Oxygen requirements of yeasts

Oxygen

Requirements of

Yeasts

WIEBE VISSER, W. ALEXANDER SCHEFFERS, WILMA H. BATENBURG-VAN DERVEGTE, AND JOHANNES P. VAN DIJKEN*

Department ofMicrobiology and Enzymology, Delft Universityof Technology, Julianalaan 67,

2628 BC Delft, The Netherlands

Received 26 July 1990/Accepted2 October 1990

Type speciesof 75yeastgenera wereexamined for their abilitytogrowanaerobically in complex and mineral media. Todefine anaerobic conditions,weaddedaredoxindicator,resazurin,tothe mediatodeterminelow redox potentials. All strains testedwerecapableof fermentingglucosetoethanolin oxygen-limited shake-flask

cultures,eventhoseofspecies generallyregarded asnonfermentative. However,only23% oftheyeastspecies

testedgrewunder anaerobic conditions. Acomparative studywithanumberofselectedstrainsrevealed that

Saccharomycescerevisiae standsoutas ayeastcapable of rapidgrowthatlow redoxpotentials. Otheryeasts, suchas Torulaspora delbrueckiiandCandida tropicalis, grewpoorly

(tLmax.

0.03 and 0.05h-1, respectively)

under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter

organismsgrewrapidlyunderoxygenlimitation and then displayedahighrateof alcoholic fermentation.Itcan

be concludedthat theseyeasts have hitherto-unidentifiedoxygenrequirementsforgrowth.

Yeasts can be divided into three groups with respect to

theirfermentative abilities, namely, obligately fermentative, facultativelyfermentative, and nonfermentative yeasts. Ap-proximately 40% of the yeast species are listed as

nonfer-mentative(4). The listing is basedupon astandardtestwith Durhamtubes, inwhich visiblegasproduction isusedasthe criterion for alcoholic fermentation. Nearly all nonfermen-tativeyeastspecies, however, have been showntoproduce ethanoltosome extentunder the conditions of the Durham tubetest,and theirfermentative capacities oftenaregreatly

enhancedundermore appropriate conditions (27).

Oneofthemostimportantparameterswithrespect tothe occurrenceofalcoholicfermentationinyeastsis the

concen-tration ofoxygeninthe culture medium. For example, the Pasteur effect, the Custers effect (23, 31;M. T. J. Custers, Ph.D. thesis, Delft University of Technology, Delft, The Netherlands, 1940), the Kluyver effect (16, 24), and the Crabtree effect (8, 10, 29) are all closely related to the availability ofoxygen.

Fermentation,inprinciple,canprovide enoughenergyfor growth.However, theabilitytogrowanaerobically depends

notonlyonthefermentativecapacity. Manyspecies require alimitedamountofoxygentofermentglucose,forexample, Hansenula nonfermentans (26). Even when fermentation does occur under anaerobic conditions, as reported for Pachysolen tannophilus, the actual growthof theorganism

onD-xyloseaswellasonD-glucoseisstilldependentonthe availability ofoxygen (19). The addition of ergosterol and

unsaturatedfatty acids,whichareconsideredtobe essential medium componentsfor anaerobic growth of

Saccharomy-cescerevisiae (1, 2),doesnoteliminate thisoxygen

require-ment. Whether yeast species other then S. cerevisiae are

able to grow anaerobically is in general unknown or the

results arecontradictory. Forexample,anaerobicgrowthof

Candida utilis has beenreported byseveralauthors(3, 13), whereasothers have stated that C. utilis isnotabletogrow anaerobically (27).

A major problem in comparing the results of different

studies with respect to anaerobic growth ofyeasts is the

*Correspondingauthor.

definition of anaerobiosis. Also, the preparation of the inoculum is important: when aerobically grown cells are

used as an inoculum for anaerobic cultures, rapid growth

may occurforanumberofgenerationseven in theabsence ofergosteroland unsaturatedfatty acids(15).

The purpose of the present study was to examine the capacity ofvariousyeastsforanaerobicgrowthunder

stan-dardized conditions. To obtainabroad spectrum of yeasts, westudiedtypespecies ofthegenera, aslistedin theListof

Cultures of the Centraalbureau voor Schimmelcultures

(CBS), Delft,TheNetherlands.

