Degradation bacterium system for repairing pesticide-polluted soil and application of degradation bacterium system

Abstract

The invention discloses a degrading bacterium system for repairing pesticide-polluted soil and application of the degrading bacterium system. The degrading bacterial system is composed of Pseudomonas plecoglossa JY-5, Klebsiella plecoglossa J2-1, Arthrobacter silvisoli JY-2 and Enterobacter ludwigii JY-1, and the degrading bacterial system is obtained by carrying out enrichment culture, screening, sequencing analysis and bacterial strain identification and purification on the soil polluted by the pesticide, and the degrading bacterial system is used for degrading the soil polluted by the pesticide, and the degrading bacterial system is used for degrading the soil polluted by the pesticide and the soil polluted by the pesticide and the soil polluted by the pesticide and the soil polluted by the pesticide and the soil polluted by the pesticide and the soil polluted by the pesticide and the soil polluted by the pesticide. According to the degrading bacterium system, the biochar is used as a carrier to prepare an immobilized bacterium agent, various common pesticides such as carbendazim and the like can be efficiently degraded, the strains have a synergistic effect, the stability in a complex soil environment is high, the anti-interference capability is outstanding, soil pesticide residues can be effectively reduced, an efficient and feasible technical scheme is provided for soil remediation in a pesticide high-pollution area, and the degrading bacterium system has wide application prospects. The method has important practical application value.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and environmental remediation microorganisms, specifically to a degrading bacterial system for remediating pesticide-contaminated soil and its application. Background Technology

[0002] During pesticide use, 80-90% enters the soil, increasing pesticide residue levels in the soil and on crops such as vegetables. Ultimately, these residues enter the human body through the food chain, impacting human health. Pesticides have always played a crucial role in agricultural production, significantly contributing to pest and disease control and crop yield increases, especially in tropical regions. Therefore, reducing pesticide residues in the soil-vegetable system to ensure the safety and quality of agricultural products and protect public health is a critical bottleneck in current agricultural safety production.

[0003] Physical and chemical methods for pesticide residue degradation are limited due to their complex and expensive facilities and high environmental safety requirements. Biological methods based on the decomposition and metabolic activity of microorganisms are the most promising and effective strategy. The essence of microbial pesticide degradation is an enzymatic reaction, where compounds enter microorganisms in some way and then undergo a series of physiological and biochemical reactions catalyzed by various enzymes, resulting in complete degradation or degradation into smaller, non-toxic or low-toxic compounds.

[0004] The most common method is to screen for strains with degradation capabilities from contaminated soil or sludge. Induced bacterial domestication has also gained attention in recent years. This involves artificially applying pesticides to sludge or soil, followed by a second application after a period, and measuring whether degradation occurs within a short time. This process of domesticating the bacterial community can yield microorganisms that effectively degrade the target pollutants. Although most microorganisms with high degradation efficiency under laboratory conditions can also exhibit degradation capabilities in the environment, their degradation effect in complex polluted field environments is not significant. This is because degrading bacteria are affected by existing microorganisms in the soil, and may even experience antagonistic effects, hindering their normal growth. Exogenous degrading bacteria may also be affected by the toxic effects of other pesticides in the environment, reducing their degradation ability. Experiments have shown that composite bacterial systems constructed using synergistic effects between bacterial species can not only efficiently decompose organic pollutants but also exist stably in the environment and are not easily contaminated by other microorganisms.

[0005] Microbial remediation technology has become a key direction for environmental governance due to its advantages such as environmental friendliness, low cost, and no secondary pollution. However, its core component—free-state functional microbial agents—faces bottlenecks in practical applications, including difficulty in survival, poor colonization, and easy loss, which severely restricts its industrialization and promotion. Specific defects of free-state microbial agents include: first, poor stress resistance, easily inactivated by extreme pH, heavy metals, etc.; second, low colonization efficiency, easily rejected by native microorganisms; and third, high risk of loss, making it difficult to maintain long-term remediation effects. These defects lead to unstable remediation effects and high costs, which are the core obstacles to engineering applications.

[0006] Single remediation technologies are insufficient for comprehensive remediation. Biochar itself has the ability to adsorb organic pollutants and heavy metals, while immobilized functional bacteria can remove pollutants through degradation and transformation. The combination of the two can form an "adsorption-degradation" synergy: biochar first enriches pollutants in the soil, providing a high concentration of substrate for immobilized bacteria and enhancing degradation efficiency; the metabolic activities of microorganisms can also reduce the toxicity of pollutants and alleviate their inhibition of biochar adsorption performance. This synergistic effect provides a new approach for the efficient remediation of complex contaminated soils and promotes research on biochar-immobilized bacteria composite systems. Summary of the Invention

[0007] Technical problem to be solved: To solve the above problems, the purpose of this invention is to isolate bacteria that can degrade multiple pesticides and form a composite bacterial system. By utilizing the synergistic effect between strains, it can simultaneously degrade multiple commonly used pesticides such as carbendazim, imidacloprid, indoxacarb, and high-efficiency cypermethrin, thus solving the problem that single bacterial agents can only degrade single pesticides and have poor remediation effects on complex pollution.

