Fermentation Processes Leading to Glycerol

I. The Influence of Certain Variables on Glycerol Formation inthe Presence of Sulfites

G. G. FREEMAN AND G. M. S. DONALD

Imperial Chemical Industries Limited, Nobel Division, Research Department, Stevenston, Ayrshire, Scotland

Received for publication June 28, 1956

The bulk of the glycerine used in industry is obtainedas a by-product of soap manufacture by hydrolysis offats; however, the synthetic process from propylenerecently developed by the Shell Chemical Corporationis now making an important contribution towards thetotal supply. This process has been described by Minerand Dalton (1953). Since World War II, it has beenevident that glycerine demand is tending to outstripproduction from saponification of fats. The main factorswhich have led to this situation are the increasingdemand for glycerine for production of explosives, tex-tiles, transparent paper (cellophane), paints, and soforth, and the impact of the synthetic detergent in-dustry upon soap production. The work described inthis series was part of a program which involved de-velopment to a pilot plant scale of a process for glycerolproduction by fermentation and isolation of the productfrom the fermented liquor undertaken by the NobelDivision of Imperial Chemical Industries Limited.

Glycerine was first reported as a minor product ofyeast fermentation of sugars in 1858 by Pasteur whoobserved that it was present in wines and beers to theextent of 2.5 and 3.6 per cent of the sugar fermented.Following the fundamental work of Neuberg and Rein-furth (1918, 1919) on the mechanism of alcoholic fer-mentation of sugars in the presence of sulfites and theindependent application of the process by Connsteinand Luidecke (1919) to the commercial scale productionof glycerine from beet sugar in Germany during WorldWar I (Protol process), developmental work on re-covery of glycerol from fermented liquors has beencarried out in Britain, in the United States and else-where at intervals since 1920. The use of a mixture ofsulfite and bisulfite instead of sodium sulfite as thesteering reagent was introduced by Cocking and Lilly(1921), and a further patent was filed in 1931 (Im-perial Chemical Industries Limited and Lilly, 1931).The sparingly soluble sulfites of calcium and magnesiumhave been recommended as steering reagents in orderto restrict the quantities of inorganic impurities in thefermented liquor (Fulmer et al., 1945; Underkofler et al.,1951a, b). The use of mixtures of ammonium sulfiteand bisulfite was also suggested by Fulmer et al. (1941)as a means of simplifying the recovery process, but

later work by these authors showed that the fermenta-tion was unsatisfactory in the presence of ammoniumsalts (Underkofler et al., 1951a).A fermentation process for glycerol based on Neu-

berg's third form of ftrmentation was described byEoff (1918) and Eoff et al. (1919). In this process yeastfermentation of sugars takes place in the presence ofabout 30 per cent of sodium carbonate, based on theweight of fermentable sugar. So far as is known thisprocedure was never developed to a commercial scale.There are numerous reviews on production of glyc-

erine by fermentation. They include the work of Pres-cott and Dunn (1949), a recent review by Underkofler(1954) and a series of abstracts of articles and patentsby Whalley (1942). An important new development inthis field is concerned with the production of glycerolby osmophilic yeasts in the absence of steering reagents.Glucose concentrations up to 29 per cent were fermentedin about 10 days with production of glycerol and D-arabitol in yields of 32 and 17 per cent respectively,in terms of glucose utilized (Spencer, 1955; Spencerand Sallans, 1956; Spencer et al., 1956). Another fer-mentation of glucose leading to glycerol (together with2,3-butyleneglycol), in the absence of steering re-agents, proceeds in the presence of Ford's strain ofBacillus subtilis (Neish et al., 1945).

In this paper experiments on the effect of variableson the kinetics of fermentation and yields of productsin the sulfite fermentation of sugars are described.These variables include sulfite dosage and sulfite con-centration, initial sugar concentration, temperature,aeration, pH, and yeast strain. In later papers in thisseries, studies on (a) the effect of sulfite on yeast growth,yeast viability, rate of fermentation and other factors(Freeman and Donald, 1957a), and (b) the effect ofyeast strain and other variables on the kinetics andyields of products of fermentation of sugars in thepresence of alkalis (Freeman and Donald, 1957b) aredescribed.

EXPERIMENTAL METHODS

Fermentation VesselsFermentations were carried out in cylindrical stain-

less steel tanks (25 cm. id. by 40 cm high) of about 12 L197

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capacity. A stainless steel lid was attached by 4 screwsand was fitted with a rubber gasket to ensure gastightness. The lid was fitted with 2 glass inspectionports and carried a paddle-type stirrer. A gas inlettube entered the vessel through the lid and was pro-vided with a cotton wad filter and sintered glass dis-perser (Aeroxl). Effluent gases were allowed to escapethrough an outlet tube, plugged with cotton. Beforesterilization by passage through the cotton filter, gasentering the vessel was passed through a Rotameter2flow-meter, calibrated to measure flows in the range of5 to 100 L/hr. Sulfite additions were normally madefrom a measuring cylinder through the effluent gasport and were accompanied by vigorous mechanicalstirring of the fermenting liquor. The fermentationvessels were immersed in electrically heated, thermo-statically controlled water baths with the liquid levelsinside and outside the fermentors approximately equal.The preferred process, developed in the course of the

work, is described below. Variations of the standardprocedure are stated in the relevant experiments.Preliminary experiments showed that pure cultureconditions were unnecessary after sulfite addition hadbegun.

Preparation of InoculumA culture medium of beer wort (sp gr 1.050; pH 6.0;

100 ml) was sterilized by autoclaving (15 lb/sq in for20 min), cooled, inoculated from a stock culture of thedesired yeast strain and incubated for 1 to 3 days at35 C with moderate aeration with air. At the end ofthis period, the cell count was 100 X 106 cells per ml.The main inoculum medium (680 ml) was prepared

from diluted Cuban blackstrap molasses and containedinitially 5 g/100 ml of reducing sugars as invert sugar.It had the following composition: Cuban blackstrapmolasses, 100 g; sodium carbonate (Na2CO3), 1 g; diso-dium hydrogen phosphate, 2.2 g; ammonium sulfate,1.3 g; and water to 1000 ml; pH 6.7. The medium wassterilized by autoclaving, inoculated from the beer wortculture (20 ml to 680 ml) and incubated for 24 to 36hr at 35 C with moderate aeration with air. At the endof this period, the culture contained approximately200 X 106 yeast cells/ml. The preferred yeast strain,B.71 in the laboratory collection, was obtained inMarch, 1950, from an industrial alcohol distillery. Thisculture was "acclimatized" to tolerate a concentrationof 6.6 g/100 ml (as Na2SO3) of sodium sulfite-bisulfitesolution at pH 6.7.

Preparation of Fermentation Medium

The Cuban blackstrap molasses used in this workhad the following composition: total copper reducing

1 Aerox Limited, Glasgow, Scotland.2 Rotameter Manufacturing Co., Ltd., Croydon, Surrey,

England.

substances as invert sugar, 53.5; unfermentable copperreducing substances as invert sugar, 3.6; and organicimpurities (by difference), 15.4 per cent.The fermentation medium had the following compo-

sition: Cuban blackstrap molasses, approx. 450 g;anhydrous sodium carbonate, approx. 5 g; disodiumhydrogen phosphate, 2.5 g; ammonium sulfate, 1.4 g;and water to make 1000 ml; pH 6.8. The molasses wasdissolved in tap water and sufficient sodium carbonatesolution (16 per cent, wt/vol) added to adjust the pHto 6.8. The concentration of molasses was adjusted sothat the initial reducing sugar concentration (as invertsugar) of the fermentation medium, after addition ofthe inoculum was 20 to 22 g/100 ml. The fermentationvessels contained 7 L of medium. Each vessel, com-plete with stirrer and air filter, was pasteurized bysteaming at 100 C for 45 min. Fermentations werenormally carried out in sets of four.

