JOURNAL OF BACTERIOLOGY

Vol. 87, No. 6, pp. 1309-1316 June, 1964

Copyright 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 cultured aerobically. Similarly, Saccharomyces

GUNNER, H. B. (Cornell University, Ithaca, cerevisiae needs certain sterols or unsaturated
N.Y.), AND M. ALEXANDER. Anaerobic growth of fatty acids for good anaerobic growth (Andreasen
Fusarium oxysporum. J. Bacteriol. 87:1309-1316. and Stier, 1954); Mlucor rouxii requires thiamine
1964.-Fusarium oxysporum, an alleged obligate and nicotinic acid (Bartnicki-Garcia and Nicker-
aerobe, was found to be capable of growth in the son, 1961); and Cytophaga succinicans needs
absence of molecular oxygen, provided the medium CO2 for development in the absence of 02 (Ander-
contained yeast extract, MnO2 , nitrate, selenite, son and Ordal, 1961).
or ferric ions. The active substance in yeast ex-
tract was not identified. The fungus possessed The present investigation was designed to
hydrogenase, and was capable of utilizing H12. determine the basis for the inability of a re-
Under anaerobic conditions, the fungus effected portedly obligately aerobic fungus to grow
the reduction of nitrate, ceric, ferric, selenite, and without molecular oxygen. Particular attention
tellurite ions, as well as the reduction of several was given to the possibility that this inability
inorganic sulfur compounds and indicators having was associated with the absence of a suitable
positive oxidation-reduction potentials. The electron acceptor and the related low oxidation-
products of anaerobic nitrate-dependent growth reduction potential.
were ethanol, C02, acetic acid, and ammonia.
Possible explanations for the apparent inability MATERIALS AND METHODS
of obligate aerobes to grow in the absence of 02
are discussed. Fusarium oxysporum f. cubense was grown in a
basal medium (medium A) consisting of: glucose,
15.0 g; NH4Cl, 4.0 g; K2HPO4, 0.8 g; KH2PO4,
Despite the importance in microbial taxonomy 0.2 g; CaC12, 0.1 g; MgSO4 7H20, 0.2 g; FeCl3*
and physiology of 02-dependent growth, little 6H20, 0.03 g; and distilled water, 1,000 ml. All
attention has been given to the biochemical basis incubations were performed at 30 C. In experi-
of the need for molecular oxygen or to the possi- ments made to determine whether the fungus
bilities that the 02 requirement of aerobic could couple anaerobic growth to the reduction of
microorganisms reflects, not a need for a specific nitrate, selenite, and ferric iron, the medium (B)
form of the element, but rather some other had the following composition: glucose, 18.0 g;
physiological characteristic or deficiency. The NH4C1, 2.0 g; KH2PO4, 1.5 g; MgSO4 7H20,
failure of the so-called strictly aerobic microor- 0.6 g; iron chelate (Jacobson, 1951), 0.025 mg;
ganism to grow in the absence of molecular 02 ZnSO4 *7H20, 0.03 g; and distilled water, 1.0
remains largely unexplained. Studies of selected liter. Potassium nitrate, Na2SeO3, and ferric
microbial strains, however, indicate that many ammonium citrate were added to final concentra-
microorganisms may, indeed, proliferate anaero- tions of 4.0 X 10-2 M 10- M, and 10-3 M, respec-
bically, but only if suitable growth factors are tively.
provided; i.e., the organism is unable to syn- In investigations of the inorganic ions suscepti-
thesize the metabolite in 02-free circumstances. ble to reduction by F. oxysporum, 10 ,umoles of
For example, Richardson (1936) noted that a the sulfur or nitrogen compounds, 200 ,umoles of
strain of Staphylococcus aureus requires uracil glucose, and 400 ,umoles of tris(hydroxymethyl)-
when grown in the absence of 02, but not when aminomethane (tris) buffer (pH 7.6) were added
