Arch Microbiol (1999) 171 : 230–236
© Springer-Verlag 1999
O R I G I N A L PA P E R
Max M. Häggblom · L. Y. Young
Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium
Received: 6 November 1998 / Accepted: 19 January 1999
Abstract A denitrifying bacterium was isolated from a river sediment after enrichment on 3-chlorobenzoate under anoxic, denitrifying conditions. The bacterium, designated strain 3CB-1, degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with stoichiometric release of halide under conditions supporting anaerobic growth by denitrification. The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor. The doubling time when growing on 3-halobenzoates ranged from 18 to 25 h. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1–2 mm in diameter) in 2 to 3 weeks. Anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values for complete oxidation of the substrate to CO2. 3-Chlorobenzoate was not degraded in the presence of oxygen. 3-Bromobenzoate and 3-iodobenzoate were also degraded under denitrifying conditions with stoichiometric release of halide, but 3-fluorobenzoate was not utilized by the bacterium. Utilization of 3-chlorobenzoate was inducible, while synthesis of enzymes for 3-hydroxybenzoate degradation was constitutively low, but inducible. Degradation was specific to the position of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate. Key words Anaerobic degradation · Denitrification · Halobenzoate · Dehalogenation
M. M. Häggblom (쾷) · L. Y. Young Biotechnology Center for Agriculture and the Environment, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA e-mail:
[email protected] Tel. +1-732-932-8165; Fax +1-732-932-0312 M. M. Häggblom Department of Biochemistry and Microbiology, Cook College, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA
Introduction Denitrifying bacteria are important in the anaerobic degradation of anthropogenic contaminants such as aromatic hydrocarbons, phenolic compounds, and benzoic acid derivatives (Elder and Kelly 1994; Frazer et al. 1995; Heider and Fuchs 1997). Nitrate is a common groundwater contaminant due to the frequent use of nitrogen fertilizers and production from ammonia by nitrification, which may promote denitrifying conditions in anoxic soils and sediments. Denitrifying microorganisms are potentially more versatile than those of other groups, in part by virtue of their facultative anaerobic nature, which allows them to remain active and viable in habitats of different oxygen concentrations. To date, detailed information on the biodegradation of halogenated benzoic acids and related compounds under denitrifying conditions is limited. Many studies have indicated that nitrate, sulfate, and other electron acceptors inhibit the degradation of halogenated aromatic compounds by anaerobic microbial communities, specifically by inhibiting reductive dehalogenation (for reviews, see Häggblom 1992; Mohn and Tiedje 1992; Fetzner and Lingens 1994; Suflita and Townsend 1995; and Häggblom and Milligan 1999). However, previous work has demonstrated that denitrifying conditions can support the degradation of monochlorinated benzoic acids (Häggblom et al. 1993, 1996; Kazumi et al. 1995), and degradation under sulfateand iron-reducing conditions has also been documented (Häggblom et al. 1993; Kazumi et al. 1995). Genthner et al. (1989b) have also observed that an anaerobic enrichment culture on 3-chlorobenzoate requires nitrate for activity. Utilization of fluorinated benzoates by denitrifying bacteria has been demonstrated; however, these strains do not utilize chlorinated or brominated substrates (Taylor et al. 1979; Schennen et al. 1985). Under methanogenic conditions, chlorobenzoates are readily dehalogenated to benzoate and are ultimately mineralized to CH4 and CO2 (Suflita et al. 1982; Horowitz et al. 1983; Gibson and Suflita 1986; Genthner et al. 1989 a, b; Gerritse and Gottschal
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1992). In addition, anaerobic phototrophic degradation of 3-chlorobenzoate by Rhodopseudomonas palustris strains has been described (Kamal and Wyndham 1990; Van der Woude et al. 1994). These studies indicate that a variety of anoxic environments can support the degradation of halogenated aromatic compounds. In previous work, we have shown that monochlorobenzoates are readily degraded in denitrifying enrichment cultures established with sediment and soil from a variety of locations (Häggblom et al. 1993, 1996). There are interesting differences in the susceptibility to biodegradation of the different halobenzoate isomers; of the chlorinated benzoic acids, the meta- and para-substituted isomers are readily degraded, while the ortho-substituted isomer is not. From initial enrichment cultures, separate denitrifying cultures utilizing either 3- or 4-chlorobenzoate as sole sources of carbon and energy have been maintained (Häggblom et al. 1996). Anaerobic halobenzoate degradation has been coupled to denitrification and does not occur in the absence of nitrate or in the presence of oxygen, suggesting a tight coupling between chlorobenzoate utilization and denitrification. In this paper, we describe the isolation of a denitrifying bacterium from a 3-chlorobenzoate enrichment culture established with sediment from the Hudson River as inoculum. The novel bacterium is capable of growing on 3-chlorobenzoate as a sole source of carbon and energy with nitrate as the terminal electron acceptor.
