alcoholic fermentation

ALCOHOLIC

FERMENTATION

BY

ARTHUR HARDEN, PH.D., D.Sc., F.R.S.

PROFESSOR OF BIOCHEMISTRY, LONDON UNIVERSITY

HEAD OP THE BIOCHEMICAL DEPARTMENT, LISTER INSTITUTE, CHELSEA

SECOND EDITION

LONGMANS, GREEN AND CO.

39 PATERNOSTER ROW, LONDON

FOURTH AVENUE " 30TH STREET, NEW YORK

BOMBAY, CALCUTTA, AND MADRAS

CONTENTS.

CHAPTER PAQB

I. HISTORICAL INTRODUCTION i

II. ZYMASE AND ITS PROPERTIES 18

III. THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION 41

IV. THE CO-ENZYME OF YEAST- JUICE 59

V. ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON

THE ENZYMES OF YEAST-JUICE 70

VI. CARBOXYLASE 81

VII. THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 85

VIII. THE CHEMICAL CHANGES INVOLVED IN FERMENTATION - - 96

IX. THE MECHANISM OF FERMENTATION 119

BIBLIOGRAPHY 136

INDEX 155

vii

CHAPTER I.

HISTORICAL INTRODUCTION.

THE problem of alcoholic fermentation,of the originand nature of that

mysterious and apparentlyspontaneous change which converted the

insipidjuiceof the grape into stimulatingwine, seems to have exerteda fascination over the minds of natural philosophers from the veryearliest times. No date can be assignedto the first observation of the

phenomena of the process. History finds man in the possession ofalcoholic liquors,and in the earliest chemical writingswe find ferment-ation,

as a familiar natural process, invoked to explain and illustrate the

hanges with which the science of those early days was concerned.

Throughout the period of alchemy fermentation plays an important

part ; it is,in fact,scarcelytoo much to say that the language of the

alchemists and many of their ideas were founded on the phenomena of

fermentation. The subtle change in propertiespermeating the whole

mass of material,the frothingof the fermentingliquid,renderingevi-dent

the vigour of the action,seemed to them the very emblems of the

mysterious process by which the long sought for philosopher'sstone

was to convert the baser metals into gold. As chemical science

emerged from the mists of alchemy, definite ideas about the natureof alcoholic fermentation and of putrefactionbegan to be formed.Fermentation was distinguishedfrom other chemical changes in which

gases were evolved, such as the action of acids on alkali carbonates

(Sylviusde le Boe, 1659) ; the gas evolved was examined and termed

gas vinorum, and was distinguishedfrom the alcohol with which ithad at first been confused (van Helmont, 1648); afterwards it wasfound that like the gas from potashes it was soluble in water (Wren,1664). The gaseous product of fermentation and putrefactionwasidentified by MacBride, in 1764, with the fixed air of Black, whilst

Cavendish in 1766 showed that fixed air alone was evolved in alcoholic

fermentation and that a mixture of this with inflammable air was pro-duced

by putrefaction.In the meantime it had been recognisedthat

only sweet liquorscould be fermented ("Ubi notandum, nihil fer-mentare quod non sit dulce," Becher, 1682), and finallyCavendish

I

2 ALCOHOLIC FERMENTATION

[1776] determined the proportion of fixed air obtainable fromsugar by fermentation and found it to be 57 per cent. It graduallybecame recognisedthat fermentation mightyieldeither spirituousor acid

liquors,whilst putrefactionwas thought to be an action of the samekind as fermentation,differingmainly in the character of the products(Becher).

As regardsthe nature of the process very confused ideas at first

prevailed,but in the time of the phlogisticchemists a definitetheoryoffermentation was proposed,firstby Willis (1659) and afterwards byStahl [1697],the fundamental idea of which survived the over-throw

of the phlogisticsystem by Lavoisier and formed the foundationof the views of Liebig. To explainthe spontaneous originof ferment-ation

and its propagationfrom one liquidto another,theysupposed thatthe process consisted in a violent internal motion of the particlesof thefermentingsubstance,set up by an aqueous liquid,whereby the com-bination

of the essential constituents of this material was loosened and

new particlesformed, some of which were thrust out of the liquid(thecarbon dioxide)and others retained in it (thealcohol).

Stahl specificallystates that a body in such a state of internal dis-quietudecan very readilycommunicate the disturbance to another,

which is itselfat rest but is capableof undergoinga similar change,sothat a putrefyinggr fermentingliquidcan set another liquidin putre-faction

or fermentation.

Taking account of the gradualaccumulation of fact and theorywefind at the time of Lavoisier,from which the modern aspect of the

problem dates,that Stahl's theoretical views were generallyaccepted.Alcoholic fermentation was known to requirethe presence of sugar andwas thought to lead to the productionof carbon dioxide, acetic acid,and alcohol.

The compositionof organiccompounds was at that time not under-stood,and it was Lavoisier who established the fact that theyconsisted

of carbon,hydrogen,and oxygen, and who made systematicanalysesof the substances concerned in fermentation (1784-1789). Lavoisier[1789]appliedthe results of these analysesto the study of alcoholicfermentation,and by employing the principlewhich he regardedas thefoundation of experimentalchemistry," that there is the same quantityof matter before and after the operation,"he drew up an equationbe-tween

the quantitiesof carbon, hydrogen, and oxygen in the originalsugar and in the resultingsubstances,alcohol,carbon dioxide,andacetic acid,showing that the productscontained the whole matter ofthe sugar, and thus for the firsttime givinga clear view of the chemical

HISTORICAL INTRODUCTION 3

change which .occurs in fermentation. The conclusion to which hecame was, we now know, very nearlyaccurate, but the research mustbe regarded as one of those remarkable instances in which the geniusof the investigatortriumphs over experimental deficiencies,for theanalyticalnumbers employed contained grave errors, and itwas onlybya fortunate compensation of these that a result so near the truth wasattained.

Lavoisier's equationor balance sheet was as follows :"

Carbon. Hydrogen. Oxygen.95*9 pounds of sugar (canesugar)consist of . 26*8 77 61*4These yield:"

577 pounds of alcohol containing . . 167 9*6 31-435*3

""

carbon dioxide containing 9*9 " 25-42*5

,,

acetic acid containing.

0*6 0*2 17

Total contained in products.... 27-2 9*8 58*5

The true composition of the sugar used was carbon 40*4, hydrogen6-1, oxygen 49-4.

Lavoisier expressed no view as to the agency by which ferment-ationwas brought about,but came to a very definite and characteristic

conclusion as to the chemical nature of the change. The sugar, whichhe regardedin harmony with his general views as an oxide, was splitinto two parts,one of which was oxidised at the expense of the other

to form carbonic acid,whilst the other was deoxygenised in favour ofthe former to produce the combustible substance alcohol," so that ifitwere possibleto recombine these two substances,alcohol and carbonicacid,sugar would result ".

From this point commences the modern study of the problem.Provided by the geniusof Lavoisier with the assurance that the hithertomysterious process of fermentation was to be ranked along withfamiliar chemical changes,and that it proceeded in harmony with thesame quantitativelaws as these simplerreactions,chemists were stimu-lated

in their desire to penetrate further into the mysteries of the phe-nomenon,and the importance and interest of the problem attracted

many workers.

So important indeed did the matter appear to Lavoisier's country-menthat in the year 8 of the French Republic(1800) a prize" con-sisting

of a gold medal, the value of which, expressed in terms of thenewly introduced metric system, was that of one kilogram of gold"was offered by the Institute for the best answer to the question :" What are the characteristics by which animal and vegetablesubstanceswhich act as ferments can be distinguishedfrom those which they arecapableof fermenting? "


This valuable prize was again offered in 1802 but was neverawarded, as the fund from which it was to be drawn was seques-trated

from the Institute in 1804. The firstresponse to this stimulat-ingoffer was an important memoir by citizen Thenard [1803],which

providedmany of the facts upon which Liebigsubsequentlybased hisviews. Thenard combats the prevailingidea, first expressed byFabroni (1787-1799),that fermentation is caused by the action ofglutenderived from grainon starch and sugar, but is himself uncertainas to the actual nature of the ferment. He pointsout that all fer-menting

liquidsdeposit a material resemblingbrewer's yeast, and heshows that this contains nitrogen,much of which is evolved asammonia on distillation. His most important result is,however,that when yeast is used to ferment pure sugar, it undergoes a gradualchange and is finallyleft as a white mass, much reduced in weight,which contains no nitrogenand is without action on sugar. Thenard,moreover, it is interestingto note, differs from Lavoisier,inasmuchas he ascribes the originof some of the carbonic acid to the carbonof the ferment,an opinionwhich was still held in various degreesbymany investigators(seeSeguin,quoted by Thenard).

Thenard's memoir was followed by a communication of funda-mentalimportance from Gay-Lussac [1810]. A process for preserv-ing

food had beeni^ntroduced by Appert,which consisted in placingthe material in bottles,closingthese very carefullyand exposingthem to the temperature of boilingwater for some time. Gay-Lussac was struck by the fact that when such a bottle was openedfermentation or putrefactionset in rapidly. Analysisof the air leftin such a sealed bottle showed that all the oxygen had been absorbed,and these facts led to the view that fermentation was set up by theaction of oxygen on the fermentable material. Experiment appearedto confirm this in the most strikingway. A bottle of preservedgrape-juice was opened over mercury and part of its contents passedthrough the mercury into a bell-jarcontainingair,the remainder intoa similar vessel free from air. In the presence of air fermentation set

in at once, in the absence of air no fermentation whatever occurred.

