3-chlorobenzoate Degradation Research Articles

Characterization of chlorobenzoate degraders isolated from polychlorinated biphenyl-contaminated soil and sediment in the Czech Republic.

Two polychlorinated biphenyl-contaminated sites in the Czech Republic, a soil at Zamberk and a sediment sludge at Milevsko, were screened for the presence of chlorobenzoate degraders. Sixteen different chlorobenzoate degraders were isolated from the soil compared with only three strains isolated from the sediment. From these strains, only four soil degraders and one strain isolated from the sediment, respectively, were shown to possess a complete chlorobenzoate (CB) pathway. Bacteria isolated from the soil have expressed more flexibility for CB degradation, namely in the case of ortho-chlorinated benzoates. They all possessed large plasmids, the restriction patterns of which were compared. Plasmids in Pseudomonas sp. A7, A8, A18 and A19, respectively, were cured and found to encode at least part of the metabolic pathway involved in the growth on ortho-chlorinated benzoates.

Degradation of chlorobenzoates in soil suspensions by indigenous populations and a specialized organism: interactions between growth and non-growth substrates

The degradation of mono- and dichlorobenzoates was followed in soil suspensions. The indigenous microflora of the soil used was able to degrade only 4-chlorobenzoic acid (4CB) and 2-chlorobenzoic acid (2CB) either added alone or present in a mixture. When the soil was inoculated with an Alcaligenes denitrificans CB strain capable of degrading 4CB, 4CB degradation was highly accelerated. The presence in the soil suspensions of other chlorobenzoates (CBs) together with 4CB affected the degradation rate of the latter compound: 2CB and 2,6-dichlorobenzoic acid (2,6-DCB) retarded the degradative process, while 3,4- or 2,3-dichlorobenzoic acids (3,4-, 2,3-DCB) totally inhibited 4CB degradation. The potential mechanisms involved in these interactions are discussed.

Degradation of chlorobenzoates in soil suspensions by indigenous populations and a specialized organism: interactions between growth and non-growth substrates

The degradation of mono- and dichlorobenzoates was followed in soil suspensions. The indigenous microflora of the soil used was able to degrade only 4-chlorobenzoic acid (4CB) and 2-chlorobenzoic acid (2CB) either added alone or present in a mixture. When the soil was inoculated with an Alcaligenes denitrificans CB strain capable of degrading 4CB, 4CB degradation was highly accelerated. The presence in the soil suspensions of other chlorobenzoates (CBs) together with 4CB affected the degradation rate of the latter compound: 2CB and 2,6-dichlorobenzoic acid (2,6-DCB) retarded the degradative process, while 3,4- or 2,3-dichlorobenzoic acids (3,4-, 2,3-DCB) totally inhibited 4CB degradation. The potential mechanisms involved in these interactions are discussed.

Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium.

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 positive of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate.

Amplification of putative chlorocatechol dioxygenase gene fragments from α- and β-Proteobacteria

Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes. These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and chlorobenzoate-degrading strains, including species of Sphingomonas. Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain. The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems.Key words: 2,4-dichlorophenoxyacetate, chlorobenzoate, biodegradation, ortho cleavage, detection.

Amplification of putative chlorocatechol dioxygenase gene fragments from alpha- and beta-Proteobacteria.

Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes. These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and cholorobenzoate-degrading strains, including species of Sphingomonas. Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain. The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems.

The Impact of Industrial Contamination on Microbial Chlorobenzoate Degradation in the Niagara Watershed

The ability of the indigenous microbial community from three streams in the Niagara River watershed to degrade chlorobenzoate congeners was studied over a two-year period using culture enrichment techniques. Sediment enrichments from Bloody Run Creek and Devil's Hole Creek, which were shown to be contaminated by groundwater run-off from the Hyde Park chemical landfill site, consistently showed a higher frequency of complete microbial degradation of 3-chloro-, 4-chloro-, and 3,4-dichlorobenzoate when compared to enrichments from control samples of Fish Creek, a tributary that is not effected by Hyde Park. The isomer showing the most frequent degradation in all sediment enrichments was 4-chlorobenzoate. Seasonal variations were observed for 3-chlorobenzoate degradation, only. Year-to-year variations in microbial chlorobenzoate degradation were noted for 4-chloro- and 3,4-dichlorobenzoate enrichments. Although 4°C batch culture enrichments consistently showed less frequent degradation of chlorobenzoates during all seasons sampled, some sites showed complete degradation at this temperature. This suggests that the culturable microbial consortia responsible for chlorobenzoate degradation include facultative psychrophiles. Phosphorous nutrient limitations in all streams limited the microbial degradation potential in situ.

