FEMS Microbiology Ecology 31 (1985) 159-170
Published by Elsevier
159
FEC 00020
Growth of a facultative anaerobe under oxygen-limiting conditions
in pure culture and in co-culture with a sulfate-reducing bacterium
(Mixed culture; coexistence of aerobes and anaerobes; glucose-fermenting bacteria; chemostat
enrichments)
Jan C. Gottschal and Regine Szewzyk *
Laboratorium ooor Microbiologie, Rijksunioersiteit Groningen, Kerklaan 30, 9751 N N Haren, The Netherlands, and * Fakultiit ff~r
Biologie, Unioersit~it Konstanz, Postfach 5560, D. 7750 Konstanz, F..R.G.
Received 17 April 1985
Revision received 23 April 1985
Accepted 24 April 1985
1. SUMMARY
2. INTRODUCTION
The occurrence and properties were studied of
glucose-metabolizing bacteria present in the
anaerobic sediment 5-10 cm below the surface of
an estuarine tidal mud-flat. Of all 'these bacteria
(104-105 per g wet sediment) 80-90~ were facultatively anaerobic species. Chemostat enrichments
on glucose under aerobic, oxygen-limited, and alternately aerobic-anaerobic conditions also yielded
cultures dominated by facultative anaerobes. One
of the dominant species, tentatively identified as a
Vibrio sp., was studied in more detail under
oxygen-limiting conditions. Fermentative and respiratory metabolisms were found to operate
simultaneously, and the ratio between the two was
regulated by the extent of oxygen limitation. A
small fraction of the acetate formed under such
growth conditions was shown to be subsequently
respired. A co~ulture was established of the Vibrio
sp. and a sulfate-reducing bacterium (Desulfovibrio
HL21) in an aerated chemostat. The importance
of these observations is discussed in relation to the
role of facultative anaerobes in anaerobic habitats.
All natural anaerobic ecosystems contain areas
which are at times in contact with oxygen. The size
of such areas may be very limited, with very steep
oxygen gradients. Some examples of such habitats
are the top layers of anoxic sediments [1], microbial mats [2,3], strongly colonized surfaces like
teeth in the oral cavity [4], soil particles [5],
bacterial aggregates in aerobic environments [6].
In some stratified water bodies with an anoxic
bottom layer oxygen gradients may extend over
much larger distances [7].
In most of these 'twilight zones' of oxygen,
conditions are not static at all. Rather is there a
dynamic equilibrium of consumption and supply
of oxygen. Fluctuations in either the rate of supply
or the rate of consumption of oxygen by the
microbial population a n d / o r the chemical oxidation of reduced compounds (sulfide in particular)
will cause considerable variation of the actual local
oxygen concentrations in such environments. It is
obvious that this will strongly affect both anaerobic
and aerobic bacteria present in such habitats. For
0168-6496/85/$03.30 © 1985 Federation of European Microbiological Societies
160
example strict anaerobes must be able to survive
aerobic, or at least 'semi-aerobic', conditions. Indeed, it has been shown that sulfate reduction did
occur in the top 5 cm of tidal sediment in which
the redox potential of the pore water ( 0 - + 100
mV) indicated oxidized conditions [6]. This was
explained by assuming that the sulfate-reducing
bacteria grew within small clumps of sediment (up
to about 100 #m) which were anoxic inside.
Methane formation was also shown to proceed
during and after addition of small amounts of
oxygen (final concentration up to 50/~M) to samples of digesting sludge [8]. Again the occurrence
of 'flocs', representing strictly anaerobic microenvironments, was suggested to explain the occurrence of a strictly anaerobic process in the presence of oxygen.
It may not be absolutely necessary for strict
anaerobes to reside in such microniches in order to
survive and grow in the presence of oxygen. First
of all, recent evidence clearly indicates that many
strictly anaerobic bacteria (including sulfate reducers, methanogens and fermentative species) are
able to withstand strongly oxygenated conditions
[9-13]. Secondly, most of the habitats mentioned
so far will probably harbour various facultative
(an)aerobes which might scavenge oxygen quite
efficiently, thus rendering their direct environment
more suitable for strict anaerobes.
The goal of the present study was to investigate
whether sulfate reduction would be possible in
aerated co-cultures of a sulfate reducer and a
facultative anaerobe. Another aim was to find out
whether facultative anaerobes play an important
role in the mineralization of sugars in such
oxygen-poor environments•
To this end, numerically dominant facultative
anaerobes were isolated from tidal mud flats in the
northern Netherlands, and one of them was studied
in pure culture and in co-culture with a sulfate
reducer isolated from the same environment•
3. MATERIALS A N D M E T H O D S
3.1. Organisms
Desulfovibrio HL21 (DSM255) was isolated
from the anaerobic intertidal sediments of the
Ems-Dollard estuary in the northern Netherlands
[141.
