Degradation of Aniline by <Emphasis Type="Italic">Delftia tsuruhatensis</Emphasis> 14S in Batch and Continuous Processes

A Delftia tsuruhatensis strain capable of consuming aniline as the sole source of carbon, nitrogen, and energy at concentrations of up to 3200 mg/l was isolated from activated sludge of the sewage disposal plants of OAO Volzhskii Orgsintez. The strain grew on catechol and p-hydroxybenzoic acid but did not consume phenol, 2-aminophenol, 3-chloroaniline, 4-chloroaniline, 2,3-dichloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline, 2-nitroaniline, 2-chlorophenol, or aminobenzoate. Aniline is degraded by cleavage of the catechol aromatic ring at the ortho position. Cells were immobilized on polycaproamide fiber. It was shown that the strain degraded aniline at 1000 mg/l in a continuous process over a long period of time.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway.

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Microbial mineralization of ring-substituted anilines through an ortho-cleavage pathway

Moraxella sp. strain G is able to utilize as sole source of carbon and nitrogen aniline, 4-fluoroaniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline (PCA), and 4-bromoaniline but not 4-iodoaniline, 4-methylaniline, 4-methoxyaniline, or 3,4-dichloroaniline. The generation time on PCA was 6 h. The pathway for the degradation of PCA was investigated by analysis of catabolic intermediates and enzyme activities. Mutants of strain G were isolated to enhance the accumulation of specific pathway intermediates. PCA was converted by an aniline oxygenase to 4-chlorocatechol, which in turn was degraded via a modified ortho-cleavage pathway. Synthesis of the aniline oxygenase was inducible by various anilines. This enzyme exhibited a broad substrate specificity. Its specific activity towards substituted anilines seemed to be correlated more with the size than with the electron-withdrawing effect of the substituent and was very low towards anilines having substituents larger than iodine or a methyl group. The initial enzyme of the modified ortho-cleavage pathway, catechol 1,2-dioxygenase, had similar characteristics to those of corresponding enzymes of pathways for the degradation of chlorobenzoic acid and chlorophenol, that is, a broad substrate specificity and high activity towards chlorinated and methylated catechols.     

Comparative studies on the metabolism of aniline and chloroanilines by Pseudomonas multivorans strain An 1

Summary Pseudomonas multivorans strain An 1 used aniline but not chloroanilines as the sole source of carbon and energy for growth. The aniline-adapted cells, however, were able to oxygenate chloroanilines. Relative oxygenation rates for aniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, and 3,4-dichloroaniline were 100, 46, 66, 20, and 3%, respectively.The first intermediates in the metabolism of chloroanilines were chlorocatechols. 3-Chlorocatechol accumulated during growth of the organism in the presence of 2-chloroaniline, whereas 4-chlorocatechol was an intermediate metabolite of 3-chloroaniline and 4-chloroaniline.Chloroanilines were able to induce synthesis of the aniline oxygenating enzyme system of Pseudomonas multivorans strain An 1. In continuous culture experiments, induction of this enzyme system appeared to depend on cell density, concentration, toxicity, and pK-values of aniline or chloroanilines.Studies with ¹⁴C-labelled 3-chloroaniline and 4-chloroaniline showed that the turnover of chloroanilines did not cease with the formation of chlorocatechols, because radioactivity was detected in the CO₂ released and in bacterial cell components. The results suggest that the turnover of chloroanilines is due to metabolism rather than to cometabolism.     

Degradation of aniline by newly isolated,extremely aniline-tolerant <Emphasis Type="Italic">Delftia </Emphasis>sp. AN3