This connection between fermentation and the presence of air was

established by numerous experiments and appeared incontestable.Fermentation, it was found, could be checked by boilingeven afterthe addition of oxygen, and hence food could be preservedin freecontact with the air,providedonly that it was raised to the tempera-ture

of boilingwater at short intervals of time. Gay-Lussac'sopinionwas that the ferment was formed by the action of the oxygen on the


liquid,and that the product of this action was altered by heat andrendered incapableof producing fermentation,as was also brewer's

yeast,which, however, he regarded,on account of its insolubility,asdifferent from the soluble ferment which initiated the change in the

limpidgrape-juice.Colin,on the other hand [1826],recognisedthatalcoholic fermentation by whatever substance it was started,resulted

in the formation of an insoluble deposit more active than theoriginalsubstance,and he suggested that this depositmight possiblyin every case be of the same nature.

So far no suspicionappears to have arisen in the minds of thosewho had occupiedthemselves with the study of fermentation that thischange differed in any essential manner from many other reactionsfamiliar to chemists. The originand propertiesof the ferment wereindeed remarkable and involved in obscurity,but the uncertaintyre-garding

this substance was no greater than that surroundingmany, ifnot all,compounds of animal and vegetableorigin. Although,how-ever,

the purelychemical view as to the nature of yeast was generallyrecognisedand adopted,isolated observations were not wanting whichtended to show that yeast might be somethingmore than a mere chemical

reagent. As early as 1680 in lettersto the Royal SocietyLeeuwen-hoek described the microscopicappearance of yeast of various originsas that of small, round, or oval particles,but no further progress seemsto have been made in this direction for nearlya century and a half,when we find that Desmazi"res [1826] examined the film formedon beer,figuredthe elongatedcells of which it was composed, anddescribed it under the name of Mycoderma Cerevisiae. He, however,regardedit rather as of animal than of vegetableorigin,and does notappear to have connected the presence of these cells with the processof fermentation.

Upon this long periodduring which yeast was regardedmerely asa chemical compound there followed,as has so frequentlyoccurred insimilar cases, a sudden outburst of discovery. No less than threeobservers hit almost simultaneouslyupon the secret of fermentation anddeclared that yeast was a livingorganism.

First among these in strict order of time was Cagniard-Latour[1838],who made a number of communications to the Academyand to the Soci6t6 Philomatiquein 1835-6,the contents of which werecollected in a paper presentedto the Academy of Sciences on 12 June,1837, ar*d published in 1838. The observations upon which this

memoir was based were almost exclusivelymicroscopical.Yeast wasrecognisedas consistingof sphericalparticles,which were capableof


reproductionby budding but incapableof motion, and it was therefore

regarded as a livingorganism probably belonging to the vegetablekingdom. Alcoholic fermentation was observed to depend on the

presence of livingyeast cells,and was attributed to some effect of their

vegetativelife(quelqueeffet de leur v6g6tation).It was also noticedthat yeast was not deprivedof its fermentingpower by exposure tothe temperature of solid carbonic acid,a sample of which was suppliedto Cagniard-Latourby Thilorier,who had onlyrecentlypreparedit forthe firsttime.

Theodor Schwann [1837], whose researches were quite inde-pendentof those of Cagniard-Latour,approached the problem from an

entirelydifferent point of view. During the year 1836 Franz Schulze

[1836] published a research on the subjectof spontaneous gener-ation,in which he proved that when a solution containinganimal or

vegetablematter was boiled,no putrefactionset in provided that allair which was allowed to have access to the liquidwas previouslypassed through strong sulphuricacid. Schwann performed a verysimilar experiment by which he showed that this same result,theabsence of putrefaction,was attained by heatingall air which came intocontact with the boiled liquid.Wishing to show that other processesin which air took part were not affected by the air being heated, hemade experiments

,

with fermentingliquidsand found, contrary to his

expectation,that a liquidcapableof undergoingvinous fermentationand containingyeast did not undergo this change after it had beenboiled,provided that, as in the case of his previous experiments,only air which had been heated was allowed to come into contactwith it.

Schwann's experiments on the preventionof putrefactionwere un-exceptionableand quitedecisive. The analogousexperiments dealing

with alcoholic fermentation were not quite so satisfactory.Yeast wasadded to a solution of cane sugar, the flask containingthe mixtureplacedin boilingwater for ten minutes, and then inverted over mer-cury.

About one-third of the liquidwas then displacedby air and theflasks corked and kept inverted at air temperature. In two flasks theair introduced was ordinaryatmosphericair,and in these flasks fermen-tation

set in after about four to six weeks. Into the other two flasks

air which had been heated was led,and in these no fermentationoccurred. As described,the experiment is quite satisfactory,butSchwann found on repetitionthat the results were irregular.Some-times

all the flasks showed fermentation,sometimes none of them.This was correctlyascribed to the experimentaldifficulties,but none


the less served as a pointof attack for hostile and damaging criticismat the hands of Berzelius (p.8).

The originof putrefactionwas definitelyattributed by Schwann tothe presence of livinggerms in the air,and the similarityof the resultobtained with yeast suggestedthe idea that alcoholic fermentation wasalso brought about by a livingorganism, a conceptionwhich was atonce confirmed by a microscopicalexamination of a fermentingliquid. The phenomena observed under the microscopewere similarto those noted by Cagniard-Latour,and in accordance with theseobservations alcoholic fermentation was attributed to the developmentof a livingorganism,the fermentative function of which was found tobe destroyedby potassium arsenite and not by extract of Nux vomica,so that the organism was regardedrather as of vegetablethan ofanimal nature. This plant received the name of " Zuckerpilz"J orsugar fungus(which has been perpetuatedin the genericterm Sac-charomyces). Alcoholic fermentation was explainedas " the decom-position

brought about by this sugar fungusremoving from the sugarand a nitrogenoussubstance the materials necessary for its growth andnourishment, whilst the remainingelements of these compounds, whichwere not taken up by the plant,combined chieflyto form alcohol ".

Kiitzing'smemoir, the third of the trio [1837],also dates from1 837, and his opinions,like those of Cagniard-Latour,are founded onmicroscopicalobservations. He recognisesyeast as a vegetableorganism and accuratelydescribes itsappearance. Alcoholic ferment-ation

depends on the formation of yeast, which is produced when the

necessary elements and the proper conditions are present and then

propagates itself. The action on the liquidthus increases and theconstituents not requiredto form the organism combine to form un-organised

substances,the carbonic acid and alcohol. " It is obvious,1'says Kutzing, in a passage which roused the sarcasm of Berzelius," that chemists must now strike yeast off the roll of chemical com-pounds,

since it is not a compound but an organised body, anorganism."

These three papers, which were publishedalmost simultaneously,were received at firstwith incredulity.Berzelius,at that time thearbiter and dictator of the chemical world, reviewed them all in his" Jahresbericht" for 1839 [1839]with impartialscorn. Th^micro-scopicalevidence was denied all value,and yeast was no more to be

regardedas an organismthan was a precipitateof alumina. Schwann's

experiment (p.6) was criticised on the ground that the fermentingpower of the added yeast had been only partiallydestroyedin the

g ALCOHOLIC FERMENTATION

flasks in which fermentation ensued, completelyin those which re-mainedunchanged, the admission of heated or unheated air being

indifferent,a criticism to some extent justifiedby Schwann's statement,alreadyquoted,of the uncertain result of the experiment.

Berzelius himself regarded fermentation as beingbrought about

by the yeast by virtue of that catalyticforce,which he had supposedto intervene in so many reactions,both between substances of mineraland of animal and vegetableorigin[1836],and which enabled "bodies,by their mere presence, and not by their affinity,to arouse affinitiesordinarilyquiescentat the temperature of the experiment, so that theelements of a compound body arrange themselves in some different

way, by which a greater degree of electro-chemical neutralisation isattained ".

To the scorn of Berzelius was soon added the sarcasm of Wohler

and Liebig [1839]. Stimulated in part by the publicationsof thethree authors alreadymentioned, and in part by the report of Turpin[1839],who at the request of the Academy of Sciences had satisfiedhimself by observation of the accuracy of Cagniard-Latour'scon-clusions,

Wohler prepared an elaborate skit on the subject,which hesent to Liebig,to whom it appealedso stronglythat he added sometouches of his own and publishedit in the " Annalen," followingim-mediately

upon a translation of Turpin'spaper. Yeast was here des-cribedwith a considerable degreeof anatomical realism as consisting

of eggs which developedinto minute animals,shaped like a distillingapparatus, by which the sugar was taken in as food and digestedintocarbonic acid and alcohol,which were separatelyexcreted, the whole

process being easilyfollowed under the miscroscope.Close upon this pleasantryfollowed a serious and importantcom-munication

from Liebig [1839],in which the nature of fermentation,putrefaction,and decay was exhaustivelydiscussed. Liebigdid not ad-mit

that these phenomena were caused by livingorganisms,nor did heattribute them like Berzelius to the catalyticaction of a substance whichitselfsurvived the reaction unchanged. As regardsalcoholic fermenta-tion,

Liebig's chief arguments may be brieflysummarised. As theresult of alcoholic fermentation,the whole of the carbon of the sugarreappears in the alcohol and carbon dioxide formed. This change is

broughtabout by a body termed the ferment,which is formed as the resultof a change set up by the access of air to plantjuicescontainingsugar,and which contains all the nitrogenof the nitrogenousconstituents of the

juice. This ferment is a substance remarkablysusceptibleof change,which undergoes an uninterruptedand progressivemetamorphosis,of


the nature of putrefactionor decay,and producesthe fermentation ofthe sugar as a consequence of the transformation which it is itself

undergoing.The decompositionof the sugar is therefore due to a condition of

instabilitytransferred to itfrom the unstable and changingferment,andonly continues so long as the decompositionof the ferment proceeds.This communication of instabilityfrom one substance undergoingchemical change to another is the basis of Liebig'sconception,and isillustratedby a number of chemical analogies,one of which will sufficeto explainhis meaning. Platinum is itself incapableof decomposingnitric acid and dissolvingin it; silver,on the other hand, possesses this

power. When platinumisalloyedwith silver,the whole mass dissolvesin nitric acid,the power possessedby the silver being transferred tothe platinum. In like manner the condition of active decompositionof the ferment is transferred to the sugar, which by itselfis quitestable.The central idea is that of Stahl (p.2) which was thus reintroducedinto scientificthought.