3. 2. Sampling site and sampling
Samples were taken from tidal flats in the
Ems-Dollard estuary at low tide in spring, summer,
and autumn 1983. The exact location of the sampling site was the so-called 'Heringplaat', a mud
flat which, at low tide is slightly above the water
level [15]. Because of the considerable influx of
fresh water from 2 rivers, the water in this area of
the estuary is brackish, with a salinity of approximately 1 0 - 1 5 ~ . Although some dissolved
and suspended organic matter, imported by both
rivers, will reach this site, the majority of this
carbon load will have been removed by this point
[16].
Sediment cores were taken using plexiglass tubes
(25 cm long; 2.5 cm inner diameter). The tubes
were sealed with rubber bungs and kept on ice
during transport to the laboratory. Under a stream
of oxygen-free N2, sediment was pushed out of the
plastic tube and subsamples from the required
depth were transferred to anaerobic medium.
3.3. Media
The anaerobic basal medium used throughout
this study was of the following composition (values in g- 1-1): Na2SO 4, 3.0; KH2PO4, 0.2; MgCI 2
• 6H20, 2.0; NaC1, 10.0; NH4C1, 0.3; KCI, 0.3;
CaC12 • 2H20, 0.15; Resazurin, 0.001. A selenium
solution (50mg Na2SeO 3 • 5 H 2 0 . l - l ) , 1 ml. 1-1;
and a trace element solution, 1 ml. 1-1 were added. The trace dement solution contained (values
in mg. 1-1): E D T A . 2 H20, 1000; FeSO 4 • 7H20,
2500; ZnSO4.7 H20, 200; MnC12.4HzO, 500;
H3BO3, 50; COC12.2H20, 150; CuSO4.5H20,
150; NiC12.6H20, 25; (NH4)rMoTO24.4H20 ,
100; Na2Wo 4 • 2H20, 50.
To the autoclaved cold basal medium the following components were added from sterile stock
solutions (values in g. 1-1): NaHCO3, 2.5; Na2S.
9H20, 0.4; yeast extract, 0.05; vitamin solution, 1
ml. 1-1. The vitamin solution contained (values in
mg. 1-1): p-aminobenzoic acid, 40; D ( + ) biotin,
10; nicotinic acid, 100; Ca-D( + )pantothenate, 50;
pyridoxaminhydrochloride, 150; thiaminhydrochloride, 100; vitamin B12, 50.
161
For aerobic cultivation, KH2PO4, NaHCO3,
Na2S. 9H20 and resazurin were omitted from the
medium and a phosphate buffer was added after
autoclaving to a final concentration of 10 raM.
The pH of the media was adjusted to 6.8-7.2 by
addition of a small amount of 1 N HCI solution.
When stored under an atmosphere of 80% N 2 and
20% CO2, the redox potential remained below
- 1 1 0 mV and the pH remained constant.
Additions were made from sterile 0.5-1.0 M
stock solutions of carbon and energy sources to
obtain the required concentrations.
3.4. Isolation and cultivation
Samples taken from just below the narrow
(0.5-5.0 cm) greyish oxic top layer of the mud
were diluted in anaerobic liquid medium. Glucose
(5 mM) served as the sole carbon and energy
source. From the most dilute tubes in which visible
growth occurred, pure cultures were obtained via
dilutions in agar-solidified media, using strictly
anaerobic techniques.
Cultures were grown in screw-cap bottles, tubes
or chemostats under an atmosphere of 80% N 2
and 20% CO 2. The chemostats had a working
volume of 500 ml and were mixed with a magnetically driven stirring-bar. The temperature was kept
constant at 28°C. The head space of the fermenter
was flushed continuously (100-300 ml/min) with
a gas mixture of 80% N 2 and 20% CO 2. Traces of
oxygen were removed from this gas mixture by
leading it over copper turnings at a temperature of
150°C. The redox potential in the chemostat cultures was monitored continuously with a platinum
electrode. The exposed platinum surface was 0.16
cm2. The reference voltage was obtained from the
Ag-AgC1 reference cell of the pH-electrode
(Schott-Ger~te GmbH, F.R.G.) used to monitor
the pH of the culture. The redox potential was
adjusted by varying the extent of aeration, which
in turn was controlled by the air-flow through the
culture (0-3 1. min -1) and by the stirring speed.
