Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100

Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).     

Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH(2))-utilizing monooxygenase, and FADH(2) was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH(2) and retarded its rapid oxidation by O(2). A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH(2)-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.     

Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100.

Burkholderia cepacia strain AC1100 can be induced for the degradation of 2,4,5-trichlorophenol (2,4,5-TCP). We have purified the active enzyme 30-fold to apparent homogeneity with a 44% yield by a two-step chromatographic procedure, and showed that it consists of a single type of subunit of 59 kDa based on SDS-PAGE using Coomassie blue and Sypro staining. This enzyme has no bound prosthetic group but requires exogenous addition of FAD and NADH to perform the dioxygen-dependent hydroxylation in the 4-position of 2,4,6-TCP. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of dioxygen per mol of 2,4,6-TCP with identification of the reaction product as 2,6-dichlorohydroquinone. Steady state kinetic parameters for cofactors and a variety of substrates were determined. Low K(m) values of 1+/-0.1 microM, 32+/-5 microM and 4+/-2 microM were found for FAD, NADH and 2,6-dichlorophenol (2,6-DCP), respectively, under saturating conditions for the two others. In the presence of 2,6-DCP as a substrate, methimazole (MMI) inhibited the enzyme competitively with a K(i)=27 microM. When other polychlorinated substrates were studied, IC(50) values for MMI were found in a range compatible with their apparent affinity. On the basis of aromatic product formation, NADH and O(2) consumption schemes for 2,4,6-TCP and 2,4,5-TCP degradation are discussed. A Blast search revealed that this enzyme has a high sequence identity (60%) with 2,4,6-TCP-4-monooxygenases from Burkholderia pickettii and from Azotobacter sp. strain GP1 which all of them catalyze para hydroxylative dehalogenation.     

2,4,5-T and 2,4,5-TCP induce oxidative damage in human erythrocytes: the role of glutathione

The molecular basis of the toxic properties of phenoxy herbicides in humans and animals has been insufficiently studied. In this study, damage parameters [levels of reduced glutathione (GSH) and total glutathione; activity of glutathione reductase (GR); activities of catalase (CAT) and superoxide dismutase (SOD); levels of adenine nucleotides and adenine energy charge (AEC)] were measured in human erythrocytes exposed in vitro to 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and its metabolite 2,4,5-trichlorophenol (2,4,5-TCP). Both 2,4,5-T and 2,4,5-TCP decreased the level of reduced glutathione (GSH) in erythrocytes in comparison to the control, but did not significantly change the total glutathione (2GSH + GSSG). This suggests that GSH concentration decreases concomitantly with an increase in oxidized glutathione (GSSG). 2,4,5-TCP at 100 ppm significantly decreased catalase and SOD activities. 2,4,5-T and 2,4,5-TCP did not significantly change the activity of glutathione reductase. 2,4,5-TCP decreased the level of ATP and increased the content of ADP and AMP, indicating a fall in AEC. 2,4,5-T and 2,4,5-TCP significantly changed the erythrocyte morphology. All these data are evidence of oxidative stress in erythrocytes incubated with 2,4,5-T and 2,4,5-TCP; the stress appears to be more intense in the case of 2,4,5-TCP.     

Degradation of 2,4,5-Trichlorophenoxyacetic acid by a Nocardioides simplex culture

A Nocardioides simplex strain 3E was isolated which totally dechlorinated 2,4,5-trichlorophenoxyacetic acid and was capable of its utilization as the sole source of carbon. The mechanism of 2,4,5-trichlorophenoxyacetic acid degradation by this strain was investigated. Chloroaromatic metabolites that occur in the lag, exponential and stationary growth phases of the strain Nocardioides simplex 3E were isolated and identified bases on a combination of TLC, GC-MS and HPLC data. Decomposition of 2,4,5-trichlorophenoxyacetic acid at the initial stage was shown to proceed by two pathways: via the splitting of the two-carbon fragment to yield 2,4,5-trichlorophenol and the reductive dechlorination to produce 2,4-dichlorophenoxyacetic acid. Hydrolytic dechlorination of 2,4,5-trichlorophenoxyacetic acid was found to yield dichlorohydroxyphenoxyacetic acid, thus pointing to the possible existence of a third branch at the initial stage of degradation of the xenobiotic. 2,4,5-Trichlorophenol and 2,4-dichlorophenoxyacetic acid produced during the metabolism of 2,4,5-trichlorophenoxyacetic acid and in experiments with resting cells are utilized by the strain Nocardioides simplex 3E as growth substrates.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2,4,5-TCP 2,4,5-trichlorophenol     