Sulfite SolutionsSulfite addition to the fermentations was either in

the form of sodium sulfite or a mixture of sodium sul-fite and sodium bisulfite. The stock solution of theformer contained 30 g/100 ml of Na2SO3 and its pHwas 9.2 to 9.4. Mixed sulfite solution was prepared (a)by passage of sulfur dioxide into a hot solution ofsodium carbonate (26 g/100 ml) until the pH fell topH 6.7 or (b) by mixing the appropriate quantitiesof sodium sulfite and sodium bisulfite, as follows:Na2SO3, 236 g; NaHSO3, 62, g; and water to 1000 ml;pH 6.7. In some experiments other proportions ofsulfite and bisulfite than the approximately 80:20 mix-ture as above, for example 50:50, were used. Sulfite-bisulfite mixtures with a total sulfite concentration of25.7 g/100 ml as Na2SO3 equivalent had a pK of 6.1.

General Fermentation TechniqueEach fermentation vessel was inoculated with 700 ml

of inoculum culture. After inoculation the fermentationmedium was mechanically stirred and aerated with air(30 L/hr, approximately 4 vol/vol/hr) for 30 min topromote yeast growth. After a further period of 3 to4 hr, vigorous fermentation had been established, thepH value had fallen to 6.0 and the hemocytometercell count had risen to about 30 X 106 cells/ml. Sulfiteadditions were then begun in portions of approximately1 per cent by volume of the fermenting liquor (initially70 ml; later 100 ml) of the 30 g/100 ml solution (asNa2SO3), equivalent to approx. 0.3 g/100 ml NasSO3equivalent in the fermentation medium. After eachsulfite addition, visible fermentation was inhibited fora period and the fermentation was allowed to becomevigorous again before a further addition was made. Theinterval between successive sulfite additions was nor-mally 30 min to 1 hr. In order to reduce to a minimumthe time of exposure of the yeast culture to sulfite-

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bisulfite mixtures at low pH values (which have a

markedly toxic effect) the first 4 to 6 additions were ofsodium sulfite solution (pH 9.2). When the pH of thefermenting medium had been restored by this means to6.6, subsequent additions were of sulfite-bisulfite mix-ture (pH 6.7). These additions were continued untilthe desired sulfite equivalent concentration (usually3.0 to 3.5 g/100 ml) was reached. This level was thenmaintained by the periodic addition of sulfite solution,as required, until the desired total quantity (usually40 per cent of the total fermentable sugar) had beenadded. During the period of sulfite addition the pH ofthe fermenting medium remained fairly constant withinthe range 6.7 to 7.0 until the sugar concentration hadfallen to about 5 g/100 ml, after which there was a

further rise to pH 7.1 to 7.4 during the final slow phaseof fermentation.

Addition of sulfite was normally complete after 27 to33 hr from inoculation and fermentation was completein approximately 120 hr. Determination of reducingsugar was carried out at intervals for observation ofthe progress of fermentation. The yields of fermentationproducts were calculated as a percentage of the fer-mentable sugar present. The latter was determined byyeast fermentation in the absence of sulfite as describedby Donald et al. (1953). The fermentation temperaturewas 35 C.

Analytical Methods

pH determinations were made by the glass electrodemethod with a Cambridge pH meter.3 Sulfite concen-

trations in fermentation liquors and steering reagentsolutions were determined by iodimetric titration inthe presence of an excess of hydrochloric acid. Resultsare expressed throughout in terms of sodium sulfiteequivalents. Non-sulfite, iodine reducing substanceswere also present in molasses fermentation liquors. A"sulfite" determination carried out on a typical fermen-tation liquor before inoculation gave an apparentsodium sulfite equivalent (as Na2SO3) of 0.32 g/100 ml.No correction has been made for this since it was notknown how the quantities of iodine reducing substancesvaried during the fermentation. Reducing sugars

were determined by a Fehling-Soxhlet-permanganatemethod. Proteins and suspended matter were removedby treatment with a slight excess of neutral lead acetatesolution and the excess lead precipitated as lead oxalate.Any sucrose present in the clarified filtrate was invertedby heating with hydrochloric acid and the reducingsugars determined as invert sugar by a combination ofthe methods of Munson and Walker (1906) and Ber-trand (1906). Cuprous oxide precipitated under theconditions specified by Munson and Walker was filteredoff, washed, redissolved in ferric sulfate solution and

3Cambridge Instrument Co., Ltd., London, England.

determined by titration with potassium permanganate.A small blank value on the reagents was deducted fromthe titers. The procedure for clarification and removalof proteins in the case of another molasses fermentationhas been described in detail by Freeman and Morrison(1946).Determination of glycerol in fermented liquors is a

difficult problem which has only recently been satis-factorily solved. In the analytical mnethods used byearlier workers, attempts were made to separate glyc-erol from organic impurities derived from molasseseither (a) by solvent extraction or (b) by distillation.Solvent extraction methods such as acetone extractionfollowed by determination of total acetyl value tendto give high results owing to incomplete elimination ofhydroxylic impurities. Methods based on separation ofglycerine by distillation give low results due to its lowvolatility. The method used for the bulk of the presentwork, a kerosene distillation method, developed in1938 by our colleagues R. A. Walmesley and R. H.Mathew and privately communicated to the authors, isbased on the following three steps: (a) extraction of aweighed amount of sample, mixed with sodium sulfate,by means of hot acetone, (b) removal of acetone andseparation of the glycerol by distillation in the presenceof kerosene and (c) extraction of glycerol from thedistillate with water, and its determination by oxida-tion with potassium dichromate. In order to determinethe accuracy of this method when applied to fermen-tation liquors, a series of "synthetic" samples was pre-pared by addition of pure glycerol and sulfite-bisulfiteliquor to molasses fermentation liquors prepared bynormal alcoholic fermentations in the absence of sulfites.A small quantity of glycerol was formed in this fer-mentation, approx. 1.0 g/100 ml, and corrections weremade for it. The "synthetic" samples were preparedas follows: Cuban blackstrap molasses was dilutedwith water and inoculated with yeast as describedabove so that the initial reducing sugar concentrationafter inoculation was 22 g/100 ml as invert sugar.Fermentation at 35 C took place for 5 days. The initialpH in this experiment was pH 5.0. The yeast was re-moved by filtration and to the fermented solution(350 ml) was added 30 per cent (wt/vol) sulfite-bisulfitesolution (pH 6.7) (100 ml), and an accurately weighedquantity of pure glycerol (20 g). The mixture wasdiluted to 500 ml. Glycerol concentrations, calculatedon this basis and found by the kerosene distillationmethod, in a series of "synthetic" samples, are tabu-lated in table 1. In calculating the concentration ofglycerol present, a value for the glycerol formed in theethanolic fermentation liquor of 0.96 g/100 ml wasfound by the kerosene distillation method and addedto the known quantity of pure glycerol. Column 2 inthe table gives the calculated glycerol values obtainedin this way. The experimental value by the kerosene

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distillation method was 7 per cent low on this basis. Onthe assumption, which may not be strictly true, thatthe error was constant irrespective of glycerol concen-tration or ratio of impurities to glycerol, the value of0.96 g/100 ml was increased by 7 per cent to give 1.03g/100 ml as the glycerol produced by fermentation andthe total glycerol content of the "synthetic" samplesvorrespondingly increased (column 3). On this basis, itwas concluded that, on the average, the kerosenedistillation method gave results on sulfite fermentationliquors which were 8.2 per cent low. Except whereotherwise stated, the analytical data quoted below(obtained by the kerosene distillation method) areuncorrected.With correction by the above factor, the kerosene

distillation method can be relied upon to give accurateand consistent values for glycerol content of sulfitefermentation liquors. When large numbers of analyseswere necessary, however, the method proved to belaborious and time consuming and, during the courseof the present investigation, work was in progress inthis laboratory on alternative methods for glyceroldetermination in fermented liquors. This led to thedevelopment of a chromatographic-periodate methodby Sporek and Williams (1954). In principle this methodis similar to that of Neish (1950), in which glycerol inmilligram amounts was separated from the impuritiespresent in fermentation liquors by chromatography anddetermined by a colorimetric procedure. Sporek andWilliams showed that the glycerol values found for aseries of 9 sulfite fermentation liquors from Cubanblackstrap molasses were in good agreement with the

TABLE 1. Determination of glycerol in "synthetic" fermenta-tion liquors by the kerosene distillation method

Known quantities of glycerol and sulfite-bisulfite wereadded to ethanolic fermentation solutions from blackstrapmolasses to simulate the product of the sulfite fermentation.

