Vol. 56, No. 12

Oxygen Requirements of YeastsWIEBE VISSER, W. ALEXANDER SCHEFFERS, WILMA H. BATENBURG-VAN DER VEGTE,

AND JOHANNES P. VAN DIJKEN*

Department of Microbiology and Enzymology, Delft University of Technology, Julianalaan 67,2628 BC Delft, The Netherlands

Received 26 July 1990/Accepted 2 October 1990

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineralmedia. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine lowredox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flaskcultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast speciestested grew under anaerobic conditions. A comparative study with a number of selected strains revealed thatSaccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts,such as Torulaspora delbrueckii and Candida tropicalis, grew poorly (tLmax. 0.03 and 0.05 h-1, respectively)under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latterorganisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It canbe concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.

Yeasts can be divided into three groups with respect totheir fermentative abilities, namely, obligately fermentative,facultatively fermentative, and nonfermentative yeasts. Ap-proximately 40% of the yeast species are listed as nonfer-mentative (4). The listing is based upon a standard test withDurham tubes, in which visible gas production is used as thecriterion for alcoholic fermentation. Nearly all nonfermen-tative yeast species, however, have been shown to produceethanol to some extent under the conditions of the Durhamtube test, and their fermentative capacities often are greatlyenhanced under more appropriate conditions (27).One of the most important parameters with respect to the

occurrence of alcoholic fermentation in yeasts is the concen-tration of oxygen in the culture medium. For example, thePasteur effect, the Custers effect (23, 31; M. T. J. Custers,Ph.D. thesis, Delft University of Technology, Delft, TheNetherlands, 1940), the Kluyver effect (16, 24), and theCrabtree effect (8, 10, 29) are all closely related to theavailability of oxygen.

Fermentation, in principle, can provide enough energy forgrowth. However, the ability to grow anaerobically dependsnot only on the fermentative capacity. Many species requirea limited amount of oxygen to ferment glucose, for example,Hansenula nonfermentans (26). Even when fermentationdoes occur under anaerobic conditions, as reported forPachysolen tannophilus, the actual growth of the organismon D-xylose as well as on D-glucose is still dependent on theavailability of oxygen (19). The addition of ergosterol andunsaturated fatty acids, which are considered to be essentialmedium components for anaerobic growth of Saccharomy-ces cerevisiae (1, 2), does not eliminate this oxygen require-ment. Whether yeast species other then S. cerevisiae are

able to grow anaerobically is in general unknown or theresults are contradictory. For example, anaerobic growth ofCandida utilis has been reported by several authors (3, 13),whereas others have stated that C. utilis is not able to growanaerobically (27).A major problem in comparing the results of different

studies with respect to anaerobic growth of yeasts is the

* Corresponding author.

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

used as an inoculum for anaerobic cultures, rapid growthmay occur for a number of generations even in the absenceof ergosterol and unsaturated fatty acids (15).The purpose of the present study was to examine the

capacity of various yeasts for anaerobic growth under stan-dardized conditions. To obtain a broad spectrum of yeasts,we studied type species of the genera, as listed in the List ofCultures of the Centraalbureau voor Schimmelcultures(CBS), Delft, The Netherlands.

MATERIALS AND METHODSOrganisms. Type strains of the yeast genera were obtained

from the CBS and are cited as listed in the List of Cultures(31st ed., 1987, or 32nd ed., 1990 [in press]): Ambrosiozymamonospora CBS 2554, Apiotrichum porosum CBS 2040,Arthroascus javanensis CBS 2555, Arxiozyma telluris CBS2685, Botryoascus synnaedendrus CBS 6161, Brettanomycesbruxellensis 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 CBS6936, Cryptococcus albidus CBS 142, Debaryomyces hans-enii CBS 767, Debaryozyma yamadae CBS 7035, Dekkerabruxellensis CBS 74, Eeniella nana CBS 1945, Endomycops-ella vini CBS 4110, Fellomyces polyborus CBS 6072, Fibu-lobasidium inconspicuum CBS 8237, Filobasidiella neofor-mans CBS 132, Filobasidium floriforme CBS 6241,Guilliermondella selenospora CBS 2562, Hanseniasporavalbyensis CBS 479, Hasegawaea japonica CBS 354, Hol-leya sinecauda CBS 8199, Holtermannia corniformis CBS6979, Hormoascus platypodis CBS 4111, Hyphopichia bur-tonfi CBS 2352, Issatchenkia orientalis CBS 5147, Kloeckeraapiculata CBS 287 (not a type strain), Kluyveromyces poly-sporus CBS 2163, Leucosporidium scottii CBS 5930, Lipo-myces starkeyi CBS 1807, Lodderomyces elongisporus CBS2605, Malassezia furfur CBS 1878, Mastigomyces philip-povii CBS 7047, Metschnikowia bicuspidata CBS 5575,Mrakia frigida CBS 5270, Myxozyma melibiosi CBS 2102,Nadsonia fulvescens CBS 2596, Nematospora coryli CBS2608, Octosporomyces octosporus CBS 371, Oosporidiummargaritiferum CBS 2531, P. tannophilus CBS 4044,

