Production and Testing of Yeast Mutants for Glycerol Formation1R. E. WRIGHT, W. F. HENDERSHOT, AND W. H. PETERSON

Departnment of Biochemistry, University of Wisconsin, Madison, Wisconsin and Forest Products Laboratory,2 Forest Service,United States Department of Agriculture, Madison, Wisconsin

Received for publication January 28, 1957

Production of glycerol by yeast fermentation in thepresence of sulfites or alkali salts has been the subject ofmuch investigation. Of the various processes proposed,only the bisulfite process has been operated industrially,and then only in time of war.

Addition of so-called steering agents has given onlyabout 60 per cent of the theoretical yield because theconcentration at which they can be used is limited bytheir toxicity. Even under practical conditions offermentation, the amount of sulfite or alkali salts is sogreat that the recovery of glycerol becomes a difficultoperation (Underkofler and Hickey, 1954).The realization that enzymes are under genetic con-

trol leads to another possible approach to obtaininghigher glycerol yields. This approach has already beenutilized with marked success in the production of peni-cillin and streptomycin, where higher yielding mutantshave been obtained after ultraviolet and nitrogen mus-tard treatment of spore suspensions.

In the case of glycerol fermentation, it seems probablethat a genetic block at one of several points might resultin higher yields of glycerol. A mutant lacking alcoholdehydrogenase would be unable to utilize acetaldehydefor the reoxidation of DPNH, and should give a higheryield of glycerol without steering agents. A mutant pro-ducing large amounts of acetic acid should give highyields of glycerol, since formation of acetic acid iscoupled with glycerol productioni. Such inutants wouldof course have to be able to reduce phosphodihy-droxyacetone to phosphoglycerol. Attempts have beenmade to find such mutants.

MATERIALS AND METHODS

Cultures. Torula utilis strain 3 (Candida utilis) wasobtained from the Department of Bacteriology andSaccharomyces cerevisiae strain WY38 from the Genet-ics Department of the IUniversity of Wisconsini; Sac-charomyces cerevisiae strain 49 from the Forest ProductsLaboratory, U. S. Department of Agriculture; andZygosaccharomyces acidifaciens from the Northern Utili-zation Research and Development Division, U. S. D. A.

1 This investigation was supporte(d in part by U. S. ArmyOrdnance.

2 Maintained at Madison, Wisconsin, in cooperation withthe University of Wisconsin.

Fermentations in the absence of sulfite. Medium no. 1was used in all such fermentations and contained com-mercial glucose, 10 g; tryptone (Difco), 5 g; yeastextract (Difco), 0.1 g; KH2PO4, 2.7 g; and K2HPO4,7.3 g per L. With less glucose and more buffer than inthe sulfite fermentation, acidic conditions could becontrolled. Without adequate buffer, T. utilis fer-mentations reached pH 3 or lower. The glucose, tryp-tone, and yeast extract were dissolved in 900 ml of water,and 18 ml of this solution was dispensed into 18 by 250mm test tubes. A 10 per cent solution of the phosphatesalts was prepared separately, and both solutions weresterilized by autoclaving. Two ml of the buffer solutionwere added to each tube before inoculation. This gavea pH of about 7.5. The tubes were incubated at 30 C forabout 3 days, and then the pH was between 6.8 and7.2, depending on the organism used. Samples for analy-sis were taken before and after incubation.

Fermentations in the presence of sulfite. The followingmedium (no. 2) with slight modifications in glucoseconcentration, as indicated in the tables, was used inall such fermentations: commerical glucose, 30 g;tryptone, 5 g; KH2PO4, 1 g; K2HPO4, 1 g; yeast extract,0.1 g; and water, 1,000 ml. Two hundred and fifty mlErlenmeyer flasks containing 100 ml of the medium wereautoclaved at 15 to 20 lb pressure for 15 min. ThepH after autoclaving was 6.4. An 18 to 24 hr culture wasused as inoculum. The amount of inoculum varied from0.2 to 1 ml, depending on the development of the organ-ism used. The flasks were incubated at 30 C, and growthwas followed by determining the per cent transmissionof light at 660 m,u in the Evelyn colorimeter3 (18 mmtubes). When the transmission reached 60 per cent(about 30 X 106 cells per ml for 7'. utilis), the pH wasadjusted to 6.8 with sterile 0.1 N- NaOH. Samples werethen removed for sugar and glycerol analyses, and 2 mlof sulfite solution were added. This solution contained11.4 g of sodium pyrosulfite (equivalent to 12.5 g ofsodium bisulfite) and 12.5 g of sodium sulfite per 100ml, and was sterilized by Seitz filtration. The sulfite con-centration of the medium was followed by iodinetitration and, when about one-third of the sulfite hadbeen bound, a second 2 ml portion of sulfite solution wasadded. Thus a total of 1 g of mixed sulfite salts was

