Bioremediation of Imidacloprid by the Local Isolate Psychrobacter celer

Pesticide biodegradation can be accomplished by the technique of bioremediation, which makes use of microorganisms’ ability to degrade pesticide residues. This study aimed to separate and identify imidacloprid-biodegradable from botanical fields soil of greenhouses in the Plant Protection Directorate /Ministry of Agriculture in Baghdad, which has been using imidacloprid pesticides for many years. Using high-performance liquid chromatography, residual imidacloprid concentrations in MSM medium at a concentration of 25 mg/L after 21 days were measured to identify the best degrading bacterial isolates. Isolate No.37 the best bacterial isolate was able to degrade 63% of imidacloprid. was identified as Psychrobacter celer according to morphological, cultural and biochemical tests. Moreover, genetic analysis for the 16S rRNA gene and given a new accession number (OP672320.1) in the GenBank of NCBI, from this study that the soil previously contaminated for long periods of time with imidacloprid can be treated by degrading the imidacloprid residues in it by isolated bacteria Psychrobacter celer.


Introduction
Pesticides have become an essential part of agriculture in order to maintain higher agricultural production and feed millions of people, but the indiscriminate use of agrochemicals has caused serious environmental problems [1,2].Due to its high potential toxicity, persistence, and slow degradation, pesticide pollution is one of the world's most serious problems.A pesticide's attitude in the ecology is therefore reflected its stability, land physicochemical characteristics, microorganism variety, and rhizospheric activities.Pesticides' long-term effects on people include carcinogenicity, mutagenicity, reproductive toxicity, and other health issues [3].Therefore, Pesticide residues are regarded as a major risk factor in society which also affects used on the farmers there that spray it in greenhouse fields [4,5].Imidacloprid (1-[(6-chloro-3-pyridinyl)-methyl]-N-nitro-2-imidazolidinimine) is a systemic neonicotinoid insecticide with the chemical compound chloronicotinyl that's used to prevent biting and sucking insects [6].Neonicotinoids manufacture more than 30% of all pesticide sales worldwide, with Imidacloprid sales exceeding $ 1x10 9 in 2009 in addition China exported in 2017 31,595 tons of imidacloprid products.Due to its physical properties, also powerful insecticidal effects with minimal application rates.It spreads quickly and lasts a long period in a variety of environments.This 1259 (2023) 012034 IOP Publishing doi:10.1088/1755-1315/1259/1/012034 2 substance can survive in the soil for 48 to 190 days and in laboratory experiments, the longest half-life of it in land was observed to be 997 days [7].In review, according to reports, the half-life of the insecticide imidacloprid in land average (28-1250) days, consisting on a soil type [8].Some pesticide will inevitably come into touch with soil flora and wildlife even after being sprinkled on plants.Thus, leaching led to pollute each of surface beside groundwater, which leads to an accumulation in the food chain.Also, because crops may absorb imidacloprid, it can enter the food chain and be harmful to both humans and aquatic life, consequently, it sometimes serves as a source of contamination for the following crop in the soil [9].It was classified the greatest likelihood of leaching by the Environmental Protection Agency in category I [10].Several studies have found that natural mechanisms like hydrolysis, photodegradation, and biodegradation can remove imidacloprid from natural environments [11].Biodegradation is the process of using biological agents to remove contaminants from the environment.It is easily accepted in the environment due to its simple operation, wide applicability, low cost, and contaminant destruction by converting pesticides into non-toxic metabolites, microbial bioremediation would reduce the detrimental impacts of pesticides on soil microflora and fauna, improving land safety.Soil microorganisms have been shown to degrade imidacloprid in several studies [12].Furthermore, several research indicates that soil microbes can degrade imidacloprid [6,13] and several pure microorganism isolates, including Pseudomonas aeruginosa [14] and P. mosselii strain NG1 [15] are discovered to breakdown imidacloprid on their own .Hydrocarbons and other of pesticides groups could bioremediation via Enterobacter sp.[16,17], who showed the ability of Enterobacter sp. to break down imidacloprid, a potent insecticide like neonicotinoid group.The purpose of this research to discover the types of imidacloprid-quickly degrading microorganisms and to pinpoint the best bacterial isolate capable of destroying imidacloprid in microbial communities in soil samples from greenhouses.