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Pachytichospora transvaalensis CBS 2186, Phaffiarhodozyma CBS 5905, Pichia membranaefaciens CBS 107,Rhodosporidium toruloides CBS 14, Rhodotorula glutinisCBS 20, S. cerevisiae CBS 1171, Saccharomycodes ludwigiiCBS 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-

num CBS 6803, Sporidiobolusjohnsonii CBS 5470, Sporobo-lomyces roseus CBS 486, Sporopachydermia lactativoraCBS 6989, Stephanoascus ciferrii CBS 6699, Sterigmatomy-ces halophilus CBS 4609, Sterigmatosporidium polymor-phum CBS 8088, Sympodiomyces parvus CBS 6147, Toru-laspora delbrueckii CBS 1146, Trichosporon beigelii CBS2466, Trigonopsis variabilis CBS 1040, Waltomyces lipoferCBS 944, Wickerhamia fluorescens CBS 4565, Wicker-hamiella domercqiae CBS 4351, Williopsis saturnus CBS5761, Wingea robertsiae CBS 2934, Yarrowia lipolytica CBS599, Zygoascus hellenicus CBS 5839 (mating type a), Zy-goascus hellenicus CBS 6736 (mating type a), Zygosaccha-romyces rouxii CBS 732, and Zygozyma oligophaga CBS7107.Media. Complex medium contained, per liter of deminer-

alized water, 10 g of yeast extract (Oxoid) and 20 g ofglucose. The initial pH of the medium was set at 5. Mineralmedium, supplemented with vitamins and trace elements,was prepared as described by Bruinenberg et al. (7), exceptthat the concentration of NaMoO4. 2H20 was increased10-fold. Glucose was added as the sole source of carbon andenergy at a concentration of 20 g. liter-'. Ergosterol andTween 80, dissolved in pure ethanol, were sterilized byheating the solution for 10 min in a nonpressurized auto-clave. These components were added to the medium atconcentrations of 6 and 660 mg. liter-', respectively. Resa-zurin was added to both media at a concentration of 0.002%to indicate low redox potentials (Eo' = -42 mV).

Inocula. Inocula for anaerobic growth tests were preparedby growing the yeasts in 100-ml cotton wool-plugged Erlen-meyer flasks containing 20 ml of medium. Cultures were

incubated on a rotary shaker at 25°C and 50 rpm. Underthese conditions, alcoholic fermentation can be triggered infacultatively fermentative yeasts because of oxygen limita-tion (28). Therefore, cells from these shake-flask culturescould be considered to be adapted to serve as inocula foranaerobic growth tests.Anaerobic growth tests. Anaerobic growth tests were con-

ducted in 30-ml serum flasks under static incubation at 25°C.To prevent the entrance of oxygen, we firmly closed theflasks with 4-mm-thick butyl rubber septa. The flasks were

almost completely filled with medium and autoclaved at110°C. During autoclaving, reducing agents in the complexmedium converted the redox indicator to colorless dihy-droresorufin. The mineral medium, treated similarly, did notbecome colorless. It was therefore deoxygenated prior tobeing autoclaved by including 8 mg of Aspergillus nigerglucose oxidase (grade III; Boehringer, Mannheim, FederalRepublic of Germany) liter-1; this enzyme preparation con-

tains, as an impurity, a catalase activity that is adequate forremoval of H202 formed in the reaction. The redox indicatorin the serum flask septa, treated this way, remained colorlessfor at least 4 months, whereas the use of ordinary rubbersepta (red rubber; BGA class 1 FDA) caused recolorizationwithin a few hours because of a high rate of oxygen diffusion.After sterilization, the flasks were not opened to prevent theentrance of oxygen. A small amount (5 to 10 ,u) of theinoculum was injected into each flask with a 2-ml syringe.