I Rubicon Co., Philadelphia, Pa.

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added. Incubation was continued unitil iodine titrationshowed no increase in bound sulfite over a 4 to 8 hrperiod.A relatively low sugar concentration was used so that

the required amount of sulfite could be added in twoaliquots without approaching the concentration of freesulfite which seriously inhibits the parent strains(about 2 per cent at pH 6.8).

ANALYTICAL MIETHODSDetermination offree and bound sulfite. Three drops of

starch indicator and 0.1 ml of 0.1 N H2SO4 were added toa 1 ml sample, and the mixture was titrated with 0.1 Niodine to an end point stable for at least 1 min. Thistiter was expressed as S03 and called "free sulfite."Excess solid sodium bicarbonate was then added and thesolution was again titrated to a slightly blue end point.This second titer was taken as a measure of the bisulfitebound as the aldehyde-bisulfite complex, and wasexpressed as sodium bisulfite, acetaldehyde, or glycerol,as was desired to follow the course of the fermentation.

Determination of glucose. Glucose was determined bythe copper reduction method of Shaffer and Somogyi(1933). In sulfite fermentations, the aldehyde-bisulfitecomplex breaks down under the alkaline conditions ofthe determination, and the components interfere withthe analysis. Addition of calcium hydroxide precipitatesthe sulfite, but the acetaldehyde remains. Tests in-dicated that at pH 1.5 the bisulfite complex could bedecomposed and the aldehyde and sulfite could be re-moved by heating on a water bath until the initialvolume was reduced by 1 to 3A. Loss of sugar under thesame conditions amounted to only 2 per cent at aninitial concentration of 30 mg per ml.

Other analyses. Glycerol was determined directly onthe broth by the colorimetric method of Lambert andNeish (1950). Correctioins for sugar were made wherethey were significant. Volatile nieutral products (VNP)were obtained by distillation of the broth, previouslyadjusted to pH 7.5, until at least half of the originalvolume had passed over. Ethyl alcohol was determinedon aliquots of the VNP distillate by oxidation withdichromate (0.2 N in 5 N H2SO4) and titration of residualdichromate with 0.1 N thiosulfate after the addition ofexcess potassium iodide.

Acetylmethylcarbinol was determined by the methodof Langlykke and Peterson (1937). Acetaldehyde wasdetermined by the colorimetric method of Desnuelleand Naudet (1945). Formaldehyde, acetic acid, ethylalcohol, and acetone have no effect on this determina-tion, even when they are present in 20 times the concen-tration of the acetaldehyde. Sodium bisulfite does-interfere, but was removed by preliminary titrationwith iodine at pH 7.5.The residue from the VNP distillation was reduced to

about one-fifth the original volume of the broth. ThepH was then adjusted to 2 with H2SO4 and the solutionvas distilled with concurrent addition of C02-free wateruntil 10 volumes had been collected. Aliquots of thisdistillate were titrated with 0.1 N Ba(OH)2 to a phenolred end point, and acidity was expressed as acetic acid.The residue from the volatile acid distillation was

made up to volume, and an aliquot was extracted withether overnight in a small liquid extractor. Tests showedthat this was sufficient time to extract all the pyruvicacid. After evaporation of the ether, the residue wasmade up to the original volume of the aliquot withC02-free water. Pyruvic acid was determined in aliquotsof this solution by the following modification of amethod that has been widely used in the determinationof a-keto acids (Lu, 1939; Friedmann and Haugen,1943). An aliquot containing 20 to 100 ,ug was made upto 3 ml. One ml of 2,4-dinitrophenylhydrazine reagent(1 mg/ml in 2 N HCI) was added. The tubes were al-lowed to stand for 5 min, and 5 ml of 2 N KOH were thenadded. Per cent transmission at 565 m,u was determinedin the Evelyn colorimeter and compared with standardsprepared from weighed amounts of sodium pyruvate.

