Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazole.

Thiabendazole (TBZ), is a persistent fungicide/anthelminthic and a serious environmental threat. We previously enriched a TBZ-degrading bacterial consortium and provided first evidence for a Sphingomonas involvement in TBZ transformation. Here, using a multi-omic approach combined with DNA-stable isotope probing (SIP) we verified the key degrading role of Sphingomonas and identify potential microbial interactions governing consortium functioning. SIP and amplicon sequencing analysis of the heavy and light DNA fraction of cultures grown on 13 C-labelled versus 12 C-TBZ showed that 66% of the 13 C-labelled TBZ was assimilated by Sphingomonas. Metagenomic analysis retrieved 18 metagenome-assembled genomes with the dominant belonging to Sphingomonas, Sinobacteriaceae, Bradyrhizobium, Filimonas and Hydrogenophaga. Meta-transcriptomics/-proteomics and non-target mass spectrometry suggested TBZ transformation by Sphingomonas via initial cleavage by a carbazole dioxygenase (car) to thiazole-4-carboxamidine (terminal compound) and catechol or a cleaved benzyl ring derivative, further transformed through an ortho-cleavage (cat) pathway. Microbial co-occurrence and gene expression networks suggested strong interactions between Sphingomonas and a Hydrogenophaga. The latter activated its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway to satisfy its B12 auxotrophy. Our findings indicate microbial interactions aligning with the 'black queen hypothesis' where Sphingomonas (detoxifier, B12 recipient) and Hydrogenophaga (B12 producer, enjoying detoxification) act as both helpers and beneficiaries.

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.

Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype.

Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, ß- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

Characterization of the biodegradation, bioremediation and detoxification capacity of a bacterial consortium able to degrade the fungicide thiabendazole.

Thiabendazole (TBZ) is a persistent fungicide used in the post-harvest treatment of fruits. Its application results in the production of contaminated effluents which should be treated before their environmental discharge. In the absence of efficient treatment methods in place, biological systems based on microbial inocula with specialized degrading capacities against TBZ could be a feasible treatment approach. Only recently the first bacterial consortium able to rapidly transform TBZ was isolated. This study aimed to characterize its biodegradation, bioremediation and detoxification potential. The capacity of the consortium to mineralize 14C-benzyl-ring labelled TBZ was initially assessed. Subsequent tests evaluated its degradation capacity under various conditions (range of pH, temperatures and TBZ concentration levels) and relevant practical scenarios (simultaneous presence of other postharvest compounds) and its bioaugmentation potential in soils contaminated with increasing TBZ levels. Finally cytotoxicity assays explored its detoxification potential. The consortium effectively mineralized the benzoyl ring of the benzimidazole moiety of TBZ and degraded spillage level concentrations of the fungicide in aqueous cultures (750 mg L-1) and in soil (500 mg kg-1). It maintained its high degradation capacity in a wide range of pH (4.5-7.5) and temperatures (15-37 °C) and in the presence of other pesticides (ortho-phenylphenol and diphenylamine). Toxicity assays using the human liver cancer cell line HepG2 showed a progressive decrease in cytotoxicity, concomitantly with the biodegradation of TBZ, pointing to a detoxification process. Overall, the bacterial consortium showed high potential for future implementation in bioremediation and biodepuration applications.

Accelerated dissipation, soil microbial toxicity and dispersal of antimicrobial resistance in soils repeatedly exposed to tiamulin, tilmicosin and sulfamethoxazole.

The application of manures leads to the contamination of agricultural soils with veterinary antibiotics (VAs). These might exert toxicity on the soil microbiota and threaten environmental quality, and public health. We obtained mechanistic insights about the impact of three VAs, namely, sulfamethoxazole (SMX), tiamulin (TIA) and tilmicosin (TLM), on the abundance of key soil microbial groups, antibiotic resistance genes (ARGs) and class I integron integrases (intl1). In a microcosm study, we repeatedly treated two soils (differing in pH and VA dissipation capacity) with the studied VAs, either directly or via fortified manure. This application scheme resulted in accelerated dissipation of TIA, but not of SMX, and accumulation of TLM. Potential nitrification rates (PNR), and the abundance of ammonia-oxidizing microorganism (AOM) were reduced by SMX and TIA, but not by TLM. VAs strongly impacted the total prokaryotic and AOM communities, whereas manure addition was the main determinant of the fungal and protist communities. SMX stimulated sulfonamide resistance, while manure stimulated ARGs and horizontal gene transfer. Correlations identified opportunistic pathogens like Clostridia, Burkholderia-Caballeronia-Paraburkholderia, and Nocardioides as potential ARG reservoirs in soil. Our results provide unprecedented evidence about the effects of understudied VAs on soil microbiota and highlight risks posed by VA-contaminated manures. ENVIRONMENTAL IMPLICATION: The dispersal of veterinary antibiotics (VAs) through soil manuring enhances antimicrobial resistance (AMR) development and poses a threat to the environment and the public health. We provide insights about the impact of selected VAs on their: (i) microbially-mediated dissipation in soil; (ii) ecotoxicity on the soil microbial communities; (iii) capacity to stimulate AMR. Our results (i) demonstrate the effects of VAs and their application-mode on the bacterial, fungal, and protistan communities, and on the soil ammonia oxidizers; (ii) describe natural attenuation processes against VA dispersal, (iii) depict potential soil microbial AMR reservoirs, essential for the development of risk assessment strategies.

