FEMS Microbiology Ecology 26 (1998) 171^179
Competition for oxygen and 3-chlorobenzoate between two
aerobic bacteria using di¡erent degradation pathways
Janneke Krooneman a; *, Edward R.B. Moore b , Jeroen C.L. van Velzen a ,
Rudolf A. Prins 1;a , Larry J. Forney a , Jan C. Gottschal a
b
a
Department of Microbiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Division of Microbiology, G.B.F., National Research Centre for Biotechnology, D-38124 Braunschweig, Germany
Received 9 October 1997; revised 24 March 1998 ; accepted 7 April 1998
Abstract
In nature a significant part of the microbial activity is concentrated at or near oxic/anoxic interfaces, where oxygen
concentrations are often low. Bacteria possessing different kinetic characteristics for oxygen and employing distinct metabolic
pathways for the degradation of (halo)aromatic substrates for which oxygen is needed as co-substrate may have to compete
with each other in such environments. In this study the competitiveness of Pseudomonas sp. strain A3 relative to Alcaligenes sp.
strain L6 was tested in batch and in continuous cultures. While both of these strains are able to metabolise 3-chlorobenzoate
(3CBA), the former was isolated under air saturating conditions and employs the catechol pathway, whereas the latter was
isolated under reduced partial pressures of oxygen and was capable of metabolising 3CBA via the gentisate pathway.
Competition experiments in batch culture resulted in pure cultures of Pseudomonas sp. strain A3 under air saturating
conditions. However, if reduced partial pressures of oxygen (2%) were used, Alcaligenes sp. strain L6 remained present in
substantial numbers after three transfers. Continuous culture experiments demonstrated that Alcaligenes sp. strain L6 was able
to outcompete Pseudomonas sp. strain A3 under oxygen- as well as under carbon-limiting conditions as long as the dilution rate
remained below 0.136 h31 (low oxygen) and below 0.178 h31 (high oxygen). These results support the hypothesis that
organisms metabolising chlorobenzoate via the gentisate pathway may play a significant role in natural ecosystems where
xenobiotic compounds and naturally produced aromatics occur at very low concentrations and in combination with limiting
oxygen tensions. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.
Keywords : Kinetic characteristics; Limiting conditions ; Pseudomonas ; Alcaligenes
1. Introduction
* Corresponding author.
Tel.: +31 (50) 3632191; Fax: +31 (50) 3632154;
E-mail: [email protected]
1
Much to our sorrow Prof. Dr. R.A. Prins passed away on
26 February 1997.
Chlorinated benzoates predominantly enter the environment because they have been used as pesticides,
e.g., 2,5-dichloro-3-aminobenzoic acid or 2,3,6-trichlorobenzoic acid, or because they are produced
as metabolites during the partial degradation of
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 3 3 - 6
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J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179
chlorinated aromatic compounds such as polychlorinated biphenyls, chlorinated phenolic compounds
and DTT [1^4]. Additionally, the natural production
of chlorinated benzoates by fungi also contributes to
their presence in the environment [5,6]. 3-Chlorobenzoate (3CBA) has been widely used as a model substrate in studies of chlorobenzoate biodegradation
[7,8]. In general, 3CBA is readily degraded aerobically through the action of dioxygenases via (chloro)catechol as the main intermediate [7^10].
Many (halo)aromatic compounds, including
3CBA, can be degraded rather easily under oxic conditions. Molecular oxygen plays a dual role in such
aerobic degradation pathways, wherein it serves as a
terminal electron acceptor in the respiratory electron
transfer chain, and also as a co-substrate in initial
oxygenation reactions wherein oxygen atoms are incorporated into the aromatic ring [11,12]. Relatively
high half saturation constants for oxygen are found
for dioxygenases (24^2000 WM), indicating that conversion rates of the (halo)aromatic compounds will
be strongly reduced at low oxygen concentrations
[13^17]. This raises the question whether these pathways have a signi¢cant role at or near oxic/anoxic
interfaces, where oxygen concentrations are often
low [18,19]. The degradation of 3CBA under reduced
partial pressures of oxygen has been shown to result
in toxic levels of chlorocatechols in cultures of Pseudomonas sp. B13 [20]. Haller and Finn [13] reported
a decreased respiration of 3CBA by Pseudomonas sp.