Substrate degradation assays and growth experiments Substrate utilization experiments were performed with dense cell suspensions of strain 3CB-1 grown on either pyruvate or 3-hydroxybenzoate in mineral salts medium with nitrate as the electron acceptor. Cells were collected by centrifugation, washed, and resuspended in fresh medium, and aliquots (5–25 ml, depending on the experiment) were supplemented with the desired substrate and electron acceptors. Individual cultures were deoxygenated by repeated evacuation and flushing with argon. The various substrates [benzoic acid, fluoro-, bromo-, and iodobenzoic acids (minimum 98% purity; Aldrich)] were added from deoxygenated stock solutions prepared in 0.1 N NaOH. All cultures were incubated at 30 °C in the dark. For growth assays, anaerobic Balch tubes (Bellco, Vineland, N.J., USA) with denitrifying medium were inoculated with a late-exponential-phase culture. 3-Halobenzoate or 3-hydroxybenzoate was supplied as the carbon source (at concentrations of 250– 1,000 M). Anaerobic conditions were generated by repeated evacuation as described above. Growth was monitored by measuring the optical density at 630 nm directly from the test tubes with a Spectronic 20 spectrophotometer (Bausch and Lomb, Rochester, N.Y., USA). To determine the stoichiometry of substrate utilization and nitrate reduction, cells were grown on pyruvate and induced for degradation by addition of either 3-chlorobenzoate or 3-hydroxybenzoate. The respective cell suspensions were washed and resuspended in fresh medium with 10 mM nitrate and were divided into aliquots of 25 ml. Duplicate cultures were supplemented with 3-chlorobenzoate (0.5 or 1.0 mM), 3-hydroxybenzoate (0.5 or 1.0 mM) or no substrate and were deoxygenated as described above. Cultures were monitored for the utilization of substrate, consumption of nitrate and production of nitrite, and N2. Due to interference with nitrite, protein values (using the Bio-Rad protein assay) were not obtained. Influence of nitrate and oxygen on 3-chlorobenzoate degradation
Materials and methods Isolation of a 3-chlorobenzoate-degrading bacterium Denitrifying enrichment cultures were established on 3-chlorobenzoate with inoculum from the Hudson River near Albany, N.Y., USA (Häggblom et al. 1993). Strict anaerobic techniques for medium preparation, culture handling, and sampling were followed throughout the study. Initial enrichment cultures were established by preparing a 10% (v/v) sediment slurry in a mineral salts medium supplemented with 30 mM nitrate, vitamins, and trace elements (Häggblom et al. 1993), dispensed under argon to a final culture volume of 50 ml, and sealed with rubber stoppers. The culture was then fed 3-chlorobenzoate (minimum 98% purity; Aldrich, Milwaukee, Wis., USA) to an initial concentration of 100 µM from a deoxygenated stock solution prepared in 0.1 N NaOH. After initial activity was observed, the culture was repeatedly diluted into fresh denitrifying medium and fed 3-chlorobenzoate over a period of 2 years (Häggblom et al. 1996). A sample of this enrichment culture was streaked onto agar (Noble Agar, Difco) plates with 1 mM 3-chlorobenzoate as the sole source of carbon with 30 mM nitrate as the electron acceptor and was incubated under N2/H2 (97:3, v/v) in an anaerobic jar. After 4 weeks of incubation, small (1–2 mm in diameter) colonies were transferred (under N2/H2) into 5 ml liquid denitrifying medium with 100 µM 3-chlorobenzoate and were tested for substrate utilization. After 5 weeks, when 3-chlorobenzoate had been depleted (8 of 12 selected cultures), the cultures were streaked onto agar plates with 1 mM 3-chlorobenzoate and 150 mg tryptic soy/l (Difco). 3-Chlorobenzoate-degrading bacteria were purified by repeated plating with a combination of 3-chlorobenzoate and tryptic soy as carbon sources under N2/H2, and one colony was chosen for further study. Purity was checked by microscopy and growth in complex medium. The strain designated 3CB-1 has been deposited in the American Type Culture Collection as ATCC 700723.
