Effect of Culture Medium pH on Chlorpyrifos Biodegradation and Metabolic Profiles of a Novel Enterobacter strain

Effect of Culture Medium pH on Chlorpyrifos Biodegradation and Metabolic Profiles of a Novel Enterobacter strain | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effect of Culture Medium pH on Chlorpyrifos Biodegradation and Metabolic Profiles of a Novel Enterobacter strain Noha Mamdoh, Reda Mohamed Taha, Karima Ali Hassan, Amany M. Reyad This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8130012/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chlorpyrifos (CPF) is a common agricultural pesticide used worldwide. As an agricultural pesticide, CPF has been used extensively and has resulted in significant contamination. Because it might leak into neighboring ditches or drains, which normally carry water to lakes and streams, it may be dangerous. The current work used the enrichment technique to isolate bacteria with a high capacity for degradation from agricultural drainage water (El-Batts drain), Fayoum, Egypt. Isolated bacteria identified as Enterobacter sp. n1 under Genbank accession number PV495863 based on morphological, biochemical, and 16S rRNA gene sequencing technique. Mineral salt media (liquid and solidified) supplemented with CPF as sole carbon and nitrogen source used for the growth of a pure culture of and Enterobacter sp. n1. Bacteria cell count and optical density were used to detect the growth. The effect of CPF concentrations (50, 75,100,125,150, and175ppm), pH values (5, 6, 7, 8, and 9), and temperatures (15, 20, 30, 35, 40℃) on the bacterial growth as well as CPF degradation rate by GC analysis were studied. Under the influence of different degrees of pH, a difference was observed in the number of metabolic products, as well as different proportions of some similar compounds in different media. Biological sciences/Biotechnology Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Chlorpyrifos (CPF) pesticide degradation Enterobacter sp. n1 GC analysis pH effect Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Pesticides are employed extensively in agriculture, which contributes to their diffuse pollution in the air, soil, and water due to spray drift and runoff [ 1 ]. For varying lengths of time, pesticide residues can remain in soil, groundwater, sediments, surface water, and atmospheric air [ 2 ]. So, when pesticide leach, ground water becomes contaminated [ 3 ]. Once pesticides are released into the environment, their fate, mobility, and transformation are influenced by a number of complex physical, chemical, and/or biological processes, such as plant or microorganism uptake, degradation, volatilization, soil accumulation, and transportation to ground and surface waters [ 1 ]. In agricultural fields, One of the most popular organophosphate insecticides is chlorpyrifos (o, o-diethyl o-(3, 5, 6-trichloro-2-pyridyl phosphonothioate) [ 4 ]. The CPF half-life could range from 60 to 120 days, depending on the kind of soil [ 5 ]. According to Thengodkar and Sivakami [ 6 ], CPF protects horticultural and plantation crops, such as citrus fruits, potatoes, tea, bananas, vegetables, cotton, coffee, wheat, cocoa, and rice, from a variety of chewing and sucking insect pests and mites. The long-lasting and broad-spectrum impacts of CPF affect non-target insect pests and pollute ground water and the environment [ 7 , 8 ]. The widespread use of CPF has a detrimental effect on the soil's native microbiota, which affects ecosystem fertility, health, and other aspects [ 9 ]. CPF blocks AChE enzymes permanently, causing the target to exhibit symptoms like agitation, hypersalivation, convulsions, etc., ultimately leading to the pest's death [ 10 , 11 ]. Numerous methods for CPF detoxification were used in many previous researches, such as charcoal adsorption [ 12 ], synthetic nanocomposites [ 13 ], titanium dioxide photocatalysis [ 14 ], and ultrasonic treatment [ 15 ]. Microbial degradation and bioremediation are advised because they are more efficient than alternative methods [ 16 ]. Researchers are interested in employing microbial methods to bioremediate from contaminated areas because the majority of chemical and physical detoxification processes are costly and inefficient [ 17 , 18 ]. Generally, bioremediation is growing in popularity because to its low cost, environmental friendliness, ability to selectively destroy, and efficiency [ 19 ]. Therefore, our study examined the effects of different pH and temperature levels on the bioremediation efficacy and the resulting byproducts in an effort to bioremediate the CPF utilizing newly isolated bacterial species with high efficiency. Numerous taxa, such as Arthrobacter , Bacillus, Microbacterium [ 20 , 21 ], Achromobacter, Ochrobactrum [ 22 ], and Alcaligenes [ 23 ], have been shown to degrade organophosphate insecticides in contaminated soil. Our investigation identified bacterial isolate as entrobacter sp. under accession numbers (PV495863) and their role as bioremediators in contaminated environments was demonstrated. Material and Methods Chemicals and reagents. Analytical grade CPF (98%, w/v) purchased from sigma alderich (Darmstadt, Germany) that was used as the standard. To prepare the stock solution for the degradation investigations, 1000 ppm of CPF was made in ethanol and diluted to the necessary quantities. Fluka AG, Buchs, Switzerland, was the supplier of all other chemicals. Bacterial isolation 400 milliliters of agricultural wastewater were collected from El-Bats agricultural drainage in Fayoum, Egypt. Then, it was shaken at 150 rpm for 30 min at 30 º C. A solution with a 10 3 :10 5 ratio was created by serially diluting one milliliter of suspension. On mineral salt medium (MSM) added with CPF at varying concentrations ranging from 50:400 ppm, the resultant solution was plated. The culture medium was prepared as documented by Cycoń et al. [ 24 ] with minor changes by adding 15 g/ L of agar agar to liquid MSM of 1.5 g/L Na 2 HPO 4 .12H 2 O, 1.5 g/L KH 2 PO 4 , 2.0 g/L (NH 4 ) 2 SO 4 , 0.01 g/L CaCl 2 .2H 2 O, 0.001 g/L FeSO 4 .7H 2 O, and 0.2 g/L MgSO 4 .7H 2 O. Purification was made to select the most tolerant bacterial isolates. Purified isolates were preserved at -80 for further study. Bacterial Identification The CPF degrading bacterial isolates were identified using 16S rRNA gene sequencing technique. According to Essa [ 25 ], the genomic DNA was extracted. Forward primer (F1; AGA GTT TGA TCC TGG CTC AG) and the reverse primer (R1; GGT TAC CTT GTT ACG ACT T) were utilized in the amplification of the 16S rRNA gene. According to Reyad et al. [ 26 ], the PCR mixture was made. The following parameters were used for the 35 cycles of PCR: denaturation at 94 º C for 40 seconds, annealing at 55 º C for 1 minute, extension at 72 º C for 2 minutes, and the final extension was at 72 º C for 10 minutes. Using a UV transilluminator, an aliquot of the PCR results (10 µL) was combined with 2 µL of DNA loading solution and electrophoresed (15 V/cm, 60 min.) on a 0.7% horizontal agarose gel in TBE buffer containing 0.5 µg/mL ethidium bromide. GATC Biotech in Constance, Germany carried out the sequencing for the amplified fragments. The NCBI Database ( www.ncbi.nlm.nlh.gov ) is where DNA sequences were aligned. Then, using TREEVIEW software (1.6.6) and the neighbor-joining approach, a phylogenetic tree was built using the 16S rRNA gene sequences of a few strains that were phylogenetically similar to the isolated strains. To obtain accession numbers, sequences were uploaded to the GenBank database. Optimization of the growth conditions Degradation of CPF by Enterobacter sp. in aqueous medium was detected. Degradation tests were conducted in 250 mL Erlenmeyer flasks with 100 mL of sterilized MSM supplemented with varying concentrations of CPF as the sole carbon and nitrogen sources (50, 75, 100, 125, 150, and 175 ppm). Then, it was inoculated with 200 µL of each bacterial inoculum (approximately 3 × 10 6 cells/mL) for six days for Enterobacter sp. Using spectrophotometry, the growth of cells in liquid media was assessed by monitoring the culture optical density at 600 nm every 24 hours [ 27 ]. As a control, inoculation media with the same amount of CPF were employed. Experiments at different temperature degrees (15, 20, 30, 35 and 40 º C) and different pH values (5, 6, 7, 8, and 9) were examined for their impacts on bacterial growth. Growth determination by monitoring the culture optical density performed in line with counting the bacterial colonies in the same concentration. Serial dilutions were carried out after a bacterial inoculum from liquid MSM was extracted every 24 hours from the test and control cultures on MSM that had been hardened by agar and supplement with the same concentration of Erlenmeyer flasks where the bacterial inoculum taken from the appropriate dilutions. As explained by Collins and Lynes [ 28 ], a colony counter was used to count the bacterial colonies (CFU/mL). A control was maintained with equal volume of MSM solidified by agar without containing bacterial culture, but with different concentration of pesticide. All the experiments were done in triplicates. Analysis of the residual CPF and bioactive compounds The capability of Enterobacter sp. was determined under different pH values, temperature degrees of optimal time incubation in order to detect the CPF biodegradation percentages and different bioactive compounds in various pH values; 6, 7, 8, and 9. The residual CPF was extracted using acetonitrile. According to Momtaz M. and Khan MS., the obtained solution was then concentrated under nitrogen flow to 1 mL for gas chromatography analysis [ 29 ]. An HP PAS-1701 column measuring 25 meters in length by 0.32 mm by 0.52 inches in thickness and an electron detector (ECD, Radioisotope Nuclide 63Ni) are features of a Hewlett-Packard, USA series 6890 gas chromatograph. The carrier gas was pure nitrogen (2 mL/min). The temperature of the detector, injector, and column was 225, 240, and 250°C, in that order. The following formula was used by Essa et al. [ 25 ] to determine CPF degradation rate: A = [Ca – Cb / Ca] x 100 where, (A) is the percentage of CPF degradation, (Ca) is the concentration of CPF (mg/L) in the medium in absence of CPF degrading strain, (Cb) is the concentration of CPF (mg/L) in presence of CPF degrading strain. Different bacterial metabolites were detected under various pH degrees using 6, 7, 8, and 9. Statistical analysis: Software called R was used to do the statistical analysis. A p-value of less than 0.05 is regarded as statistically significant. Results Bacterial characterization and identification The most CPF-resistant bacterial isolate that tolerated to 175 mg/L was selected. As shown in Table (1) , several biochemical and morphological tests were conducted in order to fully comprehend the bacterial isolate's morphological and biochemical properties. It is Gram-negative, does not form spores, and non-motile. The bacterial isolate expressed positive results to ornithine decarboxylase, arginine dihydrolase, β-galactosidase, oxidase, H 2 S production, catalase, lysine decarboxylase, urease, amylase, nitrate production, lipase, glucose, sucrose, mannitol, inositol, sorbitol, a-L-Rhamnose, acetone, and citrate utilization. In the meantime, the tests for gelatinase, tryptophane deaminase, and indole production yielded negative findings. The Blastx program (BLAST) from the National Center for Biotechnology Knowledge was used to compare the DNA sequences to unknown sequence (Fig. 1 ). As is evident, this bacterium belongs to the genus Enterobacter and shares a close relationship with the species sp. strain 24C. It showed the highest sequence similarities with Enterobacter sp. strain 24C. The sequence was submitted to GenBank database under the accession number PV495863. Table 1 Bacterial characterization Reaction Bacterial isolate Reaction Entobacter sp. n1 Fermentation of Sugars Entobacte sp. n1 Morphological Characters Gram staining Glucose + Motility + Sucrose + Cell shape Rod Mannitol + Endospore formation Inositol + Biochemical characters Sorbitol + “Enzyme profile” a-L-Rhamnose + β-galactosidase + Citrate utilization + Arginine dihydrolase + Other tests Lysine decarboxylase + H 2 S production + Orenthine decarboxylase + Acetone production + Urease + Indole production Tryptophane deaminase Gelatenase Catalase + Amylase + Lipase + Oxidase + Nitrate reduction: + Growth optimization and CPF degradation by Enterobacter sp. n1. The information gathered demonstrated Enterobacter sp. n1 has capacity to withstand high CPF levels when isolated from agricultural waste water. Data in Fig. 2 demonstrated that raising the CPF concentration to 150 ppm enhanced the development of Enterobacter sp. in the minimum media supplemented with CPF as the carbon and nitrogen source. Within 6 days of incubation, the maximum cell density (0.648) that was recorded at OD 600nm while the maximum viable cell count was 70.6×10 5 that achieved with 150 ppm of CPF after 5 days of incubation. Meanwhile, the obtained results demonstrated the effect of pH values on the growth of Enterobacter sp. n1 (Fig. 3 ). The maximum optical density (0.661) and viable cell count (72×10 5 ) was demonstrated at pH 7 after 5 days of incubation. At pH 5 a clear inhibition in the optical density and viable cell count. While at pH 9, low cell growth was demonstrated. At the same time, moderate bacterial growth and CPF biodegradation rates were demonstrated at pH 8 and 6, respectively. At the same time, the pH value adjustment showed a noteworthy