The syringe was left in place during the incubation and thusserved as an indicator of CO2 production. The gas produc-tion and turbidity of the inoculated flasks were checkedtwice daily for 1 month; prolonged incubation after thisperiod did not lead to the onset of anaerobic growth.

Batch cultivation in fermentors. Comparative studies wereperformed by use of a laboratory fermentor with a 1-literworking volume and of the type described by Harder et al.(12). The mineral medium described above was used. Toprevent foaming, we added 50 ,ul of silicone antifoamingagent per liter. pH was kept at 5.0 by automatic titration withsterile 1 M KOH, the temperature was kept at 30°C, and thecultures were stirred at 450 rpm. For exclusion of a possibleinfection, the identity of the organism was checked after-wards by the CBS.

Levels of dissolved-oxygen tension (DOT) were measuredwith a polarographic oxygen electrode (Ingold type 322756702/74247) connected to an Ingold type 170 02 amplifier.The signal of this amplifier (percent air) was monitored witha Kipp BD 41 datum recorder (Kipp & Zonen, Delft, TheNetherlands).The fermentor was continuously flushed with pure nitro-

gen gas containing less than 5 ppm of oxygen (Air Products,Waddinxveen, The Netherlands) at a flow rate of 1liter. min-'. The tubing of the fermentor was made ofNorprene (Cole-Parmer Instruments Corp., Chicago, Ill.).

Analytical methods. Ethanol concentrations were deter-mined by gas-liquid chromatography on a Varian type 3400gas chromatograph (Varian Benelux B. V., Amsterdam, TheNetherlands) with a Hayesep Q column (Chrompack, Mid-delburg, The Netherlands) at a temperature range of 150 to225°C, increasing by 15°C- min-'. Glucose concentrationswere 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, poresize, 0.45 jxm; Gelman Sciences Inc., Ann Arbor, Mich.).The filter was washed with demineralized water, dried in anR-7400 magnetron oven (Sharp Inc., Osaka, Japan) for 20min at medium power, and reweighed.Carbon analyses were done with a model 915B Tocamas-

ter total organic 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 aflow rate of 0.6 ml min-'. The detector was a Waters 441UV meter (used at 210 nm) coupled to a Waters 741 datummodule (Waters, Milford, Mass.).

Electron microscopy. Twenty-two milliliters of cell culturewas prefixed with 3 ml of 25% (vol/vol) glutaraldehyde for 10to 30 min at room temperature. After centrifugation, the cellswere fixed again with 3% (vol/vol) glutaraldehyde in 0.1 Msodium cacodylate buffer (pH 7.0) for 1 h, washed threetimes with the same buffer, and postfixed with 2% (wt/vol)aqueous KMnO4 for 2 to 24 h. After fixation, the cells werestained with 1.5% (wt/vol) aqueous uranyl acetate duringdehydration at the 50% (vol/vol) ethanol step and embeddedin Spurr resin. Ultrathin sections, poststained with Reynoldslead citrate for 10 s, were examined in a Philips EM 201electron microscope at 60 kV.

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

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

140 160

FIG. 1. Distribution of ethanol production in shake-flask cultures of fermentative ( M and ) and nonfermentative (_) strains. The

concentrations of ethanol in the cultures were determined at the end of the exponential growth phase. 1 Number of strains growinganaerobically in serum flask tests.

RESULTSEthanol production in oxygen-limited shake-flask cultures.

When grown in shake-flask cultures, all strains producedethanol, even those presently considered nonfermentativeon the basis of taxonomic tests (4). However, in such strainsethanol production was generally rather low (0 to 20 mM;Fig. 1). In some cases (for example, F. neoformans), theamount of ethanol in shake-flask cultures was as high as withtypical facultatively fermentative yeasts such as E. nana andD. hansenii.