To test for the effect of nitrate and oxygen, strain 3CB-1 was grown anaerobically on 3-hydroxybenzoate. Cells were collected by centrifugation, washed twice with nitrate-free medium, and resuspended in nitrate-free medium to approximately 108 cells/ml. The cell suspension was divided into aliquots of 5 ml and was spiked with 200 µM 3-chlorobenzoate or 3-hydroxybenzoate with or without 6 mM NO3–. The cultures were deoxygenated by repeated evacuation and flushing with argon. In addition, a set of nitrate-free cultures were incubated under a head space of air (O2). Induction To determine whether utilization of 3-chlorobenzoate and 3-hydroxybenzoate was inducible, cells grown on pyruvate were collected, washed, and resuspended in fresh denitrifying medium. Duplicate cultures received either 500 µM 3-chlorobenozoate or 3-hydroxybenzoate in the presence or absence of 1 mg streptomycin/ml to inhibit protein synthesis, and substrate utilization was monitored over time. Analytical methods Samples for chemical analyses were centrifuged if necessary, filtered through a 0.45-µm (pore size) filter, and frozen (–20 °C) until analyzed. Halobenzoates and other aromatic substrates were analyzed by high-performance liquid chromatography using a C-18 column and UV detection at 280 nm as described previously (Kazumi et al. 1995). Nitrate, nitrite, and halides were analyzed by ion chromatography with conductivity detection as described (Coschigano et al. 1994; Kazumi et al. 1995). Nitrate and nitrous oxide were analyzed by gas chromatography using a gas partitioner (Fischer Scientific, Pittsburgh, Pa., USA) as previously described (Milligan and Häggblom 1998).
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Fig. 1 Growth of strain 3CB-1 under denitrifying conditions A with different concentrations of 3-chlorobenzoate (쎲 control, 왓 0.25 mM, 왎 0.50 mM, 앳 0.75 mM, and 왖 1.00 mM) and B with different benzoic acid derivatives (쎲 control, 왓 0.50 mM 3-chlorobenzoate, 왎 0.50 mM 3-bromobenzoate, 앳 0.50 mM 3-iodobenzoate, and 왖 0.50 mM 3-hydroxybenzoate). The control cultures were inoculated but not provided with a carbon source. Data points are the means of duplicate cultures
Results Enrichment and isolation A denitrifying enrichment culture established with sediment from the Hudson River was maintained by repeated subculturing into fresh medium and refeeding with 3-chlorobenzoate (100–500 µM) as the sole source of carbon and energy. After extensive transfers of this enrichment culture, denitrifying bacteria that utilized 3-chlorobenzoate (1 mM) as the sole source of carbon with 30 mM nitrate as the electron acceptor were isolated. One colony (of eight 3-chlorobenzoate-utilizing bacteria), designated strain 3CB-1, was chosen for further study and was purified by repeated plating with a combination of 3-chlorobenzoate and tryptic soy as carbon sources. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source, strain 3CB-1 formed
Fig. 2 A Utilization of halobenzoates (쎲 3-chlorobenzoate, 쑗 3fluorobenzoate, 왔 2-chlorobenzoate, and 왓 4-chlorobenzoate) and B release of halide from 3-bromo and 3-iodobenzoate (쎲 3-bromobenzoate, 쑗 bromide, 왔 3-iodobenzoate, and 왓 iodide) by dense cell suspensions of strain 3CB-1
small colonies (1–2 mm in diameter) in 2–3 weeks. The strain is a gram-negative, short rod with motility observed transiently during early stages of growth. On the basis of 16s rRNA gene sequence analysis (complete sequence deposited in Genbank under accession no. AF 123264), strain 3CB-1 was affiliated with the genus Thauera and is closely related to Thauera aromatica with 98.5% sequence similarity (B. Song, N. J. Palleroni and M. M. Häggblom, unpublished work). Strain 3CB-1 has been deposited in the American Type Culture Collection as ATCC 700723. Growth on 3-halobenzoates Strain 3CB-1 could grow anaerobically on 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate (Fig. 1). The strain tolerated halobenzoate concentrations of up to 1 mM, but at higher concentrations growth was often inhibited depending on the inoculum density (not shown). The cultures reached stationary phase after 40–70 h of incubation.
