Microbial degradation of chlorinated phenols

The chlorinated phenols comprise a large group of toxic, man-made chemicals that are serious environmental pollutants. Microorganisms can degrade many, but not all, of the chlorinated phenols, often using chlorophenol-specific catabolic enzymes. Novel technologies are evolving for using specific microorganisms to clean contaminated soils and waters of chlorophenols.     

Microbial degradation of chlorinated phenols

Chlorophenols have been introduced into the environment through their use as biocides and as by-products of chlorine bleaching in the pulp and paper industry. Chlorophenols are subject to both anaerobic and aerobic metabolism. Under anaerobic conditions, chlorinated phenols can undergo reductive dechlorination when suitable electron-donating substrates are available. Halorespiring bacteria are known which can use both low and highly chlorinated congeners of chlorophenol as electron acceptors to support growth. Many strains of halorespiring bacteria have the capacity to eliminate ortho-chlorines; however only bacteria from the species Desulfitobacterium hafniense (formerly frappieri) can eliminate para- and meta-chlorines in addition to ortho-chlorines. Once dechlorinated, the phenolic carbon skeletons are completely converted to methane and carbon dioxide by other anaerobic microorganisms in the environment. Under aerobic conditions, both lower and higher chlorinated phenols can serve as sole electron and carbon sources supporting growth. The best studied strains utilizing pentachlorophenol belong to the genera Mycobacterium and Sphingomonas. Two main strategies are used by aerobic bacteria for the degradation of chlorophenols. Lower chlorinated phenols for the most part are initially attacked by monooxygenases yielding chlorocatechols as the first intermediates. On the other hand, polychlorinated phenols are converted to chlorohydroquinones as the initial intermediates. Fungi and some bacteria are additionally known that cometabolize chlorinated phenols.     

31P nuclear magnetic resonance studies of effects of some chlorophenols on Escherichia coli and a pentachlorophenol-degrading bacterium.

A Flavobacterium sp. that mineralizes pentachlorophenol degrades some, but not all, of the other chlorinated phenols. Whole-cell 31P nuclear magnetic resonance was used to compare and observe transmembrane pH gradients and nucleotide pools in the Flavobacterium sp. and Escherichia coli after pentachlorophenol and 3,4,5-trichlorophenol were added to the cell suspensions. The data suggest that those chlorinated phenols which are not degraded by the Flavobacterium sp. may be resistant to degradation because they act as proton dissipators.     

Phenol hydroxylases: An update

This paper reviews the enzymology of microbial degradation of chlorinated phenols, a significant group of dangerous environmental pollutants. Two groups of phenol hydroxylases responsible for hydroxylation of (halo)phenols in the ortho or para position have been described. Among ortho-hydroxylating phenol hydroxylases, one-component flavoproteins or multicomponent enzyme systems are recognized, whereas single- or two-component enzyme systems catalyze para-hydroxylation of halophenols.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Degradation and O-methylation of chlorinated phenolic compounds by Rhodococcus and Mycobacterium strains.

Three polychlorophenol-degrading Rhodococcus and Mycobacterium strains were isolated independently from soil contaminated with chlorophenol wood preservative and from sludge of a wastewater treatment facility of a kraft pulp bleaching plant. Rhodococcus sp. strain CG-1 and Mycobacterium sp. strain CG-2, isolated from tetrachloroguaiacol enrichment, and Rhodococcus sp. strain CP-2, isolated from pentachlorophenol enrichment, mineralized pentachlorophenol and degraded several other polychlorinated phenols, guaiacols (2-methoxyphenols), and syringols (2,6-dimethoxyphenols) at micromolar concentrations and were sensitive to the toxic effects of pentachlorophenol. All three strains initiated degradation of the chlorophenols by para-hydroxylation, producing chlorinated para-hydroquinones, which were then further degraded. Parallel to degradation, strains CG-1, CG-2, and CP-2 also O-methylated nearly all chlorinated phenols, guaiacols, syringols, and hydroquinones. O-methylation of chlorophenols was a slow reaction compared with degradation. The preferred substrates of the O-methylating enzyme(s) were those with the hydroxyl group flanked by two chlorine substituents. O-methylation was constitutively expressed, whereas degradation of chlorinated phenolic compounds was inducible.     