Screening for anaerobic growth. When transferred to an-aerobic conditions in serum flasks, only a few speciesexhibited clear anaerobic growth, with concomitant ethanolproduction, both in complex medium (yeast extract contain-ing) and in mineral medium (supplemented with Tween 80and 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 manycases, long lag times (5 to 10 days) elapsed before growthstarted, despite the fact that the yeasts had been pregrownunder oxygen-limited conditions. The number of generationsin the flasks with positive growth was 8 to 10, according todry-weight measurements. In this screening, it was notpossible to determine growth rates, but S. cerevisiae finishedgrowth in 24 h, leading to an estimated specific growth rateof approximately 0.3 h-1. S. cerevisiae therefore stood outas a strong fermenter that readily adapts to anaerobic growthconditions.Maintenance of anaerobic conditions in small fermentors.

The maintenance of anaerobic conditions in microbial cul-tures is usually accomplished via the addition of reducingagents such as sulfide or sulfite. For growing yeasts, suchcompounds cannot be included in the media, since theystrongly inhibit growth. As a consequence, anaerobic con-

ditions in yeast cultures are often qualified as flushed withnitrogen, and it is implicitly assumed that the actual concen-

tration of oxygen under those circumstances is zero (e.g., 5).Generally, the problems in establishing anaerobic yeast

cultures are (i) how to define and measure anaerobic condi-tions and (ii) how to achieve and maintain anaerobic condi-tions. The first problem can be solved either by measuringthe oxygen concentration directly with an oxygen probe or

by measuring the redox potential of the medium. The lastmeasurement is indirect, since the redox potential is onlypartially dependent on the oxygen concentration. The rela-tionship between the redox potential and the dissolved-oxygen concentration in microbial cultures has been de-scribed by Wimpenny (32) and Wimpenny and Necklen (33).The redox potential is taken as an appropriate parameter forthe definition of anaerobic circumstances, especially for thecultivation of strictly anaerobic bacteria (14). It can bemeasured in situ by an appropriate probe or can be indicatedby a redox dye, e.g., resazurin.

Direct measurement of low concentrations of oxygen hasbecome possible with the development of sensitive andstable polarographic oxygen sensors, which are able todetect DOT values down to 0.001% air saturation. We foundthat one must be very careful in the conversion of redoxpotentials into oxygen levels. For example, it was notpossible to obtain a redox potential low enough to decolorizethe resorufin simply by purging the medium with nitrogengas, although a DOT of 0.005% air saturation was reached.On the other hand, in a culture of C. utilis such a redoxpotential was obtained at a DOT of 0.01 to 0.02% airsaturation, indicating that the redox potential and DOT showdifferent relationships when the conditions are changed.Furthermore, the oxygen probe and, to some extent, theredox potential as well only indicate the actual activity of theoxygen in the medium. They do not provide informationabout the actual oxygen flux. Evidence in this respect is our

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observation that resorufin was colorless even in a cotton-stoppered shake-flask culture of C. utilis, conditions stillregularly considered aerobic (17). The decolorization wasdue to the rapid oxygen consumption of C. utilis andtherefore did not indicate strictly anaerobic growth. Todefine anaerobic conditions, we therefore found it necessaryto determine the actual flux of oxygen into the system. Toinvestigate the magnitude of the oxygen influx, we flushedthe fermentor with pure nitrogen until a DOT of 0.005% airsaturation was reached. Subsequently, the outlet gasflowwas blocked and an extra pressure of 0.1 atm (ca. 10 kPa)was built up by use of a 1-m-high water column. Thediffusion of oxygen into the fermentor is shown in Fig. 2. Itcould be calculated from this figure that, even when alltubing was closed as closely to the fermentor as possible, thediffusion 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 anaerobicconditions is not possible in this way because of the diffusionof oxygen.To keep the concentration of oxygen at a very low level, we

found it necessary to flush the fermentor continuously withnitrogen. We examined the effectiveness of such a procedureby monitoring the DOT as a function of time and flow rate.Although in a very short time most of the oxygen wasremoved (95% within 3 min), the concentration of oxygennevertheless continuously decreased during the next 30 h.The actual DOT finally reached depended on the flow rate ofthe nitrogen gas (Table 1). Whenever the airflow was reestab-lished, the DOT returned to the initial value of 100.0 + 0.1%

air saturation, indicating the low drift of the electrode.Anaerobic growth in fermentors. To obtain some quantita-

tive information on growth rates, we attempted to grow

various yeasts anaerobically in fermentors. Prior to inocula-tion, the fermentors were flushed vigorously with nitrogengas until the DOT did not decrease anymore. This procedureusually took 30 to 35 h. After inoculation, the nitrogen flow

was kept at 1 liter. min-1 to prevent the diffusion of oxygeninto the medium.The growth curves of the organisms tested are shown in

Fig. 3. As in the serum flask tests, S. cerevisiae was the onlyspecies capable of rapid anaerobic growth, with a lmax of 0.4h-' (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 theindicator, the redox potential in the culture decreased assoon as growth started. It could be calculated that the totalamount of oxygen that had entered the culture vessel duringthe growth period was <10 ,umol h-1.

