FORMATION OF ESTERS BY YEAST

I. THE PRODUCTION OF ETHYL ACETATE BY STANDING SURFACE CULTURESOF HANSENULA ANOMALA

JOSEPH TABACHNICK' AND M. A. JOSLYNDepartment of Food Technology, College of Agriculture, Berkeley, California

Received for publication May 8, 1952

It has been known for some time (Hansen,1891) that organic esters may be produced insmall amounts by some microorganisms as by-products in their utilization of organic com-pounds. A detailed review of the literature to1922 on aroma producing microorganisms is givenby Omelianski (1923). The taxonomy of the yeastgenus Hansenula, which contains species produc-ing the greatest yields of ester, is discussed byBedford (1942) and Gray (1949). The early ob-servations on the physiology of ester formationwere reviewed by Tabachnick (1950). While ourstudies of ester formation by yeast, begun in1946, were in progress, several papers appearedon the physiology of ester formation by H.anomala.Gray (1949), working with standing surface

cultures of H. anomala (Hansen) in a glucosemedium, showed: (1) that ester was producedunder conditions of limited aeration from someunknown anaerobically produced intermediateand (2) that the ester was utilized as rapidly asit was produced. Although the ester had beenidentified by its odor as ethyl acetate (Takahashiand Sato, 1911; Bedford, 1942), Gray was thefirst to identify it as ethyl acetate. Davies et al.(1951) presented additional proof that the esteris ethyl acetate. Peel (1951), using cell suspen-sions of H. anomala in an ethanol, acetic acidmedium, found ester formation to be an aerobicprocess with an optimum pH of 4.5 to 5. In etha-nol alone the pH optimum was difficult to definebut appeared to range from 2.5 to 3. Peel alsoshowed with growing cells and cell suspensionsthat the amount of ester formed is too large tobe accounted for by the reversal of a simpleesterase reaction, indicating a more exergonicmechanism of ester formation.

In the following report on growing cultures of'Present address: Division of Microbiology,

Albert Einstein Medical Center, Northern Divi-sion, Philadelphia 41, Pennsylvania.

1

H. anomala, some of the observations recordedby Gray (1949), Davies et al. (1951), and Peel(1951) are confirmed. These workers identifiedthe ester indirectly; we isolated and purifiedsufficient ester to determine both physical andchemical properties. In addition, it will be shownthat ethyl acetate is not produced directly fromglucose but is formed as the result of an aerobicutilization of ethanol accumulated in the fer-mentation of glucose. The influence of severalenvironmental factors on ester production will bediscussed.

METHODS

Cultures. The yeast cultures used for the majorportion of this work were Hansenula anomala var.longa type B (Naegeli) Dekker (culture no. 317in the Food Technology collection) and Pichiakluyveri Bedford (culture no. 7 in the collection).Both these yeasts have a predominantly oxida-tive metabolism and form a film on the surfaceof standing liquid cultures.

Composition of media. Medium 1-containedKH2PO4, 1 g; (NH4)2SO4, 1 g; MgSO4.2H20, 1 g;NaCl, 1 g; CaCl2 * 2H20, 0.1 g; thiamin hydrochlo-ride, 100 pg; pyridoxine hydrochloride, 200 Mg;biotin, 2.5 Mg; in 1,000 ml of distilled water. Ascarbon source glucose or ethanol was used in con-centrations given below. This medium, althoughchemically defined, was not a complete mediumfor the growth of H. anomala, the rate of glucoseutilization in medium 2 being considerably morerapid. Medium la-contained the same constitu-ents as medium 1 but the growth factors wereomitted. Medium 2-had the same salt constitu-ents as medium 1 but the growth factors werereplaced by 50 ml of liquid yeast autolysate perliter. Medium 3-Wickerham's nitrogen basemedium (Wickerham, 1946).The media were dispensed in two liter amounts

into 2,500 ml narrow necked Fernbach flasks. Adouble strength solution of the inorganic salt