Potential for bioremediation of agro-industrial effluents with high loads of pesticides by selected fungi.

Wastewaters from the fruit packaging industry contain a high pesticide load and require treatment before their environmental discharge. We provide first evidence for the potential bioremediation of these wastewaters. Three white rot fungi (WRF) (Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus) and an Aspergillus niger strain were tested in straw extract medium (StEM) and soil extract medium (SEM) for degrading the pesticides thiabendazole (TBZ), imazalil (IMZ), thiophanate methyl (TM), ortho-phenylphenol (OPP), diphenylamine (DPA) and chlorpyrifos (CHL). Peroxidase (LiP, MnP) and laccase (Lac) activity was also determined to investigate their involvement in pesticide degradation. T. versicolor and P. ostreatus were the most efficient degraders and degraded all pesticides (10 mg l⁻¹) except TBZ, with maximum efficiency in StEM. The phenolic pesticides OPP and DPA were rapidly degraded by these two fungi with a concurrent increase in MnP and Lac activity. In contrast, these enzymes were not associated with the degradation of CHL, IMZ and TM implying the involvement of other enzymes. T. versicolor degraded spillage-level pesticide concentrations (50 mg l⁻¹) either fully (DPA, OPP) or partially (TBZ, IMZ). The fungus was also able to rapidly degrade a mixture of TM/DPA (50 mg l⁻¹), whereas it failed to degrade IMZ and TBZ when supplied in a mixture with OPP. Overall, T. versicolor and P. ostreatus showed great potential for the bioremediation of wastewaters from the fruit packaging industry. However, degradation of TBZ should be also achieved before further scaling up.

Genome-Based Metabolic Reconstruction Unravels the Key Role of B12 in Methionine Auxotrophy of an Ortho-Phenylphenol-Degrading Sphingomonas haloaromaticamans.

Auxotrophy to amino acids and vitamins is a common feature in the bacterial world shaping microbial communities through cross-feeding relations. The amino acid auxotrophy of pollutant-degrading bacteria could hamper their bioremediation potential, however, the underlying mechanisms of auxotrophy remain unexplored. We employed genome sequence-based metabolic reconstruction to identify potential mechanisms driving the amino acid auxotrophy of a Sphingomonas haloaromaticamans strain degrading the fungicide ortho-phenylphenol (OPP) and provided further verification for the identified mechanisms via in vitro bacterial assays. The analysis identified potential gaps in the biosynthesis of isoleucine, phenylalanine and tyrosine, while methionine biosynthesis was potentially effective, relying though in the presence of B12. Supplementation of the bacterium with the four amino acids in all possible combinations rescued its degrading capacity only with methionine. Genome sequence-based metabolic reconstruction and analysis suggested that the bacterium was incapable of de novo biosynthesis of B12 (missing genes for the construction of the corrin ring) but carried a complete salvage pathway for corrinoids uptake from the environment, transmembrane transportation and biosynthesis of B12. In line with this the bacterium maintained its degrading capacity and growth when supplied with environmentally relevant B12 concentrations (i.e., 0.1 ng ml-1). Using genome-based metabolic reconstruction and in vitro testing we unraveled the mechanism driving the auxotrophy of a pesticide-degrading S. haloaromaticamans. Further studies will investigate the corrinoids preferences of S. haloaromaticamans for optimum growth and OPP degradation.

Bioaugmentation of thiabendazole-contaminated soils from a wastewater disposal site: Factors driving the efficacy of this strategy and the diversity of the indigenous soil bacterial community.