[0008] Technical solution: This invention provides a degrading microbial system for remediating pesticide-contaminated soil, the degrading microbial system comprising... Pseudomonas plecoglossicda JY-5 Klebsiella pneumoniae J2-1 Arthrobacter silvisoli JY-2 and Enterobacter ludwigii JY-1.

[0009] Furthermore, the aforementioned Pseudomonas plecoglossicda JY-5 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67049; Klebsiella pneumoniae J2-1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67050; Arthrobacter silvisoli JY-2 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67051; Enterobacter ludwigii JY-1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67052.

[0010] A method for constructing a degrading microbial system for remediating pesticide-contaminated soil, comprising the following steps: S1. Soil samples were inoculated into a culture medium containing mixed pesticides. After enrichment culture, the suspensions were inoculated into culture media containing pesticides for initial screening. Then, the concentration was increased for secondary screening. Continuous subculturing was carried out to obtain microbial communities with the potential to degrade pesticides. S2. High-throughput sequencing technology was used to analyze the composition and changes in microbial community during the degradation of each pesticide in single and mixed systems; S3. Strains were isolated and purified, total DNA was extracted, PCR amplification was performed, gene sequences were analyzed, homology comparisons of the strain's gene sequences were conducted, and a phylogenetic tree was constructed using biological software to ultimately obtain... Pseudomonas plecoglossicda JY-5 Klebsiella pneumoniae J2-1 Arthrobacter silvisoli JY-2 Enterobacter ludwigii JY-1.

[0011] Furthermore, in step S1, the soil sample is from the Nanfan area of ​​Hainan Province; the content of the mixed pesticide in the culture medium containing the mixed pesticide is 50-200 mg / mL; the mixed pesticide is carbendazim, imidacloprid, indoxacarb, high-efficiency cypermethrin, spirodiclofen, and bifenthrin; the pesticide content in the culture medium containing the pesticide is 50-200 mg / mL; and the concentration is increased to 100-200 mg / mL.

[0012] The above-described application of the degrading bacterial system in immobilized bacterial agents includes the degrading bacterial system and the biochar carrier.

[0013] The application of the immobilized bacterial agent described above follows these steps: Step 1: Inoculate the activated single colony into liquid culture medium, culture on a shaker, then centrifuge and discard the supernatant, add sterile water, shake thoroughly, and adjust the OD600 value of the final bacterial suspension to 1.0 for later use as seed culture; Step 2: Add biochar to MSM liquid culture medium, autoclave, cool to room temperature, and then inoculate with a mixed seed culture with an OD600 of 0.6 to obtain a mixed culture medium; Step 3: After placing the mixed culture medium on a shaker for incubation, remove it and filter it to obtain the filter residue. Then wash the filter residue with physiological saline, place the filter residue on aluminum foil to dry, and refrigerate and dry to obtain the immobilized bacterial agent.

[0014] Furthermore, the activated single colony in step one is... Pseudomonas plecoglossicda JY-5 Klebsiella pneumoniae J2-1 Arthrobacter silvisoli JY-2 Enterobacter ludwigii JY-1 colonies; the temperature for shaking culture is 25-30℃, the shaking speed is 150-200 rpm, and the shaking time is 24-48h; the centrifugation speed is 3000-4000 rpm, and the time is 15-20min.

[0015] Furthermore, in step two, the biochar is coconut shell biochar; the ratio (w:v:v) of biochar, liquid culture medium and seed liquid is (1-2):(8-10):(1-2); the autoclaving temperature is 121℃ and the time is 15-20min.

[0016] Furthermore, in step three, the temperature of the shaker culture is 25-30℃, the rotation speed of the shaker culture is 150-200 rpm, and the shaker culture time is 24-48h; the concentration of physiological saline is 0.8-0.9wt.%. Beneficial effects

[0017] This invention obtains a degradation strain from local pesticide-contaminated soil through natural screening and domestication. When applied, it has strong compatibility with the local soil micro-ecological environment, effectively avoids the antagonistic effect of native microorganisms, and can quickly colonize and form a dominant bacterial community, solving the problem of exogenous degradation bacteria being easily inactivated and difficult to survive in complex environments.

[0018] The four strains work synergistically to simultaneously degrade six commonly used pesticides, including carbendazim, imidacloprid, indoxacarb, cypermethrin, spirodiclofen, and bifenthrin. This solves the problem of single strains having a narrow degradation spectrum and being unable to cope with complex pollution. The degradation effect is more durable and has a better effect on soil remediation.

