Transformation of diphenyl ethers by Trametes versicolor and characterization of ring cleavage products

The white-rot fungi Trametes versicolor SBUG 1050, DSM 11269 and DSM 11309 are able to oxidize diphenyl ether and its halogenated derivatives 4-bromo- and 4-chlorodiphenyl ether. The products formed from diphenyl ether were 2- and 4-hydroxydiphenyl ether. Both 4-bromo- and 4-chlorodiphenyl ether were transformed to the corresponding products hydroxylated at the non-halogenated ring. Additionally, ring-cleavage products were detected by high perfomance liquid chromatography and characterized by gas chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Unhalogenated diphenyl ether was degraded to 2-hydroxy-4-phenoxymuconic acid and 6-carboxy-4-phenoxy-2-pyrone. Brominated derivatives of both these compounds were formed from 4-bromodiphenyl ether, and 4-chlorodiphenyl ether was transformed in the same way to the analogous chlorinated ring cleavage products. Additionally, 4-bromo- and 4-chlorophenol were detected as intermediates from 4-bromo- and 4-chlorodiphenyl ether, respectively. In the presence of the cytochrome-P450 inhibitor 1-aminobenzotriazole, no metabolites were formed by cells of Trametes versicolor from the diphenyl ethers investigated. Cell-free supernatants of whole cultures with high laccase and manganese peroxidase activities were not able to transform the unhydroxylated diphenyl ethers used.     

Expression of an Erwinia sp. gene encoding diphenyl ether cleavage in Escherichia coli and an isolated Acinetobacter strain PE7

Summary An Acinetobacter strain PE7 with the ability to grow on salicylic acid and to degrade diphenyl ethers was isolated from a petroleum waste pit in Louisiana. A cloned Erwinia sp. dpe gene encoding diphenyl ether cleavage was introduced into PE7 in order to enhance its degradative ability. A broad-host-range expression plasmid, pDPE2388, was constructed by inserting an SspI-HpaI fragment from a dpe gene-containing plasmid, pDPE7321, into the kanamycin resistance gene of plasmid pKT230. The DNA fragment contained the dpe gene flanked between sp6 and T7 promoters. Transconjugants of pDPE2388 plasmid into PE7 were isolated. Expression of the dpe gene in Escherichia coli or PE7 displayed a degradative ability to cleave the following diphenyl ethers: 4-chlorodiphenyl ether, 4-nitrodiphenyl ether, and 4-hydroxydiphenyl ether.Offprint requests to: V. R. Srinivasan     

Biodegradation of diphenyl ethers by a copper-resistant mutant ofErwinia sp.

Summary A bacterium tentatively identified as anErwinia sp. was isolated from sewage by enrichment on methanol and lignin. Several mutants developed from this strain were studied for their ability to degrade aromatic ethers. Different concentrations of the chemicals were incubated with the organisms and the degradation was estimated by high-performance liquid chromatography (HPLC). Among these mutants, one isolate,Erwinia sp. strain CU3614, showed resistance to copper ions (>20 mM CuSO₄) and the ability to degrade 4-hydroxydiphenyl ether (4-HDPE), 4-chlorodiphenyl ether (4-CDPE), 4-nitrodiphenyl ether (4-NDPE) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) in the presence of copper ions. Increased concentrations of copper in the medium resulted in higher degradation of 4-HDPE. Further studies with copper-sensitive mutants obtained fromErwinia sp. CU3614 by Tn5 transposon-induced mutagenesis showed a corresponding decrease in the ability to degrade 4-HDPE. These results suggest the presence of a copper-associated activity in the biotransformation of aromatic ethers.     

Degradation of diphenylether by Pseudomonas cepacia

The microbial degradation of hard coal implies the cleavage of diaryl ether linkages in the coal macromolecule. We investigated the biodegradation of diphenylether as a model compound representing this substructure of coal. A bacterial strain isolated from soil and identified as Pseudomonas cepacia, was able to grow with diphenylether as sole source of carbon. During microbial growth, three metabolites were detected in the culture supernatant by high pressure liquid chromatography. As product of ring hydroxylation and subsequent rearomatization, 2,3-dihydroxydiphenylether was identified by UV, mass and nuclear magnetic resonance spectrometry and gas chromatography analyses. The cleavage of the ether linkage led to the formation of phenol and 2-pyrone-6-carboxylic acid, the latter being not further degraded by Pseudomonas cepacia. The possible cleavage mechanism of the ether linkage is discussed.Non-standard abbreviations DPE diphenylether - PCA 2-pyrone-6-carboxylic acid - GC gas chromatography - MS mass spectrometry - HPLC high pressure liquid chromatography     

A spectrophotometric assay of trout (Salvelinus fontinalis) liver diphenyl ether hydroxylase activity

Subcellular fractions of trout (Salvelinus fontinalis) liver homogenate metabolized diphenyl ether mainly to the 4-hydroxy derivative, but with traces of other compounds, including the 3-hydroxy derivative and possibly the 4,4-dihydroxy derivative. An ultraviolet spectrophotometric method for the determination of 4-hydroxydiphenyl ether is described.     

