Differential analysis of pesticides biodegradation in soil using conventional and high-throughput technology

A potential pesticide degrading bacterial isolate (2D), showing maximum tolerance (450 ppm) for cypermethrin, fipronil, imidacloprid and sulfosulfuron was recovered from a pesticide contaminated agricultural field. The isolate degraded cypermethrin, imidacloprid, fipronil and sulfosulfuron in minimal salt medium with 94, 91, 89 and 86 % respectively as revealed by HPLC and GC analysis after 15 days of incubation. Presence of cyclobutane, pyrrolidine, chloroacetic acid, formic acid and decyl ester as major intermediate metabolites of cypermethrin biodegradation was observed in GC-MS analysis. Results based on 16S rDNA sequencing, and phylogenetic analysis showed maximum similarity of 2D with Bacillus cereus (MH341691). Stress responsive and catabolic/ pesticide degrading proteins were over expressed in the presence of cypermethrin in bacteria. Enzyme kinetics of laccase was deduced in the test isolate under normal and pesticide stress conditions. Amplification of laccase gene showed a major band of 1200bp. Maximum copy number of 16S rDNA was seenin uncontaminated soil as compared to pesticide contaminated soil using qRT-PCR. The metagenome sequencing revealed reduction in the population of proteobacteria in contaminated soil as compared to uncontaminated soil but showed dominance of actinobacteria, firmicutes and bacteriodates in pesticide spiked soil. Presence of some new phyla like chloroflexi, planctomycetes, verrucomicrobia was observed followed by extinction of acidobacteria and crenarchaeota in spiked soil. The present study highlights on the potential of 2D bacterial strain i.e., high tolerance level of pesticide, effective biodegradation rate, and presence of laccase gene in bacterial strain 2D, could become a potential biological agent for large-scale treatment of mixture of pesticide (cypermethrin, fipronil, imidacloprid and sulfosulfuron) in natural environment (soil and water).

Among the microbes used for biodegradation purposes, bacteria are most preferred as they could easily be developed into mutant strains with variety of biochemical pathways in adaptive environment [18][19].
Additionally, formulation of novel microbial consortia efficiently degrade xenobiotic compounds for their source of energy by using the co-metabolism process. The most promising and efficient technique for various biological purpose is the formation of proficient recombinant microbial strain which provides new opportunity to different biological fields [20].
Degradation of pesticides depends upon concentration, structure, solubility, soil types, soil moisture, temperature, pH, SOM (soil organic matter) and soil microbial biomass [12,21]. However, none of the studies have investigated degradation of different pesticides together through metagenomics tool to understand the mechanism behind in situ biodegradation and predict the biodegradation potential of microbes. Thus, there is a gap of knowledge regarding the functional genes and genomic potential underpinning degradation and community responses to contamination. Here we are trying to fill this knowledge gap by using advance technique like illumina sequencing of DNA isolated from pesticide contaminated agricultural soils and profiling of shifts in functional communities. Effect of pesticide stress on different category of proteins and their expression. On the basis of above reference the current study plans to the following objectives;

I.
Isolation and characterization of Pesticide degrading bacteria

II.
In vitro pesticide biodegradation in minimal medium

IV.
Estimation of pesticide degrading enzyme and their enzymatic kinetics

V.
Proteomic analysis of pesticide degrading bacterial isolate in stress and normal condition

VI.
Amplification of pesticide degrading gene

VII.
Comparative metagenomic study of pesticide contaminated and uncontaminated soil

Chemicals and media used for experiment
Standard pesticides of highest purity namely cypermethrin, fipronil, imidacloprid and sulfosulfuron (Sigma-Aldrich 99% pure) were provided by Department of Chemistry, GBPUAT Pantnagar. Stock solutions (1mg/ml) of the pesticides were prepared by dissolving standard pesticide in acetonitrile (for non volatile compound or HPLC) or hexane (for volatile compounds or GC), then sterilized with the help of bacterial filter and stored in colored bottles at 4°C in refrigerator till use. Isolation and characterization of bacterial isolates was done using Nutrient Agar medium (NA) and Mineral salt medium (MSM).