Calculated Glycerol ErrorConcentration

(1) (2) GlycerolSample Based on Based on cor- Concentra-Reference No. uncorrected rected value Foon As com- As com-

value for (X 1.07) for oun pared with pared withglycerol in glycerol in (1) (2)fermented fermentedliquor liquor

g/10O ml g/100 ml g/100 ml % %B12/41/1 4.76 4.81 4.49 -5.7 -6.7

2 4.81 4.86 4.47 -7.1 -8.03 4.80 4.85 4.45 -7.3 -8.34 4.10 4.15 3.90 -4.9 -6.05 5.21 5.26 4.94 -5.2 -6.1

B32/14/1 4.24 4.28 3.94 -7.1 -8.02 4.51 4.55 4.16 -7.9 -8.63 4.47 4.51 3.99 -10.7 -11.54 4.82 4.87 4.37 -9.3 -10.35 4.05 4.10 3.76 -7.2 -8.3

Mean error .............................. -7.2 -8.2

corresponding results by the kerosene distillationmethod, after the latter had been corrected by thefactor 1.07. Similarly, a fermentation liquor preparedfrom Cuban blackstrap molasses by the method ofEoff et al. (1919) gave uncorrected values for glycerolby the kerosene distillation method of 3.1, and 3.3per cent and values of 3.4 and 3.4 per cent by thechromatographic method. The corrected value for thekerosene distillation method (3.46 per cent) is in goodagreement with the chromatographic data.As a preliminary step in determination of acetalde-

hyde and ethanol in fermented liquors, acetaldehydewas liberated from the acetaldehyde sodium bisulfitecomplex by addition of the theoretical quantity ofbarium chloride to precipitate free sulfite, and an ex-cess of calcium carbonate. The mixture was distilledin a current of steam and the volatile components werecondensed in a long spiral tube cooled in ice. Acetalde-hyde and ethanol were determined in the distillate, theformer by Ripper's (1900) method and the latter byoxidation with excess potassium dichromate (Janke andKropacsy, 1935); in the ethanol determination a cor-rection was made for the known acetaldehyde contentof the sample.

Yeast cell counts were determined by the hemocytom-eter method. This procedure was rapid and in generalsatisfactory, although it was found that lengthy expo-sure to sulfite solutions caused clumping of the cellswhich made accurate counting difficult in certain cases.

Effect of Variables on Kinetics of Fermentationand Yields of Products

(a) Effect of sulfite dosage and concentration. A seriesof fermentations was carried out with sulfite dosagesranging from 5 to 50 per cent (based on fermentablesugar). A maximum free sulfite concentration of 3.0 to3.5 g/100 ml was established in the fermenting liquorin the experiments with sulfite dosages of 25 to 50 percent. When the dosage was less than 25 per cent cor-respondingly lower maxima were reached. The relation-ships between yields of glycerol, acetaldehyde, ethanol,and acetic acid and sulfite dosage are plotted in figure 1.This figure includes a theoretical curve for acetalde-hyde yield, (glycerol yield - acetic acid yield X 184/60)44/92, on the assumption that glycerol not formed byNeuberg's third form of fermentation is stoichio-metrically related to acetaldehyde production. Glyceroldeterminations were by the chromatographic-periodatemethod. Data for glycerol and acetaldehyde yields atsulfite dosages of 75 and 150 per cent from Neuberg andReinfurth (1918) are included in the figure. Withinthe 25 to 50 per cent range of sulfite dosage, fermen-tation time (5 days) was not significantly influenced.

In a further series of fermentations, the maximumfree sulfite concentration in the fermenting liquor wasvaried from 2.0 to 5.0 g/100 ml at a constant sulfite

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dosage of 43 per cent. The lower sulfite concentrationswere reached early in the fermentation but the highestconcentrations were attained only for a short time nearthe end of the period of sulfite addition. There was anincrease of glycerol yield from 22.3 to 25.6 per cent ofthe fermentable sugar when the maximum free sulfiteconcentration was increased from 2.0 to 4.0 g/100 ml.Fermentation time also increased with increase ofmaximum free sulfite concentration. It was concludedthat the most satisfactory maximum sulfite concen-tration was 3.5 g/100 ml.

(b) Effect of pH of sulfite solution. pH changes duringfermentation. At the end of the "prefermentation"period, when addition of sulfite was begun, the pH ofthe fermenting liquor was about pH 6.0. As the toxiceffect of sulfite-bisulfite solutions is markedly enhancedat low pH values because of the greater proportiorn ofbisulfite present, it was found necessary to restore thereaction to about pH 6.7 as rapidly as possible in orderto minimize toxic effects on the yeast.When the initial additions of steering reagent were in

the form of sodium sulfite (pH 9.2) or sodium carbonate

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solution (30 per cent wt/vol) instead of sulfite-bisul-fite (80:20; pH 6.7) a more vigorous fermentation wasobtained and the total time was reduced; the yield ofproducts was unaffected. When sodium sulfite solution(pH 9.2) was used as steering reagent throughout thefermentation, there was no significant effect on theyields of products as compared with fermentations inwhich a sulfite-bisulfite mixture (pH 6.7) was used. Inspite of the difference in pH values of these solutions,when equilibrium was established there was little effecton the pH of the fermenting liquor owing to the buffer-ing effect of carbonates derived from fermentationcarbon dioxide, organic impurities in the molasses andother components of the fermenting liquor. Thus thefinal pH of the fermented liquor was pH 7.3 whenisodium sulfite was used as steering reagent as comparedwith a mean pH range of 7.1 to 7.4 in the case of a largenumber of mixed sulfite fermentations.

Details of pH changes in the fermenting liquor undervarious conditions of fermentation are summarizedin table 2.The effect of sulfite-bisulfite solutions of pH 6.0

SULPHITE DOSCAGE

(PERPCENr 0FF' RMENTANLE SUGA.)FIG. 1. Effect of variation of sulfite equivalent dosage on yields of products in the mixed sulfite fermentation of molasses. Fermen-

tations were carried out by the standard method.

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(approx. composition: Na2SO3, 45 parts; NaHSO3, 55parts) and pH 6.3 (approx. composition: Na2SO3, 64parts; NaHSO3, 36 parts) was investigated in experi-ment 56 (table 3); the pH of the fermenting liquors wasrestored to pH 6.7 by means of sodium sulfite solution(pH 9.2). The use of these steering reagents had no

appreciable effect on the yields of products in terms ofsugar fermented, and the toxic effect of the higher bisul-fite concentrations was shown in the lowered reducingsugar attenuations and greatly increased fermentationtime. For comparative purposes, the mean values fromrepresentative mixed sulfite fermentations are also sum-marized in table 3.

(c) Effect of mode and time of initial sulfite addition.Addition of sulfite solution was normally begun as soon

as vigorous fermentation was visible. Some difficultywas experienced in determining this point precisely and,as premature addition of sulfite considerably retardedfermentation, an experiment was carried out to deter-mine the effect of a delay of 3 hr in making the illitial

addition of sulfite. In four parallel fermentations, initialsulfite additions were made 0, 1, 2, and 3 hr after vigor-ous fermentation was first observed. There was no

effect on the yields of products.In experiments 58/3 and 4, sulfite-bisulfite solution

(pH 6.7) was added continuously instead of at approxi-mately hourly intervals, the rate being adjusted so thataddition was complete in the normal period of 30 hrfrom inoculation. In spite of the lower free sulfite con-

centrations reached (maxima 2.7 and 2.1 g/100 inl,respectively) especially in the early stages, the glycerolyields (mean 25.0 per cent) were normal. The time offermentation was slightly reduced to 100 hr.