Metabolism of symmetrical dialkyl ethers by Acinetobacter sp. HO1-N

The growth of Acinetobacter species HO1-N on a homologous series of dialkyl ethers yielded characteristic cellular and extracellular ether fatty acids. Microbial growth on diheptyl ether resulted in the appearance of 7-n-heptoxy-1-n-heptanoic acid as a cellular fatty acid and 2-n-heptoxy-1-acetic acid as the sole extracellular fatty acid. The oxidation of dinonyl ether and didecyl ether by Acinetobacter resulted in the extracellular accumulation of 2-n-nonoxy-acetic acid and 2-n-decoxy-1-acetic acid, respectively. The 16-carbon ether fatty acid, 6-n-decoxy-1-n-hexanoic acid, was identified as a major cellular fatty acid in didecyl ether-grown cells. The extracellular ether fatty acids accumulated in an inverse relationship to the disappearance of the dialkyl ether and appeared to represent end products of metabolism. The carbon and energy required for cellular growth and metabolism resided in the terminal 5-carbons of diheptyl ether, 7-carbons of dinonyl ether and 8-carbons of didecyl ether. Glutarate, adipate, pimelate and suberate were identified from cells grown at the expense of diheptyl, dioctyl, dinonyl and didecyl ether, respectively, suggesting a role for dibasic acids as metabolic intermediates. A new and novel mechanism for the metabolism of symmetrical dialkyl ethers is suggested. Terminal methyl group oxidation of the dialkyl ether results in the formation of an alkoxy-fatty acid followed by an internal carbon-carbon scission reaction 2-carbons removed from the oxygen atom. The resulting endproducts are alkoxyacetic acid and the corresponding dibasic acid.Non-Standard Abbreviations TLC Thin Layer Chromatography - PS-DEGS · PS Diethylene glycol succinate - DHE Diheptyl ether - DOE Dioctyl ether - DNE Dionyl ether - DDE Didecyl ether     

Oxidation and ring cleavage of dibenzofuran by the filamentous fungus <Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>

The ability of the imperfect soil fungus Paecilomyces lilacinus to transform the environmental pollutant dibenzofuran was investigated. Transformation of dibenzofuran and related derivatives lead to 14 products, which were identified by UV spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Biotransformation was initiated by two separate hydroxylation steps, leading to the accumulation of 4-monohydroxylated and 4-dihydroxylateddibenzofurans. Hydroxylation at both aromatic rings produced 2,7-dihydroxydibenzofuran, 3,7-dihydroxydibenzofuran, and 2,8-dihydroxydibenzofuran. Further oxidation yields ring cleavage of dibenzofuran, which has not been described before for filamentous fungi. The ring fission products were identified as benzo[b]furo[3,2-d]-2-pyrone-6-carboxylic acid and [2-(1-carboxy-methylidene)-benzofuran-3-ylidene]-hydroxy-acetic acid and its derivatives hydroxylated at carbon 7 and 8 at the non-cleaved ring. Other metabolites were riboside-conjugates of 2-hydroxydibenzofuran and 3-hydroxydibenzofuran. The results showed that P. lilacinus transforms the hydrophobic compound dibenzofuran by phase I/phase II reactions to produce hydroxylated products and excretable sugar conjugates.     

Characterisation of coupling products formed by biotransformation of biphenyl and diphenyl ether by the white rot fungus Pycnoporus cinnabarinus

Cells of the white rot fungus Pycnoporus cinnabarinus grown in glucose were able to hydroxylate biphenyl and diphenyl ether, although growth was inhibited by these substrates at concentrations above 250 microM. 2- and 4-Hydroxybiphenyl were detected as products of biphenyl metabolism and 2- and 4-hydroxydiphenyl ether as products of diphenyl ether metabolism in the culture media. After addition of 2-hydroxydiphenyl ether and 2-hydroxybiphenyl to cell-free supernatants containing laccase as the only ligninolytic enzyme, different coloured precipitates were formed. HPLC analysis revealed the formation of additional hydrophobic metabolites with one major product per transformation. Mass spectrometric analysis of the methyl derivatives of the polymer mixture indicated dimers and trimers with different binding types. The main products were identified as dimers with carbon-carbon bonds in para-position to the hydroxyl group of the monomers by mass spectroscopy and nuclear magnetic resonance spectroscopy.     