Collecting Soil Samples
Land samples from Rhizosphere soils were gathered for microbe isolation from greenhouses, that was an area of 50 m 2 in the Plant Protection Directorate that had long been treated with Neonicotinoid insecticide to use in microbial isolation experiments.Soil samples were taken randomly from the top 15 to 25 centimeters, bagged in sterile containers, and sent in refrigerated containers to the lab.After the first stages of each experiment were set up, backup samples were placed in the fridge.That's according to the research [18].

Chemicals
The Plant Protection Directorate of the National Center for Pesticide Control provided imidacloprid (purity >97%).All solvents were bought from Merck KGaA in Germany (99.9% purity) and were of HPLC grade.England company Romil developed HPLC-grade water.A nylon filter (0.45) µm was used when applying HPLC-grade H 2 O and C 2 H 3 N.Using a criterion solution, analytical criterion for HPLC, standardization in the 1-50 mg/L range were produced.

Microbes Growth Culture
Following the manufacturer's recommendations, distilled water was used to make both the NA (Nutrient Agar) and MSM (Mineral Salt Medium) media, which were then both autoclave sterilized at 121 C for 15 minutes.Imidacloprid (25 ppm) was added to the media after cooling using a syringe filter and poured onto Petri dishes.Isolation and biodegradation tests were conducted in mineral salt medium (MSM) consist of (g/L) amomian sulfate 4 2.0; Potassium hydrogen phosphate ,0.625; Sodium dihydrogen phosphate 0.6; magnesium sulfate heptahydrate 0.2 and Calciumchloridehexahydrate Antarcticite 0.15 (pH 7.0), as described by [19].
IOP Publishing doi:10.1088/1755-1315/1259/1/0120343 2.4.Instruments HPLC (model LC-2010 A HT Shimadzu Japan) with a photodiode array detector (DAD) was utilized to analyze imidacloprid, and ChemStation software was utilized for processing and gathering of data.Each sample was put through a reversed-phase Orbit C18 column for separation (250 × 4.6 mm).In a binary mode at a flow rate of 1 ml/min.and an oven temperature(40°C), the mobile phase was 60 % water and 40 % acetonitrile, and the detector was set to a wavelength of 270 nm.When analyzing a sample, the calibration curve was created using external standards, and quantification was accomplished using linear regression analysis.The retention time was used to identify imidacloprid.Using a spectrophotometer set at 550 nm, the optical density (OD) of the micobes was calculated [6].

Isolation of Pesticide Degrading Bacteria
By using a flask (10 2 ml) consist of 20 ml (MSM broth) and 2.0 g of soil.After 2 hours on a rotary shaker (220 rpm), 5 ml of the mixture was transferred to a flask (500 ml) containing 200 ml of MSM broth and 25 ppm imidacloprid (pH 7).After being incubated for seven days at 28°C and 120 rpm, The purification of morphologically diverse colonies included repeatedly streaking them on pesticidetreated media.Thereafter, the pure cultures were kept in nutrient agar slants at 28°C for one to three days [20].

Biodegradation of Pesticides in Minimal Salt Medium
Falcon tubes Sterilized (50 ml) were used for all biodegradation experiments.30 ml of MSM was supplemented with pesticide (25ppm) which was considered as a source of a C&N, and vaccinated with 1 ml/one day from old microbe's culture.Incubated falcon tubes for three weeks in the dark at 28±2⁰C at 120 rpm.From the broth (1ml) was had from the flasks of ( 0, 7, 15, 21) days for extraction Uninoculated flasks, boosted with pesticide (without any bacteria) acted as the control (without any bacteria) to negate the potential for environmental impacts such as photodegradation.Quantification of pesticide was done by HPLC.and optical density (OD) at 550 nm was recorded with a spectrophotometer (Cary 50 Bio) 1.5 ml of each subsample was combined with 1.5 ml of acetonitrile in Eppendorf tubes, and the resulting mixture was centrifuged at 12,000 rpm for five minutes to separate and measure the remainder of the pesticide.Pasteur pipettes were used to transfer the supernatant to amber HPLC vials, which were then refrigerated.Each sample was injected into the HPLC in 50 microliters [6].