[0019] The immobilized microbial agent of this invention uses biochar derived from agricultural waste such as corn stalk charcoal and rice husk charcoal as a carrier. The raw materials are readily available and inexpensive, and the resource utilization of agricultural waste is realized. The carrier itself has the characteristics of well-developed pore structure and strong adsorption performance, which can not only immobilize the microbial strains, but also adsorb pollutants such as heavy metals and pesticide residues in the soil. At the same time, it improves soil permeability and water retention, enhances soil fertility, and achieves the dual effects of soil remediation and improvement. Attached Figure Description

[0020] Figure 1 The microbial community composition diagrams for different treatment groups are as follows: pesticide-free control (CK1); DMSO solvent control (CK2); acetone solvent control (CK3); imidacloprid (BCL); carbendazim (DJL); bifenthrin (LBJZ); lambda-cyhalothrin (LQJZ); spirodiclofen (LMZ); indoxacarb (YCW); and mixed pesticide (MSM-6). Figure 2 Here are colony diagrams of four degrading strains, where A represents... Klebsiella pneumoniae J2-1, B is Enterobacter ludwigii JY-1, C is Arthrobacter silvisoli JY-2, D is Pseudomonas plecoglossicda JY-5; Figure 3 Phylogenetic tree of 4 degrading bacteria: where A is Klebsiella pneumoniae J2-1, B is Enterobacter ludwigii JY-1, C is Arthrobacter silvisoli JY-2, D is Pseudomonas plecoglossicda JY-5; Figure 4 The graph shows the degradation rate of pesticides by degrading bacteria in different systems, where A is a single pesticide system and B is a mixed pesticide system. Figure 5 The graph shows the growth of seedlings under different treatment conditions. In the graph, A is the blank culture medium treatment without degrading bacteria, i.e., the control group, and B is the seed liquid treatment with degrading bacteria, i.e., the experimental group. Figure 6 Scanning electron microscope images of different biochars, where A is corn stalk charcoal, B is rice husk charcoal, C is coconut shell charcoal, D is bamboo charcoal, and E is wood charcoal. Figure 7 Figure showing the colonization rate of degrading bacterial strains on different biochar carriers; Figure 8 Infrared spectra of biochar and its immobilized bacterial agents; Figure 9 The image shows the removal effects of different treatments on six pesticides in the soil. A represents the pesticide imidacloprid, B represents the pesticide carbendazim, C represents the pesticide bifenthrin, D represents the pesticide spirodiclofen, E represents the pesticide deltamethrin, and F represents the pesticide indoxacarb. Figure 10 The diagram shows the enzyme activity in soil under different treatments, where A represents polyphenol oxidase, B represents urease, and C represents dehydrogenase. Figure 11 Figure 1 shows the plant growth in pesticide-contaminated soil under different treatments. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Example 1

[0022] Enrichment of degrading microbial communities: 10g of soil was weighed and inoculated into a 100mL MSM-6 medium (MSM-6) containing 50mg / mL of carbendazim, imidacloprid, indoxacarb, lambda-cyhalothrin, spirodiclofen, and bifenthrin in a ratio of 1:1:1:1:1:1. The mixture was incubated at 30℃ and 180 rpm for 7 days, with sterile water as a control. After enrichment culture, the suspension was inoculated into MSM-6 medium containing 100mg / mL of pesticide for initial screening, followed by secondary screening at a concentration of 200mg / mL. The suspension was then continuously passaged in glucose + MSM-6 solid medium to obtain a stable microbial community with pesticide degradation potential. Example 2

[0023] Community composition analysis of degrading bacteria: High-throughput sequencing was used to analyze the composition and changes in microbial community during the degradation of each pesticide in single and mixed systems. Bacterial suspensions were inoculated into MSM (pesticide-free control) and MSM-6 (mixed pesticide treatment group) pesticide solutions (50 mg / L) and cultured at 37°C and 180 rpm for 9 days. Genomic DNA was then extracted using the EZNA™ Mag-BindSoil DNA Kit. PCR amplification was performed using universal primers. The pesticide-free control (CK1); DMSO solvent control (CK2); acetone solvent control (CK3); imidacloprid (BCL); carbendazim (DJL); bifenthrin (LBJZ); lambda-cyhalothrin (LQJZ); spirodiclofen (LMZ); indoxacarb (YCW); and the mixed pesticide (MSM-6) were included.

[0024] The amplification system is as follows: 2×Hieff® Robust PCR Master Mix (15μL), Bar-PCR primer F (1μL), Primer R (1μL), PCR products (10~20ng), H2O (9~12μL), Total volume (30μL).

[0025] The PCR program is as follows: First, pre-denaturation is performed at 94℃ for 3 min; then, 5 cycles of amplification are performed, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 45℃ for 20 s, and extension at 65℃ for 30 s; then, 20 cycles of amplification are performed, each cycle consisting of denaturation at 94℃ for 20 s, annealing at 55℃ for 20 s, and extension at 72℃ for 30 s; after completing the cycles, a final extension is performed at 72℃ for 5 min, and finally, the reaction system is maintained at 10℃ until the program ends.

[0026] The second round of amplification was then performed (using Illumina bridge PCR compatible primers). The procedure was as follows: In a 200 μL sterile PCR tube, add 2×Hieff® Robust PCR Master Mix (15 μL), Primer F (1 μL), Index-PCR Primer R (1 μL), PCR products (10~20 ng), H2O (9~12 μL), and a total volume (30 μL) according to the system ratio. Then, gently pipette or vortex to mix the reaction solution, briefly centrifuge to collect the liquid at the bottom of the tube; next, place the PCR tube in a PCR instrument, pre-denature at 95℃ for 3 min, then perform 5 cycles of amplification (each cycle consisting of denaturation at 94℃ for 20 s, annealing at 55℃ for 20 s, and extension at 72℃ for 30 s), followed by a final extension at 72℃ for 5 min, and finally maintain at 10℃ until the program ends. The PCR products were purified by 2% agarose gel electrophoresis. After purification, both amplicones were sequenced using the Illumina MiSeq platform (Sangon Biotech Co., Ltd., Shanghai, China) according to the standard protocol of 300-base-pair reads.