233 Table 1 Utilization of 3-chlorobenzoate and 3-hydroxybenzoate by strain 3CB-1 in the presence and absence of nitrate and oxygen. Dense cell suspensions of strain 3CB-1 were incubated anaerobically under an argon head space with or without 6 mM nitrate or aerobically Incubation condition
Anoxic – nitrate Anoxic + nitrate Oxic (–nitrate) a Substrate
Substrate utilization (%) in 22 ha 3-Chlorobenzoate
3-Hydroxybenzoate
0 100 2
13 100 100
spiked to 200 µM
3-Hydroxybenzoate also served as a growth substrate for strain 3CB-1 under denitrifying conditions (Fig. 1B). The growth rates on the 3-halobenzoates were 0.04–0.06 h–1, while the growth rate on 3-hydroxybenzoate was 0.10 h–1. Increasing the concentration of 3-halobenzoates or 3-hydroxybenzoate (250–1,000 µM) resulted in a linear increase in cell yield. Growth yields were approximately 1.6-fold higher (measured as the increase in OD630) on the same concentrations of 3-hydroxybenzoate as compared to those on 3-chlorobenzoate (not shown). Degradation of halobenzoates in dense cell suspensions Dense cell suspensions of strain 3CB-1 degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate, but 3-fluorobenzoate was not degraded (Fig. 2A). 3-Bromobenzoate and 3-iodobenzoate were utilized with a stoichiometric release of halide (Fig. 2B). Due to the high background of chloride in the growth medium, release of chloride from 3-chlorobenzoate could not be measured. 2-Chlorobenzoate or 4-chlorobenzoate was not utilized, and strain 3CB-1 was thus specific to the position of the halogen substituent. Although a 50% loss of 4-chlorobenzoate was observed over 46 h, this activity was not sustained. Dense cell suspension experiments were also performed to determine the influence of nitrate and oxygen on 3-chlorobenzoate and 3-hydroxybenzoate utilization by strain 3CB-1. These experiments indicate that strain 3CB-1 only degraded 3-chlorobenzoate under denitrifying conditions in the presence of nitrate (Table 1, Fig. 3A). No loss of substrate occurred in the absence of nitrate or in the presence of oxygen. Furthermore, 3-chlorobenzoate was not degraded under oxic conditions in the presence of nitrate (data not shown). 3-Hydroxybenzoate, on the other hand, was degraded aerobically; under anoxic conditions, however, nitrate was required (Table 1, Fig. 3B). Thus, anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor, and 3-chlorobenzoate was not degraded in the presence of oxygen.