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

JOSEPH TABACHNICK AND M. A. JOSLYN

constituents was heat sterilized separatelyfrom the glucose solution, and both were mixedthen aseptically after cooling to approximately70 C. The ethyl alcohol was sterilized by passage

through a bacterial sintered glass filter, and theapproximate amount was added to the appro-

priate basal medium. The requisite amount ofgrowth factor stock solution (20 X strength) was

sterilized by filtration before addition to the me-

dium. The yeast autolysate was also sterilized byfiltration. The initial pH of all the media usedwas approimately 4.5.One per cent inoculum of young actively grow-

ing cellswas used. In the earlier experimentswherethe saponification method for ester determination(Amerine, 1944) was used, 100 ml of sample was

withdrawn aseptically for analysis, but with thecolorimetric method (Hestrin, 1949) 50 ml or lessof sample sufficed.

Analytical metwods. pH. The pH was deter-mined to :0.05 with a Beckman industrial modelglass electrode asembly.

Total acid. Ten or 25 ml aliquots were broughtto a rapid boil and titrated hot with 0.1 M NaOHusing phenolphthalein as indicator.

Deternination and identifiwtion of ester8. At thestart of this work the total ester content was

deternined by the petroleum ether extractionand saponification method described by Amerine(1944). Subsequently the Hestrin (1949) directcolorimetric method was used. Glucose and py-ruvate in concentrations from 0.2 to 1 per centinterfere with this colorimetric method, and itwas necesary to distill the ester from solutionscontaining these compounds.The esters to be identified were obtained after

12 to 16 days' growth of H. anomala and P.kluyveri in 2 L of medium 2, containing 100 g ofglucose. The yeast cells were ifitered off, and themedia were distilled under vacuum in an all glasstill to remove the esters. The distillate obtainedfrom the H. anomala culture medium, containing

8.7 mm ester per 100 ml, was concentrated byfreezing and purified by extraction (three times)with alkaline water (Porter and Stewart, 1943)and treatment with saturated calcium chloride.The purified ester then was dried with anhydrousmagnesium sulfate (Robertson, 1948). The physi-cal and chemical properties of the purified esterfrom H. anomala given in tables-1 and 2 showthe ester to be ethyl acetate. The acid portion ofthe ester also was shown to be acetic acid from

the Rf value obtained by a modification of thepaper chromatography method of Fink and Fink(1949).2

Insufficient amounts of the ester produced byP. kluyveri were recovered for complete identifi-cation. The acid portion of the ester was aceticacid, but the alcohol portion was an alcohol otherthan ethanol.

TABLE 1Physical properties of the purified ester

from Hansenula anomalaXALLIN- LITERATURE

PtTURIED CKItODT VALUES PORESTER ETHYL ETHYL

ACETATE ACETATE

Boiling Point* 75-790 74.770 770Refractive in- 1.3721t 1.3722t 1.37012tdex e.?

Density d' 0.8906 0.8943t* Micro-boiling point determination-as de-

scribed in McElvain (1949).t Bausch and Lomb-Abbe refractometer-

Tungsten filament lamp 120 v 100 watts as lightsource.

Dn2 and d': from Griswold, Chu, and Win-sauer (1949).

TABLE 21;4, ical data on the esterfrom Hansenula anomala

MLEQTAOF 1mm OF mm OF EQUIVALENT

ISOLEATS0D ALCOHOL VOL&TIL ACID WEIGHT OFSOLTED REOVEUED REOVEt ESEtESTER TAKEN RMZZ*WWZZt ZTM

1.28 1.35 1.08 92.7

* Calculated as ethanol.t Calculated as acetic acid.t Average of six determinations-literature

value - 88.

Volatile acid (acetic acid). The ester presentwas evaporated off on the steam bath underslightly alkaline conditions, and the volatile acidspresent were separated and identified by themethod of Duclaux as modified by Van der Lek(1930). The volatile acid in every instance wasacetic acid only.

Ethanol. The method used was a modificationof the acid dichromate method of Semichon and

'We wish to thank Dr. William Stepka forsuggesting the use of a mixture of 74 ml butyl al-cohol, 19 ml glacial acetic acid, and 50 ml wateras solvent for the hydroxamic acids.