Isolation of Pesticide degrading bacteria through enrichment method
Pesticides polluted agricultural soil samples were collected from Gularbhoj, Udham Singh Nagar, Uttarakhand, India. Standard methods (Methods of Soil Microbiology and Biochemistry) were used to collect the soil samples [12]. For the collection of soil sample the handheld corer of stainless steel was used. During the sampling discarded the first two cores then third cores (5 cm), taken over an area of 100 m 2 were pooled to form one composite sample. All the samples were packed in aluminium foil and sealed in sterilised plastic sampling bags to reduce the possibility of contamination. After proper labelling, samples were kept at -20°C until use. Isolation of pesticide degrading indigenous soil microorganisms was done by using enrichment technique followed by dilution method. For residual analysis of pesticides present in soil, the samples were prepared and performed according to Phartyal [22] and Negi et al. [23]. Extract of pesticides were analyzed by HPLC/GC. On the basis of the occurrence of residual pesticides in the soil sample, cypermethrin, fipronil, imidacloprid and sulfosulfuron were selected for the further study.

Screening of pesticide degrading bacterial isolates
For the screening of cypermethrin, fipronil, imidacloprid and sulfosulfuron degrading bacteria, minimal salt medium was supplemented with different concentrations of the pesticide(s). Petri plates containing 20 ml of minimal agar supplemented with pesticide(s) (10 to 450 ppm from the stock solution (1mg/ml)) and left for solidification of medium. Plates were streaked with active bacterial cultures (isolated from the soil samples) and incubated for 72 h at 30±2⁰C. . The bacterial growth was observed after 2-4 days. On the basis of maximum tolerance level of the pesticides and growth in the minimal medium, bacterial isolates were selected, purified and maintained for further studies [12].

Molecular characterization of pesticide degrading bacterial isolate
Selected bacterial isolate (2D) was characterized at morphological and molecular level. Genomic DNA was extracted from the bacterial isolate [24]. Amplification of 16S rDNA gene was done using universal primers (27f: 5′AGAGTTTGATCMTGGCTCAG3′ and 1492r: 5′TACGGYTACCTTGTTACGACTT-3′). After that the amplicon was processed for agarose gel electrophoresis and further sent for the 16S rDNA sequencing to Biotech Centre, South Campus, Delhi University. The contig of 16s rDNA gene was formed and check the percent similarity by comparing the sequences with NCBI gene database using BLAST programme. On the basis of maximum homology among the sequences the phylogeny of the organism was deduced with the help of MEGA 7.0 software [25].

In vitro pesticide biodegradation in minimal medium
Experiment on biodegradation of cypermethrin, fipronil, imidacloprid and sulfosulfuron using selected bacterial isolates was performed in an in vitro study conducted under laboratory condition in liquid medium. Fifty ml MSM, supplemented with 20 ppm (1 ml from 1000 ppm pesticide stock) of pesticide individually was inoculated with 1 ml of active bacterial culture (OD=0.8) and incubated at 30⁰C.
Further, t 2 ml of broth was withdrawn separately from all the flasks at different days interval (on 5, 10 and 15 th day) and centrifuged for 10 min at 10,000 rpm. After centrifugation, 1 ml of supernatant was added with 1 gm of sodium sulphate and 1 ml of hexane/acetonitrile. Two separate layers were formed in the separating funnel. Further the upper layer was collected and evaporated at room temperature with the help of evaporator. The dried pesticide in round bottom flask was next to mix properly with 2 ml of hexane/acetonitrile. After filtration extracted pesticide solution was collected and further analyzed with the help of GC/HPLC [12,26].