effect on Enterobacter sp. growth. Moreover, the results collected demonstrated effect of different temperature degrees on the growth. The maximum optical density (0.671) and viable cell count (73.8×10 5 ) with CPF degradation rate was demonstrated at 30 º C after 5 days of incubation. Rate of CPF degradation Our study detected that rate of CPF degradation with and without Entrobacter sp. n1 at pHs 6, 7, 8, and 9 after 5 days of incubation and at 30 º C as shown in Fig. 5 . Bioactive compounds from Enterobacter sp. n1at different pH values In the present article, the separation of compounds in MSM with CPF at different pH values (6,7, 8, and 9) by GC-MS analysis gas separation technique resulting in bioactive compounds. Eleven bioactive compounds are identified from Enterobacter sp. n1 in absence of CPF using glucose as the only carbon source at pH 7, 150 ppm and at 30 º C shown in Fig. 6 . Thirty-two bioactive compounds are identified from CPF degradation by Enterobacter sp. n1 at o pH 6 (see supplementary Table 1). Figure 7 showed CPF biodegradable major detectable metabolites at pH 6 and its GC-MS chromatogram. Forty-four bioactive compounds are identified from Enterobacter sp. at pH 7 (see supplementary Table 2) under optimum temperature and Optimum CPF concentration. Figure 8 showed major detectable metabolites at pH 7 with CPF as the only carbon and nitrogen source. Figure 8: (a) Chlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using Enterobacter Sp. n1 at pH 7 under optimum temperature and Optimum CPF concentration (150) ppm. Where (b) represents GC-MS chromatogram from Enterobacter Sp. n1 at pH 7 as a result of degradation of CPF in MSM. Thirty-two bioactive compounds are identified (see supplementary Table 3). Figure 9 showed CPF biodegradable major detectable metabolites at pH 8 and its GC-MS chromatogram. Figure 9: (a) Chlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using Enterobacter Sp. n1 at pH 8 under optimum temperature and Optimum CPF concentration (150) ppm. Where (b) represents GC-MS chromatogram from Enterobacter Sp. n1 at pH 8 as a result of degradation of CPF in MSM. Fifty-four bioactive compounds are identified in pH 9 (see supplementary Table 4), 150 ppm and at 30 º C as a result of degradation of CPF in liquid MSM after 5day. Figure 10 showed CPF biodegradable major detectable metabolites at pH 9 and its GC-MS chromatogram. DISCUSSION Bacterial breakdown of CPF has drawn a lot of attention lately since it may be an environmentally responsible and sustainable way to reduce environmental contamination from pesticides [ 47 ]. The bacterial isolates that were isolated from agricultural drainage demonstrated a high level of tolerance to high CPF concentrations. Although the hydrologic conditions in ditches can fluctuate greatly, the majority of agricultural areas have good drainage. Due to the extensive use of pesticides in agriculture, spray drift and runoff contribute to a diffuse contamination that increases the pesticides' dispersion in the air, soil, and water [ 1 ]. Certain bacterial strains that can withstand high concentrations of harmful pesticides and may be able to mineralize these substances are typically found in the microbiological communities found in agricultural wastewater [ 25 ]. Numerous elements, including as nutrient levels, soil pH, organic matter content, inoculum sizes, moisture levels, and rotations, might affect bacterial growth and survival [ 48 ]. The degradation of CPF in soil by bacteria lowers the danger of environmental and human harm [ 47 ]. Our bacterial isolate Enterobacter sp. n1 demonstrated a marked potentiality to tolerate elevated levels of CPF up to 175 ppm. Our results showed that the bioremediation of CPF is concentration dependent. This result support by research detected that bacterial species can tolerate and efficiently degrade CPF at different concentrations [ 49 , 50 , 51 , 52 , 53 ]. After 5 days growth of Enterobacter sp. n1 optimum degradation detected when initial concentration was 150 ppm where highest bacterial count (70.6×10 5 ) and the highest optical density was at (0.648). These findings are in harmony with those obtained by Chishti et al. [ 54 ] where degradation rate of Enterobacter sp. SWLC2 was 87%, initial concentration (100 mg/ L) and incubation time was 18 days. The current study demonstrated that 30 º C produced the highest levels of bacterial growth and CPF breakdown. Also, 35 º C support a good growth. These results are in agreement with those reported by Yadav et al. [ 55 ] who reported that according to the majority of research, bacterial cultures may biodegrade CPF at temperatures ranging from 30 to 37 º C. Also, Alcaligenes faecalis , Sphingomonas sp., and a co-culture of Serratia sp. and Trichosporon sp. degraded chlorpyrifos at 30°C and an initial concentration of 100 mg/L, achieving approximately 76%, 90%, and more than 90% degradation, respectively [ 49 , 56 , 57 ]. Jabeen et al. [ 53 ] reported that Mesorhizobium sp. completely biodegraded 100 mg/L of chlorpyrifos at 37°C within five days of incubation. For the same incubation period and concentration, the degradation was 55% and 85% at 30°C and 40°C, respectively. Bacillus pumilus C2A1 degraded 89% of CPF (1000 mg/L) at 37 º C [ 51 ]. Abiotic variables such as pH have one of the most significant effects on the bioremediation process. In our study, maximum of degradation rate 96.9%was observed at pH 7 and the minimum (22%) was observed at pH 9. At pH (6 and8) moderate degradation rate of CPF (76.98% and 58%, respectively) was observed. On the six days, there were differences between all of the pH treatments that had been applied. These findings are consistent with those that showed that, following 18 days of incubation at pH 8.0 and 30°C, Alcaligenes faecalis DSP3 was able to digest the main hydrolysis product TCP and almost 76% of 100 mg/L of CPF [ 49 ]. Anwar et al. [ 51 ] observed that Bacillus pumilus C2A1 could degrade 50% of CPF (50 mg/L) at an acidic pH of 5.5, whereas over 80% degradation was achieved at an alkaline pH of 8.5 in 5 days. These results are in agreement with Akbar and Sultan [ 22 ] who reported that in 10 days, Ochrobactrum sp. and Achromobacter xylosoxidans (JCp4) were able to break down 84.4% and 78.6% of the initial concentration of CPF (100 mg/L) respectively. In liquid medium, Stenotrophomonas maltophilia (RS1) and Acinetobacter calcoaceticus (RS3), biodegraded 71.3% and 73.5% of CPF in respectively where 80% degraded within 48 h when a consortium cultured [ 58 ]. Also, Bacillus cereus Ct3 was degraded 88% of CPF in 8 days at pH 8 and was resistant concentration of CPF up to 125 mg/ L [ 59 ] and our findings at high pH were in conflict with these findings. Bacillus siamensis NRRU-BW9, Bacillus amyloliquefaciens NRRU-TV11, and Priestia megaterium NRRU-BW3 can all break down CPF in an aqueous media. A degradation rate of 33–52% was observed following 14 days of incubation [ 60 ]. Among the elements that have a significant impact on the development of microbial secondary metabolites is pH [ 61 ]. The pH level has stimulatory or inhibitory effects on the secondary metabolites and influences the solubility and delivery of nutrients into the cell and routes for biosynthesis [ 62 ]. In our study there are different CPF biodegradable major detectable metabolites appeared in different pHs and sometimes disappeared as a result of biodegradation of CPF by Enterobacte r sp. n1. These metabolites show significant benefits that were studied in many previous researches. Major detectable metabolites in MSM without CPF using glucose as the only carbon source by Enterobacter Sp. n1 after 5 days at pH 7 under optimum temperature and Optimum CPF concentration (150) ppm were 1-Hexadecanol 2-methyl, Tetradecanoic acid, Oleic Acid, and Z-Docos-9-enenitrile. CPF major detectable metabolites in MSM using Enterobacter Sp. n1 after 5 days at pH 6 under optimum temperature and Optimum CPF concentration (150) ppm were Eicosane, 1-Dodecanamine, N,N-dimethyl-, Tert-Hexadecanethiol, 7,9-Di-tert-butyl-1-oxaspiro(4,5) deca-6,9-diene-2,8-dione, n-Hexadecanoic acid, Cyclohexane, 1,3,5-triphenyl, 1-Propene, 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl-, and Phenol, 2,4-bis (1,1-dimethylethyl)-, phosphite (3:1). Eicosane has therapeutic properties and anti-inflammatory [ 30 ]. 1-Dodecanamine, N, N-dimethyl-possesses antibacterial properties [ 31 ]. Tert-Hexadecanethiol has antibacterial properties and antioxidant [ 32 ]. 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione has antioxidant activity [ 33 ]. n-Hexadecanoic acid uses for treatment of malaria [ 34 ]. Also, has anti-cancer and antioxidant qualities [ 35 ]. Cyclohexane, 1,3,5-triphenyl contains three phenyl groups, so there is possibility of having antioxidant and anti-inflammatory properties [ 36 ]. Benzene, 1,1'-[2-methyl-2-(phenylthio)cyclopropylidene] has a unique structure characterized by phenyl groups and a phenylthio moiety attached to a cyclopropane ring. based on its chemical structure, it has antibacterial, antioxidant, and anticancer properties [ 37 ]. 1-Propene, 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl- has antioxidant activity [ 38 ]. Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) has anti-enterococcal and antioxidant properties [ 39 ]. Where at pH 7 major detectable metabolites were Benzene, 1,1'-(1,2-cyclobutanediyl) bis-, trans-, n-Hexadecanoic acid, Cyclohexane, 1,3,5-triphenyl-, Benzene, 1,1'-[2-methyl-2-(phenylthio)cyclopropylidene]bis-, 1-Propene, 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl, Thiocarbamic acid, N,N-dimethyl, S-1,3-diphenyl-2-butenyl ester. Benzene, 1,1'-(1,2-cyclobutanediyl) bis-, trans- has anti-inflammatory, anti-oxidants, and anti-cancer effects [ 40 ]. Thiocarbamic acid, N, N-dimethyl, S-1,3-diphenyl-2-butenyl ester used as antidiabetic medication [ 41 ]. At pH 8 major detectable metabolites were Eicosane, 1-Dodecanamine, N, N-dimethyl-, Tert-Hexadecanethiol, 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione, n-Hexadecanoic acid, Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1). As well as, at pH 9 major metabolites that detected were 7,9-Di-tert-butyl-1oxaspiro (4,5) deca-6,9-diene-2,8-dione, n-Hexadecanoic acid, Di (2-ethylhexyl) phthalate, 13-Docosenamide, (Z)-, Phenol, 2,4-bis (1,1-dimethylethyl)-phosphite, Benzenepropanoic acid, 3,5-bis. Di (2-ethylhexyl) phthalate is used in non-PVC industries for pigments, lacquers, and adhesives. It is also used in detergents, lubricating oils, industrial solvents, and wetting agents [ 42]. 13-Docosenamide, (Z)- uses biologically to treat hepatitis, muscle weakness, drowsiness, insomnia, anemia, and hyperthermia [ 43 ]. It has anticancer effect [ 44 ]. It has antioxidant effect [ 45 ]. Both cytotoxicity and antibacterial properties are demonstrated by 3,5-bis (1,1-dimethylethyl)-4-hydroxy-octadecyl ester of benzoenepropanoic acid [ 46 ]. Its antimicrobial and anticancer qualities are being investigated [ 46 ]. n-Hexadecanoic acid is one of major detectable metabolites found in different pH values (6, 7, 8, and 9) but in different concentrations (2.51, 3.23, 10.24, 5.1%, respectively). Some metabolites are restricted to defined pH values such as Cyclohexane, 1,3,5-triphenyl, Benzene, 1,1'-[2-methyl-2-(phenylthio) cyclopropylidene] bis, and 1-Propene 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl detected as major metabolites in pH (6 and 7) but they were present in different concentration where Cyclohexane, 1,3,5-triphenyl was at (3.94 and 11.38%, respectively), Benzene, 1,1'-[2-methyl-2-(phenylthio) cyclopropylidene] bis was at (5.54 and 14.08%, respectively) and 1-Propene 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl was found at (7.72 and 17.56%, respectively). Thiocarbamic acid, N, N-dimethyl, S-1,3-diphenyl-2-butenyl ester only presented in pH (7and 8). Eicosane and 1-Dodecanamine, N, N-dimethyl- are from major metabolites only presented in pH (6 and 8). Eicosane, 1-Dodecanamine and N, N-dimethyl, Tert-Hexadecanethiol were major detectable metabolites found in pH (6 and 8) but they were found at different concentrations where Eicosane (8.5 and 4.51%, respectively), 1-Dodecanamine, N, N-dimethyl ((7.21 and 4.75%, respectively), and Tert-Hexadecanethiol (2.74 and 3.21%, respectively). As well as 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione found at pH (6, 8 and 9) where concentration was (3.88, 5.69 and 3.58 respectively). Also, Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) found at pH (8 and 9) where concentrations were (3.03 and 5.89%, respectively). Our study detected that major detected metabolites may restricted to defined pH value and they could repeat at different pH values but in different concentrations. In our study effect of different pH values in present of major detectable metabolites or their concentrations result from degradation of CPF by Enterobacter sp. n1 were detected. These results are in agree with Boruta et al. [ 61 ] that examined how pH value affected the morphology and secondary metabolite production of Streptomyces rimosus and Aspergillus terreus in cocultures and axenic cultures. Secondary metabolites (6 bacterial and 4 fungal) were not produced at pH ≤ 4.0. The highest production of oxytetracycline by S. rimosus occurred at pH 5.0. Starting at pH 5.9 reduced oxytetracycline levels, but coculturing with A. terreus helped counteract this drop and increased production compared to the axenic S. rimosus culture. Coculturing at pH 5.0 or 5.9 also induced the production of oxidized rimocidin. However, A. terreus's own metabolites were not detected in the cocultures. Significant morphological differences between cocultures and axenic cultures appeared at pH 4.0. Additionally, another study showed that the production of bioactive compounds by different Streptomyces isolates varied depending on the initial pH: Streptomyces spectabilis (isolate R1) achieved maximum production at pH 5, Streptomyces purpurascens (isolate R3) reached optimal production at pH 7, Streptomyces coeruleorubidus (isolate R5) showed the highest production at pH 6, and Streptomyces lavendofoliae (isolate Y8) also produced high levels at pH 7. This indicates that different bacterial strains require specific pH levels to maximize their bioactive compound production [ 62 ]. Conclusion MSM was used to isolate Enterobacter sp. n1, a bacterium that degrade chlorpyrifos, from agricultural drainage water in Fayoum, Egypt. The strain demonstrated efficient development at ideal concentration, pH, and temperature conditions and used chlorpyrifos as the only source of carbon and nitrogen. Chlorpyrifos was significantly degraded and beneficial various metabolites were formed, as shown by gas chromatography under pH levels. These findings suggest that Enterobacter sp. n1 can be used for the bioremediation of areas contaminated with chlorpyrifos due to its great biodegradation capability. