Engineering multi-degrading bacterial communities to bioremediate soils contaminated with pesticides residues.

Parallel to the important use of pesticides in conventional agriculture there is a growing interest for green technologies to clear contaminated soil from pesticides and their degradation products. Bioaugmentation i. e. the inoculation of degrading micro-organisms in polluted soil, is a promising method still in needs of further developments. Specifically, improvements in the understanding of how degrading microorganisms must overcome abiotic filters and interact with the autochthonous microbial communities are needed in order to efficiently design bioremediation strategies. Here we designed a protocol aiming at studying the degradation of two herbicides, glyphosate (GLY) and isoproturon (IPU), via experimental modifications of two source bacterial communities. We used statistical methods stemming from genomic prediction to link community composition to herbicides degradation potentials. Our approach proved to be efficient with correlation estimates over 0.8 - between model predictions and measured pesticide degradation values. Multi-degrading bacterial communities were obtained by coalescing bacterial communities with high GLY or IPU degradation ability based on their community-level properties. Finally, we evaluated the efficiency of constructed multi-degrading communities to remove pesticide contamination in a different soil. While results are less clear in the case of GLY, we showed an efficient transfer of degrading capacities towards the receiving soil even at relatively low inoculation levels in the case of IPU. Altogether, we developed an innovative protocol for building multi-degrading simplified bacterial communities with the help of genomic prediction tools and coalescence, and proved their efficiency in a contaminated soil.

Biodegradation of a complex hydrocarbon mixture and biosurfactant production by Burkholderia thailandensis E264 and an adapted microbial consortium.

Bioremediation is considered to be an effective treatment for hydrocarbon removal from polluted soils. However, the effectiveness of this treatment is often limited by the low availability of targeted contaminants. Biosurfactants produced by some microorganisms can increase organic compound solubility and might then overcome this limitation. Two different inocula producers of biosurfactants (Burkholderia thailandensis E264 and SHEMS1 microbial consortium isolated from a hydrocarbon-contaminated soil) were incubated in Bushnell-Haas medium supplemented with hydrocarbons to investigate their biodegradation potential. Experimental results showed their ability to degrade 9.1 and 6.1% of hydrocarbons respectively after 65 days of incubation with an initial total hydrocarbon concentration of 16 g L-1. The biodegradation was more effective for the light and medium fractions (C10 to C36). B. thailandensis and SHEMS1 consortium produced surfactants after 14 days of culture during the stationary phase with hydrocarbons as the sole carbon and energy source. However, biosurfactant production did not appear to directly increase hydrocarbon degradation efficiency. The complexity and recalcitrance of hydrocarbon mixture used in this study appeared to continue to limit its biodegradation even in the presence of biosurfactants. In conclusion, B. thailandensis and SHEMS1 consortium can degrade recalcitrant hydrocarbon compounds and are therefore good candidates for the bioremediation of environments polluted by total hydrocarbons.

Effect of subtherapeutic and therapeutic sulfamethazine concentrations on transcribed genes and translated proteins involved in Microbacterium sp. C448 resistance and degradation.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into the Microbacterium sp. C448 SMZ detoxification process.

Experimental Evidence for Manure-Borne Bacteria Invasion in Soil During a Coalescent Event: Influence of the Antibiotic Sulfamethazine.

The fertilization of agricultural soil by organic amendment that may contain antibiotics, like manure, can transfer bacterial pathogens and antibiotic-resistant bacteria to soil communities. However, the invasion by manure-borne bacteria in amended soil remains poorly understood. We hypothesized that this kind of process is both influenced by the soil properties (and those of its microbial communities) and by the presence of contaminants such as antibiotics used in veterinary care. To test that, we performed a microcosm experiment in which four different soils were amended or not with manure at an agronomical dose and exposed or not to the antibiotic sulfamethazine (SMZ). After 1 month of incubation, the diversity, structure, and composition of bacterial communities of the soils were assessed by 16S rDNA sequencing. The invasion of manure-borne bacteria was still perceptible 1 month after the soil amendment. The results obtained with the soil already amended in situ with manure 6 months prior to the experiment suggest that some of the bacterial invaders were established in the community over the long term. Even if differences were observed between soils, the invasion was mainly attributable to some of the most abundant OTUs of manure (mainly Firmicutes). SMZ exposure had a limited influence on soil microorganisms but our results suggest that this kind of contaminant can enhance the invasion ability of some manure-borne invaders.