1 Agronomy paper no. 636. to the main compartment of a Thunberg tube.
2 Present address: Institute of Agricultural and In the cap was placed 1.0 ml of fungus suspension
Industrial Microbiology, University of Mas- from a 36-hr culture grown on a rotary shaker;
sachusetts, Amherst. the tube was evacuated, and the suspension was
1309
 1310 GUNNER AND ALEXANDER
tipped into the main compartment; after 12 hr,
the contents of the tube were centrifuged, and
the supernatant fluid was examined for reduced
products. 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 phosphate
buffer (pH 7.0) with 4.0 ml of a eerie-citrate
complex prepared by mixing equal volumes of
10-4 M Ce(HSO4)4 solution and 10-2 M citric acid
adjusted to pH 7.0 with NaOH; after centrifuga-
tion, the formation of cerous ions was determined
at 270 m,u. The reduction of ferric iron to the
ferrous form was determined by incubating a
washed fungal suspension in a solution containing
5.0 ,ug of ferric iron as ferric ammonium sulfate.
Reduction of the higher oxidation states of
selenium and tellurium was assayed by incubat-
ing 3.0 ml of a washed fungus suspension with 1.0
ml of 1.0% solutions of the various salts, 5.0 ml
of 0.1 M glucose solution, and 5.0 ml of 0.05 M
tris 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 a
washed F. oxysporum suspension in 0.05 M
phosphate buffer (pH 7.0) with 3.0 ml of a 0.01%
solution of the Eh indicator, 5.5 ml of 0.1 M
solution of the neutralized substrate, and 5.5 ml
of 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 continuous
monitoring with an 02 electrode (Beckman
Instruments Inc., model 11098) was devised. In
initial studies, 450 ml of medium were placed in
wide-mouthed 500-ml Erlenmeyer flasks fitted
with rubber stoppers containing inlets for N2,
sampling tube, platinum electrode, salt bridge
(3.0% agar, 1.0% NaCl), and a vacuum exhaust
line. The electrode and salt bridge were connected
to a potentiometer by means of a saturated KCl
solution in which a calomel electrode was placed.
In subsequent studies, the rubber stopper was
fitted only with gas inlet and exhaust tubes and a
Thunberg tube cap containing the inoculum. In
this way, the myeelial suspension could be
evacuated and flushed with gas together with the
rest 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 passed
through a column of reduced copper turnings
heated to 600 C, and was then introduced into a
J. BACTERIOL.
gas washing bottle containing a mixture of a
chromic salt, zinc, and sulfuric acid; this pro-
cedure for removal of traces of 02 was described
by Marshall (1960). The entire system was
evacuated to 4.0 cm of mercury, and was flushed
five times with the O2-free N2 sterilized by pas-
sage through a cotton filter. Anaerobiosis was
effected within 15 min after the growth medium
was removed from the autoclave, while all
solutions were still near 100 C, to minimize the
amount of dissolved 02 introduced into the sys-
tem with the growth media. After the 02 electrode
gave a continuous zero reading for 1 hr, a time
sufficient for the medium to cool, the inoculum
was tipped into the medium from the cap, the
inlet 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, the
pH was adjusted to 1.0 to 2.0 with concentrated
H2SO4 , and the dissolved CO2 was swept into
standard Ba(OH)2 by a stream of N2. The
fungal suspension in each flask was removed by
filtration, washed, dried at 70 C, and weighed.
The mycelium and conidia were considered to
contain 50% carbon and 5%O nitrogen. Analysis
for nitrite was by the method of Kolthoff and
Sandell (1938); ammonia was determined by
nesslerization (Wilson and Knight, 1952), ferric
iron was estimated by the method of Greweling
and Peech (1960); glucose determinations were
performed as described by Somogyi (1945); CO2
was measured titrimetrically; ethanol and lactic
acid were determined by the procedures described
by Neish (1952); acetic acid was identified by
determination of the Duclaux numbers (Mc-
Elvain, 1947); and sulfide was estimated by the
method of Delwiche (1951).