3.5. Analytical procedures
Volatile and non-volatile short chain fatty acids
and alcohols were analyzed with a Packard 437 gas
chromatograph equipped with a flame ionization
detector, as described earlier [17]. Glucose was
measured enzymatically with glucose oxidase
(Boehringer Test Combination; glucose), sulfide
was analyzed by the method of Pachmayr [18] and
organic carbon in the interstitial water was determined with a Beckman Total Carbon Analyzer
(Model 915A). The organic carbon content of dry
sediment samples was determined according to the
'Kurmies' method, a wet combustion with bichromate [19]. Total nitrogen in dry sediment samples
was determined using the Kjeldahl titration method
[20]. Sulfur and thiosulfate were measured using
the methods described by Srrbo [21] and Thorpe
[22] respectively. H 2 was measured on a Pye Unicam 104 gas chromatograph equipped with a
katharometer [17]. Maximum oxygen consumption
rates (Q~X) of cell suspensions (at 28°C) were
measured polarographically with a YSI-Biological
Oxygen Monitor. Substrates used in these measurements were present at a concentration of 0.5
mM in cell suspensions washed twice in medium
without a carbon and energy source.
Radioactivity measurements of 14CO2 evolved
during oxidation of uniformly labeled [14C]acetate
in an oxygen-limited chemostat were made with a
Nuclear Chicago Corp, Mark II liquid scintillation
counter (Des Haines, IL). To 0.5 ml of 0.2 N
NaOH sample solutions was added 10 ml of
counting fluid (Opti-Fluor; Packard Instruments,
SA, Brussels). Counting was carried out for periods of 2 min.
Total oxygen consumption in the culture was
measured by comparing the O2-content of the air
entering and leaving the culture using a 2-channel
Servomex OA184 analyzer (Servomex Control Ltd.,
Crowborough, U.K.).
4. RESULTS
4.1. Major characteristics of the sampling site
Some characteristics of the brackish [23] sediment from which the samples (5-10 cm below the
surface) were taken are presented in Table 1.
The considerable quantities of total organic
carbon and of total carbohydrates present in this
sediment are not readily available for microbial
degradation. Incubation for 15 days at 25°C did
not significantly affect the carbon content (data
162
Table 1
Some characteristics of sediment measured at different depths.
Depth
(cm)
Total organic
carbon a
Total N a
pH
Fermentation
products
0
1790
92
7.14
2250
116
7.16
3490
159
7.35
2850
84
3090
146
N.D. b /
2810
135
N.D.
2030
99
N.D.
1380
64
N.D.
1260
57
N.D.
4
8
Lactate 1-2 p.M
12
16
Acetate
7.48 25-30 #M
Ethanol
15-25/~M
20
24
28
50
80
mmol.l- 1 Wet sediment.
b N.D., Not determined.
n o t presented). Similar findings were r e p o r t e d
earlier b y L a a n e [24].
Some typical f e r m e n t a t i o n i n t e r m e d i a t e s c o u l d
be d e t e c t e d at very low c o n c e n t r a t i o n s at various
d e p t h s ( T a b l e 1). T h e r e d o x p o t e n t i a l of this sedim e n t d r o p p e d g r a d u a l l y with d e p t h to - 2 0 0 m V
at 30 c m b e l o w the surface (Fig. 1). T h e d r o p in
r e d o x p o t e n t i a l was n o t m a r k e d l y affected b y the
season, b u t was steeper in the eastern p a r t o f the
estuary, where readily d e g r a d a b l e o r g a n i c m a t t e r
e n t e r e d the e s t u a r y t h r o u g h o u t the y e a r [16].
4.2, Isolation of glucose fermenting species
Isolations were d o n e in spring, summer, a n d
a u t u m n . In all cases, growth was o b s e r v e d in the
initial a n a e r o b i c liquid culture series u p to a dilution of 1 0 4 - 1 0 5, i m p l y i n g the p r e s e n c e of at least
104-105 g l u c o s e - f e r m e n t i n g b a c t e r i a p e r g wet
sediment. D i r e c t p l a t i n g on a e r o b i c a l l y i n c u b a t e d
glucose-agar m e d i u m resulted in similar counts~
A s e v i d e n c e d b y analysis of the culture fluids o f
these dilution series the m a i n f e r m e n t a t i o n p r o d ucts in the higher d i l u t i o n s were, w i t h o u t e x c e p -
tion: acetate ( 4 - 6 m M ) , e t h a n o l ( 2 - 3 m M ) , form a t e ( 7 - 9 m M ) , a n d succinate (0.3-0.7 m M ) . N o
H E was d e t e c t e d in a n y of these cultures. I n the
0
-
'
I
'
I
'
1
'
I
~
I~
!