Fig. 3 Anaerobic utilization of A 3-chlorobenzoate and B 3-hydroxybenzoate by dense cell suspensions of strain 3CB-1 in the presence (쎲) or absence (쑗) of nitrate. Data points are the means of duplicate cultures
Stoichiometry of 3-chlorobenzoate and 3-hydroxybenzoate utilization To determine whether complete degradation of 3-chlorobenzoate and 3-hydroxybenzoate was taking place, we quantified the oxidation of substrate and the reduction of nitrate to calculate an electron balance. Cell suspensions were incubated with either 0.5 or 1 mM 3-chlorobenzoate or 3-hydroxybenzoate for 6 days until the substrate was depleted, and were then assayed for the consumption of nitrate and the production of nitrite and N2 (Table 2). The values of electrons produced were calculated from the equations for oxidation of substrate to CO2, not considering any assimilation to cell carbon. The values for electrons consumed were derived from the reduction of nitrate to nitrite and N2. Negligible amounts of N2O produced were not considered for the calculations. The ratio of electrons consumed to electrons produced (58–69%, not considering assimilation) during degradation of 3-chlorobenzoate and 3-hydroxybenzoate supports the thesis that these substrates are mineralized coupled to nitrate reduc-
234 Table 2 Balance of electron donor and acceptor during utilization of 3-chlorobenzoate and 3-hydroxybenzoate by strain 3CB-1 Substrate metabolized (µmol)a
Nitrate consumed (µmol)a
Nitrite produced (µmol)a
N2 produced (µmol)a
Nitrogen balance (%)b
Electrons produced (µmol)c
Electrons consumed (µmol)d
Electrons consumed/produced (%)e
3-Chlorobenzoate 12.9 25.2
71.2 127.7
36.9 51.5
18.2 27.0
106 71
361 706
245 484
68 69
3-Hydroxybenzoate 12.2 23.9
76.3 125.4
49.0 78.7
7.3 13.0
53 56
342 670
234 391
68 58
a Means
of duplicate cultures. Values corrected for nitrate consumption (9.6 µmol), nitrite production (0 µmol), and N2 production (10.8 µmol) in no-substrate controls b N O production negligible 2 c Calculated on the basis of the following half-reactions: C7H5O2Cl + 12 H2O → 7 CO2 + Cl– + 29 H+ + 28 e– (chlorobenzoate) C7H6O3 + 11 H2O → 7 CO2 + 28 H+ + 28 e– (hydroxybenzoate)
d Calculated
on the basis of the following half-reactions: NO3– + 2 H+ + 2 e– → NO2– + H2O NO3– + 6 H+ + 5 e– → N2 + 3 H2O (calculated from NO3– consumption less accumulated NO2–) e Conversion of carbon to cells not accounted for
tion. Anaerobic halobenzoate degradation coupled to denitrification (not accounting for assimilation to cell carbon, typically 30–40%) can thus be described by the equation: C7H5O2Cl + 5.6 NO3– + 4.6 H+ → 7 CO2 + 2.8 N2 + 4.8 H2O + Cl–. Induction To determine whether utilization of 3-chlorobenzoate and 3-hydroxybenzoate was inducible, cells of strain 3CB-1 grown on pyruvate were collected, washed, and resuspended in fresh denitrifying medium. In the absence of streptomycin, a lag period of approximately 8 h was observed before the onset of degradation (Fig. 4 A). 3-Chlorobenzoate at an initial concentration of 500 µM was depleted within 26 h and was degraded without a lag upon respiking of substrate. In the presence of 1 mg streptomycin/ml, no utilization of 3-chlorobenzoate was observed, indicating that utilization of 3-chlorobenzoate was inducible. On the other hand, 3-hydroxybenzoate was degraded without an appreciable lag period in both the presence and the absence of streptomycin (Fig. 4B). The rate of substrate utilization, however, was greater (63 µmol l–1 h–1 vs 19 mol l–1 h–1) in the absence than in the presence of streptomycin, with similar rates observed also upon respiking of the substrate. This suggests a constitutively low, but inducible synthesis of degradative enzymes.
