Composite microbial inoculant for repairing cadmium pollution mediated by polyethylene microplastics and preparation method and application thereof

Abstract

This invention provides a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics, its preparation method, and its application. The composite microbial agent comprises directed-acclimated Bacillus subtilis and Rhodopseudomonas palustris. Applied to tea garden soil contaminated with cadmium mediated by polyethylene microplastics, the directed-acclimated Bacillus subtilis and Rhodopseudomonas palustris efficiently secrete extracellular polymeric substances (EPS) in the presence of PE, achieving physical encapsulation of PE particles and chemical complexation with Cd. On the one hand, it encapsulates PE microplastics, blocking their Cd adsorption sites; on the other hand, it complexes and passivates available Cd, thus remediating cadmium pollution mediated by polyethylene microplastics. Under a concentration gradient, the optimal concentration ( ) treatment can reduce soil DTPA-Cd content by 27.27%, TCLP-Cd content by 46.88%, Cd content in tea seedling leaves by 36.67%, and Cd translocation coefficient (TF) by 53.32%, simultaneously reducing soil environmental risk and cadmium accumulation in plants, achieving systematic control from soil to plants.

Description

Technical Field

[0001] This invention relates to the field of tea garden soil remediation technology, specifically to a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics, its preparation method, and its application. Background Technology

[0002] Tea garden soils, due to their slightly acidic nature and frequent agricultural activities, easily accumulate carbon dioxide (Cd) along with polyethylene microplastic (PE) pollution. PE microplastics can act as carriers to adsorb Cd, altering its form and migration behavior in the soil, forming "PE-Cd composite pollution," significantly increasing the bioavailability and ecotoxicity of Cd. Existing remediation methods typically employ chemical passivation and soil replacement, which suffer from high costs, a high risk of secondary pollution, and disruption of soil microbial communities. While microbial remediation shows potential, existing microbial agents are mostly targeted at single pollutants, offering limited effectiveness in remediating multi-media, multi-interface composite pollution systems such as "PE-Cd composite pollution," and lacking a systemic mechanism for restoring ecological functions and ensuring crop health. Furthermore, tea gardens often suffer from combined pollution from pesticide residues and other heavy metals (such as Pd and As), making it difficult for single remediation agents to simultaneously address multiple pollutions. Therefore, there is an urgent need to develop a multifunctional composite microbial agent that can simultaneously intervene in PE-mediated behavior, efficiently passivate Cd, degrade organic pesticides, synergistically passivate multiple heavy metals, and activate systemic resistance in tea trees. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics, its preparation method, and its application, thus solving the problems mentioned in the background section.

[0004] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics is provided, comprising directed domestication of Bacillus subtilis and Rhodopseudomonas palustris.

[0005] Preferably, the weight ratio of Bacillus subtilis to Rhodopseudomonas palustris is 1:1~2.

[0006] This invention utilizes domesticated and screened Bacillus subtilis and Rhodopseudomonas palustris in a specific ratio. On one hand, it physically encapsulates PE particles by secreting extracellular polymeric substances (EPS), chemically complexing Cd heavy metals, blocking PE-mediated Cd migration and promoting the conversion of heavy metals to stable forms. On the other hand, it improves soil physicochemical properties and enzyme activity, optimizes the microbial community structure, and restores soil ecological functions. The compound microbial agent is applied through a "staged root irrigation + foliar induction" approach, enabling the microbial community to efficiently colonize the rhizosphere. This mutually activates the antioxidant, photosynthetic, and stress-resistance-related molecular pathways of tea tree roots, reducing heavy metal absorption and translocation, alleviating oxidative damage, and simultaneously achieving efficient degradation of organic pesticides. By addressing pollution source intervention, soil environmental remediation, and plant stress resistance activation from multiple dimensions, this invention breaks the chain of complex pollution, achieving comprehensive restoration of the tea garden soil environment.

[0007] Preferably, the compound microbial agent further includes alkaloid Pseudomonas aeruginosa and Fusarium, and the total amount of alkaloid Pseudomonas aeruginosa and Fusarium is 10-30% of the total amount of Bacillus subtilis and Rhodopseudomonas palustris.

[0008] Preferably, the mass ratio of the alkalogenic pseudomonocytes to Fusarium is 1:1.