MATERIALS ANDMETHODS

Organisms. Typestrains of the yeastgenerawereobtained

from the CBS and arecitedaslisted in the List of Cultures (31st ed., 1987, or32nded., 1990[in press]): Ambrosiozyma

monospora CBS 2554, Apiotrichum porosum CBS 2040, Arthroascusjavanensis CBS 2555, Arxiozyma telluris CBS 2685, BotryoascussynnaedendrusCBS6161, Brettanomyces bruxellensis CBS 72, Bullera alba CBS 501, Candida tropi-calis CBS 94, C. utilis CBS 621 (not a type strain),

Citero-myces matritensis CBS 2764, Clavispora lusitaniae CBS

6936, Cryptococcus albidus CBS 142, Debaryomyces

hans-enii CBS 767, Debaryozyma yamadae CBS 7035, Dekkera

bruxellensis CBS74,EeniellananaCBS1945, Endomycops-ella vini CBS4110, Fellomycespolyborus CBS 6072,

Fibu-lobasidium inconspicuum CBS 8237, Filobasidiella

neofor-mans CBS 132, Filobasidium floriforme CBS 6241, Guilliermondella selenospora CBS 2562, Hanseniaspora valbyensis CBS 479, Hasegawaeajaponica CBS 354,

Hol-leya sinecauda CBS 8199, Holtermannia corniformis CBS

6979, HormoascusplatypodisCBS 4111, Hyphopichia

bur-tonfi CBS2352,Issatchenkia orientalisCBS5147, Kloeckera apiculata CBS 287(notatypestrain), Kluyveromyces poly-sporus CBS 2163, Leucosporidium scottii CBS 5930,

Lipo-mycesstarkeyiCBS1807, Lodderomyces elongisporus CBS

2605, Malassezia furfur CBS 1878, Mastigomyces philip-povii CBS 7047, Metschnikowia bicuspidata CBS 5575, Mrakiafrigida CBS 5270, Myxozyma melibiosi CBS 2102, Nadsoniafulvescens CBS 2596, Nematospora coryli CBS

2608, Octosporomyces octosporus CBS 371, Oosporidium margaritiferum CBS 2531, P. tannophilus CBS 4044,

3785

0099-2240/90/123785-08$02.00/0

Rhodosporidium toruloides CBS 14, Rhodotorula glutinis

CBS 20, S. cerevisiae CBS 1171,Saccharomycodesludwigii

CBS 821, Saccharomycopsis capsularis CBS 2519,

Sarcino-sporon inkin CBS 5585, Schizoblastosporion

starkeyi-henricii CBS 2159, Schizosaccharomyces pombe CBS 356, Schwanniomyces occidentalis CBS 819, Sirobasidium mag-numCBS 6803,Sporidiobolusjohnsonii CBS 5470,

Sporobo-lomyces roseus CBS 486, Sporopachydermia lactativora

CBS6989,Stephanoascus ciferriiCBS 6699,

Sterigmatomy-ces halophilus CBS 4609, Sterigmatosporidium

polymor-phum CBS 8088, Sympodiomyces parvus CBS 6147,

Toru-laspora delbrueckii CBS 1146, Trichosporon beigelii CBS 2466, Trigonopsis variabilis CBS 1040, Waltomyces lipofer CBS 944, Wickerhamia fluorescens CBS 4565, Wicker-hamiella domercqiae CBS 4351, Williopsis saturnus CBS 5761, Wingea robertsiaeCBS 2934, Yarrowia lipolyticaCBS 599, Zygoascus hellenicus CBS 5839 (mating type a),

Zy-goascus hellenicus CBS 6736 (mating type a),

Zygosaccha-romyces rouxii CBS 732, and Zygozyma oligophaga CBS

7107.

Media. Complex medium contained, per liter of

deminer-alized water, 10 g of yeast extract (Oxoid) and 20 g of

glucose. The initial pH of the medium was set at5. Mineral medium, supplemented with vitamins and trace elements,

wasprepared asdescribed by Bruinenberg etal. (7), except

that the concentration of NaMoO4. 2H20 was increased

10-fold. Glucosewasaddedasthe solesourceof carbon and

energy at a concentration of 20 g. liter-'. Ergosterol and

Tween 80, dissolved in pure ethanol, were sterilized by

heating the solution for 10 min in a nonpressurized

auto-clave. These components were added to the medium at

concentrations of 6 and660mg. liter-', respectively.

Resa-zurinwasaddedtoboth media ataconcentration of 0.002% to indicate low redox potentials

(Eo'

= -42 mV).