10 E
20 CL
O3
¢:2
30 -l
I
,
*200
I
J
.100 *50
I
,
I
0 -50-100
Redox pofenfia[
,
I
-200
(mV)
Fig. 1. Redox profile of the sediment at 2 different locations in
the Ems-Dollard estuary during low tide. The steepest profile
(O-O) was found in the most eutrophicated south-eastern areas,
whereas at the chosen sampling location (Heringplaat) a
smoother pattern was observed ( i - I ) .
163
less dilute cultures (~< 102), abundant sulfide production was observed, and i n most cases no fermentation products other than CO 2 and some
acetate were detected.
On further examination of the various pure
cultures obtained by dilution in agar shakes it
appeared that all dilution series (15 in total) made
throughout a period of 1 year yielded facultatively
anaerobic bacteria as the dominant population in
the most dilute liquid cultures. In some of the
latter cultures, low numbers of strictly anaerobic
species were also present. Of the colonies counted
on aerobically incubated agar plates, obligate
aerobes accounted for only 10-20% of the total
plate counts.
Additional attempts were made to select for
glucose fermenting species in continuous cultures.
4 Distinctly different types of cultivation conditions were attempted: anaerobic, aerobic, oxygenlimited, and alternately aerobic-anaerobic. For all
isolation procedures in the chemostat samples were
used from the same location and depth as used
above for the dilution series. The enrichments were
run at dilution rates of 0.05 h - l - 0 . 1 h -~ and
continued for at least 10 volume changes. In all
but the aerobic cultures, complex mixed populations were obtained which were not characterized
in detail. In the aerobic cultures one species became strofigly dominant and no fermentation
products could be detected. Dilutions of these
enrichment cultures were made in anaerobic glucose-free medium from which glucose-agar plates
(incubated aerobically) and agar shakes (incubated
anaerobically) were inoculated. The numerically
dominant species were checked for aerobic and
anaerobic growth in liquid cultures. The results,
presented in Table 2, demonstrate that under all
the conditions tested facultative anaerobes could
become dominant. In order to show whether obligate aerobes could be isolated in this way, a
chemostat enrichment was set up with acetate as
the sole substrate and oxygen in excess. This time
an obligate aerobe indeed became dominant. Most
interestingly, this species could also grow on glucose.
4.3. Properties of one of the isolated facultative
anaerobes
One of the facultative (an)aerobes isolated via
direct dilution in anaerobic liquid culture series
was studied in more detail. This strain was a short,
motile rod (0.5-0.8 /am wide and 1.5-2.0 /~m
long). It exhibited a #max of 0.60-0.65 h -1 on
glucose under anaerobic conditions and a tt max of
0.75-0.80 h -1 under aerobic conditions. This
species grew optimally within a pH range of 6.3-7.9
and at sodium chloride concentrations of 0.2-2.0%.
The following compounds supported growth
Table 2
Summary of the results of different chemostat enrichments with glucose as growth-limiting substrate.
Culture conditions
Redox
potential
Dominant
population
% of
total
Major secondary
population
Dilution
rate ( h - 1)
-200
-210
- 230
+ 150
+ 230
-180
- 130
+ 300 ~ - 170
Anaerobic
Fac. anaerob.
Anaerobic
Fac. anaerob.
Fac. anaerob.
Fac. anaerob.
Fac. anaerob.
Fac. anaerob.
>
>
>
>
>
>
>
>
N.D. a
Anaerobic
Fac. anaerob.
N.D.
N.D.
Anaerobic
Anaerobic
N.D.
0.07
0.08
0.08
0.08
0.05
0.05
0.07
0.10
+ 310 ~ - 160
Fac. anaerob.
> 99
(my)
Anaerobic
Anaerobic
Anaerobic
Aerobic
Aerobic
O2-1imited
O2-1imited
Aerobic ~ Anaerobic
2h~2h
Aerobic ~ Anaerobic
6 h,,-~ 2 h
a N.D., Not determined.
95
60
90
99
99
70
80
99
' N.D.
0.10
164
Table 3
Table 4
Results of end product analysis of anaerobic cultures of Vibrio
HP1 after growth in batch culture and in steady state glucoselimited continuous culture (D = 0.08 h-1 ).
Maximum rate of oxygen consumption in the presence of
different substrates.