Process optimization and modeling of trichlorophenol degradation by Phanerochaete chrysosporium

The biodegradation of 2,4,6-trichlorophenol and 2,4,5-trichlorophenol by the white rot fungus Phanerochaete chrysosporium was studied in batch and continuous reactor systems. Experiments were conducted in shake flasks as well as in packed-bed reactors in which the fungus was immobilized. The degradation rates in the packed-bed reactors were found to be two orders of magnitude greater than those obtained in the shake flasks in which the fungus was just suspended. The degradation rate was found to be influenced by the concentrations of the carbon and nitrogen sources, pH, and fluid shear stress. Optimal ranges of these parameters to maximize biodegradation were determined. A mathematical model was developed in which the degradation process was assumed to consist of two sequential reaction steps, the first catalyzed by an extracellular enzyme system and the second requiring the presence of the mycelium. The deactivation of the extracellular enzyme system was also accounted for in the model. The Michaelis-Menten and the enzyme deactivation parameters were determined independently. Good agreement between the experimental data and the results produced by the regression was found. (c) 1995 John Wiley & Sons, Inc.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100.

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

Utilization of d-carnitine by Pseudomonas sp. AK 1

Abstract The degradation of chlorophenols by Alcaligenes eutrophus JMP134 (pJP4) was studied. The strain grew on 2,4,6-trichlorophenol or 2,4,6-tribromophenol as the sole carbon and energy source. Complete degradation of 2,4,6-trichlorophenol was confirmed by chloride release and gas chromatography analysis of supernatants from growth cultures. The 2,3,5-, 2,3,4-, 2,3,6-and 2,4,5-isomers of trichlorophenol did not support growth. However, up to 40% of 2,4,5-trichlorophenol was mineralized during growth of A. eutrophus on chemostats fed with either phenol (0.4 mM) or 2,4,6-trichlorophenol (0.4 mM) plus 2,4,5-trichlorophenol (0.1 mM). Growth on 2,4,6-trihalophenols was also observed in A. Eutrophus JMP222, the strain lacking pJP4, suggesting that this new degradative ability reported for A. eutrophus is not related to pJP4 encoded catabolic functions.     

Purification and Characterization of EDTA Monooxygenase from the EDTA-Degrading Bacterium BNC1

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O₂ were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH₂ and O₂. The FMN reductase provided EDTA monooxygenase with FMNH₂ by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35°C. K_ms were 34.1 μM for uncomplexed EDTA and 8.5 μM for MgEDTA²⁻; this difference in K_m indicates that the enzyme has greater affinity for MgEDTA²⁻. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH₂-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.     

Partial purification and characterisation of a 2,4,5-trichlorophenol detoxifying O-glucosyltransferase from wheat

An enzyme preparation with UDP-glucose-dependent O-glucosyltransferase (OGT; EC 2.4.1.-) activity toward 2,4,5-trichlorophenol has been purified 215-fold from wheat shoots. The OGT co-purified with the major extractable glucosylating activity toward the flavonol quercetin and was characterised as a monomeric 53 kDa protein. Among the xenobiotic phenols tested, the purified enzyme preparation showed at least a 10-fold preference for 2,4,5-trichlorophenol. When assayed with flavonoids, the OGT was active toward flavonols and coumestrol, showing a clear preference for 3-hydroxy flavone when incubated with a range of monohydroxylated flavonoids. It was concluded that the major 2,4,5-trichlorophenol-detoxifying OGT in wheat shoots is most probably a flavonol-3-O-glucosytransferase.     

Purification and characterization of maleylacetate reductase from Nocardioides simplex 3E utilizing phenoxyalcanoic herbicides 2,4-D and 2,4,5-T.