Inocula. Inoculaforanaerobicgrowth testswere prepared

bygrowing theyeasts in 100-ml cottonwool-plugged Erlen-meyer flasks containing 20 ml of medium. Cultures were

incubated on a rotary shaker at 25°C and 50 rpm. Under

these conditions, alcoholic fermentation can betriggered in

facultatively fermentative yeasts because ofoxygen

limita-tion (28). Therefore, cells from these shake-flask cultures could be considered to be adapted to serve as inocula for

anaerobic growth tests.

Anaerobicgrowthtests. Anaerobic growthtests were con-ductedin30-mlserumflasksunder static incubationat25°C. To prevent the entrance of oxygen, we firmly closed the

flasks with 4-mm-thick butyl rubber septa. The flasks were

almost completely filled with medium and autoclaved at 110°C. During autoclaving, reducingagents in the complex

medium converted the redox indicator to colorless

dihy-droresorufin. The mineralmedium, treatedsimilarly, didnot become colorless. It was therefore deoxygenated prior to being autoclaved by including 8 mg of Aspergillus niger

glucose oxidase (grade III; Boehringer, Mannheim,Federal

Republic of Germany)liter-1; thisenzymepreparation con-tains, asanimpurity,acatalase activitythat is adequatefor

removal of H202 formedinthe reaction. The redoxindicator

intheserumflasksepta,treatedthisway,remainedcolorless

for atleast 4 months, whereas the use of ordinary rubber

septa(red rubber; BGAclass 1 FDA) caused recolorization

withinafew hours becauseofahighrateofoxygendiffusion. Aftersterilization, theflaskswerenotopenedtopreventthe entrance of oxygen. A small amount (5 to 10 ,u) of the

inoculumwas injected into each flask with a 2-ml syringe.

tion and turbidity of the inoculated flasks were checked twice daily for 1 month; prolonged incubation after this period did not lead to the onset of anaerobicgrowth.

Batch cultivation in fermentors. Comparative studieswere

performed by use of a laboratory fermentor with a 1-liter working volume and of the type described by Harder et al. (12). The mineral medium described above was used. To prevent foaming, we added 50 ,ul of silicone antifoaming

agent perliter. pH waskeptat5.0byautomatic titration with sterile 1 M KOH, the temperature waskeptat30°C,and the cultures were stirred at450 rpm. Forexclusion ofapossible

infection, the identity of the organism was checked after-wards by the CBS.

Levels ofdissolved-oxygen tension (DOT) weremeasured with a polarographic oxygen electrode (Ingold type 322

756702/74247)connected to an Ingold type 170 02amplifier.

Thesignal of this amplifier (percentair) was monitoredwith aKipp BD 41 datum recorder (Kipp & Zonen, Delft, The Netherlands).

The fermentor was continuously flushedwith pure nitro-gen gascontaining lessthan 5 ppmofoxygen(Air Products, Waddinxveen, The Netherlands) at a flow rate of 1

liter.

min-'.

The tubing of the fermentor was made of Norprene (Cole-Parmer Instruments Corp., Chicago, Ill.).

Analytical methods. Ethanol concentrations were deter-mined bygas-liquid chromatography on a Varian type 3400 gaschromatograph (Varian Benelux B. V., Amsterdam,The Netherlands) with a Hayesep Q column (Chrompack, Mid-delburg, The Netherlands) at a temperature range of 150 to 225°C, increasing by 15°C- min-'. Glucose concentrations were determined by the GOD-PAP method of Boehringer.

Dry weight was determined by filtration of the culture sample with a weighed polysulfone filter (Supor 450, pore size, 0.45 jxm; Gelman Sciences Inc., Ann Arbor, Mich.). Thefilter was washed withdemineralized water, dried in an R-7400 magnetron oven (Sharp Inc., Osaka, Japan) for 20 min at medium power, and reweighed.

Carbon analyses were done with a model 915B Tocamas-tertotalorganic carbon analyzer (Beckman Industrial Corp., La Habra, Calif.). Organic acids were determined by high-pressure liquid chromatography with an HPX-87H column (300 by 7.8 mm; Bio-Rad, Richmond, Calif.) at room tem-perature. The column was eluted with 0.01 N H2SO4 at a flow rate of 0.6 ml

min-'.

The detector was a Waters 441 UVmeter (used at 210 nm) coupled to a Waters 741 datum module (Waters, Milford, Mass.).