Assessing the importance of interspecific interactions in the evolution of microbial communities.

Interspecific interactions play an important role in the establishment of a community phenotype. Furthermore, the evolution of a community can both occur through an independent evolution of the species composing the community and the interactions among them. In this study, we investigated how important the evolution of interspecific interactions was in the evolutionary response of eight two-bacterial species communities regarding productivity. We found evidence for an evolution of the interactions in half of the studied communities, which gave rise to a mean change of 15% in community productivity as compared to what was expected from the individual responses. Even when the interactions did not evolve themselves, they influenced the evolutionary responses of the bacterial strains within the communities, which further affected community response. We found that evolution within a community often promoted the adaptation of the bacterial strains to the abiotic environment, especially for the dominant strain in a community. Overall, this study suggested that the evolution of the interspecific interactions was frequent and that it could increase community response to evolution.

Community diversity determines the evolution of synthetic bacterial communities under artificial selection.

Artificial selection can be conducted at the community level in the laboratory through a differential propagation of the communities according to their level of expression of a targeted function. Working with communities instead of individuals as selection units brings in additional sources of variation in the considered function that can influence the outcome of the artificial selection. In this study, we wanted to assess the effect of manipulating the initial community richness on artificial selection efficiency, defined as the change in the targeted function over time. We applied artificial selection for a high productivity on synthetic bacterial communities varying for their richness (from one to 16 strains). Overall, the selected communities were 16% more productive than the control communities, but a convergence of community composition might have limited the effect of diversity on artificial selection efficiency. Community richness positively influenced community productivity and metabolic capacities and was a strong determinant of the dynamics of community evolution. We propose that applying artificial selection on communities varying for their diversity could be a way to find communities differing for their level of expression of a function but also for their responsiveness to artificial selection, provided that their initial composition is different enough.

Antibiotrophy: Key Function for Antibiotic-Resistant Bacteria to Colonize Soils-Case of Sulfamethazine-Degrading Microbacterium sp. C448.

Chronic and repeated exposure of environmental bacterial communities to anthropogenic antibiotics have recently driven some antibiotic-resistant bacteria to acquire catabolic functions, enabling them to use antibiotics as nutritive sources (antibiotrophy). Antibiotrophy might confer a selective advantage facilitating the implantation and dispersion of antibiotrophs in contaminated environments. A microcosm experiment was conducted to test this hypothesis in an agroecosystem context. The sulfonamide-degrading and resistant bacterium Microbacterium sp. C448 was inoculated in four different soil types with and without added sulfamethazine and/or swine manure. After 1 month of incubation, Microbacterium sp. (and its antibiotrophic gene sadA) was detected only in the sulfamethazine-treated soils, suggesting a low competitiveness of the strain without antibiotic selection pressure. In the absence of manure and despite the presence of Microbacterium sp. C448, only one of the four sulfamethazine-treated soils exhibited mineralization capacities, which were low (inferior to 5.5 ± 0.3%). By contrast, manure addition significantly enhanced sulfamethazine mineralization in all the soil types (at least double, comprised between 5.6 ± 0.7% and 19.5 ± 1.2%). These results, which confirm that the presence of functional genes does not necessarily ensure functionality, suggest that sulfamethazine does not necessarily confer a selective advantage on the degrading strain as a nutritional source. 16S rDNA sequencing analyses strongly suggest that sulfamethazine released trophic niches by biocidal action. Accordingly, manure-originating bacteria and/or Microbacterium sp. C448 could gain access to low-competition or competition-free ecological niches. However, simultaneous inputs of manure and of the strain could induce competition detrimental for Microbacterium sp. C448, forcing it to use sulfamethazine as a nutritional source. Altogether, these results suggest that the antibiotrophic strain studied can modulate its sulfamethazine-degrading function depending on microbial competition and resource accessibility, to become established in an agricultural soil. Most importantly, this work highlights an increased dispersal potential of antibiotrophs in antibiotic-polluted environments, as antibiotics can not only release existing trophic niches but also form new ones.

Complete Genome Sequences of Four Atrazine-Degrading Bacterial Strains, Pseudomonas sp. Strain ADPe, Arthrobacter sp. Strain TES, Variovorax sp. Strain 38R, and Chelatobacter sp. Strain SR38.

