A Catabolic Activity of Sphingomonas wittichii RW1 in the Biotransformation of Carbazole

The well-known bacterium Sphingomonas wittichii RW1 catabolically degrades dibenzo-p-dioxin and dibenzofuran, as well as their chlorinated derivatives. The catabolic degradation of dioxin is initiated by a ring-hydroxylating dioxygenase. The biotransformation of carbazole by S. wittichii RW1 was determined in the present study. Dioxin dioxygenase from the dibenzofuran induced RW1 strain was thought to be unable to attack carbazole, which includes a heterocyclic aromatic dibenzopyrrole system. However, our results showed that carbazole was transformed to anthranilic acid and catechol. The color of the culture suspension changed upon addition of carbazole due to formation of a nitrogen-containing metabolite. Relevant metabolic intermediates were identified by gas chromatographic mass spectrometry and electrospray ionization time-of-flight mass spectrometry with comparison to the corresponding authentic compounds. The dioxygenase of the dibenzofuran induced RW1 attacked at the angular position adjacent to the nitrogen atom to give a dihydroxylated metabolic intermediate. Contrary to predictions made in previous reports, S. wittichii RW1 displayed positive catabolic activity toward carbazole.     

Degradation of diphenyl ether herbicides in soils

The gas chromatographic determination of CNP (2,4,6-trichlorophenyl 4-nitrophenyl ether), nitrofen (2,4-dichlorophenyl 4′-nitrophenyI ether), chlomethoxynil (2,4-dieblorophenyl 3′-methoxy-4′-nitrophenyl ether), CFNP (2,4-dichloro-6-fluorophenyl 4-nitrophenyl ether) and their amino derivatives in soils were carried out. Good recoveries from soils were obtained for the diphenyl ethers. On the other hand, satisfactory recoveries from soils were also obtained for the amino derivatives at high concentrations, but the recoveries at lower concentrations averaged about 66% for the least recovered compound.

The degradation of several diphenyl ether herbicides in two paddy soils were compared under flooded and upland conditions. The degradation was much slower under upland than under flooded conditions. Considerable amounts of their amino derivatives were produced in soils under flooded conditions, but not under upland conditions. It was suggested that the diphenyl ethers to the amino derivatives involved both chemical and microbial processes. CNP and chlomethoxynil degraded faster at lower concentrations than at higher ones.     

Degradation of chloroacetanilide herbicides by anodic fenton treatment

Anodic Fenton treatment (AFT) is an electrochemical treatment employing the Fenton reaction for the generation of hydroxyl radicals, strong oxidants that can degrade organic compounds via hydrogen abstraction. AFT has potential use for the remediation of aqueous pesticide waste. The degradation rates of chloroacetanilides by AFT were investigated in this work, which demonstrates that AFT can be used to rapidly and completely remove chloroacetanilide herbicides from aqueous solutions. Acetochlor, alachlor, butachlor, metolachlor, and propachlor were treated by AFT, and parent compound concentrations were analyzed over the course of the treatment time. Degradation curves were plotted and fitted by the AFT kinetic model for each herbicide, and AFT model kinetic parameters were used to calculate degradation rate constants. The reactivity order of these five active ingredients toward hydroxyl radical was acetochlor approximately metolachlor > butachlor approximately alachlor > propachlor. Treatment of the chloroacetanilides by AFT removed the parent compounds but did not completely mineralize them. However, AFT did result in an increase in the biodegradability of chloroacetanilide aqueous solutions, as evidenced by an increase in the 5-day biochemical oxygen demand to chemical oxygen demand ratio (BOD5/COD) to >0.3, indicating completely biodegradable solutions. Several degradation products were formed and subsequently degraded, although not always completely. Some of these were identified by mass spectral analyses. Among the products, isomers of phenolic and carbonyl derivatives of parent compounds were common to each of the herbicides analyzed. More extensively oxidized products were not detected. Degradation pathways are proposed for each of the parent compounds and identified products.     

Effect of soil on urease inhibition by substituted urea herbicides

The effects of some substituted urea herbicides, fenuron, monuron, diuron and linuron, on soil urease were investigated. All herbicides are soil urease mixed inhibitors and the same inhibition mechanism is presumed. A kinetic relationship, which takes into account herbicide adsorption, is developed in order to calculate the inhibition constants of soil urease from adsorption constants. A linear relationship between Hammett sigma values and log K_i for fenuron, monuron and diuron is obtained, from which the formation of a complex between herbicides and enzyme is proposed By comparing kinetic constants for soil urease with those obtained for jack bean, in the presence of the same herbicides, a possible effect of the soil matrix on the enzyme-herbicide complex is also suggested.     