[0027] Microbial community analysis results as follows Figure 1 As shown, Pseudomonas It was a core dominant species in multiple groups, especially in CK1, CK2, and CK3; the MSM-6 sample, on the other hand, was... Pseudomonas , Klebsiella The community is highly diverse, with multiple species associated with it. Klebsiella The abundance was higher in CK3 and MSM-6. Overall, MSM-6 had a higher total microbial abundance. Pseudomonas , Klebsiella as well as unclassified Enterobacteriaceae These constitute the core dominant group within the community.

[0028] Based on these characteristics, we can further speculate: Pseudomonas As a core degrading bacterium, its high proportion in the pesticide-free control group indicates that the control group environment was more suitable for the metabolic activity of this bacterium. The high diversity and abundance of the MSM-6 sample reveal that "multi-bacterial synergy" may be an important structural basis for the efficient degradation system. This discovery breaks the traditional understanding that single-functional bacteria dominate and provides an important community structure reference for constructing stable and efficient composite degrading bacterial communities. Example 3

[0029] Strain isolation and purification: Mix the bacterial solution at 10 -3 Up to 10 -6Serial dilutions were performed, and appropriate amounts of the bacterial suspension were evenly spread onto glucose + MSM agar plates, then incubated at 30°C for 24 to 48 hours. Single colonies were selected and purified by streaking at least three times. The purified strains were inoculated onto LB agar plates and liquid medium, and the morphological characteristics and culture properties of the colonies were carefully observed and recorded in detail. The four purified strains are shown below. Figure 2 As shown. Example 4

[0030] 16S rDNA gene sequence analysis: Total genome sequences of the degrading strains were extracted. Using the extracted total DNA as a template, PCR amplification was performed using the universal 16S rDNA primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1429R (5'-GGTTACCTTGTTACGACTT-3'). PCR products were detected by 1% agarose gel electrophoresis. Single-band PCR products were purified using ExoSAP-IT, while PCR products with non-specific bands were excised and purified. After sequencing, homology alignment of the gene sequences of the degrading strains was performed using the Blasten software from the NCBI website (http: / / www.ncbi.nlm.nih.gov). A phylogenetic tree was constructed using the MEGA 7.0 biological software to determine the classification and nomenclature of the strains. The phylogenetic tree is shown below. Figure 3 As shown. Example 5

[0031] The preparation method of the immobilized bacterial agent includes the following steps: Step 1: Activate a single colony and inoculate it into LB liquid medium. Place the medium on a shaker and co-culture at 30°C and 150 rpm for 24 hours. Then, centrifuge at 4000 rpm for 15 minutes and discard the supernatant. Add sterile water, shake thoroughly, and adjust the OD600 value of the final bacterial suspension to 1.0. This suspension is then used as a seed culture. Step 2: Add 10% (w / v) coconut shell charcoal to MSM liquid culture medium, place in an autoclave, and sterilize at 121℃ for 20 min. After cooling to room temperature, inoculate with 10% (v / v) seed culture with an OD600 of 0.6 to obtain a mixed culture medium; Step 3: Place the mixed culture medium in a shaker (30℃, 150 rpm), fix and culture for 48 hours, then remove it and filter it with sterile gauze. Then wash the solid with 0.85wt.% physiological saline, repeating this washing process 3 times. Finally, place the immobilized microorganisms on aluminum foil, air dry at room temperature, and store them at 4℃. Indicator Test

[0032] 1. Determination of bacterial degradation rate MSM pesticide solutions of six single pesticides and pesticide mixtures with a final concentration of 50 mg / L were prepared. Parallel systems (System A and System B) for single pesticides and pesticide mixtures were set up to investigate the degradation behavior of six target pesticides (carbendazim, imidacloprid, indoxacarb, lambda-cyhalothrin, spirodiclofen, and bifenthrin) over 9 days. On days 1, 3, 5, 7, and 9, the pesticide residue content in each sample was calculated using high-performance liquid chromatography with external standard method. The degradation rate was calculated using the following formula: ρ = (1 - A1 / A0) × 100%. In the formula, A1 is the pesticide residue content (μg / mL) after inoculation with microorganisms, and A0 is the pesticide residue content (μg / mL) before inoculation with microorganisms.

[0033] Experimental results are as follows Figure 4 As shown, in system A, cypermethrin exhibited the best degradation efficiency, reaching 58.77 ± 0.93% after 9 days of cultivation. Figure 4 -A); Imidacloprid has the slowest degradation rate, with a degradation rate of only 26.60±0.25% after 9 days (-A). Figure 4 -A). In system B, spirodiclofen showed the best degradation performance, with a degradation rate of 49.64 ± 0.64% after 9 days (-A). Figure 4 -B); Compared to system A, the degradation rate of imidacloprid increased to 40.94% ± 1.80% ( Figure 4 The degradation rate of lambda-cypermethrin (-B) decreased to 34.91% ± 1.68%. These results indicate a strong correlation between the degradation efficiency of the target compounds and system conditions. The degradation stability of indoxacarb was not significantly affected by system conditions; the degradation process of imidacloprid exhibited condition dependence, with synergistic enhancement of degradation efficiency achievable under specific systems; and the degradation of lambda-cypermethrin was easily inhibited by the antagonistic effects of other components in the system. These experimental results provide data support for the environmental fate regulation of target compounds and the environmental risk assessment of their combined applications.