The amount of H2 consumed by F. oxysporum
after 7 days was determined manometrically,
the culture flasks being attached to mercury
manometers. Hydrogenase was assayed quanti-
tatively in a Warburg microrespirometer with
the use of 7-day cultures grown anaerobically in
02-free N2 . Washed mycelium suspended in 0.2%
KCl was placed in the side arm of the Warburg
flasks; the center well contained 0.2 ml of 20%
KOH, and the main compartment of the flask
contained 50 ,moles of benzyl viologen and 600
,umoles of phosphate buffer (pH 8.0), in a total
volume of 3.2 ml. The assay was performed at 36
C in an atmosphere of H2 purified of 02 by pas-
sage through chromous acid. Control flasks were
VOL. 87, 1964 ANAEROBIC GROWTH OF F. OXYSPORUM 1311
maintained in an atmosphere of N2 freed of 02 by TABLE 1. Reduction of inorganic ions by washed
passage through heated copper turnings. mycelium of Fusarium oxysporum
Anaerobically grown cultures of F. oxysporum
were tested routinely for purity by microscopic Product
Substrate Boiled
examination and by inoculation into nutrient Substrate concn mycelium
Com- Concn control*
broth containing 100 ,ug/ml of cycloheximide. pound
The tubes were incubated anaerobically under pmoles/inl Ag/ml I g/m
N2. No evidence of contamination was noted. K2S03 1.67 H 2S 3.9 1.1
RESULTS NaHSO3 1.67 H 2S 1.6 0.0
K2SO4 1.67 H 2S 1.5 0.0
F. oxysporum grew in an anaerobic environ- Na2S203 1.67 H 2S 7.0 0.0
ment, provided that higher oxidation states of Na2S206 1.67 H 2S 5.4 0.0
certain elements were included in the medium. K2S208 1.67 H 2S 4.9 0.0
For example, distinct growth was noted visually KNO3 1.67 NO2- 0.3 0.0
after 7 days of anaerobic incubation in an agar Ce(HSO4)4 0.04 Ce3+ +
medium containing 0.3 % K2S04, 0.3 % Na2SeO3, FeNH4 (SO4) 2 0.10 Fe2+ 4.0 0.0
or 0.3% Na2MoO4 2H20 with ammonium as * Concentration of product in incubation mix-
nitrogen source, or in agar containing 0.3 % tures containing boiled rather than viable my-
KNO3 as sole nitrogen source; no mycelium celium.
appeared if the ammonium medium was not
supplemented with one of these salts. Aerobically,
the fungus developed on all the media tested. containing 0.1% Vitamin Free Casamino Acids,
The results were identical whether the cultures L-arginine HCl, L-glutamic acid, DL-valine,
were incubated in an atmosphere containing DL-phenylalanine, DL-isoleucine, or DL-trypto-
N2 or H2 freed of 02 catalytically. Although the phan. The yeast extract effect on growth or
amount of residual 02 in the gas phase was not glucose disappearance also could not be replaced
determined, the absence of detectable growth in by thiamine, biotin, pyridoxine, riboflavine,
the ammonium medium containing none of these pantothenate, vitamin B12, lipoic acid, folic acid,
salts served as a biological test for the existence or ascorbic acid added singly or in various com-
Of 02 levels insufficient to support aerobic pro- binations at concentrations ranging from 0.05 to
liferation. Further, plates of Potato Dextrose 2.50 mg per liter. The morphology of the fungus
Agar (Difco) streaked with the aerobic Aspergil- grown anaerobically in the presence of yeast ex-
lus niger (Van Tieghem), Rhizobium trifolii, and tract was similar to that in the nitrate medium,
Bacillus pantothenticus, and incubated in the except for the greater abundance of microconidia
same containers, showed no growth. in the former medium.
The anaerobic cultures of F. oxysporum con- The data above indicate that growth took place
sisted of fine hair-like structures made up of short when certain inorganic ions were provided. That
hyphal fragments and many chlamydospores. the fungus does, indeed, effect a reduction of the
Upon the return to aerobic conditions, conidia inorganic ions is demonstrated by the results of
began to bud rapidly from the hyphal surface, Table 1. All of the sulfur salts and the nitrate,
and, within 24 hr, the culture assumed the ceric, and ferric ions were reduced. The rate of
characteristics of the typical F. oxysporum reduction varied markedly with the specific ion;
mycelium. Aerobically, by contrast, the dominant undoubtedly, reduced products in addition to
spore form was the microconidium, which was those listed appeared. The brick-red color of
somewhat less abundant; chlamydospores ap- selenium and the black color of tellurium de-
peared abundantly only in old cultures. veloped in mycelium incubated with Na2SeO3
The fungus also grew anaerobically in the and K2TeOs for 10 hr, and the hyphae contained
ammonium-containing liquid medium supple- red and black inclusions when provided with
mented with 0.1% yeast extract; 6.17 mg/ml of these salts. There was no selenite or tellurite
glucose disappeared in 10 days. There was neither reduction by boiled mycelium, and the viable
growth nor glucose disappearance in the absence fungus showed no activity upon selenate and
of yeast extract. Further, F. oxysporum failed to tellurate.