[0009] According to a second aspect of the present invention, a method for preparing a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics is provided, comprising the following steps: Step 1: Mix Bacillus subtilis and Rhodopseudomonas palustris in a certain ratio and inoculate them into a low-nutrient acclimatization medium. Use polyethylene microplastics and Cd composite contamination to create a target stress and set a concentration gradient. Perform stepwise subculturing at each concentration. When the strain grows stably at 2 times the concentration and the extracellular polymer secretion is ≥30% higher than before acclimatization, the acclimatization is complete. Step 2: After directional domestication, Bacillus subtilis and Rhodopseudomonas palustris were inoculated into LB liquid medium and photosynthetic cell medium, respectively, and cultured until the logarithmic growth phase to activate the strains; Step 3: Centrifuge the bacterial solution to collect the bacterial cells and resuspend them by weight. Step 4: Combine the Bacillus subtilis and Rhodopseudomonas palustris obtained in Step 3, and adjust the total bacterial concentration to 1×10⁻⁶. 9 CFU / mL.

[0010] Preferably, the activation temperature of the strain is 30~35℃, the shaking speed is 180~220rpm, and the time is 12~18h.

[0011] Preferably, alkalogenic Pseudomonas and Fusarium are also added during the compounding process.

[0012] According to a third aspect of the present invention, an application of a composite microbial agent in tea garden soil contaminated with cadmium mediated by polyethylene microplastics is provided.

[0013] Preferably, the concentration range of the compound microbial agent is 2.5 × 10⁻⁶. 7 ~1.0×10 8 CFU / g.

[0014] Preferably, the concentration range of the compound microbial agent is 5.0 × 10⁻⁶. 7 ~1.0×10 8 CFU / g.

[0015] This invention provides a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics, its preparation method, and its application. It possesses the following beneficial effects: (1) This solution provides a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics. Through targeted domestication of Bacillus subtilis and Rhodopseudomonas palustris, it can efficiently secrete extracellular polymeric substances (EPS) in the presence of PE, achieving physical encapsulation of PE particles and chemical complexation with Cd. On the one hand, it encapsulates PE microplastics, blocking their Cd adsorption sites; on the other hand, it complexes and passivates available Cd, thus achieving the remediation of cadmium pollution mediated by polyethylene microplastics. Under a concentration gradient, the optimal concentration ( Treatment can reduce soil DTPA-Cd content by 27.27%, TCLP-Cd content by 46.88%, Cd content in tea seedling leaves by 36.67%, and Cd translocation coefficient (TF) by 53.32%, simultaneously reducing soil environmental risks and plant cadmium accumulation, achieving systematic control from soil to plants.

[0016] (2) The compound microbial agent provided in this solution for remediating cadmium pollution mediated by polyethylene microplastics also has excellent passivation effects on Pd and As heavy metals commonly found in tea gardens. It can reduce the available Pb in the soil by 26.47% and the available As by 25.27%. At the same time, the compound microbial agent contains alkali-producing Pseudomonas aeruginosa and Fusarium, which can achieve a degradation rate of 46.89% for imidacloprid and a single degradation rate of 78.6% and 62.1% for commonly used pesticides in tea gardens such as acetamiprid and diflubenzuron, respectively. The simultaneous decomposition rate under the compound pesticide system exceeds 43.25%, which can realize the simultaneous treatment of heavy metals and organic pesticides and solve the problem that single microbial agents are difficult to deal with multiple pollutions in tea gardens.

[0017] (3) The application of a compound microbial agent for remediating cadmium pollution mediated by polyethylene microplastics provided in this scheme in tea garden soil. By applying it through root irrigation, the compound microbial agent colonizes in the rhizosphere. Its continuous metabolism not only directly promotes the transformation of heavy metals into stable residues, but also improves the stressed soil microenvironment. It restores the activity of soil sucrase, urease, and catalase to more than 85% of the normal level, increases the Shannon index of bacterial community by 20%, and significantly increases the abundance of beneficial bacteria such as Bacillus and Pseudomonas. It rebuilds a healthy soil microbial community and nutrient cycling function, fundamentally improving the health status and buffering capacity of the soil.