(d) Effect of initial fermentable sugar concentration.The effect of initial fermentable sugar concentrationsin the range 16 to 30 g/100 ml as invert sugar was inves-tigated in experiments 41/1-4 and 58/1 and 2 (table4). A sharp increase of time to completion of fermen-tation and of residual unfermented reducing sugar

concentration occurred in fermentations with initial

TABLE 2. pH Changes during the course of fermentations in the presence of various steering reagents

Experimental Conditions. Steering Reagent Used for: pH of Fermenting LiquorExperiment Sulfite

No.Initial additions to restore pH to 6.7 Remainder of additions

|sg0 hr 11M4 hr 12 hr 154 hr 15YAhr 23i hr 37 hr 58 hr Final: 130

hr

33/1 Na2SO3, pH 9.2 Na2SO3, pH 9.2 50 6.8 6.0 6.3 6.5 6.8 7.2 7.3 7.1 7.3

o hr 10 hr 11'4 hr 15% hr 16 hr 24 hr 35 hr 42 hr Final: 106

32/1 Na2SO3, pH 9.2 Mixed sulfite, pH 6.7 50 7.0 6.2 6.3* 6.9 7.0 6.9 7.5 7.3 7.832/2 Na2CO3 Mixed sulfite, pH 6.7 50 7.0 6.5 6.5 -1 - 6.8 7.4 7.2 7.5

ohr 11 hr 17Y4 hr 22 hr 33 hr 34 hr 58 hr Final: 144

28/1 Mixed sulfite, pH 6.7 Mixed sulfite, pH 6.7 50 7.0 6.0 6.3 6.6 7.2 7.1 7.1 7.2

* After this point, additions were of sulfite-bisulfite solution (pH 6.7) instead of sodium sulfite or sodium carbonate.

TABLE 3. Influence of pH value of sulfite-bisulfite solution in the mixed sulfite fermentationThe following steering reagents were compared (a) sodium sulfite, sodium bisulfite mixture (45/55, pH 6.0), (b) sodium sulfite,

sodium bisulfite mixture (64/36, pH 6.3) and (c) sodium sulfite, sodium bisulfite mixture (80/20, pH 6.7). The pH was restored topH 6.7 at the end of the prefermentation period by addition of sodium sulfite pH 9.2. Glycerol determinations were by the kero-sene distillation method.

Yields of Products, Per Cent ofFermentable Sugar Fermentable Time for Com-

Experiment No. Details Sulfite Dosage _ Sugar pletion of Fer-

Glycerol Ethanol Acetalde- Attenuation mentationGlyceol Etanolhyde

.% ...% . .....hr

56/1 Sulfite-bisulfite solution (pH 6.0) as steering rea- 40 23.3 15.9 9.6 86 28956/2 gent except for initial additions 21.7 17.8 8.8 97 289

56/3 Sulfite-bisulfite solution (pH 6.3) as steering rea- 40 23.1 18.6 11.3 89 28956/4 gent except for initial additions 24.3 19.0 10.5 85 121

Mean of large number of representative mixed sul- 40 25 21 10 93 120fite fermentations, with sulfite-bisulfite solution(pH 6.7) as steering reagent except for initial ad-ditions

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reducing sugar concentrations higher than 22 g/100 ml.The glycerol yield decreased slightly at this point,probably as a result of the lower sugar attenuation.The acetaldehyde yield (10 to 12 per cent) remainedfairly constant throughout and the ethanol yield,although fairly constant (21 to 23 per cent) at initialsugar concentrations up to 25 g/100 ml, fell markedlyto 15 per cent when the initial sugar concentrationwas raised to 30 g/100 ml.

(e) Effect of yeast strain. Seven strains of Saccharo-myces cerevisiae from a variety of sources in the UnitedKingdom were compared in mixed sulfite fermentationswith the preferred strain B.71. A marked variation wasobserved in the sulfite tolerance of some of the strains,which led to considerable variation in rate of fermen-tation and degree of reducing sugar attenuation (table5). Two strains of Saccharomyces thermantitonumwere also examined in mixed sulfite fermentations.These strains were listed in the Institute of BrewingYeast collection, 1949 (Institute of Brewing, 1949).These four fermentations were carried out at 40 C,which has been shown to be the optimum for fermenta-tion of S. thermantitonum in the absence of sulfite byvon Euler and Laurin (1919, 1920). This species provedto be very sensitive to sulfite and fermentation was slowand incomplete even at sulfite concentrations of 0.6g/100 ml (table 5).

In experiment 51, yeast strain B.71, "acclimatized"to the presence of 6.6 g/100 ml of sulfite-bisulfite mix-ture at pH 6.7 and subcultured for one year in thepresence of sulfite, was compared with another culturefrom the same source which had not previously beengrown in the presence of sulfite. No significant dif-ference was observed in the fermentations obtainedwith the two cultures.

TABLE 4. Effect of initial reducing sugar concentrationA series of diluted molasses media with initial reducing

sugar concentrations in the range 16 to 30 g/100 ml, as invertsugar, were fermented under comparable conditions. Theadded inorganic nutrients were kept constant as described inthe standard conditions. Glycerol determinations were bythe kerosene distillation method.

ObservedYields of Prod- Hexose

Initial ucts CeonenrtoExperi- Initial Reducing Timen- Concentration

met Reducing Sugar At- Fermen- a e

No. Sugar - tenuation tationConcentration Ac-

Glyc- Eth- etal- 73 hr 191 h rerol anol de-

hyde

g/100 ml % % % % hr gilOO ml

58/1 16.2 23.8 23.2 10.1 92.2 12158/2 18.9 25.4 23.0 9.6 92.4 121

41/1 15.7 25.2 21.7 12.2 93.7 73 1.7 1.741/2 20.8 25.7 22.6 10.0 94.4 73 2.0 2.041/3 25.8 24.8 21.2 9.7 91.6 167 4.8 2.841/4 29.8 23.5 15.2 9.9 76.8 191 10.3 6.2

(f) Effect of temperature of fermentation. A series offermentations was carried out at temperatures of 25,30 and 35 C, from which it was concluded that theoptimal temperature lay in the range of 30 to 35 C.

(g) Effect of re-use of yeast crop. It was shown in aseries of experiments that the precipitate of yeast cellswhich settled to the foot of the fermentation vesselcould be re-used to carry out at least 3 further fermen-tations without special treatment to stimulate celldivision. This procedure resulted in slight increases inglycerol yield and rate of fermentation. On completionof each successive fermentation in the series, the yeastcrop was allowed to settle to the bottom of the vesseland the fermented liquor was decanted. Fresh medium,previously pasteurized, was then added and the yeastprecipitate was dispersed in the liquid by mechanicalstirring and aeration with air. Vigorous fermentationwas observed after 2 to 4 hr.

(h) Relationship between hexose fermented and glycerolformation during the course oj the fermentation. Theintroduction of a chromatographic-periodate method ofglycerol determination (Sporek and Williams, 1954),in which it was possible to separate glycerol from rela-tively large quantities of unfermented hexose enabledthe relationship between hexose fermentation andglycerol formation to be investigated throughouttypical fermentations. Errors introduced by samplingduring the period of sulfite addition were minimized bycarrying out this work in the course of a 27,000 L pilotplant scale fermentation. Samples of the fermentingliquor were withdrawn at intervals for determinationof reducing sugar, free sulfite, and glycerol concen-trations (figure 2). In this figure, the glycerol yield isplotted both as the total yield to the time of samplingand also as the mean yield during the period betweenconsecutive samplings; in each case the yields are cal-culated as percentages of the reducing sugar fermentedin the corresponding period. The proportion of hexoseconverted to glycerol rose rapidly to a maximum (46 percent) after 20 hr from inoculation and thereafter fellsteadily to zero after 96 hr, although slow fermentationwas still in progress at that time. The maximum con-version of hexose to glycerol coincided approximatelywith the mnaximum free sulfite concentration in thefermenting liquor.

(i) Investigation of a continuous fermentation pro-cedure. A series of 12 fermentations was carried out toinvestigate a continuous fermentation procedure inin which each fermentation, after the first in the series,was inoculated with a portion of the actively fermen-ting liquor from the previous fermentor. The initialfermentation (7,000 ml) was inoculated with a culture(700 ml) of "acclimatized" yeast (strain B.71). After24 hr, addition of sulfite was almost complete. Approxi-mately one-half (3,500 mnl) of the vigorously ferment-ing liquor was then transferred to a second fermentor

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containing an equal volume of diluted molasses medium(28 g/100 ml as invert sugar) so that the reducing sugar

concentration after inoculation was 20 to 22 g/100 ml.After a further 24 hr, a portion of this liquor was usedto inoculate the next fermentor of the series in a similarmanner. Fermentation was allowed to continue in theremainder of the liquor in the usual way.

