Modeling and optimization of chlorpyrifos and glyphosate biodegradation using RSM and ANN: Elucidating their degradation pathways by GC-MS based metabolomics.

Ongoing and extensive use of pesticides negatively impact the environment and human health. Microbe-based remediation bears importance as it is an eco-friendly and cost-effective technique. The present study investigated chlorpyrifos (CHL) and glyphosate (GLY) degrading potential of Bacillus cereus AKAD 3-1, isolated from the soybean rhizosphere. Optimization and validation of different process variables were carried out by response surface methodology (RSM) and artificial neural network (ANN). Critical parameters which affect the degradation process are initial pesticide concentration, pH, and inoculum size. At optimum conditions, the bacterial strain demonstrated 94.52% and 83.58% removal of chlorpyrifos and glyphosate, respectively. Both Central-composite design (CCD-RSM) and ANN approaches proved to perform well in modeling and optimizing the growth conditions. The optimum ANN-GA model resulted in R2 ≥ 0.99 for chlorpyrifos and glyphosate, while in the case of RSM, the obtained R2 value was 0.96 and 0.95, respectively. Results indicated that the process variables significantly (p < 0.05) impact chlorpyrifos and glyphosate biodegradation. Moreover, the predicted RSM model had a "lack of fit p-value" of "0.8849" and "0.2502" for chlorpyrifos and glyphosate, respectively. GC-MS analysis revealed that the strain first converted chlorpyrifos into 3,5,6-trichloro pyridin-2-ol & O, O-diethyl O-hydrogen phosphorothiate. Later, these intermediate metabolites were broken and completely mineralized into non-toxic by-products. Similarly, glyphosate was first converted into 2-(methylamino) acetic acid and amino-oxyphosphonic acid, which were further mineralized without any toxic by-products. Taken together, the results of this study clarify the biodegradation pathways and highlights the promising potential of B. cereus AKAD 3-1 in the bioremediation of chlorpyrifos and glyphosate-polluted environments.

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.

Kinetics of aerobic and anaerobic biomineralization of atrazine in surface and subsurface agricultural soils in Ohio.

The purpose of this study was to assess atrazine mineralization in surface and subsurface samples retrieved from vertical cores of agricultural soils from two farm sites in Ohio. The Defiance site (NW-Ohio) was on soybean-corn rotation and Piketon (S-Ohio) was on continuous corn cultivation. Both sites had a history of atrazine application for at least a couple of decades. The clay fraction increased at the Defiance site and the organic matter and total N content decreased with depth at both sites. Mineralization of atrazine was assessed by measurement of (14)CO2 during incubation of soil samples with [U-ring-(14)C]-atrazine. Abiotic mineralization was negligible in all soil samples. Aerobic mineralization rate constants declined and the corresponding half-lives increased with depth at the Defiance site. Anaerobic mineralization (supplemented with nitrate) was mostly below the detection at the Defiance site. In Piketon samples, the kinetic parameters of aerobic and anaerobic biomineralization of atrazine displayed considerable scatter among replicate cores and duplicate biometers. In general, this study concludes that data especially for anaerobic biomineralization of atrazine can be more variable as compared to aerobic conditions and cannot be extrapolated from one agricultural site to another.

DNA-SIP and repeated isolation corroborate Variovorax as a key organism in maintaining the genetic memory for linuron biodegradation in an agricultural soil.

The frequent exposure of agricultural soils to pesticides can lead to microbial adaptation, including the development of dedicated microbial populations that utilize the pesticide compound as a carbon and energy source. Soil from an agricultural field in Halen (Belgium) with a history of linuron exposure has been studied for its linuron-degrading bacterial populations at two time points over the past decade and Variovorax was appointed as a key linuron degrader. Like most studies on pesticide degradation, these studies relied on isolates that were retrieved through bias-prone enrichment procedures and therefore might not represent the in situ active pesticide-degrading populations. In this study, we revisited the Halen field and applied, in addition to enrichment-based isolation, DNA stable isotope probing (DNA-SIP), to identify in situ linuron-degrading bacteria in linuron-exposed soil microcosms. Linuron dissipation was unambiguously linked to Variovorax and its linuron catabolic genes and might involve the synergistic cooperation between two species. Additionally, two novel linuron-mineralizing Variovorax isolates were obtained with high 16S rRNA gene sequence similarity to strains isolated from the same field a decade earlier. The results confirm Variovorax as a prime in situ degrader of linuron in the studied agricultural field soil and corroborate the genus as key for maintaining the genetic memory of linuron degradation functionality in that field.

Influence of different redox conditions and dissolved organic matter on pesticide biodegradation in simulated groundwater systems.

Insights into the influence of redox conditions, that is the availability of electron acceptors, and dissolved organic matter (DOM) on pesticide biodegradation in groundwater are key to understanding the environmental fate of pesticides in natural groundwater systems. Here, the influence of redox conditions and supplemental DOM addition on biodegradation of pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,6-dichlorobenzamide (BAM), mecoprop-p (MCPP) and bentazone, was tested in microcosm and subsequent column experiments. Pesticide degradation, functional genes and changes in specific fractions and quantity of DOM were systematically quantified. In aerobic microcosm experiments, the highest 2,4-D degradation rate was obtained with the presence of more assimilable DOM. In column experiments, minimal pesticide degradation (≤33.77%) in any anaerobic redox conditions was observed in the absence of DOM. However, in the presence of DOM, 2,4-D biodegradation was considerably enhanced under nitrate-reducing conditions (from 23.5 ±â€¯10.2% to 82.3 ±â€¯11.6%) and in a column without external electron acceptor amendment (from -6.3 ±â€¯12.6% to 31.1 ±â€¯36.3%). Observed preferential depletion of the fulvic acid fraction of DOM provides indications for specific functional DOM properties. The qPCR results show an increase in microbial biomass and functional genes (tfdA) in liquid phase after DOM addition. The results of this work provide insights into the interplays among DOM, redox geochemistry, and pesticide biodegradation, and show the potential of a novel approach - DOM addition to groundwater systems - for in situ biostimulation technology to remove pesticides from groundwater systems.