In a pure sugar solution the decompositionof the ferment sooncomes to an end and fermentation then ceases. In beer wort or veget-able

juices,on the other hand, more ferment is continuallyformed inthe manner alreadydescribed from the nitrogenousconstituents of the

juice,and hence the sugar is completelyfermented away and un-exhaustedferment left behind. Liebig'sviews were reiterated in his

celebrated "Chemische Briefe,"and became the generallyaccepteddoctrine of chemists. There seems little doubt that both Berzelius

and Liebigin their scornful rejectionof the results of Cagniard-Latour,Schwann and Kiitzing,were influenced,perhapsalmost unconsciously,by a desire to avoid seeingan importantchemical change relegatedtothe domain of that vital force from beneath the sway of which a large

part of organicchemistryhad justbeen rescued by Wohler's brilliantsyntheticalproductionof urea and by the less recognisedsynthesisofalcohol by Hennell (see on this point Ahrens [1902]). A strongbody of evidence,however, graduallyaccumulated in favour of thevegetable nature of yeast, so that it may be said that by 1848 apowerful minorityadhered to the views of Cagniard-Latour,Schwann,and Kiitzing[see Schrohe, 1904, p. 218, and compare Buchner,1904]* Among these must be included Berzelius [1848],who hadso forciblyrepudiatedthe idea only ten years before,-whereas Liebigin the 1851 edition of his letters does not mention the fact that yeastis a livingorganism (LetterXV).

The recognitionof the vegetablenature of yeast,however, by no

io ALCOHOLIC FERMENTATION

means disprovedLiebig'sview of the nature of the change by which

sugar was converted into carbon dioxide and alcohol,as was carefullypointed out by Schlossberger[1844] in a research on the nature ofyeast,carried out in Liebig'slaboratorybut without decisive results.

Mitscherlich was also convinced of the vegetablecharacter of yeast,and showed [1841] that when yeast was placed in a glass tubeclosed by parchment and plunged into sugar solution,the sugarentered the glasstube and was there fermented,but was not fermented

outside the tube. He regarded this as a proof that fermentation onlyoccurred at the surface of the yeast cells,and explainedthe process bycontact action in the sense of the catalyticaction of Berzelius,ratherthan by Liebig'stransference of molecular instability.Similar resultswere obtained with an animal membrane by Helmholtz [1843],who also expressed his conviction that yeast was a vegetableor-ganism.

In 1854 Schroder and von Dusch [1854,1859, 1861]stronglyrein-forcedthe evidence in favour of this view by succeedingin preventing

the putrefactionand fermentation of many boiled organicliquidsbythe simpleprocess of filteringall air which had access to them throughcotton- wool. These experiments,which were continued until 1861,led to the conclusion that the spontaneous alcoholic fermentation of

liquidswas due to livinggerms carried by the air,and that when theair was passedthrough the cotton-wool these germs were held back.

At the middle of the nineteenth century opinions with regard toalcoholic fermentation,notwithstanding all that had been done, werestill divided. On the one hand Liebig'stheory of fermentation waswidelyheld and taught. Gerhardt, for example, as late as 1856 in thearticle on fermentation in his treatise on organicchemistry [1856],givesentire support to Liebig'sviews,and his treatment of the matter af-fords

an interestingglimpseof the arguments which were then held to bedecisive. The grounds on which he rejectsthe conclusions of Schwannand the other investigatorswho shared the belief in the vegetablenatureof yeast are that,althoughin some cases animal and vegetablematterand infusions can be preservedfrom change by the methods described

by these authors,in others they cannot, a strikingcase beingthat ofmilk,which even after being boiled becomes sour even in filtered air,and this without showing any trace of livingorganisms. The actionof heat,sulphuricacid,and filtrationon the air is to remove, or destroy,not livingorganisms but particlesof decomposing matter, that is to

say, ferments which would add their activityto that of the oxygen ofthe air. Moreover, many ferments,as for example diastase,act with-

HISTORICAL INTRODUCTION n

out producingany insoluble depositwhatever which can be regardedas an organism.

" Evidemment," he concludes," la th6orie de M. Liebig expliqueseule tous les ph6nomfenes de la manifere la plus complete et la pluslogique; c'est 4 elle que tous les bons espritsne peuvent manquer dese rallier."

On the other hand it was held by many to have been shown thatLiebig1s view of the originof yeast by the action of the air on a veget-able

infusion was erroneous, and that fermentation only arose whenthe air transferred to the liquidan active agent which could be removedfrom it by sulphuricacid (Schulze),by heat (Schwann),and by cotton-wool

(Schroderand von Dusch). Accompanying alcoholic ferment-ationthere was a development of a livingorganism, the yeast,and

fermentation was believed,without any very strict proof,to be a phe-nomenondue to the lifeand vegetationof this organism. This doctrine

seems indeed [Schrohe,1904] to have been widely taught in Ger-manyfrom 1840-56, and to have established itselfin the practiceof

the fermentation industries.In 1857 commenced the classicalresearches of Pasteur which finally

decided the question as to the origin and functions of yeast and ledhim to the conclusion that " alcoholic fermentation is an act correlated

with the lifeand organisationof the yeast cells,not with the death orputrefactionof the cells,any more than it is a phenomenon of contact,in which case the transformation of sugar would be accomplishedinpresence of the ferment without yieldingup to it or taking from itanything " [1860]. It is impossiblehere to enter in detail intoPasteur's experiments on this subject,or indeed to do more than indi-cate

the generallines of his investigation.His starting-pointwas thelacticacid fermentation.

The organism to which this change was due had hitherto escapeddetection,and as we have seen the spontaneous lactic fermentation ofmilk was one of the phenomena adduced by Gerhardt (p.10)in favour ofLiebig'sviews. Pasteur [1857] discovered the lactic acid produc-ing

organism and convinced himself that it was in fact a livingorgan-ismand the active cause of the productionof lactic acid. One of the

chief buttresses of Liebig'stheorywas thus removed, and Pasteur nextproceeded to applythe same method and reasoningto alcoholic ferment-ation.

Liebig'stheoryof the originof yeastby the action of the oxygenof the air on the nitrogenousmatter of the fermentable liquidwas con-clusively

and strikinglydisprovedby the brilliant device of producinga crop of yeast in a liquidmedium containingonly comparatively


simplesubstances of known composition"sugar, ammonium tartrate and

mineral phosphate. Here there was obviouslypresentin the original

medium no matter which could be put into a state of putrefactionby

contact with oxygen and extend its instabilityto the sugar. Any such

material must firstbe formed by the vital processes of the yeast. In

the next placePasteur showed by careful analysesand estimations that,

whenever fermentation occurred,growth and multiplicationof yeast ac-companied

the phenomenon. The sugar, he proved,was not completely

decomposed into carbon dioxide and alcohol,as had been assumed by

Liebig (p. 8). A balance-sheet of materials and productswas con-structedwhich showed that the alcohol and carbon dioxide formed

amounted only to about 95 per cent, of the invert sugar fermented,the

difference being made up by glycerol,succinic acid,cellulose,and other

substances [1860, p. 347]. In every case of fermentation, evenwhen a paste of yeast was added to a solution of pure cane sugar in

water, the yeast was found by quantitativemeasurements to have taken

something from the sugar. This " something"

was indeterminate in

character,but,includingthe whole of the extractives which had passedfrom the yeast cells into the surroundingliquid,itamounted to as much

as 1-63 per cent of the weight of the sugar fermented [1860,P- 344]-

Pasteur was therefore led to consider fermentation as a physiolog-icalprocess accompanying the lifeof the yeast. His conclusions were

couched in unmistakable words : " The chemical act of fermentation is

essentiallya phenomenon correlative with a vital act,commencing and

ceasing with the latter. I am of opinion that alcoholic fermentation

never occurs without simultaneous organisation,development,multi-plicationof cells,or the continued life of cells alreadyformed. The

resultsexpressedin this memoir seem to me to be completelyopposedto the opinionsof Liebigand Berzelius. If I am asked in what con-sists

the chemical act whereby the sugar is decomposed and what isits real cause, I replythat I am completelyignorantof it.

" Ought we to say that the yeast feeds on sugar and excretes alcoholand carbonic acid ? Or should we rather maintain that yeast in its

development producessome substance of the nature of a pepsin,whichacts upon the sugar and then disappears,for no such substance is foundin fermented liquids? I have nothingto replyto these hypotheses.I neither admit them nor rejectthem, and wish only to restrain myselffrom going beyond the facts. And the facts tell me simply that alltrue fermentations are correlative with physiologicalphenomena."

Liebigfelt to the fullthe weight of Pasteur's criticisms ; his reply

I4 ALCOHOLIC FERMENTATION

Nevertheless,Liebig'sdesire to penetrate more deeply into thenature of the process of fermentation remained in many minds, and

numerous endeavours were made to obtain further insightinto theproblem. In spiteof an entire lack of direct experimentalproof,theconceptionthat alcoholic fermentation was due to the chemical actionof some substance elaborated by the cell and not directlyto thevital processes of the cell as a whole found strenuous supporters even

among those who were convinced of the vegetablecharacter of yeast.As earlyas 1833 diastase,discovered still earlier by Kirchhoff and

Dubrunfaut, had been extracted by means of water from germinatingbarleyand precipitatedby alcohol as a white powder, the solution ofwhich was capableof convertingstarch into sugar, but lost this powerwhen heated [Payen and Persoz, 1833]. Basing his ideas in partupon the behaviour of this substance,Moritz Traube [1858] enun-ciated

in the clearest possiblemanner the theory that all ferment-ations

producedby livingorganismsare caused by ferments,which aredefinitechemical substances producedin the cells of the organism. He

regardedthese substances as beingcloselyrelated to the proteinsandconsidered that their function was to transfer the oxygen and hy-drogen

of water to different parts of the molecule of the fermentable

substance and thus bring about that apparent intramolecular oxi-dationand reduction which is so characteristic of fermentative

t-

change and had arrested the attention of Lavoisier and, long after

him, of Liebig.Traube's main thesis,that fermentation is caused by definite fer-ments

or enzymes, attracted much attention,and received fresh sup-portfrom the separationof invertase in 1 860 from an extract of yeast

by Berthelot,and from the advocacy and authorityof this greatcountryman of Pasteur,who definitelyexpressed his opinion thatinsoluble ferments existed which could not be separatedfrom thetissues of the organism,and further,that the organism could not itselfbe regardedas the ferment,but only as the producer of the ferment

[1857, 1860], Hoppe-Seyler [1876] also supported the enzymetheory of fermentation,but differed in some respects from Traube

as to the exact function of the ferment [seeTraube, 1 877 ; Hoppe-Seyler,1877].