Electronmicroscopy. Twenty-two milliliters of cell culture wasprefixed with 3 ml of25% (vol/vol) glutaraldehyde for 10 to 30min at roomtemperature. After centrifugation, the cells were fixed again with 3% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.0) for 1 h, washed three times with the same buffer, and postfixed with 2% (wt/vol)

aqueous KMnO4 for 2 to 24 h. Afterfixation, the cells were stained with 1.5% (wt/vol) aqueous uranyl acetate during dehydration at the 50%(vol/vol) ethanol step and embedded in Spurr resin. Ultrathin sections, poststained with Reynolds lead citrate for 10 s, were examined in a Philips EM 201 electron microscope at 60 kV.

Chemicals. Resazurin was obtained from Janssen Chimica (Beerse, Belgium). Tween 80 was obtained from E. Merck Nederland B. V. (Amsterdam, The Netherlands). Silicone antifoaming agent was obtained from BDH (Poole, England).

5 20 40 60 80 100 120 Ethanol concentration (mM)

140 160

FIG. 1. Distribution of ethanolproductioninshake-flask cultures of fermentative(M and )andnonfermentative(_)strains. The

concentrations of ethanolin the cultures were determinedat theend of the exponential growth phase. 1 Numberofstrains growing

anaerobicallyinserumflasktests.

RESULTS

Ethanol production inoxygen-limited shake-flask cultures. When grown in shake-flask cultures, all strains produced ethanol, even those presently considered nonfermentative

onthebasis of taxonomictests(4). However, in such strains ethanol production was generally rather low (0 to 20 mM; Fig. 1). In some cases (for example, F. neoformans), the

amountof ethanol in shake-flask cultureswas ashighaswith

typical facultatively fermentativeyeastssuchasE.nanaand

D. hansenii.

Screening for anaerobic growth. When transferred to

an-aerobic conditions in serum flasks, only a few species exhibited clearanaerobicgrowth, with concomitant ethanol production, both in complex medium (yeastextract contain-ing) and in mineral medium (supplemented with Tween 80 and ergosterol). These were A. telluris, C. tropicalis, C. utilis, C. matritensis, C. lusitaniae, E. nana,H. valbyensis,

I. orientalis, K. apiculata, K. polysporus, N. coryli, P. transvaalensis, S. cerevisiae, S. ludwigii, S. pombe, T. delbrueckii, W. fluorescens, and W. saturnus. In many cases, long lag times (5 to 10days) elapsed before growth started, despite the fact that the yeastshad beenpregrown underoxygen-limited conditions. The number of generations in theflaskswithpositive growth was 8to 10, accordingto

dry-weight measurements. In this screening, it was not

possibletodeterminegrowthrates,but S.cerevisiae finished growth in 24h, leadingtoanestimated specificgrowthrate

ofapproximately 0.3 h-1. S. cerevisiae thereforestood out

as astrongfermenterthatreadilyadaptstoanaerobicgrowth conditions.

Maintenance of anaerobic conditions in small fermentors. The maintenance of anaerobic conditions in microbial

cul-tures is usually accomplished via the addition ofreducing agents suchas sulfide or sulfite. For growing yeasts, such compounds cannot be included in the media, since they strongly inhibit growth. As aconsequence, anaerobic

con-ditions in yeast cultures areoften qualified as flushed with

nitrogen, and itisimplicitly assumed that the actual

concen-trationofoxygenunderthose circumstances iszero(e.g.,5).

Generally, the problems in establishing anaerobic yeast

cultures are(i) howtodefine andmeasure anaerobic condi-tions and(ii)howto achieve andmaintain anaerobic condi-tions. The first problemcan be solvedeitherby measuring the oxygen concentration directly with anoxygen probeor

by measuring the redox potential of the medium. The last

measurement is indirect, since the redox potential is only partially dependent on the oxygen concentration. The rela-tionship between the redox potential and the dissolved-oxygen concentration in microbial cultures has been de-scribedby Wimpenny(32) and Wimpenny and Necklen (33). Theredoxpotential is takenas anappropriateparameterfor thedefinitionof anaerobic circumstances, especially for the cultivation of strictly anaerobic bacteria (14). It can be

measured in situbyanappropriate probeor canbeindicated

byaredoxdye, e.g., resazurin.