2 [VOL. 65

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

FORMATION OF ESTERS BY YEAST

Flanzy (1929) and is similar to that used by Peel(1951). When more than 0.2 mm per 100 ml ofester was present, the ester was saponified andthe ethanol present in the ester was subtractedfrom the total ethanol to obtain the free ethanolin the sample.

Glucose. Glucose was determined by themethod of Somogyi (1945).

RESULTS

(1) Rate of aerobic and anaerobic ester produc-tion in glucose yeast autolysate (medium 2). The

TABLE 3Rate of ester production by Hansenula anomala

in glucose yeast autolysate (medium 2)

GLUCOSE| ACETIC TOTAL EYLDAYS G/0 d

pH ACID, ACID, ACETATE,*mM/100 InM/100 mM/100 ML

Aerobic condition

0 5.2 4.8 1.56 0.0021 4.85 4.1 0.13 1.75 0.093 2.98 3.4 _ 2.30 0.884 2.18 3.3 0.30 2.33 1.306 0.32 3.1 0.49 2.80 4.408 0.017 3.1 0.62 2.90 6.8012 0.024 2.4 0.67 3.25 10.5014 2.5 0.92 2.64 -

Anaerobic condition

0 5.2 4.8 1.56 0.0023 4.72 4.1 0.37 1.46 0.00513 3.60 3.7 0.25 1.62 0.0124 2.45 3.7 0.27 1.75 0.00335 0.55 3.5 0.42 1.97 0.03539 0.29 3.6 - 0.05

* By the method of Amerine (1944).

anaerobic experiment was carried out under an

atmosphere of N2 in a 3 liter mercury sealedErlenmeyer flask. The data for the experimentare given in table 3. It is seen readily that underaerobic conditions forty-one per cent of the esterwas produced after all the glucose had disap-peared. The ester is not produced directly fromglucose by H. anomala but from ethanol. Themajority of ester was produced after the acidreaction had dropped to pH 3 or below. The yieldof ester after 12 days was 0.92 g from 5.2 g ofglucose or 24 per cent on the basis of carbonutilized. The volatile acid increased graduallyover a period of 12 days and then rose rapidly

from 0.67 mm per 100 to 0.92 mm per 100. Thisrise may be due to oxidation of ethanol directlybut is more likely due to hydrolysis of ester.No ester was produced by growing cells in the

absence of oxygen. The amount of ethanol formedat the completion of the experiment was 1.94 g per100 or a yield of 48 per cent on the basis of thecarbon utilized. The amount of acetic acid formedanaerobically was small; some of it probably wasintroduced with the inoculum, and the remaindermay have resulted due to an incomplete removalof oxygen from the fermentation flask. No fixed

-j

00

0

-J

n00Ul)

nw

0(f)D-JU

'U 10 20 30 40 50TIME IN DAYS

Figure 1. Rate of ester production by Hansenulaanomala in a glucose, ammonium salt mediumwithout growth factors. Ester and acetic acid inmg per 100 ml.

acid was formed anaerobically although aero-bically about 1 mm per 100 ml was found. Aero-bically the H. anomala film utilized the glucosecompletely in one-fifth the time necessary foranaerobic utilization.

(2) Ester production in a glucose synthetic me-dium (la) without growth factors. In this medium,with glucose as carbon source and ammoniumsalt as sole nitrogen source, 81 per cent of theester was produced after the glucose had beenutilized completely, as shown in figure 1. Withregard to change in pH, volatile acid, total acid,etc., the results are similar to those described forthese factors in the previous experiment. Al-

3

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

JOSEPH TABACHNICK AND M. A. JOSLYN

though this medium was a poor one for H. ano-mala, six weeks being required for complete utili-zation of glucose as contrasted with 8 days formedium 2, the yield of ester, however, remainedunaffected. The ester yield here was 20 per cent(on the basis of carbon content) as compared

TIME IN DAYSFigure B. The effect of nitrogen source and

growth factors on the yield of ester produced byHansenula anomala and Pichia kluyveri.Open circles = Hansenula anomala in 5 per cent

glucose yeast autolysate medium.Half-closed circles - Hansenula anomala in

1.8 per cent ethanol ammonium salt mediumwith growth factors.

