Biodegradation of the mixture of 2,4,6-trichlorophenol, 4-chlorophenol,and phenol by a defined mixed culture

Two new strains, Pseudomonas sp. TCP114 degrading 2,4,6-trichlorophenol (TCP) and Arthrobacter sp. CPR706 degrading 4-chlorophenol (4-CP), were isolated through a selective enrichment procedure. Both strains could also degrade phenol. The degradability of one component by a pure culture was strongly affected by the presence of other compounds in the medium. For example, when all three components (TCP, 4-CP, and phenol) were present in the medium, a pure culture of CPR706 could not degrade any of the components present. This restriction on degradability could be overcome by employing a defined mixed culture of the two strains. The mixed culture could degrade all three components in the mixture through cooperative activity. It was also demonstrated that the mixed culture could be immobilized by using calcium alginate for the semi-continuous degradation of the three-component mixture. Immobilization not only accelerates the degradation rate, but also enables reuse of the cell mass several times without losing the cells' degrading capabilities.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Biodegradation of p-nitrophenol and 4-chlorophenol by Stenotrophomonas sp

A bacterium named LZ-1 capable of utilizing high concentrations of p-nitrophenol (PNP) (up to 500 mg L(-1)) as the sole source of carbon, nitrogen and energy was isolated from an activated sludge. Based on the results of phenotypic features and phylogenetic similarity of 16S rRNA gene sequences, strain LZ-1 was identified as a Stenotrophomonas sp. Other p-substituted phenols such as 4-chlorophenol (4-CP) were also degraded by strain LZ-1, and both PNP and 4-CP were degraded via the hydroquinone pathway exclusively. Strain LZ-1 could degrade PNP and 4-CP simultaneously and the degradation of PNP was greatly accelerated due to the increased biomass supported by 4-CP. An indigenous plasmid was found to be responsible for phenols degradation. In soil samples, 100 mg kg(-1) of PNP and 4-CP in mixtures were removed by strain LZ-1 (10(6) cells g(-1)) within 14 and 16 days respectively, and degradation activity was maintained over a wide range of temperatures (4-35 degrees C). Therefore, strain LZ-1 can potentially be used in bioremediation of phenolic compounds either individually or as a mixture in the environment.     

Biodegradation of phenol and chlorophenols with defined mixed culture in shake-flasks and a packed bed reactor

Pseudomonas testosteroni CPW301 degraded phenol and 4-chlorophenol simultaneously, but degradation rates of these compounds were affected by 4-chlorophenol. Phenol increased the cell concentration and therefore the degradation efficiency of 4-chlorophenol was improved. Pseudomonas solanacearum TCP114 could degrade only 2,4,6-trichlorophenol. A defined mixed culture of P. testosteroni CPW301 and P. solanacearum TCP114 could treat phenol, 4-chlorophenol, and 2,4,6-trichlorophenol completely and overcome the inhibition of substrates to other microorganisms. The degradation capacity of the packed bed reactor (PBR) was higher than that of the continuous stirred tank reactor, but the PBR was unsuitable for oxygen-sensitive microorganisms.     

Degradation and dehalogenation of monochlorophenols by the phenol-assimilating yeast Candida maltosa

The phenol-assimilating yeast Candida maltosa is able to degrade monochlorophenols but cannot grow on these substrates. 3- and 4-chlorophenol were broken down very rapidly by phenol-grown cells under the formation of 4-chlorocatechol, 5-chloropyrogallol and 4-carboxymethylenebut-2-en-4-olide with concomitant release of chloride.2-Chlorophenol was partially converted into cis,cis-2-chloromuconic acid via 3-chlorocatechol which was also obtained from 3-chlorophenol in low amounts. No further metabolites containing chloride were found.The dehalogenation step in the chlorophenol degradation is the cycloisomerization of the cis,cis-chloromuconic acid to 4-carboxymethylenebut-2-en-4-olide in the ortho fission pathway.Dedicated to prof.Dr. E. Bayer, Tübingen, on the occasion of his 65th birthday.     

Degradation of n-alkanes and polycyclic aromatic hydrocarbons in petroleum by a newly isolated Pseudomonas aeruginosa DQ8

A bacterial isolate, designated as DQ8, was found capable of degrading diesel, crude oil, n-alkanes and polycyclic aromatic hydrocarbons (PAHs) in petroleum. Strain DQ8 was assigned to the genus Pseudomonas aeruginosa based on biochemical and genetic data. The metabolites identified from n-docosane as substrate suggested that P. aeruginosa DQ8 could oxidize n-alkanes via a terminal oxidation pathway. P. aeruginosa DQ8 could also degrade PAHs of three or four aromatic rings. The metabolites identified from fluorene as substrate suggested that P. aeruginosa DQ8 may degrade fluorene via two pathways. One is monooxygenation at C-9 of fluorene, and the other is initiated by dioxygenation at C-3 and C-4 of fluorene. P. aeruginosa DQ8 should be of great practical significance both in bioremediation of oil-contaminated soils and biotreatment of oil wastewater.     