Genetic exchange in soil between introduced chlorobenzoate degraders and indigenous biphenyl degraders.

Pseudomonas aeruginosa JB2, a chlorobenzoate degrader, was inoculated into soil having indigenous biphenyl degraders but no identifiable 2-chlorobenzoate (2CBa) or 2,5-dichlorobenzoate (2,5DCBa) degraders. The absence of any indigenous chlorobenzoate degraders was noted by the failure to obtain enrichment cultures with the addition of 2CBa, 3CBa, or 2,5DCBa and by the failure of soil DNA to hybridize to the tfdC gene, which encodes ortho fission of chlorocatechols. In contrast, DNA extracted from inoculated soils hybridized to this probe. Bacteria able to utilize both biphenyl and 2CBa as growth substrates were absent in uninoculated soil, but their presence increased with time in the inoculated soils. This increase was related kinetically to the growth of biphenyl degraders. Pseudomonas sp. strain AW, a dominant biphenyl degrader, was selected as a possible parental strain. Eight of nine recombinant strains, chosen at random, had high phenotypic similarity (90% or more) to the inoculant; the other, strain JB2-M, had 78% similarity. Two hybrid strains, P. aeruginosa JB2-3 and Pseudomonas sp. JB2-M, were the most effective of all strains, including strain AW, in metabolizing polychlorinated biphenyls (Aroclor 1242). Repetitive extragenic palindromic-PCR analysis of putative parental strains JB2 and AW and the two recombinant strains JB2-3 and JB2-M showed similar fragments among the recombinants and JB2 but not AW. These results indicate that the bph genes were transferred to the chlorobenzoate-degrading inoculant from indigenous biphenyl degraders.

Anaerobic Dehalogenation of 2-Chlorophenol by Mixed Bacterial Cultures in Absence of Methanogenesis

This study investigated the 2-chlorophenol (OCP) degradation potential of mixed fermentative (non-methanogenic) anaerobic cultures derived from sewage sludge. Under mesophilic conditions OCP was degraded in mineral salts medium at concentrations five times greater than those utilized by known anaerobic chlorophenol and chlorobenzoate degraders. Acetate was found to be the end product of OCP-degradation in these cultures. Carbon dioxide could not be shown to be an end product. OCP degradation was fermentative, i.e. did not require sulphate or nitrate as terminal electron acceptor.

Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains.

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 microM) and 3,4-CBA (50 microM). Although 2,5-cba and 2,6-cba were not utilised as carbon sources by strain jhr22, they did not inhibit 2-cba utilisation at the concentrations studied, whereas 2,4-cba was co-metabolised with 2-cba. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.

Use of ans-triazine nitrogen source to select for and isolate a recombinant chlorobenzoate-degradingPseudomonas

A novel mating system, which uses the s-triazine nitrogen source (cyanuric acid, CA) as a selective nutritional marker, was developed to evaluate mobility and facilitate identification of chlorobenzoate (CBA) degradation genes in Pseudomonas aeruginosa strain JB2. Matings were done between strain JB2, which is unable to grow with CA, and a CA-utilizer Pseudomonas sp. strain D on 2-CBA-CA and 3-CBA-CA media. Isolates were recovered only from the 2-CBA-CA and were all identified as strain D derivatives. Subtractive hybridization analysis of genomic DNAs from the parental strains and a selected isolate, Pseudomonas sp. strain JPL, was used to determine that the latter organism had acquired approx. 16.1 kb of DNA from strain JB2.