Maleylacetate reductase was isolated and purified from the Gram-positive strain Nocardioides simplex 3E which is able to utilize the phenoxyalcanoic herbicides 2,4-D and 2,4,5-T. Cells were grown on 2,4-D as the sole carbon source. The enzyme was purified by 380-fold with 3.0% yield. The purified maleylacetate reductase is a homodimer with subunit molecular mass of 37 kD. The enzyme required NADH as a cofactor; the Km for maleylacetate is 25 microM; Vmax (with NADH as cofactor) and kcat are 185 U/mg and 6845 min-1, respectively. The enzyme is very unstable; its pH and temperature optima are at 7.0-7.1 and 50 degrees C, respectively.     

Chlorinated phenoxyacetic acids and chlorophenols in the modified Allium test

The chlorinated phenoxyacetic acids 2-methyl-4-chlorophenoxyacetic acid (MCPA), 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4,5-T butoxyethyl ester and the chlorophenols 2,4-dichlorophenol and 2,4,5-trichlorophenol were tested for genotoxicity in the modified Allium test, which is based on exposure to the test chemicals of growing onions. The mean length of growing roots were measured and chromosome damage was recorded. Of the substances tested, MCPA was the most toxic and the chlorophenoxyacetic acids were more toxic than the chlorophenols. The lower threshold values for growth retardation were below 0.1 ppm for the acids, approx. at 0.1 ppm for the ester and less than 5 ppm for the phenols. Though a monocotyledon, Allium cepa was sensitive enough to respond to even low concentrations of these dicotyledon-selecting pesticides.     

Vanillate hydroxylase from the white rot basidiomycete Phanerochaete chrysosporium

A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN₃, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.Abbreviations MHQ Methoxy-p-hydroquinone - GLC gas liquid chromatography - TMSi trimethylsilane - TLC thin layer chromatography     

Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates.     

Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.     

Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures.

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.     

Purification and characterization of phenylacetaldehyde reductase from a styrene-assimilating Corynebacterium strain, ST-10.

A novel phenylacetaldehyde reductase was purified about 50-fold to homogeneity from Corynebacterium sp. strain ST-10, which can assimilate gaseous styrene as the sole carbon and energy source. The enzyme was inductively synthesized when grown on gaseous styrene and had an important role in styrene metabolism in vivo. The enzyme had a molecular weight of 155,000 and was composed of four identical subunits (molecular weight, 42,000). The enzyme catalyzed the reduction of not only phenylacetaldehyde but also various aldehydes and ketones; however, it did not catalyze the reverse reaction, the dehydrogenation of 2-phenylethanol. The enzyme required NADH as a cofactor and showed no activity with NADPH; therefore, it was defined as an NADH-dependent phenylacetaldehyde reductase. The enzyme stereospecifically produced (S)-(-)-1-phenylethanol from acetophenone; therefore, it would be useful as a biocatalyst.     

Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from Pseudomonas putida

4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines. This enzyme was purified to homogeneity from Pseudomonas putida by affinity chromatography. The protein had a molecular weight of 91,000 and was a dimer of identical subunits. It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity. The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2 x 10(-4) M and 5.9 x 10(-5) M respectively. It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.     

Biodegradation of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by dichlorophenol-adapted microorganisms from freshwater,anaerobic sediments

Reductive dechlorination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated in anaerobic sediments by non-adapted microorganisms and by microorganisms adapted to either 2,4- or 3,4-dichlorophenol (DCP). The rate of dechlorination of 2,4-D was increased by adaptation of sediment microorganisms to 2,4-DCP while dechlorination by sediment microorganisms adapted to 3,4-DCP displayed a lag phase similar to non-adapted sediment slurries. Both 2,4- and 3,4-DCP-adapted microorganisms produced 4-chlorophenoxyacetic acid by ortho-chlorine removal. Lag phases prior to dechlorination of the initial addition of 2,4,5-T by DCP-adapted sediment microorganisms were comparable to those from non-adapted sediment slurries. However, the rates of dechlorination increased upon subsequent additions of 2,4,5-T. Biodegradation of 2,4,5-T by sediment microorganisms adapted to 2,4- and/ or 3,4-DCP produced 2,5-D as the initial intermediate followed by 3-chlorophenol and phenol indicating a para > ortho > meta order of dechlorination. Dechlorination of 2,4,5-T, by either adapted or non-adapted sediment microorganisms, progressed without detection of 2,4,5-trichlorophenol as an intermediate.