Closed circles = Hansenula anomala in 5 percent glucose ammonium salt medium.Open triangles - Pichia kluyveri in 5 per cent

glucose yeast autolysate medium.Closed triangles - Pichia kluyveri in Wicker-

ham's nitrogen base medium with 5 per centglucose.

with 24 per cent for growth in medium 2. Thusit appears that no specific nitrogen source orgrowth factor is especially beneficial to esterproduction, or what is more likely, H. anomalacan synthesize all the enzyme complexes neces-sary for ester production from glucose, am-monium ion, and the usual essential inorganicelements. As a result of the poor growth in

medium la, however, the time necesary formaximum ester production was prolonged. Theeffect on ester production of various growthmedia for H. anomala and P. kluyveri is shownin figure 2.

(3) Ethanol as an "intermediate" in ethylacetate production from glucose. Yamada (1927)and Gordon (1950) postulate ethanol as thesource of ethyl acetate but present no experi-mental evidence for their views. Figure 2 inGray's paper (1949) shows that approximately

TIME IN DAYSFigure 8. Illustrating the direct production of

ethyl acetate from ethanol accumulated in thefermentation of glucose by Hansenula anomala inmedium 1. Ethanol and ethyl acetate in mg per100 ml.

40 per cent of the ester in his experiment wasformed after all the glucose had been utilized,but this fact is not mentioned in his discussion.Peel (1951) was the first to report the productionof ester from ethanol.

Figure 3 illustrates clearly the direct formationof ethyl acetate from the ethanol produced inthe utilization of glucose in medium 1. At firstthere was a rapid increase in ethanol productionup to the 20th day; all of the glucose was utilizedduring this interval. Twenty per cent of the ethylacetate was formed slowly during this time. Afterthis period, there were a relatively rapid utiliza-tion of the ethanol produced and a correspondingfivefold increase in the rate of ethyl acetate pro-

4 [VOL. 65

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

FORMATION OF ESTERS BY YEAST

duction. Since ethanol accumulated in the medi-um during glucose utilization and there is a threeday lag before the ethanol is actively respired, itis highly probable that some of the enzymes in-volved in ethanol utilization are adaptive innature.The maximum yield of ethanol including that

transformed into ethyl acetate was 50.6 per cent.The yield of ethyl acetate was 17.8 per cent. Theyeast film apparently utilized some of the freeacetic acid since its value here never rose above0.48 mM per 100 ml. The total acid and pHchanges were similar to those in the previousexperiments.

(4) The production of ethyl acetate with ethuanolas sole carbon source. The changes occurring inmedium 1, with 1.8 per cent ethyl alcohol as solecarbon source, typical of the data obtained forthe direct production of ethyl acetate fromethanol, are shown in figure 4. The curve ofinterest in figure 4 is the acetic acid curve. Upto the 17th day there was a slow increase to 75 mgper 100 ml of acetic acid. For the next nine daysthere was a slow utilization of acetic acid andfinally a sharp increase coincidental with therapid hydrolysis of ethyl acetate. The largestamount of free acetic acid obtained was 348 mgper 100 ml. The major portion of this acid appearsto arise directly from the hydrolysis of the esteras can be seen readily from comparing the ethanoland acetic acid curves in figure 4. Figure 4 showsno accumulation of ethanol but instead an in-crease in acetic acid during ester hydrolysis. Theleveling off of the acetic acid curve after the 44thday is probably caused either by utilization ofacetate or death of the cells in the yeast film.

Since 5 mm per 100 ml of acetic acid at pH2.4 to 2.6 (pH for the last 24 days of the experi-ment) is usually toxic to H. anomala (as deter-mined from cell suspension experiments), thecells in the film were examined periodically anda vital stain was made using 1:10,000 methyleneblue at pH 4.6. At the conclusion of both thisand the glucose experiment (figure 3), 90 percent of the cells grown on glucose was found tobe viable by vital stain, the rest being granularand distorted. The cells grown in the ethanolmedium were 100 per cent dead after 35 days. Acheck by viable count on wort agar plates,however, showed 16 per cent of these ethanolgrown cells to be alive. Except for these fewresistant cells, it can be said that about 5 mm per

100 ml of acetic acid at pH 2.4 is toxic to H.anomala.The fixed acid produced from ethanol was

1.6 mm per 100 ml. The maximum yield of esterwas 44.5 per cent on the basis of carbon utilized.

