Biodegradation of the chlorophenoxy herbicide (R)-(+)-mecoprop by Alcaligenes denitrificans

An Alcaligenes denitrificans strain capable of utilizing theherbicide (R)-(+)-2(2-methyl-4-chlorophenoxy)propionicacid (mecoprop) as a sole carbon source was isolated fromsoil and cultured in liquid medium. Crude cell extracts of thebacterium were utilized in spectrophotometric assays toelucidate a biochemical pathway for degradation ofmecoprop. Results indicated a reaction sequence analogousto the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D).GC-MS analysis provided direct evidence for thebiotransformation of mecoprop to the transient metabolite4-chloro-2-methylphenol (MCP). No NADPH-dependentactivity was observed during this reaction. Pyruvate wasverified as the second product derived from the aliphatic sidechain of mecoprop. MCP was subsequently transformed to asubstituted catechol by an NADPH-dependentmonooxygenase. When grown on mecoprop, A.denitrificans was adapted to oxidize catechol and its 4- and3-methylated derivatives indicating the broad substratespecificity of catechol dioxygenase. The microorganism wasdemonstrated to adopt the ortho mechanism of aromaticcleavage which resulted in the formation of2-methyl-4-carboxymethylene but-2-en-4-olide, a reactionintermediate of the -ketoadipate pathway.     

Simultaneous degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 2-(2-methyl-4-chlorophenoxy)propionic acid by mixed bacterial cultures

The simultaneous degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) was achieved by two mixed cultures in the absence of any additional carbon or energy substrates. Mecoprop was not completely degraded by either of the two cultures, nor did addition of 2,4-D affect the degradation of mecoprop. The cultures completely degraded 2,4-D, and the degradation was uninfluenced by the addition of mecoprop. Nearly complete dechlorination of the mixture of two herbicides was achieved by both cultures, on the basis of the total amount of the two herbicides degraded. During the course of the reaction, however, the expected values of chloride were not met. Cell growth continued after the degradation of the parent substrates ceased. Although the mecoprop degradation did not continue to completion, spectral and growth data indicated that the metabolites which had accumulated during the reaction were degraded upon further incubation.     

Bacterial diversity in soil enrichment cultures amended with 2 (2-methyl-4-chlorophenoxy) propionic acid (mecoprop)

Summary The tfdA gene encodes for an alpha-ketoglutarate-dependent dioxygenase enzyme which catalyses the first step of the degradation of phenoxyalkanoic acid herbicides such as 2 (2-methyl-4-chlorophenoxy) propionic acid (mecoprop). The bacterial diversity of soil enrichment cultures containing mecoprop was examined by Denaturing Gradient Gel Electrophoresis (DGGE) and clone libraries of both 16S rRNA genes and tfdA genes. The 16S rRNA gene sequences were diverse and clustered with either the Beta- or Gammaproteobacteria. The 16S rRNA gene sequence from a bacterial strain isolated from an enrichment culture, grown on R-mecoprop, which represented a dominant band in the DGGE profiles, had a high 16S rRNA sequence identity (100%) to Burkholderia glathei. This is the first report that B. glathei is implicated in mecoprop degradation. PCR amplification of the tfdA genes detected class III tfdA genes only, and no class I or class II tfdA sequences were detected. To understand the genes involved the degradation of specific mecoprop (R-) and (S-) enantiomers, oligonucleotide probes targeting the tfdA, rdpA, sdpA and cadA genes were hybridized to DNA extracted from enrichment cultures grown on either R-mecoprop or (R/S) racemic mecoprop. Strong hybridization signals were obtained with sdpA and tfdA probes using DNA extracted from cultures grown on racemic mecoprop. A strong hybridization signal was also obtained with the rdpA probe with DNA extracted from the cultures grown on R-mecoprop. This suggests the rdpA gene is involved in R-mecoprop degradation while tfdA, sdpA and cadA genes are involved in the degradation of both R- and S-mecoprop.     