develop anaerobically in the ammonium medium Because one hypothesis to account for the
1312 GUNNER AND ALEXANDER J. BACTERIOL.

TABLE 2. Reduction by Fusar ium oxysporum of dyes (Table 3). The MnO2 included in the basal me-
of different oxidation-reduction potentials dium to poise it at a higher initial Eh permitted
almost as active a glucose dissimilation (4.6 mg/
l Substrates used as
electron donorst ml of glucose disappearance in 72 hr) as did the
Indicator Eh of inl yeast extract. Parallel nutritional studies failed to
dicator* t
reveal any response of the fungus to manganese
_ I as such. Thus, maintenance of a high Eh, a
treatment which may act physiologically by
Benzenoneindo-3 '- providing the fungus with oxidized materials
chlorophenol ........ 0.233 + + + + that could serve as electron acceptors in the
2,6-Dichlorobenzen- absence of 02, or the provision of factors in
oneindophenol .. .... 0.217 + + + +
Benzenoneindo-2'- yeast extract, permit anaerobic activity and
methylphenol ....... 0.208 + + + + growth of the fungus.
2,6-Dichlorobenzen- Further trials were made to determine whether
oneindo-3'-methyl the fungus could couple anaerobic growth to
phenol .............. 0.181 + + + + the reduction of nitrate, selenite, and ferric iron.
2,6-Dibromobenzen- Although F. oxysporum failed to grow without 02
oneindo-3'-methoxy- in the simple medium, anaerobic proliferation
phenol .............. 0.159 + - + + did take place if the fungus was provided with
Benzenoneindo-3 '-sul- nitrate, selenite, or ferric iron (Table 4). Sur-
fonaphthol .......... 0.123 - - + -
prisingly, yeast extract alone did not permit
Thionin ............... 0.063 - - + -
Methylene blue ....... 0.011 + + + + anaerobic growth in this instance, although it did
Indigo carmine ....... -0.125 - - - - stimulate the fungus in the absence of 02 when
Phenosafranine. -0.252 - - - - nitrate, selenite, or ferric ions were present.
Neutral red ....... -0.325 - - - - Selenite allowed the fungus to increase in cell mass
in 02-free circumstances, even when, as indicated
* From Handbook of Indicators, National by the aerobic cultures, the concentration was
Aniline Division, Allied Chemical Corp., New high enough to be markedly toxic; such anaero-
York. bically grown fungi formed considerable quanti-
t Symbols: + denotes reduction was complete ties of selenium. In nitrate-grown anaerobic
within 10 hr; - denotes no detectable reduction at
the end of 10 hr.
TABLE 3. Effect of manganese dioxide and yeast
extract on anaerobic activity of Fusarium
inability of aerobes to grow without 02 is the oxysporum
need by the organism for an electron acceptor of
high oxidation-reduction potential, an examina- Addition to basal Glucose
Time Eh pH meta-
tion was made of the coupling of substrate oxida- medium bolized
tion by the fungus with indicators of different
hr mv mg/ml
Eh. The substrates included glucose and suc-
cinate, one with a low potential, and the other None ............. 0 -208 7.25 0.0
with a high potential in their respective coupled 24 -281 7.60 1.9
systems. The data are summarized in Table 2. 48 -289 7.49 2.1
72 -299 7.57 2.0
Only substances possessing potentialsof + 0.011 v
or greater were reduced by the intact organism. Yeast extract
It is apparent that the fungus is not capable of (0.5%) ......... 0 -238 7.65 0.0
coupling oxidations with externally supplied 24 -339 7.70 0.8
electron acceptors having a negative Eh , regard- 48 -380 7.68 5.2