Degradation of Mono-Fluorophenols by an Acclimated Activated Sludge

Acclimated activated sludge was examined for its ability to degrade mono-fluorophenols as the sole carbon source in aerobic batch cultures. The acclimated activated sludge degraded fluorophenol efficiently. It degraded 100 mg/l 3-fluoropheno and 4-fluorophenol in 16 h with, respectively, 99.85% and 99.91% fluoride anion release and it degraded 50 mg/l 2-fluorophenol in 15 h with 99.26% fluoride anion release. The aerobic biodegradability of the mono-fluorophenols decreased in the order: 4-fluorophenol > 3-fluorophenol > 2-fluorophenol, resulting mainly from a different octanol/water partition coefficient and different steric parameter of the fluorophenols. The mechanism study revealed that the initial step in the aerobic biodegradation of mono-fluorophenols by the activated sludge was their transformation to fluorocatechol. Following transformation of the fluorophenol to fluorocatechol, ring cleavage by catechol 1, 2-dioxygenases proceeded via an ortho-cleavage pathway, then defluorination occurred.     

Microbial transformations of N-(4-chlorphenyl)-benzoisothiazolone

Summary The microbial degradation of N-(4-chlorphenyl)-benzoisothiazolone (1) was studied by Streptomyces species in analogy to the metabolism of this drug in animals. As main metabolite 2-thiomethyl-N-(4-chlorphenyl)-benzamide (3) was found. The corresponding sulfoxide (4) and the sulfone (5) were obtained as further transformation products. The unmethylated 2-sulfhydryl compound (2) could be isolated in only small amounts. The degradation pathway follows by these results via a reductive cleavage of the S-N-bond of the isothiazolone ring with subsequent methylation of the sulfhydryl group and further oxidation of the resulting thiomethyl substituent.     

Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol beta-aryl ether substructure of lignin.

This study examined the ligninase-catalysed degradation of lignin model compounds representing the arylglycerol beta-aryl ether substructure, which is the dominant one in the lignin polymer. Three dimeric model compounds were used, all methoxylated in the 3- and 4-positions of the arylglycerol ring (ring A) and having various substituents in the beta-ether-linked aromatic ring (ring B), so that competing reactions involving both rings could be compared. Studies of the products formed and the time courses of their formation showed that these model compounds are oxidized by ligninase (+ H2O2 + O2) in both ring A and ring B. The major consequence with all three model compounds is oxidation of ring A, leading primarily to cleavage between C(alpha) and C(beta) (C(alpha) being proximal to ring A), and to a lesser extent to the oxidation of the C(alpha)-hydroxy group to a carbonyl group. Such C(alpha)-oxidation deactivates ring A, leaving only ring B for attack. Studies with C(alpha)-carbonyl model compounds corresponding to the three basic model compounds revealed that oxidation of ring B leads in part to dealkoxylations (i.e. to cleavage of the glycerol beta-aryl ether bond and to demethoxylations), but that these are minor reactions in the model compounds most closely related to lignin. Evidence is also given that another consequence of oxidation of ring B in the C(alpha)-carbonyl model compounds is formation of unstable cyclohexadienone ketals, which can decompose with elimination of the beta-ether-linked aromatic ring. The mechanisms proposed for the observed reactions involve initial formation of aryl cation radicals in either ring A or ring B. The cation radical intermediate from one of the C(alpha)-carbonyl model compounds was identified by e.s.r. spectroscopy. The mechanisms are based on earlier studies showing that ligninase acts by oxidizing appropriately substituted aromatic nuclei to aryl cation radicals [Kersten, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 2609-2612; Hammel, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 8348-8353].     

Degradation of d,l-syringaresinol,a β-β′ linked lignin model compound,by Fusarium solani M-13-1

A - linked lignin model compound, d,l-syringaresinol monobenzyl ether (Ib) was incubated with Fusarium solani M-13-1 in a shaking culture. From the culture filtrates, three compounds II, IIIb and IV were isolated and identified. Substrate Ib was oxidized at the -position of the side chain to give a hemiketal, an -hydroxylated compound IIA, which was then transformed to the ketoalcohol, 3-hydroxymethyl-2-(4-benzyloxy-3,5-dimethoxyphenyl)-4-(4-hydroxy-3,5-dimethoxybenzoyl)-tetrahydrofuran (IIB). These products were converted to a -lactone derivative, 6-oxo-2-(4-benzyloxy-3,5-dimethoxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane (IIIb), via alkyl-aryl cleavage. The syringyl moiety released from II by the cleavage reaction was identified as 2,6-dimethoxy-p-benzoquinone (IV). Incubation of 2,6-dimethoxyphenol (V) in fungal culures did not give the p-quinone IV. d,l-Syringaresinol dimethyl ether was not degraded and the etherated moiety of Ib was not attacked by the fungus, indicating that the degradation of d,l-syringaresinol was catalyzed by phenol oxidizing enzymes. The oxidation products of Ib with peroxidase/H₂O₂ was investigated and discussed in relation to the degradation products of the fungus.Abbreviation TLC thin layer chromatography     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

