JOURNAL OF BACTERIOLOGYVol. 87, No. 6, pp. 1309-1316 June, 1964Copyright © 1964 by the American Society for Microbiology

Printed in U.S.A.

ANAEROBIC GROWTH OF FUSARIUM OXYSPORUM'H. B. GUNNER2 AND M. ALEXANDER

Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York

Received for publication 10 December 1963

ABSTRACT

GUNNER, H. B. (Cornell University, Ithaca,N.Y.), AND M. ALEXANDER. Anaerobic growth ofFusarium oxysporum. J. Bacteriol. 87:1309-1316.1964.-Fusarium oxysporum, an alleged obligateaerobe, was found to be capable of growth in theabsence of molecular oxygen, provided the mediumcontained yeast extract, MnO2 , nitrate, selenite,or ferric ions. The active substance in yeast ex-tract was not identified. The fungus possessedhydrogenase, and was capable of utilizing H12.Under anaerobic conditions, the fungus effectedthe reduction of nitrate, ceric, ferric, selenite, andtellurite ions, as well as the reduction of severalinorganic sulfur compounds and indicators havingpositive oxidation-reduction potentials. Theproducts of anaerobic nitrate-dependent growthwere ethanol, C02, acetic acid, and ammonia.Possible explanations for the apparent inabilityof obligate aerobes to grow in the absence of 02are discussed.

Despite the importance in microbial taxonomyand physiology of 02-dependent growth, littleattention has been given to the biochemical basisof the need for molecular oxygen or to the possi-bilities that the 02 requirement of aerobicmicroorganisms reflects, not a need for a specificform of the element, but rather some otherphysiological characteristic or deficiency. Thefailure of the so-called strictly aerobic microor-ganism to grow in the absence of molecular 02remains largely unexplained. Studies of selectedmicrobial strains, however, indicate that manymicroorganisms may, indeed, proliferate anaero-bically, but only if suitable growth factors areprovided; i.e., the organism is unable to syn-thesize the metabolite in 02-free circumstances.For example, Richardson (1936) noted that astrain of Staphylococcus aureus requires uracilwhen grown in the absence of 02, but not when

1 Agronomy paper no. 636.2 Present address: Institute of Agricultural and

Industrial Microbiology, University of Mas-sachusetts, Amherst.

cultured aerobically. Similarly, Saccharomycescerevisiae needs certain sterols or unsaturatedfatty acids for good anaerobic growth (Andreasenand Stier, 1954); Mlucor rouxii requires thiamineand nicotinic acid (Bartnicki-Garcia and Nicker-son, 1961); and Cytophaga succinicans needsCO2 for development in the absence of 02 (Ander-son and Ordal, 1961).The present investigation was designed to

determine the basis for the inability of a re-portedly obligately aerobic fungus to growwithout molecular oxygen. Particular attentionwas given to the possibility that this inabilitywas associated with the absence of a suitableelectron acceptor and the related low oxidation-reduction potential.

MATERIALS AND METHODS

Fusarium oxysporum f. cubense was grown in abasal medium (medium A) consisting of: glucose,15.0 g; NH4Cl, 4.0 g; K2HPO4, 0.8 g; KH2PO4,0.2 g; CaC12, 0.1 g; MgSO4 7H20, 0.2 g; FeCl3*6H20, 0.03 g; and distilled water, 1,000 ml. Allincubations were performed at 30 C. In experi-ments made to determine whether the funguscould couple anaerobic growth to the reduction ofnitrate, selenite, and ferric iron, the medium (B)had the following composition: glucose, 18.0 g;NH4C1, 2.0 g; KH2PO4, 1.5 g; MgSO4 7H20,0.6 g; iron chelate (Jacobson, 1951), 0.025 mg;ZnSO4 *7H20, 0.03 g; and distilled water, 1.0liter. Potassium nitrate, Na2SeO3, and ferricammonium citrate were added to final concentra-tions of 4.0 X 10-2 M 10- M, and 10-3 M, respec-tively.