Production and Isolation of MutantsAll media were sterilized at 15 to 20 lb pressure for

15 miii unless otherwise specified. Solid media wereprepared by addition of 1.7 per cent of agar. Difco agarwas used in all "minimal" media.

Basal salts solution. This solution contained the in-organic constituents as used by Olson and Johnson(1949) with the followving changes: KH2PO4 was reducedto 0.7 g, 0.4 g of K2HPO4 was included, and the saltswere dissolved in 900 instead of 1,000 ml.

Glucose minimal medium (no. 3) was prepared by add-ing 1 volume of 10 per cent glucose solution to 9 vol-umes of the basal salts solution before autoclaving.Vitamins and casein hydrolyzates (Difco) in manydifferent combinations were added to the glucose mini-mal medium to determine which yeasts needed thesenutrients.

Complete medium (no. 4). This medium was the sameas medium 2, except that glucose was reduced to 10 gand KH2PO4 was increased to 1.7 g per L.

Acid indicator medium (no. 5). This medium containedglucose, 50 g; tryptone, 20 g; yeast extract, 1 g; K2HPO4,3.5 g; KH2PO4, 6.5 g; agar, 17 g; and bromeresol green,0.2 g per L.

Ultraviolet treatment. T. utilis was grown overnight inthe glucose minimal medium. The cells were centrifuged,washed with sterile distilled water, and resuspended indistilled water to give about 5 X 106 cells/ml (90 percent transmission at 660 m,u). Fifteen ml of this suspen-sion (in a 9 cm diameter Petri plate) were irradiated by a

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15 w-att Geineral Electric4 sterilamp at a distanice of 50cm. The suspension w%as agitated durinig irradiation bymeans of a rotary table making one revolution persecond. Irradiationi wvas conitinued for various timesbetween 4 and 5.5 min, to give, in the absence of anypostirradiation treatmenit, survivals of from 3 to 0.01per cent as designed in particular experiments.

Light reactivation. In experiments on light reactiva-tion, the irradiated suspensioni was centrifuged and thecells were suspended in the basal salts solution. An18 mm diameter test tube containing this suspeinsion(90 per cent transmission) was placed oni a reciprocatingshaker at 30 C and about 50 cm from a 150 watt flood-lamp. Controls were shielded from the light.

Viable counts were made by spreading suitable dilu-tions of the suspensions on the surface of the completemedium with a glass rod. Plates were incubated at 30 C.The replica plating method of Lederberg and Lederberg(1952) was used in the selection of nutritional mutantsand possible high glycerol producing strains afterirradiation.

Y'. utilis wA-as used in these investigationis because it is

4General Electric Corporationi, Clevelanid, Ohio.

thought to be a stable haploid culture. It also hassimple growth requirements, and its existence pre-dominiaintly as single cells in liquid culture facilitates(uantitative studies.

Various postirradiation treatments were investigatedin an attempt to find the treatment that would yieldthe largest percenitage of mutants among the survivors.The niumber of auxotrophic mutants, or mutants withmore complex growth requirements than the parentstraini, was used as an inidex of the relative mutagenicefficiencies of the different treatments. These mutantswere easily detected by replication from complete tomiinimal medium. No comparable test for high glycerolproducing strains was available.The treatments investigated are outlined in figure 1.

Method 1 gave survivors with large differences ingrowth rate on solid medium so that new coloniescontinued to appear, even after 3 days of incubation.The size of the colonies after 3 days varied from 0.5 to4 mm, and this reduced the efficiency of the replicationprocedure.Method 2 is similar to the oine most commonly used

in isolating bacterial mutants. The cells are incubatedin complete liquid medium before plating to allow seg-

CEL L SUSPEAISION(ABOUT 5 x /06 PER ML.)

U V TREA TMEIV T

r- - A/QTO

ALLIQxL IQUIDFOR? X

1ALI/OUOL - -

COM1PL IE

3 PERCEN T SURVIVAL

MET/HOD 2

I ME THOD 4

JOT TO COMPLETEMEDIUM. INCUBA TED -- -

4BOUT 16 HR.