Direct experimental evidence was, however, still wanting, andPasteur's reiterated assertion [1875] that all fermentation phenomenawere manifestations of the lifeof the organism remained uncontroverted

by experience.Numerous and repeateddirect experimentalattacks had been made


from time to time upon the problem of the existence of a fermentation

enzyme, but allhad yieldednegativeor unreliable results.As earlyas 1846 a bold attempt had been made by Liidersdorff

[1846]to ascertain whether fermentation was or was not bound upwith the lifeof the yeastby grindingyeast and examining the groundmass. A singlegram of yeast was thoroughly ground, the processlastingfor an hour, and the product was tested with sugar solution.Not a singlebubble of gas was evolved. A similar result wasobtained in a repetitionof the experiment by Schmidt in Liebig'slaboratory[1847],the grinding being continued in this case for sixhours, but the natural conclusion that livingyeast was essential forfermentation was not accepted,on the ground that duringthe lengthyprocess of trituration in contact with air the yeast had become altered

and now no longer possessedthe power of producing alcoholic fer-mentation,but instead had acquiredthat of changing sugar into lactic

acid [seeGerhardt, 1856, p. 545],Similar experimentsmade in 1871 by Marie von ManasseYn [1872,

1897], in which yeast was ground for six to fifteen hours withpowdered rock crystal,yieldedproductswhich fermented sugar, but

they contained unbroken yeast cells,so that the results obtained could

not be considered decisive [Buchner and Rapp, 1898, i],althoughFrau von Manassefn herself drew from them and from others in which

sugar solution was treated with heated yeast,but not under asepticcon-ditions,the conclusion that livingyeast cells were not necessary for

fermentation.

Quite unsuccessful were also the attempts made to accomplish theseparationof fermentation from the livingcell by Adolf Mayer [1879,p. 66],and, as we learn from Roux, by Pasteur himself,grinding,freezing,and plasmolysingthe cells,having in his hands proved alikein vain. Extraction by glycerolor water, a method by which manyenzymes can be obtained in solution,gave no better results [Nageliand Loew, 1878],and the enzyme theory of alcoholic fermentationappearedquiteunjustifiedby experiment.

Having convinced himself of this,Nageli[1879] suggested anew explanationof the facts based on molecular-physicalgrounds.According to this view, which unites in itself some of the conceptionsof Liebig,Pasteur,and Traube, fermentation is the transference of astate of motion from the molecules,atomic groups, and atoms of the

compounds constitutingthe livingplasma of the cell to the ferment-ablematerial,whereby the equilibriumexistingin the molecules of the

latter is disturbed and decompositionensues [1879,P- 29\


This somewhat complex idea,whilst including,as did Liebig'stheory,Stahl's fundamental conceptionof a transmission of a state of

motion, satisfiesPasteur's contention that fermentation cannot occurwithout life,and at the same time explainsthe specificaction of differ-ent

organismsby differences in the constitution of their cell contents.The reallyessential part of Nageli'stheoryconsisted in the limitationof the power of transference of molecular motion to the livingplasma,by which the failure of all attempts to separatethe power of ferment-ation

from the livingcell was explained. This was the specialphe-nomenonwhich requiredexplanation; to account for this the theory

was devised,and when this was experimentallydisproved,the theorylost all significance.

For nearlytwenty years no further progress was made, and thenin 1 897 the questionwhich had aroused so much discussion and con-jecture,

and had given rise to so much experimentalwork, was finallyanswered by Eduard Buchner, who succeeded in preparingfrom yeasta liquidwhich,in the completeabsence of cells,was capableof effectingthe resolution of sugar into carbon dioxide and alcohol [1897,i].

In the lightof this discoverythe contribution to the truth made

by each of the great protagonistsin the prolonged discussion on theproblem of alcoholic fermentation can be discerned with some degreeof clearness. Liebig'smain contention that fermentation was essen-tially

a chemical act was correct,althoughhis explanationof the natureof this act was inaccurate. Pasteur,in so far as he considered the act

of fermentation as indissolublyconnected with the life of the organism,was shown to be in error, but the function of the organism has onlybeen restricted by a singlestage, the active enzyme of alcoholic fer-mentation

has so far only been observed as the productof the livingcell. Nearest of all to the truth was Traube, who in 1858 enunciatedthe theorem, which was only proved for alcoholic fermentation in1897, that all fermentations produced by livingorganismsare due toferments secreted by the cells.

Buchner's discoveryof zymase has introduced a new experimentalmethod by means of which the problem of alcoholic fermentation canbe attacked,and the result has been that since 1897 a considerable

amount of information has been gainedwith regard to the nature andconditions of action of the enzymes of the yeast cell. It has been

found that the machineryof fermentation is much more complex thanhad been surmised. The enzyme zymase, which is essential for fer-mentation,

cannot of itselfbringabout the alcoholic fermentation of

sugar, but isdependenton the presence of a second substance,termed,for


want of a more reasonable name, the co-enzyme.The chemical nature

and function of this mysterious coadjutor are still unknown, but as it

withstands the temperature of boiling water and is dialysable, it is

probably more simple in constitution than the enzyme. This, however,

is not all ; for the decomposition of sugar a phosphate is also indis-pensable.

Itappears that in yeast-juice, and therefore also most pro-bably

in the yeast cell, the phosphorus present takes an active part in

fermentation and goes through a remarkable cycle of changes. The

breakdown ofsugar into alcohol and carbon dioxide is accompanied

by the formation ofa complex hexosephosphate, and the phosphate

is split off from this compound and thus again rendered available

for action by means of a special enzyme, termed hexosephosphatase.

In addition to this complex of ferments, the cell also possesses special

enzymes by which the zymase and the co-enzyme can be destroyed, and,

further, at least one substance, known as an anti-enzyme, which

directly checks this destructive action. It seems probable, moreover,

that the decomposition of thesugar molecule takes place in stages,

although much doubt yet exists as to the nature of these.

At the present moment the subject remains one of the most

interesting in the whole field of biological chemistry, the limited

degree of insight which has already been gained into the marvellous

complexity of the cell lending additional zest to the attempt to pene-trate

the darkness which shrouds the still hidden mysteries.

CHAPTER II.

ZYMASE AND ITS PROPERTIES.

Discovery of Zymase.

THE history of Buchner's discovery is of great interest [Gruber, 1908 ;Hahn, 1908]. As early as 1893 Hans and Eduard Buchner foundthat the cells of even the smallest micro-organism could be broken

by being ground with sand [Buchner, E. and H., and Hahn, 1903,

p. 20], and in 1896 the same process was applied by these two inves-tigatorsto yeast, with the object of obtaining a preparation for

therapeutic purposes. Difficulties arose in the separation of the cell

contents from the ground-up mixture of cell membranes, unbroken

cells,and sand, but these were overcome by carrying out the sugges-tionof Martin Hahn (at that time assistant to Hans Buchner) that

kieselguhr should be added and the liquid squeezed out by means of

a hydraulic press [Buchner, E. and H., and Hahn, 1903, p. 58]. The

yeast-juice thus obtained was, in the first instance, employed for animal

experiments, but underwent change very rapidly. The ordinary anti-septics

were found to be unsuitable, and hence sugar was added as a

preservative, and it was the marked action of the juice upon this addedcane sugar that drew Eduard Buchner's attention to the fact that

fermentation was proceeding in the absence of yeast-cells.As in the case of so many discoveries, the new phenomenon was

brought 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 which enabled him to realise its

importance and give to it the true interpretation.In his first papers [1897, I, 2; 1898], Buchner established the

following facts : (i) yeast-juice free from cells is capable of producingthe alcoholic fermentation of glucose, fructose, cane sugar, and

maltose ; (2) the fermenting power of the juice is neither destroyedby the addition of chloroform, benzene, or sodium arsenite [HansBuchner, 1897], by filtration through a Berkefeld filter,by evaporationto dryness at 30" to 35", nor by precipitation with alcohol; (3) thefermenting power is completely destroyed when the liquid is heatedto 50".

18

ZYMASE AND ITS PROPERTIES 19

From these facts he drew the conclusion "that the productionofalcoholic fermentation does not requireso complicatedan apparatusas the yeast cell,and that the fermentative power of yeast-juiceis dueto the presence of a dissolved substance ". To this active substance

he gave the name of zymase.

Buchner's discoverywas not received without some hesitation. Anumber of investigatorsprepared yeast-juice,but failed to obtain anactive product [Will,1897; Delbruck, 1897; Martin and Chapman,1898 ; ReynoldsGreen, 1897 ; Lintner,1899]. A more accurate know-ledge

of the necessary conditions and of the propertiesof yeast-juice,however, led to more successful results [Will,1 898 ; Reynolds Green,1898; Lange, 1898],and it was soon established that,given suitableyeast,an active preparationcould be readilyprocured by Buchner'smethod. Criticism was then directed to the effect of the admitted

presence of a certain number of micro-organisms in yeast-juice[Staven-hagen, 1897],but Buchner [Buchnerand Rapp, 1897]was able to showby experiments in the presence of antisepticsand with juice filteredthrough a Chamberland candle that the fermentation was not dueto livingorganismsof any kind.