In investigations of the inorganic ions suscepti-ble to reduction by F. oxysporum, 10 ,umoles ofthe sulfur or nitrogen compounds, 200 ,umoles ofglucose, and 400 ,umoles of tris(hydroxymethyl)-aminomethane (tris) buffer (pH 7.6) were addedto the main compartment of a Thunberg tube.In the cap was placed 1.0 ml of fungus suspensionfrom a 36-hr culture grown on a rotary shaker;the tube was evacuated, and the suspension was

1309

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GUNNER AND ALEXANDER

tipped into the main compartment; after 12 hr,the contents of the tube were centrifuged, andthe supernatant fluid was examined for reducedproducts. The reduction of ceric ions was meas-ured by incubation of 1.0 ml of washed F. oxy-sporum mycelium suspended in 0.05 M phosphatebuffer (pH 7.0) with 4.0 ml of a eerie-citratecomplex prepared by mixing equal volumes of10-4 M Ce(HSO4)4 solution and 10-2 M citric acidadjusted to pH 7.0 with NaOH; after centrifuga-tion, the formation of cerous ions was determinedat 270 m,u. The reduction of ferric iron to theferrous form was determined by incubating awashed fungal suspension in a solution containing5.0 ,ug of ferric iron as ferric ammonium sulfate.Reduction of the higher oxidation states ofselenium and tellurium was assayed by incubat-ing 3.0 ml of a washed fungus suspension with 1.0ml of 1.0% solutions of the various salts, 5.0 mlof 0.1 M glucose solution, and 5.0 ml of 0.05 Mtris buffer (pH 7.6).The capacity of the organism to use electron

acceptors of various oxidation-reduction po-tentials was assessed by incubating 3.0 ml of awashed F. oxysporum suspension in 0.05 Mphosphate buffer (pH 7.0) with 3.0 ml of a 0.01%solution of the Eh indicator, 5.5 ml of 0.1 Msolution of the neutralized substrate, and 5.5 mlof 0.05 M phosphate buffer (pH 7.0). Dye reduc-tion was recorded at hourly intervals.For growth experiments, a culture system that

permitted rigorous 02 removal and continuousmonitoring with an 02 electrode (BeckmanInstruments Inc., model 11098) was devised. Ininitial studies, 450 ml of medium were placed inwide-mouthed 500-ml Erlenmeyer flasks fittedwith rubber stoppers containing inlets for N2,sampling tube, platinum electrode, salt bridge(3.0% agar, 1.0% NaCl), and a vacuum exhaustline. The electrode and salt bridge were connectedto a potentiometer by means of a saturated KClsolution in which a calomel electrode was placed.In subsequent studies, the rubber stopper wasfitted only with gas inlet and exhaust tubes and aThunberg tube cap containing the inoculum. Inthis way, the myeelial suspension could beevacuated and flushed with gas together with therest of the system prior to addition of the inocu-lum to the fresh medium.

Before entering the culture vessels, the N2(Seaford grade, rated as 99.99% pure) was passedthrough a column of reduced copper turningsheated to 600 C, and was then introduced into a

gas washing bottle containing a mixture of achromic salt, zinc, and sulfuric acid; this pro-cedure for removal of traces of 02 was describedby Marshall (1960). The entire system wasevacuated to 4.0 cm of mercury, and was flushedfive times with the O2-free N2 sterilized by pas-sage through a cotton filter. Anaerobiosis waseffected within 15 min after the growth mediumwas removed from the autoclave, while allsolutions were still near 100 C, to minimize theamount of dissolved 02 introduced into the sys-tem with the growth media. After the 02 electrodegave a continuous zero reading for 1 hr, a timesufficient for the medium to cool, the inoculumwas tipped into the medium from the cap, theinlet and outlet tubes of each flask were closed,and the flasks were incubated at 30 C.To determine the products of fermentation,

samples of the culture filtrate were removed, thepH was adjusted to 1.0 to 2.0 with concentratedH2SO4 , and the dissolved CO2 was swept intostandard Ba(OH)2 by a stream of N2. Thefungal suspension in each flask was removed byfiltration, washed, dried at 70 C, and weighed.The mycelium and conidia were considered tocontain 50% carbon and 5%O nitrogen. Analysisfor nitrite was by the method of Kolthoff andSandell (1938); ammonia was determined bynesslerization (Wilson and Knight, 1952), ferriciron was estimated by the method of Grewelingand Peech (1960); glucose determinations wereperformed as described by Somogyi (1945); CO2was measured titrimetrically; ethanol and lacticacid were determined by the procedures describedby Neish (1952); acetic acid was identified bydetermination of the Duclaux numbers (Mc-Elvain, 1947); and sulfide was estimated by themethod of Delwiche (1951).The amount of H2 consumed by F. oxysporum