H1 and Pseudomonas sp. H2 at oxygen concentrations below 20^48 WM. In fact, Pseudomonas sp. H1
was unable to grow on 3CBA in poorly aerated media. Hence, it is to be expected that aerobic metabolism of (halo)aromatic compounds will be restricted
by the availability of oxygen in deeper layers of the
soil and in groundwater where levels of dissolved
oxygen are usually much lower than at the surface.
Recently, a bacterium identi¢ed as an Alcaligenes
sp. was enriched and isolated on 3CBA as the sole
growth substrate under low partial pressures of oxygen [21]. It was shown that this organism does not
degrade 3CBA via chlorocatechol but rather via the
gentisate or the protocatechuate pathway (Fig. 1).
Furthermore, this Alcaligenes sp. strain L6 possessed
a relatively high a¤nity for oxygen (11^30 ml min31
mg protein31 ). These observations resemble those of
Olsen et al. [22] who isolated bacteria on toluene
Fig. 1. Various pathways for the degradation of 3-chlorobenzoate
via (chloro)catechol, gentisate and protocatechuate.
from hypoxic petroleum aquifers. These organisms
compensated for limited availabilities of oxygen by
the production and use of catechol-dioxygenases
with improved oxygen a¤nities [23]. Similarly, the
half-saturation constant for oxygen during the
growth of a Mycobacterium sp. on pyrene in a fermentor was also relatively low (5.9 WM). Yet, at oxygen concentrations below 3.4 WM, growth was two
times slower than expected from the kinetic data and
was probably due to the limited activity of an oxygenase needed for pyrene degradation [16].
This illustrates the di¤culty of predicting the
growth of organisms under various conditions based
solely on the kinetics of substrate conversion. The
outcome of competition experiments between haloaromatics-utilising aerobes is likely to be more relevant
for predicting growth under nutrient-limiting conditions. In natural environments, microbes degrading
3CBA must compete for both the aromatic substrate
and for oxygen, since oxygen is required for ring
¢ssion and may be required for the initial conversion
reactions. Organisms with the highest a¤nity for a
particular substrate will win the competition as long
as no other interfering interactions occur and no
toxic end products are produced. This a¤nity can
be calculated from the slope of the W vs. S relationship at low substrate concentrations of the individual
species. This holds even for the simultaneous limitation of 3CBA and oxygen, if double substrate
Monod-kinetics are assumed [24^28].
In this report, we describe the physiological characteristics of a Pseudomonas sp., isolated on 3CBA
under air-saturating conditions. The data on the ki-
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J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179
netic characteristics for oxygen and 3CBA were then
compared with those of Alcaligenes sp. strain L6,
which had been previously isolated using low oxygen
concentrations [21]. Competition experiments between the Pseudomonas sp. and Alcaligenes sp.
were performed under oxygen-limiting and 3CBAlimiting conditions at various growth rates to gain
insight into which type of organism or metabolic
route may play a more signi¢cant role in situ. The
experimental data are compared with the outcome
predicted based on the kinetic parameters.
2. Materials and methods
2.1. Media and growth conditions
The medium used for the cultivation of strain A3
and strain L6 was a low-chloride minimal medium
(LMM-medium) described earlier by Gerritse et al.
[29]. Yeast extract (10 mg l31 ) was added to the
medium before autoclaving. Vitamins (1 ml l31 )
[30], 25 mM potassium ammonium phosphate bu¡er,
pH 7 (from autoclaved 1 M stock solution), and
substrate were added after autoclaving. Cultures
were incubated at 30³C in the dark under a 20%
O2 atmosphere in a rotary incubator.
Batch cultures, with a reduced oxygen tension,
were grown in 250-ml serum bottles with butyl rubber stoppers. Sterile air was added to the nitrogen
gas phase to a ¢nal concentration of 2% oxygen (24
WM). The oxygen concentration in the liquid phase
was kept equal to the gas-phase concentration by
incubating in a rotary incubator at 150 rpm. Stock
solutions of (chloro)benzoates (0.1 M) were added to
make a ¢nal concentration of 0.7 mM. The ratio
between the gas phase volume and the volume of
the liquid phase was 6.5 to ensure that the oxygen
added was enough for total consumption of the substrate. Batch cultures, both in pure and mixed cultures, were transferred after complete depletion of
the substrate.