A bacterial strain, AN3, which was able to use aniline or acetanilide as sole carbon, nitrogen and energy sources was isolated from activated sludge and identified as Delftiasp. AN3. This strain was capable of growing on concentrations of aniline up to 53.8 mM (5000 mg/l). Substituted anilines such as N-methylaniline, N, N-dimethylaniline, 2-methylaniline, 4-methylaniline, 2-chloroaniline, 3-chloroaniline, o-aminoaniline, m-aminoaniline, p-aminoaniline, and sulfanilic acid did not support the growth of strain AN3. The optimal temperature and pH for growth and degradation of aniline were 30 degrees C and 7.0, respectively. The activities of aniline dioxygenase, catechol 2,3-dioxygenase and other enzymes involved in aniline degradation were determined, and results indicated that all of them were inducible. The K (m) and V (max) of aniline dioxygenase were 0.29 mM and 0.043 mmol/mg protein/min, respectively. The K (m) and V (max) of catechol 2, 3-dioxygenase for catechol were 0.016 mM and 0.015 mmol/mg protein/min, respectively. Based on the results obtained, a pathway for the degradation of aniline by Delftiasp. AN3 was proposed. The importance of the strain to the operation of municipal wastewater treatment plants is discussed.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

Degradation of chloroanilines in soil slurry by specialized organisms

The microbial degradation of 2-chloro-, 3-chloro-, 4-chloro-, and 3,4-dichloroaniline was examined as single compounds as well as a mixture in soil slurries. At 30°C the degradation of chloroanilines by indigenous soil populations in soil slurries was observed when soil slurry was freshly contaminated or precontaminated to allow binding of chloroanilines to the soil matrix. Within 6 weeks, 3-chloro- and 3,4-dichloroaniline (each 2 mm) were degraded more rapidly (about 50% chloride elimination) than 4-chloro- and 2-chloroaniline, due to stronger adsorption of 4-chloroaniline and greater resistance of 2-chloroaniline. The addition of various supplements such as buffer, mineral salts and acetate only slightly influenced the degradation of chloroanilines by the indigenous soil populations. The mineralization was drastically enhanced when laboratory-selected chloroaniline-degraders (8·10⁶ cells/g) such as Pseudomonas acidovorans strain BN3.1 were supplemented to the soil slurries so that complete elimination of chloride from the chloroanilines occurred within 10 days. Correspondence to: F. R. Brunsbach     

Biodegradation of 4-chloroaniline by bacteria enriched from soil

4-Chloroaniline has been released into the environment due to extensive use in chemical industries and intensive agriculture; hence, it becomes one of the hazardous pollutants in the priority pollutant list. In this study, three gram-negative bacteria were enriched and isolated from agricultural soil as 4-chloroaniline-degrading bacteria. They were identified as Acinetobacter baumannii CA2, Pseudomonas putida CA16 and Klebsiella sp. CA17. They were able to utilize 4-chloroaniline as a sole carbon and nitrogen source without stimulation or cocultivation with aniline or another cosubstrate. The biodegradation in these bacteria was occurred via a modified ortho-cleavage pathway of which the activity of chlorocatechol 1, 2-dioxygenase was markedly induced. They grew well on 0.2-mM 4-chloroaniline exhibiting a 60-75% degradation efficiency and equimolar liberation of chloride. The isolates were able to survive in the presence of 4-chloroaniline at higher concentrations (up to 1.2 mM). 2-Chloroaniline, 3-chloroaniline and aniline, but not 3, 4-dichloroaniline, were also growth substrates for these isolates. The results of cosubstrate supplementation illustrated the suitable conditions of each isolate to improve growth rate and 4-chloroaniline biodegradation efficiency. These results suggest that these isolates have a potential use for bioremediation of the site contaminated with 4-chloroaniline.     

Degradation of 3,4-dichloroaniline in synthetic and industrially produced wastewaters by mixed cultures freely suspended and immobilized in a packed-bed reactor