Products
(mmol/100 mmol glucose)
Batch
Continuous culture
D = 0.08 h- ]
Formate a
Acetate
Ethanol
Succinate b
Lactate
Cells
(mmol C/100 mmol glucose)
Carbon recovery
(including cells)
Redox balance O/P,
143.0
65.5
56.2
32.2
2.8
109.8
80.4
48.0
29.0
0.5
84
91
96%
0.94
97%
1.02
No H 2 w a s detected in any culture.
b Succinate was assumed to be formed via carboxylation of
phosphoenolpyruvate with exogenous CO2.
a
u n d e r a n a e r o b i c c o n d i t i o n s : glucose, g l u c o s a m i n e
N - a c e t y l - g l u c o s a m i n e gluconate, galactose, fructose, arabinose, maltose, mannitol, amylose, xylan
a n d inulin. U n d e r a e r o b i c conditions, some add i t i o n a l c o m p o u n d s were used: acetate, p r o p i o n a t e , b u t y r a t e , lactate, pyruvate, succinate,
glutamate, a s p a r t a t e , glycerol, e t h a n o l a n d chitin.
M e t h a n o l , formate, sorbitol, xylose, lactose, cellulose a n d x y l a n were also tested, b u t d i d n o t
s u p p o r t growth. F u m a r a t e r e d u c t i o n with l a c t a t e
as electron d o n o r d i d not occur u n d e r a n a e r o b i c
conditions. N i t r a t e was not r e d u c e d with a n y of
the s u b s t r a t e s tested.
T h e f e r m e n t a t i o n of glucose in b a t c h cultures
y i e l d e d formate, acetate, ethanol, succinate a n d
lactate. In c o n t i n u o u s culture u n d e r glucose-limiting c o n d i t i o n s the same end p r o d u c t s were f o r m e d
( T a b l e 3).
Based o n these p r o p e r t i e s this strain was tentatively identified as a Vibrio species [25-27], a n d
was p r o v i s i o n a l l y n a m e d Vibrio HP1.
4.4. Growth in the presence of limiting amounts of
oxygen
A n a e r o b i c a l l y g r o w n cultures of Vibrio HP1
were able to switch over to a e r o b i c m e t a b o l i s m
w i t h o u t a m e a s u r a b l e lag. In T a b l e 4 it can b e seen
that strictly a n a e r o b i c cultures still possessed a
These values were obtained with washed cell suspensions prepared from samples taken from various O2-1imitedsteady state
cultures. The millivolt data given in this table refer to the
steady state redox values measured in the chemostat cultures,
corresponding to those in Fig. 3. N.D., Not determined.
Substrate
Maximum rate of oxygen consumption
(/LI O2/h. mg dr. wt.)
-80mV
-50mV
-20mV
+190mV
1 Glucose
2 Formate
3 Ethanol
4 Succinate
5 Acetate
6 Lactate
108.9
52.3
17.9
78.4
47.9
34.8
254.0
54.0
24.3
86.4
86.2
43.2
227.6
59.8
6.9
55.9
46.3
27.0
152.7
55.5
17.0
38.6
54.9
11.0
Mixture 2-6
181.5
N.D.
118.8
115.7
glucose-oxidizing p o t e n t i a l of 70% of the p o t e n t i a l
o f the oxygen-sufficient cultures. A l s o the c a p a c i t y
to oxidize all of its o w n f e r m e n t a t i o n p r o d u c t s was
p r e s e n t in b o t h a n a e r o b i c a n d a e r a t e d cultures. If
this c a p a c i t y varied at all, it was greatest in the
a n a e r o b i c cultures, p a r t i c u l a r l y in the case of succ i n a t e a n d lactate oxidation. M o r e o v e r , it can be
seen from Fig. 2 t h a t u p o n s u d d e n t r a n s i t i o n f r o m
a n a e r o b i c to aerobic c o n d i t i o n s all f e r m e n t a t i o n
p r o d u c t s were r a p i d l y oxidized to c o m p l e t i o n , inc l u d i n g glucose, which was still c o n t i n u o u s l y fed
to the culture.
In the following e x p e r i m e n t the g r o w t h of this
facultative a n a e r o b e was s t u d i e d u n d e r c o n d i t i o n s
of a limited s u p p l y of oxygen. A series of c o n t i n u ous cultures was set up in which the air flow rate
t h r o u g h the culture a n d the stirring speed were
a d j u s t e d to o b t a i n the desired o x y g e n a t i o n o f the
culture. This was r e c o r d e d with a redox e l e c t r o d e
which allowed c o n t r o l l e d cultivation at oxygen
c o n c e n t r a t i o n s b e l o w the d e t e c t i o n level of conv e n t i o n a l oxygen electrodes. These cultures were
r u n at a dilution rate of 0.08 h - ] a n d a reservoir
glucose c o n c e n t r a t i o n of 10 raM. T h e results, summ a r i z e d in Fig. 3, i n d i c a t e d that with an increasing
o x y g e n s u p p l y (increasing Eh-values) the cell density increased a n d the c o n c e n t r a t i o n of fermentation p r o d u c t s decreased. A m a x i m u m value o f 525
I
I
11
I
I
l
10
9
8
E
7
6
•
g 6,
% 3
g
succ¢nafe
acetate
2
E
c_
0
I
I
l
I
I
1
2
3
~,
5
165
mg dry w t . / l was obtained when oxygen was in
excess and fermentation products were no longer
present in the culture. Only in these oxygen-sufficient cultures could oxygen be detected with a
lead-silver type O2-electrode.