Metabolism of stevioside by chickens

In intubation experiments (643-1168 mg per animal), most of the stevioside administered to chickens was recovered unchanged in the excreta, and only about 2% was converted into steviol. Neither stevioside nor steviol could be found in the blood. In chronic studies (667 mg of stevioside/kg of feed) with laying hens and meat-type chickens, no significant differences were found in feed uptake, weight gain, and feed conversion as the result of stevioside administration. The egg production and egg composition of laying hens were not influenced. Most of the stevioside taken up was found untransformed in the excreta, and about 21.5% or 7.3% was converted to steviol by meat-type chickens or laying hens, respectively. No stevioside or steviol could be detected in the blood or in the eggs of the different groups of animals. In anaerobic incubation experiments with chicken excreta, only a 20% conversion of stevioside into steviol was found. No harmful effects were observed in the chronic stevioside supplementation experiments nor in the intubation experiments in which very high stevioside doses were given.     

Gas chromatography of uracil herbicides by on-column methylation with trimethylanilinium hydroxide

Substituted uracil herbicides injected into a gas chromatograph react with trimethylanilinium hydroxide to give N-methyl derivatives with good gas chromatographic properties. Maximum methylation is obtained when the molar ratio of methylating reagent to herbicide is ca 4:1. This technique for preparing derivatives provides rapid qualitative and quantitative chromatography of the substances examined. Chromatographic response was linear with increased concentration for the synthetic standard and the on-column product of uracil herbicide. The proposed derivatization method was used to analyze herbicides in formulations. The methyl derivatives were identified spectroscopically.     

Degradation of phenyl carbamate herbicides by Pseudomonas alcaligenes isolated from soil

A bacterial strain isolated from soil and identified as Pseudomonas alcaligenes, was able to hydrolyze four phenylcarbamate herbicides (CIPC, BIPC, IPC and swep) to corresponding anilines and alcohols by co-metabolism. The pH of the growth medium had little influence on bacterial growth and rate of herbicide transformation. Increase in CIPC and BIPC concentrations resulted in a significant inhibition of bacterial growth and herbicide degradation. Using a range of antibiotics it was shown that the enzyme involved in degradation was inducible.     

Metabolism of fluorodifen by soil microorganisms

The metabolism of fluorodifen (p-nitrophenyl α,α,α,-trifluoro-2-nitro-p-tolyl ether) by soil microorganisms in the presence or absence of other carbon and nitrogen sources was studied. The degradation of this herbicide continued for 5 days, when benzoate or acetate and ammonium sulphate were included in the cultures, and for more than 5 weeks when fluorodifen was used as a sole source of carbon and nitrogen.Under all conditions nitrite ions were produced at concentrations ranging between 5 and 80 per cent of the nitro-nitrogen of the fluorodifen present. The highest concentration of nitrite was obtained when added carbon sources were used with fluorodifen. The lowest nitrite concentration accumulated when the fluorodifen was used as sole source of carbon and nitrogen. The nitrite reached a maximum value after a few days of incubation, followed by rapid disappearance.p-Nitrophenol and quinol were identified in the acid-ether extract of cultures. It is suggested that the first step in the degradation of fluorodifen is the hydrolysis of the ether linkage followed by the direct elimination of the nitro-groups as nitrite ions.     

Analysis of herbicides in olive oil by liquid chromatography time-of-flight mass spectrometry

The application of liquid chromatography time-of-flight mass spectrometry (LC/TOF-MS) for the identification and quantitation of four herbicides (simazine, atrazine, diuron, and terbuthylazine) in olive oil samples is reported here. The method includes a sample treatment step based on a preliminary liquid-liquid extraction followed by matrix solid-phase dispersion (MSPD) using aminopropyl as a sorbent material. A final cleanup step is performed with florisil using acetonitrile as an eluting solvent. The identification by LC/TOF-MS is accomplished with the accurate mass (and the subsequent generated empirical formula) of the protonated molecules [M + H]+, along with the accurate mass of the main fragment ion and the characteristic chlorine isotope cluster present in all of them. Accurate mass measurements are highly useful in this type of complex sample analyses since they allow us to achieve a high degree of specificity, often needed when other interferents are present in the matrix. The mass accuracy typically obtained is routinely better than 2 ppm. The sensitivity, linearity, precision, mass accuracy, and matrix effects are studied as well, illustrating the potential of this technique for routine quantitative analyses of herbicides in olive oil. Limits of detection (LODs) range from 1 to 5 microg/kg, which are far below the required maximum residue level (MRL) of 100 microg/kg for these herbicides in olive oil.     