Metabolism of phenylbenzoate by Pseudomonas sp. strain TR3

A bacterial isolate, tentatively identified as Pseudomonas sp. strain TR3, was found to utilize the diaryl ester phenylbenzoate as sole source of carbon and energy. This strain has the ability to productively degrade phenylbenzoate and some substituted derivatives by a catabolic sequence which was characterized biochemically. The biodegradation of phenylbenzoate is thus initiated by an inducible esterase, effectively hydrolyzing the diaryl esters to produce stoichiometric amounts of two monoaromatic metabolites, identified as benzoate and phenol in the case of phenylbenzoate. The diaryl ester p-tolylbenzoate was hydrolyzed to yield benzoate and 4-methylphenol while 4-chlorophenylbenzoate gave rise to the production of benzoate and 4-chlorophenol. These monoaromatic catabolites were further degraded via the oxoadipate pathway.     

Anaerobic biodegradation of chlorophenols in fresh and acclimated sludge.

We investigated the anaerobic biodegradation of mono- and dichlorophenol isomers by fresh (unacclimated) sludge and by sludge acclimated to either 2-chlorophenol, 3-chlorophenol, or 4-chlorophenol. Biodegradation was evaluated by monitoring substrate disappearance and, in selected cases, production of 14CH4 from labeled substrates. In unacclimated sludge, each of the monochlorophenol isomers was degraded. The relative rates of disappearance were in this order: ortho greater than meta greater than para. For the dichlorophenols in unacclimated sludge, reductive dechlorination of the Cl group ortho to phenolic OH was observed, and the monochlorophenol compounds released were subsequently degraded. 3,4-Dichlorophenol and 3,5-dichlorophenol were persistent. Sludge acclimated to 2-chlorophenol cross-acclimated to 4-chlorophenol but did not utilize 3-chlorophenol. This sludge also degraded 2,4-dichlorophenol. Sludge acclimated to 3-chlorophenol cross-acclimated to 4-chlorophenol but not to 2-chlorophenol. This sludge degraded 3,4- and 3,5-dichlorophenol but not 2,3- or 2,5-dichlorophenol. The specific cross-acclimation patterns observed for monochlorophenol degradation demonstrated the existence of two unique microbial activities that were in turn different from fresh sludge. The sludge acclimated to 4-chlorophenol could degrade all three monochlorophenol isomers and 2,4- and 3,4-dichlorophenol. The active microbial population in this sludge appeared to be a mixture of populations present in the 2-chlorphenol- and 3-chlorophenol-acclimated sludges, both of which could utilize 4-chlorophenol. Experiments with 14C-radiolabeled p-chlorophenol, o-chlorophenol, and 2,4-dichlorophenol demonstrated that these compounds were converted to 14CH4 and 14CO2.     

Degradation of 4-Chlorophenol at Low Temperature and during Extreme Temperature Fluctuations by Arthrobacter chlorophenolicus A6

Low average temperatures and temperature fluctuations in temperate soils challenge the efficacy of microbial strains used for clean up of pollutants. In this study, we investigated the cold tolerance of Arthrobacter chlorophenolicus A6, a microorganism previously shown to degrade high concentrations of 4-chlorophenol at 28°C. Luciferase activity from a luc-tagged derivative of the strain (A6L) was used to monitor the metabolic status of the population during 4-chlorophenol degradation. The A6L strain could degrade 200–300 g mL^–1 4-chlorophenol in pure cultures incubated at 5°C, although rates of degradation, growth and the metabolic status of the cells were lower at 5°C compared to 28°C. When subjected to temperature fluctuations between 5 and 28°C, A6L continued to degrade 4-chlorophenol and remained active. In soil microcosm experiments, the degradation rates were significantly faster the first week at 28°C, compared to 5°C. However, this difference was no longer seen after 7 days, and equally low 4-chlorophenol concentrations were reached after 17 days at both temperatures. During 4-chlorophenol degradation in soil, CFU and luciferase activity values remained constant at both 5 and 28°C. However, once most of the 4-chlorophenol was degraded, both values decreased by 1–1.5 logarithmic values at 28°C, whereas they remained constant at 5°C, indicating a high survival of the cells at low temperatures. Because of the ability of A. chlorophenolicus A6 to degrade high concentrations of 4-chlorophenol at 5°C, together with its tolerance to temperature fluctuations and stress conditions found in soil, this strain is a promising candidate for bioaugmentation of chlorophenol-contaminated soil in temperate climates.This revised version was published online in November 2004 with corrections to Volume 48.     

Novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6, a previously described 4-chlorophenol-degrading strain, was found to degrade 4-chlorophenol via hydroxyquinol, which is a novel route for aerobic microbial degradation of this compound. In addition, 10 open reading frames exhibiting sequence similarity to genes encoding enzymes involved in chlorophenol degradation were cloned and designated part of a chlorophenol degradation gene cluster (cph genes). Several of the open reading frames appeared to encode enzymes with similar functions; these open reading frames included two genes, cphA-I and cphA-II, which were shown to encode functional hydroxyquinol 1,2-dioxygenases. Disruption of the cphA-I gene yielded a mutant that exhibited negligible growth on 4-chlorophenol, thereby linking the cph gene cluster to functional catabolism of 4-chlorophenol in A. chlorophenolicus A6. The presence of a resolvase pseudogene in the cph gene cluster together with analyses of the G+C content and codon bias of flanking genes suggested that horizontal gene transfer was involved in assembly of the gene cluster during evolution of the ability of the strain to grow on 4-chlorophenol.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain.

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Enrichment, isolation, and characterization of 4-chlorophenol-degrading bacterium Rhizobium sp. 4-CP-20

The objectives of this research were to monitor the variations of species in mixed cultures during the enrichment period, isolate species and identify and characterize the pure 4-chlorophenol (4-CP) degrading strains from enriched mixed cultures. Strain Rhizobium sp. 4-CP-20 was isolated from the acclimated mixed culture. The DGGE result indicated that strain Rhizobium sp. 4-CP-20 was undetectable at the beginning but detectable after 2 weeks of enrichment. The optimum growth temperatures for Rhizobium sp. 4-CP-20 were both 36°C using 350 mg l⁻¹ glucose or sodium acetate as the substrate. The optimum pH range for degrading 100 mg l⁻¹ 4-CP was between 6.89 and 8.20. Strain Rhizobium sp. 4-CP-20 could degrade 4-CP completely within 3.95 days, as the initial 4-CP concentration was 100 mg l⁻¹. If the initial 4-CP concentration was higher than 240 mg l⁻¹, the growth of bacterial cells and the activity of degrading 4-CP were both inhibited.     

Degradation of 4-chlorophenol in municipal wastewater by adsorptiv immobilized Alcaligenes sp. A 7-2

Summary Alcaligenes sp. A 7-2 has been applied in a packed-bed fermenter to degrade 4-chlorophenol in municipal wastewater continuously. With sterile wastewater degradation rates up to 300 mol/l/h were reached when precultivated Alcaligenes sp. A 7-2 had been adsorbed onto the Lecaton-packed-bed-material.The natural microbial population of the wastewater was not able to degrade 4-chlorophenol. Beside an accumulation of the haloaromatic compound a yellow-greenish substance exhibiting the spectral characteristics of 5-chloro-2-hydroxymuconic acid semialdehyde was found.This compound caused a rapid decrease in metabolic activity of the microbial culture.With non-sterile wastewater Alcaligenes sp. A 7-2 could not be established as member of the natural mixed population. Due to the poor retainment of the specialized strain in the packed-bed the degradation capacity of the fermentation system decreased and 4-chlorophenol was accumulated.     

Substrate-dependent autoaggregation of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1 during the degradation of mono-chlorophenols and phenol

A bacterium, CP1, identified as Pseudomonas putida strain, was investigated for its ability to grow on and degrade mono-chlorophenols and phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1.56 mM 2- and 3-chlorophenol, 2.34 mM 4-chlorophenol and 8.5 mM phenol using an ortho-cleavage pathway. P. putida CP1, acclimated to degrade 2-chlorophenol, was capable of 3-chlorocatechol degradation, while P. putida, acclimated to 4-chlorophenol degradation, degraded 4-chlorocatechol. Growth of P. putida CP1 on higher concentrations of the mono-chlorophenols, ≥1.56 mM 4-chlorophenol and ≥0.78 mM 2- and 3-chlorophenol, resulted in decreases in cell biomass despite metabolism of the substrates, and the formation of large aggregates of cells in the culture medium. Increases in cell biomass with no clumping of the cells resulted from growth of P. putida CP1 on phenol or on lower concentrations of mono-chlorophenol. Bacterial adherence to hydrocarbons (BATH) assays showed cells grown on the higher concentrations of mono-chlorophenol to be more hydrophobic than those grown on phenol and lower concentrations of mono-chlorophenol. The results suggested that increased hydrophobicity and autoaggregation of P. putida CP1 were a response to toxicity of the added substrates. Journal of Industrial Microbiology & Biotechnology (2002) 28, 316–324 DOI: 10.1038/sj/jim/7000249 Received 27 June 2001/ Accepted in revised form 09 February 2002     

Biodegradation of 4-chlorophenol by adsorptive immobilized Alcaligenes sp. A 7-2 in soil

