The natural biotic capacity of soils to degrade ␥-hexachlorocyclohexane (␥-HCH, lindane) was estimated using an enrichment technique based on the ability of soil bacteria to develop on synthetic media and degrade the xenobiotic compound, used as the sole source of carbon and energy. Bacterial inocula from relatively highly contaminated soils (from wood treatment factories) were found to promote efficiently the degradation of ␥-HCH, which subsequently permitted isolation of a competent ␥-HCH-degrading microorganism. The decrease of ␥-HCH concurrently with the release of chloride ions and the production of CO 2 demonstrated the complete mineralization of ␥-HCH mediated by the isolate. This was confirmed by gas chromatography-mass spectrometry analyses showing that degradation subproducts of ␥-HCH included an unidentified tetrachlorinated compound and subsequently 1,2,4-trichlorobenzene and 2,5-dichlorophenol. The two linA-and linB-like genes coding, respectively, for a ␥-HCH dehydrochlorinase and a dehalogenase were characterized by using a PCR strategy based on sequence homologies with previously published sequences from Sphingomonas paucimobilis UT26. Nucleotide sequence analysis of the linA-like region revealed the presence of a 472-bp open reading frame exhibiting high homology with the linA gene from S. paucimobilis, while a preliminary study also indicated strong homology among the two linB genes. All enzymes involved in the ␥-HCH degradative pathway appear to be extracellular and encoded by genes located on the chromosome, although numerous cryptic plasmids have been detected.
When Mycobacterium aurum MO1 was grown with morpholine, the release of ammonia into the supernatant was proportional to the disappearance of morpholine, showing that this compound was mineralized. MO1 was able to grow in high concentrations of morpholine but accumulation of ammonia inhibited growth and degradation of morpholine. Immobilization of bacterial cells in carrageenan gel beads showed that morpholine degradation in these conditions began earlier and was faster than in free culture. One of the two branches of the lower pathway of morpholine biodegradation was induced while the other branch was inhibited in the presence of morpholine. Strain MO1 grew on heterocyclic compounds similar to morpholine, demonstrating that MO1 is able to degrade heterocyclic compounds containing nitrogen atoms (piperidine and pyrrolidine). Compounds containing sulphur or oxygen atoms or compounds with double bonds were not degraded.
A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring.
A method based on the polymerase chain reaction (PCR) was developed for a rapid and specific detection of toluene degradative genes in soil. The xylE gene coding for catechol 2,3-dioxygenase was chosen as a target gene. The detection threshold was evaluated in microcosms using a sterilized standard soil inoculated with various amounts of a degradative strain of Pseudomonas putida (mX). The extracted DNA was used as a template to amplify the xylE gene. PCR followed by hybridization with an internal probe allowed us to detect 10' bacteria per g of soil. In polluted soils, quantification of target DNA by competitive PCR was compared with enumeration of degradative microflora. This molecular method appeared to be rapid, sensitive and more suitable than the microbiological approach to estimate the biodegradative potential of a polluted soil.
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