Biodegradation of chloroacetamide herbicides by Paracoccus sp. FLY-8 in vitro

A butachlor-degrading strain, designated FLY-8, was isolated from rice field soil and was identified as Paracoccus sp. Strain FLY-8 could degrade and utilize six chloroacetamide herbicides as carbon sources for growth, and the degradation rates followed the order alachlor > acetochlor > propisochlor > butachlor > pretilachlor > metolachlor. The influence of molecular structure of the chloroacetamide herbicides on the microbial degradation rate was first analyzed; the results indicated that the substitutions of alkoxymethyl side chain with alkoxyethyl side chain greatly reduced the degradation efficiencies; the length of amide nitrogen's alkoxymethyl significantly affected the biodegradability of these herbicides: the longer the alkyl was, the slower the degradation efficiencies occurred. The phenyl alkyl substituents have no obvious influence on the degradation efficiency. The pathway of butachlor complete mineralization was elucidated on the basis of the results of metabolite identification and enzyme assays. Butachlor was degraded to alachlor by partial C-dealkylation and then converted to 2-chloro-N-(2,6-dimethylphenyl)acetamide by N-dealkylation, which subsequently transformed to 2,6-diethylaniline, which was further degraded via the metabolites aniline and catechol, and catechol was oxidized through an ortho-cleavage pathway. This study highlights an important potential use of strain FLY-8 for the in situ bioremediation of chloroacetamide herbicides and their metabolite-contaminated environment.     

Metabolism of metolachlor by fungal cultures.

Metabolism of metolachlor was studied using a mixed fungal culture isolated from a metolachlor-acclimated field soil. The culture rapidly degraded metolachlor with a half-life of 3.5 days in broth. Aspergillus flavus and A. terricola purified from the mixed culture also metabolized metolachlor effectively. Five metabolites obtained were identified by co-chromatography on HPLC by comparing with authentic standards and by GC-MS. Hydrolytic dechlorination, N-dealkylation, and amide bond cleavage appeared to be the dominant transformations involved in the metabolism. Metabolites, 6-methyl 2-ethyl acetanilide and 6-methyl 2-ethyl aniline, identified in this study are new metabolites of metolachlor being reported from any mixed or pure microbial cultures. The mixed culture could degrade 99% of metolachlor at a fortification level as high as 100 microg mL(-)(1).     

Inhibition of soil humus-laccase complexes by some phenoxyacetic and s-triazine herbicides

Soil laccase was extracted with 0. 1 m sodium pyrophosphate and purified by Sephadex G-25 gel chromatography. Oxygen consumption during the enzymatic reaction was determined by a polarographic method with catechol or p-phenylenediamine as the substrates. The fraction showed laccase activity toward both the substrates. The activity toward p-phenylenediamine was six times higher than that toward catechol. The properties of soil laccase suggest that the enzyme is associated with humic compounds and forms a humus-laccase complex (Ruggiero and Radogna, 1984). The effect of three phenoxyacetic and three s-triazine herbicides on the activity of the humus-laccase complex was assessed. All the herbicides inhibited laccase activity to varying degrees. The inhibition of humus-laccase activity by 2,4-D was investigated in detail. The data, plotted in the form of a Lineweaver-Burk plot, showed that the 2,4-D acted as a non-competitive inhibitor. The apparent K_m values were 28.7 and 6.0 nm for catechol and p-phenylenediamine, respectively. Some aspects of the relationship between herbicide, substrate and humus-laccase complex are discussed.     

Biotransformation of the neonicotinoid insecticide thiacloprid by the bacterium Variovorax boronicumulans strain J1 and mediation of the major metabolic pathway by nitrile hydratase

A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a β-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.     

Infection and colonization of aseptically micropropagated sugarcane seedlings by nitrogen-fixing endophytic bacterium, Herbaspirillum sp. B501gfp1

In this study, we used Herbaspirillum sp. B501gfp1 (B501gfp1), an isolate from wild rice, to investigate the interaction between a non-host nitrogen-fixing endophytic bacterium and micropropagated sugarcane plants under aseptic condition. Two Japanese sugarcane plants (Saccharum sp.) cultivars (cvs) NiF8 and Ni15 were inoculated using B501gfp1 in two inoculum doses of 10⁸ and 10² bacterial-cells-per-milliliter suspension. The results showed that bacterial cells colonized both the root and stem tissues, and colonization was apparent in the intercellular spaces. Higher bacterial numbers were detected in plant tissues inoculated with the higher inoculum concentration treatment. Bacterial numbers also varied between the two cultivars, with the higher values determined in cv Ni15. This study provides evidence that Herbaspirillum sp. B501gfp1, a rice isolate, could colonize sugarcane tissues, suggesting non-specificity of host plant among endophytes.