The most weighty criticism of Buchner's conclusion consisted in anattempt to show that the propertiesof yeast-juiceipightbe due to thepresence, suspended in it,of fragments of livingprotoplasm,which,although severed from their originalsurroundingsin the cell,mightretain for some time the power of producing alcoholic fermentation.This, it will be seen, was an endeavour to extend Nageli'stheorytoinclude in it the newly discovered fact.

In favour of this view were adduced the similaritybetween theeffects of many antisepticson living yeast and on the juice,theephemeral nature of the fermentingagent present in the juice,the effectof dilution with water, and the phenomenon of autofermentation whichis exhibited by the juicein the absence 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, IQOO; Wroblewski, 1899, 1901].

A brief generaldescriptionof the actual propertiesof yeast-juiceand of the phenomena of fermentation by its means is sufficient toshow the great improbabilityof this view.

The juicepreparedby Buchner's method forms a somewhat viscousopalescentbrownish-yellowliquid,which is usuallyfaintlyacid inreaction [compare Ahrens, 1900] and almost opticallyinactive. Ithas a specificgravityof 1*03 to ro6, contains 8*5 to 14 per cent.

2*


of dissolved solids,and leaves an ash amounting to i -4 to 2 per cent.About 07 to 1 7 per cent, of nitrogenis present,nearlyall in the formof protein,which coagulatesto a thick white mass when the juiceisheated.

A powerfuldigestiveenzyme of the type of trypsinis also present,sothat when the juiceispreserveditsalbumin undergoesdigestionat a ratewhich depends on the temperature [Hahn, 1898 ;Geret and Hahn, 1898,1,2; 1900 ; Buchner, E. and H.,and Hahn, 1903, pp. 287-340],and isconverted into a mixture of bases and amino-acids. After about six daysat 37",or i o to 14 days at the ordinarytemperature,the digestionis socompletethat no coagulationoccurs when the juiceisboiled. As this pro-teoclasticenzyme, like the alcoholic enzyme, cannot be extracted from

the livingcells,it is termed yeast endotrypsinor endotryptase.Fresh

yeast-juiceproducesa slow fermentation of sugar, which lasts for forty-eightto ninety-sixhours at 25"to 30",about a week at the ordinary

temperature,and then ceases, owing, not to exhaustion of the sugar,but to the disappearanceof the fermentingagent. When the juiceis preservedor incubated in the absence of a fermentable sugar this

disappearanceoccurs considerablysooner, so that even after standingfor a singleday at room temperature, or two days at o",no fermen-tation

may occur when sugar is added. The reason for this behaviour

has not been definitelyascertained. As will be seen later on (p.64)the phenomenon is a complex one, but the disappearanceof the

enzyme was originallyascribed by Buchner to the digestiveaction

upon itof the endotrypsinof the juice[1897,2],and no better explana-tionhas yet been found. Confirmation of this view is afforded by the

fact that the addition of a trypticenzyme of animal origingreatlyhastens the disappearanceof the alcoholic enzyme [Buchner,E. and H.,and Hahn, 1903, p. 126],and that some substances which hinder thetrypticaction favour fermentation [Harden,1903]. The amount offermentation produced is almost unaffected by the presence of such

antisepticsas chloroform or toluene,although some others,such asarsenites and fluorides,decrease it when added in comparativelyhighconcentrations,and it is only slightlydiminished by dilution withthree or four volumes of sugar solution,somewhat more considerablyby dilution with water. When it is filtered through a Chamberlandfilterthe first portionsof the filtrateare capableof bringing aboutfermentation,but the fermentingpower diminishes in the succeedingportionsand finallydisappears.The juicecan be spun in a centrifugalmachine without being in any way altered,and no separationintomore or less active layerstakes placeunder these conditions.


mixed with an equal weight of silver sand and O'2 to 0-3 parts ofkieselguhr,care being taken that this is free from acid. The correctamount of kieselguhrto be added can only be ascertained by experi-ence,

and varies with different samples of yeast. The dry powderthus obtained is brought in portionsof 300 to 400 grams into a largeporcelainmortar and ground by hand by means of a porcelainpestlefastened to a long iron rod which passes through a ring fixed in the

wall (Fig. i). The mortar used byBuchner has a diameter of 40 cm. and

the pestleand rod togetherweigh 8kilos.

As the grindingproceedsthe light-coloured powder graduallydarkensand becomes brown, and the massbecomes moist and adheres to the

pestle,until finally,after two to threeminutes' grinding,it takes the con-sistency

of dough, at which stage the

process is stopped. The mass is nextenveloped in a press cloth and sub-mitted

to a pressure of 90 kilos, per

sq. cm. in a hydraulichand press, the

pressure being very graduallyraisedin order to avoid rupture of the cloth.

The cloth requiredfor I ooo grams of

yeast measures 60 by 75 cm. and is

previouslysoaked in water and thensubmitted to a pressure of 50 kilos,

per sq. cm., retainingabout 35 to 40c.c. of water.

The juiceruns from the press onto a folded filter paper, to remove kieselguhrand yeast cells,and

passes into a vessel standingin ice water.The yieldof juice obtained by Buchner in an operationof this

kind from i kilo, of yeast amounts to 320 to 460 c.c. It may be in-creased

by re-grindingthe press cake and againsubmittingit to pres-sure,and then amounts on the average to 450 to 500 c.c.

Since the cell membranes constitute about 20 per cent, of the

weightof the dry yeast,this yieldcorrespondsto more than 60 percent, of the total cell contents of the yeast. It has been computedby Will [quotedby Buchner, E. and H.? and Hahn, 1903, p. 66]that

FIG. i.

ZYMASE AND ITS PROPERTIES

only about 20 per cent, of the cells are left unaltered by one grindingand pressing,and only 4 per cent, after a repetitionof the process,at least 57 per cent of the cells beingactuallyrupturedby the doubleprocess, and the remainder to some extent altered. It seems probablefrom these figuresthat a certain amount of the juicemay be derived

bmfSiSn"iSPACE oc-

"

-

CUPIHD BY

.,

MASS TO BE'

PRESSED.

FIG. 2.

from the unbroken cells,and Will expresslystates that many un-brokencells have lost their vacuoles.

If the yeast be submitted to a process of regeneration,whichconsists in exposure to a well-aerated solution of sugar and mineral

salts until fermentation is complete,the juicesubsequentlyob*

24 ALUJHUL1C FJiKMJKJNTATlUJN

tained is more active than that yieldedby the originalyeast [Albert1899, i].

A modified method of grinding yeast was introduced by Macfadyen,Morris,and Rowland [1900],who placed a mixture of yeasand sand in a jacketedand cooled vessel,in which a spindlecarryingbrass flangeswas rapidly rotated [Rowland, 1901]. One kilo, oyeast ground in this way for 3-5 hours yielded350 c.c. of juice.

This grindingprocess was at firstadopted by Harden and Younjin their experimentsbut was afterwards abandoned in favour of Buchner's hand-grindingprocess, as it was found liable to yieldjuicesolow fenpentingpower, probablyon account of inefficient cooling dur

ing the grindingprocess. A slightmodification of Buchner's proceshas, however, been introduced, the hand-ground mass being mixecwith a further quantity of kieselguhr until a nearly dry powder iiformed, and the mass packed between two layersof chain cloth itsteel filterplatesand pressedout in a hydraulicpress at about 2 ton:to the square inch (300 kilos, per sq. cm.). The press and platesanshown in section in Fig. 2. It has also been found convenient t"remove yeast cells and kieselguhrfrom the freshlypressedjuice b]centrifugalisationinstead of by filtration through paper, and to waslthe yeast before grindingby means of a filter-press.

Working with English top yeasts Harden and Young have founcthe yieldof juiceextremely variable,the generalrule being that th"amount of juiceobtainable from freshlyskimmed yeast is smaller tharthat yieldedby the same yeast after standingfor a day or two afteibeing skimmed. The yield for 1000 grams of pressedbrewer's yeasvaries from 150 to 375 c.c., and is on the average about 250 c.c.

Very fresh yeast occasionallypresents the peculiarphenomenorthat scarcelyany juice can be expressed from the ground massalthough the latter does not differ in appearance or consistencyfrom "mass which givesa good yield.

Extraction of Zymase from Unground Yeast.

/. Maceration of Dried Yeast.

A valuable addition to the methods of obtainingan active solutionof zymase was made in 1911 by Lebedeff [1911,2 ; 1912, 2 ; see alsc191*1 3" 7i and 1912, i]. This investigatorhad been in the habit o1grindingdried yeast with water for preparingsamplesof yeast-juiceof uniform character and observed that when the dried yeast was

digestedwith sugar solution and the mixture heated,coagulation


took place throughout the whole liquid,the proteinsof the yeasthaving passed out of the cells. Further examination revealed theinterestingfact that dried yeast readilyyielded an active extractwhen macerated in water for some time. The qualityof the resulting" maceration extract " depends on a considerable number of factors,the chief of which are : (i)the temperature of dryingof the yeast ; (2)the temperature of maceration ; (3)the duration of maceration ; and (4)the nature of the yeast,as well as, of course, the amount of water

added in maceration.

In generalthe yeast should be dried at 25"-3O"and then maceratedwith 3 parts of water for 2 hours at 35".

The temperature of maceration may as a rule be varied,withoutdetriment to the productprovidedthat the time of maceration is alsosuitablyaltered; thus with dried Munich yeast, maceration for 4-5hours at 25" is about as effective as 2 hours at 35",whereas treatmentfor a shorter time at 25" or a longertime at 35"produces in generalaless efficacious extract. Yeast dried at a lower temperature than 25"tends to yieldan extract poor in co-enzyme (p.59) and hence of lowfermentingpower, this being especiallymarked at air temperature.