Directmeasurementoflowconcentrations ofoxygen has become possible with the development of sensitive and stable polarographic oxygen sensors, which are able to

detectDOTvalues downto0.001%airsaturation. We found that one must be very careful in the conversion ofredox potentials into oxygen levels. For example, it was not

possibletoobtainaredoxpotentiallowenoughtodecolorize the resorufin simply by purging the medium with nitrogen gas, althoughaDOTof0.005% air saturationwasreached. On the other hand, in a culture of C. utilis such a redox

potential was obtained at a DOT of 0.01 to 0.02% air

saturation, indicatingthat the redoxpotentialandDOT show

different relationships when the conditions are changed.

Furthermore, the oxygen probe and, to some extent, the

redoxpotentialaswellonlyindicate the actualactivityof the

oxygen in the medium. They do not provide information

about the actualoxygenflux. Evidence in thisrespectisour v,) (oU1) 4-0 a1) .0 E z 35 30 25 20 15 10 5 0

0 .+ .0 4--0 o ol

F-0

C]

0.05

0.04 0.03

0.02

0.01

0.00 '

'Ia

I --I a I I I 0 5 10 15 20 25 30 TIME

(hours)

FIG. 2. Oxygen diffusion intoa2-literlaboratoryfermentor indicatedbyrecordertracingsof theoxygenprobe.The measurementswere

determined in a1.5-litervolume of mineral medium.(A) Equilibrium situation withflushingwithnitrogen (1 liter. min-'). (B) Fermentor

pressurizedto1.1atm(ca. 111kPa)and alltubingclosed.(C)Fermentorkeptunderanitrogen atmosphere byuseofa1-m-highwatercolumn.

(D) Flushing with nitrogenresumed.

observation that resorufin was colorless even in a

cotton-stoppered shake-flask culture of C. utilis, conditions still regularly considered aerobic (17). The decolorization was

due to the rapid oxygen consumption of C. utilis and therefore did not indicate strictly anaerobic growth. To defineanaerobic conditions,wethereforefounditnecessary

to determine the actual flux ofoxygen into the system. To investigate the magnitude of the oxygen influx, we flushed

thefermentor withpure nitrogen untilaDOT of 0.005% air

saturation was reached. Subsequently, the outlet gasflow was blocked and an extrapressure of 0.1 atm(ca. 10 kPa)

was built up by use of a 1-m-high water column. The diffusion ofoxygeninto thefermentor is shown inFig. 2. It could be calculated from this figure that, even when all tubingwasclosedascloselytothe fermentoraspossible,the diffusion of oxygen was still approximately 2 x

10'

,umol- h-1. We confirmed that gas

leakage

did not occur.

These data clearly show that maintaining strictly anaerobic conditions isnotpossibleinthiswaybecause of the diffusion ofoxygen.

Tokeeptheconcentration ofoxygenataverylow level,we

found it necessary to flush the fermentor continuously with nitrogen.Weexamined the effectiveness of suchaprocedure

by monitoring the DOTas afunction of time and flowrate.

Although in a very short time most of the oxygen was

removed (95% within 3 min), the concentration of oxygen

nevertheless continuously decreased during the next 30 h. Theactual DOT finallyreached dependedontheflowrateof thenitrogengas(Table1). Whenever theairflowwas

reestab-lished, the DOT returnedtothe initialvalue of 100.0 + 0.1% airsaturation, indicating the low driftof the electrode.

Anaerobicgrowth in fermentors. To obtainsome

quantita-tive information on growth rates, we attempted to grow

variousyeastsanaerobically in fermentors. Priorto inocula-tion, the fermentors were flushed vigorously with nitrogen

gasuntiltheDOT didnotdecreaseanymore.Thisprocedure

usually took 30to 35h. Afterinoculation, thenitrogen flow

waskept at1liter. min-1topreventthediffusion of oxygen into themedium.

The growthcurves of theorganisms tested are shown in Fig. 3. As in theserumflasktests,S.cerevisiae wastheonly species capableofrapid anaerobic growth,withalmaxof 0.4

h-'

(Table 2). The other species tested (C. utilis, T.

del-brueckii, and C. tropicalis)grew poorly under these condi-tions

(lmax,

<0.05

h-').

In all cases, according to the

indicator, the redox potential in the culture decreased as soon asgrowth started. It could be calculated that the total amountofoxygenthat had entered the culture vesselduring thegrowthperiod was <10 ,umol

h-1.