[0034] 2. Investigation of microbial community promotion experiments The experiment selected commercially available *Chicken Feather Cabbage* seeds from the same package that were plump, brightly colored, and free from insect infestation and mold as the test material. The seeds were first soaked in pure water for 4 hours, rinsed 2-3 times to remove impurities, and then surface-sterilized by soaking in sterile water for 3 minutes, 75% alcohol for 1 minute, and 3.5% sodium hypochlorite for 10 minutes. They were then rinsed 3 times with sterile water for 1 minute each time. The sterile water from the final rinse was spread on an LB agar plate to test the sterilization effect. The seed solution from Example 5 was diluted to 0.4 at OD600nm. The experimental group was treated with the seed solution for 4 hours, while the control group was treated with LB medium diluted by the same factor. The treated seeds were then transferred to Hoagland medium for seedling cultivation. After 24 hours, the seeds germinated, and the germination rate was recorded. Seedlings with uniform germination were selected and transferred to sterile nutrient soil substrate for cultivation in a plant greenhouse at 25℃ with 12 hours of alternating light and dark conditions. The experimental and control groups were irrigated with the seed solution and LB medium diluted solution, respectively. The experiment lasted 28 days, with 6 replicates per group.

[0035] The results are shown in Table 1. The normal germination potential of the seeds was 94.00±0.58%. The fresh weight of the seedlings in the experimental group reached 5.06±0.68 g, and the root length was 15.22±1.53 cm, which were significantly increased by 65.90% and 48.20% respectively compared with the control group (3.05±0.68 g and 10.27±0.74 cm). The differences between the two core indicators were statistically significant (p<0.05). In addition, regarding auxiliary indicators, the germination potential of the experimental group was 89±1.87%, and that of the control group was 80±1.58%, both of which were superior to the control group and the differences were significant (p<0.05). The above results fully demonstrate that this strain has outstanding effects in promoting seed germination (improving germination rate and germination potential) and enhancing seedling growth vitality (increasing fresh weight and root length). Figure 5 The overall performance was significantly better than that of the control group, which fully verified the application value of the strain.

[0036] Table 1 Summary of indicators for promoting growth Note: Different lowercase letters indicate significant differences between groups. p <0.05)3. Basic physicochemical properties of biochar.

[0037] 3. Basic physicochemical properties of biochar

[0038] The main characteristic of biochar is its high carbon content. As shown in Table 2, compared to other elements, the C content (≥42.655) of all three types of biochar is the highest. Atomic ratios are commonly used to reflect the physicochemical properties of biochar. (N+O) / C represents the polarity of biochar; a larger (N+O) / C ratio indicates greater polarity. H / C represents the aromaticity of biochar; a smaller H / C ratio indicates higher aromaticity. (C+H) / O represents the reducing power of biochar; a larger (C+H) / O ratio indicates greater reducing power. O / C represents the stability of biochar; O / C < 0.2 indicates a half-life greater than 1000 years, and a smaller ratio indicates greater stability.

[0039] The H / C ratios of corn stalk charcoal, rice husk charcoal, coconut shell charcoal, bamboo charcoal, and wood charcoal were 0.046, 0.037, 0.022, 0.036, and 0.056, respectively. This indicates that rice husk charcoal, coconut shell charcoal, and bamboo charcoal all have high levels of aromatization, with coconut shell charcoal exhibiting the highest level. Simultaneously, rice husk, coconut shell, and bamboo charcoal all possess high reducing properties and stability, but coconut shell charcoal exhibits even higher reducing properties and stability. Elemental analysis suggests that the differences in polarity, reducing properties, and stability among the three types of biochar are likely the primary reason for their different properties and functions.

[0040] Table 2 Analysis of elemental content in different biochar samples

[0041] 4. Structural characteristics analysis of biochar The basic physicochemical properties of different biomass materials are shown in Table 3, and their electron micrographs are shown in […]. Figure 6 After carbonization, biomass exhibits a clear porous structure, which significantly increases the specific surface area of ​​biochar. Coconut shell biochar has the highest specific surface area and total pore volume, reaching 1,072.8604 m² / g and 0.478540 cm³ / g, respectively; followed by rice husk biochar, with a specific surface area of ​​204.1520 m² / g and a total pore volume of 0.103070 cm³ / g. In contrast, bamboo charcoal and wood charcoal have finer particles and relatively fewer pores after carbonization. Wood charcoal has the smallest specific surface area at only 84.6781 m² / g, while bamboo charcoal has the smallest total pore volume at 0.013455 cm³ / g.