ME TH/OD 3

SUSPENSIONEXPOSED TO LIGH/T(/50 W. FLOODLAMP A TABOUT 50 CM.) INABSENCE OF ORGANICENVERGY SOURCE

ITS PLATED ON

'TE SOL ID MEDIUM

TEST FOR MUTA TION.REPL ICA TE ON TO GLUCOSEMINIMAL MEDIUM. NVO GROWTHON THIS MEDIUM INDICA TESNU TRI TIONIAL MUTA TION.

X 109 7145

FIG. 1. Outline of methods used in obtaining mutants

METHOD /

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regation of nuclei in multinucleate cells, separation ofjoined cells, and to overcome the effect of phenotypiclag (Newcombe, 1948).

Visible light has been shown to decrease the lethaleffects of ultraviolet light (Dulbecco, 1949; Kelner,1949), and its effect on T. utilis was investigated byMethods 3 and 4. Irradiated samples were plated outbefore light treatment and after 2, 4, and 23 hr exposureto light in the basal medium. Count plates were incu-bated in the dark, and a control suspension was kept inthe dark and counted at the same times. Uniformity ofgrowth was assessed by counting the colonies visible onthe plates at 24, 48, and 96 hr. Light treatment resultsin a shorter lag, a progressive evening of colony size,and a greater over-all survival. Holding the cells inminimal medium in the dark for the same time has asimilar effect, although not to the same extent.

RESULTS WITH TORULA UTILIS MUTANTS

Approximately 5,000 colonies were screened for mu-tants by the four methods described. Three mutantswere obtained by Method 1, one each by Methods 2and 4, and 15 by Method 3 after exposure to light for10 to 24 hr. Exposure to light for 24 hr. increased thesurvival figure from 2.2 per cent to 34.5 per cent, orabout 16 times (not shown in table 1). Growth in liquidmedium with or without light treatment reduced therecovery of mutants, possibly because the mutantsgrew more slowly than the unchanged cells and hencewere lost in the increased population.Mutants were classified in 2 groups, nutritional (N I to

N8) and morphological (MI to M12). Five of the nu-

TABLE 1. Or-igin, character, and glycerol produtcing ability of7'orula utilis morphological mutants

TreatmentGlc__________________GlucoseGlcNumber Character use erolMethod Initial Illumi- Use Yieldtsurvival nation

% hr % %I'arent D)iscrete cells 98 16Ml Discrete cells 1 2.2 0 45 17M3 l)iscrete cells 3 2.2 10 99 19M4 Clumped cells 3 2.2 10 98 18M5 Clumped cells 3 2.2 24 33 28M6 D)iscrete cells 3 2.2 24 97 12M7 Clumped cells 3 0.18 24 60 28M9 I)iscrete cells 3 0.18 24 35 23M1O D)iscrete cells 3 0.18 24 71 18Mul Clumped cells 3 0.18 24 32 28M12 Discrete cells 3 0.18 24 69 22

* Glticose used is the sugar fermented after beginning sul-fite addition. At this time the glucose content was about 2 percent and growth was equivalent to 60 per cent transmission oflight (660 m,). The time of fermentation was usually 100 hror more.

t Glycerol yields are based on glucose used after beginningaddition of sulfite and corrected for glycerol produced beforeaddition of sulfite.

tritional mutants required biotin (1 pg per L of mediumfor optimal growth). The other three nutritional mu-tants apparently required amino acids, because growthoccurred when casein hydrolyzate (Difco) was addedto the basal medium. No strains requiring other vita-mins were found. The range of types of nutritional mu-tants was unexpectedly small. None of the nutritionalmutants produced more glycerol than the parentstrain, which was run as a control. No further workwas done on these mutants.The morphological mutants formed small, rough