(5) Effect of excess aeration on ester production.This experiment was conducted with 1,000 mlof medium 1 using the shake culture techniqueidentical with that used in obtaining active cell

7 ~~~~8

oS0~~~~~~~C4-j

0

Cd 2~~~:

C1 10 20 30 40TIME IN DAYS

Figure 4. The direct production of ethylacetate from ethanol by Hansenula anomala andthe increase in acetic acid concentration asthe ester is utilized. Ethyl acetate and aceticacid in mg per 100 ml.

suspensions. The relatively rapid glucose utiliza-tion in this experiment (figure 5) demonstratesthat availability of oxygen, as well as lack ofcertain growth factors, may have been one of thelimiting factors with regard to glucose utilizationin the previous experiments with medium 1.The total ethanol produced from 2 per cent

glucose was only 13 per cent of that formed inthe standing culture experiment (figure 3). Theproduction of ethyl acetate was also lower thanwith the standing culture experiments, being57 per cent of that formed in the standing cultureexperiment with medium 1. The lower yield ofester is not surprising since ethanol productionalso is lower under the above experimental condi-tions. One liter of a water solution containing3.78 mm per 100 of ethanol and 1.60 mm per 100ethyl acetate, respectively, was shaken for 12hours. After 12 hours the results showed that

5

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

JOSEPH TABACHNICK AND M. A. JOSLYN

2 per cent of the ester had been lost by evapora-tion and 9.3 per cent of the ethanol. This loss isnot large enough to account entirely for therapid decreases in these compounds within the4 hour sampling periods of the actual experiment.There was a relatively large amount of free

acetic acid (4.28 mu/100) produced in 50 hours.From the ethanol and ethyl acetate curves offigure 5, it would appear that much of the acetatearose from incomplete oxidation of ethanol. Theamount of fixed acid produced here was 1.72mm per 100 and the pH dropped to a low of 2.2.

TIME IN HOURS

Figure 5. The effect of excess aeration on esterproduction by Hanmenula anomala.

The results of this experiment combined withthose for the standing cultures show, as was

pointed out by Gray (1949), that there is a critical02 tension at which maxmal production of estercan occur. The other very important factor inhigh yields of ester, i.e., pH, will be discussed ina later publication dealing with cell suspensionexperiments.

(6) Ester production by Pichia kluyveri inmedia 2 and 3. The results obtained for standingsurface cultures of P. kluyveri in media 2 and 3are shown in figures 6 and 7. These show that theester formed by P. kluyeri was produced directlyfrom glucose or from some product formed earlyin glucose utilization.

The ester odor emitted by these cultures re-minded one of the heady odor of gardenias, butodor alone is not sufficient for distinguishingesters. The typical sharp odor of ethyl acetatewas easy to identify when H. anomala was grownin an ethanol medium 1, but this odor wasmasked upon growth in other media. In medium 1with glucose, the odor was flower-like, whereasin medium 2 containing yeast autolysate, theapple-like odor described by earlier investigators(Hansen, 1891) was noted. The type of odoremitted during growth of the organism appears

a:

4~~~~~~~~~~~U3.0

ui XYXFiur 6.Etrpouto b ihaku fr

in a5prcnglcsyes uoyaemdum.

Lu0

UU)

<0

4 8 12 16TIME IN DAYS

Figure 6. Ester production by Pichia kluyveriin a 5 per cent glucose yeast autolysate medium.

to depend upon both the carbon and nitrogensources present in the medium.Although the glucose was utilized at a slightly

more rapid rate in medium 2, twice as much esterwas produced from the 5 per cent glucose inmedium 3 (figure 7), which may contain one ormore factors beneficial to ester production. Themaximum yield of ester produced by P. kluyveriwas approximately one-fifth of that formed byH. anomala. Absolute values for ester cannotbe determined since the identity of the alcoholportion of the ester remains unknown. The esterproduced by P. kluyvri does not appear to beethyl acetate (see above). This organism canproduce the ester with ethanol as sole carbon

6 [voL. 65

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

FORMATION OF ESTERS BY YEAST

source in medium 2, but the ester formed has notbeen identified.