Summary Degradation of 3,4-dichloroaniline (34DCA) in aqueous by undefined cultures of free and immobilized cells was examined. Batch cultures of freely suspended cells and continuous degradation in a packed-bed reactor were studied using both synthetically concocted and industrially produced waste-waters. 34DCA was found to be degraded with a concomitant evolution of chloride ions into the bulk medium. The [acked bed reactor with biomass immobilized on celite diatomaceous earth was found to be capable of degrading over 98% of the 34DCA present in a synthetically concocted inlet stream at a concentration of 250 mg l^–1. Residence times of less than 4 h were employed, giving an overall volumetric degradation rate for the packed bed of 90 mg l^–1 h^–1. The industrially produced wastewater contained, in addition to 34DCA, aniline, 4-chloroaniline, 2,3-dichloroaniline (23DCA) and 3,4-dichloronitrobenzene. The biomass enriched on the synthetic 34DCA waste-water was found to be capable of degrading these compounds in addition to 34DCA with the exception of 23DCA. 34DCA degradation efficiencies of over 95% were obtained for the industrial waste-water at reactor residence times of 4.6 h, giving volumetric degradation rates of 24 mg l^–1 h^–1. Offprint requests to: A. G. Livingston     

Characterization of an extracellular laccase of Leptosphaerulina chartarum

Laccase-producing fungi were isolated from air, using selective media with a chromogenic substrate to indicate enzyme activity. The best laccase producer strain proved to be a Leptosphaerulina chartarum isolate. Laccase production was investigated in the presence of various inducers in different cultivation conditions. The extracellular laccase was purified for further investigations. SDS-PAGE showed that this laccase is a monomeric protein of 38 kDa molecular weight. The enzyme is active in the pH-range of 3.5–6, with an optimum at pH 3.8. It is active in the 10–60 °C temperature range, with an optimum at 40 °C. After 20 min incubation at temperatures above 70 °C the enzyme lost its activity. Degradation of seven aniline and phenol compounds (2,4-dichlorophenol; 2-methyl-4-chlorophenol; 3-chloroaniline; 4-chloroaniline; 2,6-dimethylaniline; 3,4-dichloroaniline and 3-chloro-4-methylaniline) was investigated, with or without guaiacol (2-methoxyphenol) as mediator molecule. Addition of a mediator to the system significantly increased the degradation levels. These results confirmed that the isolated laccase is able to convert these harmful xenobiotics at in vitro conditions.     

Degradation of aniline and monochlorinated anilines by soil-born Pseudomonas acidovorans strains

Four bacterial strains (CA26, CA28, CA37, and CA45), which all were able to use aniline, 3-chloroaniline (3-CA), and 4-chloroaniline (4-CA) as sole sources of carbon, nitrogen and energy, were isolated after enrichment in aerated soil columns and identified as Pseudomonas acidovorans strains. In addition strains CA26 and CA45 were able to degrade 2-chloroaniline (2-CA) at very low rates. At 25°C strain CA28 was grown on aniline and 3-CA with generation times of 3.0 and 7.7 h, respectively, and exhibited complete mineralization of these substrates in degradation rates of 2.25 mmol aniline and 1.63 mmol 3-CA g^-1 of biomass per hour, respectively. Degradation of 4-CA occurred at 1.54 mmol 4-CA g^-1 of biomass per hour and a generation time of 18.7 h but, in contrast, was not complete due to formation of minor amounts of chlorohydroxymuconic semialdehyde, a meta-cleavage product of 4-chlorocatechol. The initial attack on the substrate, the formation of corresponding chlorocatechols from 3-CA and 4-CA, was found to be the rate-limiting degradation step. Evidence for two different aniline-oxygenase systems in strain CA28 with distinct activity pattern on chlorinated and nonsubstituted anilines was demonstrated by oxygen uptake rate experiments with aniline and chloroaniline pregrown cells. Further degradation was shown to be initialized by catechol dioxygenases.Non-standard abbreviations CA chloroaniline - DCA dichloroaniline - ECM enrichment and cultivation medium - CFU colony forming unit     

Cometabolic degradation of 2- and 3-chloroaniline because of glucose metabolism byRhodococcus sp. An 117

