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.     

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.     

Biodegradation of chlorinated phenolic compounds

Chlorophenolic compounds are generated from a number of industrial manufacturing processes including pulp and paper manufacture. These compounds are found to be toxic and recalcitrant and hence their discharge into the environment must be regulated. Slow and partial degradation of chlorophenols under aerobic and anaerobic natural environment has been observed. Aerobic biodegradation of chlorophenols proceeds through the formation of catechols while under anaerobic conditions, reductive dehalogenation is the preferred metabolic pathway. Number and position of chlorine substituents on the phenolic ring has influence on the rate and extent of biodegradation of chlorophenols. In engineered systems, acclimatization of biomass to chlorophenols markedly enhances the biodegradation ability by reducing the initial lag phase and by countering inhibition. Partial removal of chlorophenols between 40-60% is usually observed in aerobic and anaerobic processes. Removal can be enhanced by a combination of aerobic and anaerobic operations.     

Degradation of chlorinated nitroaromatic compounds

Chlorinated nitroaromatic compounds (CNAs) are persistent environmental pollutants that have been introduced into the environment due to the anthropogenic activities. Bacteria that utilize CNAs as the sole sources of carbon and energy have been isolated from different contaminated and non-contaminated sites. Microbial metabolism of CNAs has been studied, and several metabolic pathways for degradation of CNAs have been proposed. Detoxification and biotransformation of CNAs have also been studied in various fungi, actinomycetes and bacteria. Several physicochemical methods have been used for treatment of wastewater containing CNAs; however, these methods are not suitable for in situ bioremediation. This review describes the current scenario of the degradation of CNAs.     

Degradation of pyridine by Arthrobacter crystallopoietes and Rhodococcus opocus strains

Abstract Two pyridine-degrading microorganisms Arthrobacter crystallopoietes (VKM Ac-1334D) and Rhodococcus opacus (VKM Ac-1333D) were isolated from soil. The Gas chromatography-mass spectroscopy analysis showed that the former species formed 3-hydroxypyridine, 2,3- and 2,6-dihydroxypyridines during its growth in media containing pyridine, while the latter formed 2-hydroxy- and 2,6-dihydroxypyridines as degradation intermediates. Products of the pyridine ring cleavage (5-amino-2-oxo-4-pentenoic acid and 3-pentenoic acid monoamide) were also detected.     

Degradation of chlorinated biphenyls and products of their bioconversion by Rhodococcus sp. B7a strain

Strain Rhodococcus sp. B7a isolated from artificially polluted soil destructs mono- and di-substituted ortho- and/or para-chlorinated biphenyls with utilization of chlorinated benzoic acids and shows high degradation activity as regards trichlorinated biphenyls. It is shown that p-hydroxybenzoic and protocatehoic acids are the products of p-chlorobenzoic acid catabolism.     

Transformation of chlorinated phenolic compounds in the genusRhodococcus

The ability of strains of the genusRhodococcus to transform chlorinated phenolic compounds was studied. Noninduced cells of several strains ofRhodococcus, covering at least eight species, were found to attack mono-, di-, and trichlorophenols by hydroxylation at theortho position to chlorocatechols. 3-chlorophenol and 4-chlorophenol were converted to 4-chlorocatechol, 2,3-dichlorophenol to 3,4-dichlorocatechol, and 3,4-di-chlorophenol to 4,5-dichlorocatechol. The chlorocatechols accumulated to nearly stoichiometric amounts. Other mono- and dichlorophenols were not transformed. The ability of the strains to hydroxylate chlorophenols correlated with the ability to grow on unsubstituted phenol as the sole source of carbon and energy. SeveralRhodococcus strains attacked chlorophenolic compounds by both hydroxylation and O-methylation. 2,3,4-, 2,3,5- and 3,4,5-trichlorophenol were hydroxylated to trichlorocatechol and then sequentially O-methylated to chloroguaiacol and chloroveratrole. Tetrachlo-rohydroquinone was O-methylated sequentially to tetrachloro-4-methoxy-phenol and tetrachloro-1,4-dimethoxybenzene. Several of the active strains had no known history of exposure to any chloroaromatic compound. Rhodococci are widely distributed in soil and sludge and these results suggest that this genus may play an important role in transformation of chlorinated phenolic compounds in the environment.     