after 7 days was determined manometrically,the culture flasks being attached to mercurymanometers. Hydrogenase was assayed quanti-tatively in a Warburg microrespirometer withthe use of 7-day cultures grown anaerobically in02-free N2 . Washed mycelium suspended in 0.2%KCl was placed in the side arm of the Warburgflasks; the center well contained 0.2 ml of 20%KOH, and the main compartment of the flaskcontained 50 ,moles of benzyl viologen and 600,umoles of phosphate buffer (pH 8.0), in a totalvolume of 3.2 ml. The assay was performed at 36C in an atmosphere of H2 purified of 02 by pas-sage through chromous acid. Control flasks were

1310 J. BACTERIOL.

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ANAEROBIC GROWTH OF F. OXYSPORUM

maintained in an atmosphere of N2 freed of 02 bypassage through heated copper turnings.

Anaerobically grown cultures of F. oxysporumwere tested routinely for purity by microscopicexamination and by inoculation into nutrientbroth containing 100 ,ug/ml of cycloheximide.The tubes were incubated anaerobically underN2. No evidence of contamination was noted.

RESULTS

F. oxysporum grew in an anaerobic environ-ment, provided that higher oxidation states ofcertain elements were included in the medium.For example, distinct growth was noted visuallyafter 7 days of anaerobic incubation in an agarmedium containing 0.3 % K2S04, 0.3% Na2SeO3,or 0.3% Na2MoO4 2H20 with ammonium asnitrogen source, or in agar containing 0.3 %KNO3 as sole nitrogen source; no myceliumappeared if the ammonium medium was notsupplemented with one of these salts. Aerobically,the fungus developed on all the media tested.The results were identical whether the cultureswere incubated in an atmosphere containingN2 or H2 freed of 02 catalytically. Although theamount of residual 02 in the gas phase was notdetermined, the absence of detectable growth inthe ammonium medium containing none of thesesalts served as a biological test for the existenceOf 02 levels insufficient to support aerobic pro-liferation. Further, plates of Potato DextroseAgar (Difco) streaked with the aerobic Aspergil-lus niger (Van Tieghem), Rhizobium trifolii, andBacillus pantothenticus, and incubated in thesame containers, showed no growth.The anaerobic cultures of F. oxysporum con-

sisted of fine hair-like structures made up of shorthyphal fragments and many chlamydospores.Upon the return to aerobic conditions, conidiabegan to bud rapidly from the hyphal surface,and, within 24 hr, the culture assumed thecharacteristics of the typical F. oxysporummycelium. Aerobically, by contrast, the dominantspore form was the microconidium, which was

somewhat less abundant; chlamydospores ap-peared abundantly only in old cultures.The fungus also grew anaerobically in the

ammonium-containing liquid medium supple-mented with 0.1% yeast extract; 6.17 mg/ml ofglucose disappeared in 10 days. There was neithergrowth nor glucose disappearance in the absenceof yeast extract. Further, F. oxysporum failed todevelop anaerobically in the ammonium medium

TABLE 1. Reduction of inorganic ions by washedmycelium of Fusarium oxysporum

Substrate

K2S03NaHSO3K2SO4Na2S203Na2S206K2S208KNO3Ce(HSO4)4FeNH4 (SO4) 2

Substrateconcn

pmoles/inl1.671.671.671.671.671.671.670.040.10

Product

Com- Concnpound

H 2SH 2SH 2SH 2SH 2SH 2SNO2-Ce3+Fe2+

Ag/ml3.91.61.57.05.44.90.3+4.0

Boiledmyceliumcontrol*

I g/m

1.10.00.00.00.00.00.0

0.0

* Concentration of product in incubation mix-tures containing boiled rather than viable my-celium.