In both media 2 and 3, the acetic acid decreasedsimultaneously with ester utilization (figures 6and 7). Apparently both ester and acetic acidmay be utilized readily by P. kluyveri when allthe glucose has disappeared. Twice as much aceticacid was formed in Wickerham's medium 3. Verylittle total acid was formed in either medium,and in medium 2 some of this acid was utilizedduring the last six days. The pH of the medium

TIME IN DAYS

Figure 7. Ester production by Pichia kluyveriin 5 per cent glucose Wickerbam's nitrogenbase medium.

dropped rapidly to 2.5 but in medium 2 there wasa slight rise to pH 3 during the last six days.

DISCUSSION

The experiments described above with growingcultures of H. anomala show that ethyl acetateis a direct product of ethanol oxidation. Appa-rently any carbon source which H. anomala can

utilize with a resultant production of ethanol willresult also in ethyl acetate production, providedsufficient ethanol is formed and the other re-

quirements (02 tension, low pH) for ester pro-duction are met.The fact that oxygen tension has an important

influence on ester production was confirmed. Aliterature survey by Tabachnick (1950) of pure

cultures of ester producing organisms showed allsuch organisms to be predominantly aerobic.In a survey of various ester producing yeastsand molds it was found that the acid portion ofthe isolated esters was in all instances acetic acid.Strict anaerobic conditions result in poor growthwith H. anomala and no ester production, whilean excess of air may result in either low ethanolproduction or perhaps a rapid oxidation of bothethanol and any ester which may be formed.With the proper amount of aeration, H. anomalacan give yields of ester as high as 48 per cent oftheoretical with ethanol as substrate. Some ofthe energy made available by ethanol oxidationprobably is used in forming the ester linkage.The aerobic production of ethanol observed

with this organism is not an unusual phenome-non. The aerobic production of ethanol from glu-cose by various molds was noted by Foster(1949) and in yeast by Custers (1940) underconditions of aeration even more efficient thanthose used here. With H. anomala approximatelyone-third as much ethanol was produced pergram of glucose consumed aerobically. In thestanding culture experiments semiaerobic condi-tions prevailed, for some of the yeast film settledto the bottom of the Fernbach flasks and gasbubbles were seen to rise from the bottom ofthe flask. The cells in the surface fihm, however,were more numerous and probably carried outthe major portion of both ethanol and ethylacetate production from glucose, for, as has beenobserved, a very low oxygen tension results invery slow glucose utilization and little esterformation.

Peel (1951) calculated that the concentrationof ethyl acetate in equilibrium with the observedconcentrations of ethanol and acetic acid presentin a culture medium would be 1.5 X 10-5 M in-stead of 46 X 10-3M actually found. Our valuesfor ethyl acetate production similarly are onethousand times greater than calculated equilib-rium values, but since there is no evidence of theexistence of a true equilibrium between the eth-anol formed from glucose or initially added andthe ethyl acetate and acetic acid produced, thisis not sufficient evidence against a mechanisminvolving esterase catalyzed hydrolysis. Otherevidence against a mechanism involving a re-versal by an esterase consists of: (1) the directproduction of large amounts of ethyl acetatewith ethanol as sole carbon source and (2) the

71953]