We report here the complete genome sequences of four atrazine-degrading bacteria. Their genomes will serve as references for determining the genetic changes that have occurred during an evolution experiment.

Insights into the Function and Horizontal Transfer of Isoproturon Degradation Genes (pdmAB) in a Biobed System.

Biobeds, designed to minimize pesticide point source contamination, rely mainly on biodegradation processes. We studied the interactions of a biobed microbial community with the herbicide isoproturon (IPU) to explore the role of the pdmA gene, encoding the large subunit of an N-demethylase responsible for the initial demethylation of IPU, via quantitative PCR (qPCR) and reverse transcription-PCR (RT-qPCR) and the effect of IPU on the diversity of the total bacterial community and its active fraction through amplicon sequencing of DNA and RNA, respectively. We further investigated the localization and dispersal mechanisms of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. strain SH (previously isolated from the soil used in the biobed) compared with the abundance of the pdmA gene and metagenomic fosmid library screening. pdmA abundance and expression increased concomitantly with IPU mineralization, verifying its major role in IPU transformation in the biobed system. DNA- and RNA-based 16S rRNA gene sequencing analysis showed no effects on bacterial diversity. The pdmAB-harboring plasmid pSH showed a consistently lower abundance than pdmA, suggesting the localization of pdmAB in replicons other than pSH. Metagenomic analysis identified four pdmAB-carrying fosmids. In three of these fosmids, the pdmAB genes were organized in a well-conserved operon carried by sphingomonad plasmids with low synteny with pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potentially crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon.IMPORTANCE Our study provides novel insights into the interactions of IPU with the bacterial community of biobed systems, reinforces the assumption of a transposable nature of IPU-degrading genes, and verifies that on-farm biobed systems are hot spots for the evolution of pesticide catabolic traits.

Assessing the Effects of ß-Triketone Herbicides on the Soil Bacterial and hppd Communities: A Lab-to-Field Experiment.

Maize cultivators often use ß-triketone herbicides to prevent the growth of weeds in their fields. These herbicides target the 4-HPPD enzyme of dicotyledons. This enzyme, encoded by the hppd gene, is widespread among all living organisms including soil bacteria, which are considered as "non-target organisms" by the legislation. Within the framework of the pesticide registration process, the ecotoxicological impact of herbicides on soil microorganisms is solely based on carbon and nitrogen mineralization tests. In this study, we used more extensive approaches to assess with a lab-to-field experiment the risk of ß-triketone on the abundance and the diversity of both total and hppd soil bacterial communities. Soil microcosms were exposed, under lab conditions, to 1× or 10× the recommended dose of sulcotrione or its commercial product, Decano®. Whatever the treatment applied, sulcotrione was fully dissipated from soil after 42 days post-treatment. The abundance and the diversity of both the total and the hppd bacterial communities were not affected by the herbicide treatments all along the experiment. Same measurements were led in real agronomical conditions, on three different fields located in the same area cropped with maize: one not exposed to any plant protection products, another one exposed to a series of plant protection products (PPPs) comprising mesotrione, and a last one exposed to different PPPs including mesotrione and tembotrione, two ß-triketones. In this latter, the abundance of the hppd community varied over time. The diversity of the total and the hppd communities evolved over time independently from the treatment received. Only slight but significant transient effects on the abundance of the hppd community in one of the tested soil were observed. Our results showed that tested ß-triketones have no visible impact toward both total and hppd soil bacteria communities.

Environmental risk assessment of antibiotics in agroecosystems: ecotoxicological effects on aquatic microbial communities and dissemination of antimicrobial resistances and antibiotic biodegradation potential along the soil-water continuum.

Antibiotics have a wide application range in human and veterinary medicines. Being designed for pharmacological stability, most antibiotics are recalcitrant to biodegradation after ingestion and can be persistent in the environment. Antibiotic residues have been detected as contaminants in various environmental compartments where they cause human and environmental threats, notably with respect to the potential emergence and proliferation of antibiotic-resistant bacteria. An important component of managing environmental risk caused by antibiotics is to understand exposure of soil and water resources to their residues. One challenge is to gain knowledge on the fate of antibiotics in the ecosystem along the soil-water continuum, and on the collateral impact of antibiotics on environmental microorganisms responsible for crucially important ecosystem functions. In this context, the ANTIBIOTOX project aims at studying the environmental fate and impact of two antibiotics of the sulfonamide class of antibiotics, sulfamethazine (SMZ), and sulfamethoxazole (SMX).