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding No funding Author Contribution A.M.R. and R.M.T. conceived, designed, and coordinated the study. N.M. carried out the experimental studies. A.M.R., N.M., K.A.H., and R. M.T. wrote, organized, and revised the manuscript. All authors have read and approved the final manuscript. Data Availability "The DNA sequence has been deposited in GenBank, a member of the International Nucleotide Sequence Database Collaboration (INSDC), and is publicly accessible under the assigned accession number PV495863" References Tudi, M. et al. Agriculture development, pesticide application and its impact on the environment. Int. J. Environ. Res. Public. Health . 18 (3), 1112 (2021). Jara, E. A. & Winter, C. K. Safety levels for organophosphate pesticide residues on fruits, vegetables, and nuts. Int. J. Food Contam. 6 , 1–8 (2019). Farhan, M. et al. Biodegradation of chlorpyrifos using isolates from contaminated agricultural soil, its kinetic studies. Sci. Rep. 11 (1), 10320 (2021). Kaushal, J., Khatri, M. & Arya, S. K. A treatise on organophosphate pesticide pollution: current strategies and advancements in their environmental degradation and elimination. Ecotoxicol. Environ. Saf. 207 , 111483 (2021). Bosu, S., Rajamohan, N., Al Salti, S., Rajasimman, M. & Das, P. Biodegradation of chlorpyrifos pollution from contaminated environment – a review on operating variables and mechanism. Environ. Res. 248 , 118212 (2024). Thengodkar, R. R. M. & Sivakami, S. Degradation of chlorpyrifos by an alkaline phosphatase from the cyanobacterium Spirulina platensis. Biodegradation 21 , 637–644 (2010). Ruan, Q. L. et al. Chlorpyrifos exposure reduces reproductive capacity owing to a damaging effect on gametogenesis in the nematode Caenorhabditis elegans . J. Appl. Toxicol. 32 (7), 527–535 (2012). Silva, M. H. Effects of low-dose chlorpyrifos on neurobehavior and potential mechanisms: a review of studies in rodents, zebrafish, and Caenorhabditis elegans . Birth Defects Res. 112 (6), 445–479 (2020). Dar, M. A., Kaushik, G. & Villarreal-Chiu, J. F. Pollution status and bioremediation of chlorpyrifos in environmental matrices by the application of bacterial communities: a review. J. Environ. Manage. 239 , 124–136 (2019). King, A. M. & Aaron, C. K. Organophosphate and carbamate poisoning. Emerg. Med. Clin. North. Am. 33 (1), 133–151 (2015). Kumar, A., Margekar, S. L., Margekar, P. & Margekar, V. Recent advances in management of organophosphate and carbamate poisoning. Indian J. Med. Spec. 9 (3), 154–159 (2018). Lei, W., Tang, X. & Zhou, X. Biochar amendment effectively reduces the transport of 3,5,6-trichloro-2-pyridinol (a main degradation product of chlorpyrifos) in purple soil: experimental and modeling. Chemosphere 245 , 125651 (2020). Soltani-Nezhad, F., Saljooqi, A., Mostafavi, A. & Shamspur, T. Synthesis of Fe₃O₄/CdS–ZnS nanostructure and its application for photocatalytic degradation of chlorpyrifos pesticide and brilliant green dye from aqueous solutions. Ecotoxicol. Environ. Saf. 189 , 109886 (2020). Sivagami, K., Vikraman, B., Krishna, R. R. & Swaminathan, T. Chlorpyrifos and endosulfan degradation studies in an annular slurry photo reactor. Ecotoxicol. Environ. Saf. 134 , 327–331 (2016). Liu, Y. et al. Effects of electrolyzed water-ultrasound treatment on pesticide removal in fresh-cut potatoes with different cutting methods. J. Food Process. Eng. 46 (11), e14423 (2023). Bose, S., Kumar, P. S. & Vo, D. V. N. A review on the microbial degradation of chlorpyrifos and its metabolite TCP. Chemosphere 283 , 131447 (2021). Horne, I., Sutherland, T. D., Harcourt, R. L., Russell, R. J. & Oakeshott, J. G. Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate. Appl. Environ. Microbiol. 68 (7), 3371–3376 (2002). Rudakiya, D. M., Patel, Y. & Chhaya, U. Strategies to improve remediation technology using fungi. In: Microbial Rejuvenation of Polluted Environment. Vol. 1. pp. 185–202. (2021). Fuentes, M. S., Raimondo, E. E., Amoroso, M. J. & Benimeli, C. S. Removal of a mixture of pesticides by a Streptomyces consortium: influence of different soil systems. Chemosphere 173 , 359–367 (2017). Ahirwar, U., Kollah, B., Dubey, G. & Mohanty, S. R. Chlorpyrifos biodegradation in relation to metabolic attributes and 16S rRNA gene phylogeny of bacteria in a tropical vertisol. SN Appl. Sci. 1 , 1–11 (2019). Srinivasan, P. et al. Chlorpyrifos degradation efficiency of Bacillus sp. laccase immobilized on iron magnetic nanoparticles. 3 Biotech. 10 , 1–11 (2020). Akbar, S. & Sultan, S. Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement. Braz J. Microbiol. 47 (3), 563–570 (2016). Yadav, U. et al. Chlorpyrifos degradation by plant growth-promoting Alcaligenes faecalis bacteria isolated from oil-contaminated soil. Bioremediat. J. 25 (1), 80–88 (2020). Cycoń, M., Żmijowska, A., Wójcik, M. & Piotrowska-Seget, Z. Biodegradation and bioremediation potential of diazinon-degrading Serratia marcescens to remove other organophosphorus pesticides from soils. J. Environ. Manage. 117 , 7–16 (2013). Essa, A. M. Effect of continuous mercury stress on mercury reducing community of some characterized bacterial strains. Afr. J. Microbiol. Res. 6 (6), 1255–1261 (2012). Reyad, A., Radwan, T., Ibrahim, W. M. & Essa, A. Biodegradation of atrazine by Ochrobactrum oryzae isolated from the agricultural wastewater. Wulfenia 21 (4), 286–310 (2014). Beal, J. et al. Robust estimation of bacterial cell count from optical density. Commun. Biol. 3 (1), 512 (2020). Collins, C. H. & Lyne, P. M. Microbiological methods 5th edn, p.137–140 (Butterworth & Co (Publishers) Ltd, 1984). Momtaz, M. & Khan, M. S. Analysis of chlorpyrifos pesticide residue in locally grown cauliflower, cabbage, and eggplant using gas chromatography–mass spectrometry (GC-MS) technique: a Bangladesh perspective. Foods 13 (11), 1780 (2024). Chuah, X. Q., Okechukwu, P. N., Amini, F. & Teo, S. S. Eicosane, pentadecane and palmitic acid: the effects in in vitro wound healing studies. Asian Pac. J. Trop. Biomed. 8 (10), 490–499 (2018). Bespalova, Y., Kwon, D. & Vasanthan, N. Surface modification and antimicrobial properties of cellulose nanocrystals. J Appl. Polym. Sci ; 134 (18). (2017). Elshaarawy, R. et al. Preliminary assessment of bioactive ingredients and antioxidant activity of some Red Sea invertebrates' extracts. Egypt J. Aquat. Biol. Fish ; 27 (4). (2023). Jayathri, P. M. P. et al. Antioxidant activity and chemical constituents of methanolic extract of Durio zibethinus Murr. (durian) peels. (2021). Afolayan, F. I. D., Odeyemi, R. A. & Salaam, R. A. In silico and in vivo evaluations of multistage antiplasmodial potency and toxicity profiling of n-Hexadecanoic acid derived from Vernonia amygdalina . Front. Pharmacol. 15 , 1445905 (2024). Bharath, B., Perinbam, K., Devanesan, S., AlSalhi, M. S. & Saravanan, M. Evaluation of the anticancer potential of Hexadecanoic acid from brown algae Turbinaria ornata on HT–29 colon cancer cells. J. Mol. Struct. 1235 , 130229 (2021). Zhang, L. et al. Antioxidant and anti-inflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds. J. Agric. Food Chem. 59 (23), 12361–12367. 10.1021/jf203146e (2011). Khoshbakht, A., Shiran, J. A., Miran, M. & Sepehri, S. Synthesis and evaluation of in vitro antioxidant, anticancer, and antibacterial properties of new benzylideneiminophenylthiazole analogues. BMC Chem. 18 (1), 173 (2024). Hinampas, R. & Walag, A. M. A review of marine natural products as potential source of antioxidants. BIOTROPIA 31 (3), 438–448 (2024). Taher, M. A. et al. Bioactive compounds extracted from leaves of G. cyanocarpa using various solvents in chromatographic separation showed anti-cancer and anti-microbial potentiality in in silico approach. Chin. J. Anal. Chem. 51 (12), 100336 (2023). Zainuddin, N. A., Mohamad, N. D. & Rodzay, R. M. Rapid expansion supercritical solution (RESS) of CO₂ as a green technology method for pineapple peels solid oil particle formation. In: IOP Conf Ser Mater Sci Eng . ;1053(1):012086. IOP Publishing. (2021). Ajilore, B. S., Olorunnisola, O. S. & Owoade, A. O. Tetracarpidium conophorum seed extract reduces intestinal absorption and increases cellular trapping of glucose. Bull. Natl. Res. Cent. 45 , 1–10 (2021). Koch, H. M., Preuss, R. & Angerer, J. D. Di(2-ethylhexyl) phthalate (DEHP): human metabolism and internal exposure–an update and latest results. Int. J. Androl. 29 (1), 155–165 (2006). Duraisamy, M. & Selvaraju, R. Analysis of chemical compounds by using gas chromatography and mass spectrum analysis, in vitro antioxidant and antibacterial activity of methanolic extracts of seaweed Ulva flexuosa Wulfen (green algae). Aegaeum J. 8 , 1438–1454 (2020). Sonbol, H. S., Hosawi, S. B. & Hosawi, M. B. GC-MS analysis of the bioactive phytochemical compounds with anticancer activity in the Capparis cartilaginea fruit extracts. J. Adv. Pharm. Educ. Res. 13 (2), 64–70 (2023). Khan, S., Kaur, H. & Jhamta, R. Evaluation of antioxidant potential and phytochemical characterization using GC-MS analysis of bioactive compounds of Achillea filipendulina (L.) leaves. J. Pharmacogn Phytochem . 8 (3), 258–265 (2019). Amer, M. H. et al. The potential of Brachionus plicatilis extract against bacterial infection and cancer: In vitro and in silico studies. Egypt. J. Chem. 67 (9), 713–725 (2024). Dar, M. A. & Kaushik, G. Optimizing the malathion degrading potential of a newly isolated Bacillus sp. AGM5 based on Taguchi design of experiment and elucidation of degradation pathway. Biodegradation 33 (5), 419–439 (2022). Saengsanga, T. & Phakratok, N. Biodegradation of chlorpyrifos by soil bacteria and their effects on growth of rice seedlings under pesticide-contaminated soil. Plant. Soil. Environ. ; 69 (5). (2023). Yang, L., Zhao, Y. H., Zhang, B. X., Yang, C. H. & Zhang, X. Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacterium. FEMS Microbiol. Lett. 251 (1), 67–73 (2005). Ghanem, I., Orfi, M. & Shamma, M. Biodegradation of chlorpyrifos by Klebsiella sp. isolated from an activated sludge sample of wastewater treatment plant in Damascus. Folia Microbiol. (Praha) . 52 , 423–427 (2007). Anwar, S., Liaquat, F., Khan, Q. M., Khalid, Z. M. & Iqbal, S. Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by Bacillus pumilus strain C2A1. J. Hazard. Mater. 168 (1), 400–405 (2009). Awad, N. S., Sabit, H. H., Abo-Aba, S. E. & Bayoumi, R. A. Isolation, characterization and fingerprinting of some chlorpyrifos-degrading bacterial strains isolated from Egyptian pesticides-polluted soils. Afr. J. Microbiol. Res. 5 (18), 2855–2862 (2011). Jabeen, H., Iqbal, S. & Anwar, S. Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by a novel rhizobial strain Mesorhizobium sp. HN3. Water Environ. J. 29 (1), 151–160 (2015). Chishti, Z., Ahmad, Z., Zhang, X. & Jha, S. K. Optimization of biotic and abiotic factors liable for biodegradation of chlorpyrifos and their modeling using neural network approaches. Appl. Soil. Ecol. 166 , 103990 (2021). Yadav, M., Shukla, A. K., Srivastva, N., Upadhyay, S. N. & Dubey, S. K. Utilization of microbial community potential for removal of chlorpyrifos: a review. Crit. Rev. Biotechnol. 36 (4), 727–742 (2016). Li, X., He, J. & Li, S. Isolation of a chlorpyrifos-degrading bacterium, Sphingomonas sp. strain Dsp-2, and cloning of the mpd gene. Res. Microbiol. 158 (2), 143–149 (2007). Xu, G. et al. Mineralization of chlorpyrifos by co-culture of Serratia and Trichosporon spp. Biotechnol. Lett. 29 , 1469–1473 (2007). Omeiri, M., Khnayzer, R., Yusef, H., Tokajian, S. & Salloum, T. Biodegradation of chlorpyrifos by bacterial strains isolated from Lebanese soil and its association with plant growth improvement. Bioremediat. J. 28 (1), 14–33 (2024). Farhan, M. et al. Biodegradation of chlorpyrifos using isolates from contaminated agricultural soil, its kinetic studies. Sci. Rep. 11 (1), 10320 (2021). Saengsanga, T. & Phakratok, N. Biodegradation of chlorpyrifos by soil bacteria and their effects on growth of rice seedlings under pesticide-contaminated soil. Plant Soil. & Environment , 69 (5). (2023). Boruta, T., Foryś, M., Pawlikowska, W., Englart, G. & Bizukojć, M. Initial pH determines the morphological characteristics and secondary metabolite production in Aspergillus terreus and Streptomyces rimosus cocultures. Arch. Microbiol. 