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

JOSEPH TABACHNICK AND M. A. JOSLYN

need for some unspecified amount of oxygen formaximum yields of ester. Both Gray (1949) andDavies et al. (1951) found acetaldehyde presentin small amounts in the spent glucose mediumupon which H. anomala had grown. This is notsufficient evidence for implicating acetaldehydeas an intermediate in ethyl acetate formation.Peel (1951) reported that acetaldehyde is notdirectly converted to ester but in low concentra-tions does stimulate ester production fromethanol. Contrary to previous reports, theamounts of growth factors and the nitrogensource do not affect significantly the yields ofethyl acetate produced by H. anomala. In acomplete medium, however, the substrate isconsumed more rapidly, and as a consequencethe maximum yield of ester may appear twoweeks sooner than in a less favorable medium.Although ester production appears to occur

over a fairly wide pH range (4.4 to 2.2), thehighest yields of ester are obtained below pH 3.The importance of low pH in the production ofhigh yields of ester was not made clear until workwith cell suspensions had been conducted (un-published data). At this low pH, relatively lowconcentrations of acetic acid are toxic to thisorganism, and as suggested by Gordon (1950)for Endoconidiophora moniliformis, the formationof ethyl acetate may protect the organism fromthe toxic effects of acetic acid. In this instanceethanol would be acting as the detoxifying agentrather than acetate, which is known to have adetoxifying function in animal metabolism.The rapid utilization of the ethyl acetate

produced by H. anomala has been observed byother workers (Kayser and Demalon, 1909; Will,1913; Gray, 1949; Peel, 1951). The fact thatacetic acid accumulates during ester utilizationimplies that the ethanol portion of the hydrolyzedester is preferred by the yeast. Very little experi-mental evidence (Will, 1913) is available on themechanism for ethyl acetate utilization byH. anomala.The fixed acid formed by H. anomala in the

media used averaged 1 mm per 100 ml. A frac-tionation of the acids present (Hyams, 1950) bypaper chromatography resulted in the identifica-tion of spots for fumaric and citric acids and aspot which may be succinic or lactic acid. Underanaerobic conditions no fixed acid was formed.Aerobic C1402 fixation experiments' with cellular

' We wish to thank Dr. V. H. Lynch for per-forming these experiments.

suspensions of this organism resulted in radio-activity appearing in spots identified as succinic,malic, citric, and fumaric acids, these acids ofcourse being extracted from within the cells.The above data appear to indicate that a Krebs-like respiratory mechanism may exist in H.anomala for the oxidation of ethanol as well asother substrates.The experiments with growing cultures of

P. kluyveri show that the results obtained withH. anomala are unique for this organism and arenot applicable to other ester forming yeasts.The ester formed by P. kluyveri has not beenidentified. The amount of ester formed was small(h that produced by H. aromala). The significantincrease in ester yield when the cells were grownin Wickerham's synthetic medium as comparedto the lower yield with equally good growth in ayeast autolysate medium implies that P. kluyverimay be a good organism to use in investigatingthe need for cofactors which may be involved inester bond synthesis.

ACKNOWLEDGMENTS

We wish to express our thanks to ProfessorH. A. Barker and M. Doudoroff for helpful sug-gestions made throughout the course of thiswork.

SUMMARY

With growing cultures of Hansenula anomalaand glucose as carbon source, it was shown thatethyl acetate, the ester produced by this or-ganism, was formed as a result of the aerobicutilization of ethanol accumulated in the fer-mentation of glucose. The ester, in turn, wasutilized rapidly under aerobic conditions.The same high yields of ester were obtained

regardless of the type of nitrogen source added.However, in a nutritionally complex mediumcontaining glucose, which favored a high growthrate, the maximum yield of ester was obtainedwithin 8 days as contrasted to 50 days in amedium in which ammoniuim salts were used assole source of nitrogen.The high yields of ester obtained leave no

doubt that the formation of ester is an energyconsuming reaction and not the reversal of asimple esterase hydrolysis.

Since free acetic acid is toxic to H. anomalaat the acid pH (2.4 to 3.0) for optimal esteryields, it would appear that the formation ofethyl acetate has survival value for this or-

8 [VOL. 65

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

FORMATION OF ESTERS BY YEAST

ganism by preventing the accumulation of toxicamounts of acetie acid in the medium.

Experiments with growing cultures of Pichiakluyveri indicate that other microorganisms mayproduce esters other than ethyl acetate directlyfrom glucose or some early product of glucose.

REFERENCESAMERINE, M. A. 1944 Determination of esters

in wines. Food Research, 9, 392-395.BEDFORD, C. L. 1942 A taxonomic study of the

genus Hansenula. Mycologia, XXXIV, 628-649.