Use of an s-triazine nitrogen source to select for and isolate a recombinant chlorobenzoate-degrading Pseudomonas

A novel mating system, which uses the s-triazine nitrogen source (cyanuric acid, CA) as a selective nutritional marker, was developed to evaluate mobility and facilitate identification of chlorobenzoate (CBA) degradation genes in Pseudomonas aeruginosa strain JB2. Matings were done between strain JB2, which is unable to grow with CA, and a CA-utilizer Pseudomonas sp. strain D on 2-CBA-CA and 3-CBA-CA media. Isolates were recovered only from the 2-CBA-CA and were all identified as strain D derivatives. Subtractive hybridization analysis of genomic DNAs from the parental strains and a selected isolate, Pseudomonas sp. strain JPL, was used to determine that the latter organism had acquired approx. 16.1 kb of DNA from strain JB2.

Diversity of anaerobic microbial processes in chlorobenzoate degradation: Nitrate, iron, sulfate and carbonate as electron acceptors

The utilization of monochlorobenzoate isomers (2-, 3- and 4-chlorobenzoate) by anaerobic microbial consortia in River Nile sediments was systematically evaluated under denitrifying, Fe-reducing, sulfidogenic and methanogenic conditions. Loss of all three chlorobenzoates was noted in denitrifying cultures; furthermore, the initial utilization of chlorobenzoates was fastest under denitrifying conditions. Loss of 3-chlorobenzoate was seen under all four reducing conditions and the degradation of chlorobenzoates was coupled stoichiometrically to NO3- loss, Fe2+ production, SO4(2-) loss or CH4 production, indicating that the chlorobenzoates were oxidized to CO2. To our knowledge, this is the first observation of halogenated aromatic degradation coupled to Fe reduction.

Diversity of anaerobic microbial processes in chlorobenzoate degradation: nitrate, iron, sulfate and carbonate as electron acceptors

Diversity of anaerobic microbial processes in chlorobenzoate degradation: nitrate, iron, sulfate and carbonate as electron acceptors

Simultaneous degradation of 3-chlorobenzoate and phenolic compounds by a defined mixed culture ofPseudomonas spp.

A mixed culture of a chlorobenzoate-(3-CBA)-degradingPseudomonas aeruginosa, strain 3mT, and a phenol/cresols-degradingPseudomonas sp., strain CP4, simultaneously and efficiently degraded mixtures of 3-CBA and phenol/cresols. However, strains 3mT and CP4 usedortho- andmeta-ring cleavage pathways, respectively. Degradation of 3-CBA was complete when the 3-CBA was equal in amount to or less than that of phenol. CP4/3mT inoculum ratios (w/w) of 1:1 or 1:2 gave the most effective degradation of both the substrates in the mixture. The mixed culture degraded equimolar mixtures of 3-CBA/phenol up to 10MM. Equimolar mixtures of 3-CBA ando-, m- orp-cresol were also degraded by the mixed culture.

Formation of chlorocatechol meta cleavage products by a pseudomonad during metabolism of monochlorobiphenyls

Pseudomonas cepacia P166 was able to metabolize all monochlorobiphenyls to the respective chlorobenzoates. Although they transiently accumulated, the chlorobenzoate degradation intermediates were further metabolized to chlorocatechols, which in turn were meta cleaved. 2- and 3-Chlorobiphenyl both produced 3-chlorocatechol, which was transformed to an acyl halide upon meta cleavage. 3-Chlorocatechol metabolism was toxic to the cells and impeded monochlorobiphenyl metabolism. In the case of 2-chlorobiphenyl, toxicity was manifested as a diminished growth rate, which nevertheless effected rapid substrate utilization. In the case of 3-chlorobiphenyl, which generates 3-chlorocatechol more rapidly than does 2-chlorobiphenyl, toxicity was manifested as a decrease in viable cells during substrate utilization. 4-Chlorobenzoate was transformed to 4-chlorocatechol, which was metabolized by a meta cleavage pathway leading to dehalogenation. Chloride release from 4-chlorocatechol metabolism, however, was slow and did not coincide with rapid 4-chlorocatechol turnover. Growth experiments with strain P166 on monochlorobiphenyls illustrated the difficulties of working with hydrophobic substrates that generate toxic intermediates. Turbidity could not be used to measure the growth of bacteria utilizing monochlorobiphenyls because high turbidities were routinely measured from cultures with very low viable-cell counts.