Various organic compounds were tested for their ability to stimulate degradation of monochloroanilines by aniline-grown cells ofRhodocuccus sp. An 117 in 0.1 M phosphate buffer, pH 6.9. Among them, glucose proved to be the most effective. In its presence both 2- and 3-chloroaniline were degraded with a transient accumulation of 3- and 4-chlorocatechnol, respectively. The turnover rates were 43 and 57% compared with the unsubstituted aniline, whereas in the case of 4-chloroaniline the rates did not exceed 5%. Aniline, when used as the additional carbon source, stimulated 3-chloroaniline degradation. With the exception of a certain initial delay in removal of the monochloroaniline, the respective kinetics resembled those seen with glucose as the cosubstrate. Evidence is presented that the enzymes involved in monochloroaniline turnover were identical with those responsible for aniline catabolism. The results obtained suggest that the cometabolic effect of glucose was due to certain products derived from the metabolism of the cosubstrate. The molar ratio between the amount of glucose added, on the one hand, and the amount of 2- or 3-chloroaniline converted, on the other hand, was found to be 0.4–0.5∶1.0.     

Point source detoxification of an industrially produced 3,4-dichloroaniline-manufacture wastewater using a membrane bioreactor

A membrane bioreactor has been used to treat an industrially produced waste-water containing aniline, 4-chloroaniline, 2,3-dichloroaniline and 3,4-dichloroaniline. Conventional direct biological treatment of such effluents cannot be implemented without some form of pretreatment or dilution because of the hostile inorganic composition of the waste-water. In order to overcome this problem a membrane separation step selectively removes the organics from the waste-water and subsequent biodegradation takes place in the biological growth compartment of the reactor system. At a waste-water flow rate of 69 ml h^–1 (corresponding to a contact time of approximately 1.5 h) over 99% of the organic compounds quoted above were removed and biodegraded. Correspondence to: A. G. Livingston     

3,4-dichloroaniline--a nitrogen and carbon source for a mixed culture of microorganisms

A mixed microbial culture assimilating 4-chloroaniline and 3,4-dichloroaniline as sole sources of carbon and nitrogen was isolated from soil treated with propanide for a long period of time. The process is accompanied with a 100% liberation of chloride ions. Aniline cannot serve as a growth substrate for the mixed culture. The authors have studied whether the mixed culture utilizing chloroanilines can decompose these compounds in model experiments with natural waters and soil suspensions. The culture actively decomposes 3,4-dichloroaniline (20 mg/litre) in natural water samples into which it has been added at a concentration of 10(3)--10(4) cells/ml. The decomposition is accelerated in experiments with suspensions of meadow-chernozem soils when cells of the mixed culture are added to the suspensions. The rate of 3,4-dichloroaniline degradation in a grey forest soil suspension remains nearly unchanged when the mixed culture is added to it. Degradation of the chloroaniline is accompanied with equivalent liberation of chloride ions when the mixed culture is added to natural water samples.     

Mineralization of 3-chloro-4-methylaniline via an ortho-cleavage pathway by Pseudomonas cepacia strain CMA1

Pseudomonas cepacia strain CMA1, which was isolated from soil, utilized 3-chloro-4-methylaniline (3C4MA) in concentrations up to 1.4 mm (0.2 g·l^–1) as the sole source of carbon, nitrogen, and energy. In addition, 3-chloroaniline, 4-chloroaniline and phenol, but not aniline or methylanilines, were degraded by strain CMA1. Biodegradation of the anilines was coupled to the liberation of ammonium and chloride. The broad specificities of the aniline- and catechol-oxidizing enzymes were demonstrated in oxygen uptake experiments, which in addition showed higher activities for ring-cleaving than for aniline-oxidizing enzymes. Two ring-cleaving catechol 1,2-dioxygenases, which were induced selectively after growth on 3C4MA (pyrocatechase type II) and phenol (pyrocatechase type I), respectively, were discerned after partial purification by DEAE-cellulose chromatography. Correspondence to: F. Streichsbier     

Standardized plant cell suspension test systems for an ecotoxicologic evaluation of the metabolic fate of xenobiotics

A rapid assay for the metabolic behaviour of organic chemicals in plants has been developed using soybean (Glycine max L.) and wheat (Triticum aestivum L.) cell suspension cultures. The test was performed with the following ¹⁴C-labelled compounds: 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloroaniline (4-CA), 3,4-dichloroaniline (3,4-DCA), pentachlorophenol (PCP), diethylhexylphthalate (DEHP), perylene (PR) and benzo[a]pyrene (BaP), which were applied at 1 mg · 1⁻¹ for 48 h during the logarithmic growth phase. All chemicals were catabolized The predominant fractions found were polar conjugates and non-extractable (bound) residues, with significantly less non-polar conversion products. Soybean cells often released large amounts of polar conjugates into the medium while in wheat cultures metabolites were mainly cell-associated. High rates of pentachlorophenol and chlorinated aniline incorporation into non-extractable residues were found in wheat suspension cells.     