The initial free sulfite concentration in fermentations3 to 12 was in the range 1.7 to 1.9 g/100 ml. The yeastcell population in the fermenting liquors fell from initialand final counts of 30 X 106 and 60 X 106 cells/nil,respectively, in the first fermentation to 10 X 106and 20 X 106f cells/ml, respectively, from the thirdfermentation onward. The limited cell division observedin the later fermentations occurred during the first 24 hrafter inoculation and thereafter little further growthoccurred. The time for completion of fermentationincreased rapidly from 120 hr in the first fermentationto about 288 hr in the third and subsequent fermen-tations. The yields of products were normal. It was

clear from these experiments that owing to the slow

rate of yeast cell division in the presence of sulfite,satisfactory continuous fermentation procedures were

not possible. The question of yeast cell multiplicationin the presence of sulfite has been investigated byFreeman and Donald (1957a).

(j) Effect of nature of substrate. Blackstrap molasses as

compared with glucose, sucrose, raw sugar. Experimentsin which Cuban blackstrap molasses was compared withglucose, sucrose, and raw sugar as substrates in themixed sulfite fermentation showed that the impuritiespresent in blackstrap molasses play an important rolein the fermentation. Media containing only pure sugars

or raw sugar and salts fermented at markedly slowerrates under comparable conditions of free sulfite con-

centration and sulfite dosage (table 6). Blackstrapmolasses media with initial reducing sugar concen-

trations of about 20 g/100 ml fermented satisfactorilyin the presence of maximum free sulfite concentrationsof 3.5 g/100 ml whereas in media containing raw sugaror sucrose as substrates, under the same conditions,fermentation was slow and incomplete. (The raw sugar

TABLE 5. Comparison of 8 strains of Saccharomyces cerevisiae and 2 strains of Saccharomyces thermantitonum in mixedsulfite fermentations

The S. cerevisiae fermentations were carried out under normal conditions at 35 C and the S. thermantitonum fermentations at40 C. With the exception of those used in experiments 51/1 and 81, all the cultures were "acclimatized" to the presence of sulfite-bisulfite mixture (6.6 g/100 ml at pH 6.7). Glycerol determinations were by the kerosene distillation method.

'Culture No. Yields of Products Reducing Time ofExperiment No. ry Collec- Details of Culture Source of Culture Sge - Attentua- Fertina

tion Glycerol Ethanol Acetael io

% % % % % days

54/2-3 (mean) B.63 S. cerevisiae Brewers' yeast, acquired 1947 40 25.0 20.0 7.8 93 554/1 B.71 Industrial alcohol distillery 40 24.9 21.5 7.5 93 5

47/2 B.66 S. cerevisiae Bakers' yeast 37 26.0 16.6 9.4 94 847/3 B.67 Brewers' yeast, acquired 1950 29 11.1 6.1 3.1 50 1247/4 B.71 40 25.7 17.9 8.7 90 12

48/1 B.68 S. cerevisiae Prof. R. H. Hopkins, No. 1* 40 22.5 20.8 7.7 95 548/2 B.69 Prof. R. H. Hopkins, No. 2* 40 23.6 18.2 8.1 88 948/3 B.70 Prof. R. H. Hopkins, No. 3* 40 20.9 14.2 7.9 76 948/4 B.71 40 23.7 15.7 11.3 84 9

49/1 B.72 S. cerevisiae Brewers' yeast, acquired 1950 40 24.8 18.8 11.0 81 849/2 B.71 40 25.7 19.8 8.8 92 6

51/2 B.71 S. cerevisiae "Acclimatized" to presence of sul- 40 23.7 20.0 8.4 94.1 4fite-bisulfite, pH 6.7

51/1 B.71 Unacclimatized culture 40 24.6 22.0 9.3 93.8 4

81/1 B.76 S. thermantitonum The Institute of Brewing yeast col- 1.8 9.7 30.0 1.5 91.1 20Lorgensen strain lection No. 790

81/3 B.76 The Institute of Brewing yeast col- 1.8 10.4 30.3 Nil 88.4 20lection No. 790

81/2 B.77 S. thermantitonum The Institute of Brewing yeast col- 33.2 28.7 21.5 6.6 96.8 20Chapman strain lection

81/4 B.77 The Institute of Brewing yeast col- 13.2 18.5 18.5 3.0 98.5 16lection

* Acquired March 1950.

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used in this work is the startiing material for refiningcane sugar in the Uniited Kingdom; average specimenscontained approx 97 per cent of sucrose, and 1.6 percent of invert sugar). In experiments 84/1 and 86/1,with raw sugar as the substrate, the prefermentationperiod was prolonged to 512 to 612 hr. At this stagecell populations were 51 X 106 and 36 X 106 cells/ml,respectively, and the reaction of the medium had fallento pH 5.6 in both cases. When the maximum free sul-fite concentration was restricted to 1.6 to 1.7 g/100 mlas in experiments 103/1-4 with glucose and sucrose assubstrates, the fermentation proceeded at a rate approx-imately comparable to that obtained with molassesmedia, and it is clear that sulfite concentration is acritical factor in unaerated fermentations of glucoseand sucrose.The effect of aeration with air at 4 vol/vol/hr was

investigated in a series of fermentations of raw sugarin which maximum free sulfite concentrations of 1.0,1.5, 2.0, and 3.0 g/100 ml were reached. Under these

A-0~~~~~/4.0

Li

kiiIL 50

4ZN

(Q hlaoIi

?_0~ ~ ~

conditions, high free sulfite concentrations were toler-ated and the fermentations proceeded to completion inperiods as short as 192 hr and resulted in yields ofproducts comparable to those obtained with black-strap molasses as substrate under aerated or unaeratedconditions. Comparison of the effects of aeration withair, nitrogen, and carbon dioxide showed that the stim-ulative effect of aeration with air in raw sugar fermen-tations depended both upon the presence of an excess ofoxygen in the fermenting medium and upon removal oftoxic volatile fermentation products by the gas stream.Thus the rate of fermentation during aeration withnitrogen was considerably lower than during aerationwith air but significantly higher than in the absence ofaeration. When carbon dioxide was passed through thefermenting medium, fermentation proceeded at adiminishing rate during the initial 24 hr and ceasedafter 48 hr owing to the death of the yeast cells.

(k) Oxidation-reduction potentials offermenting liquors.The importance of aeration with air in influencing the

c

Is

C)0

~'-410

- P.~~~~~~~~~~~~~~~P

TIMvE (HOUp')FIG. 2. Relationship between hexose fermented, glycerol formation, and free sulfite concentration during the course of

the fermentation. A, Glycerol yield: mean between consecutive samplings; B, Glycerol yield: total to time of sampling; C,Glycerol concentration corrected to final volume; D, Observed free sulfite concentration (as Na2SO3); E, Reducing sugar con-centration corrected to final volume.

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rate of fermentation of raw sugar, glucose, and sucrose

in the presence of sulfites led to a study of the oxida-tion-reduction potentials of raw sugar and molassesliquors during the fermentation. Reproducible resultswere obtained by means of colorimetric determinationwith B. D. H. "Redox indicators4." They showed thatthe presence of sulfite is the major factor in determin-ing the oxidation-reduction potential of the fermentingliquors. The potential fell from above 0.227 (the limitof the available range of indicators) to between 0.118 and0.063 v immediately after the initial addition of sul-fite and remained within this range until fermentationwas complete. No significant effect on the potential byaeration with air was detectable by the methodsemployed.

(1) Minor fermentation products and components of thefermented liquor. (1) Acetic acid: Volatile acids were

determined in the fermented liquors by a modificationof the method of Pregl (1945), consisting essentially ofacidification of the fermented liquor (10 ml) with 25per cent p-toluenesulfonic acid (4 ml) and distillationin vacuo into an excess of sodium hydroxide. The dis-tillate contained sulfur dioxide as well as volatile organicacids; the former was determined iodimetrically and a

correction applied to the gross titer.Nelson (1929) stated that a sample of Puerto Rican

molasses contained formic acid (0.1 per cent) and aceticacid (0.2 per cent). The specimen of Cuban blackstrapmolasses used in our work contained volatile acids,

4The British Drug Houses Ltd., Poole, Dorset, England.

calculated as acetic acid (0.38 per cent). It was con-

cluded that the bulk of the volatile acids found in themixed sulfite fermentation liquors were formed in thefermentations since only 0.2 g/100 ml as acetic acid isaccounted for by the volatile acids present in the molas-ses whereas, for example, the concentration in thefermented liquor in experiment 126/1 was 0.6 g/100 ml.The influence of sulfite dosage on acetic acid productionin the fermentation is shown in figure 1.The bulk of the volatile acids present in a typical

fermentation liquor was identified as acetic acid byisolation of the p-phenylphenacyl ester, mp and mixedmp 108 to 110 C.