Statistical Analysis
One-way analysis of variance (ANOVA) was carried out.on HPLC data showing variations in imidacloprid concentration among treatments.Differences in means were evaluated using the LSD test (p 0.05) in SPSS 19.Controls were not included in the data analysis and were simply used to monitor the development of the isolates.

Molecular Characterization
Bacterial isolate which was the most efficient and responsible for degrading imidacloprid that had been discovered by 16S ribosomal DNA.Following the amplification of bacterial ribosomal RNA, sequence analysis and validation of homogenic data were performed using the rRNA database at the National Center for Biotechnology Information (NCBI).Via used these procedures while extracting DNA.

Primer's Sequence
Primers that were utilized are listed in Table (1).[21].The bacterial culture has grown in 5 ml LB broth and, then oneml from it was poured toEppendorf tubes.These tubes were centrifuged for (3 min/8000 rpm) the particle was left alone while the supernatant was discarded.Genomic DNA extracted according to ABC DNA Isolation Kit.The amount and integrity of DNA were determined using the spectrophotometer (Nanodrop) 2.9.2.Quantitation of DNA A Quantus Fluorometer was used to measure the concentration of DNA that had been extracted to confirm the samples' quality for further applications.Quantifluor Dye was diluted to a volume of 200 µl for each l µl of DNA.DNA concentrations were measured after a 5-minute incubation at room temperature.

Primer Preparation
Supplied primers in a lyophilized Quantus Fluorometer from Promega company.After that, dissolve in water devoid of nuclease to form a stock solution with a final concentration of 100 pmol/l.To create a workable primer solution with a 10 pmol/l concentration, 90 l of nuclease-free water were combined with 10 l of primer stock solution, which was kept at 20 C in the freezer.

PCR Reaction Mix Preparation and PCR Thermocycling Conditions
Aseptically prepared the PCR reaction mix by using Taq Ready master mix Kit according to the manufacturer's instructions (Promega, USA).Polymerase chain reaction assays were performed at a reaction volume of 50 μl (table 2), and according to the PCR program Table (3).Table 2. Master mix kit according to the manufacturer's instructions.

Gel Electrophoresis and Sample Preparation
DNA and PCR products were detected using gel electrophoresis, which was observed using ethidium bromide and a UV transilluminator documentation system.Mixed 7 μL of DNA extract sample with 3 μL of loading dye solution and put into the wells before electrophoresis.Whereas for PCR, each well was loaded with 10μL of PCR products on the gels.DNA ladders (100bp) were always performed concurrently with each electrophoretic run to detect PCR product size.UV Transilluminator documentation system was used to visualize DNA bands [22].

HPLC Calibration
Under experimental conditions, imidacloprid retention times were 4.8 minutes, Figure (1).To establish the sensitivity of the HPLC, working standard solutions of imidacloprid were prepared at various dilutions (1, 5, 25, and 50 ppm) and used to calibrate the instrument before any sample analyses were injected.For imidacloprid, the correlation between the quantity of standard solution injected and the resultant peak area was 0.999, suggesting a linear connection.

