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Competition for oxygen and 3-chlorobenzoate between two aerobic bacteria using different degradation pathways
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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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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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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
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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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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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Table 2 Enzyme activities in cell free extracts of Pseudomonas sp. strain A3 grown on 3CBA, benzoate or protocatechuate Growth substrate
Speci¢c activity (nmol min31 mg protein31 ) of : Catechol dioxygenase
Gentisate dioxygenase
Protocatechuate dioxygenase
3CBA Benzoate Protocatechuate
1750 450 22
0 0 0
57 nda 3295
a
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 concentrations the Qmax values increased with an apparent 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.
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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
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0.188 0.188 0.154 0.130 0.100 0.025 0.010 0.025
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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 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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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).
References [1] Swanson, C.R. (1969) The benzoic acid herbicides. In: Degradation of Herbicides (Kearny and Kaufman, Eds.), pp. 299^ 320. Marcel Dekker, New York, NY. [2] Safe, S.H. (1984) Microbial degradation of poly-chlorinated biphenyls. In: Microbial Degradation of Organic Compounds (Gibson, D.T., Ed.). Marcel Dekker, New York, NY. [3] Abramowitz, D.A. (1990) Aerobic and anaerobic biodegradation of PCBs : a review. Crit. Rev. Biotechnol. 10, 241^251. [4] Londry, K.L. and Fedorak, P.M. (1993) Fluorophenols and 3£uorobenzoate in phenol-degrading methanogenic cultures. Arch. Microbiol. 160, 137^143. [5] Gribble, G.W. (1994) The natural production of chlorinated compounds. Environ. Sci. Technol. 28, 310^319. [6] Swarts, H.J., Verhagen, F.J.M., Field, J.A. and Wijnberg, J.B.P.A. (1996) Novel chlorometabolites produced by Bjerkandera species. Phytochemistry (Oxford) 42, 1699^1701. [7] Haëggblom, M. (1990) Mechanisms of bacterial degradation and transformation of chlorinated monoaromatic compounds. J. Basic Microbiol. 30, 115^141. [8] Haëggblom, M.M. (1992) Microbial breakdown of halogenated aromatic pesticides and related compounds. FEMS Microbiol. Rev. 103, 29^72. [9] Reineke, W. and Knackmuss, H.J. (1988) Microbial degradation of haloaromatics. Annu. Rev. Microbiol. 42, 263^287. [10] Chaudry, G.R. and Chapalamadugu, S. (1991) Biodegradation of halogenated organic compounds. Microbiol. Rev. 55, 59^79. [11] Fetzner, S. and Lingens, F. (1994) Bacterial dehalogenases : biochemistry, genetics and biotechnological applications. Microbiol. Rev. 58, 641^685. [12] Fukuda, M. (1993) Diversity of chloroaromatic oxygenases. Curr. Op. Biotech. 4, 339^343. [13] Haller, H.D. and Finn, R.K. (1979) Biodegradation of 3chlorobenzoate and formation of black colour in the presence and absence of benzoate. Eur. J. Appl. Microbiol. Biotechnol. 8, 191^205. [14] Shaler, T.A. and Klecíka, G.M. (1986) E¡ects of dissolved oxygen concentration on biodegradation of 2,4-dichlorophenoxyacetic acid. Appl. Environ. Microbiol. 51, 950^955. [15] Viliesid, F. and Lilly, M.D. (1992) In£uence of dissolved oxygen tension on the synthesis of catechol 1,2-dioxygenase by Pseudomonas putida. Enzyme Microbiol. Technol. 14, 561^ 565. [16] Fritzsche, C. (1994) Degradation of pyrene at low de¢ned oxygen concentrations by a Mycobacterium sp. Appl. Environ. Microbiol. 60, 1687^1689.