Ultrastructureofanaerobicallygrowncells. The ultrastruc-tureofthefour yeasts shown inFig. 3wasinvestigatedwith regard to the presence of mitochondria (Fig. 4). Special

attention was paid to the staining procedures, because

inadequate staining often precludes the visualization of

mitochondrialstructures(9).Indeed, fixationwithpotassium

permanganatealonedidnotreveal mitochondrialstructures in S. cerevisiae (results not shown), whereas these struc-tures were clearly visible when

glutaraldehyde-potassium

permanganatefixationwasused(Fig. 4a). The otherspecies

also contained mitochondrial structures, although the fine

TABLE 1. Influenceofthenitrogenflow rate on theDOT ina

2-liter fermentor

N2flowrate DOT

(liters min-') (% airsaturation)

0.5 0.075 1.0 0.023 1.5 0.020 2.0 0.015 3.0 0.012 5.0 0.010 10.0 0.007 20.0 0.005

Dry

weight

(g1I)

,'C)

°

*

(IQ 0 . ' 0 tz _.. t ~~~D.O.T. (S 0 a 2 a

DOQT.

(06

Dry

welght

(gA)

0 a I.A 0 la I ,. . a s _

D.O.T.

(90

a

FIG. 4. Electronmicrographs ofanaerobicallygrown yeasts(bars,1,um). (a)S.cerevisiae.(b)T.delbrueckii. (c)C.tropicalis. (d,e,and

f)C.utilis. Note thepresence ofpromitochondria (small arrows). Thelargearrow(panelf)indicatesaconnection of theringstructure toother membrane structuresinthe cell.

TABLE 2.Maximalgrowth rates and cellyieldsinanaerobic

batch cultures

Species

~~ILmax

Yield(gigof

Species

(h-')

glucose)

S. cerevisiae 0.40 0.10

C. utilis 0.01 0.03

T.delbrueckii 0.03 0.06

C. tropicalis 0.05 0.07

structure was definitely less developed than that in

aerobi-cally grown cells (results not shown), in which the cristae were clearly visible. Most remarkable was the difference between the cells of C. utilis and the cells of the other

species. Coexistingwith themitochondrialstructuresin this yeast wererelatively largemembranousstructures,eitherin theform ofalaminarmembrane systembranchingout atthe ends orin the form ofaring structure ofconcentric mem-branes. The membranous structures seemed tobe intercon-nected in the cells (Fig. 4f). Single membranes were also found (Fig.4d), and these werealsoconnected tothelarger

FIG. 4-Continued structures. The membranous structures were not found in

cells grown aerobically with glucose (results not shown). DISCUSSION

Forthe screening of the property of anaerobic growth of yeasts, itwas necessary toselect representative strains. We

decided to choose type species of genera rather than

ran-domlyselected strains.

Although all strains tested were able to produce some

ethanol, only 18 grew anaerobically to some extent in

mineralmediumsupplementedwith ergosterol and Tween 80 orincomplex medium. Allofthese belonged to the group of

facultatively fermentative yeasts as listed by Barnett et al.

(4).

Itisimportant to note that a good fermentative capacity is a prerequisite for anaerobic growth, since none of the

so-called nonfermentativeyeasts grew under strictly

anaer-obic conditions. However, a good fermentative capacity alone is not sufficient to fulfill all the requirements of anaerobically.grown cells, since many rapidly fermenting

species lacked the abilitytogrow anaerobically. The latter propertyisrelativelyrareamong yeasts. Evenwhengrowth occurred,itwasratherslow. S. cerevisiae,however,seemed tobeapositive exception inthis respect(Fig. 3).

Theinability ofmanyfacultatively fermentative yeasts to grow anaerobically may be caused by a variety offactors.

For example, anaerobic alcoholic fermentation of xylose maybeprevented byadisturbedredox balance(6). Itseems

unlikely,however, thatthe absence of anaerobicgrowthon glucose is generally due to redoxproblems. In thecaseofP.

tannophilus,it has been shown thathydrogen acceptors such asdiacetyloracetoincannotreplace oxygen(19).P.

tanno-philusis unabletogrowunless oxygen is available.Although yeastssuchasC. utilis canslowly growanaerobically (Fig.