Ultrastructure of anaerobically grown cells. The ultrastruc-ture of the four yeasts shown in Fig. 3 was investigated withregard to the presence of mitochondria (Fig. 4). Specialattention was paid to the staining procedures, becauseinadequate staining often precludes the visualization ofmitochondrial structures (9). Indeed, fixation with potassiumpermanganate alone did not reveal mitochondrial structuresin S. cerevisiae (results not shown), whereas these struc-tures were clearly visible when glutaraldehyde-potassiumpermanganate fixation was used (Fig. 4a). The other speciesalso contained mitochondrial structures, although the fine

TABLE 1. Influence of the nitrogen flow rate on the DOT in a2-liter fermentor

N2 flow rate DOT(liters min-') (% air saturation)

0.5 0.0751.0 0.0231.5 0.0202.0 0.0153.0 0.0125.0 0.010

10.0 0.00720.0 0.005

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FIG. 4. Electron micrographs of anaerobically grown yeasts (bars, 1 ,um). (a) S. cerevisiae. (b) T. delbrueckii. (c) C. tropicalis. (d, e, andf) C. utilis. Note the presence of promitochondria (small arrows). The large arrow (panel f) indicates a connection of the ring structure to othermembrane structures in the cell.

TABLE 2. Maximal growth rates and cell yields in anaerobicbatch cultures

Species ~~ILmax Yield (gig ofSpecies (h-') glucose)

S. cerevisiae 0.40 0.10C. utilis 0.01 0.03T. delbrueckii 0.03 0.06C. tropicalis 0.05 0.07

structure was definitely less developed than that in aerobi-cally grown cells (results not shown), in which the cristaewere clearly visible. Most remarkable was the differencebetween the cells of C. utilis and the cells of the otherspecies. Coexisting with the mitochondrial structures in thisyeast were relatively large membranous structures, either inthe form of a laminar membrane system branching out at theends or in the form of a ring structure of concentric mem-branes. The membranous structures seemed to be intercon-nected in the cells (Fig. 4f). Single membranes were alsofound (Fig. 4d), and these were also connected to the larger

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OXYGEN REQUIREMENTS OF YEASTS 3791

FIG. 4-Continued

structures. The membranous structures were not found incells grown aerobically with glucose (results not shown).

DISCUSSIONFor the screening of the property of anaerobic growth of

yeasts, it was necessary to select representative strains. Wedecided to choose type species of genera rather than ran-domly selected strains.Although all strains tested were able to produce some

ethanol, only 18 grew anaerobically to some extent inmineral medium supplemented with ergosterol and Tween 80or in complex medium. All of these belonged to the group offacultatively fermentative yeasts as listed by Barnett et al.(4).

It is important to note that a good fermentative capacity isa prerequisite for anaerobic growth, since none of theso-called nonfermentative yeasts grew under strictly anaer-obic conditions. However, a good fermentative capacityalone is not sufficient to fulfill all the requirements ofanaerobically. grown cells, since many rapidly fermentingspecies lacked the ability to grow anaerobically. The latterproperty is relatively rare among yeasts. Even when growthoccurred, it was rather slow. S. cerevisiae, however, seemedto be a positive exception in this respect (Fig. 3).The inability of many facultatively fermentative yeasts to

grow anaerobically may be caused by a variety of factors.For example, anaerobic alcoholic fermentation of xylosemay be prevented by a disturbed redox balance (6). It seemsunlikely, however, that the absence of anaerobic growth onglucose is generally due to redox problems. In the case of P.tannophilus, it has been shown that hydrogen acceptors suchas diacetyl or acetoin cannot replace oxygen (19). P. tanno-philus is unable to grow unless oxygen is available. Althoughyeasts such as C. utilis can slowly grow anaerobically (Fig.3), the rates of growth and alcoholic fermentation are greatlyenhanced in oxygen-limited shake-flask cultures. Underthese conditions, growth and alcoholic fermentation are asrapid as with S. cerevisiae (28). Hence, it seems likely that in