In all of these cultures glucose was undetectahie, i.e., < 50 #M. Addition of more glucose to
the medium reservoir resulted in an increase in cell
density. Similarly, increased aeration resulted in
elevated cell densities and in a drop in fermentation product formation in all but the oxygen-sufficient steady-state cultures. Therefore, one must
conclude that these cultures grew under a dual
limitation of glucose and oxygen. The aerobic
6
Time ( h r s . )
Fig. 2. Growth of Vibrio H P I in continuous culture following a
transition from anaerobic to aerobic conditions at a dilution
rate of 0.08 h -~. Concentrations of fermentation products
following anaerobic to aerobic transition: succinate, (I-m);
acetate (O-O); formate, (A-A); ethanol, ( O - O ) .
'
'
'
'
i
.
.
.
I
.
.
.
.
.
t
A
i
i
2QO00
~50
500~
)
~I00
BO
60
3.0
IEO00
/
//
$
go
21)0
I formate
o
3
c~
10
9
2.o
1~000
8
8
7
1.0
~ooo
g 6
~s
acetate
3
2
I
0
succmate
ethanol
r
i
,
I
I
i
I
0
I
-100 -BO -60 -40 -20
0
20
40
Redox pofentiat
60 80 100 120 1/+0 160 180 200
(mY)
Fig. 3. Growth of Yibrio H P I in continuous Culture at a
dilution rate of 0.08 h - 1 limited by both oxygen and glucose at
various levels of oxygen supply. (A) Pereentag¢ glucose fermented, relative to total consumption ( O - O ) and c¢11 density
(~-O) at different levels of oxygen supply. (B) Fermentation
products detacted in the steady state cultures as a function of
the oxygen supply: succinate, (m-I); acetate, (Gb-O); formate,
(A-&); ethanol, ( O - O ) .
Time
(hrs }
Fig. 4. Accumulation o f 14CO2 as a result of the oxydation of
added [14C]acctat¢ (see text) to an oxygen and glucose-limited
chemostat culture operated at a dilution rate of 0.08 h - 1. The
t4CO2 evolving from the culture was trapped in 30 ml of 0.2 N
NaOH, and 0.5-ml aliquots of this solution were measured by
liquid scintillation, counting. The dashed line represents #mol
CO 2 present per ml NaOH solution calculated on the basis of
the measured increase in radioactivity (O-O; cpm 14CO2/ml
NaOH solution).
166
respiration of glucose was limited by the availability of oxygen whereas the fermentative metabolism
was restricted by the limiting supply of glucose.
The simultaneous occurrence of respiration and
fermentation was further confirmed by measurement of the actual oxygen consumption in the
culture (data not shown). It was also considered
possible that fermentation products produced in
these cultures were subsequently respired, thus
competing with glucose for the available oxygen.
This possibility was investigated, for acetate only,
in a steady-state culture grown under glucose and
oxygen limitation (Eh = - 5 0 mV) at a dilution
A
rate of 0.08 h-5. At zero time, 100/tl of uniformly
labeled [14C]acetate (0.17 ~tmol; 59 /tCi/~mol)
was injected into the culture and the ]4CO2 evolved
was trapped by passing the airstream from the
culture through a bottle containing 30 ml of 0.2N
NaOH. In Fig. 4 it can be seen that the amount of
CO 2, calculated from the 14C-radioactivity measured in the N a O H solution, increased linearly
over the first 24 h. This was not due to the
presence of [~4C]acetate in the N a O H solution, as
no acetate ( < 1 /~M) could be detected therein.
Given a steady-state acetate concentration in this
culture of 5.6 mM, it was calculated that at least
B
C
g
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2
g
140
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~o 120
L.J
i-
100
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E
,-
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ua
o
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.
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ANAEROB:IC
I
ANAEI~OBIC
.