Evidence of cytochrome P450-catalyzed cleavage of the ether bond of phenoxybutyrate herbicides in Rhodococcus erythropolis K2-3

Bacterial strain Rhodococcus erythropolis K2-3 can cleave theether bond of the phenoxybutyrate herbicides, i.e., 4-(2,4-dichlorophenoxy)butyrate(2,4-DB) and 4-(4-chloro-2-methylphenoxy)butyrate (MCPB), by anenzyme system that is constitutively expressed. The enzyme(s) involved were investigated in this study. The rate ofdisappearance of 2,4-DB determined in a whole cell assay amounted to0.6 mmol/h ¶ g_{dry mass}.Carbon monoxide difference spectra of dithionite-reduced wholecells and crude cell extracts suggested that strain K2-3 contains a soluble cytochrome P450(P450), named P450_PB-1. The addition of various phenoxybutyrate substrates to crude cell extracts resulted in typical difference spectra following the type I pattern ofsubstrate binding with P450. The rate of 2,4-DB cleavage was reduced by inhibitors of P450: 5 mM metyrapone and carbon monoxide at a CO/O₂ ratio of 10 reduced the activity by about 20%, and 70%, respectively. The ether cleaving activity completely disappearedafter disruption of the cells and could not be detected in crude extracts. To elucidate theenzymatic basis of this reaction, P450 was partially purified. With the resulting enzyme preparation,2,4-DB cleavage activity was re-established, becoming measurable after the addition of eitherphenazine methosulfate or ferredoxin and ferredoxin/NADP oxidoreductase from spinach. We detected no activities attributable to -ketoglutarate-dependent dioxygenase orNAD(P)H-dependent monooxygenase. These results collectively indicatethat cleavage of the ether bond of phenoxybutyrate herbicides is catalyzed by P450-mediated activityin this strain. One of the products derived from this reaction is dichlorophenol, and comparativechromatographic analyses suggest that the other product is a C4-carbonicacid, most likely succinic semialdehyde/succinate.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

Aromatic ring cleavage of β-O-4 lignin model dimers without prior demeth(ox)ylation by lignin peroxidase

Methyl oxalate of arylglycerol was formed as an aromatic ring cleavage product in degradation of arylglycerol-β-aryl ether (β-O-4) type lignin substructure model dimers by extracellular lignin peroxidase of Phanerochaete chrysosporium. The enzymatic cleavage of arylglycerol-β-(o-[²H₃]methoxyphenyl) ether indicated that the methyl group of the methyl ester was derived from the methoxy group of the β-O-4 model dimer. It is thus concluded that demeth(ox)ylation was not essential for the enzymatic aromatic ring cleavage of the methoxylated aromatic substrates, β-O-4 lignin substructure models.     

Synthesis of 4-Deoxy-4-C-hydroxymethyl-α-L-Lyxo-Pyranosyl Thymine

Abstract

The synthesis of 1-[4-deoxy-4-C-hydroxymethyl-α-L-lyxopyranosyl]thymine has been accomplished by two synthetic routes both starting from methyl 2, 3-O-isopropylidene-β-D-ribopyranoside. The first route makes use of a ring opening, ring closure reaction sequence to increase the proportion of the desired L-isomers. The second route utilizes the soft nucleophilic character of malonyl anions and ozonolytic cleavage of enol ether to introduce the branched chain. The newly obtained pyranosyl nucleoside obtains a ⁴C₁ conformation with an equatorially oriented thymine moiety.     

Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48

The degradation pathways for cyclic alkanes (c-alkanes) in Rhodococcus sp. NDKK48 were investigated. Strain NDKK48 used dodecylcyclohexane as a sole carbon and energy source, and five metabolites in the dodecylcyclohexane degradation pathway were detected by gas-chromatography/mass spectra. The metabolites were identified as cyclohexanecarboxylic acid, cyclohexylacetic acid, 1-cyclohexene-1-acetic acid, 4-dodecylcyclohexanol, and 4-dodecylcyclohexanone. The strain degrades dodecylcyclohexane via a ring oxidation pathway and an alkyl side chain oxidation pathway. Cyclohexanecarboxylic acid was further oxidized to muconic acid via 1-cyclohexene-1-carboxylic acid and benzoic acid, and the muconic acid was finally used by strain NDKK48 for growth. Methylcyclohexane and cyclohexane were co-oxidized with hexadecane by strain NDKK48. Methylcyclohexane was degraded via a ring oxidation pathway, and the degradation pathway contained part of the Baeyer-Villiger oxidation for ring cleavage. Cyclohexane was also degraded by the same pathway as methylcyclohexane. Thus, strain NDKK48 has two pathways for the complete degradation of c-alkanes.     

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.