[0042] The electron microscopic structural characteristics of different biomass biochars differed significantly: corn straw biochar ( Figure 6 -A) mostly exhibits a fibrous / tubular interwoven structure, retaining the vascular bundle framework of straw, with pores mainly consisting of long, narrow channels, and the surface accompanied by the dissociation of fine fibers; rice husk biochar ( Figure 6 -B) has a honeycomb porous structure with interlocking silica particles, retaining the hollow pores of rice husks while having a large number of regular silica microcrystals attached to its surface; coconut shell biochar ( Figure 6 -C) is characterized by a dense microporous-mesoporous network, with pores that are nearly circular and evenly distributed, thick walls, and a high degree of structural compactness; bamboo charcoal ( Figure 6 -D) exhibits layered, long tubular pores, continuing the fibrous vascular structure of bamboo, with a large aspect ratio and fine cracks between layers; wood biochar ( Figure 6 -E) These pores are mostly irregular, sheet-like or blocky, with diverse pore shapes and relatively smooth walls. In some areas, a dense layer of lignin residue can be seen. These structural differences are directly related to their application characteristics such as specific surface area and adsorption performance.

[0043] Table 3 Structural characteristics of different biomass

[0044] 5. Colonization of degrading bacteria in different biochars Weigh 1g of the biochar agent (wet weight) into a 10mL centrifuge tube, add 5mL of 0.85% physiological saline to wash the solid, centrifuge at 3000rpm for 5min, discard the supernatant, repeat this process 3 times, add 5mL of sterile water to the precipitate, vortex for 5min, sonicate for 1min, let stand, and then dilute the supernatant by 10%. -2 Up to 10 -4 Gradient dilutions were prepared in 5 g / L glucose + MSM solid medium and incubated at 30°C for 5 days. Colony formation was observed and counted.

[0045] Microbial colonization in different types of biochar, such as Figure 7 As shown: Rice husk inoculant (2.71×10⁻⁶) 5 ±0.75×10 5 ) and coconut shell fungicide (2.53×10 5 ±0.20×10 5 The cell count of rice husks and coconut shells was significantly higher than that of corn straw, bamboo charcoal, and wood biochar carriers. This indicates that rice husks and coconut shells are more conducive to the colonization and proliferation of bacterial strains as biochar carriers, and their viable cell loading capacity is superior to that of corn straw, bamboo charcoal, and wood biochar carriers. Considering the structural characteristics of the carriers ( Figure 6 The pore morphology and size of rice husk and coconut shell biochar are more suitable for the growth space of bacterial strains. For example, the honeycomb pores of rice husk can provide dispersed attachment sites for bacterial strains, while the dense micropores of coconut shell can reduce bacterial strain loss. These factors together support a higher survival rate of viable bacteria. On the other hand, the fibrous pores of corn stalks are easy to dissociate, and the long tubular pores of bamboo charcoal have too strong connectivity, which is not conducive to the stable colonization of bacterial strains, resulting in a lower viable bacterial count.

[0046] 6. Fourier Transform Infrared Spectroscopy Analysis Infrared spectroscopy analysis revealed that the core evidence for the successful adsorption of microorganisms into the pores of biochar was the change in multidimensional spectral characteristics: after biochar modification, a 3293.00 cm⁻¹ (aminoNH) pore appeared. Figure 8 Peaks of microbial-specific functional groups, such as 1629.14 cm⁻¹ (amide carbonyl C=O) and 1617.57 cm⁻¹ (olefin double bond C=C), were observed. Characteristic peaks in the original material, such as 3328.19 cm⁻¹ (hydroxyl OH) and 1628.66 cm⁻¹ (carbonyl C=O), underwent peak shifts, broadening, or intensity changes. Additionally, a new peak, 432.96 cm⁻¹, was added. Figure 8 A), 410.30 cm⁻¹ Figure 8 B), 472.01 cm⁻¹ Figure 8 D) Inorganic functional group peaks, some original functional groups (such as hydroxyl OH at 334.21 cm⁻¹) were replaced, and the absorption peak intensity in the low wavenumber region (<1000 cm⁻¹) was significantly improved and the peak shape was more complex. These changes together confirm that microorganisms do not simply attach to the surface of biochar, but enter the pores and form stable interactions with pore functional groups through hydrogen bonding, electrostatic interaction or complexation reaction, thus confirming the successful adsorption of microorganisms in the pores of biochar.

[0047] Although no obvious characteristic absorption peaks were observed in coconut shell biochar and its inoculant system, this phenomenon itself can serve as indirect evidence that microorganisms successfully adsorbed into the pores of coconut shell biochar. On the one hand, coconut shell biochar has a high specific surface area and a well-developed pore structure, and its surface functional groups (such as hydroxyl and carbonyl groups) content is inherently low, resulting in no significant characteristic peaks in the infrared spectrum of the original material. On the other hand, when microorganisms adsorb into the pores, the characteristic functional groups such as amino (NH), amide carbonyl (C=O), and olefin double bond (C=C) on their cell surface are wrapped by the pore structure of coconut shell biochar, and the microorganisms form a stable bond with the inner wall of the pores through hydrogen bonds and electrostatic interactions, which shields or weakens the vibrational signals of these functional groups, ultimately resulting in no obvious absorption peaks in the spectrum. Based on the spectral characteristics of bamboo charcoal and wood charcoal inoculant systems (the appearance of microbial-specific peaks and peak shifts), the absence of peaks in coconut shell biochar and its inoculant is essentially due to the efficient adsorption and encapsulation of microorganisms by the pores, resulting in the inability to detect characteristic signals, rather than the lack of microbial adsorption. This further confirms the stable fixation effect of coconut shell biochar pores on microorganisms.