Mineralization and co-metabolic degradation of phenoxyalkanoic acid herbicides by a pure bacterial culture isolated from an aquifer

A mecoprop [(+/-)-2-(4-chloro-2-methylphenoxy)propionic acid; MCPP]-degrading bacterium identified as Stenotrophomonas maltophilia PM was isolated from a Danish aquifer. Besides mecoprop, the bacterium was also able to degrade MCPA [(4-chloro-2-methylphenoxy)acetic acid)], MCPB [(4-chloro-2-methylphenoxy)butyric acid], 4-CPA [(4-chlorophenoxy)acetic acid], 2, 4-D [(2, 4-dichlorophenoxy)acetic acid], 2, 4-DP [(+/-)-2-(2, 4-dichlorophenoxy)propionic acid] and 2, 4-DB [(2, 4-dichlorophenoxy)butyric acid]. The bacterium was able to grow using these individual phenoxyalkanoic acids as the sole source of carbon and energy. In addition, it was able to co-metabolically degrade the phenoxyalkanoic acid 2, 4, 5-T [(2, 4, 5-trichlorophenoxy)acetic acid)] in the presence of mecoprop. At high 2, 4, 5-T concentrations (100 and 52 mg/l), however, only partial degradation of both mecoprop and 2, 4, 5-T was obtained, thus indicating the production of toxic metabolites. Bacterial yields were highest when grown on the monochlorinated phenoxyalkanoic acids as compared to the dichlorinated analogues, an exception being growth on 4CPA, which resulted in the lowest yield at all. Using [ring-U-14C]-labeled herbicides it was shown that the lower yield on 2, 4-D than on mecoprop was accompanied by greater CO2 generation, thus indicating that less energy is available from the complete oxidation of the dichlorinated phenoxyalkanoic acids than the monochlorinated analogues.     

Complete microbial degradation of both enantiomers of the chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propionic acid] in an enantioselective manner by Sphingomonas herbicidovorans sp. nov.

Sphingomonas herbicidovorans MH (previously designated Flavobacterium sp. strain MH) was able to utilize the chiral herbicide (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) as the sole carbon and energy source. When strain MH was offered racemic mecoprop as the growth substrate, it could degrade both the (R) and the (S) enantiomer to completion, as shown by biomass formation, substrate consumption, and stoichiometric chloride release. However, the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. These results suggest the involvement of specific enzymes for the degradation of each enantiomer. This view was substantiated by the fact that resting cells of strain MH grown on (S)-mecoprop were able to degrade the (S) but not the (R) enantiomer of mecoprop. Accordingly, resting cells of strain MH grown on (R)-mecoprop preferentially metabolized the (R) enantiomer. Nevertheless, such cells could transform (S)-mecoprop at low rates. Oxygen uptake rates with resting cells confirmed the above view, as oxygen consumption was strongly dependent on the growth substrate. Cells grown on (R)-mecoprop showed oxygen uptake rates more than two times higher upon incubation with the (R) than upon incubation with the (S) enantiomer and vice versa.     

Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp.CS1 and Ralstonia sp. CS2 isolated from agricultural soils

The herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad-leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade ( R/S )-mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4- D (2,4-dichlorophenoxyacetic acid), MCPA [4-chloro-2-methyl phenoxy acetic acid] and ( R/S )-2,4-DP [2-(2,4-dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene-specific probes derived from the 2,4- D -degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd -like genes are present in mecoprop-degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA , tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA , tfdB and tfdC genes of V. paradoxus . It is therefore likely that tfd -like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.     

Degradation of some phenoxy acid herbicides by mixed cultures of bacteria isolated from soil treated with 2-(2-methyl-4-chloro)phenoxypropionic acid

Mixed bacterial cultures capable of using 2-methyl-4-chIorophenoxyacetic acid (MCPA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) as the sole source of carbon and energy were isolated from field soil treated with the herbicide (±)2-(2-methyl-4-chloro)phenoxypropionic acid (mecoprop). An enrichment technique with two aromatic compounds as sources of carbon was used. Effects of temperature and substrate concentration were studied. The mixed cultures retained their ability to degrade MCPA although the bacteria were grown for 3 months (32 successive passages) with glucose as the sole source of carbon and energy. With benzoic acid as co-substrate, one of the cultures was also able to degrade mecoprop and (±)2-(2, 4-dichloro)phenoxypropionic acid (dichlorprop). This ability was not maintained, however, over more than 10 passages.     