Assessment of the ecotoxicological impact of natural and synthetic ß-triketone herbicides on the diversity and activity of the soil bacterial community using omic approaches.

The emergence of pesticides of natural origin appears as an environmental-friendly alternative to synthetic pesticides for managing weeds. To verify this assumption, leptospermone, a natural ß-triketone herbicide, and sulcotrione, a synthetic one, were applied to soil microcosms at 0× (control), 1× or 10× recommended field dose. The fate of these two herbicides (i.e. dissipation and formation of transformation products) was monitored to assess the scenario of exposure of soil microorganisms to natural and synthetic herbicides. Ecotoxicological impact of both herbicides was explored by monitoring soil bacterial diversity and activity using next-generation sequencing of 16S rRNA gene amplicons and soil metabolomics. Both leptospermone and sulcotrione fully dissipated over the incubation period. During their dissipation, transformation products of natural and synthetic ß-triketone were detected. Hydroxy-leptospermone was almost completely dissipated by the end of the experiment, while CMBA, the major metabolite of sulcotrione, remained in soil microcosms. After 8 days of exposure, the diversity and structure of the soil bacterial community treated with leptospermone was significantly modified, while less significant changes were observed for sulcotrione. For both herbicides, the diversity of the soil bacterial community was still not completely recovered by the end of the experiment (45 days). The combined use of next-generation sequencing and metabolomic approaches allowed us to assess the ecotoxicological impact of natural and synthetic pesticides on non-target soil microorganisms and to detect potential biomarkers of soil exposure to ß-triketones.

Assessment of the effects of oxamyl on the bacterial community of an agricultural soil exhibiting enhanced biodegradation.

Modern agricultural practices largely rely on pesticides to protect crops against various pests and to ensure high yields. Following their application to crops a large amount of pesticides ends up in soil where they may affect non-target organisms, among which microorganisms. We assessed the effects of the carbamate nematicide oxamyl on the whole bacterial diversity of an agricultural soil exhibiting enhanced biodegradation of oxamyl through 16S rRNA amplicon next generation sequencing (NGS) and on the oxamyl-degrading bacterial community through cehA q-PCR analysis and 14C-oxamyl mineralization assays. Oxamyl was rapidly mineralized by the indigenous microorganisms reaching >70% within a month. Concomitantly, a significant increase in the number of oxamyl-degrading microorganisms was observed. NGS analysis of the total (DNA) and active (RNA) bacterial community showed no changes in α-diversity indices in response to oxamyl exposure. Analysis of the ß-diversity revealed significant changes in the composition of the soil bacterial community after 13 and 30 days of oxamyl exposure only when the active fraction of the bacterial community was considered. These changes were associated with seven OTUs related to Proteobacteria (5), Acidobacteria (1) and Actinobacteria (1). The relative abundance of the dominant bacterial phyla were not affected by oxamyl, except of Bacteroidetes and Gemmatimonadetes which decreased after 13 and 30 days of oxamyl exposure respectively. To conclude, oxamyl induced changes in the abundance of oxamyl-degrading microorganisms and on the diversity of the soil bacterial community. The latter became evident only upon RNA-based NGS analysis emphasizing the utility of such approaches when the effects of pesticides on the soil microbial community are explored.

Lab to Field Assessment of the Ecotoxicological Impact of Chlorpyrifos, Isoproturon, or Tebuconazole on the Diversity and Composition of the Soil Bacterial Community.

Pesticides are intentionally applied to agricultural fields for crop protection. They can harm non-target organisms such as soil microorganisms involved in important ecosystem functions with impacts at the global scale. Within the frame of the pesticide registration process, the ecotoxicological impact of pesticides on soil microorganisms is still based on carbon and nitrogen mineralization tests, despite the availability of more extensive approaches analyzing the abundance, activity or diversity of soil microorganisms. In this study, we used a high-density DNA microarray (PhyloChip) and 16S rDNA amplicon next-generation sequencing (NGS) to analyze the impact of the organophosphate insecticide chlorpyrifos (CHL), the phenyl-urea herbicide isoproturon (IPU), or the triazole fungicide tebuconazole (TCZ) on the diversity and composition of the soil bacterial community. To our knowledge, it is the first time that the combination of these approaches are applied to assess the impact of these three pesticides in a lab-to-field experimental design. The PhyloChip analysis revealed that although no significant changes in the composition of the bacterial community were observed in soil microcosms exposed to the pesticides, significant differences in detected operational taxonomic units (OTUs) were observed in the field experiment between pesticide treatments and control for all three tested pesticides after 70 days of exposure. NGS revealed that the bacterial diversity and composition varied over time. This trend was more marked in the microcosm than in the field study. Only slight but significant transient effects of CHL or TCZ were observed in the microcosm and the field study, respectively. IPU was not found to significantly modify the soil bacterial diversity or composition. Our results are in accordance with conclusions of the Environmental Food Safety Authority (EFSA), which concluded that these three pesticides may have a low risk toward soil microorganisms.