The subsequent treatment of the yeast during maceration may,however, be of great influence in such cases. Thus a yeast dried at

15" gave by maceration at 25" for 4-5 hours a weak extract (yieldingwith excess of sugar 0*33 g. CO2),whereas when macerated at 35" for2 hours it yieldeda normal extract (1*36g. CO2).

The nature of the yeast is of paramount importance. Thus whileMunich (bottom)yeast usuallygivesa good result,a top yeast from aParis brewery was found to yieldextracts containingneither zymasenor its co-enzyme in whatever way the preparationwas conducted.The existence of such yeasts is of great interest,and it was probablydue to the unfortunate selection of such a yeast for his experimentsthat Pasteur was unable to prepare active fermenting extracts andtherefore failed to anticipateBuchner by more than 30 years (see p.1 5). The English top yeasts as a rule give poor results [seeDixonand Atkins, 1913] and sometimes yield totallyinactive macerationextract. It is not understood why the enzyme passes out of the cell

duringthe process of maceration and the whole method givesrise toa number of extremelyinterestingproblems.

Method."

A suitable yeast is washed by decantation, filtered

through a cloth,lightlypressedby means of a hand press, and then

passedthrough a sieve of 5 mm. mesh, spreadout in a layeri-i -5 cm.thick and leftat 25"-35"for two days. Fiftygrams of the dried yeastis

26 ALCJUJtiUJLlC

thoroughlyand carefullymixed with 1 50 c.c. of water in a basin by meansof a spatulaand the whole digestedfor two hours at 3 5". The mass oftenfroths considerably.It is then filtered throughordinaryfolded filter

paper, preferablyin two portions,and collected in a vessel cooled byice. The separationmay also be effected by centrifugingor pressingout the mass, and the maceration may be convenientlyconducted ina flask immersed in the water of a thermostat. It is not advisable to

macerate more than 50 grams in one operation. Under these con-ditions

25-30 c.c. of extract are obtained after 20 minutes' filtration,70-80 c.c. in twelve hours. Dried Munich yeast can be bought fromMessrs. Schroder of Munich and serves as a convenient source of the

extract.1

This extract closelyresembles in propertiesthe juiceobtained bygrindingthe same yeast,but it is usuallymore active and containsmore inorganicphosphate(seep. 46).

2. Other Methods.

Attempts to prepare active extracts from undried yeast in an

analogousmanner have so far not been very successful. Thus Rinckle-ben [1911] found that plasmolysisby glycerol(8 per cent.)or sodiumphosphate(5per cent.)sometimes yieldedan active juiceand sometimesa juicewhich contained enzyme but no co-enzyme, but more often aninactive juice incapable of activation (p. 64) [see also Kayser,19"].

Giglioli[1911 ]by the addition of chloroform also obtained an activeliquid. It appears in fact as though almost any method of plasmoly-sing the yeast cell may yield a certain proportionof zymase in theexudate.

An ingeniousprocess has been devised by Dixon and Atkins[1913]who appliedthe method of freezingin liquidair which theyhad found efficacious for obtainingthe sap from various plantorgans.They thus succeeded in obtainingfrom yeast, derived from Guinness*brewery in Dublin, liquidscapable of fermentingsugar and of aboutthe same efficacyas the maceration extracts prepared by Lebedeff smethod from the same yeast. The results were, however, in both

cases very low,the maximum total productionof CO2 by 25 c.c. of liquidfrom excess of sugar being 32-5 c.c. (airtemperature)or about cro6 g.Munich yeast on the other hand yields,either by maceration orgrinding,a liquidgivingas much as I -5-2 g. of CO2 per 25 c.c., whilst

1 The material suppliedis occasionallyfound to yield an inactive extract and everysampleshould be tested,


Englishyeast-juicepreparedby grindingoften givesas much as 0*5-07 g. ofC02.

No direct comparison with the juiceprepared by grindingwasmade by Dixon and Atkins,but it may be concluded from their resultsthat the best method of obtainingan active preparationfrom the topyeasts used in this country is that of grinding. Maceration,freezingand plasmolysisalike yieldpoor results. With Munich yeast on theother hand the maceration process yieldsexcellent results,whilst theliquidair process has not so far been tried.

Practical Methods for the Estimation of the FermentingPower of Yeast-Juice.

In order to estimate the amount of carbon dioxide evolved in a

given time and the total amount evolved by the action of yeast-juiceon sugar, Buchner adopted an extremelysimplemethod, which con-sisted

in carryingout the fermentation in an Erlenmeyer flask pro-videdwith a small wash-bottle,which contained sulphuricacid and was

closed by a Bunsen valve,and ascertainingthe loss of weight duringthe experiment. Corrections are necessary for the carbon dioxide

present in the originaljuiceand retained in the liquidat the close ofthe experimentand for that present in the air space of the apparatus,but it was found that for most purposes these could be neglected. Incases in which greater accuracy was desired,the carbon dioxide wasdisplacedby air before the weighings were made. A typicalexperi-ment

of this kind,without displacementof carbon dioxide,is the follow-ing:"

March 22, 1899,Berlin bottom yeast V. 20 c.c. juice+ 8 grams cane sugar +0*2 c.c. toluene as antisepticat 16". Grams of carbon dioxide after

24 48 72 96 hours.

0*40 0*64 0*99 z*ii

The total weight of carbon dioxide evolved under these conditionsis termed the fermentingpower of the juice(Buchner).

A more accurate method [Macfadyen,Morris,and Rowland, 1900]consists in passing the carbon dioxide into caustic soda solutionand estimatingit by titration. The yeast-juice,sugar, and antisepticare placedin an Erlenmeyer flask providedwith a straightglasstube,through which air can be passedover the surface of the liquid,and aconductingtube leadinginto a second flask which contains 50 c.c. of10 per cent, caustic soda solution and is connected with the air by aguard tube containingsoda lime. The juicecan be freed from carbondioxide by agitationin a current of air before the flaskis connected to


that containingthe caustic soda solution,and at the end of the periodof incubation air is passed through the apparatus,the liquidbeingboiled out if great accuracy is required. The absorptionflask is thendisconnected and the amount of absorbed carbon dioxide estimated bytitration. This is carried out by making up the contents of the flaskto 200 c.c., takingout an aliquotportion,renderingthisexactlyneutralto phenophthaleinby the addition firstof normal and finallyof deci-normal acid,adding methyl orange and titratingwith decinormal acidto exact neutrality.Each c.c. of decinormal acid used in this last titra-tion

represents0*0044 gram of carbon dioxide in the quantityof solutiontitrated.

These methods are only suitable for observations at considerableintervals of time. For the continuous observation of the course of fef-

Fio. 3.

mentation Harden, Thompson and Young [1910]connect the ferment-ationflaskwith a Schiff s azotometer filledwith mercury and measure the

volume of gas evolved, the liquidhaving been previouslysaturatedwith carbon dioxide (Fig.3). The level of the mercury in the reser-voir

is kept constant by a syphon overflow,as shown in the figure,or,accordingto a modification introduced by S. G. Paine,by a speciallyconstructed bottle providedwith two tubulures near the bottom. Thisensures that no change in the pressure in the flask occurs, and thevolume of gas observed is reduced to normal pressure by means of atable. Before making a readingit is necessary to shake the fermentingmixture thoroughly,as the albuminous liquidvery readilybecomesgreatlysupersaturatedwith carbon dioxide,so much so in fact thatvery littlegas is evolved in the intervals between the shakings.Theexact procedurein making an observation consists in shakingthe flask


about fortytimes as quickly. The total carbon dioxide obtainablefrom the yeast-juice,moreover, correspondsto the fermentation of only2 to 3 grams of sugar, whilst the livingyeast will readilyferment amuch largerquantity,althoughthe exact limit in this respect has notbeen accuratelydetermined. The reasons for this great difference in

behaviour will be discussed later on, after the various factors concerned

in fermentation have been considered (p.123).

(S)Relation of Alcohol to Carbon Dioxide.

In all cases of fermentation by yeast-juiceand zymin, the relativeamounts of carbon dioxide and alcohol produced are substantiallyinthe ratio of the molecular weightsof the compounds, that is as 44 : 46,so that for I part of carbon dioxide I -04 of alcohol are formed. This

has been shown for the juiceand zymin from bottom yeasts by Buchner[Buchner,E. and H., and Hahn, 1903, pp. 210, 21 1],who obtained theratios I'OI, 0*98, 1*01, and 0^99 from experiments in which from 8 to

15 grams of alcohol were produced. Similar numbers, 0*90, 1*12,O'95, 0*91 and 0*92, have been obtained for the juicefrom top yeasts byHarden and Young [1904],who worked with much smaller quantities.The variable results obtained with juicefrom top yeast by Macfadyen,Morris and Rowland [1900],have not been confirmed.

(c)Relation of Carbon Dioxide and Alcohol Produced to the Amountof Sugar Fermented.

The construction of a balance-sheet between the sugar fermented

and the productsformed is of specialinterest in the case of alcoholicfermentation by yeast-juice,because,there being no cell growth as inthe case of livingyeast, an opportunityappears to be afforded of as-certaining

whether the whole of the sugar is converted into alcohol

and carbon dioxide,or whether some fraction of the sugar passes into

any of the well-known subsidiaryproductsof alcoholic fermentation byyeast, such as glycerol,fusel oil,or succinic acid. Unfortunatelythequestion cannot be settled in this way. When the loss of sugarduringthe fermentation is estimated directly,it is usuallyfound to beconsiderablygreater than the sum of the alcohol and carbon dioxideproducedfrom it. This fact was firstobserved by Macfadyen,Morris andRowland [1900],and was then confirmed by Buchner [Buchner,E.and H., and Hahn, 1903, p. 212],in one instance,the excess of sugarlost over productsbeing in this case about 1 5 per cent, of the total

sugar which had disappeared. The matter was then more thoroughlyinvestigatedby Harden and Young [1904].


