Introduction

Chlorpyrifos is an organophosphorus pesticide used as a broad-spectrum insecticide in various agricultural and non-agricultural conditions¹. Chlorpyrifos is a persistent pesticide that contaminates soil and water, harms non-target organisms, disrupts soil biodiversity, and bioaccumulates through the food chain. Its residues can damage soil health and lead to pest resistance, posing long-term environmental risks. Extensive use of chlorpyrifos adversely affects the environment and human health, inhibiting the activity of acetylcholinesterase enzyme in nervous system, leading to the accumulation of acetylcholine, overstimulation of nerve cells, paralysis and death². Now a days, researchers have been exploring eco-friendly solutions to mitigate the adverse effects of pesticides. Plant growth-promoting rhizobacteria along with plant growth promotion have the ability to degrade a wide range of xenobiotic compounds³. Interaction of PGPR and plants enhances the degradation of chlorpyrifos in soil. The production of organophosphorus hydrolase enzyme by PGPR converts chlorpyrifos into its metabolites, 3, 5, 6-trichloro-2-pyridinol (TCP) and diethylthiophosphate (DETP)⁴. These metabolites are further metabolized by other enzymes, such as monooxygenases, dehydrogenases and dehalogenases into non-toxic compounds that can be utilized by other microorganisms present in the soil⁵.

Bacillus tropicus and related PGPR strains play a dual role in remediating chlorpyrifos-contaminated soils and supporting plant growth. Their enzymatic breakdown of chlorpyrifos, combined with nutrient cycling, stress tolerance, and growth promotion, makes them valuable allies for sustainable agriculture and soil health restoration. Exopolysaccharides produced by PGPR enhance plant tolerance towards pesticides by acting as chelating agents, sequestering and detoxifying harmful chemicals⁶. Additionally, surfactants secreted by PGPR promote the degradation of pesticides and reduce their toxic effects on plants. Furthermore, biofilm synthesis by PGPR offer a protective shield against pesticide-induced stress, preventing their entry into plant tissues. These findings highlight the potential of PGPR synthesizing exopolysaccharide, surfactant and biofilm as promising biocontrol agents for sustainable agriculture in pesticide-contaminated environments⁷. Maize inoculation with PGPR resulted in plant growth promotion with a decreased chlorpyrifos residue in plant tissues⁸. In this study PGPR, DK5 was examined for synthesizing exopolysaccharide, surfactant, biofilm and ability to degrade chlorpyrifos in spiked soil.

Materials and Methods

The bacterial isolate DK5 identified as Bacillus tropicus, was isolated from pesticide contaminated soil by enrichment method. Commercial-grade chlorpyrifos was obtained from a local market in Kurukshetra. The reference material was procured from Sigma-Aldrich, India. HPLC grade solvents and water were purchased from HiMedia.

Biofilm production

The overnight grown DK5 bacteria isolate was transferred to a 96-well plate containing different concentrations of pesticides (P1 (20 µg/ml), P2 (40 µg/ml) and P3 (100 µg/ml))and incubated for 24 hr at 30 ℃. After incubation, the wells were washed with sterile phosphate-buffered saline to remove the unattached bacteria. Bacterial isolates adhering to the wall were stained with 0.1% crystal violet solution and incubated for 10 minutes. After incubation 200 µL of 95% ethanol was added to each well to dissolve the crystal violet bound to the biofilm. The biofilm synthesis was estimated by calculating the optical density of crystal violet-stained biofilm at 490 nm by using a microplate reader⁹.

Exopolysaccharide synthesis

The overnight grown bacterial isolate DK5 in presence of different concentrations of pesticides (P1 (20 µg/ml), P2 (40 µg/ml) and P3 (100 µg/ml)) was centrifuged at 8000 rpm. The cell-free supernatant of DK5 was mixed with equal volume of ice-cold ethanol and incubated for 24 hr at 4 °C. The mixture was centrifuged for 15 minutes, followed by the addition of an equal volume of 5 % phenol and concentrated sulfuric acid. The solution was kept at room temperature for 30 minutes.  The mixture was centrifuged and pellet was washed with ethanol before drying at 60 °C. Pellet was suspended in ethanol and absorbance of mixture was taken at 490 nm by using a spectrophotometer¹⁰.

Surfactant analysis

The overnight grown bacterial isolate DK5 in presence of different concentrations of pesticides (P1 (20 µg/ml), P2 (40 µg/ml) and P3 (100 µg/ml))  was treated with dichloromethane for extraction of surfactant. The extracted surfactant was dried with anhydrous sodium sulphate and concentrated using a rotary evaporator. The extract was dissolved in small amount of methanol and spotted on TLC plate coated with silica gel¹¹. The absorbance of dissolved extract was taken at 540 nm by using a spectrophotometer.

Ex-situ chlorpyrifos degradation

The 120g soil was airdried and autoclaved; soil sample was then spiked with chlorpyrifos (35 ppm g^-1) and inoculated with bacterial isolate DK5 (10⁸ CFU g^-1). The soil samples were incubated at 30 ℃ with 45 % water-holding capacity in dark conditions. Then 20 g soil samples were drawn at an interval of 0, 10, 20 and 30 days. The soil samples were treated with 0.25 ml of 25 % ammonia solution, 0.5 g activated charcoal and 0.5 g florisil and packed in the column. The solvent (1:1 ethyl acetate: acetonitrile) was passed through the column.  The eluted solvent was dried and reconstituted in 20 µl of solvent for HPLC analysis. HPLC for detection of chlorpyrifos a mobile phase of Acetonitrile–ultrapure water(50:50 v/v) at a flow rate of 1.2 mL/min, 20 μL of the sample was injected at 30°C and detected at 254nm. The pesticide degradation was estimated by considering the area under the peak specified for chlorpyrifos. Removal efficiency of pesticides by was calculated by using formula. Where, (R %, removal percentage), (A_0, peak area of under pesticide residue) and (A, peak area of 100 µg/ml concentration of pesticide)¹².