Biodegradation of toxic and environmental pollutants.

Organic chemicals that are toxic to humans and to the environment can be transformed and metabolized by a variety of microorganisms. Such chemicals include trichloroethylene, chloroform, carbon tetrachloride, toluene, phenols, chlorinated phenols, polychlorinated biphenyls and polyaromatic hydrocarbons. This review focuses on some of the most important recent developments in the biodegradation of these toxic chemicals. Depending on the compound and the organism, the extent of our understanding ranges from the molecular level to the conceptual.     

Degradation of chlorinated phenols by a pentachlorophenol-degrading bacterium

A pentachlorophenol (PCP)-degrading Flavobacterium sp. was tested for its ability to dechlorinate other chlorinated phenols by using resting cells that had been grown in the presence or absence of PCP. Phenols with chlorine atoms at positions 2 and 6 of the phenol ring were dechlorinated completely by PCP-induced cells. Other chlorinated phenols were not significantly mineralized. When PCP was added to a culture growing on L-glutamate, there was a lag period before the start of PCP degradation. When similar cells were treated with chloramphenicol prior to the addition of PCP, they did not degrade added PCP, even after prolonged incubations. Thus, the enzymes necessary for PCP degradation appeared to be inducible. Suspensions of cells grown in the presence of 2,4,6-trichlorophenol or 2,3,5,6-tetrachlorophenol did not show a lag period for mineralization of PCP, 2,4,6-trichlorophenol, or 2,3,5,6-tetrachlorophenol, indicating that one enzyme system probably was induced for the biodegradation of all three compounds. Nondegradable chlorophenols were toxic toward the Flavobacterium sp., probably acting as uncouplers of oxidative phosphorylation.     

Degradation of chlorinated phenols by a pentachlorophenol-degrading bacterium.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. was tested for its ability to dechlorinate other chlorinated phenols by using resting cells that had been grown in the presence or absence of PCP. Phenols with chlorine atoms at positions 2 and 6 of the phenol ring were dechlorinated completely by PCP-induced cells. Other chlorinated phenols were not significantly mineralized. When PCP was added to a culture growing on L-glutamate, there was a lag period before the start of PCP degradation. When similar cells were treated with chloramphenicol prior to the addition of PCP, they did not degrade added PCP, even after prolonged incubations. Thus, the enzymes necessary for PCP degradation appeared to be inducible. Suspensions of cells grown in the presence of 2,4,6-trichlorophenol or 2,3,5,6-tetrachlorophenol did not show a lag period for mineralization of PCP, 2,4,6-trichlorophenol, or 2,3,5,6-tetrachlorophenol, indicating that one enzyme system probably was induced for the biodegradation of all three compounds. Nondegradable chlorophenols were toxic toward the Flavobacterium sp., probably acting as uncouplers of oxidative phosphorylation.     

Chlorinated phenols control the expression of the multidrug resistance efflux pump MexAB-OprM in Pseudomonas aeruginosa by interacting with NalC

NalC is a TetR type regulator that represses the multidrug efflux pump MexAB-OprM in Pseudomonas aeruginosa. Here we explain the mechanism of NalC-mediated regulation of MexAB-OprM. We show that NalC non-covalently binds chlorinated phenols and chemicals containing chlorophenol side-chains such as triclosan. NalC-chlorinated phenol binding results in its dissociation from promoter DNA and upregulation of NalC's downstream targets, including the MexR antirepressor ArmR. ArmR upregulation and MexR-ArmR complex formation have previously been shown to upregulate MexAB-OprM. In vivo mexB and armR expression analyses were used to corroborate in vitro NalC-chlorinated phenol binding. We also show that the interaction between chlorinated phenols and NalC is reversible, such that removal of these chemicals restored NalC promoter DNA binding. Thus, the NalC-chlorinated phenol interaction is likely a pertinent physiological mechanism that P. aeruginosa uses to control expression of the MexAB-OprM efflux pump.     