Identification of intermediate metabolites of biodegraded pesticide using GC-MS
On the basis of residual analysis of the pesticides in the medium using HPLC/ GC, percent biodegradation of cypermethrin was maximum among all the pesticides when treated with bacterial isolate (2D).
Therefore the intermediate metabolites of cypermethrin biodegradation were analyzed with the help of GC-MS [27]. Degradation products of cypermethrin were extracted (50 ml of Mineral salt medium supplemented with 20 ppm of pesticide in 250ml flask containing) and analysed by GC-MS [12].
Uninoculated flask spiked with pesticide was used as control.

Estimation of laccase enzyme in bacterial strain 2D.
One ml of bacterial culture (12-14h old) inoculated into TYE broth (Tryptone yeast extract: tryptone, 2%; yeast extract, 2%; pH, 7.2) was incubated at 37 °C at 150 rpm for 5 days with (20 ppm) and without pesticide. Further, centrifugation at 6,000g was performed for 20 min at 4 °C. Before the sonication (at 20 MHz; five times, each for 45 s and 30 s gap between each step), 10 mM of PMSF (Phenylmethylsulfonyl fluoride) was added in 0.1 M phosphate buffer with pH 6.5 for washing cell pellets and inhibit protease activity in the supernatant. Again centrifugation at 14,000 g for 20 min at 4 °C was done to obtain cell extract which contains crude intracellular laccase enzyme. Enzyme activity can be measured at 465 nm using guaiacol as it forms reddish brown color in presence of laccase [28].

Kinetics of laccase by Lineweaver-Burk model
Lineweaver-Burke plots were used to derive kinetic parameters of the laccase enzyme [12,29,30].

Equation 1 indicates Michaelis-Menton equation (
where Vmax is maximum rate of reaction, r is substrate degradation in mM/min, Km is growth rate constant, S is initial substrate concentration).

Proteomic analysis of pesticide degrading bacterial isolate
To perform whole cell proteome analysis, bacterial strain was grown in minimal medium supplemented with 0.1% glucose in the presence/absence of the pesticide. Twenty ml of log phase bacterial culture, grown in minimal medium was used for the extraction of extracellular proteins. Bacterial culture was centrifuged at 12,000 rpm for 10 min at 4 o C. Pellets were discarded and supernatant was used for further examination. Ammonium sulphate was added to precipitate the extracellular proteins. Suspension was kept at 4 0 C overnight. Precipitated protein was concentrated by using dialysis. Concentration of protein was estimated [31]. Lyophilized protein samples were analysed using 2D gel electrophoresis. In silico, analysis of protein spots separated on 2D gel was carried with Expasy (TagIdent database) based on their molecular weight and isoelectric point (pI) [32].

Amplifcation of laccase gene.
Presence of laccase, a major pesticide degrading gene was targeted in the bacterial isolate and checked by using the primers [33]. The primers used for laccase amplification were CulAF-5′ACMWCBGTYCAYTGGCAYGG3′ and Cu4R-5′TGCTCVAGBAKRTGGCAGTG-3′. Reaction mixture constitutes: 20 pm/µl Primers (Forward and Reverse), 10 mM dNTPs mix, 10x Assay buffer with MgCl 2 , 3.0 U/µl TaqDNA polymerase, 10 mg/ml BSA 3 and 50ng/µl Template DNA. Reaction conditions maintained were: Initial denaturation at 94 0 C for 3min followed by denaturation at 94 0 C for30 sec, annealing at 50 0 C for 30 sec, and from second phase repeat of 35 cycles, followed by extension at 72 0 C for 1min and final extension at 72 0 C for 5 min.