Transcriptional analysis reveals the metabolic state of Burkholderia zhejiangensis CEIB S4-3 during methyl parathion degradation.

Burkholderia zhejiangensis CEIB S4-3 has the ability to degrade methyl parathion (MP) and its main hydrolysis byproduct p-nitrophenol (PNP). According to genomic data, several genes related with metabolism of MP and PNP were identified in this strain. However, the metabolic state of the strain during the MP degradation has not been evaluated. In the present study, we analyzed gene expression changes during MP hydrolysis and PNP degradation through a transcriptomic approach. The transcriptional analysis revealed differential changes in the expression of genes involved in important cellular processes, such as energy production and conversion, transcription, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, among others. Transcriptomic data also exhibited the overexpression of both PNP-catabolic gene clusters (pnpABA'E1E2FDC and pnpE1E2FDC) present in the strain. We found and validated by quantitative reverse transcription polymerase chain reaction the expression of the methyl parathion degrading gene, as well as the genes responsible for PNP degradation contained in two clusters. This proves the MP degradation pathway by the strain tested in this work. The exposure to PNP activates, in the first instance, the expression of the transcriptional regulators multiple antibiotic resistance regulator and Isocitrate Lyase Regulator (IclR), which are important in the regulation of genes from aromatic compound catabolism, as well as the expression of genes that encode transporters, permeases, efflux pumps, and porins related to the resistance to multidrugs and other xenobiotics. In the presence of the pesticide, 997 differentially expressed genes grouped in 104 metabolic pathways were observed. This report is the first to describe the transcriptomic analysis of a strain of B. zhejiangensis during the biodegradation of PNP.

The pesticide mineralization capacity in sand filter units of drinking water treatment plants (DWTP): Consistency in time and relationship with intake water and sand filter characteristics.

Sand filters (SFs) are commonly applied in drinking water treatment plants (DWTPs) for removal of iron and manganese but also show potential for microbial degradation of pesticide residues. The latter is advantageous in case the intake water contains pesticide residues. However, whether this involves mineralization suggesting no generation of harmful transformation products, its consistency over time, and how this ability relates to physicochemical and biological characteristics of the DWTP intake water and the SFs is unknown. The capacity to mineralize the herbicides bentazon and 2-methyl-4-chlorophenoxyacetic acid (MCPA) was examined in SF samples from 11 DWTPs differing in operation, intake water composition and pesticide contamination level. MCPA was mineralized in all biologically active SFs while mineralization of bentazon occurred rarely. Mineralization of both compounds was consistent in time and across samples taken from different SF units of the same DWTP. Kinetic modelling of mineralization curves suggested the occurrence of growth linked bentazon and MCPA mineralization in several SF samples. Multivariate analysis correlating intake water/SF characteristics with pesticide mineralization indicated that pesticide mineralization capacity depended on a range of intake water characteristics, but was not necessarily explained by the presence of the pesticide in the intake water and hence the in situ exposure of the SF community to the pesticide. This was supported by testing a sample from DWTP Kluizen for its capacity to mineralize 5 other pesticides including pesticides not present or occasionally present in the intake water. All of those pesticides were mineralized as well.

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.

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.

In situ response of the linuron degradation potential to linuron application in an agricultural field.

To assess the involvement of the genus Variovorax and the linuron hydrolase gene libA in in situ linuron degradation in agricultural fields, changes in Variovorax community size and composition, in libA abundance and in linuron mineralization capacity were monitored in field soil plots either treated or not with a linuron-containing herbicide mixture. Changes in Variovorax community composition, due to the proliferation of a hereto unknown Variovorax phylotype D, and increases in libA numbers occurred concomitant to increases in linuron mineralization capacity in the plot treated with the herbicide mixture. The observations suggest that Variovorax and libA proliferated as a response to linuron and hence their contribution to in situ linuron degradation. The involvement of Variovorax phylotype D and libA in linuron degradation in the examined soil was supported by laboratory soil microcosm experiments. Attempts to enrich in suspended cultures and isolate the organism corresponding to phylotype D from the soil were unsuccessful as the enrichment resulted in replacement of Variovorax phylotype D by other Variovorax phylotypes. This illustrates that linuron-degrading strains isolated by liquid enrichment cultures are not always representatives of those responsive to linuron in the field, although the genus specificity of linuron degradation was retained.

Fungal hyphae stimulate bacterial degradation of 2,6-dichlorobenzamide (BAM).

Introduction of specific degrading microorganisms into polluted soil or aquifers is a promising remediation technology provided that the organisms survive and spread in the environment. We suggest that consortia, rather than single strains, may be better suited to overcome these challenges. Here we introduced a fungal-bacterial consortium consisting of Mortierella sp. LEJ702 and the 2,6-dichlorobenzamide (BAM)-degrading Aminobacter sp. MSH1 into small sand columns. A more rapid mineralisation of BAM was obtained by the consortium compared to MSH1 alone especially at lower moisture contents. Results from quantitative real-time polymerase chain reaction (qPCR) demonstrated better spreading of Aminobacter when Mortierella was present suggesting that fungal hyphae may stimulate bacterial dispersal. Extraction and analysis of BAM indicated that translocation of the compound was also affected by the fungal hyphae in the sand. This suggests that fungal-bacterial consortia are promising for successful bioremediation of pesticide contamination.