206 (12), 1–12 (2024). Bundale, S., Begde, D., Nashikkar, N., Kadam, T. & Upadhyay, A. Optimization of culture conditions for production of bioactive metabolites by Streptomyces spp. isolated from soil. Adv. Microbiol. 5 (6), 441 (2015). Additional Declarations No competing interests reported. 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10:20:58","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":284052,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/330f3e7412695cc7b96cdc06.png"},{"id":97248335,"identity":"8bdda3e3-3c6e-4a78-ada5-d659a133f3e7","added_by":"auto","created_at":"2025-12-02 12:54:04","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110002,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/a69b06fa308624aa92f6b19c.png"},{"id":97148968,"identity":"7d8e1fab-ebf0-4e02-8e2e-c036f586bf12","added_by":"auto","created_at":"2025-12-01 10:20:58","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":23469,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/36ba544fd41ceb96fa7b0721.png"},{"id":97248597,"identity":"1d25b520-76fd-4b38-806c-01e49a3d9f38","added_by":"auto","created_at":"2025-12-02 13:03:55","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134547,"visible":true,"origin":"","legend":"","description":"","filename":"038f02e36c4b4a529c578c5c2240eae61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/9e9ce8ec584ea1d94d9c2b31.xml"},{"id":97149042,"identity":"ec3d435b-9655-4413-8ea4-fc92be47cc69","added_by":"auto","created_at":"2025-12-01 10:20:59","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":149589,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/05cd4ff313fc031daf83391e.html"},{"id":97148944,"identity":"e3012129-d625-4705-a4a1-4eeda215b9c7","added_by":"auto","created_at":"2025-12-01 10:20:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74701,"visible":true,"origin":"","legend":"\u003cp\u003eThe location of our CPF-resistant bacterial strain (the yellow highlighted one) within the related genera that is displayed by a neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences. The sequence was submitted to GenBank database under the accession number \u003cstrong\u003ePV495863.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/ae23b061c45791e9130ee514.jpg"},{"id":97248417,"identity":"c15f6992-8d4a-46ff-91e1-a669e9669e3f","added_by":"auto","created_at":"2025-12-02 12:58:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96908,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different CHF concentrations on bacterial growth, where (a) represents viable cell count (CFU/mL×10\u003csup\u003e5\u003c/sup\u003e) and (b) represents optical density (OD\u003csub\u003e600\u003c/sub\u003e). The standard errors of the means are shown by the error bars, and three replicates' means are used to create the data.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/b463787a7afc4ec8892ba244.jpg"},{"id":97148946,"identity":"7a7d5833-a794-411a-902d-8c66d146ea83","added_by":"auto","created_at":"2025-12-01 10:20:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74136,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different pH values on bacterial growth where (a) represents viable cell count (CFU/ml×10\u003csup\u003e5\u003c/sup\u003e) and (b) represents optical density (OD\u003csub\u003e600\u003c/sub\u003e). The standard errors of the means are shown by the error bars, and three replicates' means are used to create the data. (N) indicates that there was no detectable bacterial growth.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/1f0db25f40001d239a29ca69.jpg"},{"id":97148942,"identity":"63a6ad35-bfed-4d09-a194-d83f87c7467a","added_by":"auto","created_at":"2025-12-01 10:20:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67282,"visible":true,"origin":"","legend":"\u003cp\u003eWhere (N) indicated that there was no bacterial growth.\u003c/p\u003e\n\u003cp\u003eEffect of different temperature dgrees on bacterial growth where (a) represents viable cell count (CFU/ml×10\u003csup\u003e5\u003c/sup\u003e) and (b) represents optical density(OD\u003csub\u003e600\u003c/sub\u003e). The standard errors of the means are shown by the error bars, and three replicates' means are used to create the data.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/b00736a8864da3170e9e73e1.jpg"},{"id":97248362,"identity":"f6e395ca-f9e9-4be2-994e-c0f76971fdf8","added_by":"auto","created_at":"2025-12-02 12:55:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":51177,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation rate of CPF with and without \u003cem\u003eEntrobacter \u003c/em\u003esp. n1 after 5 days of incubation, 30\u003csup\u003eº\u003c/sup\u003eC and at pH (6, 7, 8, and 9).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/b9baacd13994199baf85cee1.jpg"},{"id":97148953,"identity":"cf650b74-5b31-4b77-990b-aa4c32066e14","added_by":"auto","created_at":"2025-12-01 10:20:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":87359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e represents major detectable metabolites in MSM using \u003cem\u003eEnterobacter \u003c/em\u003eSp. n1 at pH 7 at 30\u003csup\u003eº\u003c/sup\u003eC without CPF. As well as, \u003cstrong\u003e(b)\u003c/strong\u003e represents GC-MS chromatogram from\u003cem\u003e Enterobacter\u003c/em\u003e Sp. n1 at pH 7 as a result of degradation of glucose in MSM.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/fed7b3350de1f5aa5b9ffa40.jpg"},{"id":97249061,"identity":"fb2f81ae-5934-47e0-9e8d-fd04aa059b84","added_by":"auto","created_at":"2025-12-02 13:10:06","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":172608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eChlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 at pH 6 under optimum temperature and optimum CPF concentration (150) pp. Where \u003cstrong\u003e(b)\u003c/strong\u003e represents GC-MS chromatogram from\u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 at pH 6 as a result of degradation of CPF in MSM.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/3813a3538384a88699ba8509.jpg"},{"id":97249642,"identity":"649a3b45-e88d-41c2-8db9-be136ebd1780","added_by":"auto","created_at":"2025-12-02 13:13:09","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":101524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eChlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using \u003cem\u003eEnterobacter \u003c/em\u003eSp. n1 at pH 7 under optimum temperature and Optimum CPF concentration (150) ppm. Where \u003cstrong\u003e(b)\u003c/strong\u003e represents GC-MS chromatogram from\u003cem\u003e Enterobacter\u003c/em\u003e Sp. n1 at pH 7 as a result of degradation of CPF in MSM.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/5b6cec4bbc24203c16a15cdf.jpg"},{"id":97148964,"identity":"c076b851-4733-4054-a063-f58d914b28c2","added_by":"auto","created_at":"2025-12-01 10:20:58","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":107518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eChlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using \u003cem\u003eEnterobacter\u003c/em\u003eSp. n1 at pH 8 under optimum temperature and Optimum CPF concentration (150) ppm. Where \u003cstrong\u003e(b)\u003c/strong\u003e represents GC-MS chromatogram from\u003cem\u003e Enterobacter\u003c/em\u003e Sp. n1 at pH 8 as a result of degradation of CPF in MSM.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/7766628625e4c0cf79065596.jpg"},{"id":97249140,"identity":"789276ca-fc20-43eb-851f-f1284786c8fc","added_by":"auto","created_at":"2025-12-02 13:10:42","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":92390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eChlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 at pH 8 under optimum temperature and optimum CPF concentration (150) ppm. Where \u003cstrong\u003e(b)\u003c/strong\u003e represents GC-MS chromatogram from\u003cem\u003e Enterobacter\u003c/em\u003e Sp. n1 at pH 9 as a result of degradation of CPF in MSM.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/440c311d1b96430ab4dbff22.jpg"},{"id":106402587,"identity":"1184baac-757c-471b-97ae-d29eea142e02","added_by":"auto","created_at":"2026-04-08 09:12:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1971110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/757de286-1731-4665-9c50-ce3b4a57af0e.pdf"},{"id":97148943,"identity":"aa675c2a-dc59-4039-949e-3aa6e8c6d253","added_by":"auto","created_at":"2025-12-01 10:20:58","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":40752,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8130012/v1/54012e4a82a9a7e38fd93c09.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Culture Medium pH on Chlorpyrifos Biodegradation and Metabolic Profiles of a Novel Enterobacter strain","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePesticides are employed extensively in agriculture, which contributes to their diffuse pollution in the air, soil, and water due to spray drift and runoff [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. For varying lengths of time, pesticide residues can remain in soil, groundwater, sediments, surface water, and atmospheric air [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSo, when pesticide leach, ground water becomes contaminated [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Once pesticides are released into the environment, their fate, mobility, and transformation are influenced by a number of complex physical, chemical, and/or biological processes, such as plant or microorganism uptake, degradation, volatilization, soil accumulation, and transportation to ground and surface waters [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn agricultural fields, One of the most popular organophosphate insecticides is chlorpyrifos (o, o-diethyl o-(3, 5, 6-trichloro-2-pyridyl phosphonothioate) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The CPF half-life could range from 60 to 120 days, depending on the kind of soil [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. According to Thengodkar and Sivakami [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], CPF protects horticultural and plantation crops, such as citrus fruits, potatoes, tea, bananas, vegetables, cotton, coffee, wheat, cocoa, and rice, from a variety of chewing and sucking insect pests and mites. The long-lasting and broad-spectrum impacts of CPF affect non-target insect pests and pollute ground water and the environment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The widespread use of CPF has a detrimental effect on the soil's native microbiota, which affects ecosystem fertility, health, and other aspects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CPF blocks AChE enzymes permanently, causing the target to exhibit symptoms like agitation, hypersalivation, convulsions, etc., ultimately leading to the pest's death [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNumerous methods for CPF detoxification were used in many previous researches, such as charcoal adsorption [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], synthetic nanocomposites [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], titanium dioxide photocatalysis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and ultrasonic treatment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicrobial degradation and bioremediation are advised because they are more efficient than alternative methods [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Researchers are interested in employing microbial methods to bioremediate from contaminated areas because the majority of chemical and physical detoxification processes are costly and inefficient [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Generally, bioremediation is growing in popularity because to its low cost, environmental friendliness, ability to selectively destroy, and efficiency [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, our study examined the effects of different pH and temperature levels on the bioremediation efficacy and the resulting byproducts in an effort to bioremediate the CPF utilizing newly isolated bacterial species with high efficiency.\u003c/p\u003e\u003cp\u003eNumerous taxa, such as \u003cem\u003eArthrobacter\u003c/em\u003e, \u003cem\u003eBacillus, Microbacterium\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], \u003cem\u003eAchromobacter, Ochrobactrum\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and \u003cem\u003eAlcaligenes\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], have been shown to degrade organophosphate insecticides in contaminated soil. Our investigation identified bacterial isolate as \u003cem\u003eentrobacter\u003c/em\u003e sp. under accession numbers (PV495863) and their role as bioremediators in contaminated environments was demonstrated.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cb\u003eChemicals and reagents.\u003c/b\u003e Analytical grade CPF (98%, w/v) purchased from sigma alderich (Darmstadt, Germany) that was used as the standard. To prepare the stock solution for the degradation investigations, 1000 ppm of CPF was made in ethanol and diluted to the necessary quantities. Fluka AG, Buchs, Switzerland, was the supplier of all other chemicals.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial isolation\u003c/h2\u003e\u003cp\u003e400 milliliters of agricultural wastewater were collected from El-Bats agricultural drainage in Fayoum, Egypt. Then, it was shaken at 150 rpm for 30 min at 30\u003csup\u003e\u0026ordm;\u003c/sup\u003eC. A solution with a 10\u003csup\u003e3\u003c/sup\u003e:10\u003csup\u003e5\u003c/sup\u003e ratio was created by serially diluting one milliliter of suspension. On mineral salt medium (MSM) added with CPF at varying concentrations ranging from 50:400 ppm, the resultant solution was plated.