Continuous culture experiments were done in a
chemostat with a culture vessel volume of 500 ml.
The oxygen concentration in the culture liquid was
continuously monitored using a polarographic electrode (Ingold, Urdorf, Switzerland) and regulated
automatically by coupling the stirring rate to the
173
O2 -monitor. The 3CBA concentration in the medium
reservoir was 2.5 mM. The pH was maintained at a
constant value of 7.0 by the automatic addition of
KOH. Kinetic parameters in pure cultures were
measured after at least ¢ve volume changes and a
steady state was reached. During competition experiments in continuous cultures, at least ten volume
changes were performed before the dominant strain
was characterised.
Since metabolic characteristics for degradation of
some aromatic compounds are known to be located
on transferable genetic elements (plasmids) [7,8]
mixed cultures were routinely checked to determine
if catabolic gene transfer occurred between Pseudomonas sp. strain A3 and Alcaligenes sp. strain L6.
This was done by determining if either strain had
acquired the ability to metabolise catechol (representing Pseudomonas sp. strain A3) and protocatechuate (representing Alcaligenes sp. strain L6).
2.2. Chlorobenzoate-degrading bacteria
Pseudomonas sp. strain A3 is a motile, polar,
mono£agellated Gram-negative rod, able to grow
on 3CBA as sole source of carbon and energy. The
organism was maintained and routinely cultivated
with 3CBA as the sole substrate. The sequence of
the 16S rDNA gene was determined and compared
with sequences available in the RDP and EMBL
databases [31,32]. Strain A3 clustered with species
of the genus Pseudomonas (sensu stricto) [33,34]
and most closely to P. citronellolis (99.5% sequence
similarity). Based upon these data, strain A3 was
recognised as a species of the genus Pseudomonas.
The sequence for the 16S rRNA gene of strain A3
has been deposited with the EMBL under the accession number Y13246. The isolation procedure, identi¢cation, and characterisation of Alcaligenes sp.
strain L6 have been described earlier by Krooneman
et al. [21].
2.3. Analytical procedures
Growth was monitored by measuring turbidity at
433 nm. Cell carbon was analysed with a total organic carbon analyser (Shimadzu TC-500) using
biphthalate as a standard. The method of Lowry
[35] was used to measure protein concentrations of
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J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179
cell suspensions. Bovine serum albumin was used as
a standard. Protein concentrations in cell-free extracts were detected with the method according to
Bradford [36]. Chloride measurements were done
colorimetrically using the method of Bergmann and
Sanik [37] and NaCl was used as a standard. Concentrations of benzoate and chlorinated benzoates
were detected by gas chromatography as described
by Gerritse and Gottschal [29] after methylation with
methanol and extraction with chloroform using 2bromobenzoate as an internal standard.
2.4. Oxygen uptake rates
Cells of exponentially growing cultures were centrifuged (10 min, 4³C, 11 000Ug) and the pellet was
washed twice in LMM-bu¡er (pH 7) which contained 25 mM potassium ammonium phosphate
bu¡er, 0.1 g l31 MgSO4 W7H2 O and 0.05 g l31 Ca(NO3 )2 W4H2 O. The cell pellets were resuspended in
LMM-bu¡er and stored on ice until use within 2 h.
An oxygen YSI-type polarographic electrode in a
biological oxygen monitor was used to measure respiration rates. Test substrates were prepared as
100-mM stock solutions and added to the cell
suspensions to ¢nal concentrations of 1 mM. Apparent Km -values for O2 were obtained from both Direct Linear and Eadie Hofstee plots. Half-saturation
constants for the growth substrate 3CBA were obtained by measuring respiration rates at air-saturating conditions and various concentrations of 3CBA
[27,28].