A rapid method to screen degradation ability in chlorophenoxyalkanoic acid herbicide-degrading bacteria

AIMS: An agar medium containing a range of related chlorophenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop and racemic 2,4-DP (2-(2,4-dichlorophenoxy) propionic acid) was developed to assess the catabolic activity of a range of degradative strains. METHODS AND RESULTS: The medium was previously developed containing 2,4-D as a carbon source to visualise degradation by the production of dark violet bacterial colonies. Strains isolated on mecoprop were able to degrade 2,4-D, MCPA, racemic mecoprop, (R)-mecoprop and racemic 2,4-DP, whereas the 2,4-D-enriched strains were limited to 2,4-D and MCPA as carbon sources. Sphingomonas sp. TFD44 solely degraded the dichlorinated compounds, 2,4-D, racemic 2,4-DP and 2,4-DB (2,4-dichlorophenoxybutyric acid). However, Sphingomonas sp. AW5, originally isolated on 2,4,5-T, was the only strain to degrade the phenoxybutyric compound MCPB (4-chloro-2-methylphenoxybutyric acid). CONCLUSION: This medium has proved to be a very effective and rapid method for screening herbicide degradation by bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This method reduces the problem of assessing the biodegradability of this family of compounds to an achievable level.     

Enantioselective degradation of the herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] by mixed and pure bacterial cultures

Abstract A consortium of three bacteria was isolated from top soil through their capacity to utilise the chlorinated, aromatic herbicide mecoprop as a single growth substrate. The consortium constituted a tight association of Alcaligenes denitrificans, Pseudomonas glycinea and Pseudomonas marginalis . The culture exclusively degraded the ( R )-(+)-isomer of the herbicide while the ( S )-(−)-enantiomer remained unaffected. The mecoprop-degrading community could also degrade 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid and racemic 2-phenoxypropionic acid. Initially, no single member of the consortium was able to degrade mecoprop as a pure culture but after prolonged incubation, A. denitrificans was able to grow on the herbicide as the sole source of carbon and energy.     

Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.     

Genetic and phenotypic diversity of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy)propionic acid]-degrading bacteria isolated from soils

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer.     

Fate of the herbicides mecoprop, dichlorprop, and 2,4-D in aerobic and anaerobic sewage sludge as determined by laboratory batch studies and enantiomer-specific analysis

Aerobic degradation experiments with the racemic mixtures of mecoprop and dichlorprop revealed that activated sludge collected from the aeration tank of a municipal waste water treatment plant degraded both enantiomers of mecoprop and dichlorprop within 7 days, albeit in an enantioselective manner; the (S) enantiomers were preferentially degraded. Mecoprop, dichlorprop, and 2,4-D were completely metabolized under aerobic conditions, as shown by the 86–98% elimination of dissolved organic carbon. Under anaerobic conditions, the concentration of 2,4-D decreased exponentially with a first-order reaction rate constant of 0.24 per day and without a lag-phase. After an incubation time of 17 days, 2,4-D was completely removed. 2,4-Dichlorophenol was the main metabolite of anaerobic 2,4-D degradation; only traces of 4-chlorophenol were detected. In contrast, the chiral phenoxypropionic acid herbicides mecoprop and dichlorprop persisted under anaerobic conditions during 49 days of incubation.     

Comamonas acidovorans strain MC1: a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the herbicides 2,4-D and MCPA

A gram-negative prototrophic bacterial species, strain MC1, was isolated from the vicinity of herbicide-contaminated building rubble and identified by 16S rDNA sequence analysis, its physiological properties, GC content, and fatty acid composition as Comamonas acidovorans. This strain displays activity for the productive degradation of the two enantiomers of dichlorprop [(RS)-2-(2,4-dichlorophenoxy-)propionate; (RS)-2,4-DP] and mecoprop [(RS)-2-(4-chloro-2-methyl-) phenoxypropionate; (RS)-MCPP] in addition phenoxyacetate herbicides, i.e. 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), and various chlorophenols were utilized. Rates amounted to 1.2 mmoles/h g dry mass (2,4-D) and 2.7 mmoles/h g dry mass [(RS)-2,4-DP]. Degradation of (RS)-2,4-DP was not inhibited up to concentrations of 500 mg/l, nor of 2,4-D up to 200 mg/l. The optimum pH value of (RS)-2,4-DP degradation was around 8. The application of respective primers for PCR amplification revealed the presence of tfdB and tfdC genes.     