~#ii
ANAEROBIC
I
02-
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300
I
LIMIT~
Fig. 5. Co-cultivation of Vibrio HP1 and Desulfooibrio HL21 in continuous culture under strictly anaerobic conditions and under
conditions of oxygen, lactate and glucose limitation. Product formation in steady state cultures (D = 0.05 h - l ) is presented together
with the redox potential measured in these cultures. Values for sulfide represent the sum of the quantities measured in the gas phase
and in the culture liquid. (A) Anaerobic pure culture of Vibrio HPI: glucose limited; reservoir concentration; Sr = 10 mM glucose. (B)
Anaerobic coculture of Vibrio HP1 and Desulfooibrio HL21: glucose limited; Sr=10 mM glucose. (C) As B, but Sr=10 mM
glucose + 10 mM lactate. (D) as C, but in the presence of limiting oxygen concentrations (see text).
167
2.8% of the m o u n t of acetate formed per unit of
time was further oxidized to CO2 and H20.
4.5. Co-cultivation of Vibrio HP1 and Desulfovibrio HL21 under oxygen-limiting conditions
The isolated facultative anaerobes coexist with
strictly anaerobic sulfate-reducing bacteria in the
estuarine mud (see also above). In the topmost
part of the anaerobic estuarine sediments, molecular oxygen will at times diffuse down to areas
where facultative anaerobes and sulfate reducers
may then compete for potential electron donors,
albeit with different electron acceptors (Oe and
SO2-, respectively). The feasibility of this situation was studied in mixed cultures of Vibrio HP1
and Desulfovibrio HL21, a sulfate reducer isolated
from the estuarine mud at some earlier date (see
section 3). Fig. 4 summarizes the results of experiments with continuous cultures of these species
grown on glucose alone and on glucose + lactate
as model substrates. Comparison of Figure 5A and
5B shows the ability of Desuifooibrio HL21 to
thrive on formate and ethanol produced by the
facultative anaerobe under strictly anaerobic conditions. In Fig. 5C it can be seen that if lactate is
included in the medium this substrate is also
oxidized completely to acetate by the sulfate reducer (Vibrio HP1 is unable to use lactate under
anaerobic conditions). When at this stage air was
introduced via a narrow needle (diameter 0.1 cm)
at a rate of 170 m l / h a new steady state could be
obtained with both species present (Fig. 5D). The
cell density had increased from 270 to 355 mg dry
wt./l. The number of facultative anaerobes had
increased from 2.6-109/ml to 3.7. 109/ml,
whereas the number of viable sulfate reducers had
not changed significantly (2.5-3.0. 109/ml) as indicated by plate- and agar-shake counts, respectively. Neither sulfur nor thiosulfate, possible
products of the chemical reaction of sulfide and
oxygen, could be detected.
When the rate of aeration was increased to such
an extent that oxygen was no longer limiting, the
sulfate reducers were washed out of the culture.
In subsequent experiments with fresh inocula
from the upper anoxic sediment layer enrichments
were obtained repeatedly of mixed cultures of
sulfate reducers and facultative anaerobes in con-
tinuous cultures grown on glucose under oxygenlimiting conditions.
5. DISCUSSION
The above results demonstrate the abundance
of facultative glucose metabolizing anaerobes in
the top layer of the anoxic mud at the chosen
location in the Ems-Dollardestuary. At the depth
at which these facultative anaerobes thrive (5-10
cm below the surface), the redox potential of the
pore water ranges from - 5 0 - + 50 inV. 02 will
diffuse down from the air during low tide and
oxygen supply to this sediment layer will be facilitated further by bioturbation [15] thus causing
partially oxidized conditions. Apparently, such
conditions strongly favour facultative anaerobes.
This notion is further supported by the outcome of
the chemostat enrichments of glucose-metabolizing
bacteria under conditions of a continuous limited
supply of oxygen. In such enrichments, facultative
anaerobes clearly had a selective advantage over
both strict anaerobes and aerobes.
