of formation of, 57, 117,

Hexoses, action of alkalis on, 96,

ISOAMYL alcohol, formation from

leucine, 87.Isoleucine, decomposition of, by yeast,

87.

Α-KETONIC acids, fermentation of, 81.

LACTIC acid, destruction of, by yeast-juice, 102.

— — formation from sugars byalkalis, 97.

— — — of, in yeast-juice, 102.— — non-fermentability of, by

yeast, 103.— — theory of fermentation, 102.

Leucine, decomposition of, by yeast, 87.

MACERATION extract, preparation of, 25.Mannose, fermentation of, by yeast, 131.

— — of by yeast-juice, 32.

Methylglyoxal, conversion of, into lacticacid, 101.

— non-fermentability of, 104.— as an intermediate product of

alcoholic fermentation, 113.

OXALACETIC acid, formation of, fromtartaric acid, 101.

PERMANENT yeast, 38.Phenylethyl alcohol, 88.Phosphate, changes of, in alcoholic

fermentation, 47.— effect of, on fermentation by

yeast-juice, 42.— — — — — by zymin, 46.— — — — — of fructose, 73.— — of on total fermentation of

yeast-juice, 54.

— influence on fermentation ofconcentration of, 71.

— inhibition by, 71.Phosphates, essential for alcoholic

fermentation, 55.Proteoclastic enzyme of yeast, 20.Protoplasmic theory of activity of yeast-

juice, 19.Pyruvic acid, fermentation of, 81.

— — theory of fermentation, 109.

RATE of fermentation, controlling factorsof, 119.

Reductase, intervention of, in alcoholicfermentation, 111.

SERUM, effect of, on fermentation byyeast-juice, 41.

Succinic acid, formation of, in

fermentation, 89.— — formed from glutamic acid by

yeast, 90.Synthetic enzyme in yeast-juice, 32.

TEMPERATURE coefficient of fermentationby yeast, 129.

— — — — by zymin, 122.— — — esterification of

phosphoric acid by yeast extract,58.

Tryptophol, 88.Tyrosol, 88.

WOHL'S theory of fermentation, 101.

YEAST, action of toluene on, 124.— and yeast-juice, fermentation by,

compared, 29, 124.

— discovery of the vegetable natureof, 5.

— fermentation by, 127.— — of different sugars by, 130.— influence of concentration of

dextrose on fermentation by, 128.— — — — of, on rate of

fermentation, 129.— — of toluene on

autofermentation of, 126.— nature of the process of

fermentation by, 123.— temperature coefficient of

fermentation by, 129.— theories of fermentation by, 133.

Yeast-juice and yeast, fermentingpowers compared, 29, 124.

— co-enzyme of, 59.

— dialysis of, 59, 62.— effect of arsenate on

fermentation by, 75.— — of concentration of sugar on

fermentation by, 34.— — of dilution on fermentation

by, 35.— — of phosphate on total

fermentation, produced by, 54.— estimation of fermenting power

of, 27.— evaporation of, 37.— filtration of through gelatin filter,

59.— precipitation of, 38.— preparation of, 21.— properties of, 19.— ratio of alcohol and carbon

dioxide, produced by, 30.— synthesis of complex

carbohydrate by, 31.— variation of rate of fermentation

by, with concentration of sugar,121.

ZYMASE, Buchner's discovery of, 16.— enzymic destruction of, 64.— properties of, 18.— regeneration of inactive, 64.— separation from co-enzyme, 59.

Zymin, 21, 38.— fermentation by, 39.— rate of fermentation by, 39.— temperature coefficient of

fermentation by, 122.

ABERDEEN: THE UNIVERSITY PRESS

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