Decolorization of the textile dyes using purified banana pulp polyphenol oxidase

Polyphenol oxidase (PPO) purified using DEAE-cellulose and Biogel P-100 column chromatography from banana pulp showed 12.72-fold activity and 2.49% yield. The optimum temperature and pH were found to be 30 degrees C and 7.0, respectively for its activity. Catechol was found to be a suitable substrate for banana pulp PPO that showed V(max), 0.041 mM min(-1) and K(m), 1.6 mM. The enzyme activity was inhibited by sodium metabisulfite, citric acid, cysteine, and beta-mercaptoethanol at 10 mM concentration. The purified enzyme could decolorize (90%) Direct Red 5B (160 microg mL(-1)) dye within 48 h and Direct Blue GLL (400 microg mL(-1)) dye up to 85% within 90 h. The GC-MS analysis indicated the presence of 4-hydroxy-benzenesulfonic acid and Naphthalene-1,2,3,6-tetraol in the degradation products of Direct Red 5B, and 5-(4-Diazenyl-naphthalene-1-ylazo)-8-hydroxy-naphthalene-2-sulfonic acid and 2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acid in the degradation products of Direct Blue GLL.     

Effects of Mecoprop (an Auxin Analogue) on Ethylene Evolution and Epinasty in Two Biotypes of Stellaria media

Petiolar epinasty and the production of ethylene (ethene) werestudied in chickweed biotypes, Stellaria media, treated withthe herbicide and auxin analogue (RS)-2-(4-chloro-o-tolyloxy)propionicacid, potassium salt, common name mecoprop. This compound causedsevere epinasty and stimulated the production of ethylene fromshoot explants. However, when intact plants were treated withethylene, the leaves became only slightly epinastic. The ethyleneprecursor, 1-aminocyclopropane-I-carboxylic acid (ACC), at concentrationswhich stimulated the release of ethylene, was equally ineffectivein causing epinasty. Furthermore, 2, 5-norbornadiene, a specific,competitive inhibitor of ethylene action, only partly alleviatedmecoprop-induced epinasty. The responses observed in chickweedwere compared with those produced in tomato plants. ACC inducedepinasty in tomato within 2 h and these symptoms were completelyinhibited by norbornadiene. However, as in chickweed, the inhibitorgave only partial reversal of mecoprop-induced epinasty, implyingthat the epinastic response caused by the herbicide was notattributable to ethylene alone. We therefore suggest that mecoprop-inducedepinasty is a result of the combined ethylene-stimulating andgrowth-promoting properties of the herbicide. Mecoprop-stimulated ethylene evolution was initially significantlygreater in a herbicide-resistant, compared with a more susceptiblebiotype of chickweed. The significance of this finding is discussedin relation to the mechanism of mecoprop resistance in chickweed. Epinasty, ethylene, (RS)-2-(4-chloro-o-tolyloxy)propionic acid, mecoprop, herbicide resistance, chickweed, Stellaria media L., tomato, Lycopersicon esculentum L.     

Separation of two dichlorprop/α-ketoglutarate dioxygenases with enantiospecific properties from Comamonas acidovorans MC1

Comamonas acidovorans MC1, which is capable of degrading the chiral phenoxypropionate herbicides 2-(2,4-dichlorophenoxy)propionate [dichlorprop, (RS)-2,4-DP] and 2-(4-chloro-2-methylphenoxy)propionate [mecoprop, (RS)-MCPP] and of degrading the phenoxyacetate herbicides 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), was investigated with respect to the enzymatic basis of this broad substrate specificity. The initial steps of the degradation pathway of (RS)-2,4-DP and 2,4-D were studied. By applying either ion exchange chromatography or hydrophobic interaction chromatography it was possible to separate two enzyme fractions with etherolytic activity, which exhibited pronounced substrate specificity. One enzyme fraction was highly specific for the degradation of the R-enantiomer of 2,4-DP and did not essentially attack the S-configuration. The other enzyme fraction showed pronounced activity toward the cleavage of the S-enantiomer and additionally utilized 2,4-D with almost equal velocity; (R)-2,4-DP was even cleaved at a low rate by this enzyme. These results confirm the existence of phenoxyalkanoatedegrading enzymes with enantiospecific properties in strain MC1.     