2.5. Enzyme assays
Cell free extracts were obtained from exponentially growing cells. Cell suspensions were washed
twice in 25 mM ammonium phosphate bu¡er, pH
7 and concentrated 25-fold by centrifugation (10
min, 4³C, 11 000Ug). Crude cell extracts were obtained by using a French pressure cell (3 times at
1000 psi). Cell debris was removed by centrifugation
(11 000Ug, 10 min, 4³C) and the supernatants were
stored on ice until use within 2 h. Catechol dioxygenase activity was measured according to Dorn and
Knackmuss [20] with catechol as the substrate. Gentisate dioxygenase activity was detected with gentisate as the substrate [38] and protocatechuate dioxygenase activities were detected with protocatechuate
as the substrate as described earlier by Stanier and
Ingraham [39].
3. Results
3.1. Isolation and characterisation of strains A3 and
L6
The objective of this study was to determine the
outcome of competition experiments between Alcaligenes sp. strain L6 and Pseudomonas sp. strain A3
that di¡er in their a¤nity for O2 when 3CBA was
the sole carbon source. Pseudomonas sp. strain A3
was isolated under air-saturating conditions in the
presence of 3CBA and benzoate from a mixture of
sewage sludge and soil polluted with pesticides. Pseudomonas sp. strain A3 was able to grow on 3CBA as
the sole source of carbon and energy with a maximum speci¢c growth rate of 0.27 h31 and a molar
growth yield of 20 g of cell carbon per mol of 3CBA
utilised. Stoichiometric release of chloride from
3CBA was measured during growth. Oxygen uptake
rates by washed cell suspensions using intermediates
of known metabolic pathways for 3CBA degradation
Table 1
Maximum oxygen uptake rates of batch- and continuous grown cells of Pseudomonas sp. strain A3 under fully aerobic conditions
Test substrate
3CBA
Benzoate
Hydroxybenzoatesa
Catechol
Maximum oxygen uptake rates (nmol min31 mg protein31 ) of cells pregrown on :
3CBA (batch culture)
3CBA (continuous culture)
BA (batch culture)
55.3
508
0
750
82
290
0
1018
46.2
359
0
494
a
Hydroxybenzoates tested: 3-hydroxybenzoate, 4-hydroxybenzoate, 3,4-dihydroxybenzoate (protocatechuate), and 2,5-dihydroxybenzoate
(gentisate).
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J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179
175
Table 2
Enzyme activities in cell free extracts of Pseudomonas sp. strain A3 grown on 3CBA, benzoate or protocatechuate
Growth substrate
3CBA
Benzoate
Protocatechuate
a
Speci¢c activity (nmol min31 mg protein31 ) of :
Catechol dioxygenase
Gentisate dioxygenase
Protocatechuate dioxygenase
1750
450
22
0
0
0
57
nda
3295
nd=not determined.
indicated that the catechol pathway was the predominant route used by Pseudomonas sp. strain A3 during the degradation of 3CBA and benzoate (Table
1). None of the intermediates from the gentisate (3hydroxybenzoate and 2,5-dihydroxybenzoate) or
protocatechuate (4-hydroxybenzoate and 3,4-dihydroxybenzoate) pathways were respired. 3CBA- and
benzoate-grown cells possessed high catechol-1,2-dioxygenase activities, low protocatechuate 3,4-dioxygenase activities, and no gentisate dioxygenase activity, indicating that the (chloro)catechol pathway was
used for the metabolic utilisation of 3CBA and benzoate. By contrast, protocatechuate-grown cells possessed low catechol-1,2-dioxygenase activity, high
protocatechuate-3,4-dioxygenase activity, and no
gentisate dioxygenase activity (Table 2). Alcaligenes
sp. strain L6 was isolated on 3CBA as the sole
growth substrate under low partial pressures of oxygen. It was shown that this organism does not degrade 3CBA via chlorocatechol but rather via the
gentisate or the protocatechuate pathway [21]. Furthermore, this Alcaligenes sp. strain L6 possessed
relatively high a¤nities for oxygen (11^30 ml
min31 mg protein31 ) [21].