colonies on the complete medium (no. 4) and grewpoorly on minimal medium (no. 3). In liquid medium,some of the strains tended to clump, but the others re-tained the discrete type .of growth of the parent. Theover-all number of mutants obtained was much lowerthan would have been expected on the basis of studieswith bacteria where, for example, 1 to 3 per cent of thesurvivors after a 99.95 per cent kill of Escherichia coliby ultraviolet light were found to be nutritional mu-tants (Adelburg, 1953). The larger volume of cytoplasmsurrounding the nucleus of yeast cells may be responsi-ble for some protection against ultraviolet radiation.The greater yield of mutants found in studies on bac-teria may also be an expression of the more efficientselective methods available for these organisms (seebelow). Figures given by Reaume and Tatum (1949) forrecovery of mutants from a haploid strain of S. cere-visiae indicate a spontaneous mutation rate of about Iper cent and a 5- to 10-fold increase on treatment withnitrogen mustard. Thus T. utilis appears to be veryresistant to the mutagenic action of ultraviolet light.How-ever, in view of (1) its extreme stability under or-dinary cultural conditions, when compared with otheryeasts definitely known to be haploid; (2) the low num-ber of nutritional mutants recovered after ultraviolettreatment; and (3) the fact that most of these mutantsare of the same type, it is possible that T. utilis may notbe haploid as is generally believed.The glycerol producing power of the morphological

mutants was tested several times, and typical resultsare given in table 1. Most of the mutants were poorerglucose fermenters than the parent, but several gavedistinctly higher yields of glycerol. Poor utilization ofglucose is often associated with a high conversion ofglucose to glycerol, but such data must be discountedbecause of the relatively large effect of analytical errorsand the known high production of glycerol during theearly stage of a fermentation. Mutant NI is an exampleof this early high production. When better utilizationof glucose was obtained, the relative yield of glycerolwas much less. Further tests than those recorded intable 1 indicated that M 1I was among the best of themutants, and it was accordingly used in a more com-plete study of products with and without added sulfite.

Fermentation products in the absence of sulfite. In

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table 2 (columns 1 and 3) are listed the main productsobtained with the parent culture and mutant M 1lwhen no sulfite was added to the fermentationi, butwith added CaCO3. The two cultures do not differ sig-nificantly in their fermentation products. Theoretically,1 mm of glycerol for each mm of pyruvic acid and 2 mmfor each mm of acetic acid should be found in the fer-mentation products. With T. utilis strain 3, 20.4 mMof glycerol would therefore be expected and 17.0 mMwere found, while in the strain M 1I fermentation, 18.7mm of glycerol would be expected and 17.0 mm werefound. The recovery may have been lower than thetheoretical because the fermentations were not carriedout under strictly anaerobic conditions. The molecularratios of pyruvic to acetic acid were 7.6:1 for T. utilisstrain 3 and 6.8: 1 for strain MlI 1. Thus the formationof pyruvic acid from approximately 8 per cent of thesugar utilized accounts for the high glycerol productionby these organisms.

Pyruvic acid has been demonstrated as an intermedi-ate product in the fermentation of glucose by yeastjuice (Grab, 1921) and by toluene-treated yeast cells(Neuberg and Kobel, 1929). Neuberg and Kobel (1930)later showed that pyruvic acid is produced from glucoseby intact cells of S. cerevisiae in the presence ofNa2HPO4. A conversion of 0.88 per cent of glucose topyruvic acid was obtained. In the absence of Na2HPO4,the pH dropped from about 6 to 3 and no pyruvic acidcould be detected. A comparison of these findings withthe results in this paper shows that T. utilis producesapproximately 9 times as much pyruvic acid, on thebasis of glucose consumed, as does S. cerevisiae.

Pyruvic acid was identified in the acid-ether extractby treating the extract with 2, 4-dinitrophenylhydrazineand chromatographing the resultant hydrazone on asilica column. The method used was a slight modifica-tion of that described by Brummond (1953) and the

TABLE 2. Fermentation products formed in the absence andpresence of sulfite by Torula utilis, strains 3 and Mll

T. utilis strain 3 T. utilis strain Mil

mM/l00 mM/l100 ml/t100 mM/l100Products mM of mm of mm of mms of

glucose glucose glucose glucoseused in the used in the used in the used in theabsence presence absence presenceof sulfite of sulfite of sulfite of sulfite

Glycerol ............... 17.0 29.0 17.0 31.2Ethyl alcohol .......... 88.2 79.2 85.3 69.3Acetoin ................ 0.07 3.6 0.04 5.3Acetic acid ............ 2. 1 5.9 2.1 4.7Pyruvic acid........... 16.2 8.4 14.5 4.0Acetaldehyde .......... none 9.0 none 9.6Carbon dioxide -.106.8 92.0Yeast carbon**. 46.2 44.8Carbon recovery (%c) - 78.2 71.8O/R ratio .- 1.07 - 0.99

* The yeast carbon was calculated on the basis of 46 percent carbon in dry yeast.

finidings are illustrated in figure 2. The nonvolatile acidformed by culture M11 was also shown to be pyruvicacid by the same procedure.

