Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles

The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides.     

Degradation of Chlorbromuron and Related Compounds by the Fungus Rhizoctonia solani

The ability of the soil fungus Rhizoctonia solani to degrade phenyl-substituted urea herbicides was investigated. The fungus was able to transform chlorbromuron [3-(3-chloro-4-bromophenyl)-1-methyl-1-methoxyurea] to the demethylated product [3-(3-chloro-4-bromophenyl)-1-methoxyurea], which was isolated and identified. Evidence was obtained that further degradation of chlorbromuron occurred. Several other phenylurea compounds (chloroxuron, diuron, fenuron, fluometuron, linuron, metobromuron, neburon, and siduron) were also metabolized by the fungus, indicating that R. solani may possess a generalized ability to attack this group of herbicides.     

The quantity and quality of dissolved organic matter as supplementary carbon source impacts the pesticide-degrading activity of a triple-species bacterial biofilm

Effects of environmental dissolved organic matter (eDOM) that consists of various low concentration carbonic compounds on pollutant biodegradation by bacteria are poorly understood, especially when it concerns synergistic xenobiotic-degrading consortia where degradation depends on interspecies metabolic interactions. This study examines the impact of the quality and quantity of eDOM, supplied as secondary C-source, on the structure, composition and pesticide-degrading activity of a triple-species bacterial consortium in which the members synergistically degrade the phenylurea herbicide linuron, when grown as biofilms. Biofilms developing on 10 mg L^?1 linuron showed a steady-state linuron degradation efficiency of approximately 85 %. The three bacterial strains co-localized in the biofilms indicating syntrophic interactions. Subsequent feeding with eDOM or citrate in addition to linuron resulted into changes in linuron-degrading activity. A decrease in linuron-degrading activity was especially recorded in case of co-feeding with citrate and eDOM of high quality and was always associated with accumulation of the primary metabolite 3,4-dichloroaniline. Improvement of linuron degradation was especially observed with more recalcitrant eDOM. Addition of eDOM/citrate formulations altered biofilm architecture and species composition but without loss of any of the strains and of co-localization. Compositional shifts correlated with linuron degradation efficiencies. When the feed was restored to only linuron, the linuron-degrading activity rapidly changed to the level before the mixed-substrate feed. Meanwhile only minor changes in biofilm composition and structure were recorded, indicating that observed eDOM/citrate effects had been primarily due to repression/stimulation of linuron catabolic activity rather than to biofilm characteristics.     

Effects of enrichment with phthalate on polycyclic aromatic hydrocarbon biodegradation in contaminated soil

The effect of enrichment with phthalate on the biodegradation of polycyclic aromatic hydrocarbons (PAH) was tested with bioreactor-treated and untreated contaminated soil from a former manufactured gas plant (MGP) site. Soil samples that had been treated in a bioreactor and enriched with phthalate mineralized (14)C-labeled phenanthrene and pyrene to a greater extent than unenriched samples over a 22.5-h incubation, but did not stimulate benzo[a]pyrene mineralization. In contrast to the positive effects on (14)C-labeled phenanthrene and pyrene, no significant differences were found in the extent of biodegradation of native PAH when untreated contaminated soil was incubated with and without phthalate amendment. Denaturing-gradient gel electrophoresis (DGGE) profiles of bacterial 16S rRNA genes from unenriched and phthalate-enriched soil samples were substantially different, and clonal sequences matched to prominent DGGE bands revealed that beta-Proteobacteria related to Ralstonia were most highly enriched by phthalate addition. Quantitative real-time PCR analyses confirmed that, of previously determined PAH-degraders in the bioreactor, only Ralstonia-type organisms increased in response to enrichment, accounting for 89% of the additional bacterial 16S rRNA genes resulting from phthalate enrichment. These findings indicate that phthalate amendment of this particular PAH-contaminated soil did not significantly enrich for organisms associated with high molecular weight PAH degradation or have any significant effect on overall degradation of native PAH in the soil.     

Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading variovorax strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

Degradation of iprodione by a soil Arthrobacter-like strain.

A bacterial strain able to transform iprodione was isolated from a fast iprodione-degrading soil by enrichment procedures. Transformation was detected through 3,5-dichloroaniline production as measured by a rapid colorimetric method. The strain, MA6, was tentatively identified as an Arthrobacter sp. When it was incubated with MA6 in a minimum mineral medium (pH 6.5), iprodione (8.8 mumol/liter) was transformed into two major metabolites that were identified by high-performance liquid chromatography analysis: 3,5-dichlorophenylcarboximide (metabolite 1) and (3,5-dichlorophenylurea) acetic acid (metabolite 2), which was produced after ring cleavage of the former product. These products were synthesized in the laboratory and compared with metabolites 1 and 2 which were formed during iprodione degradation. Small quantities of 3,5-dichloroaniline also appeared in the bacterial culture but did not substantially increase between the first and second days of incubation. In contrast, in the sterile control medium, iprodione was spontaneously transformed into hydantoic acid and an iprodione isomer. Chemical and biological transformations of iprodione seem to occur through two different pathways. One biological degradation pathway is proposed.     

Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading Variovorax Strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

Microbial community profiling in cis- and trans-dichloroethene enrichment systems using denaturing gradient gel electrophoresis

The effective and accurate assessment of the total microbial community diversity is one of the primary challenges in modem microbial ecology, especially for the detection and characterization of unculturable populations and populations with a low abundance. Accordingly, this study was undertaken to investigate the diversity of the microbial community during the biodegradation of cis- and trans-dichloroethenes in soil and wastewater enrichment cultures. Community profiling using PCR targeting the 16S rRNA gene and denaturing gradient gel electrophoresis (PCR-DGGE) revealed an alteration in the bacterial community profiles with time. Exposure to cis- and trans-dichloroethenes led to the disappearance of certain genospecies that were initially observed in the untreated samples. A cluster analysis of the bacterial DGGE community profiles at various sampling times during the degradation process indicated that the community profile became stable after day 10 of the enrichment. DNA sequencing and phylogenetic analysis of selected DGGE bands revealed that the genera Acinetobacter, Pseudomonas, Bacillus, Comamonas, and Arthrobacter, plus several other important uncultured bacterial phylotypes, dominated the enrichment cultures. Thus, the identified dominant phylotypes may play an important role in the degradation of cis- and trans-dichloroethenes.     

Purification and Properties of an Aryl Acylamidase of Bacillus sphaericus, Catalyzing the Hydrolysis of Various Phenylamide Herbicides and Fungicides

From Bacillus sphaericus ATCC 12123 an aryl acylamidase (EC 3.5.1.13) was purified to homogeneity by ion exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. The enzyme is inducible by various phenylamides of the acylanilide, phenylcarbamate, and methoxysubstituted phenylurea type. It has a molecular weight of 75,000. Enzyme activity was inhibited by sulfhydryl reagents, several metal ions, and 3,4-dichloroaniline (a product of linuron degradation). A requirement for divalent metal ions in enzyme activity could not be demonstrated. In the presence of 6 M urea an irreversible inactivation of the enzyme occurred. The hydrolysis of L-alanine-4-nitroanilide was competitively inhibited by puromycin.     

Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon.

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the alpha-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

3,4-dichloroaniline cometabolism by representatives of the genus Pseudomonas

The effect of propanide, linuron and 3,4-dichloroaniline on soil organisms was studied. Two strains of Pseudomonas aurantiaca 1 and 7, were isolated from soil; they decomposed propanide yielding 3,4-dichloroaniline. These strains, as well as a number of collection cultures belonging to the Pseudomonas genus, could transform 3,4-dichloroaniline at a rate of 0-100 per cent during 48 hours. A certain correlation existed between this transformation ability and the level of total oxidase activity. All the strains of Pseudomonas studied in this work were characterized by a low peroxidase activity, and no strict correlation was detected between its level and the ability to transform 3,4-dichloroaniline.     