The application of the fungicide thiabendazole (TBZ) in fruit packaging plants (FPP) results in the production of effluents which are often disposed in adjacent field sites. These require remediation to prevent further environmental dispersal of TBZ. We assessed the bioaugmentation potential of a newly isolated TBZ-degrading bacterial consortium in a naturally contaminated soil (NCS) exhibiting a natural gradient of TBZ levels (12000, 400, 250 and 12 mg kg-1). The effect of aging on bioaugmentation efficacy was comparatively tested in a soil with similar physicochemical properties and soil microbiota, which was artificially, contaminated with the same TBZ levels (ACS). The impact of bioaugmentation and TBZ on the bacterial diversity in the NCS was explored via amplicon sequencing. Bioaugmentation effectively removed TBZ from both soils at levels up to 400 mg kg-1 but failed at the highest contamination level (12000 mg kg-1). Dissipation of TBZ in bioaugmented samples showed a concentration-dependent pattern, while aging of TBZ had a slight effect on bioaugmentation efficiency. Bioaugmentation had no impact on the soil bacterial diversity, in contrast to TBZ contamination. Soils from the hotspots of TBZ contamination (12000 mg kg-1) showed a drastically lower α-diversity driven by the dominance of ß- and γ-proteobacteria at the expense of all other bacterial phyla, especially Actinobacteria. Overall, bioaugmentation with specialized microbial inocula could be an effective solution for the recovery of disposal sites contaminated with persistent chemicals like TBZ.

Metabolic and Evolutionary Insights in the Transformation of Diphenylamine by a Pseudomonas putida Strain Unravelled by Genomic, Proteomic, and Transcription Analysis.

Diphenylamine (DPA) is a common soil and water contaminant. A Pseudomonas putida strain, recently isolated from a wastewater disposal site, was efficient in degrading DPA. Thorough knowledge of the metabolic capacity, genetic stability and physiology of bacteria during biodegradation of pollutants is essential for their future industrial exploitation. We employed genomic, proteomic, transcription analyses and plasmid curing to (i) identify the genetic network of P. putida driving the microbial transformation of DPA and explore its evolution and origin and (ii) investigate the physiological response of bacterial cells during degradation of DPA. Genomic analysis identified (i) two operons encoding a biphenyl (bph) and an aniline (tdn) dioxygenase, both flanked by transposases and (ii) two operons and several scattered genes encoding the ortho-cleavage of catechol. Proteomics identified 11 putative catabolic proteins, all but BphA1 up-regulated in DPA- and aniline-growing cells, and showed that the bacterium mobilized cellular mechanisms to cope with oxidative stress, probably induced by DPA and its derivatives. Transcription analysis verified the role of the selected genes/operons in the metabolic pathway: DPA was initially transformed to aniline and catechol by a biphenyl dioxygenase (DPA-dioxygenase); aniline was then transformed to catechol which was further metabolized via the ortho-cleavage pathway. Plasmid curing of P. putida resulted in loss of the DPA and aniline dioxygenase genes and the corresponding degradation capacities. Overall our findings provide novel insights into the evolution of the DPA degradation pathway and suggests that the degradation capacity of P. putida was acquired through recruitment of the bph and tdn operons via horizontal gene transfer.

Author Correction: Metabolic pathway and cell adaptation mechanisms revealed through genomic, proteomic and transcription analysis of a Sphingomonas haloaromaticamans strain degrading ortho-phenylphenol.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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.

Metabolic pathway and cell adaptation mechanisms revealed through genomic, proteomic and transcription analysis of a Sphingomonas haloaromaticamans strain degrading ortho-phenylphenol.

Ortho-phenylphenol (OPP) is a fungicide contained in agro-industrial effluents produced by fruit-packaging plants. Within the frame of developing bio-strategies to detoxify these effluents, an OPP-degrading Sphingomonas haloaromaticamans strain was isolated. Proteins/genes with a putative catabolic role and bacterium adaptation mechanisms during OPP degradation were identified via genomic and proteomic analysis. Transcription analysis of all putative catabolic genes established their role in the metabolism of OPP. The formation of key transformation products was verified by chromatographic analysis. Genomic analysis identified two orthologous operons encoding the ortho-cleavage of benzoic acid (BA) (ben/cat). The second ben/cat operon was located in a 92-kb scaffold along with (i) an operon (opp) comprising genes for the transformation of OPP to BA and 2-hydroxypenta-2,4-dienoate (and genes for its transformation) and (ii) an incomplete biphenyl catabolic operon (bph). Proteomics identified 13 up-regulated catabolic proteins when S. haloaromaticamans was growing on OPP and/or BA. Transcription analysis verified the key role of the catabolic operons located in the 92-kb scaffold, and flanked by transposases, on the transformation of OPP by S. haloaromaticamans. A flavin-dependent monoxygenase (OppA1), one of the most up-regulated proteins in the OPP-growing cells, was isolated via heterologous expression and its catabolic activity was verified in vitro.

Isolation and characterisation of a Sphingomonas strain able to degrade the fungicide ortho-phenylphenol.