Fermentation products in the presence of sulfite. Thefermentations were ruin in a closed system so that theCO2 would be absorbed and determined. Each flaskwas equipped with a magnetic stirrer to keep the yeastin suspension. Medium 2 was used, and the volumeof centrifuged cells after incubation at 30 C for 12 hramounted to approximately 1 per cent of the volumeof the medium. (One ml of wet yeast is equivalent toabout 200 mg dry weight.) The pH of the medium wasthen adjusted to 6.8, and the addition of sterile 25 percent sulfite was begun. The additions were made asoften as was needed to maintain a concentration ofabout 0.5 per cent of free sulfite. Five 1 ml additionswere made over a period of 50 hr. Data on the productsformed by the parent culture and mutant Ml 1 aregiven in table 2 (columns 2 and 4).

In both fermentations, about 90 per cent of the glu-cose was utilized. The yields of products were about thesame in both fermentations. Carbon dioxide was thelargest product, ethanol second, and glycerol third. On

0.7 y

L EGEND:* H YDRAZOIVE FROM /00 Y

0.6 _ PYRl V/C ACIDO HYDRAZOAIE FROM MIXTURE 507

PYRUV/C AND 50r UtNOWINE0.5_ ACID

04

0/ 2

/1OLDBAC/< VOL UM4ES

x 1091 6

FlIG. 2. Chromatographic comparison of 2,4-dinitrophenyl-hydrazones of pyruivic acid and the nonvolatile acid producedby Torlltla uttilis.

Column: 4 g silica, 2.8 ml lm phosphate buffer, pH 7.2.Solvent: 95 per cent chloroform, 5 per cent butanol, sat-

uirated with buffer.Holdback volume (Hbv): 4.9 ml.Twenty drop (0.057 Hbv") fractions were collected and optical

density at 350 m,u determined after addition of 2.5 ml of solvent.The large peak that moves with the front is the excess 2,4-

dinitrnnhe.nvlhydraz.7ine .

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YEAST MUTANTS AND GLYCEROL FORMATION27

a wNeight basis, glycerol accounted for 15 per cent ofthe glucose fermented l)y the parent, culture and 16.2per cent of that, uitilized in the mutant MIII fermenta-tioni.

If the data are compared oIn a molar basis, the agree-ment between glycerol and its linked products is fairlygood. Assuming that there should be I mm of glycerolfor each mm of acetaldehyde and pyruvic acid and 2mm of glycerol for each mm of acetoin and acetic acid,the calculated glycerol yield is 36.4 mM (found 29.4) forT. utilis strain 3 and 33.6 (found 31.2) for strain Ml1.The oxidation-reduction balance (0/1t) is good but therecovery of carbon is poor. Approximately 25 per centis missiing. Since the O/R balance is so close to 1, themissing products must also have an O/R ratio of 1.Lactic acid or a polysaccharide would satisfy this O/Rrequirement, but neither (could be detected in themedium.

In comparisoi with the sulfite free fermentation, moreglycerol anid acetaldehyde, as would be expected, wereproduced per 100 mm of glucose. More acetoin andacetic acid anid less pyruvic acid were produced in thepresence of sulfite. Acetoin is formed by condensationanid decarboxylation of pyruvic acid. If the mm ofpyruvic reqjuired to form the acetoin are added to thepyruvic acid present, the total is 15.6 mm for the parentcultuie anid 14.6 mm for the mutant. These figures areabout, the same as those found in the sulfite-free fer-mentation.