\u003c/p\u003e\u003cp\u003eThe culture medium was prepared as documented by Cycoń \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] with minor changes by adding 15 g/ L of agar agar to liquid MSM of 1.5 g/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.12H\u003csub\u003e2\u003c/sub\u003eO, 1.5 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.0 g/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.01 g/L CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO, 0.001 g/L FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, and 0.2 g/L MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO. Purification was made to select the most tolerant bacterial isolates. Purified isolates were preserved at -80 for further study.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBacterial Identification\u003c/h3\u003e\n\u003cp\u003eThe CPF degrading bacterial isolates were identified using 16S rRNA gene sequencing technique.\u003c/p\u003e\u003cp\u003eAccording to Essa [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], the genomic DNA was extracted. Forward primer (F1; AGA GTT TGA TCC TGG CTC AG) and the reverse primer (R1; GGT TAC CTT GTT ACG ACT T) were utilized in the amplification of the 16S rRNA gene. According to Reyad \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the PCR mixture was made. The following parameters were used for the 35 cycles of PCR: denaturation at 94\u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 40 seconds, annealing at 55\u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 1 minute, extension at 72\u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 2 minutes, and the final extension was at 72\u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 10 minutes. Using a UV transilluminator, an aliquot of the PCR results (10 \u0026micro;L) was combined with 2 \u0026micro;L of DNA loading solution and electrophoresed (15 V/cm, 60 min.) on a 0.7% horizontal agarose gel in TBE buffer containing 0.5 \u0026micro;g/mL ethidium bromide. GATC Biotech in Constance, Germany carried out the sequencing for the amplified fragments. The NCBI Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.ncbi.nlm.nlh.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nlh.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) is where DNA sequences were aligned. Then, using TREEVIEW software (1.6.6) and the neighbor-joining approach, a phylogenetic tree was built using the 16S rRNA gene sequences of a few strains that were phylogenetically similar to the isolated strains. To obtain accession numbers, sequences were uploaded to the GenBank database.\u003c/p\u003e\n\u003ch3\u003eOptimization of the growth conditions\u003c/h3\u003e\n\u003cp\u003eDegradation of CPF by \u003cem\u003eEnterobacter\u003c/em\u003e sp. in aqueous medium was detected. Degradation tests were conducted in 250 mL Erlenmeyer flasks with 100 mL of sterilized MSM supplemented with varying concentrations of CPF as the sole carbon and nitrogen sources (50, 75, 100, 125, 150, and 175 ppm). Then, it was inoculated with 200 \u0026micro;L of each bacterial inoculum (approximately 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL) for six days for \u003cem\u003eEnterobacter\u003c/em\u003e sp.\u003c/p\u003e\u003cp\u003eUsing spectrophotometry, the growth of cells in liquid media was assessed by monitoring the culture optical density at 600 nm every 24 hours [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As a control, inoculation media with the same amount of CPF were employed. Experiments at different temperature degrees (15, 20, 30, 35 and 40\u003csup\u003e\u0026ordm;\u003c/sup\u003eC) and different pH values (5, 6, 7, 8, and 9) were examined for their impacts on bacterial growth. Growth determination by monitoring the culture optical density performed in line with counting the bacterial colonies in the same concentration. Serial dilutions were carried out after a bacterial inoculum from liquid MSM was extracted every 24 hours from the test and control cultures on MSM that had been hardened by agar and supplement with the same concentration of Erlenmeyer flasks where the bacterial inoculum taken from the appropriate dilutions. As explained by Collins and Lynes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], a colony counter was used to count the bacterial colonies (CFU/mL). A control was maintained with equal volume of MSM solidified by agar without containing bacterial culture, but with different concentration of pesticide. All the experiments were done in triplicates.\u003c/p\u003e\n\u003ch3\u003eAnalysis of the residual CPF and bioactive compounds\u003c/h3\u003e\n\u003cp\u003eThe capability of \u003cem\u003eEnterobacter\u003c/em\u003e sp. was determined under different pH values, temperature degrees of optimal time incubation in order to detect the CPF biodegradation percentages and different bioactive compounds in various pH values; 6, 7, 8, and 9. The residual CPF was extracted using acetonitrile. According to Momtaz M. and Khan MS., the obtained solution was then concentrated under nitrogen flow to 1 mL for gas chromatography analysis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. An HP PAS-1701 column measuring 25 meters in length by 0.32 mm by 0.52 inches in thickness and an electron detector (ECD, Radioisotope Nuclide 63Ni) are features of a Hewlett-Packard, USA series 6890 gas chromatograph. The carrier gas was pure nitrogen (2 mL/min). The temperature of the detector, injector, and column was 225, 240, and 250\u0026deg;C, in that order. The following formula was used by Essa \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] to determine CPF degradation rate:\u003c/p\u003e\u003cp\u003eA = [Ca \u0026ndash; Cb / Ca] x 100\u003c/p\u003e\u003cp\u003ewhere, (A) is the percentage of CPF degradation, (Ca) is the concentration of CPF (mg/L) in the medium in absence of CPF degrading strain, (Cb) is the concentration of CPF (mg/L) in presence of CPF degrading strain. Different bacterial metabolites were detected under various pH degrees using 6, 7, 8, and 9.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis:\u003c/h2\u003e\u003cp\u003eSoftware called R was used to do the statistical analysis. A p-value of less than 0.05 is regarded as statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eBacterial characterization and identification\u003c/h2\u003e\u003cp\u003eThe most CPF-resistant bacterial isolate that tolerated to 175 mg/L was selected. As shown in \u003cb\u003eTable\u0026nbsp;(1)\u003c/b\u003e, several biochemical and morphological tests were conducted in order to fully comprehend the bacterial isolate's morphological and biochemical properties. It is Gram-negative, does not form spores, and non-motile. The bacterial isolate expressed positive results to ornithine decarboxylase, arginine dihydrolase, β-galactosidase, oxidase, H\u003csub\u003e2\u003c/sub\u003eS production, catalase, lysine decarboxylase, urease, amylase, nitrate production, lipase, glucose, sucrose, mannitol, inositol, sorbitol, a-L-Rhamnose, acetone, and citrate utilization. In the meantime, the tests for gelatinase, tryptophane deaminase, and indole production yielded negative findings. The Blastx program (BLAST) from the National Center for Biotechnology Knowledge was used to compare the DNA sequences to unknown sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As is evident, this bacterium belongs to the genus \u003cem\u003eEnterobacter\u003c/em\u003e and shares a close relationship with the species sp. strain 24C. It showed the highest sequence similarities with \u003cem\u003eEnterobacter\u003c/em\u003e sp. strain 24C. The sequence was submitted to GenBank database under the accession number \u003cb\u003ePV495863.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBacterial characterization\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eReaction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBacterial isolate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eReaction\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eEntobacter\u003c/em\u003e sp. n1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFermentation of\u003c/p\u003e\u003cp\u003eSugars\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eEntobacte\u003c/em\u003e sp. n1\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMorphological\u003c/p\u003e\u003cp\u003eCharacters\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGram staining\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGlucose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMotility\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSucrose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCell shape\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRod\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMannitol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEndospore formation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInositol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBiochemical characters\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSorbitol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e\u0026ldquo;Enzyme profile\u0026rdquo;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ea-L-Rhamnose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eβ-galactosidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCitrate utilization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArginine dihydrolase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eOther tests\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLysine decarboxylase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrenthine decarboxylase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAcetone production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUrease\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIndole production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTryptophane deaminase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGelatenase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCatalase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmylase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLipase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOxidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNitrate reduction:\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth optimization and CPF degradation by\u003c/b\u003e \u003cb\u003eEnterobacter\u003c/b\u003e \u003cb\u003esp. n1.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe information gathered demonstrated \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 has capacity to withstand high CPF levels when isolated from agricultural waste water. Data in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e demonstrated that raising the CPF concentration to 150 ppm enhanced the development of \u003cem\u003eEnterobacter\u003c/em\u003e sp. in the minimum media supplemented with CPF as the carbon and nitrogen source. Within 6 days of incubation, the maximum cell density (0.648) that was recorded at OD\u003csub\u003e600nm\u003c/sub\u003e while the maximum viable cell count was 70.6\u0026times;10\u003csup\u003e5\u003c/sup\u003e that achieved with 150 ppm of CPF after 5 days of incubation.\u003c/p\u003e\u003cp\u003eMeanwhile, the obtained results demonstrated the effect of pH values on the growth of \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The maximum optical density (0.661) and viable cell count (72\u0026times;10\u003csup\u003e5\u003c/sup\u003e) was demonstrated at pH 7 after 5 days of incubation. At pH 5 a clear inhibition in the optical density and viable cell count. While at pH 9, low cell growth was demonstrated. At the same time, moderate bacterial growth and CPF biodegradation rates were demonstrated at pH 8 and 6, respectively. At the same time, the pH value adjustment showed a noteworthy effect on \u003cem\u003eEnterobacter\u003c/em\u003e sp. growth.\u003c/p\u003e\u003cp\u003eMoreover, the results collected demonstrated effect of different temperature degrees on the growth. The maximum optical density (0.671) and viable cell count (73.8\u0026times;10\u003csup\u003e5\u003c/sup\u003e) with CPF degradation rate was demonstrated at 30\u003csup\u003e\u0026ordm;\u003c/sup\u003eC after 5 days of incubation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRate of CPF degradation\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOur study detected that rate of CPF degradation with and without \u003cb\u003eEntrobacter\u003c/b\u003e \u003cb\u003esp. n1\u003c/b\u003e at pHs 6, 7, 8, and 9 after 5 days of incubation and at 30\u003csup\u003e\u0026ordm;\u003c/sup\u003eC as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBioactive compounds from\u003c/b\u003e \u003cb\u003eEnterobacter\u003c/b\u003e \u003cb\u003esp. n1at different pH values\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the present article, the separation of compounds in MSM with CPF at different pH values (6,7, 8, and 9) by GC-MS analysis gas separation technique resulting in bioactive compounds.\u003c/p\u003e\u003cp\u003eEleven bioactive compounds are identified from \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 in absence of CPF using glucose as the only carbon source at pH 7, 150 ppm and at 30\u003csup\u003e\u0026ordm;\u003c/sup\u003eC shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThirty-two bioactive compounds are identified from CPF degradation by \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 at o pH 6 (see supplementary Table\u0026nbsp;1). Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e showed CPF biodegradable major detectable metabolites at pH 6 and its GC-MS chromatogram.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eForty-four bioactive compounds are identified from \u003cem\u003eEnterobacter\u003c/em\u003e sp. at \u003cb\u003epH 7 (see supplementary Table\u0026nbsp;2)\u003c/b\u003e under optimum temperature and Optimum CPF concentration. Figure\u0026nbsp;8 showed major detectable metabolites at pH 7 with CPF as the only carbon and nitrogen source.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;8: (a)\u003c/b\u003e Chlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 at pH 7 under optimum temperature and Optimum CPF concentration (150) ppm. Where \u003cb\u003e(b)\u003c/b\u003e represents GC-MS chromatogram from \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 at pH 7 as a result of degradation of CPF in MSM.\u003c/p\u003e\u003cp\u003eThirty-two bioactive compounds are identified (see supplementary Table\u0026nbsp;3). Figure\u0026nbsp;9 showed CPF biodegradable major detectable metabolites at pH 8 and its GC-MS chromatogram.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;9: (a)\u003c/b\u003e Chlorpyrifos [CPF] biodegradable major detectable metabolites in MSM using \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 at pH 8 under optimum temperature and Optimum CPF concentration (150) ppm. Where \u003cb\u003e(b)\u003c/b\u003e represents GC-MS chromatogram from \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 at pH 8 as a result of degradation of CPF in MSM.\u003c/p\u003e\u003cp\u003eFifty-four bioactive compounds are identified in \u003cb\u003epH 9\u003c/b\u003e (see supplementary Table\u0026nbsp;4), 150 ppm and at 30\u003csup\u003e\u0026ordm;\u003c/sup\u003eC as a result of degradation of CPF in liquid MSM after 5day. Figure\u0026nbsp;10 showed CPF biodegradable major detectable metabolites at pH 9 and its GC-MS chromatogram.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBacterial breakdown of CPF has drawn a lot of attention lately since it may be an environmentally responsible and sustainable way to reduce environmental contamination from pesticides [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The bacterial isolates that were isolated from agricultural drainage demonstrated a high level of tolerance to high CPF concentrations. Although the hydrologic conditions in ditches can fluctuate greatly, the majority of agricultural areas have good drainage. Due to the extensive use of pesticides in agriculture, spray drift and runoff contribute to a diffuse contamination that increases the pesticides' dispersion in the air, soil, and water [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Certain bacterial strains that can withstand high concentrations of harmful pesticides and may be able to mineralize these substances are typically found in the microbiological communities found in agricultural wastewater [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Numerous elements, including as nutrient levels, soil pH, organic matter content, inoculum sizes, moisture levels, and rotations, might affect bacterial growth and survival [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The degradation of CPF in soil by bacteria lowers the danger of environmental and human harm [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Our bacterial isolate \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 demonstrated a marked potentiality to tolerate elevated levels of CPF up to 175 ppm. Our results showed that the bioremediation of CPF is concentration dependent. This result support by research detected that bacterial species can tolerate and efficiently degrade CPF at different concentrations [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. After 5 days growth of \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 optimum degradation detected when initial concentration was 150 ppm where highest bacterial count (70.6\u0026times;10\u003csup\u003e5\u003c/sup\u003e) and the highest optical density was at (0.648). These findings are in harmony with those obtained by Chishti \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] where degradation rate of \u003cem\u003eEnterobacter\u003c/em\u003e sp. SWLC2 was 87%, initial concentration (100 mg/ L) and incubation time was 18 days. The current study demonstrated that 30\u003csup\u003e\u0026ordm;\u003c/sup\u003eC produced the highest levels of bacterial growth and CPF breakdown. Also, 35\u003csup\u003e\u0026ordm;\u003c/sup\u003eC support a good growth. These results are in agreement with those reported by Yadav \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] who reported that according to the majority of research, bacterial cultures may biodegrade CPF at temperatures ranging from 30 to 37\u003csup\u003e\u0026ordm;\u003c/sup\u003eC. Also, \u003cem\u003eAlcaligenes faecalis\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e sp., and a co-culture of \u003cem\u003eSerratia\u003c/em\u003e sp. and \u003cem\u003eTrichosporon\u003c/em\u003e sp. degraded chlorpyrifos at 30\u0026deg;C and an initial concentration of 100 mg/L, achieving approximately 76%, 90%, and more than 90% degradation, respectively [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Jabeen \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] reported that \u003cem\u003eMesorhizobium\u003c/em\u003e sp. completely biodegraded 100 mg/L of chlorpyrifos at 37\u0026deg;C within five days of incubation. For the same incubation period and concentration, the degradation was 55% and 85% at 30\u0026deg;C and 40\u0026deg;C, respectively. \u003cem\u003eBacillus pumilus\u003c/em\u003e C2A1 degraded 89% of CPF (1000 mg/L) at 37 \u003csup\u003e\u0026ordm;\u003c/sup\u003eC [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAbiotic variables such as pH have one of the most significant effects on the bioremediation process. In our study, maximum of degradation rate 96.9%was observed at pH 7 and the minimum (22%) was observed at pH 9. At pH (6 and8) moderate degradation rate of CPF (76.98% and 58%, respectively) was observed. On the six days, there were differences between all of the pH treatments that had been applied. These findings are consistent with those that showed that, following 18 days of incubation at pH 8.0 and 30\u0026deg;C, Alcaligenes faecalis DSP3 was able to digest the main hydrolysis product TCP and almost 76% of 100 mg/L of CPF [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Anwar \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] observed that \u003cem\u003eBacillus pumilus\u003c/em\u003e C2A1 could degrade 50% of CPF (50 mg/L) at an acidic pH of 5.5, whereas over 80% degradation was achieved at an alkaline pH of 8.5 in 5 days. These results are in agreement with Akbar and Sultan [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] who reported that in 10 days, \u003cem\u003eOchrobactrum\u003c/em\u003e sp. and \u003cem\u003eAchromobacter xylosoxidans\u003c/em\u003e (JCp4) were able to break down 84.4% and 78.6% of the initial concentration of CPF (100 mg/L) respectively. In liquid medium, \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e (RS1) and \u003cem\u003eAcinetobacter calcoaceticus\u003c/em\u003e (RS3), biodegraded 71.3% and 73.5% of CPF in respectively where 80% degraded within 48 h when a consortium cultured [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Also, \u003cem\u003eBacillus cereus\u003c/em\u003e Ct3 was degraded 88% of CPF in 8 days at pH 8 and was resistant concentration of CPF up to 125 mg/ L [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and our findings at high pH were in conflict with these findings. \u003cem\u003eBacillus siamensis\u003c/em\u003e NRRU-BW9, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e NRRU-TV11, and \u003cem\u003ePriestia megaterium\u003c/em\u003e NRRU-BW3 can all break down CPF in an aqueous media. A degradation rate of 33\u0026ndash;52% was observed following 14 days of incubation [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Among the elements that have a significant impact on the development of microbial secondary metabolites is pH [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The pH level has stimulatory or inhibitory effects on the secondary metabolites and influences the solubility and delivery of nutrients into the cell and routes for biosynthesis [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study there are different CPF biodegradable major detectable metabolites appeared in different pHs and sometimes disappeared as a result of biodegradation of CPF by \u003cem\u003eEnterobacte\u003c/em\u003er sp. n1. These metabolites show significant benefits that were studied in many previous researches. Major detectable metabolites in MSM without CPF using glucose as the only carbon source by \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 after 5 days at \u003cb\u003epH 7\u003c/b\u003e under optimum temperature and Optimum CPF concentration (150) ppm were 1-Hexadecanol 2-methyl, Tetradecanoic acid, Oleic Acid, and Z-Docos-9-enenitrile.\u003c/p\u003e\u003cp\u003eCPF major detectable metabolites in MSM using \u003cem\u003eEnterobacter\u003c/em\u003e Sp. n1 after 5 days at \u003cb\u003epH 6\u003c/b\u003e under optimum temperature and Optimum CPF concentration (150) ppm were Eicosane, 1-Dodecanamine, N,N-dimethyl-, Tert-Hexadecanethiol, 7,9-Di-tert-butyl-1-oxaspiro(4,5) deca-6,9-diene-2,8-dione, n-Hexadecanoic acid, Cyclohexane, 1,3,5-triphenyl, 1-Propene, 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl-, and Phenol, 2,4-bis (1,1-dimethylethyl)-, phosphite (3:1).\u003c/p\u003e\u003cp\u003eEicosane has therapeutic properties and anti-inflammatory [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. 1-Dodecanamine, N, N-dimethyl-possesses antibacterial properties [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Tert-Hexadecanethiol has antibacterial properties and antioxidant [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione has antioxidant activity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. n-Hexadecanoic acid uses for treatment of malaria [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Also, has anti-cancer and antioxidant qualities [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Cyclohexane, 1,3,5-triphenyl contains three phenyl groups, so there is possibility of having antioxidant and anti-inflammatory properties [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Benzene, 1,1'-[2-methyl-2-(phenylthio)cyclopropylidene] has a unique structure characterized by phenyl groups and a phenylthio moiety attached to a cyclopropane ring. based on its chemical structure, it has antibacterial, antioxidant, and anticancer properties [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 1-Propene, 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl- has antioxidant activity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) has anti-enterococcal and antioxidant properties [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhere at pH 7 major detectable metabolites were Benzene, 1,1'-(1,2-cyclobutanediyl) bis-, trans-, n-Hexadecanoic acid, Cyclohexane, 1,3,5-triphenyl-, Benzene, 1,1'-[2-methyl-2-(phenylthio)cyclopropylidene]bis-, 1-Propene, 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl, Thiocarbamic acid, N,N-dimethyl, S-1,3-diphenyl-2-butenyl ester. Benzene, 1,1'-(1,2-cyclobutanediyl) bis-, trans- has anti-inflammatory, anti-oxidants, and anti-cancer effects [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thiocarbamic acid, N, N-dimethyl, S-1,3-diphenyl-2-butenyl ester used as antidiabetic medication [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt pH 8 major detectable metabolites were Eicosane, 1-Dodecanamine, N, N-dimethyl-, Tert-Hexadecanethiol, 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione, n-Hexadecanoic acid, Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1).\u003c/p\u003e\u003cp\u003eAs well as, at pH 9 major metabolites that detected were 7,9-Di-tert-butyl-1oxaspiro (4,5) deca-6,9-diene-2,8-dione, n-Hexadecanoic acid, Di (2-ethylhexyl) phthalate, 13-Docosenamide, (Z)-, Phenol, 2,4-bis (1,1-dimethylethyl)-phosphite, Benzenepropanoic acid, 3,5-bis. Di (2-ethylhexyl) phthalate is used in non-PVC industries for pigments, lacquers, and adhesives. It is also used in detergents, lubricating oils, industrial solvents, and wetting agents [ 42]. 