3),theratesofgrowthandalcoholic fermentationaregreatly

enhanced in oxygen-limited shake-flask cultures. Under these conditions, growth and alcoholic fermentation are as

rapidaswith S.cerevisiae(28). Hence,itseemslikelythat in

manyfacultatively fermentative yeasts, anunimpaired mito-chondrial function is required for growth. The existence of mitochondria inanaerobically grown cells has been disputed in the literature for a long time. It was stated by several authors that S. cerevisiae showed a complete absence of

mitochondria when grown under anaerobic conditions (18, 30). This observation, however, is now known to be the resultofinadequate electron microscopy techniques (9, 22).

Therefore, the theoryof de novo synthesis of mitochondria incellsgrownanaerobically and subsequentlytransferredto

highlevelsofoxygen(18, 30) had to berejected. However, the mitochondria in anaerobically grown cells do not have the same ultrastructure as do those in aerobically grown

cells; however, upon aeration they start to become fully organized (21). Indeed, in the four yeasts studied here, promitochondriacould be detected(Fig. 4).Remarkableare the membranous structuresin the yeast C. utilis, asalready

reported by Linnane and co-workers (18). These authors

suggestedthat suchstructuresshouldberegardedas precur-sorsofmitochondria. This ideaseemsunlikely, however, in view of the fact that these structures were notobserved in theother yeasts (Fig. 4).

Whether promitochondria fulfill a physiological function

remains to be elucidated. It is known that some of the

assimilatoryprocesses

required

forcell

synthesis

take

place

within the mitochondria (20);hence, the transport of certain intermediates over the mitochondrial membrane remains a

necessity under anaerobic conditions. These transport pro-cesses mustbeenergizedin theabsenceof electrontransfer. The results of

Subik

et al. (25) and Gbelska et al.

(11)

strongly suggest that underanaerobic

conditions,

transport processes andother

energy-requiring

reactions in mitochon-driaareenergized bytheimportof

cytoplasmic

ATPvia the reversal of adenosine nucleotide translocation. Anaerobic

growth of S. cerevisiae was shown to be arrested in the presence of

bongkrekic acid,

a

specific

inhibitor of the

mem-Sofar, it is unclear why in avariety ofyeasts the role of

mitochondria inanabolic reactions isapparentlymore

impor-tantthan itis inS. cerevisiae. Our resultsdemonstratethat in

studies onalcoholic fermentationby yeastsgreatcareshould

betaken withrespectto culture conditions. The serumflask

test used here can be considered a useful system for a qualitative estimation of the anaerobic behavior of yeasts. However, for quantitative aspects, fermentor cultures are required, even though the entrance of oxygen cannot be totallyprevented (Fig. 2).Nevertheless, it isclear that yeasts

likeC. utilis, C. tropicalis, and T. delbrueckii donotgrowas

wellasS.cerevisiaewhenonlytracesofoxygenareavailable.

The biochemical basis for this difference between S.

cerevi-siae andthe otheryeasts remainsto be elucidated.

Inourstudy, only type species ofgeneraweretested. We

thereforecannotexcludethepossibilitythat inadditiontoS. cerevisiae, other yeasts may possess the capacity for fast

anaerobic growth. However, on the basis of the limited

amount of data presented here it seems likely that this property is not widespread among yeasts.

ACKNOWLEDGMENTS

Weareindebted totheCentraalbureauvoorSchimmelculturesfor providinguswith the strainstested andforcarryingout

identifica-tiontests. Wethank Maudy Smithforstimulating discussionsand

MarcRijneveenforperforming partof theexperimental work.

Theinvestigationsweresupported bytheFoundationfor

Biolog-icalResearch,which issubsidizedbyTheNetherlandsOrganization

forScientific Research.

LITERATURECITED

1. Andreasen,A.A.,and T.J. B.Stier. 1953. Anaerobicnutrition

of Saccharomyces cerevisiae. I. Ergosterol requirement for

growthinadefinedmedium. J. Cell.Comp. Physiol. 41:23-26.

2. Andreasen,A.A.,andT.J.B.Stier.1954.Anaerobicnutrition

of Saccharomyces cerevisiae. II. Unsaturated fatty acid re-quirement for growth in a defined medium. J. Cell. Comp. Physiol.43:271-281.

3. Babi,T.,F.J.Moss,and B.J.Ralph. 1969. Effectsofoxygen

andglucoselevelson lipid compositionof yeastCandida utilis grownincontinuous culture. Biotechnol. Bioeng. 11:593-603.

4. Barnett,J.A., R. W. Payne,and D. Yarrow. 1983. Aguideto

identifyingandclassifying yeasts. Cambridge University Press, Cambridge.

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