Aerobic versus anaerobic metabolism of halogenated anilines by aParacoccus sp.

AParacoccus sp. which transforms aniline and different halogen-substituted derivatives under aerobic and anaerobic conditions was isolated from the soil. In experiments with¹⁴C-ring-labeled 4-chloroaniline, approximately 60% of the radioactive material disappeared from the growth medium after incubation under anaerobiosis within 48 hr, but under aerobic conditions no decrease of radioactivity in the growth medium was observed, although 4-chloroaniline was completely metabolized. Acetylation appears to constitute, especially under aerobic conditions, a major transformation mechanism by the bacterium, since almost 50% of the acetylated compound could be detected and identified if aniline, 2-, 3-, and 4-chloroaniline served as substrate. The formation of different metabolites under aerobic and anaerobic conditions clearly indicates the existence of two separate pathways in the metabolism of aniline compounds depending on the oxygen status of the environment.     

Effect of Sulfate and Organic Carbon Supplements on Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries

When di-, tri-, and tetrachloroaniline were incubated in methanogenic groundwater slurries, they were reductively dehalogenated by the aquifer microbiota. 2,3,4-Trichloroaniline was metabolized by two pathways. Primary dehalogenation occurred at either the meta or ortho position of this substrate to form 2,4- and 3,4-dichloroaniline, respectively. The latter chemical could be stoichiometrically converted to 3-chloroaniline. 2,3,4,5-Tetrachloroaniline was degraded by the sequential removal of halogens from the para and then the ortho position to form 3,5-dichloroaniline. An additional pathway was observed with this substrate when the aquifer slurries were amended with butyrate. That is, halogens could be removed from both the meta and ortho positions of tetrachloroaniline. The amendment of sulfate to methanogenic aquifer slurries slowed the rate of 2,3,4,5-tetrachloroaniline degradation and increased the amount of substrate channeled through the additional pathway. The reported intermediates or end products are identified by their chromatographic mobility and mass-spectral profiles.     

Characterization of isofunctional ring-cleaving enzymes in aniline and 3-chloroaniline degradation by Pseudomonas acidovorans CA28

During degradation of aniline and 3-chloroaniline, respectively, by Pseudomonas acidovorans CA28, selective induction of two catechol 1,2-dioxygenases (C12O) was observed. C12O I activity was the sole ring-cleaving enzyme detectable in cell-free extracts after growth on aniline, while C12O II was exclusively found after growth on 3-chloroaniline. Both enzymes were clearly differentiated by their elution behaviour on DEAE-cellulose and their substrate specificities. For C12O I high activity was demonstrable only with unsubstituted catechol, while C12O II showed preference for and high affinity towards chlorinated catechols. Therefore, evidence of different ortho-cleavage enzymes in Pseudomonas acidovorans CA28 involved in aniline and 3-chloroaniline metabolism, respectively, is indicated.     

Stimulation of 3,4-dichloroaniline mineralization by aniline.

Mineralization of free and of humus-bound 3,4-dichloroaniline (DCA) by a Pseudomonas putida strain isolated by analog enrichment was greatly enhanced in the presence of aniline. The addition of aniline to soil that contained 0.2 to 100 micrograms of DCA per g in free or in humus-bound form increased the mineralization rates of DCA severalfold. Within the concentration ranges tested, absolute mineralization of DCA per unit time was positively correlated with both increasing DCA and increasing aniline concentrations. The specific enrichment of microbial populations and the induction of pathways that can co-metabolize DCA are the most plausible explanations for the effect of aniline. The observed phenomenon points to a potential approach for eliminating xenobiotic pollutants from contaminated soils.