Extraction of soil DNA
Collection of soil samples (pesticide contaminated and uncontaminated sites) was done from two different agricultural fields of Gularbhoj, Uttarakhand. Uncontaminated soil sample was taken as a control. DNA extraction from both samples was performed using HiPurA TM soil DNA Purification Kit. Soil (500g), from each sample was processed for DNA extraction. After quantification, purity of extracted DNA was tested in a NanoDrop spectrophotometer at 260 and 280 nm and by electrophoresis using 1% agarose.
DNA concentration of the sample was 50 ng/L.
The microbiota of the soil samples was examined by targetingV3-V4 region of 16S rRNA through Illumina Miseq platform. Paired end reads obtained were further processed, checked (for score distribution, base quality, average base content and GC distribution) and merged using FLASH program [34]. Multiple filters were applied to generate high quality reads (~350 to ~ 450bp). UCHIME algorithm was used to detect and remove chimera. Entire downstream analysis was done using QIIME (Version 1.9.1) program [35]. All pre-processed reads obtained were pooled and clustered into OTUs (Operational Taxonomic Units) using Uclust program at 97% similarity. Representative sequences for each OTU were selected by aligning the sequences against Green gene data set via PyNAST [36]. Taxonomic classification was done by RDP classifier against SILVA 16S RNA genes database. Based on reads and OTU distribution of phyla and genera for each sample the reads were categorized. Alpha diversity was analyzed via three indices:chao1, shannon and observed-species matrix. All the indices were calculated using QIIME (Version 1.9.1).

Results
Four pesticide utilizing bacterial isolates were recovered from pesticide contaminated agricultural fields using plate assay. Three bacterial isolates (2A, 2B and 2C) were able to grow at 300 ppm of the selected pesticides but only bacterial isolate (2D) was able to grow upto 450 ppm of the pesticides. Performance of 2D bacteria under laboratory condition (grew on cypermethrin, fipronil, imidacloprid and sulfosulfuron at 450 ppm) enabled us to investigate the biodegradation potential of the bacteria.

In vitro biodegradation of pesticides in minimal medium
Gas chromatography (GC) results of standard of imidacloprid, fipronil and sulfosulfuron revealed the presence of major single peak of each pesticide at 4.3, 4.9, and 6.3 min (retention time) respectively under optimized conditions of the instrument. On the other hand cypermethrin showed four independent major peaks during a retention time of 16-17.65 (Fig S1 a,b&c). These major peaks indicated at different retention time includes at 16.13 min indicate cis α , 17.00 min indicate cis β, 17.40 min indicate trans α and 17.62 min indicate trans β isomers of cypermethrin, (Fig S1 d). Isolate 2D showed maximum degradation of cypermethrin (94%) fo llowed by imidacloprid (91%), fipronil (89%) and sulfosulfuron (86%) after 15 days of incubation in minimal medium (Fig 1).

Characterization of the Bacterial Isolate
Phylogenetic analysis of amplified 16S rDNA gene sequences revealed 99% homology with Bacillus cereus of bacterial strain 2D. Gene sequences were submitted to GenBank and the organism was provided with an accession numbers (Accession No. MH341691) (Fig S3 &S4).

Enzyme kinetics
Laccase enzyme activity of test bacterial isolate was examined quantitatively and qualitatively for its possible role in degradation of cypermethrin. Biodegradation of pesticides is an enzyme-catalyzed transformation of organic compounds into their simpler products. Specificity of laccase enzyme make it crucial for degradation of huge variety of toxic environmental pollutants.
Michaelis Menten equation was solved in best way by plotting the data as 1/r vs 1/S to get a straight-line equation [29,[38][39] (Fig 2). In the presence and absence of cypermethrin, bacterial isolate (2D) produced

Laccase gene Amplification
Laccase amplification was found positive in test bacterial isolate and the size of amplicon for laccase was approximately 1200 bp (Fig 3). Bacterial strain (2D) expressed 14% proteins for stress response, 32% proteins involved in protein synthesis and modification, 14% in gene regulation, 20% in energy production, 18% catabolic/pesticide degrading proteins and 2% were uncharacterized proteins under normal condition. Under stress, percent expression of protein was different and the order was: 30% stress response proteins, 22% of proteins involved in protein synthesis and modification, 12% in gene regulation, 14% in energy production, 22% catabolic/pesticide degrading protein and 2% uncharacterized proteins (Fig 4).