containing 0.1% Vitamin Free Casamino Acids,L-arginine HCl, L-glutamic acid, DL-valine,DL-phenylalanine, DL-isoleucine, or DL-trypto-phan. The yeast extract effect on growth orglucose disappearance also could not be replacedby thiamine, biotin, pyridoxine, riboflavine,pantothenate, vitamin B12, lipoic acid, folic acid,or ascorbic acid added singly or in various com-binations at concentrations ranging from 0.05 to2.50 mg per liter. The morphology of the fungusgrown anaerobically in the presence of yeast ex-tract was similar to that in the nitrate medium,except for the greater abundance of microconidiain the former medium.The data above indicate that growth took place

when certain inorganic ions were provided. Thatthe fungus does, indeed, effect a reduction of theinorganic ions is demonstrated by the results ofTable 1. All of the sulfur salts and the nitrate,ceric, and ferric ions were reduced. The rate ofreduction varied markedly with the specific ion;undoubtedly, reduced products in addition tothose listed appeared. The brick-red color ofselenium and the black color of tellurium de-veloped in mycelium incubated with Na2SeO3and K2TeOs for 10 hr, and the hyphae containedred and black inclusions when provided withthese salts. There was no selenite or telluritereduction by boiled mycelium, and the viablefungus showed no activity upon selenate andtellurate.Because one hypothesis to account for the

VOL. 87, 1964 1311

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GUNNER AND ALEXANDER

TABLE 2. Reduction by Fusarium oxysporum of dyesof different oxidation-reduction potentials

l Substrates used aselectron donorst

Indicator Eh of inldicator* t

_ I

Benzenoneindo-3 '-chlorophenol ........ 0.233 + + + +

2,6-Dichlorobenzen-oneindophenol .. .... 0.217 + + + +

Benzenoneindo-2'-methylphenol ....... 0.208 + + + +

2,6-Dichlorobenzen-oneindo-3'-methylphenol .............. 0.181 + + + +

2,6-Dibromobenzen-oneindo-3'-methoxy-phenol.............. 0.159 + - + +

Benzenoneindo-3 '-sul-fonaphthol.......... 0.123 - - + -

Thionin ............... 0.063 - - + -

Methylene blue ....... 0.011 + + + +Indigo carmine ....... -0.125 - - - -

Phenosafranine. -0.252 - - - -

Neutral red ....... -0.325 - - - -

* From Handbook of Indicators, NationalAniline Division, Allied Chemical Corp., NewYork.

t Symbols: + denotes reduction was completewithin 10 hr; - denotes no detectable reduction atthe end of 10 hr.

inability of aerobes to grow without 02 is theneed by the organism for an electron acceptor ofhigh oxidation-reduction potential, an examina-tion was made of the coupling of substrate oxida-tion by the fungus with indicators of differentEh. The substrates included glucose and suc-cinate, one with a low potential, and the otherwith a high potential in their respective coupledsystems. The data are summarized in Table 2.Only substances possessing potentialsof + 0.011 vor greater were reduced by the intact organism.It is apparent that the fungus is not capable ofcoupling oxidations with externally suppliedelectron acceptors having a negative Eh , regard-less of the initial energy source.

In the basal medium, the fungus brought abouta moderate drop in Eh and a small disappearanceof sugar. If the medium contained yeast extract,however, both the fall in Eh and the disappear-ance of glucose became appreciably more marked

(Table 3). The MnO2 included in the basal me-dium to poise it at a higher initial Eh permittedalmost as active a glucose dissimilation (4.6 mg/ml of glucose disappearance in 72 hr) as did theyeast extract. Parallel nutritional studies failed toreveal any response of the fungus to manganeseas such. Thus, maintenance of a high Eh, atreatment which may act physiologically byproviding the fungus with oxidized materialsthat could serve as electron acceptors in theabsence of 02, or the provision of factors inyeast extract, permit anaerobic activity andgrowth of the fungus.

Further trials were made to determine whetherthe fungus could couple anaerobic growth tothe reduction of nitrate, selenite, and ferric iron.Although F. oxysporum failed to grow without 02in the simple medium, anaerobic proliferationdid take place if the fungus was provided withnitrate, selenite, or ferric iron (Table 4). Sur-prisingly, yeast extract alone did not permitanaerobic growth in this instance, although it didstimulate the fungus in the absence of 02 whennitrate, selenite, or ferric ions were present.Selenite allowed the fungus to increase in cell massin 02-free circumstances, even when, as indicatedby the aerobic cultures, the concentration washigh enough to be markedly toxic; such anaero-bically grown fungi formed considerable quanti-ties of selenium. In nitrate-grown anaerobic

TABLE 3. Effect of manganese dioxide and yeastextract on anaerobic activity of Fusarium

oxysporum

Addition to basalmedium

None .............