3.2. Substrate kinetics of Pseudomonas sp. strain A3
and Alcaligenes sp. strain L6
The kinetic parameters for oxygen consumption
(Qmax
O2 , apparent Km values, and oxygen a¤nity
(Qmax
O2 =Km ) were determined using washed cell suspensions of strain A3 or strain L6 that had been
grown in continuous culture with di¡erent steady
state concentrations of oxygen in the culture medium. Steady state cultures of strain A3 were grown
on 3CBA, with varying concentrations of oxygen in
the culture medium. With decreasing O2 concentravalues increased with an apparent
tions the Qmax
O2
decrease in Km values (Table 3). At low oxygen concentrations (v10 WM O2 ) cultures of Pseudomonas
sp. strain A3 grew at a speci¢c growth rate that was
lower than the dilution rate (0.01 h31 ). The cells
aggregated, the culture liquid turned brownish, and
the cells were washed out from the chemostat vessel.
Table 3
Apparent Km -values for O2 , maximum oxygen uptake rates on 3CBA, and oxygen a¤nities of washed cells of Pseudomonas sp. strain A3
and Alcaligenes sp. strain L6 grown in continuous culture on 3CBA with di¡erent concentrations of oxygen in the culture liquid (Sr =
2.5 mM 3CBA, D = 0.025 h31 )
Organism
O2 (WM)
Km (WM)
Qmax
O2
(nmol min31 mg protein31 )
A¤nity (Qmax
O2 =Km )
(ml min31 mg protein31 )
Alcaligenes sp. strain L6a
143
24
0.1
17 (1.3)c
7 (1.1)
8 (0.9)
187
238
240
11
34
30
Pseudomonas sp. strain A3
143
60
24
10
24 (2.2)
15 (1.3)
16 (1.1)
^b
82
99
133
^
a
Data taken from Krooneman et al. (1996).
b
Not determined, no growth possible of strain A3 at oxygen concentrations 6 10 WM O2 .
c
Mean values out of 10 measurements are shown and the standard deviation is shown in parentheses.
FEMSEC 919 2-7-98
3.4
6.6
8.3
^
176
J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179
Incubation of the brownish-coloured culture under
oxic conditions did not result in increasing cell densities, neither with additional substrate, nor with the
addition of freshly grown cells.
With respect to the substrate kinetics for oxygen
uptake, Alcaligenes sp. strain L6 demonstrated the
same general pattern as Pseudomonas sp. strain A3:
an increase in Qmax
O2 with a corresponding decrease in
Km values with decreasing oxygen concentrations in
the culture liquid, which resulted in increased overall
a¤nities for oxygen (see [21]). Pseudomonas sp.
strain A3 possessed an apparent Km value for
3CBA of 200 WM.
3.3. Competition experiments in batch culture
Competition experiments between Alcaligenes sp.
strain L6 and Pseudomonas sp. strain A3 were carried out in batch culture under air-saturating conditions and at low partial pressures of oxygen using
3CBA as the growth substrate. By measuring respiration rates on catechol or gentisate and protocatechuate it was possible to determine the proportion
of strains A3 and L6. Under air-saturating conditions Pseudomonas sp. strain A3 outcompeted Alcaligenes sp. strain L6 after three transfers (made after
depletion of the substrate). There was no respiratory
activity with protocatechuate or gentisate which indicated that strain L6 was absent. However, at low
oxygen tensions (2% O2 ) both Pseudomonas sp.
strain A3 and Alcaligenes sp. strain L6 were present
after three transfers. This conclusion is based on the
observation that the respiration rate was 525 nmol
min31 mg protein31 with catechol and 15 nmol
min31 mg protein31 with protocatechuate. These
rates were 70% and 15% of the oxygen uptake rates
measured in pure cultures for Pseudomonas sp. strain
A3 and Alcaligenes sp. strain L6, respectively.
the kinetic characteristics for the metabolism of oxygen and 3-chlorobenzoate may allow one to predict
which strain would dominate under given conditions.
The dominant strain of the culture was monitored by
measuring the oxygen uptake rate using catechol and
protocatechuate as substrates. Absence of catechol
respiration activity inferred that Pseudomonas sp.
strain A3 was absent from the culture. The opposite,
which is no respiratory activity of protocatechuate,
inferred the absence of Alcaligenes sp. strain L6. Stable respiration rates of both protocatechuate and
catechol during ten volume changes re£ected coexistence of both strains.