Real time PCR (qRTPCR) analysis
Maximum copy number of 16S rDNA per gram of soil sample was 1.96×10 8 in pesticide contaminated soil while uncontaminated soil had 5.25×10 8 copy number.

Comparative analysis of microbial diversity in pesticide contaminated and uncontaminated soil samples
High-throughput Metagenomic approach was used to study the potential functional microbiome and composition of taxonomic community in pesticide contaminated soils.  samples (Fig S8).

Species diversity/ Alpha diversity
Relative abundance of top 25 classified OTUs was studied at genus level. Value of Shannon species diversity index was high (3.198) in pesticide contaminated (2G) soil. A sum of 1627 species were identified genotypically as compared to control (uncontaminated soil 2GC) where the Shannon species diversity index was 2.739 and 1,850 species were identified genotypically (Fig 6).

Fig 6.
The bar graph show alpha diversity, the relative abundance of the top 25 classification OTUs results for genus taxonomic level of 2G and 2GC soil samples.

Hierarchal clustering
The hierarchial clustering heat map displays OTUs count per sample. Higher the relative abundance of an OTU in a sample, the more intense the colour at the corresponding position in the heat map (Fig 7). The population size for Clostridium was larger in 2G but in 2GC soil sample the population size of Koribacter was large. Bacteria categorized as unclassified at genus level were dominantly present in both the soils but their percentage of abundance decreased in 2GC soil sample.

Discussion
In the present study, interactions with the local farmers, survey of the nearby areas and the literature citations led to the findings that imidacloprid, cypermethrin, fipronil and sulfosulfuron are some common pesticides which are being used by the farmers in different cropping systems [40]. Presence of imidacloprid (0.42µg/g), cypermethrin (0.35µg/g) and fipronil (0.26 µg/g) and sulfosulfuron (0.18 µg/g) was found in soil samples. Degree of contamination in soil varies from less polluted (less then ≤ 0.5 mg/kg) to moderately polluted (in between 0.5 to 1 mg/kg) to heavily polluted (more then≥1 mg/kg) based on environmental quality standards [41]. Hence four pesticides (cypermethrin, fipronil, imidacloprid and sulfosulfuron) were selected in the present study for microbial biodegradation. The similar residual analysis of pesticides (carbendazim, endosulfan and imidacloprid) from the agricultural soil was done by the several researchers [22][23]. Our pesticide utilizing/ tolerant bacterial isolates were recovered from pesticide polluted agricultural soil using plate assay.
Performance of 2D bacteria under laboratory condition (grew on cypermethrin, fipronil, imidacloprid and sulfosulfuron at 450 ppm) enabled us to investigate the biodegradation potential of the bacteria.
Cypermethrin, endosulfan, imidacloprid, fipronil and sulfosulfuron degrading bacterial and fungal strains were also isolated from the rhizospheric fields by several authors [12,[42][43][44].  [23,45]. Therefore the bacterial isolate used in present study showed more tolerance for the pesticide(s) than those used by Nawab et al.
and Negi et al. [23,45]. Bacterial strain 2D could degrade all the tested pesticides at higher concentration.
Changes of regular expression of some enzyme(s) involved in degradation of imidacloprid, fipronil, cypermethrin and sulfosulfuron at increased concentrations by 2D cannot be ignored. On the basis of our experiment on tolerance for pesticides by 2D, we presume that metabolic activity of our strain was not subjected to catabolic repression at higher concentration of the pesticides. Bacillus cereus (strain 2D) potential of degradation and tolerance to pesticides (higher concentration) proved it as a suitable strain for remediation of polluted sites.
Isolated bacterial strain Bacillus cereus 2D showed higher range of pesticide degradation potential (94-86%) within shorter period of time (15 days). Pankaj et al. and Bhatt et al. [46,44] have reported biodegradation of cypermethrin using Bacillus spp. and found 85% degradation after 15 days under similar conditions. Bacillus subtilis BSF01 has been also reported for their schematic growth linked biodegradation pathway of β-cypermethrin [47]. Microorganisms used in biodegradation need an acclimatization period to induce synthesis of enzymes for biodegradation that may account for prolonged lag phase, which was observed at higher concentrations of cypermethrin, fipronil and sulfosulfuron [48].
Bacillus sp. FA3 degrades fipronil (76%) within 15 days, isolated from agricultural field and effective for its removal from water and soil environment [39]. Hence our bacterial strain Bacillus cereus 2D is more efficient than the previously reported bacteria for the removal of mixture of pesticide from the natural environment [39].
Toxicity of metabolites produced during cypermethrin degradation was checked by inoculating it (fifteen days old broth with metabolites) with fresh bacterial culture. Results proved these metabolites as nontoxic due to no effects on bacterial growth even after complete breakdown of pesticide. In the condition. Production of laccase is high which helps bacterial isolates to overcome from the pesticide stress and make them comfortable to breakdown and utilize pesticides as carbon and energy source.

