Biopurification system as a source of pesticide-tolerant bacteria able to degrade the commonly used pesticides chlorpyrifos and iprodione

Intensive use of pesticides applied simultaneously in field to improve the effectiveness of pest control increase the environmental contamination, affecting the soil and water quality. Some of the commonly used pesticides are the insecticide chlorpyrifos and the fungicide iprodione; being thus critically essential to develop bioremediation methods to remove these contaminants by tolerant-bacteria. In this study we selected and characterized different pesticides-tolerant bacteria isolated from a biomixture of a biopurification system that had received continuous applications of a mixture of the pesticides chlorpyrifos and iprodione. Out of the 10 isolated bacterial colonies, only six strains presented adequate growth in presence of the both pesticides at 100 mg L−1. Biochemical and enzymatic characterization using API ZYM showed that all isolates (100%) were positive for esterase, leucine aminopeptidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. According to the molecular level study of the 16S ribosomal gene and MALDI TOF/TOF MS, it was possible to determine that the isolated bacteria belong to the genera Pseudomonas, Rhodococcus and Achromobacter. Bacterial growth decreased proportionally (R2 > 0.96) as been as both pesticide concentrations increased from 10 to 100 mg L−1. Achromobacter sp. strain C1 showed the best chlorpyrifos removal (between 56–29%) after 120 h of incubation. On the other hand, the highest iprodione removal (between 91.2–98.9%) was observed for the Pseudomonas sp. strain C9, which was not detected after 48 h of incubation. According with their identification and ability to remove the contaminants, Achromobacter sp. strain C1 and Pseudomonas sp. strain C9 appear as promising microorganisms for their use in the treatment of matrices contaminated with chlorpyrifos, iprodione or their mixture. The results of this study will help to improve current technologies for the biodegradation of this commonly used insecticide and fungicide, in order to give a response to the problem of contamination by pesticides.


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Different pesticides are applied simultaneously in the field to improve the effectivity of pest 68 control, thus increasing environmental contamination and affecting the soil and water quality TCP produced was 10 times lower compared to that produced by Streptomyces sp. AC7 strain 122 (4.32 mg L -1 ). 123 Further, several studies have reported the potential of indigenous microbial consortia isolated 124 from contaminated soils to degrade different pesticides and pesticide mixtures. In this 125 context, Fuentes et al. [28] reported a Streptomyces sp. consortium able to remove an 126 organochlorine pesticide mixture composed of lindane, methoxychlor, and chlordane.  The previous studies mentioned have reported the ability of selected bacteria isolated from 137 pesticide-contaminated soils to remove pesticides. However, the isolation and 138 characterization of pesticide-degrading microorganisms from a biopurification system used 139 for pesticide treatment have been scarcely studied. Therefore, the goal of this study was to 140 select and characterize bacterial species isolated from a biopurification system and with the 141 ability to degrade the fungicide IPR and the insecticide CHL.  g MnSO 4 · H 2 O, 0.2 g ZnSO 4 , 0.1 g CuSO 4 , 0.25 g Na 2 MoO 4 , 1000 mL distilled water) was 161 used for pesticide degradation assay. The initial pH of the medium was adjusted to 7.0 prior 162 to sterilization by autoclaving (121 °C for 20 min). Subsequently, cycloheximide (0.05 g L -163 g NaCl, 2.5 g yeast extract, and 10.0 g casein peptone was used for routine cultivation of the 165 isolated bacteria. The pH of LB was adjusted to 7.0 prior to autoclaving. Plate count agar 166 (PCA) containing (per L) 5.0 g tryptone, 2.5 g yeast extract, 1.0 g glucose, and 15.0 g agar- 167 agar was adjusted to pH 7.2 prior to sterilization, and 0.05 g cycloheximide was added to 168 avoid fungal contamination. Finally, R2A agar containing (per L) 0.5 g casein acid 169 hydrolysate, 0.5 g yeast extract, 0.5 proteose peptone, 0.5 g dextrose, 0.5 g soluble starch, For strain isolation, biomixture subsamples were collected from different parts of the BPS, 182 and a composite sample (500 g) was stored at 4 °C for no longer than 12 hours. 183 Microorganisms in the biomixture were counted using the serial dilution method. For this, 184 10 g biomixture was added to 90 mL saline solution (0.9%), and the suspension was shaken 185 vigorously. Subsequently, 150-µL aliquots of each dilution were inoculated on Petri dishes 186 containing PCA medium. Incubation was performed at 28 ± 2 °C for 48 h, following which, 187 the colonies formed were counted.  To examine the ability of the strains to grow in the presence of pesticides (CHL and IPR), a 199 quantitative assay was performed. The study consisted of evaluating biomass growth in flasks 200 containing 50 mL of LB broth supplemented with each pesticide at 10 mg L -1 concentration.

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The flasks were incubated at 28 ± 2 °C and 130 rpm under constant shaking during 48 h, and 202 bacterial growth was measured by measuring the absorbance at 600 nm. Thereafter, 203 absorbance values were converted to biomass dry weight (g L -1 ) using a calibration curve (R 2 204 > 0.999). 206 The selected bacterial strains were characterized by a combination of phenotypic tests as 207 described by Krishnapriya et al. [30], which are based mainly on colony morphology, Gram 208 staining reaction, and colony pigmentation. in the equipment sampler and dried at 30°C, followed by microscopic observations.