With a high concentration of oxygen in the culture
(143 WM) Alcaligenes sp. strain L6 outcompeted
Pseudomonas sp. strain A3 at dilution rates as high
as 0.175 h31 as indicated by no detectable respiration
of catechol. Coexistence between the two strains occurred at a higher dilution rate of 0.186 h31 , and
Pseudomonas sp. strain A3 outcompeted Alcaligenes
sp. strain L6 at rates above 0.186 h31 . However, at
the lower oxygen concentration (24 WM of O2 in the
culture liquid) the Alcaligenes sp. strain L6 dominated Pseudomonas sp. strain A3 only at low dilution
rates (0.1^0.130 h31 ). At the lowest oxygen concentrations (v10 WM), Alcaligenes sp. strain L6 outcompeted Pseudomonas sp. strain A3, since the Pseudomonas could not grow under these conditions
(Table 4).
The experimental data demonstrated that stable
Table 4
Dominant strains in continuous culture with 3CBA as the growth
substrate at di¡erent dilution rates with 143 WM O2 , 24 WM O2 ,
or 10 WM O2 in the culture liquid
O2 (WM)
Dilution rate (h31 )
Dominant strain
143
0.136
0.143
0.154
0.175
0.186
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6 +
Pseudomonas sp. strain A3
Pseudomonas sp. strain A3
Pseudomonas sp. strain A3
Pseudomonas sp. strain A3
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6
Alcaligenes sp. strain L6
3.4. Competition experiments in continuous culture
In order to manipulate the environmental conditions such as the O2 concentration and the growth
rate more precisely, competition experiments between Alcaligenes sp. strain L6 and Pseudomonas
sp. strain A3 were also done in continuous cultures.
Since both oxygen and 3-chlorobenzoate are required and used for the growth of these organisms,
24
10
6 0.1
FEMSEC 919 2-7-98
0.188
0.188
0.154
0.130
0.100
0.025
0.010
0.025
J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179
Fig. 2. The 3-chlorobenzoate concentration and the oxygen
concentration as a function of the growth rate according to the
double substrate Monod-equation
W ˆ Wmax …3CBAO2 †=……Km;3CBA ‡ 3CBA†…Km;O2 ‡ O2 ††
in which Km…3CBA† is 200 WM for strain A3 and 30 WM for strain
L6, Km…O2 † is 20 WM for Pseudomonas sp. strain A3 and 10 WM
for Alcaligenes sp. strain L6. The grey area represents Alcaligenes
sp. strain L6 and the white area represents Pseudomonas sp.
strain A3.
coexistence at high oxygen levels occurred between
dilution rates of 0.175 and 0.186 h31 and at low
oxygen concentrations occurred between dilution
rates of 0.130 and 0.154 h31 . These experimental
results ¢t the theoretical predictions using the equation for double substrate Monod-kinetics, assuming
a Km…3CBA† of 200 WM for strain A3 and 30 WM for
strain L6 and a Km…O2 † of 20 WM for strain A3 and 10
WM for strain L6 (data not shown).
4. Discussion
The aerobic microbial degradation of 3CBA can
proceed via a variety of metabolic pathways. In most
bacteria isolated and described so far, 3CBA is degraded via chlorocatechol under aerobic conditions
[7,8]. Alternative degradation pathways via gentisate
or protocatechuate as intermediates have been reported previously for a few Alcaligenes spp.
[21,40,41]. Since oxygen serves as a co-substrate for
3CBA degradation, aerobic metabolism in natural
habitats may be restricted by low substrate concentrations or O2 -limited conditions. The ecological
relevance of the metabolic utilisation of chlorobenzoate via the (chloro)catechol pathway and the heterotrophic metabolism of 3CBA via one of the alter-
177
native pathways can be tested by studying the
competition between these di¡erent groups of bacteria at low oxygen and low 3CBA concentrations.