(2) Lactic acid: The lactic acid content of sulfitefermentation liquors was determined semiquantitativelyby a modification of the method of Boyland (1928) andit was concluded that a small amount of lactic acidequivalent to approx 1.8 per cent of the hexose fer-mented was formed during typical fermentations of mo-lasses in the presence of sulfites.

(3) Aconitic acid: Aconitic acid was determined bythe decarboxylation method of Roberts and Ambler(1947), and a correction was made for the carbonatecontent of the fermented liquors. A representativefermentation liquor from Cuban blackstrap molasses(experiment 64/3) contained 0.82 g/100 ml of aconiticacid, and an unfermented molasses solution, corre-

spondingly diluted, contained 0.7 g/100 ml of the acid.It was concluded that aconitic acid was not a productof the sulfite fermentation.

TABLE 6. Anaerobic, mixed sulfite fermentations of glucose, sucrose, and raw sugar

Anaerobic fermentations of glucose, sucrose, and raw sugar were carried out under the normal conditions. The fermentationmedia contained ammonium sulfate (1.4 g/L) and disodium hydrogen phosphate (2.5 g/L) at pH 6.8. Glycerol determinations wereby the kerosene distillation method.

Sugar Concentration Sulfite as Na2SO3 Times torodctas Invert Sugar Te to Yields of Poducts Rate ofExperiment No. Substrate Completion Fermentation

Maximum of During InitialInitial Final concen- Dosage Fermentation Glycerol Ethanol Acet- 24 hr

tration alehyde

g/100 ml g/100 ml % days % % % g hexose/L/hr

84/1 Raw sugar 20.9 0.1 1.2 20 13 15.6 31.0 5.6 0.686/1 9.8 0.4 1.6 40 5 24.8 18.1 11.3 1.3

68/1 and 2 (mean) Sucrose 21.3 4.3 3.1 37 31 24.5 16.1 10.0 1.368/3 and 4 (mean) 21.4 4.1 1.9 19 31 16.2 30.5 6.2 1.4103/1 19.8 0.2 1.7 33 8 24.2 24.8 8.9 2.8103/2 10.5 0.0 1.6 40 4 23.7 20.6 10.3 2.6

103/3 Glucose 19.6* 0.2* 1.6 35 4 23.2 21.4 9.5 3.3103/4 10.2* 0.0 1.6 40 3 22.0 21.0 8.4 2.8

Mean of representative 20-22 2.0 3.5 40 5 25 21 10 2.4Cuban blackstrapmolasses fermenta-tions

* As glucose.

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(m) Effect of scale of fermentation. Comparison of thekinetics and yields of products in fermentations carriedout (a) in the laboratory with an initial volume of 7 L,(b) on a semitechnical scale with an initial volume of2,700 L and (c) on a pilot plant scale with initial vol-umes of 27,000 L showed that within these limits thescale of fermentation had no detectable effect.

Fermentations with Washed Yeast Suspensions

The experiments described above were carried outunder laboratory conditions approximating as closelyas possible those which could be used in industrialscale fermentations. Blackstrap molasses, a complexmixture of fermentable sugars containing numerous

organic and inorganic impurities, was used as startingmaterial in the bulk of the work, and under the experi-mental conditions yeast growth, as well as fermentation,took place. In the following experiments, to determinethe influence of pH and free sulfite concentration on

the sulfite fermentation, the experimental conditionswere simplified by use of glucose (initially 9 to 10 g/100ml) as substrate in the presence of sodium phosphateand ammonium sulfate. The fermentations were inocu-lated with sufficient quantities, corresponding toabout 80 X 106 cells per ml, of a washed suspension ofyeast (strain B.71, previously "acclimatized" to 6.6 per

cent free sulfite equivalent concentration) to promoterapid fermentation without significant increase of cellpopulation.

(a) Effect of free sulfite concentration. At the time ofinoculation, a sufficient solution of sodium sulfite andbisulfite (pH 6.7) was added to the fermentation vesselsto give free sulfite equivalent concentrations of 0, 1, 2and 3 g/100 ml, respectively, and further additions

were made at intervals to maintain these concen-

trations. The change in concentration of glucose due tothese latter sulfite additions was small and the resultshave not been corrected for this factor. The pH valuewas initially 6.7; in the sulfite containing liquors thepH remained in the range 6.7 to 8.3 during the fermen-tation. In the absence of sulfite the pH rapidly fell to2.5 to 3.0. The glucose concentrations of the fermentingliquors were determined at intervals and are reproducedin table 7. There was no initial lag in onset of fermen-tation. The initial approximately linear rates of fer-mentation were 0.57, 0.33, 0.27, and 0.15 g glucose/100ml/hr in the presence of respective free sulfite concen-

trations of 0, 1, 2, and 3 g/100 ml, that is, in the ratios100:59:47:27. After 35 to 60 hr of fermentatioin,dependent upon the free sulfite concentration, a finalslow phase of fermentation set in, during which only5 per cent or less of the original glucose was fermented.After 118 hr of fermentation, 1.9, 2.3, 4.5, and 13.8per cent of the original glucose, respectively, remainedunfermented.

Glycerol yields by the kerosene distillation method interms of total glucose were 26.5, 28.7, and 26.0 per centin the presence of respective free sulfite concentrationsof 1, 2, and 3 g/100 ml, that is, glycerol yield was prac-

tically independent of free sulfite concentration underthese conditions. In terms of hexose fermented, how-ever, the glycerol yields were 27.2, 30.2, and 30.5 per

cent. It was concluded that under these conditions a

free sulfite concentration of 2 to 3 g/100 ml was optimalfor the major period of the fermentation but that thefree sulfite concentration should be allowed to fall toless than 1 per cent at the completion of fermentation

TABLE 7. Relationship between glucose concentration and time, in fermentations containing 0 to S g/100 ml of free sulfiteequivalent

To a solution (625 ml) containing glucose, 119 g/L; Na2HPO4, 2.0 g/L; (NH4)2SO4, 1.2 g/L; MgSO4-7H20, 0.5 g/L and KCI, 5.0g/L at pH 6.7, the following additions were made:

Experiment No. Sodium Sulfite-Bisulfite Solution, pH 6.7 Inoculum Containing 3 X 109 Cells/ml Water

ml ml ml

WS3/1 0 14 702 24 14 463 47 14 234 70 14 0

Temperature 35 C, pH 6.7 to 8.3, except in experiment WS3/1, where the pH value rapidly fell to pH 2.5 to 3.0. The free sulfiteconcentrations in expts. WS3/2, 3, and 4 were maintained at 1.0, 2.0, and 3.0 g/100 ml respectively by periodic addition of sulfite-bisulfite solution, pEI 6.7.

Glucose Concentration pH ValuesExperiment No. Concentration

| hr |4 hr 7,4 hr j 10%4 hr 22 hr 30 hr 46 hr 54 hr 70 hr 118 hr 0.5 hr 22 hr 46¼ hr 118 hr

g/J00 ml gI/O1 ml

WS3/1 0 9.54 6.99 5.16 4.08 1.64 0.67 0.44 0.27 0.27 0.18 4.26 2.64 3.04 2.792 1.0 9.46 8.28 7.03 5.90 3.16 1.41 0.41 0.22 0.24 0.22 6.96 6.78 7.12 8.303 2.0 9.54 8.48 7.44 6.61 4.64 3.13 1.61 0.87 0.66 0.43 7.14 6.94 7.08 7.714 3.0 9.60 9.10 8.29 7.63 6.29 5.32 4.26 3.45 1.86 1.31 7.20 6.76 7.10 7.40

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in order to obtain maximum utilization of fermentablesugar.