CUSTERS, M. TH. J. 1940. Investigations on theyeast genus Brettanomyces. Ph.D. Disserta-tion, Delft.

DAVIES, R., FALKINER, E. A., WILKINSON, J. F.,AND PEEL, J. L. 1951 Ester formation byyeasts. 1. Ethyl acetate formation by Han-senula species. Biochem. J., 49, 58-61.

FINK, FAY, AND FINK, R. M. 1949 Applicationof fiter paper partition chromatography toqualitative analysis of volatile and non-vola-tile organic acids. Proc. Soc. Exptl. Biol.Med., 70, 654-656.

FOSTER, J. W. 1949 Chemical activities of fungi.Academic Press, Inc., New York, N. Y.

GORDON, M. A. 1950 The physiology of a bluestain mold with special influence to produc-tion of ethyl acetate. Mycologia, XLII (1),167-185.

GRAY, W. D. 1949 Initial studies on the me-

tabolism of Hansenula anomala (Hansen) Sy-dow. Am. J. Botany, 36, 475-480.

GRISWOLD, J., CHU, PAO L., AND WINSAUER, W. 0.

1949 Phase equilibria in ethyl alcohol-ethylacetate-water system. Ind. Eng. Chem., 41,2352-2358.

HANSEN, EMIL C. 1891 Undersogelser over Al-koholyurs vamperisis Fysiologi og MorpologiVIII. Compt. rend. trav. lab. Carlsberg,3,44

HESTRIN, S. 1949 The reaction of acetylcholineand other carboxylic acid derivatives withhydroxylamine and its analytical applica-tions. J. Biol. Chem., 180, 249-261.

HYAMS, RICHARD 1950 Unpublished data.

KAYSER, E., AND DEMALON, A. 1909 Influencede I'aeration sur la formation des produitsvolatils dans la fermentation alcolique. Rev.viticult., 31, 61-64.

McELVAIN, S. M. 1949 The characterization oforganic compounds. The Macmillan Co.,New York, N. Y.

OMELIANSKI, V. L. 1923 Aroma producing mi-croorganisms. J. Bact., 8, 393-419.

PEEL, J. L. 1951 Ester formation by yeasts.2. Formation of ethyl acetate by washed sus-pensions of Hansenula anomala. Biochem.J., 49, 62-67.

PORTER, C. W., AND STEWART, T. D. 1943 Or-ganic chemistry for the laboratory. Ginn andCo., New York, N. Y.

ROBERTSON, G. R. 1948 Laboratory practice oforganic chemistry. The Macmillan Co., NewYork, N. Y.

SEMICHON, L., AND FLANZY, M. 1929 Dosage del'alcohol dans les vins et les spiriteaux parl'emplair du melange sulfichromique. Ann.fals et fraudes, 22, 139-153. See also ibid, 22,414-415; 23, 347-349

SOMOGTI, M. 1945 A new reagent for the deter-mination of sugars. J. Biol. Chem., 160, 61-73.

TABACHNICK, J. 1950 The chemistry and physi-ology of ester production by Hansenula ano-mala. Ph.D. Thesis, Univ. of Calif.

TAKAHASHI, T., AND SATO, H. 1911 Some newvarieties of Willia anomala as aging yeast ofsaki. J. Univ. Tokyo, Coll. of Agric., I, (3),227-269.

VAN DER LEK, J. B. 1930 Onderziekengen overde butyl alkoholgisting. Ph.D. Dissertation,Delft.

WICKERHAM, L. J. 1946 A critical evaluation ofthe nitrogen assimilation tests commonlyused in the classification of yeasts. J. Bact.,52, 293-301.

WILL, H. 1913 Einwerkung von Estern aufHefen und ondere Sprosspilze. Zentr. Bakt.Parasitenk., Abt. II, 38, 539-576.

YAMADA, M. 1927 On the mechanism of fruitester formation by Willia anomala sp. Bull.Agric. Chem. Soc. Japan, 3, (4-6), 73-76.

1953] 9

on February 3, 2020 by guest

http://jb.asm.org/

Dow

nloaded from