Yeast extract(0.5%) .........

MnO2 (0.05 M)....

Time

hr

0

244872

0

244872

0

244872

Eh

mv

-208-281-289-299

-238-339-380-400

-105-165-162

pH

7.257.607.497.57

7.657.707.687.60

7.257.807.57

-190 7.70

Glucosemeta-bolized

mg/ml

0.01.92.12.0

0.00.85.25.1

0.00.84.74.6

1312 J. BACTERIOL.

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ANAEROBIC GROWTH OF F. OXYSPORUM11

cultures, 35.0 and 19.0 ,ug/ml of ammoniumnitrogen were found in the presence and absenceof yeast extract at the end of 7 days, but thenitrite-nitrogen levels were less than 0.1 ,ug/ml.Ferric-grown anaerobic cultures had, at the endof 21 days, 22.8 and 12.6 ,ug/ml of ferrous iron inthe presence and absence of yeast extract. Noferrous iron was found in any of the aerobiccultures, but 1.3 ,ug/ml of ferrous iron werefound in the anaerobic vessels receiving heat-in-activated mycelium.Approximately 0.47 mg of ferrous iron was

produced per mg of mycelium formed; assumingthe mycelium to contain 5% nitrogen derivedfrom the ammonium produced by nitrate reduc-tion, about 0.6 mg of ammonium-nitrogen wasproduced from nitrate per mg of mycelium. Therespective oxidized ions, ferric and nitrate, werenot the sole electron acceptors for anaerobicgrowth, however. For example, after 7 days in anitrate-containing medium, nitrate and 100.8 mgof glucose-C had disappeared, and 4.5 mg ofcell-C, 11.0 mg of C02-C, 66.0 mg of ethanol-C,and 18.0 mg of lactic acid-C appeared; little ornone of these products was found in nitrate-freesolutions, the fermentation of glucose generatingelectron acceptors utilizable by the fungus.Although an oxidation-reduction balance wasnot made in this instance because of the com-plexity of the medium (1% peptone, 1% glucose,1% KNO3, 0.01 % yeast extract), the molarratios of the products suggested an anomalousfermentation, namely the formation of 6 molesof ethanol, 2 moles of C02, and 1 mole of lacticacid in the course of disappearance of 3.0 molesof glucose.A possible explanation for the small yield of

CO2 compared to ethanol, assuming a typicalalcoholic fermentation, is the reassimilation of aportion of the evolved gas. To test the possibilityof an effect of CO2 on growth of the fungus,various concentrations of NaHCO3 were addedto medium B (450 ml per flask) containing either0.4% KNO3 or 0.2% NH4Cl as sole nitrogensource. Yeast extract (5.0 mg per liter) wasincluded in the medium, the pH was adjusted to6.5 to 6.8, and the flasks were incubated anaero-bically for 7 days. No detectable growth tookplace in the NH4Cl series, regardless of thebicarbonate concentration. The results of thenitrate series (Table 5) indicate a clear stimula-tion by bicarbonate with an optimal response atabout 20 mmoles per 450 ml of medium. Higher

TABLE 4. Growth of Fusar-ium oxysporum inpresence of nitrate, selenite, or

ferric iiron

Fungus (mg, dry wt)

Addition to Incubation No yeast extract Yeast extractmedium (days) _

Aero- Anaero- Aero- Anaero-bic bic bic bic

None 7 119.2 1.0 252.8 1.7Nitratet 68.3 16.1 139.8 28.5Selenite 30.4 4.6 34.3 6.0

None 21 103.5 1.6 158.3 2.2Ferric iron 81.5 11.5 187.6 23.4Ferric iron t 0.0 0.0 0.0 0.0

* Added at a rate of 0.5% in cultures grown for7 days and 0.1% in those grown for 21 days.

t No ammonium in the medium.t Inoculum inactivated by heating.