[0018] (4) The application of a compound microbial agent for remediating cadmium pollution mediated by polyethylene microplastics provided in this scheme in tea garden soil. The compound microbial agent interacts with the roots of tea trees, activates the systemic resistance of tea trees at the physiological and molecular levels, reduces the MDA content of tea seedling leaves by 41.79%, increases Chl-a content by 81.37%, upregulates the expression of key photosynthetic genes (psbA, rbcL), activates jasmonic acid synthesis, lignin synthesis and antioxidant-related molecular pathways, effectively alleviates oxidative damage and photosynthetic inhibition, significantly reduces the translocation of Pb and As to leaves, and ensures the yield and quality safety of tea.

[0019] (5) The application of a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics provided in this scheme in tea garden soil, through the application mode of "staged root irrigation + foliar induction", improves the colonization efficiency and functional sustainability of functional microbial communities in the rhizosphere, providing clear and verifiable quantitative basis and operation scheme for the large-scale field application of the technology, and ensuring the stability and repeatability of the remediation effect. Detailed Implementation

[0020] To better illustrate the content of this invention, the following description is provided in conjunction with specific embodiments.

[0021] The raw materials used in the embodiments and comparative examples of this invention are polyethylene microplastics (PE), specifically polyethylene particles with a particle size <5mm; and Bacillus subtilis (…). Bacillus subtilis,B.s Select Bacillus subtilis TAX-001; Rhodopseudomonas palustris ( Rhodopseudomonas palustris,R.p ), select Rhodopseudomonas palustris RP-002.

[0022] The proper nouns used in this invention are explained as follows: Cd: Cadmium, a heavy metal pollutant; CM: Compound microbial inoculant, an inoculant composed of Bacillus subtilis and Rhodopseudomonas palustris; CFU / g: Colony forming units per gram of soil, used to indicate the concentration of inoculant. DTPA-Cd: Diethylenetriaminepentaacetic acid-extractable cadmium, representing plant-available cadmium in the soil; TCLP-Cd: Toxicity characteristics of cadmium extracted through leaching process, representing hazardous cadmium in soil; EPS: Extracellular polymeric substances secreted by microorganisms that can complex heavy metals; MDA: Malondialdehyde, a marker of oxidative damage; Chl-a: Chlorophyll a, photosynthetic pigment; Pb: Lead, a heavy metal pollutant; As: Arsenic, a heavy metal pollutant; IM: Imidacloprid, a common pesticide in tea gardens; ACE: Acetamiprid, a common pesticide used on tea plants; DIF: Diflubenzuron, a common pesticide used on tea plants.

[0023] Example 1 A composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics is prepared through the following steps: Strains were prepared as follows: Bacillus subtilis TAX-001 and Rhodopseudomonas palustris RP-002; these strains were previously verified to produce EPS and be resistant to Cd. 2+ And the ability to colonize PE surfaces.

[0024] Targeted acclimatization: The two bacteria were mixed at a 1:1 live cell ratio and inoculated into a low-nutrient acclimatization medium. A concentration gradient was established to target polyethylene microplastic (PE) and cadmium (Cd) combined pollution as the stress. The baseline concentration (1x) was: PE 1.65 mg·kg⁻¹. -1 Dry soil, Cd 0.5 mg·kg -1 Dry soil was used. Five concentration gradient levels (1 / 10, 1 / 5, 1 / 2, 1, 2) were established (i.e., the concentrations of each pollutant changed proportionally). Activated Bacillus subtilis and Rhodopseudomonas palustris were inoculated into liquid culture media at the corresponding stress concentrations and passaged sequentially for acclimatization. Each concentration level was passaged three times, with strain activity monitored using plate counting each time. Acclimatization was considered complete when the strains grew stably at twice the concentration and the secretion of extracellular polymeric substances (EPS) increased by ≥30% compared to before acclimatization. The acclimatized strains were purified and stored at -80℃ as inoculum for subsequent inoculum preparations.