The conditions under which the experiment must be carried outare not very favourable to the attainment of extreme accuracy. Yeast-

juicecontains glycogen and a diastatic enzyme which converts thisinto dextrins and finallyinto sugar. This process goes on throughoutfermentation,tendingto increase the sugar present and to make the ap-parent

loss of sugar less than the sum of the products. In spiteof this itwas found that a certain amount of sugar invariablydisappearedwithoutbeingaccounted for as alcohol or carbon dioxide,and this whether thefermentation lasted sixtyor a hundred and eighthours, and inde-pendently

of the dilution of the juice. This disappearingsugaramounted in some cases to 44 per cent, of the total loss of sugar, and

on the average of twenty-fiveexperimentswas 38 per cent. Furtherinformation was sought by converting all the sugar-yieldingcon-stituents

of the juiceinto sugar by hydrolysisbefore and after thefermentation. This process revealed the fact that when the glucoseequivalentof the juicebefore and after fermentation was determinedafter hydrolysiswith three times normal acid for three hours (anda cor-rection

made for the loss of reducing power experiencedby glucoseitself when submitted to this treatment),the difference was almostexactlyequalto the alcohol and carbon dioxide produced. In otherwords, accompanying fermentation,a change proceedsby which sugaris converted into a less reducing substance, reconvertible into sugarby hydrolysiswith acids. Similar results were subsequentlyobtainedby Buchner and Meisenheimer [1906],who employed 1-5 normal acidand observed a small nett loss of sugar. Stillmore recentlyLebedeff

[1909, 1910, see also 1913, 2] has carried out similar estimationswith the same result. It is doubtful whether the experimentswhichhave so far been made on this pointare sufficientlyaccurate to decidewith certaintywhether or not the loss of sugar is exactlyequal to thesum of the carbon dioxide and alcohol produced. It has been shown

by Buchner and Meisenheimer [1906]that glycerolis a constant pro-ductof alcoholic fermentation by yeast-juice(p.95),and no other source

for this than the sugar has yet been found, so that it is not improbablethat a small amount of sugar is converted into non-carbohydratesub-stances

other than carbon dioxide and alcohol.

It has also been shown [Harden and Young, 1913] that the deficitof sugar is not due to the formation of hexosephosphate(p.47),which has a lower reduction than glucose,and that the solution fromwhich the sugar (eitherglucoseor fructose)has disappearedactuallycontains some substance of relativelyhigh dextrorotation and of lowreducingpower.


However this may be, it may be considered as Establishedthat

during alcoholic fermentation sugar is converted by an enzyme intosome compound of less reducing power, which againyieldssugar onhydrolysiswith acids. The exact nature of this substance has notbeen ascertained,but it appears likelythat the process is a syntheticalone resultingin the formation of some polysaccharide,possiblyinter-mediate

between the hexoses and glycogen.A similar phenomenon has been observed with livingyeast by

Euler and Johansson[1912,i],and Euler and Berggren [1912],whoseinterpretationof the observation is discussed later on (p.57).

("/)Fermentation of DifferentCarbohydrates.Autofermentation.

Yeast-juiceand zyrpin ferment all the sugars which are fermentedby the yeast from which theyare prepared,and, in addition,a numberof colloidal substances which cannot pass throughthe membrane of the

livingyeast cell,but which are hydrolysedby enzymes in the juiceand thus converted into simplersugars capableof fermentation [Buchnerand Rapp, 1 898, 3 ; 1 899, 2], Of the simplesugars which have been ex-amined,

glucose,fructose,and mannose are freelyfermented,1-arabinose

not at all,whilst the case of galactoseisdoubtful. Galactose is,however,fermented by juicepreparedfrom a yeast which has been " trained " toferment galactose[Harden and Norris,1910]. As regardsboth therate of fermentation and the total amount of carbon dioxide evolved

from glucoseand fructose by the action of a definite arpount of yeast-juice,Buchner and Rapp obtained practicallyidentical numbers.Harden and Young [1909],using juice from top yeast, found thatfructose was slightlymore rapidlyfermented and gave a somewhat

largertotal than glucose,whilst mannose was initiallyfermented atalmost the same rate as glucose,but gave a decidedlylower total,the

followingbeingthe average result : "

Sugar. Relative Rates. Relative Totals.Glucose

....

i I

Fructose.... 1*29 1*15

Mannose.... 1*04 0-67

Among the disaccharides,cane sugar and maltose are freelyfer-mented,and the juicecan be shown like livingyeast to contain inver-

tase and maltase. The extent of fermentation does not differmateriallyfrom that attained with glucose. Lactose is not fermented.

Of the higher sugars raffinose is fermented by juicefrom bottomyeast, but more slowlythan cane sugar or maltose. No experimentsseem to have been made with juicefrom top yeast.


As regardsthe fermentation of the higher carbohydrates,very littleexperimentalwork has been carried out. Buchner and Rapp foundthat the fermentation of starch paste was doubtful,but that solublestarch and commercial dextrin were fermented with some freedom. No

specialstudyhas been made of the diastaticenzymes which bringaboutthe hydrolysisof these substances.

The fermentation of glycogen by yeast-juiceis of considerableinterest,since it is known that the characteristic reserve carbo-hydrate

of the yeast cell is glycogen [seeHarden and Young, 1902,where the literature is cited],and moreover that in livingyeast theintracellular fermentation of glycogen proceeds readily,whereasglycogen added to a solution in which yeast is suspended is notaffected. Yeast-juicecontains a diastatic enzyme which hydrolysesglycogen to a reducingand fermentable sugar, so that in a juicepoorin zymase to which glycogenhas been added, the amount of sugar isfound to increase,the hydrolysisof the glycogen proceedingmorequickly than the fermentation of the resultingsugar [Harden andYoung, 1904],but the course of this enzymic hydrolysisof glycogenby yeast-juicehas not yet been studied. As a rule,it is found bothwith juicesfrom top and bottom yeast that the evolution of carbondioxide from glycogenproceedsless rapidlyand reaches a lower totalthan from an equivalentamount of glucose.

Since nearlyall samples of yeast contain glycogen,yeast-juiceandalso zymin usuallycontain this substance as well as the products of itshydrolysis.These provide a source of sugar which enters into alco-holic

fermentation,so that a slow spontaneous production of carbondioxide and alcohol proceedswhen yeast-juiceis preserved without anyaddition of sugar. The extent of this autofermentation varies consider-ably,

as might be expected,with the nature of the yeast employed *orthe preparationof the material,but is generallyconfined within thelimits of O'o6 to 0*5 gram of carbon dioxide for 25 c.c. of juice.

In juicefrom bottom yeast it amounts to about 5 to 10 per cent,of the total fermentation obtainable with glucose[Buchner,1900, 2],whereas in juicefrom top yeasts,which givesa smaller total ferment-ation

with glucose,it may occasionallyequal,or even exceed, the

glucose fermentation, and frequentlyamounts to 30 to 50 per cent ofit. It is therefore generallyadvisable in studyingthe effectof yeast-juice on any particularsubstance to ascertain the extent of auto-fermentation by means of a parallelexperiment.

The maceration extract of Lebedeff (p.24) is usually,but not in-variably[Oppenheimer,1914, 2],free from glycogen,which is hydro

3


lysedand fermented during the processes of drying and macerating,and therefore as a rule shows no appreciableautofermentation.

(e)EffectofConcentration of Sugar on the Total Amount ofFermentation.

The kinetics of fermentation by zymase will be considered later on

(p.120),but the effect on the total fermentation of different concentra-tionsof sugar, this substance beingpresent throughoutin considerable

excess, may be advantageouslydiscussed at this stage. The subjecthas been investigatedby Buchner [Buchner,E. and H.,and Hahn, 1903,pp. 1 50-8 ; Buchner and Rapp, 1 897] using cane sugar, and he hasfound both for yeast-juiceand for dried yeast-juicedissolved in waterthat (a)the total amount of fermentation increases with the concentra-tion

of the sugar ; (")the initialrate of fermentation decreases withthe concentration of the sugar. The followingare the results of a

typicalexperiment,20 c.c. of yeast-juicebeingemployed in presenceof toluene at 22" :"

The results as to the total fermentations in experimentsof thiskind are liable to be vitiated by the circumstance that when a lowinitialconcentration of sugar isemployed, the supplyof sugar may beso greatlyexhausted before the close of the experiment as to cause amarked diminution in the rate of fermentation and hence an undulylow total. Even allowing,however, for any effect of this kind,the

foregoingtable clearlyshows the increase in total fermentation andthe decrease in initial rate accompanying the increase of sugar concen-tration

from I o to 40 per cent. Working with a greater range ofconcentrations (S'3-53'3grin, per 100 c.c.)Lebedeff has obtainedsimilar results with maceration extract [1911,4],but has found thatthe total amount fermented diminishes after a certain optimum con-centration

(about33-3 grm. per 100 c.c.)is reachedA practicalconclusion from these experimentsis that a high


concentration of sugar tends to preserve the enzyme in an active state

for a longer time. Simultaneouslyit prevents the development ofbacteria and yeast cells.

(/) Effectof Varying Concentration of Yeast-Juice.This subject,which is of considerable importance with reference to

the questionof the protoplasmicor enzymic nature of the active agentin yeast-juice,has been examined in some detail by Buchner [Buchner,E. and H., and Hahn, 1903, pp. 158-65]and by Meisenheimer [1903]for juicesfrom bottom yeast,by Harden and Young [1904] for thosefrom top yeast, and by Lebedeff [1911,4] for maceration extract, theresults obtained being in substantial agreement.