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J. Krooneman et al. / FEMS Microbiology Ecology 26 (1998) 171^179 [17] Van der Woude, B.J., Gottschal, J.C. and Prins, R.A. (1995) Degradation of 2,5-dichlorobenzoic acid by Pseudomonas aeruginosa JB2 at low oxygen tensions. Biodegradation 6, 39^46. [18] Wimpenny, J.W.T. (1981) Spatial order in microbial ecosystems. Biol. Rev. 56, 295^342. [19] Koch, A.L. (1992) Di¡usion : the crucial process in many aspects of the biology of bacteria. Adv. Microbiol. Ecol. 11, 37^ 70. [20] Dorn, E. and Knackmuss, H.J. (1978) Chemical structure and biodegradability of halogenated aromatic compounds. Two catechol 1,2-dioxygenases from a 3-chlorobenzoate-grown pseudomonad. Biochem. J. 174, 73^84. [21] Krooneman, J., Wieringa, E.B.A., Moore, E.R.B., Gerritse, J., Prins, R.A. and Gottschal, J.C. (1996) Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols. Appl. Environ. Microbiol. 62, 2427^2434. [22] Olsen, R.H., Mikesell, M.D. and Kukor, J.J. (1994) Enumeration and characterisation of BTEX-degrading bacteria from hypoxic environments functional with mixed electron acceptors. Res. Microbiol. 145, 47^49. [23] Kukor, J.J. and Olsen, R.H. (1996) Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments. Appl. Environ. Microbiol. 62, 1728^1740. [24] Harder, W. and Dijkhuizen, L. (1982) Strategies of mixed substrate utilization in microorganisms. Philos. Trans. R. Soc. Lond. Ser. B 297, 459^480. [25] Veldkamp, H. (1977) Ecological studies with the chemostat. Adv. Microb. Ecol. 1, 59^94. [26] Gottschal, J.C. (1985) Some re£ections on microbial competitiveness among heterotrophic bacteria. Antonie van Leeuwenhoek 51, 473^494. [27] Button, D.K. (1991) Biochemical basis for whole-cell uptake kinetics: speci¢c a¤nity, oligotrophic capacity, and the meaning of the Michaelis constant. Appl. Environ. Microbiol. 57, 2033^2038. [28] Button, D.K. (1993) Nutrient-limited microbial growth kinetics : overview and recent advances. Antonie van Leeuwenhoek 63, 225^235. [29] Gerritse, J. and Gottschal, J.C. (1992) Mineralization of the herbicide 2,3,6-trichlorobenzoic acid by a co-culture of anaerobic and aerobic bacteria. FEMS Microbiol. Ecol. 101, 89^ 98. [30] DeWeerd, K.A., Mandelco, L., Tanner, R.S., Woese, C.R. and Su£ita, J.M. (1990) Desulfomonile tiedjei gen. nov. and spec. nov., a novel, anaerobic, dehalogenating, sulfate-reducing bacterium. Arch. Microbiol. 154, 23^30.
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[31] Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R., McCaughey, M.J. and Woese, C.R. (1997) The RDP (ribosomal database project). Nucleic Acids Res. 25, 109^110. [32] Stoesser, G., Sterk, P., Tuli, M.A., Stoehr, P.J. and Cameron, G.N. (1997) The EMBL nucleotide sequence database. Nucleic Acids Res. 25, 7^13. [33] Moore, E.R.B., Mau, M., Arnscheidt, A., Boëttger, E.C., Hutson, R.A., Collins, M.D., Van de Peer, Y., De Wachter, R. and Timmis, K.N. (1996) The determination and comparison of the 16S rRNA gene sequences of the genus Pseudomonas (sensu stricto) and estimation of the natural intrageneric relationships. Syst. Appl. Microbiol. 19, 478^492. [34] Anzai, Y., Kudo, Y. and Oyaizu, H. (1997) The phylogeny of the genera Chryseomonas, Flavimonas and Pseudomonas supports synonymy of these three genera. Int. J. Syst. Bacteriol. 47, 240^251. [35] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin reagent. J. Biol. Chem. 193, 265^275. [36] Bradford, M.M. (1976) A rapid sensitive method for quanti¢cation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [37] Bergman, J.G. and Sanik, J. (1957) Determination of trace amounts of chlorine in naphtha. Anal. Chem. 29, 241^243. [38] Wheelis, M.L., Palleroni, N.J. and Stanier, R.Y. (1967) The metabolism of aromatic acids by Pseudomonas testosteroni and P. acidovorans. Arch. Mikrobiol. 59, 302^314. [39] Stanier, R.Y. and Ingraham, J.L. (1954) Protocatechuic acid oxidase. J. Biol. Chem. 210, 799^808. [40] Fava, F., Di Gioia, D., Marchetti, L., Quattroni, G. and Marra¤a, V. (1993) Aerobic mineralization of chlorobenzoates by a natural polychlorinated biphenyl-degrading mixed bacterial culture. Appl. Microbiol. Biotechnol. 40, 541^548. [41] Nakatsu, C.H. and Wyndham, R.C. (1993) Cloning and expression of the transposable chlorobenzoate-3,4-dioxygenase genes of Alcaligenes sp. strain BR60. Appl. Environ. Microbiol. 59, 3625^3633. [42] Fava, F., Di Gioia, D., Romagnoli, C., Marchetti, L. and Mares, D. (1993) Biosynthesis and cytoplasmic accumulation of a chlorinated catechol pigment during 3-chlorobenzoate aerobic co-metabolism in Pseudomonas £uorescens. Arch. Microbiol. 160, 350^357. [43] Dorn, E., Hellwig, M., Reineke, W. and Knackmuss, H.J. (1974) Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad. Arch. Microbiol. 99, 61^70.
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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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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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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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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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Table 2 Enzyme activities in cell free extracts of Pseudomonas sp. strain A3 grown on 3CBA, benzoate or protocatechuate Growth substrate
Speci¢c activity (nmol min31 mg protein31 ) of : Catechol dioxygenase
Gentisate dioxygenase
Protocatechuate dioxygenase
3CBA Benzoate Protocatechuate
1750 450 22
0 0 0
57 nda 3295
a
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 concentrations the Qmax values increased with an apparent 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.
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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
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0.188 0.188 0.154 0.130 0.100 0.025 0.010 0.025
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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 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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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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