(b) Effect of pH. A series of fermentations was carriedout in which the pH values were maintained at pH 6.2,6.5, 6.7, and 7.0. The free sulfite equivalent concen-tration was kept constant at 2.5 g/100 ml by inter-mittent addition of sulfite-bisulfite solution of theappropriate pH value. The results are summarized intable 8; they show that glucose attenuation and rate offermentation fell off rapidly at and below pH 6.5. Itwas concluded that the optimal pH for the sulfitefermentation under these conditions was in the rangeof 6.7 to 7.0.

DISCUSSION

Under the standard conditions of fermentation, whichhave been described, sulfite dosage was probably themost important factor in determining yields of productsand kinetics of fermentation. The dependence of theyields of glycerol, ethanol, acetaldehyde, and aceticacid on sulfite dosage is shown in figure 1. The graphshows that at 50 per cent sulfite dosage, acetaldehydefound and "theoretical acetaldehyde" approach equal-ity, and acetic acid production is practically zero. Thesefacts are consistent with the conclusion that, at sulfitedosages in excess of 50 per cent, glycerol production isas predicted by Neuberg's second equation (1), whereasat lower sulfite dosages a proportion of the glycerol isformed by another route as predicted by equation (2).The sulfite dosages which are necessary for high glycerol

TABLE 8. Influence of pH on rate and completeness of fermenta-tion of glucose and glycerol yields in the presence of 2.5 g/100

ml of free sulfite equivalentTo the glucose medium described in table 7 (625 ml), the

following additions were made:

Experiment Sodium Sulfite-Bisulfite Solution InoculumNo. Containing Water

pH Concentration Volume 6 x lO Cells/ml

g/100 ml ml ml ml

WS5/1 7.6 26.0 63 10 02 7.2 30.4 53 10 103 6.7 33.4 50 10 134 6.2 39.3 43 10 20

Temperature 35 C. Free sulfite concentration, 2.5 g/100ml. The fermented liquors were harvested after 144 hr. Glyc-erol determinations were by the kerosene distillation method.

EMperimentnMH Intia rat of Glucose Glycerol YieldExperient ean Initial rate of Attenuation

No. Value Fermentation after 144 Hr of Of total Of glucoseFermentation Ofglucosefeend

g glucosel % % %100 ml/hr

WS5/1 7.0 0.32 97.8 25.9 26.52 6.7 0.21 98.2 23.9 24.33 6.5 0.09 75.0 22.4 29.94 6.2 0.08 9.3

yields have important effects on rate of fermentation,time to completion of fermentation and sugar attenua-tion in the later stages of the process. This is clearlyshown by comparison of the initial rates of fermen-tation and the residual glucose concentrations (after118 hr fermentation) in experiments WS3/1-4 (table7) in the presence of free sulfite concentrations of 0, 1,2, and 3 g/100 ml.Comparison of the kinetics of fermentation, yields of

products and pH changes during fermentations in thepresence of (a) sodium sulfite and (b) various mixturesof sodium sulfite and sodium bisulfite showed that thenature of the sulfite steering reagent was unimportantuntil the proportion of bisulfite to sulfite exceeded0.25:1; addition of mixtures containing higher propor-tions of bisulfite caused partial inhibition of the fer-mentation owing to the toxicity of this component.The results of the present work and that of earlier

workers (Neuberg and Reinfurth, 1918, 1919) supportthe view that the sulfite fermentation may be regardedas a yeast fermentation in which the products aredominated by the sulfite steering reagent in a mannerwhich may be predicted from the laws of mass action.Maximum yields of glycerol and acetaldehyde resultedfrom a high concentration of free sulfite in the ferment-ing liquor, which favored the formation of the acetal-dehyde-bisulfite complex and suppressed its subsequentdissociation. The concentration of sulfite which may beemployed under practical conditions is, however,limited by the tolerance of the yeast and normally alittle less than half of the acetaldehyde formed as anintermediate is reduced to ethanol as in the case ofethanolic fermentation of hexose.

Production of glycerol in the sulfite fermentationmay be expressed by equation (1), from which it fol-lows that theoretically glycerol production is stoichio-metrically equivalent to bisulfite fixation as acetal-dehyde-bisulfite compound.

C6H1206 + NaHSO3= CH,3CHO-NaHSO3 + C3H803 + CO2

This relationship has been used by some workers, forexample, Underkofler et al. (1951a), as a means of ascer-taining the glycerol content of fermented liquors bymeans of determination of sulfite fixation. In our expe-rience, this proved to be a fairly satisfactory procedurebut should be used with caution since (a) glycerolequivalent to 2.5 to 3.6 per cent of the fermentablesugar is formed in normal ethanolic fermentations inthe absence of sulfite and under these conditions glyc-erol formation is greatly influenced by the pH of themedium and (b) a portion of the added free sulfite islost from the system by separation as insoluble calciumsulfite by reaction with calcium salts in the molasses.

It follows, from the Embden-Meyerhof-Parnas scheme

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for the dissimilation of glucose, that glycerol andacetaldehyde are formed in equimolecular proportionswhen the hexose is fermented by yeast in the presence

of an acetaldehyde fixing reagent such as sulfite. It hasbeen shown (figure 1) that when the quantity of sul-fite added, as Na2SO3, was equivalent to 50 per cent or

more of the fermentable hexose, the yield of acetal-dehyde agreed closely with the theoretical but at lowersulfite dosages the yield of acetaldehyde was less thanpredicted by the theory and the discrepancy increasedprogressively as the sulfite dosage was diminished. It issuggested that when the quantity of added sulfite isless than half that of the fermentable hexose a pro-

portion of the latter is fermented, with the productionof glycerol, ethanol, acetic acid, and carbon dioxide, inaccordance with equation (2), in addition to that whichundergoes normal ethanolic fermentation in accordancewith equation (3):

2C6H1206 + H20 = 2C3H803 + CH3COOH

+ C2H50H + 2CO2 (2)

C6H1206 = 2C2H50H + 2CO2 (3)

This view is supported by the presence of acetic acid as

a minor product of the fermentation under these condi-tions. (Production of glycerol and acetic acid by yeastfermentation of hexose can also be expressed as:

3C6H2206 + 2H20

= 4C3H803 + 2CH3COOH + 2CO2 (4)

When acetaldehyde is dissimilated in presence of yeastequimolar quantities of ethanol and acetic acid are

formed (Freeman and Donald, 1957b) and for thisreason equation (2) is preferred.)The theoretical yield of 51 per cent of glycerol, in the

sulfite fermentation, as required by equation (1) hasnever been realized. The major products of the fermen-tation are glycerol, acetaldehyde, ethanol, and carbondioxide. In addition it has been shown that acetic acidand lactic acid are found as minor products and it isknown that a small proportion of the hexose is con-

sumed in building up yeast cell tissue. If these minorlosses of hexose be neglected it is reasonable to assume

that hexose molecules not fermented according toequation (1) will undergo normal ethanolic fermen-tation (Neuberg's first form of yeast fermentation) as

required by equation (3). The yields of the major fer-mentation products will then be represented by thesum of equations (1) and (3), according to the relativenumbers of hexose molecules which are fermented bythe two routes. If, for example, equal numbers of hexosemolecules were fermented in each of the two ways theover-all yields of products would be represented byequation (5) as follows:

from

C6H1206 = C3H803 + CH3CHO + C02 (1)C6HI206 = 2C2H50H + 2C02 (3)2C6H1206 = C3H803 + CH3CHO+ 2C2H50H + 3C02 (5)Parts by weight:

360 92 44 92 132Parts by weight:

100 25.6 12.3 25.6 36.7

The yields found by experiment, with a sulfite dosageof 40 per cent, are of approximately the same order,namely, glycerol, 27.2; ethanol, 21; and acetaldehyde10 per cent (table 6; the glycerol value is based on thekerosene distillation method and corrected by the fac-tor 1.09).By means of a triangular diagram (prepared by our

colleagues Dr. K. Luckhurst and J. V. Gregg) relatingthe principal products of hexose fermented by theroutes of Neuberg's three forms of fermentation, therelative proportions of hexose molecules fermented bythe three routes in the presence of sulfite equivalentdosages of 5, 25, and 50 per cent have been determined.At these sulfite dosages, the percentages of hexosemolecules were as follows:

Hexose Molecules Fermented by Neuberg's Forms ofSulfite Dosage Fermentation

First Second Third

5 75.5 7.0 17.525 59.1 24.8 16.150 42.4 54.2 3.4

SUMMARY

The following factors, which influence the kinetics offermentation of hexoses in the presence of sulfites,have been investigated: pH, sulfite dosage and freesulfite concentration, initial substrate concentration,temperature, nature of yeast strain, and aeration.The pH optimum was 6.7 to 7.0 and the optimal

temperature 30 to 35 C. The quantity of added sulfite interms of total fermentable hexose has been shown to bean important factor in determining the relative yieldsof glycerol, acetaldehyde, ethanol, and acetic acid. Theoptimal conditions for Cuban blackstrap molassesfermentations were: initial reducing sugar concen-tration, 20 to 22 g/100 ml; maximum free sulfite con-centration, 3 to 3.5 g/100 ml as Na2SO3 equivalent; andaeration with air restricted to 30 min during the "pre-fermentation" period. Under these conditions and witha sulfite dosage of 40 per cent, the fermentation wascomplete in 5 days with a fermentable hexose attenua-tion of about 93 per cent. The fermented liquor con-tained glycerol, 4.5 (corrected value determined bykerosene distillation method); acetaldehyde, 1.7;ethanol, 3.5; and acetic acid, 0.3 g/100 ml; equivalentto yields of 27.2, 10, 21, and 1.1 per cent respectively

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in terms of total fermentable hexose. Acetic acid andlactic acid (about 1.8 per cent of hexose fermented) areminor products of the sulfite fermentation.

REFERENCESBERTRAND, G. 1906 Les dosage des sucres r6ducteurs.

Bull. Soc. Chim. Paris, 35, 1285-1299.BOYLAND, E. 1928 Chemical changes in muscle. I. Meth-

ods of analysis. Biochem. J., 22, 236-244.COCKING, A. T. AND LILLY, C. H. 1921 Improvements in

the production of glycerine by fermentation. BritishPatent 164,034.

CONNSTEIN, W. AND LUDECKE, K. 1919 tUber Glycerin-Gewinnung durch Garung. Ber. deut. chem. Ges., 52,1385-1391.

DONALD, G. M. S., FREEMAN, G. G., AND CUNNINGHAM, M. N.1953 The determination of unfermentable reducing sub-stances in molasses. Analyst, 78, 321-322.

EOFF, J. R. 1918 Process of manufacturing glycerol. U. S.Patent 1,288,398.

EOFF, J. R., LINDER, W. V., AND BEYER, G. F. 1919 Pro-duction of glycerine from sugar by fermentation. Ind.Eng. Chem. (Ind.), 11, 842-845.

FREEMAN, G. G. AND MORRISON, R. 1. 1946 The determina-tion of some products of sugar and molasses fermenta-tions. Analyst, 71, 511-520.

FREEMAN, G. G. AND DONALD, G. M. S. 1957a Fermentationprocesses leading to glycerol. II. Studies on the effectof sulfites on viability, growth, and fermentation ofSaccharomyces cerevisiae. AppI. Microbiol., 5, 211-215.

FREEMAN, G. G. AND DONALD, G. M. S. 1957a Fermentationprocesses leading to glycerol. III. Studies on glycerolformation in the presence of alkalis. Appl. Microbiol., 5,216-220.

FULMER, E. I., UNDERKOFLER, L. A., AND HICKEY, R. J.1941 Fermentative glycerol production. U. S. Patent2,416,745.

FULMER, E. I., HICKEY, R. J. AND UNDERKOFLER, L. A. 1945Glycerol production. U. S. Patent 2,388,840.

Imperial Chemical Industries Limited, and Lilly, C. H. 1931Improvements in processes for the manufacture of glyc-erine by fermentation. British Patent 349,192.

Institute of Brewing Yeast Collection 1949 J. Inst. Brew-ing, 55, 1-6.

JANKE, A. AND KROPACSY, S. 1935 tYber die Bestimmungdes Athylalkohols und seiner Oxydationsprodukte inbiologischen Substraten. Biochem. Z., 278, 30-36.

MINER, C. S. AND DALTON, N. N. 1953 Glycerol. ReinholdPublishing Corporation, New York City, New York.

MUNSON, L. S. AND WALKER, P. H. 1906 Table reproducedin Handbook of Chemistry and Physics, 1955-1956, 37thedition, Chemical Rubber Publishing Co., Cleveland,Ohio. p. 1665.

NEISH, A. C. 1950 Determination of glycerol and relatedcompounds in fermentation solutions by partition chro-matography. Can. J. Research, B28, 535-540.

NEISH, A. C., BLACKWOOD, A. C., AND LEDINGHAM, G. A.1945 Dissimilation of glucose by Bacillus subtilis (Ford'sstrain). Can. J. Research, B23, 290-296.

NELSON, E. K. 1929 Some organic acids of sugar canemolasses. J. Am. Chem. Soc., 51, 2808-2810.

NEUBERG, C. AND REINFURTH, E. 1918 Natuirliche underzwungene Glycerin-bildung bei der alkoholischen Ga-rung. Biochem. Z., 92, 234-266.

NEUBERG, C. AND REINFURTH, E. 1919 Weitere Untersuch-ungen uber die korrelative Bildung von Acetaldehyd undGlycerin bei der Zuckerspaltung und neue Beitrage zurtheorie der alkoholischen Garung. Ber. deut. chem. Ges.,52, 1677-1703.

PREGL, F. 1945 Quantitative Organic Microanalysis, 4th ed.J. & A. Churchill Ltd., London, England.

PRESCOTT, S. C. AND DUNN, C. G. 1949 Industrial Micr-o-biology, 2nd ed. McGraw-Hill Book Co., Inc., London, Eng-land.

RIPPER, M. 1900 Eine allgemeine anwendbare massanalyt-ische Bestimmung der Aldehyde. Monatsh. Chem., 21,1079-1084.

ROBERTS, E. J. AND AMBLER, J. A. 1947 Quantitativemethod for aconitic acid and aconitates. Ind. Eng. Chem.(Anal. Ed.), 19, 118-120.

SPENCER, J. F. T. 1955 Glycerol production by osmophilicyeasts. Thesis submitted to University of Saskatchewanin May, 1955.

SPENCER, J. F. T. AND SALLANS, H. R. 1956 Production ofpolyhydric alcohols by osmophilic yeasts. Can. J. Mi-crobiol., 2, 72-79.

SPENCER, J. F. T., NEISH, A. C., BLACKWOOD, A. C., ANDSALLANS, H. R. 1956 Polyhydric alcohol production byosmophilic yeasts: Studies with C14-labelled glucose.Can. J. Biochem. Physiol., 34, 495-501.

SPOREK, K. AND WILLIAMS, A. F. 1954 Chromatographicdetermination of glycerol in fermentation solutions.Analyst, 79, 63-69.

UNDERKOFLER, L. A., FULMER, E. I., HICKEY, R. J., ANDLEES, T. M. 1951a Production of glycerol by fermenta-tion. I. Fermentation of dextrose. Iowa State Coll. J.Sci., 26, 111-133.

UNDERKOFLER, L. A., FULMER, E. I., LEES, T. M., AND Fi-GARD, P. H. 1951b Production of glycerol by fermenta-tion. II. Fermentation of disaccharides and starch.Iowa State Coll. J. Sci., 26, 135-147.

UNDERKOFLER, L. A. 1954 Chapter 8 of Industrial Fermen-tations. Edited by Underkofler, L. A. and Hickey, R. J.1, Chemical Publishing Co., Inc., New York City, NewYork.

VON EULER, H. AND LAURIN, I. 1919 Zur Kenntnis der HefeSaccharomyces thermantitonum. Biochem. Z., 97, 156-169.

VON EULER, H. AND LAURIN, I. 1920 Zur Kenntnis der HefeSaccharomyces thermantitonum. II. Mitteilung. Biochem.Z., 102, 258-267.

WHALLEY, M. E. 1942 Abstracts of Articles and Patents onthe Production of Glycerol by Fermentation. NationalResearch Council Report No. 1070, Ottawa, Canada.

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