TABLE 5. Anaerobic growth of Fusarium oxysporumin a nitrate medium containing various

concentrations of bicarbonate

NaHCO, concn* Growtht Final pH(dry wt)

0 3.7 6.810 7.8 7.720 15.7 8.050 10.9 8.6

* Expressed as mmoles per 450 ml.t Expressed as mg per 450 ml.

bicarbonate concentrations appeared to inhibitthe fungus. A stimulation of F. oxysporummultiplication in soil and a specific incorporationof C14-CO2 into the mycelium of the funguswere reported by Stover and Freiberg (1958).The observed response to NaHCO3 does notresult from a pH effect, because the fungus grewreadily from pH 3.0 to 9.0.To test the possibility that an additional

oxidizable substrate may have been providedto the fungus in the form of the H2 generated inthe chromous acid deoxygenating treatment,cultures were incubated under N2 purified of 02only by passage over the heated reduced copperand under H2 freed of 02 by passage through thechromous acid scrubbing bottle. Medium B wasused, the nitrogen source being either KNO3 orNH4C1 (0.2%). In cultures incubated under N2 ,

growth was noted in solutions containing nitrate

VOL. 87, 1964 1313

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GUNNER AND ALEXANDER

TABLE 6. Produzcts of anaerobic growth of Fusariuinmoxysporum in atmospheres of hydrogen

and nitrogen

N2 atmosphere H2 atmosphere

DeterminationAnmo- Nitrate Anmmo Nitratenium* nium

Gas consumed,mmoles ........... O. 0.0 0.96 1.26

Growth, mg (dry wt). O. 5 6.6 3.8 7.8Glucose utilized,

mmoles ........... <0.01 1.18 0.18 0.84Products, mmolesC02. <0.01 1.73 <0.01 1.16Ethanol ...... <0..<.01 1.92 0.44 1.87Acetic acid ......... O.00O 0.44 0.00 0.00Ammonia...... 0..O.00 0.34 0.00 0.24

* Nitrogen source for growth. Cultures grownin 450 ml of Medium B supplemented with 4.0g per liter of KNO3 .

but not in those receiving ammonium-nitrogen.Surprisingly, however, the fungus developed inthe H2 atmbsphere when either nitrate or am-monium was supplied to it. This suggests thatthe fungus may have an H2 metabolism.The ability of the organism to oxidize H2 was

examined by growing the fungus in medium Bwith H2 as the gas phase and recording theamount of gas consumed. Parallel culture vesselswere incubated under N2 to serve as controls.Although there was no N2 disappearance, signifi-cant amounts of H2 were consumed, whetherthe sole fixed nitrogen source was ammonium ornitrate (Table 6). In each instance, the carbonsupplied as substrate was quantitatively re-covered in the various products, but the oxida-tion-reduction balance was incomplete. It is ofinterest that the fungal mass relative to glucoseconsumed was greater in the presence of H2,suggesting that the fungus obtained energy forgrowth by H2 oxidation.The products of nitrate-grown F. oxysporum

incubated in an N2 atmosphere were C02,ethanol, and acetate; nitrate-grown culturesunder H2 contained no acetate, but the yield ofethanol relative to C02 was correspondinglyhigher. No CO2 was detected in H2-grown cul-tures receiving ammonium as nitrogen source,although ethanol accumulated. It would thusappear that H2 oxidation is coupled with thereduction of some oxidized product of fermenta-tion.

The presence of hydrogenase in the fungus wasdemonstrated with washed cell suspensions thathad been grown aerobically and anaerobically inmedium B amended with nitrate. Myceliumrepresenting 1.6 mg of dry weight was added tothe caps of Thunberg tubes, and 50,moles ofbenzyl viologen and 600 /Amoles of phosphate(pH 8.0) were placed in the tube. The totalvolume was 4.0 ml. The tubes were evacuated,flushed five times with H2 purified of 02 by thechromous acid method, mixed, and incubated at30 C. By this procedure, fungal suspensionsprepared from 3- and 7-day cultures grown eitheraerobically or anaerobically were found to reducebenzyl viologen. There was no reduction when thefungal suspension included in the tubes wasboiled, nor was there activity when unheatedfungi were incubated with buffer and dye in thepresence of N2 rather than H2, except for a veryslow dye reduction in one instance with 3-daysuspensions grown in air. Methylene blue at aconcentration of 0.01% was not reduced underidentical circumstances; the results were similarto those of Gest (1954) with Clostridium butyli-cum.