Degradation of 2-methylaniline and chlorinated isomers of 2-methylaniline by Rhodococcus rhodochrous strain CTM

Rhodococcus rhodochrous strain CTM co-metabolized 2-methylaniline and some of its chlorinated isomers in the presence of ethanol as additional carbon source. Degradation of 2-methylaniline proceeded via 3-methylcatechol, which was metabolized mainly by meta-cleavage. In the case of 3-chloro-2-methylaniline, however, only a small proportion (about 10%) was subjected to meta-cleavage; the chlorinated meta-cleavage product was accumulated in the culture fluid as a dead-end metabolite. In contrast, 4-chloro-2-methylaniline was degraded via ortho-cleavage exclusively. Enzyme assays showed the presence of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase as inducible enzymes in strain CTM. Extended cultivation of strain CTM with 2-methylaniline and 3-chloro-2-methylaniline yielded mutants, including R. rhodochrous strain CTM2, that had lost catechol 2,3-dioxygenase activity; these mutants degraded the aromatic amines exclusively via the ortho-cleavage pathway. DNA hybridization experiments using a gene probe revealed the loss of the catechol 2,3-dioxygenase gene from strain CTM2.     

Degradation of phenol and phenolic compounds by Pseudomonas putida EKII

Summary The phenol-degrading strain Pseudomonas putida EKII was isolated from a soil enrichment culture and utilized phenol up to 10.6 mM (1.0 g·1 ^-1) as the sole source of carbon and energy. Furthermore, cresols, chlorophenols, 3,4-dimethylphenol, and 4-chloro-m-cresol were metabolized as sole substrates by phenol-grown resting cells of strain EKII. Under conditions of cell growth, degradation of these xenobiotics was achieved only in co-metabolism with phenol. Phenol hydroxylase activity was detectable in whole cells but not in cell-free extracts. The specificity of the hydroxylating enzyme was found during transformation of cresols and chlorophenols: ortho- and meta-substituted phenols were degraded via 3-substituted catechols, while degradation of para-substituted phenols proceeded via 4-substituted catechols. In cell-free extracts of phenol-grown cells a high level of catechol 2,3-dioxygenase as well as smaller amounts of 2-hydroxymuconic semialdehyde hydrolyase and catechol 1,2-dioxygenase were detected. The ring-cleaving enzymes were characterized after partial purification by DEAE-cellulose chromatography.     

Hydroxylation and dechlorination of chlorinated guaiacols and syringols by Rhodococcus chlorophenolicus.

We show that Rhodococcus chlorophenolicus PCP-I, a polychlorophenol degrader, also degrades various chlorine-substituted guaiacols (2-methoxyphenols) and syringols (2,6-dimethoxyphenols). The substrates investigated were tetrachloroguaiacol, 3,4,6- and 3,5,6-trichloroguaiacol, 3,5- and 3,6-dichloroguaiacol, trichlorosyringol, and 3,5-dichlorosyringol. The first step was a hydroxylation, probably in a position para to the preexisting hydroxyl. Tetrachloroguaiacol and trichlorosyringol, with a chlorine substituent in the para position, were both hydroxylated and dechlorinated. The optimum temperature for degradation of polychlorinated guaiacols and syringols was 37 to 41 degrees C. Degradation of polychlorinated phenols, guaiacols, and syringols by R. chlorophenolicus was inducible, and induction was controlled coordinately.     

Degradation of phenol and phenolic compounds by a defined denitrifying bacterial culture

Phenol, a major pollutant in several industrial waste waters is often used as a model compound for studies on biodegradation. This study investigated the anoxic degradation of phenol and other phenolic compounds by a defined mixed culture of Alcaligenes faecalis and Enterobacter species. The culture was capable of degrading high concentrations of phenol (up to 600 mg/l) under anoxic conditions in a simple minimal mineral medium at an initial cell mass of 8 mg/l. However, the lag phase in growth and phenol removal increased with increase in phenol concentration. Dissolved CO₂ was an absolute requirement for phenol degradation. In addition to nitrate, nitrite and oxygen could be used as electron acceptors. The kinetic constants, maximum specific growth rate _max; inhibition constant, K _i and saturation constant, K _s were determined to be 0.206 h^–1, 113 and 15 mg phenol/l respectively. p-Hydroxybenzoic acid was identified as an intermediate during phenol degradation. Apart from phenol, the culture utilized few other monocyclic aromatic compounds as growth substrates. The defined culture has remained stable with consistent phenol-degrading ability for more than 3 years and thus shows promise for its application in anoxic treatment of industrial waste waters containing phenolic compounds.     