Pseudomonas sp. strain A3 was unable to grow on
3CBA under oxygen-limiting conditions in a chemostat. Calculation of the growth rate of Pseudomonas
sp. strain A3 at low oxygen concentrations (10 WM)
in the presence of 2 mM 3CBA led to the prediction
that strain A3 should still be able to grow at a
growth rate of approximately 0.014 h31 . However,
even at a dilution rate of 0.01 h31 , Pseudomonas sp.
strain A3 washed out from the culture vessel, possibly caused by limitation of oxygenase activity which
is necessary for 3CBA degradation. Accumulation of
chlorocatechols may be enhanced at low oxygen concentrations due to the relatively low oxygen a¤nities
of catechol dioxygenases and then could undergo
oxidative polymerisation to form polychlorocatechol
[42]. The observed brownish colour of the medium
during growth of Pseudomonas sp. strain A3 at low
oxygen concentrations points towards oxidative polymerisation of chlorinated catechols, as shown before for other Pseudomonas spp. [13,42,43]. Polymerisation of the chlorocatechols may reduce the
concentration of chlorocatechol available for the
growth of Pseudomonas sp. strain A3 and they also
appear toxic as indicated by the inability of A3 to
grow in its own supernatant once a brownish colour
developed.
The growth rates of Alcaligenes sp. strain L6 and
Pseudomonas sp. strain A3 calculated at various
3CBA and oxygen concentrations can be used to
predict which organism may be expected to predominate under various oxygen and chlorobenzoate concentrations. When the speci¢c growth rates were
plotted vs. 3CBA and oxygen concentrations, it can
be seen that at low oxygen and low 3CBA concentrations Alcaligenes sp. strain L6 will outcompete
Pseudomonas sp. strain A3 (Fig. 2). However, this
is true only if the growth rate remains below 0.136
h31 at low oxygen concentrations (excess of 3CBA)
and below 0.178 h31 at low 3CBA concentrations
(excess of oxygen). In batch cultures, bacteria are
able to grow at maximum growth rate, because all
substrates are initially present in excess. Competition
experiments in batch culture under air-saturating
conditions resulted in a pure culture of Pseudomonas
sp. strain A3 after three transfers indicating that
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J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179
Pseudomonas sp. strain A3 was more competitive
than Alcaligenes sp. strain L6 at high growth rates.
Even at lower oxygen concentrations (2% O2 ), Pseudomonas sp. strain A3 dominated the mixed batch
culture after three transfers. However, competition
experiments performed in a chemostat showed
clearly that, at high oxygen concentrations (143
WM) and 3CBA limitation, Alcaligenes sp. strain L6
outcompeted Pseudomonas sp. strain A3 so long as
the dilution rate was kept below 0.186 h31 , whereas
at higher dilution rates, pure cultures of Pseudomonas sp. strain A3 were obtained. At low dilution
rates (0.1^0.13 h31 ) and a low oxygen concentration
(24 WM), Alcaligenes sp. strain L6 outcompeted
Pseudomonas sp. strain A3. Higher dilution rates resulted in the opposite outcome. There were no apparent toxicity problems caused by chlorocatechols
during O2 -limited growth.
In natural habitats, low growth rates are the rule
rather than the exception, due to growth-limiting
concentrations of nutrients, including carbon, oxygen and other factors. An important ¢nding of the
present study for the role of chloroaromatics-degrading bacteria in natural ecosystems is that organisms
similar to Alcaligenes sp. strain L6, may play a much
more active and signi¢cant role than hitherto
thought. These bacteria, degrading 3CBA via the
gentisate pathway are characterised by a relatively
low Wmax and Km value for oxygen and chlorobenzoate. Organisms that possess the gentisate pathway
may even be characteristic of environments with low
concentrations of O2 and (halo)aromatic compounds, since they appear to possess higher substrate
a¤nities. This is particularly important in view of
the fact that most of our current knowledge on the
role of aerobic chlorobenzoate-degrading bacteria is
based on the type of bacteria that use the catechol
pathway, characterised by relatively high Wmax and
high Km values.
Acknowledgments
We thank Teresa M.D. Pedro Gomes and Angelika Arnscheidt for their technical assistance, and
Gert-Jan Euverink for his assistance in creating
Fig. 2. This investigation was ¢nancially supported
by The National Institute of Public Health and En-
vironmental Protection, The Netherlands. In addition, this project was supported by the Human Capital Mobility Network Programme of the European
Community for the study of microbial diversity
(Contract CHRX-CT93-0194).
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