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The strains were subjected to biochemical characterization using the APIZYM kit 217 (Biomerieux, France) according to the manufacturer's instructions. This microbial 218 identification system consists of 19 substrates in a microplate, which was incubated at 28 °C 219 for up to 4 days. The enzyme activity was detected based on the intensity of color developed 220 following the addition of reagents.
Average recoveries for the pesticide were: IPR, 92 ± 2.2%; CHL, 101 ± 0.7%. Limit of 298 quantification (LOQ) was determined using the smallest concentration of the analyte in the 299 test sample, which induced a signal that was ten times higher than the background noise level  The strains selected for their tolerance and ability to grow in the presence of pesticides were 338 characterized based on some phenotypic and biochemical characteristics (   (Table 2).   (Table 3).

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To identify the phylogeny of the isolates, strains from different genera were chosen to 388 construct the phylogenetic tree. Phylogenetic analysis (Fig. 2) based on the 16S rDNA using 389 MEGA7 software indicated that the isolates had higher similarity with the 16S rDNA 390 sequence from pesticide-degrading bacteria, i.e., Pseudomonas caspiana (strains C4 and C9), 391 Rhodococcus jialingiae (strain C8), and Achromobacter spirinitus (C1, C7 and C10).  Direct analysis of intact cells by MALDI-TOF/TOF MS showed a very good spectral quality 407 with score identification of 2.04 to 2.54 (Table 3), safely allowing accurate identification to 408 the genus level. Genus identification of the different strains was in agreement with the 16S 409 rDNA sequence identification. The dendrogram constructed using the MALDI Biotyper data 410 of the six bacteria in the presence of CHL and IPR showed that Achromobacter sp. strains 411 C1, C7, and C10 were differentiated and grouped separately when exposed to different 412 pesticides, and a similar response was observed for Pseudomonas sp. strains C4 and C9 (Fig.   413 3).  420 Biomass growth of the six tolerant-pesticides strains was evaluated at different incubation 421 times and increasing pesticide concentrations, observing that bacterial growth decreased 422 proportionally (R 2 > 0.96) as both pesticide concentrations increased from 10 to 100 mg L -1 .

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As observed in Fig. 4, all bacteria exposed to CHL concentrations from 10 to 50 mg L -1

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showed an increase in biomass over time. However, a high inhibition of biomass growth was 425 observed in all strains cultivated in liquid medium supplemented with 100 mg L -1 CHL. In  (Table 4).  Table 4. First-order kinetics parameter for chlorpyrifos (CHL) and iprodione (IPR) removal and specific growth rate (µ) of strains C1, 443 C4, C7, C8, C9 and C,10 in liquid medium supplemented with 0-100 mg L -1 of pesticide individually.
0 Similar to that observed for CHL, biomass of bacteria exposed to IPR increased over time,  (Table 4).     (Table 4).

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The results showed that product levels slightly increased over time, reaching a maximum Nonetheless, the removal of 10 mg L -1 and 20 mg L -1 of CHL was effectively performed by 582 the Achromobacter sp. strain C1, probably requiring only few days more to achieve complete 583 CHL elimination. A study reported that A. xylosoxidans JCp4 was able to mineralize 100 mg 584 L -1 CHL completely after ten days with only a transient accumulation of TCP [40]. Our work 585 constitutes one of the few reports of Achromobacter as CHL-degraders.

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The appearance of 3,5-DCA, recognized as the major metabolite of IPR degradation, their at 587 9 h of incubation was observed at concentrations lower than 0.5 mg L -1 . The appearance of 588 3,5-DCA was coincident with the fastest decrease of IPR levels. After this time, 3,5-DCA 589 concentrations were slightly increased, such that no IPR residues were found after 48 h of 590 incubation. Although IPR is a common fungicide frequently used in crops and with a 591 classification of "probable carcinogen to humans," treatment to eliminate IPR using 592 microorganisms has been poorly studied. Some studies reported IPR and 3,5-DCA 593 degradation by microorganisms isolated from soil, Arthrobacter sp. strains C1, and 594 Achromobacter sp. strains C2 from liquid medium, showing a T 1/2 of 2.3 h and 19.5 h, 595 respectively [9]. In our study, a small amount of time was required for Achromobacter sp. 596 strains C1 to remove 50% of the contaminant from liquid medium (T 1/2 between 4-9 h), 597 which might signify the environmental adaptation of this bacteria being exposed to continued 598 pesticide application in the biomixture used for their isolation. According to Campos et al.

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[9], IPR removal could occur via initial hydrolysis to isopropylamine and metabolite I (3,5-600 dichlorophenyl-carboxamide) and then to metabolite II (3,5-dichlorophenylurea-acetate) 601 before being hydrolyzed to 3,5-DCA and probably glycine. Similar results were reported by In this study, we described different bacteria isolated from a biomixture used in a