less of the initial energy source. 72 -400 7.60 5.1
In the basal medium, the fungus brought about
a moderate drop in Eh and a small disappearance MnO2 (0.05 M).... 0 -105 7.25 0.0
of sugar. If the medium contained yeast extract, 24 -165 7.80 0.8
however, both the fall in Eh and the disappear- 48 -162 7.57 4.7
72 -190 7.70 4.6
ance of glucose became appreciably more marked
VOL. 87, 1964 ANAEROBIC GROWTH OF F. OXYSPORUM11
1313
cultures, 35.0 and 19.0 ,ug/ml of ammonium TABLE 4. Growth of Fusar-ium oxysporum in
nitrogen were found in the presence and absence presence of nitrate, selenite, or
of yeast extract at the end of 7 days, but the ferric iiron
nitrite-nitrogen levels were less than 0.1 ,ug/ml. Fungus (mg, dry wt)
Ferric-grown anaerobic cultures had, at the end
of 21 days, 22.8 and 12.6 ,ug/ml of ferrous iron in Addition to Incubation No yeast extract Yeast extract
medium (days) _
the presence and absence of yeast extract. No
ferrous iron was found in any of the aerobic Aero- Anaero- Aero- Anaero-
bic bic bic bic
cultures, but 1.3 ,ug/ml of ferrous iron were
found in the anaerobic vessels receiving heat-in- None 7 119.2 1.0 252.8 1.7
activated mycelium. Nitratet 68.3 16.1 139.8 28.5
Approximately 0.47 mg of ferrous iron was Selenite 30.4 4.6 34.3 6.0
produced per mg of mycelium formed; assuming
the mycelium to contain 5% nitrogen derived None 21 103.5 1.6 158.3 2.2
from the ammonium produced by nitrate reduc- Ferric iron 81.5 11.5 187.6 23.4
Ferric iron t 0.0 0.0 0.0 0.0
tion, about 0.6 mg of ammonium-nitrogen was
produced from nitrate per mg of mycelium. The *
Added at a rate of 0.5% in cultures grown for
respective oxidized ions, ferric and nitrate, were 7 days and 0.1% in those grown for 21 days.
not the sole electron acceptors for anaerobic t No ammonium in the medium.
growth, however. For example, after 7 days in a t Inoculum inactivated by heating.
nitrate-containing medium, nitrate and 100.8 mg
of glucose-C had disappeared, and 4.5 mg of TABLE 5. Anaerobic growth of Fusarium oxysporum
cell-C, 11.0 mg of C02-C, 66.0 mg of ethanol-C, in a nitrate medium containing various
and 18.0 mg of lactic acid-C appeared; little or concentrations of bicarbonate
none of these products was found in nitrate-free NaHCO, concn* Growtht Final pH
solutions, the fermentation of glucose generating (dry wt)
electron acceptors utilizable by the fungus. 0 3.7 6.8
Although an oxidation-reduction balance was 10 7.8 7.7
not made in this instance because of the com- 20 15.7 8.0
plexity of the medium (1% peptone, 1 % glucose, 50 10.9 8.6
1 % KNO3, 0.01 % yeast extract), the molar
ratios of the products suggested an anomalous * Expressed as mmoles per 450 ml.
fermentation, namely the formation of 6 moles t Expressed as mg per 450 ml.
of ethanol, 2 moles of C02, and 1 mole of lactic
acid in the course of disappearance of 3.0 moles bicarbonate concentrations appeared to inhibit
of glucose. the fungus. A stimulation of F. oxysporum
A possible explanation for the small yield of multiplication in soil and a specific incorporation
CO2 compared to ethanol, assuming a typical of C14-CO2 into the mycelium of the fungus
alcoholic fermentation, is the reassimilation of a were reported by Stover and Freiberg (1958).
portion of the evolved gas. To test the possibility The observed response to NaHCO3 does not
of an effect of CO2 on growth of the fungus, result from a pH effect, because the fungus grew
various concentrations of NaHCO3 were added readily from pH 3.0 to 9.0.