It is not at all clear why these growth conditions
select so strongly for facultative anaerobic species,
but it may be due to the same principle encountered with other 'versatile' microbial species growing under mixed substrate limited conditions. Thus,
facultatively chemolithotrophic thiobacilli have
been shown to possess a competitive advantage
over more specialized autotrophic and heterotrophic species when growing mixotrophically, at
the expense of inorganic sulfur compounds and
organic substrates [28]. Chemostat enrichments
under such growth conditions have supported this
principle [29,30]. This principle may also apply
under strictly anaerobic conditions. The specialized Clostridium cochleareum, which grows only on
glutamate, glutamine and histidine, was outcompeted by the more versatile Clostridium
tetanomorphum, which grows on some other substrates, e.g. glucose, when glucose was supplied
together with glotamate in a carbon-limited chemostat [31]. For this principle of 'mixed physiology'
to offer any competitive advantage, facultative
anaerobes must be capable of respiring and fermenting at the same time in the presence of limit-
168
ing amounts of oxygen. This property has indeed
been found in some facultative anaerobes [32-35],
and has now been demonstrated once more for the
Vibrio sp. isolated in the course of this study. In
fact, this species seems extremely well-adapted to
growth under oxygen-limiting conditions. With a
fermentable substrate like glucose, Vibrio HP1 respires as much glucose as possible with the amount
of oxygen available, and ferments all glucose which
is left. Moreover, even during complete anaerobiosis the maximum respiring capacity for the various
substrates tested was not markedly depressed. This
property has been observed with some other
facultative anaerobes [34,35] and has at least two
interesting consequences. Firstly, while fermenting
glucose under oxygen-limiting conditions in pure
culture, this species has the potential to simultaneously respire its own fermentation products. Although we have shown only that this indeed occurred in the case of acetate, there is no reason
why the other products would not be used as well.
The ecological significance and possible advantage
of this property, though, is as yet difficult to
assess. In the natural environment accompanying
anaerobic and aerobic species will compete for
fermentation products, keeping their concentration
very low, and thus strongly reduce the possible
beneficial effect of their oxidation.
Probably a more significant result of the sustained high respiration potentials is the ability it
offers to switch over immediately from fermentation to aerobic growth (Fig. 2). This trait may add
considerably to the competitiveness of facultative
anaerobes in an environment with a fluctuating
oxygen supply. This notion is indeed supported by
the observation that these bacteria could be isolated selectively in chemostats run under a regime
of aerobic-anaerobic transitions. Whereas this
latter result may seem logical in view of the flexible metabolism of facultative anaerobes, it is
surprising to see that this physiological type of
bacteria could also gain dominance under completely aerobic conditions. Only with a non-fermentable substrate, like acetate, as the growthlimiting factor could an obligate aerobe be enriched for from the estuarine mud in continuous
culture. These results, plus the fact that only low
numbers of aerobes were found in enumerations
using aerobically incubated glucose-containing
agar plates, suggest that the upper anoxic sediment
layer constitutes an environment strongly selective
for sugar-metabolizing facultative anaerobes.
Although with respect to fermentable sugars
strict aerobes may well be outcompeted in such
environments, they have possibly become specialized in rapid growth on a very large spectrum of
less easily fermentable substrates. This property
has been found repeatedly with obligately aerobic
species and may well be responsible for their competitiveness in permanently aerobic environments
[361.
In the sediment layer from which we have isolated Vibrio HP1, facultative anaerobes must
coexist with strict anaerobes. Many of these strictly
anaerobic species have in fact been shown to
survive strongly oxygenated conditions [9-13].
Moreover, in some cases these species have been
shown to be metabolically active even under 'in
situ' conditions which on a macroscopic scale must
be considered aerobic. This has been explained by
assuming the presence of microhabitats providing
strictly anaerobic conditions [6,8], which may represent an important mechanism by which
anaerobes are able to extend their niches into
aerobic environments. Our present results suggest
that this is not the only possible way by which
sulfate reducers may remain metabolically active
under conditions of a continued supply of oxygen.
Apparently facultative anaerobes are able to maintain oxygen partial pressure so low that even in
completely mixed liquid cultures, free of particulate material, conditions are made anaerobic
(fig. 5). Clearly, these latter bacteria might represent a somewhat special case, as they produce
hydrogen sulfide, which itself could provide some
protection against the effects of oxygen, although
it has also been shown that the presence of sulfide
and other sulfhydryl group-containing compounds
may enhance oxygen sensitivity [11]. It is noteworthy that Pitt and Lee [37] reported enhanced
methane formation in small batch cultures of mixed
populations upon introduction of small amounts
of oxygen in the headspace of these cultures. This
effect may perhaps be explained by assuming that
oxygen stimulated the initial breakdown of complex polymeric substrates by (facultative) aerobes
169
present in these cultures. Also, in an anaerobic
digestor, methane production was reported in the
presence of measurable amounts of dissolved
oxygen [8].
In conclusion it may be emphasized that aerobic
and anaerobic metabolism are not at all mutual
exclusive. Not only are both physiological modes
compatible within a single organism, but strict
anaerobes can also coexist with facultative
anaerobes in aerated mixed cultures. It is more
than likely that the same principle may apply to
many naturally occurring interfaces between
aerobic and anaerobic habitats.
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