Degradation of o-toluidine by Rhodococcus rhodochrous

Abstract A Gram-positive bacterium with the ability to utilize o -toluidine as sole source of carbon and nitrogen was isolated from soil. The organism was identified as Rhodococcus rhodochrous Sb 4. 3-Methylcatechol and the meta-fission product of 3-methylcatechol were identified as metabolites. A pathway for the degradation of o -toluidine is proposed.     

Rifampicin inactivation by Rhodococcus and Mycobacterium species

When prokaryotes are exposed to inhibitory concentrations of the antibiotic rifampicin, the only means hitherto identified by which cells overcome this inhibition is through mutational alteration in the target moiety, DNA-dependent RNA polymerase. In the nocardioform bacterium Rhodococcus erythropolis a novel mechanism has been identified, consisting of an inducible rifampicin-inactivating mechanism. Changes in the drug absorbance spectrum paralleled the decline in bacteriostatic activity of the antibiotic.     

Metabolism of the herbicide atrazine by Rhodococcus strains.

Rhodococcus strains were screened for their ability to degrade the herbicide atrazine. Only rhodococci that degrade the herbicide EPTC (s-ethyl-dipropylthiocarbamate) metabolized atrazine. Rhodococcus strain TE1 metabolized atrazine under aerobic conditions to produce deethyl- and deisopropylatrazine, which were not degraded further and which accumulated in the incubation medium. The bacterium also metabolized the other s-triazine herbicides propazine, simazine, and cyanazine. The N dealkylation of triazine herbicides by Rhodococcus strain TE1 was associated with a 77-kb plasmid previously shown to be required for EPTC degradation.     

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.     

Hydroxylation and dechlorination of chlorinated guaiacols and syringols by Rhodococcus chlorophenolicus

We show that Rhodococcus chlorophenolicus PCP-I, a polychlorophenol degrader, also degrades various chlorine-substituted guaiacols (2-methoxyphenols) and syringols (2,6-dimethoxyphenols). The substrates investigated were tetrachloroguaiacol, 3,4,6- and 3,5,6-trichloroguaiacol, 3,5- and 3,6-dichloroguaiacol, trichlorosyringol, and 3,5-dichlorosyringol. The first step was a hydroxylation, probably in a position para to the preexisting hydroxyl. Tetrachloroguaiacol and trichlorosyringol, with a chlorine substituent in the para position, were both hydroxylated and dechlorinated. The optimum temperature for degradation of polychlorinated guaiacols and syringols was 37 to 41 degrees C. Degradation of polychlorinated phenols, guaiacols, and syringols by R. chlorophenolicus was inducible, and induction was controlled coordinately.     

Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus

Summary An actinomycete, Rhodococcus chlorophenolicus, isolated from a pentachlorophenol-degrading mixed bacterial culture is a polychlorophenol degrader. It was shown to oxidize pentachlorophenol into carbon dioxide and to metabolize also 2,3,4,5-,2,3,4,6-, and 2,3,5,6-tetrachlorophenol, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,6-, and 2,4,5-trichlorophenol, 2,5-, and 2,6-dichlorophenol and tetrachloro-p-hydroquinone in an inducible manner. Pentachlorophenol set on the synthesis of enzymes required for the metabolism of all these chlorophenols and of tetrachloro-p-hydroquinone. 2,4,5-, and 2,4,6-trichlorophenol and 2,5-, and 2,6-dichlorophenol were degraded by R. chlorophenolicus cells only if these had previous contact to pentachlorophenol. Other chlorophenols mentioned were able to set on the synthesis of enzymes for their own degradation. 2,3,4,5-, and 2,3,4,6-tetrachlorophenol, and 2,3,5-, 2,4,5-, and 3,4,5-trichlorophenol were more toxic to R. chlorophenolicus than the other chlorophenols, but nevertheless 2,3,4,5-, and 2,3,4,6-tetrachlorophenol and 2,3,5-trichlorophenol were readily degraded by the bacteria.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - TeCH tetrachloro-p-hydroquinone An example of numeration: 2345-TeCP, 2,3,4,5-tetrachlorophenol