O-methylation of chlorinated phenols in the genus Rhodococcus

The ability to O-methylate chlorinated phenols and phenol derivatives in the genus Rhodococcus was studied. Several species and strains O-methylated chlorophenols to the corresponding anisoles, namely R. equi, R. erythropolis, R. rhodochrous, and Rhodococcus sp. strains P1 and An 117. The ability for a strain to O-methylate chlorophenols did not require that it had been isolated from an environment containing a chlorinated aromatic compound. O-methylation activity was stimulated by the presence of carbohydrate. All strains preferentially O-methylated a substrate with the hydroxyl group flanked by two chlorine substitunts.     

Interaction of chlorinated phenols with thyroxine binding sites of human transthyretin, albumin and thyroid binding globulin

Previous results (Brouwer and van den Berg, Toxicol. Appl. Pharmacol., 85 (1986) 301) indicated preferential binding of a hydroxylated metabolite of tetrachlorobiphenyl to transthyretin (TTR) a carrier of thyroxine (T4). In the present study it was investigated whether the T4 binding site of TTR could be occupied specifically by hydroxylated chlorinated aromatic compounds using chlorinated phenol congeners as model compounds in a competition assay with [125I]T4. Chlorinated aromatics such as 2,3-dichlorobenzene and 3,4,3',4'-tetrachlorobiphenyl, and phenols such as 4-hydroxybiphenyl and phenol were inefficient competitors. All chlorinated phenols tested were competitors for the T4 binding site of TTR. The ranking in competition was pentachlorophenol (PCP) greater than trichlorophenols greater than dichlorophenols greater than monochlorophenols. Structures with chlorine in both ortho positions to the hydroxyl group were more efficient competitors. The relative affinity of binding of pentachlorophenol (PCP) to TTR was about twice that of T4. Scatchard analysis showed that PCP mainly decreased the affinity constant K11 while the binding capacity R1 was not altered, indicating a competitive type of inhibition. PCP was also able to compete with T4 sites on albumin with a relative affinity of 0.25. T4 binding to thyroid binding globulin (TBG) was much less affected by interference of PCP (relative affinity 0.001). The results indicate a specific interaction of chlorophenols with the T4 binding site of TTR.     

Isolation and characterization of a 2,4-dichlorophenoxyacetic acid-degrading soil bacterium

Summary A 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, Xanthobacter sp. CP, was isolated after enrichment in aerated soil columns. A limited number of chlorinated phenols and chlorinated phenoxyalkanoic acids with an even number of carbon atoms in the side chain served as substrates for growth, although whole cells exhibited oxygen uptake with a wide range of those compounds. The maximal growth rate with 2,4-D was 0.13·h^-1 at a growth yield of 0.1 g biomass/g 2,4-D. Chloride ions were released quantitatively from 2,4-D and related chlorinated aromatic compounds which served as growth substrates. No by-products of 2,4-D metabolism were detected in oxygen-sufficient cultures of Xanthobacter sp. CP and catechols were cleaved exclusively by catechol 1,2-dioxygenase.     

Environmental biosensors for organochlorines, cyanobacterial toxins and endocrine disrupting chemicals

Environmental biosensors and related techniques for monitoring organochlorines, endocrine disrupting chemicals and cyanobacterial toxins are described. The practical requirements for an ideal environmental biosensor are analyzed. Specific case studies for environmental applications are reported for triazines chlorinated phenols, PCBs, microcystins, and endocrine disrupting chemicals. A new promising approach is reported for microcystins and alkylphenols that utilize electrooptical detection.     

Mutants of luminous bacteria selected for bioluminescent toxicity tests

Mutants of the luminescent bacterial strain NRRL B-11177 were isolated with pleiotropic hypersensitivity towards hydrophobic antimicrobial agents. SDS-PAGE analyses of outer membrane proteins and lipopolysaccharides revealed that the outer membrane structure of the ahs-mutants was altered. QSAR analysis showed that the inhibitory effect of chloro-substituted phenols on bioluminescence of the ahs-mutants depended on their hydrophobicity. The effect of chlorinated phenols and detergents on bioluminescence was increased in the ahs-mutants. The potential use of these mutants in bioluminescent toxicity tests was discussed.     