many facultatively fermentative yeasts, an unimpaired mito-chondrial function is required for growth. The existence ofmitochondria in anaerobically grown cells has been disputedin the literature for a long time. It was stated by severalauthors that S. cerevisiae showed a complete absence ofmitochondria when grown under anaerobic conditions (18,30). This observation, however, is now known to be theresult of inadequate electron microscopy techniques (9, 22).Therefore, the theory of de novo synthesis of mitochondriain cells grown anaerobically and subsequently transferred tohigh levels of oxygen (18, 30) had to be rejected. However,the mitochondria in anaerobically grown cells do not havethe same ultrastructure as do those in aerobically growncells; however, upon aeration they start to become fullyorganized (21). Indeed, in the four yeasts studied here,promitochondria could be detected (Fig. 4). Remarkable arethe membranous structures in the yeast C. utilis, as alreadyreported by Linnane and co-workers (18). These authorssuggested that such structures should be regarded as precur-sors of mitochondria. This idea seems unlikely, however, inview of the fact that these structures were not observed inthe other yeasts (Fig. 4).Whether promitochondria fulfill a physiological function

remains to be elucidated. It is known that some of theassimilatory processes required for cell synthesis take placewithin the mitochondria (20); hence, the transport of certainintermediates over the mitochondrial membrane remains anecessity under anaerobic conditions. These transport pro-cesses must be energized in the absence of electron transfer.The results of Subik et al. (25) and Gbelska et al. (11)strongly suggest that under anaerobic conditions, transportprocesses and other energy-requiring reactions in mitochon-dria are energized by the import of cytoplasmic ATP via thereversal of adenosine nucleotide translocation. Anaerobicgrowth of S. cerevisiae was shown to be arrested in thepresence of bongkrekic acid, a specific inhibitor of theATP-ADP translocator of the mitochondrial inner mem-

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brane. This inhibition could not be relieved by the additionof a variety of growth factors.So far, it is unclear why in a variety of yeasts the role of

mitochondria in anabolic reactions is apparently more impor-tant than it is in S. cerevisiae. Our results demonstrate that instudies on alcoholic fermentation by yeasts great care shouldbe taken with respect to culture conditions. The serum flasktest 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 betotally prevented (Fig. 2). Nevertheless, it is clear that yeastslike C. utilis, C. tropicalis, and T. delbrueckii do not grow aswell as S. cerevisiae when only traces of oxygen are available.The biochemical basis for this difference between S. cerevi-siae and the other yeasts remains to be elucidated.

In our study, only type species of genera were tested. Wetherefore cannot exclude the possibility that in addition to S.cerevisiae, other yeasts may possess the capacity for fastanaerobic growth. However, on the basis of the limitedamount of data presented here it seems likely that thisproperty is not widespread among yeasts.

ACKNOWLEDGMENTS

We are indebted to the Centraalbureau voor Schimmelcultures forproviding us with the strains tested and for carrying out identifica-tion tests. We thank Maudy Smith for stimulating discussions andMarc Rijneveen for performing part of the experimental work.The investigations were supported by the Foundation for Biolog-

ical Research, which is subsidized by The Netherlands Organizationfor Scientific Research.

LITERATURE CITED1. Andreasen, A. A., and T. J. B. Stier. 1953. Anaerobic nutrition

of Saccharomyces cerevisiae. I. Ergosterol requirement forgrowth in a defined medium. J. Cell. Comp. Physiol. 41:23-26.

2. Andreasen, A. A., and T. J. B. Stier. 1954. Anaerobic nutritionof 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. Effects of oxygenand glucose levels on lipid composition of yeast Candida utilisgrown in continuous culture. Biotechnol. Bioeng. 11:593-603.

4. Barnett, J. A., R. W. Payne, and D. Yarrow. 1983. A guide toidentifying and classifying yeasts. Cambridge University Press,Cambridge.

5. Brown, C. M., and B. Johnson. 1971. Influence of oxygentension on the physiology of Saccharomyces cerevisiae incontinuous culture. Antonie van Leeuwenhoek J. Microbiol.Serol. 37:477-487.

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