Manometric measurement of hydrogenaseactivity revealed a linear consumption of H2with time. From the linear portion of the curve,the rate of gas disappearance was calculated tobe 38 ,uliters of H2 per hr per mg of cell nitro-gen.

It should be pointed out that the cell yieldobtained was consistently low, although theincrease in mass over that used as inoculum wasalways appreciable. For example, after 7 days,the weight of fungal material in a typical instancewas 16.1 mg in 450 ml of medium containing 1.0%glucose in the absence of yeast extract, and 28.5mg with 0.5% yeast extract. If the latter mediumwas fortified with 10-3 M ferric ammonium citrate,the fungal weight even after 21 days was notincreased.

DISCUSSIONIt is surprising that many microorganisms are

incapable of anaerobic proliferation, because theyappear capable of performing all essentialmetabolic processes common to the obligate orfacultative anaerobes. Several mechanisms can beadvanced to account for this apparent inabilityto grow in environments devoid of 02: the failureof the organism to synthesize an essential metab-olite in the absence of 02 which it makes under

1314 J. BACTERIOL.

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

ANAEROBIC GROWTH OF F. OXYSPORUM1

aerobic circumstances; the inability to couplethe anaerobic oxidation of reduced pyridinenucleotides or flavins with an inorganic ororganic compound that serves as an alternate to02 as the terminal electron acceptor in cellmetabolism; the accumulation of toxic productswhich would not be produced or would not reachinhibitory levels were 02 present; the failure toobtain sufficient energy to sustain growth fromenergy-liberating reactions carried out in theabsence of 02 ; an obligate requirement formolecular oxygen such as that reported duringolefin utilization by Candida lipolytica (Ishikuraand Foster, 1961); and the necessity for a highEh for the initiation or maintenance of growth,the converse to the postulate of Hanke and Katz(1943) for the basis of the apparent inability ofClostridiumi sporogenes to grow in air. Thesehypotheses can be tested experimentally, andevidence is provided herein that there is anincrease in F. oxysporum cell mass anaerobicallyunder the appropriate conditions. Certain of thehypotheses could account for the existence of thetrue obligate aerobe; e.g., although the failure ofan organism to grow without 02 may result fromits inability to synthesize an essential metaboliteunder anaerobiosis, inclusion of the factor in themedium might not overcome the deficiency,because the exogenously supplied molecule maynot penetrate into the cell. Similarly, the cellmay be impermeable to exogenously suppliedelectron acceptors, although 02 enters with littledifficulty.

Jacobs, Johantges, and Deibel (1963) recentlyreported that an electron carrier for nitratereduction was not synthesized by anaerobicallygrown staphylococci. On the basis of such find-ings, which tend to support the second mechanismadvanced above, it is not difficult to postulatethe existence of an obligate aerobe whose in-ability to grow anaerobically results solely fromits failure to produce an organic cofactor neededfor the reduction of some inorganic or organicelectron acceptor.The data with F. oxysporum suggest that the

fungus will grow in the absence of 02, providedthat some unidentified substance or substances inyeast extract are supplied to it, or that MnO2or nitrate, selenite, or ferric salts are includedin the medium. It is not clear whether the higheroxidation states of these elements serve asacceptors coupled with the electron transportchain or as agents to poise the Eh at a more

positive potential, or both. It thus appears thatthe fungus requires growth factors for prolifera-tion at low oxidation-reduction potentials,whereas the presence of the various oxidants,including 02, overcomes this requirement.Hence, the aerobic habit of this organism isassociated with the need for a suitable oxidantor for certain growth substances.The evidence presented indicates that, even in

a simple medium, the fungus utilizes MnO2,nitrate, ferric, or selenite ions as terminal electronacceptors for growth. This capability may pos-sibly be extended to other microorganisms andto the higher oxidation states of other elements.The potential for such an activity is suggested bythe large number of elements which, in theirhigher oxidation states, can be reduced in thepresence of H2 by cell extracts of Micrococcuslactilyticus (Woolfolk and Whitely, 1962). Thepresent communication demonstrates that ceriumshould be included among the elements subjectedto microbiological reduction. Which of theseelements can serve as terminal electron acceptorsto sustain anaerobic proliferation is not known;undoubtedly, certain of them can thus function,provided they exist at a higher potential than dothe coenzymes that must be reoxidized, and theypenetrate into the cell.As yet unexplained is the incomplete oxidation-

reduction balance and the cessation of fungalgrowth when adequate sugar and yeast extractwere still available to F. oxysporum. Because onlyoxidation-reduction indicators having positiveEh values were reduced by the intact mycelium,regardless of the carbon substrate, and, as theEh during anaerobic growth consistently fell tonegative potentials, the cessation of growth mayhave resulted from an inhibitory effect of lowpotential or of reduced products of fermentation.