Most-probable-number technique for the enumeration of aromatic degraders in natural environments

A most-probable-number (MPN) method is described for the enumeration of heterotrophic populations capable of utilizing chlorinated and nonchlorinated benzoates and phenols as sole carbon sources. A correlation coefficient of 0.91 was obtained between the numbers determined by the MPN technique and the standard plate count. The MPN method gave realistic cell counts when population densities were low, and the presence of oligocarbophiles did not give spurious results.     

Biotechnological applications of peroxidases

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while co-immobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance.     

Molecular characteristics of xenobiotic-degrading sphingomonads

The genus Sphingomonas (sensu latu) belongs to the α-Proteobacteria and comprises strictly aerobic chemoheterotrophic bacteria that are widespread in various aquatic and terrestrial environments. The members of this genus are often isolated and studied because of their ability to degrade recalcitrant natural and anthropogenic compounds, such as (substituted) biphenyl(s) and naphthalene(s), fluorene, (substituted) phenanthrene(s), pyrene, (chlorinated) diphenylether(s), (chlorinated) furan(s), (chlorinated) dibenzo-p-dioxin(s), carbazole, estradiol, polyethylene glycols, chlorinated phenols, nonylphenols, and different herbicides and pesticides. The metabolic versatility of these organisms suggests that they have evolved mechanisms to adapt quicker and/or more efficiently to the degradation of novel compounds in the environment than members of other bacterial genera. Comparative analyses demonstrate that sphingomonads generally use similar degradative pathways as other groups of microorganisms but deviate from competing microorganisms by the existence of multiple hydroxylating oxygenases and the conservation of specific gene clusters. Furthermore, there is increasing evidence for the existence of plasmids that only can be disseminated among sphingomonads and which undergo after conjugative transfer pronounced rearrangements.     

Abbau chlorierter Benzole,Phenole und Cyclohexan-Derivate durch Benzol und Phenol verwertende Bodenbakterien unter aeroben Bedingungen

From soil samples of different origin (field, grassland and forest soils) small numbers ofNocardin andPseudomonas spec., able to utilize benzene and phenol could be isolated. Organisms which could only utilize phenol and phenolcarboxylic acids were more numerous and consisted mainly ofArthrobacter spec. It was tested to what extent these organisms could also utilize chlorinated aromatic and cyclohexane derivatives. For the degradation studies the bacteria were precultivated on benzene or p-hydroxybenzoic acid and then the compounds used were added. These compounds were labeled by¹⁴C and their degradation rates determined by measuring the¹⁴CO₂ release.Pseudomonas andNocardia spec. precultivated on benzene could also degrade the chlorinated derivatives of benzene and phenol. The monochlorinated derivates were degraded more easily than the di- and trichlorinated derivates. The chlorinated benzenes, especially in higher concentrations, were less degraded than the chlorinated phenols, but with lower concentrations their degradation rates were about similar. This was due to a higher toxicity of the benzenes. The phenol utilizingArthrobacter spec. were only able to degrade phenol and the chlorinated phenols. Benzoic and m-chlorobenzoic acid were degraded to CO₂ by thePseudomonas andNocardia spec. only. The benzene utilizing pseudomonads released more CO₂ from γ-pentachlorocyclohexane than from γ-hexachlorocyclohexane, but none from cyclehexane. Upon precultivation of benzene utilizing pseudomonads in glucose, the aromatic compounds were also degraded, but especially the chlorinated derivatives to a lower extent. In comparison with these soil organisms in pure culture, experiments with soil samples showed a degradation of all compounds which were used by the isolated organisms after variable induction periods. Cyclohexane was degraded slowly to CO₂ by the mixed soil flora in contrast to the benzene or phenol utilizing pure cultures.     

Metabolism of Halophenols by 2,4,5-trichlorophenoxyacetic acid-degrading Pseudomonas cepacia.

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 were able to completely and rapidly dechlorinate several chlorine-substituted phenols, including 2,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol. Several other trichlorophenols were only partially dechlorinated. The evidence suggests that 2,4,5-trichlorophenol is an intermediate in the degradation of 2,4,5-trichlorophenoxyacetic acid by strain AC1100. Moreover, although strain AC1100 was isolated by selection for growth on a chlorinated aromatic compound, brominated and fluorinated analogs were efficiently dehalogenated by strain AC1100 resting cells, whereas an iodinated analog was poorly dehalogenated.