13-Docosenamide, (Z)- uses biologically to treat hepatitis, muscle weakness, drowsiness, insomnia, anemia, and hyperthermia [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. It has anticancer effect [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. It has antioxidant effect [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Both cytotoxicity and antibacterial properties are demonstrated by 3,5-bis (1,1-dimethylethyl)-4-hydroxy-octadecyl ester of benzoenepropanoic acid [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Its antimicrobial and anticancer qualities are being investigated [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003cp\u003en-Hexadecanoic acid is one of major detectable metabolites found in different pH values (6, 7, 8, and 9) but in different concentrations (2.51, 3.23, 10.24, 5.1%, respectively). Some metabolites are restricted to defined pH values such as Cyclohexane, 1,3,5-triphenyl, Benzene, 1,1'-[2-methyl-2-(phenylthio) cyclopropylidene] bis, and 1-Propene 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl detected as major metabolites in pH (6 and 7) but they were present in different concentration where Cyclohexane, 1,3,5-triphenyl was at (3.94 and 11.38%, respectively), Benzene, 1,1'-[2-methyl-2-(phenylthio) cyclopropylidene] bis was at (5.54 and 14.08%, respectively) and 1-Propene 3-(2-cyclopentenyl)-2-methyl-1,1-diphenyl was found at (7.72 and 17.56%, respectively). Thiocarbamic acid, N, N-dimethyl, S-1,3-diphenyl-2-butenyl ester only presented in pH (7and 8). Eicosane and 1-Dodecanamine, N, N-dimethyl- are from major metabolites only presented in pH (6 and 8). Eicosane, 1-Dodecanamine and N, N-dimethyl, Tert-Hexadecanethiol were major detectable metabolites found in pH (6 and 8) but they were found at different concentrations where Eicosane (8.5 and 4.51%, respectively), 1-Dodecanamine, N, N-dimethyl ((7.21 and 4.75%, respectively), and Tert-Hexadecanethiol (2.74 and 3.21%, respectively). As well as 7,9-Di-tert-butyl-1-oxaspiro (4,5) deca-6,9-diene-2,8-dione found at pH (6, 8 and 9) where concentration was (3.88, 5.69 and 3.58 respectively). Also, Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) found at pH (8 and 9) where concentrations were (3.03 and 5.89%, respectively). Our study detected that major detected metabolites may restricted to defined pH value and they could repeat at different pH values but in different concentrations.\u003c/p\u003e\u003cp\u003eIn our study effect of different pH values in present of major detectable metabolites or their concentrations result from degradation of CPF by \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 were detected. These results are in agree with Boruta \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] that examined how pH value affected the morphology and secondary metabolite production of \u003cem\u003eStreptomyces rimosus\u003c/em\u003e and \u003cem\u003eAspergillus terreus\u003c/em\u003e in cocultures and axenic cultures. Secondary metabolites (6 bacterial and 4 fungal) were not produced at pH\u0026thinsp;\u0026le;\u0026thinsp;4.0. The highest production of oxytetracycline by \u003cem\u003eS. rimosus\u003c/em\u003e occurred at pH 5.0. Starting at pH 5.9 reduced oxytetracycline levels, but coculturing with \u003cem\u003eA. terreus\u003c/em\u003e helped counteract this drop and increased production compared to the axenic \u003cem\u003eS. rimosus\u003c/em\u003e culture. Coculturing at pH 5.0 or 5.9 also induced the production of oxidized rimocidin. However, \u003cem\u003eA. terreus's\u003c/em\u003e own metabolites were not detected in the cocultures. Significant morphological differences between cocultures and axenic cultures appeared at pH 4.0. Additionally, another study showed that the production of bioactive compounds by different \u003cem\u003eStreptomyces\u003c/em\u003e isolates varied depending on the initial pH: Streptomyces spectabilis (isolate R1) achieved maximum production at pH 5, Streptomyces purpurascens (isolate R3) reached optimal production at pH 7, \u003cem\u003eStreptomyces coeruleorubidus\u003c/em\u003e (isolate R5) showed the highest production at pH 6, and \u003cem\u003eStreptomyces lavendofoliae\u003c/em\u003e (isolate Y8) also produced high levels at pH 7. This indicates that different bacterial strains require specific pH levels to maximize their bioactive compound production [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMSM was used to isolate \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1, a bacterium that degrade chlorpyrifos, from agricultural drainage water in Fayoum, Egypt. The strain demonstrated efficient development at ideal concentration, pH, and temperature conditions and used chlorpyrifos as the only source of carbon and nitrogen. Chlorpyrifos was significantly degraded and beneficial various metabolites were formed, as shown by gas chromatography under pH levels. These findings suggest that \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 can be used for the bioremediation of areas contaminated with chlorpyrifos due to its great biodegradation capability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNo funding\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.M.R. and R.M.T. conceived, designed, and coordinated the study. N.M. carried out the experimental studies. A.M.R., N.M., K.A.H., and R. M.T. wrote, organized, and revised the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003e\"The DNA sequence has been deposited in GenBank, a member of the International Nucleotide Sequence Database Collaboration (INSDC), and is publicly accessible under the assigned accession number PV495863\"\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTudi, M. et al. Agriculture development, pesticide application and its impact on the environment. \u003cem\u003eInt. J. Environ. Res. Public. Health\u003c/em\u003e. \u003cb\u003e18\u003c/b\u003e (3), 1112 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJara, E. A. \u0026amp; Winter, C. K. Safety levels for organophosphate pesticide residues on fruits, vegetables, and nuts. \u003cem\u003eInt. J. Food Contam.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 1\u0026ndash;8 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFarhan, M. et al. Biodegradation of chlorpyrifos using isolates from contaminated agricultural soil, its kinetic studies. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (1), 10320 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaushal, J., Khatri, M. \u0026amp; Arya, S. K. A treatise on organophosphate pesticide pollution: current strategies and advancements in their environmental degradation and elimination. \u003cem\u003eEcotoxicol. Environ. Saf.\u003c/em\u003e \u003cb\u003e207\u003c/b\u003e, 111483 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBosu, S., Rajamohan, N., Al Salti, S., Rajasimman, M. \u0026amp; Das, P. Biodegradation of chlorpyrifos pollution from contaminated environment \u0026ndash; a review on operating variables and mechanism. \u003cem\u003eEnviron. Res.\u003c/em\u003e \u003cb\u003e248\u003c/b\u003e, 118212 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThengodkar, R. R. M. \u0026amp; Sivakami, S. Degradation of chlorpyrifos by an alkaline phosphatase from the cyanobacterium Spirulina platensis. \u003cem\u003eBiodegradation\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 637\u0026ndash;644 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuan, Q. L. et al. Chlorpyrifos exposure reduces reproductive capacity owing to a damaging effect on gametogenesis in the nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. \u003cem\u003eJ. Appl. Toxicol.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e (7), 527\u0026ndash;535 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva, M. H. Effects of low-dose chlorpyrifos on neurobehavior and potential mechanisms: a review of studies in rodents, zebrafish, and \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. \u003cem\u003eBirth Defects Res.\u003c/em\u003e \u003cb\u003e112\u003c/b\u003e (6), 445\u0026ndash;479 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDar, M. A., Kaushik, G. \u0026amp; Villarreal-Chiu, J. F. Pollution status and bioremediation of chlorpyrifos in environmental matrices by the application of bacterial communities: a review. \u003cem\u003eJ. Environ. Manage.\u003c/em\u003e \u003cb\u003e239\u003c/b\u003e, 124\u0026ndash;136 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKing, A. M. \u0026amp; Aaron, C. K. Organophosphate and carbamate poisoning. \u003cem\u003eEmerg. Med. Clin. North. Am.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e (1), 133\u0026ndash;151 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar, A., Margekar, S. L., Margekar, P. \u0026amp; Margekar, V. Recent advances in management of organophosphate and carbamate poisoning. \u003cem\u003eIndian J. Med. Spec.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (3), 154\u0026ndash;159 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLei, W., Tang, X. \u0026amp; Zhou, X. Biochar amendment effectively reduces the transport of 3,5,6-trichloro-2-pyridinol (a main degradation product of chlorpyrifos) in purple soil: experimental and modeling. \u003cem\u003eChemosphere\u003c/em\u003e \u003cb\u003e245\u003c/b\u003e, 125651 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoltani-Nezhad, F., Saljooqi, A., Mostafavi, A. \u0026amp; Shamspur, T. Synthesis of Fe₃O₄/CdS\u0026ndash;ZnS nanostructure and its application for photocatalytic degradation of chlorpyrifos pesticide and brilliant green dye from aqueous solutions. \u003cem\u003eEcotoxicol. Environ. Saf.\u003c/em\u003e \u003cb\u003e189\u003c/b\u003e, 109886 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSivagami, K., Vikraman, B., Krishna, R. R. \u0026amp; Swaminathan, T. Chlorpyrifos and endosulfan degradation studies in an annular slurry photo reactor. \u003cem\u003eEcotoxicol. Environ. Saf.\u003c/em\u003e \u003cb\u003e134\u003c/b\u003e, 327\u0026ndash;331 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, Y. et al. Effects of electrolyzed water-ultrasound treatment on pesticide removal in fresh-cut potatoes with different cutting methods. \u003cem\u003eJ. Food Process. Eng.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e (11), e14423 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBose, S., Kumar, P. S. \u0026amp; Vo, D. V. N. A review on the microbial degradation of chlorpyrifos and its metabolite TCP. \u003cem\u003eChemosphere\u003c/em\u003e \u003cb\u003e283\u003c/b\u003e, 131447 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHorne, I., Sutherland, T. D., Harcourt, R. L., Russell, R. J. \u0026amp; Oakeshott, J. G. Identification of an \u003cem\u003eopd\u003c/em\u003e (organophosphate degradation) gene in an \u003cem\u003eAgrobacterium\u003c/em\u003e isolate. \u003cem\u003eAppl. Environ. Microbiol.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e (7), 3371\u0026ndash;3376 (2002).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRudakiya, D. M., Patel, Y. \u0026amp; Chhaya, U. Strategies to improve remediation technology using fungi. In: Microbial Rejuvenation of Polluted Environment. Vol. 1. pp. 185\u0026ndash;202. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuentes, M. S., Raimondo, E. E., Amoroso, M. J. \u0026amp; Benimeli, C. S. Removal of a mixture of pesticides by a \u003cem\u003eStreptomyces\u003c/em\u003e consortium: influence of different soil systems. \u003cem\u003eChemosphere\u003c/em\u003e \u003cb\u003e173\u003c/b\u003e, 359\u0026ndash;367 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhirwar, U., Kollah, B., Dubey, G. \u0026amp; Mohanty, S. R. Chlorpyrifos biodegradation in relation to metabolic attributes and 16S rRNA gene phylogeny of bacteria in a tropical vertisol. \u003cem\u003eSN Appl. Sci.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 1\u0026ndash;11 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSrinivasan, P. et al. Chlorpyrifos degradation efficiency of \u003cem\u003eBacillus\u003c/em\u003e sp. laccase immobilized on iron magnetic nanoparticles. \u003cem\u003e3 Biotech.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 1\u0026ndash;11 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkbar, S. \u0026amp; Sultan, S. Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement. \u003cem\u003eBraz J. Microbiol.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e (3), 563\u0026ndash;570 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav, U. et al. Chlorpyrifos degradation by plant growth-promoting \u003cem\u003eAlcaligenes faecalis\u003c/em\u003e bacteria isolated from oil-contaminated soil. \u003cem\u003eBioremediat. J.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (1), 80\u0026ndash;88 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCycoń, M., Żmijowska, A., W\u0026oacute;jcik, M. \u0026amp; Piotrowska-Seget, Z. Biodegradation and bioremediation potential of diazinon-degrading \u003cem\u003eSerratia marcescens\u003c/em\u003e to remove other organophosphorus pesticides from soils. \u003cem\u003eJ. Environ. Manage.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e, 7\u0026ndash;16 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEssa, A. M. Effect of continuous mercury stress on mercury reducing community of some characterized bacterial strains. \u003cem\u003eAfr. J. Microbiol. Res.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e (6), 1255\u0026ndash;1261 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReyad, A., Radwan, T., Ibrahim, W. M. \u0026amp; Essa, A. Biodegradation of atrazine by \u003cem\u003eOchrobactrum oryzae\u003c/em\u003e isolated from the agricultural wastewater. \u003cem\u003eWulfenia\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (4), 286\u0026ndash;310 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBeal, J. et al. Robust estimation of bacterial cell count from optical density. \u003cem\u003eCommun. Biol.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (1), 512 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCollins, C. H. \u0026amp; Lyne, P. M. \u003cem\u003eMicrobiological methods\u003c/em\u003e 5th edn, p.137\u0026ndash;140 (Butterworth \u0026amp; Co (Publishers) Ltd, 1984).