An attempt to control the polychlorocatechol pigment production during 3-chlorobenzoate aerobic co-metabolism in growing-cell batch culture

A strain of Pseudomonas fluorescens, designed CP30, was capable of co-metabolizing 3-chlorobenzoic acid via chlorocatechols, thus producing a polychlorocatechol black pigment. The pigment production was found to be enzyme-mediated in P. fluorescens cultures; however, the results of the autoxidation studies suggested that a large amount of pigment could derive from the simple autoxidation of the chlorocatechols accumulated in the CP30 culture during the 3-chlorobenzoate degradation. The polymerization of chlorocatechols should reduce the toxicity of the culture and generally resulted in a large production of a water-insoluble (i.e. a easily removable) pigment with the release a large amount of chloride ions. Both 3-chlorobenzoate degradation and pigment production were stimulated and inhibited in the same way by high and low pH values, respectively, in CP30 cultures; the culture conditions which permitted an extensive 3-chlorobenzoate degradation with the lowest pigment production were not identified. However, on the basis of the detoxification property of the pigment production process, high pH and dissolved oxygen concentrations appears to be the most suitable culture conditions for an enhanced detoxicant treatment of 3-clorobenzoate-contaminated waste water.

Degradation of chlorobenzoates in soil slurry by special organisms

The microbial degradation of 2-chloro-, 3-chloro-, 4-chloro-, 3,4-dichloro- and 3,5-dichlorobenzoate was examined either as single compounds or as mixtures in soil slurry. The influence of water content, rotation frequency, supplementation of mineral salts and acetate and the addition of specialized bacterial cultures (Pseudomonas sp. strain B13, P. ruhlandii strain WR912, P. cepacia strain JH230) to the soil on the degradation rate was studied. Only 4-chlorobenzoate was completely degraded within 17 days by the original soil population. In contrast, the total degradation of all other compounds, i.e. stoichiometric elimination of chloride, was only observed when specialized organisms were inoculated into the soil slurry. Small amounts of specialized bacteria (500 colony-forming units/ml) led to rapid and complete degradation. Even 3,4-dichlorobenzoate, which cannot be used by our strains as a growth substrate, was degraded in a mixture with other chlorobenzoates. Nitrite, which sometimes appeared during the incubations, inhibited the degradation.

Enhanced mineralization of polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading bacteria.

An Altamont soil containing no measurable population of chlorobenzoate utilizers was examined for the potential to enhance polychlorinated biphenyl (PCB) mineralization by inoculation with chlorobenzoate utilizers, a biphenyl utilizer, combinations of the two physiological types, and chlorobiphenyl-mineralizing transconjugants. Biphenyl was added to all soils, and biodegradation of 14C-Aroclor 1242 was assessed by disappearance of that substance and by production of 14CO2. Mineralization of PCBs was consistently greatest (up to 25.5%) in soils inoculated with chlorobenzoate degraders alone. Mineralization was significantly lower in soils receiving all other treatments: PCB cometabolizer (10.7%); chlorobiphenyl mineralizers (8.7 and 14.9%); and mixed inocula of PCB cometabolizers and chlorobenzoate utilizers (11.4 and 18.0%). However, all inoculated soils had higher mineralization than did the uninoculated control (3.1%). PCB disappearance followed trends similar to that observed with the mineralization data, with the greatest degradation occurring in soils inoculated with the chlorobenzoate-degrading strains Pseudomonas aeruginosa JB2 and Pseudomonas putida P111 alone. While the mechanism by which the introduction of chlorobenzoate degraders alone enhanced biodegradation of PCBs could not be elucidated, the possibility that chlorobenzoate inoculants acquired the ability to metabolize biphenyl and possibly PCBs was explored. When strain JB2, which does not utilize biphenyl, was inoculated into soil containing biphenyl and Aroclor 1242, the frequency of isolates able to utilize biphenyl and 2,5-dichlorobenzoate increased progressively with time from 3.3 to 44.4% between 15 and 48 days, respectively. Since this soil contained no measurable level of chlorobenzoate utilizers yet did contain a population of biphenyl utilizers, the possibility of genetic transfer between the latter group and strain JB2 cannot be excluded.