[0025] Strain activation: Bacillus subtilis was inoculated into LB liquid medium, and Rhodopseudomonas palustris was inoculated into photosynthetic bacteria medium. The cultures were shaken at 30°C and 180 rpm to the logarithmic growth phase, respectively. Preparation and counting of bacterial suspensions: The activated bacterial suspensions were centrifuged to collect the bacterial cells, which were then resuspended in sterile buffer containing metabolic induction components. The concentration of each bacterial suspension was determined by plate counting. Compound formulation: Activated Bacillus subtilis TAX-001 and Rhodopseudomonas palustris RP-002 were compounded at a live cell ratio of 1:1. Then, alkali-producing Pseudomonas and Fusarium (the Fusarium used has undergone safety assessment and does not produce known mycotoxins and is non-pathogenic to plants) were added at a mass ratio of 1:1. The amount added was 10% of the total amount of Bacillus subtilis and Rhodopseudomonas palustris, adjusting the total bacterial concentration to 1×10⁻⁶. 9 The CFU / mL compound bacterial agent stock solution was then mixed with an appropriate amount of humic acid carrier to prepare a compound microbial agent.

[0026] Example 2 The preparation method of this embodiment is the same as that of Example 1. The difference is that, in the compounding process, the activated Bacillus subtilis TAX-001 and Rhodopseudomonas palustris RP-002 are compounded at a live bacteria ratio of 1:2.

[0027] Example 3 The preparation method of this embodiment is the same as that of Example 1, except that alkalogenic pseudomonocytes and Fusarium are omitted in the compounding process.

[0028] Comparative Example 1 The preparation method of this comparative example is the same as that of Example 1, except that during the compounding process, the activated Bacillus subtilis TAX-001 and Rhodopseudomonas palustris RP-002 are compounded at a live bacteria ratio of 1:0.5.

[0029] Comparative Example 2 The preparation method of this comparative example is the same as that of Example 1, except that during the compounding process, the activated Bacillus subtilis TAX-001 and Rhodopseudomonas palustris RP-002 are compounded at a live bacteria ratio of 1:2.5.

[0030] Comparative Example 3 The preparation method of this comparative example is the same as that of Example 1, except that no directional domestication step was performed.

[0031] Performance testing I. Potted plant experiment to verify the repair effect and dose effect (1) Preparation of contaminated soil: Tea garden soil was collected, air-dried and sieved. Different types of soil were prepared by adding polyethylene microplastics and cadmium chloride solution: ① Single Cd pollution (Cd content 0.5 mg / kg dry soil); ② Single PE pollution (PE content 1.65 mg / kg dry soil); ③ PE-mediated Cd pollution (PE 1.65 mg / kg dry soil, Cd 0.5 mg / kg dry soil). The contaminated soil was aged at room temperature for 60 days. (2) Experimental design: A pot experiment was used to study the remediation effect and mechanism of different concentrations of compound microbial agents on soil under single pollution and PE-mediated Cd pollution conditions. A total of 9 treatment groups were designed, as shown in Table 1. Specifically, there were pollution control (CK), single Cd pollution (Cd), single PE pollution (PE), and PE-mediated Cd pollution (PE+Cd) groups, none of which were added with microbial agents to assess the background pollution effect. Under single Cd or PE pollution conditions, fixed concentrations of microbial agents were applied to evaluate their remediation effect on single pollution. Under PE-mediated Cd pollution conditions, three concentrations of microbial agents were set to explore the dose relationship between the remediation effect of the compound microbial agent in Example 1 and the amount of microbial agent used. All treatments were replicated at least 3 times and a completely randomized block design was used to ensure statistical reliability. Table 1

[0032] (3) Application of microbial agents: One-year-old Longjing 43 tea seedlings were transplanted in contaminated soil. 60 days after transplanting, compound microbial agent suspension was applied by root irrigation according to Table 1. The non-microbial treatment group was given an equal amount of sterile water. (4) Cultivation and detection: After the inoculant was applied, the plants were cultivated for another 60 days, and then samples of rhizosphere soil and tea seedlings were collected for systematic analysis. (5) Results data analysis: Soil cadmium passivation: Under PE-mediated Cd pollution conditions, compared with the pollution control group without microbial treatment, the application of high-concentration compound microbial agents can reduce the content of toxic leached cadmium in the soil by 46.88% and the content of plant-available cadmium by 27.27%, while promoting the conversion of cadmium to a stable residual state. Cadmium control in tea trees: Compared with the pollution control group without microbial treatment, the application of compound microbial agents significantly reduced the translocation of Cd from the roots to the leaves. All three concentrations of microbial agents reduced the Cd content in the leaves by about 36%, with the high concentration treatment reducing the cadmium translocation coefficient by 53.32%. Repair effect: The microbial agent treatment also significantly improved soil pH, nutrient content and the activities of sucrase, urease and catalase, and effectively alleviated oxidative damage and photosynthetic inhibition of tea seedlings. The leaf MDA content decreased by 41.79%, and the chlorophyll content and net photosynthetic rate were close to normal levels.