to medium B (450 ml per flask) containing either To test the possibility that an additional
0.4% KNO3 or 0.2% NH4Cl as sole nitrogen oxidizable substrate may have been provided
source. Yeast extract (5.0 mg per liter) was to the fungus in the form of the H2 generated in
included in the medium, the pH was adjusted to the chromous acid deoxygenating treatment,
6.5 to 6.8, and the flasks were incubated anaero- cultures were incubated under N2 purified of 02
bically for 7 days. No detectable growth took only by passage over the heated reduced copper
place in the NH4Cl series, regardless of the and under H2 freed of 02 by passage through the
bicarbonate concentration. The results of the chromous acid scrubbing bottle. Medium B was
nitrate series (Table 5) indicate a clear stimula- used, the nitrogen source being either KNO3 or
tion by bicarbonate with an optimal response at NH4C1 (0.2%). In cultures incubated under N2 ,
about 20 mmoles per 450 ml of medium. Higher growth was noted in solutions containing nitrate
1314 GUNNER AND ALEXANDER J. BACTERIOL.

TABLE 6. Produzcts of anaerobic growth of Fusariuinm The presence of hydrogenase in the fungus was
oxysporum in atmospheres of hydrogen demonstrated with washed cell suspensions that
and nitrogen had been grown aerobically and anaerobically in
N2 atmosphere H2 atmosphere medium B amended with nitrate. Mycelium
Determination
representing 1.6 mg of dry weight was added to
Anmo- Nitrate Anmmo Nitrate the caps of Thunberg tubes, and 50 ,moles of
nium* nium
benzyl viologen and 600 /Amoles of phosphate
Gas consumed, (pH 8.0) were placed in the tube. The total
mmoles ........... O. 0.0 0.96 1.26 volume was 4.0 ml. The tubes were evacuated,
Growth, mg (dry wt). O. 5 6.6 3.8 7.8 flushed five times with H2 purified of 02 by the
Glucose utilized, chromous acid method, mixed, and incubated at
mmoles ........... <0.01 1.18 0.18 0.84 30 C. By this procedure, fungal suspensions
Products, mmoles prepared from 3- and 7-day cultures grown either
C02 . <0.01 1.73 <0.01 1.16 aerobically or anaerobically were found to reduce
Ethanol ...... <0.. <.01 1.92 0.44 1.87 benzyl viologen. There was no reduction when the
Acetic acid ......... O.00O 0.44 0.00 0.00 fungal suspension included in the tubes was
Ammonia ...... 0.. O.00 0.34 0.00 0.24 boiled, nor was there activity when unheated
* Nitrogen source for growth. Cultures grown fungi were incubated with buffer and dye in the
in 450 ml of Medium B supplemented with 4.0 presence of N2 rather than H2, except for a very
g per liter of KNO3 . slow dye reduction in one instance with 3-day
suspensions grown in air. Methylene blue at a
but not in those receiving ammonium-nitrogen. concentration of 0.01 % was not reduced under
Surprisingly, however, the fungus developed in identical circumstances; the results were similar
the H2 atmbsphere when either nitrate or am- to those of Gest (1954) with Clostridium butyli-
monium was supplied to it. This suggests that cum.
the fungus may have an H2 metabolism. Manometric measurement of hydrogenase
The ability of the organism to oxidize H2 was activity revealed a linear consumption of H2
examined by growing the fungus in medium B with time. From the linear portion of the curve,
with H2 as the gas phase and recording the the rate of gas disappearance was calculated to
amount of gas consumed. Parallel culture vessels be 38 ,uliters of H2 per hr per mg of cell nitro-
were incubated under N2 to serve as controls. gen.
Although there was no N2 disappearance, signifi- It should be pointed out that the cell yield
cant amounts of H2 were consumed, whether obtained was consistently low, although the
the sole fixed nitrogen source was ammonium or increase in mass over that used as inoculum was
nitrate (Table 6). In each instance, the carbon always appreciable. For example, after 7 days,
supplied as substrate was quantitatively re- the weight of fungal material in a typical instance
covered in the various products, but the oxida- was 16.1 mg in 450 ml of medium containing 1.0%
tion-reduction balance was incomplete. It is of glucose in the absence of yeast extract, and 28.5
interest that the fungal mass relative to glucose mg with 0.5 % yeast extract. If the latter medium
consumed was greater in the presence of H2, was fortified with 10-3 M ferric ammonium citrate,
suggesting that the fungus obtained energy for the fungal weight even after 21 days was not
growth by H2 oxidation. increased.