RI ESULTS WVITH SACCHAROMYCES MUTANTS

Jlultants unable to use alcohol. The haploid strain ofS. cereisiae, WY38, was irradiated with ultravioletlight for 4 mimi, which gave a survival of 0.5 to 1 percent. The yield of mutanits that would not grow on theglucose minimal medium (no. 3) supplemented withbiotin, thiamine, pyridoxine, and pantothenic acidvaried from 1 to 2.5 per cent, which is a higher per-

TABi,E 3. Glycerol production by muiitants of haploid strain ofSaccha rootiycCes cerevisiae 11 Y38 screened for ethyl alcohol

utilization,*

GlucoseMutants Alcoholzati Glycerol YieldUiiain Before sulfite Final

addition

_mg/Inl ing/minWYNV38 Parelit 18.1 1.4 12.0

63 Yes 18.1 1.2 11.864 Yes 18.1 (.5 11.48( Yes 21.9 0.6 13.272 No 21.9 0.8 10.473 No 19.5 5.6 10.079 No 15.5 2.0 14. 1

* Original gltucose wbas 29.2 mg/ml. Yield of glycerol is cal-culated on gltucose uise(d after addition of stllfite and correctedfor glycerol prodlulcedl l)efore suilfite adlditions. Fermentationtime was 72 hr.

centage than was obtained with T. utilis. In addition,19 colonies grew on replication from complete to theabove glucose-vitamin medium but not on this mediumwhen the glucose was replaced by ethyl alcohol (2 percent). Three of these and three colonies that grew wellon the alcohol medium, and also the parent, were testedfor glycerol production (table 3). Glucose utilizationwas good for all the strains with the exception of mutant73. Glycerol production was about the same for mutantsthat could use alcohol and those that could not, andnone of the mutants produced significantly higheryields of glycerol than the parent. It was later shownthat these cultures were respiratory (petite) mutants(Slonimski, 1949), which lack enzymes of the terminaloxidative chain, and hence, like mutants lacking alcoholdehydrogenase, would be unable to growv, on alcohol asa sole source of carbon.

Another screening test was devised on the assump-tion that high acid production other than lactic acidshould be linked to glycerol production. Acid indicatormedium (no. 7) was used for this purpose. The pH ofthis medium was 6.3 to 6.5 after it was autoclaved. Inthe buffered medium, yeast WY38 forms smooth, lightgreen colonies under either aerobic or anaerobic condi-tions. The colonies do not give a distinct yellow zonein the surrounding medium, but they do so if the bufferis omitted. After irradiation, no colony giving a largezone of acid was detected, but there were differencesin colony color. Three types of colonies were observed(dark green, light green, and yellow) after 28 hr ofaerobic or anaerobic incubation. Most of these cultureswere stable on subculture, indicating some type ofmutation. These cultures were tested for production oftitratable acid in complete medium (3 per cent glucose),and for glycerol production in the standard sulfitemedium. The data are given in table 4.

TABLE 4. Glycerol products by colony-type mutants* of Saccha-o-myces cerevisiae XW'Y38

Mutants

WY385056654451574558

Colony Color

ParentD)ark greenDark greenDark greenLight greenLight greenLight greenYellowYellow

AcidProduction

ml 0.1N/mI

0.280.210.230.240.240.230.270.25

Glucose

Beforesulfite Finaladdition

mIg/ml

30.725.135.014.532.120.728.321.929.8

ing/nil

1.71.71.31.31.61.81.73.31.8

GlycerolYield

13.915.46.019.710.717.720.620.513.3

* The color of colony is that obtained on agar mediuim con-taining bromeresol green. Original gltucose was 38.4 mg/mi.Glycerol yield is calculated as in table 3. Fermentation timewas 60 hr.

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There was no correlation between colony color, ti-tratable acid, or glycerol production. Four of the eightmutants gave from 20 to 50 per cent higher yields ofglycerol than the parent, but they did not follow anycolony color pattern. The mutants giving the highestconversion of glucose to glycerol in the sulfite periodalso utilized glucose in the pre-sulfite growth phasemuch faster than the parent. Conversely, the slowestglucose users were the poorest glycerol producers.