Discussion Fig. 4 Induction of A 3-chlorobenzoate and B 3-hydroxybenzoate utilization by strain 3CB-1. Cells were pregrown on pyruvate and incubated 쎲 without or 쑗 with streptomycin to inhibit protein synthesis. 3-Chlorobenzoate and 3-hydroxybenzoate were respiked at 26 and 23 h, respectively. Data points are the means of duplicate cultures
A 3-chlorobenzoate-utilizing bacterium was isolated from a river sediment after enrichment under denitrifying conditions. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1–2 mm in diameter) in 2–3 weeks. The bacterium, designated strain
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3CB-1, is a gram-negative, short rod and, based on 16S rRNA sequence analysis, is a member of the genus Thauera (B. Song, N.J. Palleroni and M.M. Häggblom, unpublished work). A detailed taxonomic description is in preparation. Under anoxic, denitrifying conditions, strain 3CB-1 degrades 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with a stoichiometric release of halide. Degradation activity was specific to the position of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate. The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor. The doubling time when growing on 3chloro-, 3-bromo-, and 3-iodobenzoate ranged from 18 to 25 h. Anaerobic degradation of both 3–chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values expected for complete oxidation of the substrate to CO2. Degradation of chlorobenzoate by strain 3CB-1 did not occur in the absence of nitrate or in the presence of oxygen, suggesting that some enzyme or regulatory component of the degradation pathway is sensitive to oxygen. This strict requirement for denitrification and inhibition by oxygen has been previously observed in halobenzoatedegrading enrichment cultures (Häggblom et al. 1996). The reason why oxygen inhibits 3-chlorobenzoate utilization is unclear at this time. Interestingly, 3-hydroxybenzoate or benzoate was degraded either aerobically or under denitrifying conditions. Utilization of both 3-chlorobenzoate and 3-hydroxybenzoate was inducible in strain 3CB-1; however, 3-hydroxybenzoate degradation had constitutively low expression. Although a number of denitrifying bacteria (many members of the genera Thauera or Azoarcus) capable of degrading different benzoic acid derivatives have been described (Heising et al. 1991; Anders et al. 1995; CheeSanford et al. 1996; Gallus et al. 1997; Song et al. 1998, 1999; Springer et al. 1998), the utilization of chloro-, bromo-, or iodobenzoates as growth substrates under denitrifying conditions by an environmental isolate has not been reported. However, Coschigano et al. (1994) have previously described the utilization of 4-chlorobenzoate by a constructed bacterial strain. In this case, the 4-chlorobenzoate dehalogenase genes from Pseudomonas sp. strain CBS3 (Savard et al. 1986; Löffler et al. 1991) were transferred into and expressed in strain T1, recently identified as Thauera aromatica (Song et al. 1998). The transconjugant strain dehalogenated 4-chlorobenzoate to 4-hydroxybenzoate, which was used as a growth substrate under denitrifying conditions (Coschigano et al. 1994). A coryneform bacterium, strain NTB-1, is able to dehalogenate 4-chlorobenzoate to 4-hydroxybenzoate hydrolytically under anoxic conditions in the presence of nitrate (Groenewegen et al. 1990, 1992). In this case, it was suggested that nitrate drives an energy-dependent hydroxylation of 4-chlorobenzoate, although further degradation of 4-hydroxybenzoate by this bacterium requires oxygen (Groenewegen et al. 1992).
Reductive dechlorination of 2,4-dichlorobenzoate via a 2,4-dichlorobenzoyl-coenzyme A intermediate to 4-chlorobenzoate in an NADPH-dependent reaction has been demonstrated for two aerobic coryneform bacteria (Romanov and Hausinger 1996). 2-Chlorobenzoate, however, is not degraded by these strains. Schennen et al. (1985) have demonstrated that a denitrifying bacterium now classified as Azoarcus evansii (Anders et al. 1995) degrades 2-fluorobenzoate via a coenzyme A derivative with a fortuitous defluorination. This denitrifying bacterium does not degrade chlorinated benzoic acids. We have previously demonstrated that 3- and 4-chlorobenzoate can be degraded by denitrifying enrichment cultures established with soils and sediments from a variety of environments and geographical locations (Häggblom et al. 1996). These findings suggest that denitrifying bacteria with the ability to degrade halobenzoate are widely distributed in nature – an interesting point since most other chlorinated aromatic compounds (e.g., chlorophenols) are recalcitrant under denitrifying conditions. It is, therefore, useful to examine these halobenzoate-degrading organisms in greater detail and to determine their metabolic and taxonomic diversity. Acknowledgements This work was supported in part by grant nos. R820686 and R822487 from the U.S. Environmental Protection Agency.
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