BACKGROUND: Ortho-phenylphenol (OPP) is a fungicide used in fruit packaging plants for the control of fungal infestations during storage. Its application leads to the production of large wastewater volumes which according to the European legislation should be treated on site. In spite of this, no efficient treatment systems are currently available, and the development of biological systems based on tailored-made pesticide-degrading inocula for the treatment of these wastewaters is an appealing solution. RESULTS: Enrichment cultures from a soil collected from a wastewater disposal site resulted in the isolation of a pure Sphingomonas haloaromaticamans strain P3 able to degrade rapidly OPP and use it as an energy source. Its degrading capacity was dependent on the external supply of amino acids or on the presence of other bacteria that did not contribute to fungicide degradation. The isolated S. haloaromaticamans strain was able to metabolise up to 150 mg L(-1) of OPP within 7 days, in a wide range of pH (4.5-9) and temperatures (4-37 °C), and in the presence of other pesticides (thiabendazole and diphenylamine) co-used in the fruit packaging industry. CONCLUSION: Overall, the OPP-degrading bacterium isolated showed high potential for use in future biodepuration treatment systems and bioremediation strategies.

Integrated biodepuration of pesticide-contaminated wastewaters from the fruit-packaging industry using biobeds: Bioaugmentation, risk assessment and optimized management.

Wastewaters from fruit-packaging plants contain high loads of toxic and persistent pesticides and should be treated on site. We evaluated the depuration performance of five pilot biobeds against those effluents. In addition we tested bioaugmentation with bacterial inocula as a strategy for optimization of their depuration capacity. Finally we determined the composition and functional dynamics of the microbial community via q-PCR. Practical issues were also addressed including the risk associated with the direct environmental disposal of biobed-treated effluents and decontamination methods for the spent packing material. Biobeds showed high depuration capacity (>99.5%) against all pesticides with bioaugmentation maximizing their depuration performance against the persistent fungicide thiabendazole (TBZ). This was followed by a significant increase in the abundance of bacteria, fungi and of catabolic genes of aromatic compounds catA and pcaH. Bioaugmentation was the most potent decontamination method for spent packing material with composting being an effective alternative. Risk assessment based on practical scenarios (pome and citrus fruit-packaging plants) and the depuration performance of the pilot biobeds showed that discharge of the treated effluents into an 0.1-ha disposal site did not entail an environmental risk, except for TBZ-containing effluents where a larger disposal area (0.2ha) or bioaugmentation alleviated the risk.

Isolation of a diphenylamine-degrading bacterium and characterization of its metabolic capacities, bioremediation and bioaugmentation potential.

The antioxidant diphenylamine (DPA) is used in fruit-packaging plants for the control of the physiological disorder apple scald. Its use results in the production of DPA-contaminated wastewater which should be treated before finally discharged. Biological treatment systems using tailored-made microbial inocula with specific catabolic activities comprise an appealing and sustainable solution. This study aimed to isolate DPA-degrading bacteria, identify the metabolic pathway of DPA and evaluate their potential for future implementation in bioremediation and biodepuration applications. A Pseudomonas putida strain named DPA1 able to rapidly degrade and utilize DPA as the sole C and N source was enriched from a DPA-contaminated soil. The isolated strain degraded spillage-level concentrations of DPA in liquid culture (2000 mg L(-1)) and in contaminated soil (1000 mg kg(-1)) and metabolized DPA via the transient formation of aniline and catechol. Further evidence for the bioremediation and biodepuration potential of the P. putida strain DPA1 was provided by its capacity to degrade the post-harvest fungicide ortho-phenylphenol (OPP), concurrently used by the fruit-packaging plants, although at slower rates and DPA in a wide range of pH (4.5-9) and temperatures (15-37 °C). These findings revealed the high potential of the P. putida strain DPA1 for use in future soil bioremediation strategies and/or as start-up inocula in wastewater biodepuration systems.

Effects of systemic pesticides imidacloprid and metalaxyl on the phyllosphere of pepper plants.

Microbes inhabiting the phyllosphere of crops are exposed to pesticides applied either directly onto plant foliage or indirectly through soil. Although, phyllosphere microbiology has been rapidly evolving, little is still known regarding the impact of pesticides on the epiphytic microbial community and especially on fungi. We determined the impact of two systemic pesticides (metalaxyl and imidacloprid), applied either on foliage or through soil, on the epiphytic fungal and bacterial communities via DGGE and cloning. Both pesticides induced mild effects on the fungal and the bacterial communities. The only exception was the foliage application of imidacloprid which showed a more prominent effect on the fungal community. Cloning showed that the fungal community was dominated by putative plant pathogenic ascomycetes (Erysiphaceae and Cladosporium), while a few basidiomycetes were also present. The former ribotypes were not affected by pesticides application, while selected yeasts (Cryptococcus) were stimulated by the application of imidacloprid suggesting a potential role in its degradation. A less diverse bacterial community was identified in pepper plants. Metalaxyl stimulated an Enterobacteriaceae clone which is an indication of the involvement of members of this family in fungicide degradation. Further studies will focus on the isolation of epiphytic microbes which appear to be stimulated by pesticides application.