ACKNOWLEDGMENT

This work was supported in part by a grantfrom the United Fruit Co., Boston, Mass.

LITERATURE CITED

ANDERSON, R. L., AND E. J. ORDAL. 1961. CO2 -dependent fermentation of glucose by Cyto-phaga succinicans. J. Bacteriol. 81:139-146.

ANDREASEN, A. A., AND T. J. B. STIER. 1954.Anaerobic nutrition of Saccharomyces cere-visiae. II. Unsaturated fatty acid requirementfor growth in a defined medium. J. CellularComp. Physiol. 43:271-281.

VOL. 87, 1964 1315

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

GUNNER AND ALEXANDER

BARTNICKI-GARCIA, S., AND W. J. NICKERSON.1961. Thiamine and nicotinic acid: anaerobicgrowth factors for Mucor rouxii. J. Bacteriol.82:142-148.

DELWICHE, E. A. 1951. Activators for the cysteinedesulfhydrase system of an Escherichia colimutant. J. Bacteriol. 62:717-722.

GEST, H. 1954. Oxidation and evolution of molec-ular hydrogen by microorganisms. Bacteriol.Rev. 18:43-73.

GREWELING, T., AND M. PEECH. 1960. Chemicalsoil tests. Cornell Univ. Agr. Expt. Sta. Bull.960.

HANKE, M. E., AND Y. J. KATZ. 1943. An elec-trolytic method for controlling oxidation-reduction potential and its application in thestudy of anaerobiosis. Arch. Biochem. 2:183-200.

ISHIKURA, T., AND J. W. FOSTER. 1961. Incorpora-tion of molecular oxygen during microbialutilization of olefins. Nature 192:892-893.

JACOBS, N. J., J. JOHANTGES, AND R. H. DEIBEL.1963. Effect of anaerobic growth on nitratereduction by Staphylococcus epidermidis. J.Bacteriol. 85:782-787.

JACOBSON, L. 1951. Maintenance of iron supply innutrient solutions by a single addition offerric potassium ethylenediamine tetra-acetate. Plant Physiol. 26:411-413.

KOLTHOFF, I. M., AND E. B. SANDELL. 1938. Text-book of quantitative inorganic analysis. TheMacmillan Co., New York.

McELVAIN, S. M. 1947. The characterization oforganic compounds. The Macmillan Co., NewYork.

MARSHALL, J. H. 1960. The production of ana-erobic conditions with chromous salts. J. Gen.Microbiol. 22:645-648.

NEISH, A. C. 1952. Analytical methods for bac-terial fermentations. Natl. Res. Council Can.Rept. 46-8-3, 2nd revision.

RICHARDSON, G. M. 1936. The nutrition of Staphy-lococcus aureus. Necessity for uracil in ana-erobic growth. Biochem. J. 30:2184-2190.

SOMOGYI, M. 1945. A new reagent for the deter-mination of sugars. J. Biol. Chem. 160:61-68.

STOVER, R. H., AND S. R. FREIBERG. 1958. Effect ofcarbon dioxide on multiplication of Fusariumnin soil. Nature 181:788-789.

WILSON, P. W., AND S. G. KNIGHT. 1952. Experi-ments in bacterial physiology, 3rd ed. BurgessPublishing Co., Minneapolis.

WOOLFOLK, C. A., AND H. R. WHITELY. 1962.Reduction of inorganic compounds withmolecular hydrogen by Micrococcus lactilyticusI. Stoichiometry with compounds of arsenic,selenium, tellurium, transition and otherelements. J. Bacteriol. 84:647-658.

1316 J. BACTERIOL.

on January 9, 2020 by guesthttp://jb.asm

.org/D

ownloaded from