Michaelis-Menten equation defines
Km as the total substrate present at half of its maximum velocity (Vmax) [51]. Results indicate that to overcome the pesticide stress, the bacterial strain induces laccase enzyme. Oxidation of various inorganic and organic compounds (both phenolic and non-phenolic substrates) was catalysed by laccase via reduction of molecular oxygen to water [52]. Several biotechnological applications of laccase includes plastic degradation, xenobiotics bioremediation, bioleaching, decolourization of textile dyes, biosensors, lignin degradation, food industry, and biofuels production etc. [53][54]. Gangola and co-workers [12], reported efficient detoxification and degradation of cypermethrin by soil inhabitant Bacillus subtilis 1D with esterase and laccase activity. Laccase concentration in presence of cypermethrin was 62 μg/μL with Km value 61.57 M and in its absence was 42 μg/ μL with Km value 83 M after 15 days [12]. Pseudomonas putida MTCC 7525 was isolated from the soil sample containing sawdust and dairy effluent showed optimum laccase production (94.10 U/ml) at pH 8 and incubation at 30 0 C with 1mM sodium nitrate as N source and 10% skim milk [55].
Presence of laccase gene was found in Bacillus cereus 2D and the size of amplicon for laccase was approximately 1200 bp (Fig. 3). Our results were similar to findings of Ausec et al. [33] as amplicon size of 1200 bp in B. cereus 2D was observed. By using primers it was confirmed that laccase encoding genes exists in bacterial genome and may get activated in stressed conditions in presence of pesticides). This gene is major regulatory gene and found responsible for the degradation of imidacloprid, fipronil, sulfosulfuron and cypermethrin. These pesticide have some common chemical bonds in their structures hence could be degraded by similar laccase. Presence of laccase gene has been already reported in Azospirillum sp. [56], Pseudomonas syringae [57] and B. subtilis [12]. Fungal laccase enzyme for degradation of liluron, chlorpyrifos and metribuzin was reported by Gouma, [58]. The release of extracellular laccase from Bacillus cereus allows degradation and decolorization of dye [59]. More studies were available on pesticide degradation by fungal laccase as compared to bacterial laccase. So, bacterial species with laccase activity makes them prominent candidate for degradation of variety of pollutants. The presence of laccase in Bacillus cereus (2D) is the first report in context of pesticide degradation.
Proteomic study revealed that under stress condition, percentage of stress responsive proteins and catabolic/pesticide degrading proteins was high, which make the organism more comfortable under such environment. There is direct involvement of proteins in pesticide degradation. Expression of such protein encoding genes have essential role in breakdown of toxic organic pollutant [60]. Proteomics explored degradation of PCB (polychlorinated bipheny) by microbes at molecular level [61]. Moreover, Pankaj and co-workers [62], Bacteria with potential to survive in pesticide treated soil were Clostridium sp., which is reported to degrade many pesticides like Alachlor, chlorpropham, DDT, Lindane etc. [68].
In 2G soil sample (pesticide contaminated), majority of genera were Clostridium (8.30%), Nocardioides Phycicoccus (2.21%). Nocardioides was isolated from pesticide contaminated soil which utilized atrazine as a sole C and N source [69]. Presence of enzyme TrzN in Nocardioides sp. was responsible for biodegradation of pesticide chloro-s-trazina [70]. Bellilinea and Longilinea sp. are still not reported for pesticide biodegradation but Bellilinea sp. has shown catalytic breakdown of 2-methyl-naphthalene (a carcinogenic hydrocarbon) [67,71]. Thermophilic (Bellilinea caldifistulae) and mesophilic (Longilinea arvoryzae) strains were isolated from thermophilic digester sludge and rice paddy soil and found responsible for propionate-degradation. Role of Anaerolineae has been reported for wastewater treatment which had different recalcitrant hydrocarbons [72].
Shannon species diversity index was high (3.198) in pesticide contaminated (2G) soil as compared to uncontaminated soil (2GC) i.e., 2.739. Degradation effects of chlorpyrifos at variable concentrations on microbial diversity in soil has been studied in vitro which demonstrated similar variation in the diversity indices as found in biolog assay, showing chlorpyrifos inhibited microbial population in initial 2 weeks and later reached to same level as control [73].
Difference in microbial communities and population size indicates the repeated application of the pesticides in the agriculture field which forces the system to adopt the new population with replacement of the older one. The number of pesticide sensitive species decreased with time while pesticide tolerating species survived for longer. It is deduced from our results that pesticide is toxic for some species of the same phylum while other species utilize pesticide as a C and energy source and survived longer. Increase in population size of the Proteobacteria, Actinobacteria, Firmicutes and Chloroflexi showed that these are actively dividing bacteria in pesticide contaminated soil. Positive association of microbes in consortia may also intensify its abilities. We observed that communities with high metabolic potential for pesticide degradation were present in soils where pesticides retained for a longer time. Comparative investigation of two soil type suggested that abundance of high metabolic communities were present in rapidly degrading soil which indicates high functional capacity of the microbes in terms of nutrient cycling. We can say the microbial community present in 2GC was sensitive towards the pesticide while microbial community in 2G soil was resistant to pesticides. Dehalococcoidetes, Caldineae, Thermodesulfobacteria and Thermoprotei in pesticide treated soil sample (2G).