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMomtaz, M. \u0026amp; Khan, M. S. Analysis of chlorpyrifos pesticide residue in locally grown cauliflower, cabbage, and eggplant using gas chromatography\u0026ndash;mass spectrometry (GC-MS) technique: a Bangladesh perspective. \u003cem\u003eFoods\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (11), 1780 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChuah, X. Q., Okechukwu, P. N., Amini, F. \u0026amp; Teo, S. S. Eicosane, pentadecane and palmitic acid: the effects in in vitro wound healing studies. \u003cem\u003eAsian Pac. J. Trop. Biomed.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (10), 490\u0026ndash;499 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBespalova, Y., Kwon, D. \u0026amp; Vasanthan, N. Surface modification and antimicrobial properties of cellulose nanocrystals. \u003cem\u003eJ Appl. Polym. Sci\u003c/em\u003e ;\u003cb\u003e134\u003c/b\u003e(18). (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElshaarawy, R. et al. Preliminary assessment of bioactive ingredients and antioxidant activity of some Red Sea invertebrates' extracts. \u003cem\u003eEgypt J. Aquat. Biol. Fish\u003c/em\u003e ;\u003cb\u003e27\u003c/b\u003e(4). (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJayathri, P. M. P. et al. Antioxidant activity and chemical constituents of methanolic extract of Durio zibethinus Murr. (durian) peels. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAfolayan, F. I. D., Odeyemi, R. A. \u0026amp; Salaam, R. A. In silico and in vivo evaluations of multistage antiplasmodial potency and toxicity profiling of n-Hexadecanoic acid derived from \u003cem\u003eVernonia amygdalina\u003c/em\u003e. \u003cem\u003eFront. Pharmacol.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 1445905 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBharath, B., Perinbam, K., Devanesan, S., AlSalhi, M. S. \u0026amp; Saravanan, M. Evaluation of the anticancer potential of Hexadecanoic acid from brown algae \u003cem\u003eTurbinaria ornata\u003c/em\u003e on HT\u0026ndash;29 colon cancer cells. \u003cem\u003eJ. Mol. Struct.\u003c/em\u003e \u003cb\u003e1235\u003c/b\u003e, 130229 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, L. et al. Antioxidant and anti-inflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e59\u003c/b\u003e (23), 12361\u0026ndash;12367. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/jf203146e\u003c/span\u003e\u003cspan address=\"10.1021/jf203146e\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhoshbakht, A., Shiran, J. A., Miran, M. \u0026amp; Sepehri, S. Synthesis and evaluation of in vitro antioxidant, anticancer, and antibacterial properties of new benzylideneiminophenylthiazole analogues. \u003cem\u003eBMC Chem.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (1), 173 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHinampas, R. \u0026amp; Walag, A. M. A review of marine natural products as potential source of antioxidants. \u003cem\u003eBIOTROPIA\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e (3), 438\u0026ndash;448 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaher, M. A. et al. Bioactive compounds extracted from leaves of \u003cem\u003eG. cyanocarpa\u003c/em\u003e using various solvents in chromatographic separation showed anti-cancer and anti-microbial potentiality in in silico approach. \u003cem\u003eChin. J. Anal. Chem.\u003c/em\u003e \u003cb\u003e51\u003c/b\u003e (12), 100336 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZainuddin, N. A., Mohamad, N. D. \u0026amp; Rodzay, R. M. Rapid expansion supercritical solution (RESS) of CO₂ as a green technology method for pineapple peels solid oil particle formation. In: \u003cem\u003eIOP Conf Ser Mater Sci Eng\u003c/em\u003e. ;1053(1):012086. IOP Publishing. (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAjilore, B. S., Olorunnisola, O. S. \u0026amp; Owoade, A. O. \u003cem\u003eTetracarpidium conophorum\u003c/em\u003e seed extract reduces intestinal absorption and increases cellular trapping of glucose. \u003cem\u003eBull. Natl. Res. Cent.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, 1\u0026ndash;10 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoch, H. M., Preuss, R. \u0026amp; Angerer, J. D. Di(2-ethylhexyl) phthalate (DEHP): human metabolism and internal exposure\u0026ndash;an update and latest results. \u003cem\u003eInt. J. Androl.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (1), 155\u0026ndash;165 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuraisamy, M. \u0026amp; Selvaraju, R. Analysis of chemical compounds by using gas chromatography and mass spectrum analysis, in vitro antioxidant and antibacterial activity of methanolic extracts of seaweed \u003cem\u003eUlva flexuosa\u003c/em\u003e Wulfen (green algae). \u003cem\u003eAegaeum J.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 1438\u0026ndash;1454 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSonbol, H. S., Hosawi, S. B. \u0026amp; Hosawi, M. B. GC-MS analysis of the bioactive phytochemical compounds with anticancer activity in the \u003cem\u003eCapparis cartilaginea\u003c/em\u003e fruit extracts. \u003cem\u003eJ. Adv. Pharm. Educ. Res.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (2), 64\u0026ndash;70 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan, S., Kaur, H. \u0026amp; Jhamta, R. Evaluation of antioxidant potential and phytochemical characterization using GC-MS analysis of bioactive compounds of \u003cem\u003eAchillea filipendulina\u003c/em\u003e (L.) leaves. \u003cem\u003eJ. Pharmacogn Phytochem\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e (3), 258\u0026ndash;265 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmer, M. H. et al. The potential of \u003cem\u003eBrachionus plicatilis\u003c/em\u003e extract against bacterial infection and cancer: In vitro and in silico studies. \u003cem\u003eEgypt. J. Chem.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e (9), 713\u0026ndash;725 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDar, M. A. \u0026amp; Kaushik, G. Optimizing the malathion degrading potential of a newly isolated Bacillus sp. AGM5 based on Taguchi design of experiment and elucidation of degradation pathway. \u003cem\u003eBiodegradation\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e (5), 419\u0026ndash;439 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaengsanga, T. \u0026amp; Phakratok, N. Biodegradation of chlorpyrifos by soil bacteria and their effects on growth of rice seedlings under pesticide-contaminated soil. \u003cem\u003ePlant. Soil. Environ.\u003c/em\u003e ;\u003cb\u003e69\u003c/b\u003e(5). (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, L., Zhao, Y. H., Zhang, B. X., Yang, C. H. \u0026amp; Zhang, X. Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacterium. \u003cem\u003eFEMS Microbiol. Lett.\u003c/em\u003e \u003cb\u003e251\u003c/b\u003e (1), 67\u0026ndash;73 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhanem, I., Orfi, M. \u0026amp; Shamma, M. Biodegradation of chlorpyrifos by \u003cem\u003eKlebsiella\u003c/em\u003e sp. isolated from an activated sludge sample of wastewater treatment plant in Damascus. \u003cem\u003eFolia Microbiol. (Praha)\u003c/em\u003e. \u003cb\u003e52\u003c/b\u003e, 423\u0026ndash;427 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnwar, S., Liaquat, F., Khan, Q. M., Khalid, Z. M. \u0026amp; Iqbal, S. Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by \u003cem\u003eBacillus pumilus\u003c/em\u003e strain C2A1. \u003cem\u003eJ. Hazard. Mater.\u003c/em\u003e \u003cb\u003e168\u003c/b\u003e (1), 400\u0026ndash;405 (2009).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAwad, N. S., Sabit, H. H., Abo-Aba, S. E. \u0026amp; Bayoumi, R. A. Isolation, characterization and fingerprinting of some chlorpyrifos-degrading bacterial strains isolated from Egyptian pesticides-polluted soils. \u003cem\u003eAfr. J. Microbiol. Res.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e (18), 2855\u0026ndash;2862 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJabeen, H., Iqbal, S. \u0026amp; Anwar, S. Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by a novel rhizobial strain \u003cem\u003eMesorhizobium\u003c/em\u003e sp. HN3. \u003cem\u003eWater Environ. J.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (1), 151\u0026ndash;160 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChishti, Z., Ahmad, Z., Zhang, X. \u0026amp; Jha, S. K. Optimization of biotic and abiotic factors liable for biodegradation of chlorpyrifos and their modeling using neural network approaches. \u003cem\u003eAppl. Soil. Ecol.\u003c/em\u003e \u003cb\u003e166\u003c/b\u003e, 103990 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav, M., Shukla, A. K., Srivastva, N., Upadhyay, S. N. \u0026amp; Dubey, S. K. Utilization of microbial community potential for removal of chlorpyrifos: a review. \u003cem\u003eCrit. Rev. Biotechnol.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e (4), 727\u0026ndash;742 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi, X., He, J. \u0026amp; Li, S. Isolation of a chlorpyrifos-degrading bacterium, \u003cem\u003eSphingomonas\u003c/em\u003e sp. strain Dsp-2, and cloning of the \u003cem\u003empd\u003c/em\u003e gene. \u003cem\u003eRes. Microbiol.\u003c/em\u003e \u003cb\u003e158\u003c/b\u003e (2), 143\u0026ndash;149 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu, G. et al. Mineralization of chlorpyrifos by co-culture of \u003cem\u003eSerratia\u003c/em\u003e and \u003cem\u003eTrichosporon\u003c/em\u003e spp. \u003cem\u003eBiotechnol. Lett.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 1469\u0026ndash;1473 (2007).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOmeiri, M., Khnayzer, R., Yusef, H., Tokajian, S. \u0026amp; Salloum, T. Biodegradation of chlorpyrifos by bacterial strains isolated from Lebanese soil and its association with plant growth improvement. \u003cem\u003eBioremediat. J.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (1), 14\u0026ndash;33 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFarhan, M. et al. Biodegradation of chlorpyrifos using isolates from contaminated agricultural soil, its kinetic studies. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (1), 10320 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaengsanga, T. \u0026amp; Phakratok, N. Biodegradation of chlorpyrifos by soil bacteria and their effects on growth of rice seedlings under pesticide-contaminated soil. \u003cem\u003ePlant Soil. \u0026amp; Environment\u003c/em\u003e, \u003cb\u003e69\u003c/b\u003e(5). (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBoruta, T., Foryś, M., Pawlikowska, W., Englart, G. \u0026amp; Bizukojć, M. Initial pH determines the morphological characteristics and secondary metabolite production in \u003cem\u003eAspergillus terreus\u003c/em\u003e and \u003cem\u003eStreptomyces rimosus\u003c/em\u003e cocultures. \u003cem\u003eArch. Microbiol.\u003c/em\u003e \u003cb\u003e206\u003c/b\u003e (12), 1\u0026ndash;12 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBundale, S., Begde, D., Nashikkar, N., Kadam, T. \u0026amp; Upadhyay, A. Optimization of culture conditions for production of bioactive metabolites by Streptomyces spp. isolated from soil. \u003cem\u003eAdv. Microbiol.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e (6), 441 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chlorpyrifos (CPF), pesticide degradation, Enterobacter sp. n1, GC analysis, pH effect","lastPublishedDoi":"10.21203/rs.3.rs-8130012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8130012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChlorpyrifos (CPF) is a common agricultural pesticide used worldwide. As an agricultural pesticide, CPF has been used extensively and has resulted in significant contamination. Because it might leak into neighboring ditches or drains, which normally carry water to lakes and streams, it may be dangerous. The current work used the enrichment technique to isolate bacteria with a high capacity for degradation from agricultural drainage water (El-Batts drain), Fayoum, Egypt. Isolated bacteria identified as \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1 under Genbank accession number \u003cb\u003ePV495863\u003c/b\u003e based on morphological, biochemical, and 16S rRNA gene sequencing technique. Mineral salt media (liquid and solidified) supplemented with CPF as sole carbon and nitrogen source used for the growth of a pure culture of and \u003cem\u003eEnterobacter\u003c/em\u003e sp. n1. Bacteria cell count and optical density were used to detect the growth. The effect of CPF concentrations (50, 75,100,125,150, and175ppm), pH values (5, 6, 7, 8, and 9), and temperatures (15, 20, 30, 35, 40℃) on the bacterial growth as well as CPF degradation rate by GC analysis were studied. Under the influence of different degrees of pH, a difference was observed in the number of metabolic products, as well as different proportions of some similar compounds in different media.\u003c/p\u003e","manuscriptTitle":"Effect of Culture Medium pH on Chlorpyrifos Biodegradation and Metabolic Profiles of a Novel Enterobacter strain","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 10:20:53","doi":"10.21203/rs.3.rs-8130012/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9587e2fe-9a13-48e7-bd99-d3b0bb674d5f","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58805219,"name":"Biological sciences/Biotechnology"},{"id":58805220,"name":"Earth and environmental sciences/Environmental sciences"},{"id":58805221,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-06T08:26:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 10:20:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8130012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8130012","identity":"rs-8130012","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}