The products of nitrate-grown F. oxysporum
incubated in an N2 atmosphere were C02, DISCUSSION
ethanol, and acetate; nitrate-grown cultures It is surprising that many microorganisms are
under H2 contained no acetate, but the yield of incapable of anaerobic proliferation, because they
ethanol relative to C02 was correspondingly appear capable of performing all essential
higher. No CO2 was detected in H2-grown cul- metabolic processes common to the obligate or
tures receiving ammonium as nitrogen source, facultative anaerobes. Several mechanisms can be
although ethanol accumulated. It would thus advanced to account for this apparent inability
appear that H2 oxidation is coupled with the to grow in environments devoid of 02: the failure
reduction of some oxidized product of fermenta- of the organism to synthesize an essential metab-
tion. olite in the absence of 02 which it makes under
VOL. 87, 1964 ANAEROBIC GROWTH OF F. OXYSPORUM11315
aerobic circumstances; the inability to couple
the anaerobic oxidation of reduced pyridine
nucleotides or flavins with an inorganic or
organic compound that serves as an alternate to
02 as the terminal electron acceptor in cell
metabolism; the accumulation of toxic products
which would not be produced or would not reach
inhibitory levels were 02 present; the failure to
obtain sufficient energy to sustain growth from
energy-liberating reactions carried out in the
absence of 02 ; an obligate requirement for
molecular oxygen such as that reported during
olefin utilization by Candida lipolytica (Ishikura
and Foster, 1961); and the necessity for a high
Eh for the initiation or maintenance of growth,
the converse to the postulate of Hanke and Katz
(1943) for the basis of the apparent inability of
Clostridiumi sporogenes to grow in air. These
hypotheses can be tested experimentally, and
evidence is provided herein that there is an
increase in F. oxysporum cell mass anaerobically
under the appropriate conditions. Certain of the
hypotheses could account for the existence of the
true obligate aerobe; e.g., although the failure of
an organism to grow without 02 may result from
its inability to synthesize an essential metabolite
under anaerobiosis, inclusion of the factor in the
medium might not overcome the deficiency,
because the exogenously supplied molecule may
not penetrate into the cell. Similarly, the cell
may be impermeable to exogenously supplied
electron acceptors, although 02 enters with little
difficulty.
Jacobs, Johantges, and Deibel (1963) recently
reported that an electron carrier for nitrate
reduction was not synthesized by anaerobically
grown staphylococci. On the basis of such find-
ings, which tend to support the second mechanism
advanced above, it is not difficult to postulate
the existence of an obligate aerobe whose in-
ability to grow anaerobically results solely from
its failure to produce an organic cofactor needed
for the reduction of some inorganic or organic
electron acceptor.
The data with F. oxysporum suggest that the
fungus will grow in the absence of 02, provided
that some unidentified substance or substances in
yeast extract are supplied to it, or that MnO2
or nitrate, selenite, or ferric salts are included
in the medium. It is not clear whether the higher
oxidation states of these elements serve as
acceptors coupled with the electron transport
chain or as agents to poise the Eh at a more
positive potential, or both. It thus appears that
the 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 is
associated with the need for a suitable oxidant
or 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 electron
acceptors for growth. This capability may pos-
sibly be extended to other microorganisms and
to the higher oxidation states of other elements.
The potential for such an activity is suggested by
the large number of elements which, in their
higher oxidation states, can be reduced in the
presence of H2 by cell extracts of Micrococcus
lactilyticus (Woolfolk and Whitely, 1962). The
present communication demonstrates that cerium
should be included among the elements subjected
to microbiological reduction. Which of these
elements can serve as terminal electron acceptors
to sustain anaerobic proliferation is not known;
undoubtedly, certain of them can thus function,
provided they exist at a higher potential than do
the coenzymes that must be reoxidized, and they
penetrate into the cell.
As yet unexplained is the incomplete oxidation-
reduction balance and the cessation of fungal
growth when adequate sugar and yeast extract
were still available to F. oxysporum. Because only
oxidation-reduction indicators having positive
Eh values were reduced by the intact mycelium,
regardless of the carbon substrate, and, as the
Eh during anaerobic growth consistently fell to
negative potentials, the cessation of growth may
have resulted from an inhibitory effect of low
potential or of reduced products of fermentation.
ACKNOWLEDGMENT
This work was supported in part by a grant
from the United Fruit Co., Boston, Mass.
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