Isolation and Screening of Pesticide Degrading Bacteria
Vary bacterial strains were separated to test imidacloprid biodegradation for 21 days.A one-way ANOVA was employed to examine variations between Thirty-seven isolates taken from MSM medium.One-way assay of variance (ANOVA) utilized to examine if differences between groups had statistically significant.It was found that the daily concentration means (7, 15, and 21) days were all significantly variations at the confidence level of 95%.Where significant between groups on day 7 (df=4, F=28.672, p≤ 0.000).also significant between groups on day 15 (df=4, F= 56.866, p≤ 0.000) and significant between groups on day 21 (df=4, F=49.837, p≤ 0.000) (Table 4).In combination with ANOVA, a post hoc test was employed to identify the subset of pairs that showed statistically significant differences.According to the results of LSD on days 15 and 21, the isolates were divided into 8 categories; on day 21, they were separated into 5 groups.At 2 weeks, six distinct microbes separated (Nos.45, 9, 32, 8, 17, 37) had through the primary 3 groups, and on day 21, four different isolates (Nos.9, 15, 45, and 37) were among the first three groups.The LSD test was used to select the final biodegradable isolates for3 weeks because of to little treatment concentration.Tasted isolates demonstrated the ability to degrade imidacloprid at 25 mg/l in MSM medium.Imidacloprid biodegradation in a spiking medium showed wide variation with four bacterial strains Figure (2).Imidacloprid biodegradation by four isolates was between 35.1% and 52.4% on day 7 of incubation, although it increased afterward to 41.6% and 53.8% of the spiking 3 weeks to 43.4% and 63% by incubation.
Four bacterial isolates (No.4,15,37,45) showed continuous growth over the course of the incubation period, which was clearly correlated with the ability for degradation.Figure (3) shows the average growth rates of the four biodegradable isolates throughout 21 days of incubation.Isolate No.37 grew at a faster and higher growth recorded by (O.D. at 600 nm) after incubation pace than the other isolates and reached this maximum size by day 7 Compared to other isolates that continued to grow at a lower rate on the 7 days and the following days.Moreover, it reached the maximum level of biodegradation of imidacloprid (63%), compared to other isolates, Figure (2).Table 4.A one-way ANOVA test of the Significance for Days (1,7,15,21).

Morphological and Biochemical Characterization of Isolate No.37
The best-isolated bacterium for imidacloprid breakdown was found to be No.37 after being analyzed using HPLC.Gram staining and culture characteristics were used to determine the isolate's identity, which was then confirmed using traditional techniques based on growth.

Molecular Characterization of Pesticide-Degrading Bacteria No.37
16S rRNA gene sequence analysis allowed for the molecular identification of the chosen isolates.Using ntBlast, similar sequences of other microorganisms were retrieved from GenBank and compared to the 16S rRNA sequence of the isolates.The PCR results showed that the replicated isolate had a 1500 bp band ,Figure (5).The 16S ribosomal RNA gene was sequenced using this band [24].The newly obtained 16S rRNA fragment with known bacterial sequences in the GenBank database using BLASTN analysis showed sequence similarity to nitrogenous bases with a percentage of 94.36% of Psychrobacter celer, and this sequence was published in GenBank (NCBI) under the accession numbers (OP672320.1).

Conclusion
The results of this research demonstrated that imidacloprid might be degraded by certain bacterial species.The chosen soil bacterial isolates were able to degrade between 45 and 63 % of imidacloprid by 21 days.However, the bacteria (Psychrobacter celer) introduced in this study was the first report in the world of the degradation of imidacloprid by Psychrobacter celer in MSM media from greenhouse soil samples.These bacterial conversions of imidacloprid open up new avenues for its chemical degradation in soil.More research is needed to determine how exactly these isolates degrade imidacloprid.More experiments are required.

Figure 3 .
Figure 3. Growth of different biodegradation isolates during 21 days.

Figure 4 .
After 24 hours of incubation at 28°C on a selective medium (MacConkey agar, mannitol agar plates), and general medium (Nutrient agar).The cells of bacterial strain Psychrobacter celer were non-endospore-forming, Gram-negative, motile, and light pink color in MacConkey agar as shown in Figure (4 A).Colonies formed by the strain of Psychrobacter celer on nutrient agar are coccobacilli Figure (4B, C) [23].Fig. 4. Isolate No.37 on MacConkey and Nutrient agar Medium.Isolate No.37 on MacConkey and Nutrient agar Medium.

Figure 6 .
Figure 6.Result of alignments of sample local isolate no.37 psychrobacter celer by phylogenetic structuring tree of NCBI.

Table 1 .
Primers utilized in this Study.
PCR products were delivered to Macrogen Corporation -Korea, which used an automated DNA sequencer called the ABI3730XL, for Sanger sequencing.Geneious software was used to gather and evaluate the data.