Dilution of yeast-juicewith sugar solution,so that the concentra-tionof the sugar remains constant, produces a small progressive

diminution in the total fermentation, which only becomes markedwhen more than 2 volumes are added, and this independentlyof theactual concentration of the sugar. Dilution with water produces asomewhat more decided diminution,which, however, does not exceed

50 per cent, of the total for the addition of 3 volumes of water. The

effect on maceration extract is somewhat greater but of the same kind.

The autofermentation of juicefrom top yeast is scarcelyaffected bydilution with 4 volumes of water.


On the whole, therefore,yeast-juicemaybe said to be only slightlyaffected by dilution even with pure water, and the effect of the latter

can in no way be regarded as comparable with the poisonous effectwhich it exerts on livingprotoplasm,as suggested by Macfadyen,Morris,and Rowland [1900].

Effectof Antisepticson the Fermentation of Sugars byYeast-Juice.

Buchner has paid specialattention to the effect of antisepticson the course of fermentation by yeast-juice[Buchner and Rapp, 1897 ;1898, 2, 3; 1899, I ; Buchner and Antoni, 1905, I ; Buchner and

Hoffmann, 1907 ;Buchner, E. and H.,and Hahn, 1903, pp. 169-205 ;seealso Albert, 1899, 2 ; Gromoff and Grigorieff,1904; Duchafcek,1909]in order (i)to obtain evidence as to the possibilityof the active agentin yeast-juiceconsistingof fragments of protoplasm and not of asoluble enzyme, and (2) also to provide a safe 'method of avoidingcontamination,by the growth of bacteria or yeasts,of the liquidsusedwhich were often kept at 25" for several days. The results of these

experiments are brieflysummarised in the followingtable,in whichthe effect of each substance on the total fermentation produced isnoted :"

Substance.

Concentrated solution of glycerol)" *" i, sugar

Toluene (tosaturation or excess)Chloroform 0*5 per cent.

"

0-8 per cent, (saturation)"

Large excess (17per cent.)Chloral hydrate0-7 per cent. .

"3'5-5*4 Per cent.

Phenol 0*1 per cent.

Thymol

Benzoic acid

Salicylicacid 0*1""

""27Formaldehyde 0-12

ii 0*24Acetone 6

14Alcohol 6

14Sodium fluoride 0*5

ii

Ammonium fluoride 0*55 per cent.Sodium azoimide,NaN3, 0*36per cent.

*i "f 071 "Quininehydrochloride x "Ozone 10*4-34*8milligramsper 20 c.c.Hydrocyanicacid ra per cent.

Effect on Total Fermentation.

Slightdiminution"

increaseLess than 10 per cent, diminution

SlightincreaseNo change64 per cent, diminutionIncrease up to 27 per cent.

Completely destroyedNo change40 per cent, diminutionCompletely destroyedSlightdiminutionMarked

7 per cent, diminution2610

3520

30-6020

80

0-20

75QO

Almost completelydestroyedCompletelydestroyedSlightdiminutionMarked

"

SlightincreaseMarked diminution

Completelydestroyed


thoroughlydry is found to retain its propertiesalmost unimpairedforat least a year, and can be heated to 85"foreighthours without under-going

any serious loss of fermentingpower [Buchner and Rapp, 1898,4; 1901 ; Buchner, E. and H., and Hahn, 1903, pp. 132-9],

Active powders can also be obtained by precipitatingyeast-juicewith alcohol,alcohol and ether,or acetone. The preparationis besteffected by bringingthe juiceinto 10 volumes of acetone, centrifug-ing at once and as rapidlyas possible,washing,firstwith acetone andthen with ether,and finallydrying over sulphuricacid. The whitepowder thus obtained is not completelysoluble in water but is almost

entirelydissolved by aqueous glycerol(2*5to 20 per cent),forminga solution which has practicallythe same fermenting power as theoriginaljuice. The precipitationcan be repeatedwithout any seriousloss of fermentingpower. Prolonged contact of the precipitatewiththe supernatant liquid,especiallywhen alcohol or alcohol and etherare used, causes a rapid loss of the characteristic property [Albertand Buchner, 1900, 1,2; Buchner, E. and H., and Hahn, 1903,

pp. 228-246 ; Buchner and Duchafcek,1909].Dry preparationscapableof fermentingsugar can also be readily

obtained from yeast without any preliminaryrupture of the cells.Heat alone (yieldinga product known as hefanol)or treatment withdehydratingagents may be used for this purpose, and a brief allusionhas alreadybeen made (p.21) to the different varieties of permanentyeast (Dauerhefe)obtainable in these ways. The most important of theseproducts are the dried Munich yeast (Lebedeff,see p. 25),and thematerial known as zymin, which is now made under patent rightsformedicinal purposes by Schroder of Munich. The latter has proved ofvalue in the investigationof the productionof zymase in the yeast cell

[Buchnerand Spitta,1902],and of many other problemsconcerned withalcoholic fermentation. In order to prepare it 500 grams of finelydivided pressedbrewer's yeast,containingabout 70 per cent, of water,are brought into 3 litres of acetone, stirred for ten minutes, andfilteredand drained at the pump. The mass is then well mixed with

I litre of acetone for two minutes and again filtered and drained.The residue isroughlypowdered,well kneaded with 250 c.c. ofether forthree minutes, filtered,drained, and spread on filter paper or porousplates. After standingfor an hour in the air it is dried at 45" for

twenty-fourhours. About 150 grams of an almost white powdercontainingonly 5 -5to 6-5 per cent, of water are obtained. This is quiteincapableof growth or reproductionbut producesa very considerableamount of alcoholic fermentation,far greater indeed than a correspond-


ing quantityof yeast-juice.Two grams of the powder correspondingto 6 grams of yeast and about 3-5 to 4 c.c. of yeast-juice,are capableoffermentingabout 2 grams of sugar, whereas the 4 c.c. of yeast-juicewould on the average only ferment from one-quarter to one-sixth ofthis amount of sugar. The rate produced by this amount of zymin isabout one-eighthof that given by the correspondingamount of liv-ing

yeast [Albert,1900; Albert, Buchner, and Rapp, 1902]. Theproteoclasticferment is stillpresent in zymin, which undergoes auto-lysisin presence of water in a similar manner to yeast-juice[Albert,1901, 2].

As alreadymentioned an active juicecan be preparedby grindingacetone-yeastwith water, sand,and kieselguhr,and this process presentsthe advantagethat samplesof yeast-juiceof approximatelyconstantcompositioncan be preparedat intervals from successive portionsof auniform supplyof acetone-yeast.

Preparationsof acetone-yeast,made from yeast freed from glycogenby exposure in a thin layerto the air for three or four hours at 35" to45",or eighthours at the ordinarytemperature [Buchnerand Mitscher-lich,1904],show practicallyno autofermentation and may be usedanalyticallyfor the estimation of fermentable sugars.

All the foregoingpreparationsexhibit the same generalpropertiesas yeast-juice,as regardstheir behaviour towards the various sugars,antiseptics,etc.

When zymin is mixed with sugar solution without beingpreviouslyjjround,it exhibits a peculiaritywhich is of some practicalinterestThe time which elapsesbefore the normal rate of fermentation isattained and the total fermentation obtainable vary with the amount of

sugar solution added, the time increasingand the total diminishingasthe quantityof this increases. This phenomenon appears to have beennoticed by Trommsdorff [1902],and a singleexperiment of Buch-ner

shows the influence of the same conditions [Buchner,E. and H.,andHahn, 1903, p. 265, Nos. 700-1]. Harden and Young have foundthat when 2 grams of zyipinare mixed with varyingquantitiesof 10

per cent, sugar solution the followingresults are obtained :"


This behaviourappears to

be due to the removal of soluble matter

essential for fermentation from the cell, which is discussed later on. It

follows that when zymin is being tested for fermenting power, a uni-form

method should be adopted, and all comparative tests should be

made with thesame

volumes of addedsugar

solution. Ground zymin

appears to begin to ferment somewhat more slowly than unground

(2 grm. to 12*4 c.c. of sugar solution in each case), but eventually

produces the same total volume of gas [Buchner and Antoni, 1905, i].

CHAPTER III.

THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION.

IN the course of some preliminary experiments (commenced by thelate Allan Macfadyen, but subsequently abandoned) on the productionof anti-ferments by the injection of yeast-juice into animals, theserum of the treated animals was tested for the presence of such anti-bodies

both for the alcoholic and proteoclastic enzymes of yeast-juice,and it was then observed that the serum of normal and of treated

animals alike greatly diminished the autolysis of yeast-juice.As the explanation of the comparatively rapid disappearance of

the fermenting power from yeast-juice had been sought, as alreadymentioned (p. 20), in the hydrolytic action of the tryptic enzymewhich always accompanies zymase, the experiment was made of

carrying out the fermentation in the presence of serum, with the result

that about 60 to 80 per cent, more sugar was fermented than in the

absence of the serum [Harden, 1903].This fact was the starting-point of a series of attempts to obtain a

similar effect by different means, in the course of which a boiled and

iltered solution of autolysed yeast-juice was used, in the hope that theproducts formed by the action of the tryptic enzyme on the proteinsrf the juice would, in accordance with the general rule, prove to be aneffective inhibitant of that enzyme. This solution was, in fact, found

:o produce a very marked increase in the total fermentation effected

)y yeast-juice,the addition of a volume of boiled juice equal to that)f the yeast-juice doubling the amount of carbon dioxide evolvedHarden and Young, 1905, i]. This effect was found to be commono the filtrates from boiled fresh yeast-juice and from boiled autolysed'east-juice,and was ultimately traced in the main, not to the anti-ryptic effect which had been surmised, but to two independent factors,dther of which was capable in some degree of bringing about the

"bserved result.

Boiled yeast-juice was indeed found to possess a decided anti-.utolyticeffect, as determined by a comparison of the amounts of

litrogen rendered non-precipitable by tannic acid in yeast-juicealone4*


and in a mixture of yeast-juiceand boiled juice on

alcoholic fermentation

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gas evolved

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