Survival and performance of two cellulose-degrading microbial systems inoculated into wheat straw-amended soil

A cellulose-degrading composite microbial system containing a mixture of microbes was previously shown to demonstrate a high straw-degrading capacity. To estimate its potential utilization as an inoculant to accelerate straw biodegradation after returning straw to the field, two cellulose-degrading composite microbial systems named ADS3 and WSD5 were inoculated into wheat straw-amended soil in the laboratory. The microbial survival of the inoculant was confirmed by a denaturing gradient gel electrophoresis (DGGE) analysis, whereas the enhancement of straw degradation in soil was assessed by measuring the mineralization of the soil organic matter and the soil cellulase activity. The results indicated that most of the DGGE bands from ADS3 were detected after inoculation into straw-amended autoclaved soil, yet only certain bands from ADS3 and WSD5 were detected after inoculation into straw-amended non-autoclaved soil during five weeks of incubation; some bands were detected during the first two weeks after inoculation, and then disappeared in later stages. Organic matter mineralization was significantly higher in the soil inoculants ADS3 and WSD5 than in the uninoculated controls during the first week, yet the enhanced degradation did not persist during the subsequent incubation. Similar to the increase in soil organic matter, the cellulase activity also increased during the first week in the ADS3 and WSD5 treatments, yet decreased during the remainder of the incubation period. Thus, it was concluded that, although the survival and performance of the two inoculants did not persist in the soil, a significant enhancement of degradation was present during the early stage of incubation.     

Investigation on the regulation of aniline utilization in Pseudomonas multivorans strain An 1

Summary Pseudomonas multivorans strain An 1 was isolated from forest soil after enrichment in a medium containing 0.1% aniline as the sole source of carbon and energy. Increasing aniline concentrations increasingly inhibited bacterial growth. At pH 7, aniline concentrations greater than 16mM were toxic enough to completely arrest growth. The optimal pH for growth on aniline was 6.5.On binary mixtures of aniline and additional carbon sources, diauxic growth was observed. The addition carbon sources caused various degrees of repression of the aniline catabolizing enzyme system. The fastest induction of this system occurred at pH 4, suggesting that protonization of the aniline molecule is crucial.     

Isolation from Agricultural Soil and Characterization of a Sphingomonas sp. Able To Mineralize the Phenylurea Herbicide Isoproturon

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

Effect of propanil,linuron, and dicamba on the degradation kinetics of 2,4‐dichlorophenoxyacetic acid by Burkholderia sp. A study by differential analysis of 2,4‐dichlorophenoxyacetic acid degradation data

The successive application of distinct pesticides, or mixtures of them, is a frequent practice that could adversely affect the microbial species inhabiting soil and aquatic ecosystems. The ability of soil or aquatic microbiota to degrade a pesticide could be affected by the presence of another. If the degradation rate of the first compound is inhibited, its dissipation half‐life in the environment could be hazardously enlarged. Few studies have been made to quantify the impact on the biodegradation rate of pesticides in soils or water by the presence of other pesticides. In this work, a method for assessing the effect of a pesticide on the biodegradation rate of another, measuring its effect on the biodegradation kinetics of a single bacterial strain is presented. The mathematical analysis is a powerful tool to study the stoichiometry and kinetics of microbial processes, which was used to evaluate independently, in detail, the effect of three pesticides (propanil, linuron, and dicamba) on the biodegradation kinetics of 2,4‐dichlorophenoxyacetic acid by a strain of Burkholderia sp. It was evidenced that linuron and dicamba caused a decay of more than 40% in the top instantaneous degradation rate of 2,4‐dichlorophenoxyacetic acid, while propanil showed a minimal effect.     