Summary Alcaligenes sp. A 7-2 immobilized on granular clay has been applied in a percolator to degrade 4-chlorophenol in sandy soil. Good adsorption rates on granular clay were achieved using cell suspensions with high titres and media at pH 8.0. The influence of various parameters such as aeration rate, pH, temperature, concentration of 4-chlorophenol and size of inoculum on the degradation rate were investigated. During fedbatch fermentations under optimal culture conditions, concentrations of 4-chlorophenol up to 160 mg·1^–1 could be degraded. Semicontinuous culture experiments demonstrated that the degradation potential in soil could be well established and enhanced by the addition of immobilized bacteria. Continuous fermentation was performed with varying 4-chlorophenol concentrations in the feed and different input levels. The maximum degradation rate was 1.64 g·1^–1·day^–1. Offprint requests to: H.-J. Rehm     

Degradation of mono-chlorophenols by a mixed microbial community via a meta- cleavage pathway

A mixed microbial community, specially designed todegrade a wide range of substituted aromaticcompounds, was examined for its ability to degrademono-chlorophenols as sole carbon source in aerobicbatch cultures. The mixed culture degraded 2-, 3-, and4 -chlorophenol (1.56 mM) via a meta- cleavagepathway. During the degradation of 2- and3-chlorophenol by the mixed culture, 3-chlorocatecholproduction was observed. Further metabolism was toxicto cells as it led to inactivation of the catechol2,3-dioxygenase enzyme upon meta- cleavage of3-chlorocatechol resulting in incomplete degradation.Inactivation of the meta- cleavage enzyme led toan accumulation of brown coloured polymers, whichinterfered with the measurement of cell growth usingoptical denstiy. Degradation of 4-chlorophenol by themixed culture led to an accumulation of5-chloro-2-hydroxymuconic semialdehyde, themeta- cleavage product of 4-chlorocatechol. Theaccumulation of this compound did not interfere withthe measurement of cell growth using optical density.5-chloro-2-hydroxymuconic semialdehyde was furthermetabolized by the mixed culture with a stoichiometricrelease of chloride, indicating complete degradationof 4-chlorophenol by the mixed culture via ameta- cleavage pathway.     

Improved Degradation of Monochlorophenols by a Constructed Strain

Pseudomonas sp. strain B13, a strain able to degrade 3-chlorobenzoate and, after prolonged adaptation (40 days), 4-chlorophenol, could transfer the ability to degrade chlorocatechols to a recipient, Alcaligenes sp. strain A7, which is able to grow with benzoate and phenol. Representative transconjugants, such as Alcaligenes sp. strain A7-2, were able to utilize all three isomeric chlorophenols; this property was not possessed by the donor or the recipient. The ability to grow readily with 4-chlorophenol may be attributable to a more rapid induction of phenol hydroxylase by Alcaligenes sp. strain A7-2 than by Pseudomonas sp. strain B13, a property which correlates with the greater level of resistance to chlorophenols shown by the transconjugant.     

Degradation of prochloraz and 2,4,6-trichlorophenol by environmental bacterial strains

 Eight bacterial isolates from enrichment with 2,4,6-trichlorophenol (TCP) as sole carbon source were tested for their potential to degrade prochloraz. None of them could grow on prochloraz. Strain C964, identified as Aureobacterium sp., effectively reduced the fungitoxic activity of prochloraz in a bioassay and degradation was confirmed by HPLC. Two other isolates, strain C611 and C961, using TCP as a carbon source, belong to the β subclass of the proteobacteria and presumely degrade TCP via 2,4-dichlorohydroquinone and hydroxyhydroquinone as indicated by oxygen-consumption tests. Received: 3 July 1995/Received revision: 27 July 1995/Accepted: 31 July 1995     

Novel 4-Chlorophenol Degradation Gene Cluster and Degradation Route via Hydroxyquinol in Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6, a previously described 4-chlorophenol-degrading strain, was found to degrade 4-chlorophenol via hydroxyquinol, which is a novel route for aerobic microbial degradation of this compound. In addition, 10 open reading frames exhibiting sequence similarity to genes encoding enzymes involved in chlorophenol degradation were cloned and designated part of a chlorophenol degradation gene cluster (cph genes). Several of the open reading frames appeared to encode enzymes with similar functions; these open reading frames included two genes, cphA-I and cphA-II, which were shown to encode functional hydroxyquinol 1,2-dioxygenases. Disruption of the cphA-I gene yielded a mutant that exhibited negligible growth on 4-chlorophenol, thereby linking the cph gene cluster to functional catabolism of 4-chlorophenol in A. chlorophenolicus A6. The presence of a resolvase pseudogene in the cph gene cluster together with analyses of the G+C content and codon bias of flanking genes suggested that horizontal gene transfer was involved in assembly of the gene cluster during evolution of the ability of the strain to grow on 4-chlorophenol.