Results and Discussion

Biofilm, exopolysaccharide and biosurfactant production by bacterial isolate DK5

The biofilm, exopolysaccharide and biosurfactant production by DK5 is shown in figure 1. Biofilm formation is a complex and adaptive mechanism used by many bacteria to protect themselves in challenging environments¹³. Biofilm synthesis by bacterial isolate DK5 exponentially increase with rise in the concentration of chlorpyrifos. The maximum synthesis of biofilm by DK5 was found at 100 µg/ml concentration of chlorpyrifos. This suggests the resilience and adaptation to of DK5 towards environmental stress caused by the pesticide. The biofilm produced by PGPR helps to colonize plant roots, which lead to improved nutrient uptake, increased plant growth, and enhanced resistance to diseases and pests¹⁴.

The maximum synthesis of EPS by DK5 was 213.47 µg/ml at 100 µg/ml concentration of chlorpyrifos. EPS provides structural integrity to bacterial communities, serves as a protective barrier against adverse environmental conditions. The increased EPS production was found in the presence of pollutants¹⁵. PGPR can promote plant growth, enhance plant tolerance to biotic and abiotic stresses and improve soil structure with water-holding capacity¹⁶.

Qualitative analysis of biosurfactant was analysed by spot inoculating all the samples on TLC labelled as c, p1, p2 and p3. The surfactant synthesis by DK5 was slightly decline with the increase concentration of chlorpyrifos. The biosurfactants produced by Pseudomonas aeruginosa increased the solubility of chlorpyrifos in water and facilitated its breakdown, leading to a reduction in its persistence in the environment¹⁷. Biosurfactants also improves the plant growth and reduces the toxicity of pesticides towards plants by forming micelles¹⁸.

In-situ chlorpyrifos degradation

In laboratory experiment, DK5 was showing higher rate of chlorphrifos degradation in soil. In HPLC analysis, after 30 days of incubation 96.1% chlorpyrifos was degraded by DK5 as shown in figure 2. In control only 16.33 % chlorpyrifos degradation was observed. In previous study a Bacillus strain was able to remove 68.14% of 30 mg L-1 chlorpyrifos in 96 h²³. These results suggest that the inoculation of PGPR, DK5 into soil enhance the degradation of chlorpyrifos. Inoculation of PGPR reduce the harmful effects of pesticides on the environment¹⁹. Bacillus species helps in the degradation of various organic pollutants, including chlorpyrifos²⁰.

The unintentional adverse effects of chlorpyrifos breakdown by Bacillus tropicus include 3,5,6-Trichloro-2-pyridinol (TCP), an environmentally stable and moderately toxic chemical that has a negative impact on terrestrial and marine ecosystems. Other metabolites that appear are Diethyl Thiophosphate (DETP) and Diethyl Phosphate (DEP); though less lethal, they may bioaccumulate if not completely eliminated²¹. The inoculation of PGPR into soil significantly enhanced the degradation of chlorpyrifos and alter the soil microbial community composition²².

The ability of DK5 to synthesize biofilm, EPS, and biosurfactants in the presence of chlorpyrifos indicates its potential to enhance plant growth, improve soil health, and reduce pesticide toxicity. These findings highlight the promising role of DK5 as a potential biocontrol agent for mitigating the adverse impacts of chlorpyrifos in agriculture. Further field studies are necessary to explore the practical application of DK5 as a PGPR and its long-term effects on soil quality, crop productivity and environmental sustainability.

Figure 1: a) Biofilm, b) EPS and c) biosurfactant production by DK5 at P1 (20 µg/ml), P2 (40 µg/ml) and P3 (100 µg/ml) concentration of chlorpyrifos. Click here to view Figure

Figure 2: Chlorpyrifos degradation in soil by DK5. Click here to view Figure

Conclusion

In conclusion, this study demonstrates the potential of the Bacillus tropicus strain DK5 as a promising biocontrol agent for mitigating the environmental and agricultural impacts of chlorpyrifos. Biosurfactants and exopolysaccharides synthesized by DK5 help in the growth of plants and concurrently aid in the degradation of chlorpyrifos in the soil. The result demonstrates that 96.1% of chlorpyrifos could be degraded after incubation for 30 days. Therefore, it has been proved that DK5 reduces pesticide residues and enhances the health of soils. Results show a crucial role for DK5 in sustainable agriculture. Soil improvements of microbial communities together with reduced pesticide toxicity will promote the use of such a microbial synergist. However, more field studies must be performed under real agricultural conditions to validate its long-term efficacy as well as potential adverse environmental impacts.

Acknowledgement  

The first author acknowledges Kurukshetra University for providing assistantship to carry out research work. We acknowledge the University Institute of Engineering and Technology for providing lab facilities with high end instruments.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Author Contributions

Deepak Kumar Malik: Conceptualization, Methodology, Writing – Original Draft.

Vivek Singh: Data Collection, Analysis, Writing – Review & Editing.

Rajesh Agnihotri: Visualization

Meenu Rathi: Resources and Supervision

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