[0048] 7. Remediation of contaminated soil by immobilized microbial agents Uncontaminated pure soil was selected, and the target pesticide solution was added. The soil was first placed in a ventilated environment, and after the acetone had completely evaporated, it was aged in the dark for 7 days to prepare artificially contaminated soil with a concentration of 50 mg / kg. 5% (V / V) of immobilized microbial agent granules were added to this contaminated soil. The soil was grouped as follows: corn straw biochar (01), rice husk biochar (02), coconut shell biochar (03), bamboo charcoal (04), and wood charcoal (05); corn straw biochar microbial agent (J1), rice husk biochar microbial agent (J2), coconut shell biochar microbial agent (J3), bamboo charcoal microbial agent (J4), and wood charcoal microbial agent (J5); a blank control group (CK1) and a free microbial community control group (CK2) were also set up, with 5 replicates in each group.

[0049] (1) The content of six pesticides in the soil of each treatment group was measured 28 days after remediation. The removal rate was calculated.

[0050] The results are as follows Figure 9 As shown, compared with the blank control group (CK1), both biochar and inoculant had a certain removal effect on the target pesticide. Among them, according to... Figure 9 The pesticide content was calculated, and the highest removal rates of imidacloprid, carbendazim, bifenthrin, spirodiclofen, and indoxacarb were achieved by treatment with rice husk charcoal fungicide (J2), with removal rates of 78.96%, 69.94%, 70.97%, 84.14%, and 86.67%, respectively; while the removal rate of high-efficiency cypermethrin by treatment with coconut shell charcoal fungicide (J3) was 69.92%.

[0051] Compared with the CK2 group, the immobilized bacterial agent improved the pesticide removal rate in the soil to a certain extent. Figure 9 The removal rates of immobilized microorganisms increased by 26.86%-73.12%, 19.88%-65.47%, 17.37%-69.67%, 10.15%-42.45%, 21.32%-59.07%, and 44.88%-47.44%, respectively. Imidacloprid showed the most significant removal effect, reaching a maximum of 73.12%, followed by spirodiclofen and carbendazim, with increases of 69.67% and 65.47%, respectively. This indicates that the immobilized microorganisms exhibited good removal effects on different types of pesticides.

[0052] This result may be closely related to the protective effect of biochar carriers on microorganisms and their adsorption and enrichment effect on pesticides: by providing a stable micro-ecological environment, biochar reduces the inhibition of functional microorganisms by unfavorable factors in the soil, and significantly improves the survival ability and metabolic activity of strains; at the same time, the rich pore structure and large specific surface area of ​​biochar can quickly adsorb pesticide molecules in the soil, so that pollutants form a local high concentration area around microorganisms, thereby promoting the secretion of degradation enzymes and the efficiency of catalytic reaction.

[0053] The above results fully demonstrate that biochar-immobilized microbial agents, through a synergistic "adsorption-degradation" effect, effectively enhance the remediation efficacy of soil contaminated with compound pesticides, providing a theoretical basis and technical support for the green remediation of pesticide pollution in farmland soil. From the overall removal effect, the J2 treatment group showed outstanding performance in removing multiple pesticides, achieving high removal rates for carbendazim, bifenthrin, spirodiclofen, and indoxacarb. The J3 treatment group showed significant effects in removing imidacloprid and lambda-cyhalothrin. The J4 treatment group only showed good results in removing spirodiclofen. The changes in the content of target pesticides in the soil by different treatment groups in this experiment are shown below. Figure 9 The results clearly show that the combined treatment of biochar and degrading microorganisms has different effects in the remediation of pesticide-contaminated soil, providing an important basis for further optimization of remediation schemes.

[0054] (2) The enzyme activity in the soil was detected, and the results are as follows: Figure 10 As shown, in soil polyphenol oxidase ( Figure 10 -A), urease ( Figure 10 -B), dehydrogenase ( Figure 10 In the detection of C) activity, the biochar immobilized inoculants (J1-J5) showed significantly better effects on enhancing the activity of the three enzymes than biochar alone (01-05) and the control group (CK1, CK2). The biochar immobilized inoculants (J1-J5), especially groups J2 and J3, could significantly activate enzyme activity, with polyphenol oxidase, urease, and dehydrogenase activities increasing by 61.52%, 14.46%, and 39.88%, respectively. This indicates that the treatment with biochar immobilized inoculants can comprehensively enhance the metabolic function of soil microorganisms, with rice husk charcoal inoculants (J2) and coconut shell charcoal inoculants (J3) showing the best effects.

[0055] This result also suggests that immobilized microbial agent treatment may enhance metabolic pathways related to enzyme synthesis by regulating the structure of soil microbial communities and enriching functional microbial groups. Combining the functional characteristics of the three enzymes, the enhancement of polyphenol oxidase is conducive to the conversion of phenolic substances, the enhancement of urease activity can promote nitrogen cycling, and the activation of dehydrogenase directly reflects the total metabolic intensity of microorganisms. The synergistic effect of the three indicates that immobilized microbial agents can not only activate soil biological activity, but also improve soil nutrient conversion efficiency and pollutant degradation potential.