Biodegradation Kinetics of Phenanthrene by a Fusant Strain

The fusant strain (F14), which produced by protoplast fusion between Sphingomonas sp. GY2B (GenBank DQ139343) and Pseudomonas sp. GP3A (GenBank EU233280), was tested for phenanthrene biodegradation at 30 °C and pH of 7.0. The kinetics of phenanthrene biodegradation by F14 was investigated over a wide range of initial concentration (15-1,000 mg l(-1)). The rate and the extent of phenanthrene degradation increased with the increase of concentration up to 230 mg l(-1), which indicated negligible inhibition effect at low concentrations. The non-competitive inhibition model was found to be fit for the process. GC-MS analysis showed that biodegradation of phenanthrene by F14 was via dioxygenation at both 1,2- and 3,4-positions and followed by 2-hydroxy-1-naphthoic acid and 1-hydroxy-2-naphthoic acid. The relative intensity of 2-hydroxy-1-naphthoic acid was approximately 3-4 times higher than that of 1-hydroxy-2-naphthoic acid, indicating the 2-hydroxy-1-naphthoic acid was the predominant product in the phenanthrene degradation by fusant strain F14.     

Reductive dehalogenation mediated initiation of aerobic degradation of 2-chloro-4-nitrophenol (2C4NP) by Burkholderia sp. strain SJ98

Burkholderia sp. strain SJ98 (DSM 23195) was previously isolated and characterized for degradation and co-metabolic transformation of a number nitroaromatic compounds. In the present study, we evaluated its metabolic activity on chlorinated nitroaromatic compounds (CNACs). Results obtained during this study revealed that strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon, nitrogen, and energy under aerobic conditions. The cells of strain SJ98 removed 2C4NP from the growth medium with sequential release of nearly stoichiometric amounts of chloride and nitrite in culture supernatant. Under aerobic degradation conditions, 2C4NP was transformed into the first intermediate that was identified as p-nitrophenol by high-performance liquid chromatography, LCMS-TOF, and GC-MS analyses. This transformation clearly establishes that the degradation of 2C4NP by strain SJ98 is initiated by "reductive dehalogenation"; an initiation mechanism that has not been previously reported for microbial degradation of CNAC under aerobic conditions.     

Identical ring cleavage products during anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin indicate a new metabolic pathway

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C(1) unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C(11)H(16)O(4) (C(11)H(16)O(4)-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by beta-oxidation of one of the side chains of the C(11)H(16)O(4)-diacid. Stable isotope-labeling growth experiments with either (13)C-labeled naphthalene, per-deuterated naphthalene-d(8), or a (13)C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.     

Effects of phenoxy acid herbicides and glyphosate on nitrogenase activity (acetylene reduction) in root-associated Azospirillum, Enterobacter and Klebsiella

Abstract Nitrogenase activity (C₂H₂ reduction) in root-associated Azospirillum lipoferum, Klebsiella pneumoniae, Enterobacter agglomerans and Pseudomonas sp. isolated from roots of Finnish grasses was assayed in the presence of glyphosate, the phenoxy acid herbicides 2-methyl-4-chlorophenoxy acetic acid (MCPA), 2,4-dichlorophenoxy acetic acid (2,4-D), (±)-2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) and (±)-2-(2,4-dichlorophenoxy)propionic acid (dichlorprop), and the commercial products Roundup, Nurmikko-Hedonal, Mepro, and Dipro. In the presence of the phenoxy acid herbicides the nitrogenase activity of K. pneumoniae was significantly inhibited, but that of E. agglomerans was stimulated. With the exception of Mepro and mecoprop no phenoxy acid herbicides inhibited the nitrogenase activity of A. lipoferum and none that of Pseudomonas sp. Nurmikko-Hedonal considerably stimulated the nitrogenase activity of E. agglomerans , and Pseudomanas sp. On the other hand, the nitrogenase activity of both K. pneumoniae and E. agglomerans was considerably repressed by glyphosate and Roundup, which also inhibited the growth of the bacteria. These chemicals had no effect on the growth of A. lipoferum and Pseudomonas sp., but stimulated their nitrogenase activity.