Evidence for photolytic and microbial degradation processes in the dissipation of leptospermone, a natural ß-triketone herbicide.

Bioherbicides appear as an ecofriendly alternative to synthetic herbicides, generally used for weed management, because they are supposed to have low side on human health and ecosystems. In this context, our work aims to study abiotic (i.e., photolysis) and biotic (i.e,. biodegradation) processes involved in the fate of leptospermone, a natural ß-triketone herbicide, by combining chemical and microbiological approaches. Under controlled conditions, the photolysis of leptospermone was sensitive to pH. Leptospermone has a half-life of 72 h under simulated solar light irradiations. Several transformation products, including hydroxy-leptospermone, were identified. For the first time, a bacterial strain able to degrade leptospermone was isolated from an arable soil. Based on its 16S ribosomal RNA (rRNA) gene sequence, it was affiliated to the Methylophilus group and was accordingly named as Methylophilus sp. LS1. Interestingly, we report that the abundance of OTUs, similar to the 16S rRNA gene sequence of Methylophilus sp. LS1, was strongly increased in soil treated with leptospermone. The leptospermone was completely dissipated by this bacteria, with a half-life time of 6 days, allowing concomitantly its growth. Hydroxy-leptospermone was identified in the bacterial culture as a major transformation product, allowing us to propose a pathway of transformation of leptospermone including both abiotic and biotic processes.

Ecotoxicological Impact of the Bioherbicide Leptospermone on the Microbial Community of Two Arable Soils.

The ecotoxicological impact of leptospermone, a ß-triketone bioherbicide, on the bacterial community of two arable soils was investigated. Soil microcosms were exposed to 0 × (control), 1 × or 10 × recommended dose of leptospermone. The ß-triketone was moderately adsorbed to both soils (i.e.,: K fa ~ 1.2 and K oc ~ 140 mL g(-1)). Its dissipation was lower in sterilized than in unsterilized soils suggesting that it was mainly influenced by biotic factors. Within 45 days, leptospermone disappeared almost entirely from one of the two soils (i.e., DT50 < 10 days), while 25% remained in the other. The composition of the microbial community assessed by qPCR targeting 11 microbial groups was found to be significantly modified in soil microcosms exposed to leptospermone. Pyrosequencing of 16S rRNA gene amplicons showed a shift in the bacterial community structure and a significant impact of leptospermone on the diversity of the soil bacterial community. Changes in the composition, and in the α- and ß-diversity of microbial community were transient in the soil able to fully dissipate the leptospermone, but were persistent in the soil where ß-triketone remained. To conclude the bacterial community of the two soils was sensitive to leptospermone and its resilience was observed only when leptospermone was fully dissipated.

Draft Genome Sequence of Pseudomonas sp. Strain ADP, a Bacterial Model for Studying the Degradation of the Herbicide Atrazine.

We report here the 7,259,392-bp draft genome of Pseudomonas sp. strain ADP. This is a bacterial strain that was first isolated in the 1990s from soil for its ability to mineralize the herbicide atrazine. It has extensively been studied as a model to understand the atrazine biodegradation pathway. This genome will be used as a reference and compared to evolved populations obtained by experimental evolution conducted on this strain under atrazine selection pressure.

s-triazine degrading bacterial isolate Arthrobacter sp. AK-YN10, a candidate for bioaugmentation of atrazine contaminated soil.