Conclusion
Present study recommends the application of indigenous microorganisms in biodegradation of common agriculture pesticides. Because in stress conditions bacteria could change their genetic profile and easily induce mutant strains, which can adopt in different environmental condition by activating their vast range of biochemical metabolism diversity, hence need to indepth study in this regard. The use of proteomics tools for the purpose of environmental bioremediation gives detailed information of microbial cells protein and composition. Hence it offers a valuable approach to decipher the mechanisms involved in bioremediation at molecular level. Diversity, composition and metabolic potential of soil microbiome is of crucial importance in bioremediation. In addition soil microbial communities also regulate biogeochemical cycling. Hence, detailed analysis of microbial communities at functional and structural level will guide to monitor and assess the effect of pesticides on soil health and their biological status respectively. Therefore, based on the biodegradation potential and wide range of activity for different substrates, the Bacillus cereus 2D could be a solid recommendation for the biodegradation of pesticides.
While the Metagenomic study of contaminated sample (soil or water) could unrevealed the type of microbial population and the interaction among them for the need of pesticide biodegradation in natural environment. For future prospects the activity of bacterial isolate could be check against the other xenobiotic compounds and the mechanism of gene regulation.

Additional information
Supplementary files: Supplementary file is attached for the support of data. The Accession number of the isolated strain 2D is MH341691 provided from NCBI