Responses of the anaerobic bacterial community to addition of organic C in chromium(VI)- and iron(III)-amended microcosms

Chromium (VI) is toxic to microorganisms and can inhibit the biodegradation of organic pollutants in contaminated soils. We used microcosms amended with either glucose or protein (to drive bacterial community change) and Fe(III) (to stimulate iron-reducing bacteria) to study the effect of various concentrations of Cr(VI) on anaerobic bacterial communities. Microcosms were destructively sampled based on microbial activity (measured as evolution of CO2) and analyzed for the following: (i) dominant bacterial community by PCR-denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene; (ii) culturable Cr-resistant bacteria; and (iii) enrichment of iron-reducing bacteria of the Geobacteraceae family by real-time PCR. The addition of organic C stimulated the activities of anaerobic communities. Cr(VI) amendment resulted in lower rates of CO2 production in glucose microcosms and a slow mineralization phase in protein-amended microcosms. Glucose and protein amendments selected for different bacterial communities. This selection was modified by the addition of Cr(VI), since some DGGE bands were intensified and new bands appeared in Cr(VI)-amended microcosms. A second dose of Cr(VI), added after the onset of activity, had a strong inhibitory effect when higher levels of Cr were added, indicating that the developing Cr-resistant communities had a relatively low tolerance threshold. Most of the isolated Cr-resistant bacteria were closely related to previously studied Cr-resistant anaerobes, such as Pantoea, Pseudomonas, and Enterobacter species. Geobacteraceae were not enriched during the incubation. The studied Cr(VI)-contaminated soil contained a viable anaerobic bacterial community; however, Cr(VI) altered its composition, which could affect the soil biodegradation potential.     

Responses of the Anaerobic Bacterial Community to Addition of Organic C in Chromium(VI)- and Iron(III)-Amended Microcosms

Chromium (VI) is toxic to microorganisms and can inhibit the biodegradation of organic pollutants in contaminated soils. We used microcosms amended with either glucose or protein (to drive bacterial community change) and Fe(III) (to stimulate iron-reducing bacteria) to study the effect of various concentrations of Cr(VI) on anaerobic bacterial communities. Microcosms were destructively sampled based on microbial activity (measured as evolution of CO₂) and analyzed for the following: (i) dominant bacterial community by PCR-denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene; (ii) culturable Cr-resistant bacteria; and (iii) enrichment of iron-reducing bacteria of the Geobacteraceae family by real-time PCR. The addition of organic C stimulated the activities of anaerobic communities. Cr(VI) amendment resulted in lower rates of CO₂ production in glucose microcosms and a slow mineralization phase in protein-amended microcosms. Glucose and protein amendments selected for different bacterial communities. This selection was modified by the addition of Cr(VI), since some DGGE bands were intensified and new bands appeared in Cr(VI)-amended microcosms. A second dose of Cr(VI), added after the onset of activity, had a strong inhibitory effect when higher levels of Cr were added, indicating that the developing Cr-resistant communities had a relatively low tolerance threshold. Most of the isolated Cr-resistant bacteria were closely related to previously studied Cr-resistant anaerobes, such as Pantoea, Pseudomonas, and Enterobacter species. Geobacteraceae were not enriched during the incubation. The studied Cr(VI)-contaminated soil contained a viable anaerobic bacterial community; however, Cr(VI) altered its composition, which could affect the soil biodegradation potential.     

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.     

Elucidating the key member of a linuron-mineralizing bacterial community by PCR and reverse transcription-PCR denaturing gradient gel electrophoresis 16S rRNA gene fingerprinting and cultivation

A bacterial community from Danish agricultural soil was enriched with linuron [N-(3,4-dichlorophenyl)-N'-methoxy-N'-methylurea] as the sole carbon and nitrogen source. The community mineralized [ring-U-14C]linuron completely to 14CO2 and 14C-biomass. Denaturing gradient gel electrophoresis analysis and cultivation revealed that a Variovorax sp. was responsible for the mineralization activity.