The Arthrobacter sp. strain AK-YN10 is an s-triazine pesticide degrading bacterium isolated from a sugarcane field in Central India with history of repeated atrazine use. AK-YN10 was shown to degrade 99 % of atrazine in 30 h from media supplemented with 1000 mg L(-1) of the herbicide. Draft genome sequencing revealed similarity to pAO1, TC1, and TC2 catabolic plasmids of the Arthrobacter taxon. Plasmid profiling analyses revealed the presence of four catabolic plasmids. The trzN, atzB, and atzC atrazine-degrading genes were located on a plasmid of approximately 113 kb.The flagellar operon found in the AK-YN10 draft genome suggests motility, an interesting trait for a bioremediation agent, and was homologous to that of Arthrobacter chlorophenolicus. The multiple s-triazines degradation property of this isolate makes it a good candidate for bioremediation of soils contaminated by s-triazine pesticides.

Isolation and characterization of Bradyrhizobium sp. SR1 degrading two ß-triketone herbicides.

In this study, a bacterial strain able to use sulcotrione, a ß-triketone herbicide, as sole source of carbon and energy was isolated from soil samples previously treated with this herbicide. Phylogenetic study based on16S rRNA gene sequence showed that the isolate has 100 % of similarity with several Bradyrhizobium and was accordingly designated as Bradyrhizobium sp. SR1. Plasmid profiling revealed the presence of a large plasmid (>50 kb) in SR1 not cured under nonselective conditions. Its transfer to Escherichia coli by electroporation failed to induce ß-triketone degrading capacity, suggesting that degrading genes possibly located on this plasmid cannot be expressed in E. coli or that they are not plasmid borne. The evaluation of the SR1 ability to degrade various synthetic (mesotrione and tembotrione) and natural (leptospermone) triketones showed that this strain was also able to degrade mesotrione. Although SR1 was able to entirely dissipate both herbicides, degradation rate of sulcotrione was ten times higher than that of mesotrione, showing a greater affinity of degrading-enzyme system to sulcotrione. Degradation pathway of sulcotrione involved the formation of 2-chloro-4-mesylbenzoic acid (CMBA), previously identified in sulcotrione degradation, and of a new metabolite identified as hydroxy-sulcotrione. Mesotrione degradation pathway leads to the accumulation of 4-methylsulfonyl-2-nitrobenzoic acid (MNBA) and 2-amino-4 methylsulfonylbenzoic acid (AMBA), two well-known metabolites of this herbicide. Along with the dissipation of ß-triketones, one could observe the decrease in 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition, indicating that toxicity was due to parent molecules, and not to the formed metabolites. This is the first report of the isolation of bacterial strain able to transform two ß-triketones.

Evidence for cooperative mineralization of diuron by Arthrobacter sp. BS2 and Achromobacter sp. SP1 isolated from a mixed culture enriched from diuron exposed environments.

Diuron was found to be mineralized in buffer strip soil (BS) and in the sediments (SED) of the Morcille river in the Beaujolais vineyard repeatedly treated with this herbicide. Enrichment cultures from BS and SED samples led to the isolation of three bacterial strains transforming diuron to 3,4-dichloroaniline (3,4-DCA) its aniline derivative. 16S rRNA sequencing revealed that they belonged to the genus Arthrobacter (99% of similarity to Arthrobacter globiformis strain K01-01) and were designated as Arthrobacter sp. BS1, BS2 and SED1. Diuron-degrading potential characterized by sequencing of the puhA gene, characterizing the diuron-degradaing potential, revealed 99% similarity to A. globiformis strain D47 puhA gene isolated a decade ago in the UK. These isolates were also able to use chlorotoluron for their growth. Although able to degrade linuron and monolinuron to related aniline derivatives they were not growing on them. Enrichment cultures led to the isolation of a strain from the sediments entirely degrading 3,4-DCA. 16S rRNA sequence analysis showed that it was affiliated to the genus Achromobacter (99% of similarity to Achromobacter sp. CH1) and was designated as Achromobacter sp. SP1. The dcaQ gene encoding enzyme responsible for the transformation of 3,4-DCA to chlorocatechol was found in SP1 with 99% similarity to that of Comamonas testosteroni WDL7. This isolate also used for its growth a range of anilines (3-chloro-4-methyl-aniline, 4-isopropylaniline, 4-chloroaniline, 3-chloroaniline, 4-bromoaniline). The mixed culture composed of BS2 and SP1 strains entirely mineralizes (14)C-diuron to (14)CO2. Diuron-mineralization observed in the enrichment culture could result from the metabolic cooperation between these two populations.