Three of the most promising mutants were tested in20 per cent glucose fermentations because this concen-tration is usually in industrial production. The completemedium (no. 2) was used, but with 20 instead of 3 percent glucose. One hundred ml of medium in a 500 mlErlenmeyer flask was inoculated with 0.4 ml of a 24 hrculture grown aerobically in medium no. 2. The flaskwas shaken on a reciprocating shaker at 90 cycles permin during incubation at 30 C. The yeast was allowedto grow for 24 hr before any sulfite was added. Sulfitewas added manually to maintain from 0.5 to 0.75 percent free sulfite until no more sulfite was bound duringa 6 hr period. Table 5 gives the results of two such ex-periments. In the first experiment, WY38 and threemutants derived from it are compared. Glucose utiliza-tion was rapid and about the same for all the cultures,but glycerol yields differed greatly. Mutant 45 waspoorer than the parent, but strains 57 and 65 gave morethan twice as much glycerol as the parent.

In table 5, it should be noted that glycerol yield isbased on total glucose used instead of on glucose usedafter beginning the addition of sulfite. This method ofcalculation would necessarily be followed in fermenta-tion on an industrial scale.

In experiment 2, culture 57 was compared to S.cerevisiae strain 49, a yeast that has been used exten-sively in the last 5 years for glycerol production at theForest Products Laboratory. In this experiment, theflasks were given a heavy inoculum of preformed cells.This inoculum was built up in 3 stages, and in the laststage was grown in medium no. 2 containing 20 percent of glucose and 0.5 per cent of sulfite. The cellswere centrifuged from the medium and 1 ml of packed

TABLE 5. Comparison of superior cultures of Saccharomlycescerevisiae in sulfite fer-mentation with 20 per cent glucose

Glucose Glycerol BasedMutants Time on Total

Before sulfite Final Glucose Usedaddition

hr mg/ml mg/ml %

Experiment 1WY38 72 170 29 10.1

45 72 174 29 6.157 72 171 22 25.465 72 163 28 22.0

Experiment 2WY57 108 200 2.4 20.6SC49 108 200 2.4 23.2

cells was used per 100 ml of medium. This type ofinoculum permitted immediate addition of sulfite andmade all the glucose available for glycerol production.Determination of bound sulfite during fermentationshowed that mutant 57 bound sulfite more rapidly thanmutant 49 for the first 96 hr, when it reached a maxi-mum. Culture 49 continued to bind sulfite, and at 108hr had bound more sulfite than 57. The yield of glycerolat this time was 10 per cent greater for mutant 49, butit is possible that mutant 57, because of its higher rateof glucose utilization, had run out of glucose and turnedto glycerol as a source of energy. Some other testsshowed that mutant 57 grew about 4 times as fast asmutant 49 in the presence of 0.5 per cent sulfite. In 1per cent sulfite medium, mutant 49 grew very littlebut mutant 57 still made excellent growth. At 1.5 percent sulfite, neither yeast made much growth. Whilemutant 57 is a promising yeast, no claim to superiorityover mutant 49 can be made on the basis of presentdata. This question can only be answered by extensivepilot plant tests.

SUMMARYThirteen morphological and eight nutritional mu-

tants of Torula utilis strain 3 (Candida utilis) were ob-tained by ultraviolet irradiation, light reactivation,and subsequent plating.The mutants were tested for glycerol production, and

none of them produced more than the parent.T. utilis strain 3 and one of the mutants, Ml 1, were

compared with respect to formation of several productsfrom glucose with and without added sulfite. Therewas no difference between the two cultures in yield ofproducts on a given medium, but added sulfite increasedthe yield of glycerol, acetaldehyde, acetoin, and aceticacid, and decreased the production of pyruvic acid. Inthe sulfite-free fermentations, pyruvic acid accountedfor about 8 per cent of the glucose utilized.

Only about 75 per cent of the glucose was recoveredin cells and products. The unknown products musthave an oxidation-reduction ratio of about 1 since thedetermined products had such a ratio.A haploid strain of Saccharomyces cerevisiae, WY38,

gave a higher percentage of mutants than T. utilis.Fifteen mutants were obtained and tested for glycerolproducing ability. Two of the mutants gave from 50 to250 per cent more glycerol than the parent in sulfitefermentations. One of the mutants gave about the sameyield of glycerol but grew faster and tolerated sulfitebetter than a lonig-used, high-yielding strain of Sac-charomyces cerevisiae, 49.

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