[0056] This experiment, based on previous research, involved planting plants and comparing them with plants grown in soil treated with biochar and immobilized bacterial agents.

[0057] Potted plant experiment photos as follows Figure 11As shown, the differences in growth phenotypes of leafy vegetables under different treatments are evident. Groups CK1 and CK2 showed smaller plant size and sparser leaves, indicating relatively weaker growth. In contrast, groups J2, J3, and J4 exhibited the most abundant and darkest green leaves, demonstrating the best overall growth among all groups. This clearly reflects the significant differences in the growth-promoting effects of different treatments on leafy vegetables, with groups J2, J3, and J4 (rice husk charcoal inoculant, coconut shell charcoal inoculant, and bamboo charcoal inoculant, respectively) showing the most significant growth-enhancing effects.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A degrading microbial system for remediating pesticide-contaminated soil, characterized in that: The degrading bacterial strain includes Pseudomonas plecoglossicda JY-5 Klebsiella pneumoniae J2-1 Arthrobacter silvisoli JY-2 and Enterobacter ludwigii JY-1.

2. The degrading microbial system for remediating pesticide-contaminated soil according to claim 1, characterized in that: The Pseudomonas plecoglossicda JY-5 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67049; Klebsiella pneumoniae J2-1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67050; Arthrobacter silvisol i JY-2 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67051; Enterobacter ludwigii JY-1 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 29, 2025, with accession number GDMCC No. 67052.

3. A method for constructing a degrading microbial system for remediating pesticide-contaminated soil, characterized in that, The steps are as follows: S1. Soil samples were inoculated into a culture medium containing mixed pesticides. After enrichment culture, the suspensions were inoculated into culture media containing pesticides for initial screening. Then, the concentration was increased for secondary screening. Continuous subculturing was carried out to obtain microbial communities with the potential to degrade pesticides. S2. High-throughput sequencing technology was used to analyze the composition and changes in microbial community during the degradation of each pesticide in single and mixed systems; S3. Strains were isolated and purified, total DNA was extracted, PCR amplification was performed, gene sequences were analyzed, homology comparisons of the strain's gene sequences were conducted, and a phylogenetic tree was constructed using biological software to ultimately obtain... Pseudomonas plecoglossicda JY-5 Klebsiella pneumoniae J2-1 Arthrobacter silvisoli JY-2 Enterobacter ludwigii JY-1.

4. The method for constructing a degrading microbial system for remediating pesticide-contaminated soil according to claim 3, characterized in that: In step S1, the soil sample came from the Nanfan area of ​​Hainan Province; the mixed pesticide content in the culture medium containing mixed pesticides was 50-200 mg / mL; the mixed pesticides were carbendazim, imidacloprid, indoxacarb, high-efficiency cypermethrin, spirodiclofen, and bifenthrin; the pesticide content in the culture medium containing pesticides was 50-200 mg / mL; the concentration was increased to 100-200 mg / mL.

5. The application of the degrading bacterial system according to claim 1 or 2 in immobilized bacterial agents, characterized in that: The immobilized bacterial agent includes a degrading bacterial strain and a biochar carrier.

6. The application according to claim 5, characterized in that, The preparation steps of the immobilized bacterial agent are as follows: Step 1: Inoculate the activated single colony into liquid culture medium, culture on a shaker, then centrifuge and discard the supernatant, add sterile water, shake thoroughly, and adjust the OD600 value of the final bacterial suspension to 1.0 for later use as seed culture; Step 2: Add biochar to MSM liquid culture medium, autoclave, cool to room temperature, and then inoculate with a mixed seed culture with an OD600 of 0.6 to obtain a mixed culture medium; Step 3: After placing the mixed culture medium on a shaker for incubation, remove it and filter it to obtain the filter residue. Then wash the filter residue with physiological saline, place the filter residue on aluminum foil to dry, and refrigerate and dry to obtain the immobilized bacterial agent.

7. The application of the degrading bacterial system according to claim 6 in immobilized bacterial agents, characterized in that: The activated single colonies in step one are Pseudomonas plecoglossicda JY-5 Klebsiella pneumoniae J2-1 Arthrobacter silvisoli JY-2 Enterobacter ludwigii JY-1 colonies; the temperature for shaking culture is 25-30℃, the shaking speed is 150-200 rpm, and the shaking time is 24-48h; the centrifugation speed is 3000-4000 rpm, and the time is 15-20min.

8. The application of the degrading bacterial system according to claim 6 in immobilized bacterial agents, characterized in that: In step two, the biochar is coconut shell biochar; the mass-to-volume ratio of biochar, liquid culture medium, and seed liquid is (1-2):(8-10):(1-2); the autoclaving temperature is 121℃ and the time is 15-20 min.

9. The application of the degrading bacterial system according to claim 6 in immobilized bacterial agents, characterized in that: In step three, the temperature of the shaker culture is 25-30℃, the shaking speed is 150-200 rpm, and the shaking time is 24-48h; the concentration of physiological saline is 0.8-0.9wt.%.

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