[0033] II. Determination of the Optimal Concentration of Compound Microbial Agents (1) Preparation of contaminated soil: The actual cultivated layer soil (0~20cm) of the tea garden was used, air-dried, and sieved through a 2mm sieve. By adding analytical grade imidacloprid standard and analytical grade Pb(NO3)2 and Na3AsO4 salt solutions, soils with different contamination types were prepared: ① single Pb contamination (Pb content 200 mg / kg); ② single As contamination (As content 50 mg / kg); ③ single imidacloprid contamination (IM content 2 mg / kg); ④ combined contamination of imidacloprid and heavy metals (Pb, As) (IM 2 mg / kg dry soil, Pb 200 mg / kg dry soil, As 50 mg / kg dry soil). All contaminated soils were aged and equilibrated in a constant temperature and humidity incubator (25±2°C, field water holding capacity 60%) for 60 days to simulate the natural aging process. (2) Experimental Design: This experiment adopted the pot experiment method to systematically study the remediation effect and mechanism of the compound microbial agent prepared in Example 1 on soil under single pollution and combined pollution conditions of imidacloprid and heavy metals lead and arsenic. There were 11 treatment groups in total, as shown in Table 2, namely, unpolluted control (CK), single Pb pollution (Pb), single As pollution (As), single imidacloprid pollution (IM), and imidacloprid and heavy metal combined pollution (IM+Pb+As). No microbial agent was added to any of them to assess the background pollution effect. Under single pollution conditions of Pb, As, or imidacloprid, a fixed concentration of microbial agent was applied to evaluate its remediation effect on single pollution. Under combined pollution conditions of imidacloprid and heavy metals (Pb, As), three concentrations of microbial agent were set up to explore the dose-response relationship between the remediation effect and the amount of microbial agent. All treatments were repeated at least 3 times and a completely randomized block design was adopted to ensure the reliability of the system. Table 2

[0034] (3) Application of microbial agents: Transplant one-year-old “Longjing 43” tea seedlings with suppressed growth into aged and contaminated soil. 60 days after transplanting, apply the corresponding microbial agent suspension or sterile water by root irrigation according to the dosage in Table 2. The volume of each pot should be 200 mL, ensuring uniform penetration. (4) Cultivation and detection: After the inoculant was applied, the plants were cultivated at room temperature for 60 days. After the cultivation was completed, the rhizosphere soil and complete upper leaves, stems and roots of each pot of tea seedlings were systematically analyzed. (5) Results data analysis: Soil heavy metal passivation and pre-pesticide degradation: Under conditions of combined pollution with imidacloprid and heavy metals, compared with the control group without microbial treatment, the use of high-concentration compound microbial agents can reduce the content of available Pb by 26.47% and the content of available As by 25.27%, while promoting the conversion of cadmium to a stable residual state, with an IM residue degradation rate of 46.89%. Heavy metal control in tea plants: Compared with the pollution control group without microbial treatment, the application of compound microbial agents significantly reduced the translocation of Pb and As from roots to leaves. All three concentrations of the agent reduced the Pb content in leaves by approximately 35.72% and the As content by approximately 34.81%. Among them, the high concentration of the agent reduced the translocation coefficient of Pb by 53.92% and the translocation coefficient of As by 54.21%. Repair effect: The microbial agent treatment also significantly improved soil pH, nutrient content and the activities of sucrase, urease and catalase, and effectively alleviated oxidative damage and photosynthetic inhibition of tea seedlings. The leaf MDA content decreased by 39.47%, and the chlorophyll content and net photosynthetic rate returned to normal levels.

[0035] III. Verification of the degradation effect of the compound microbial agent prepared in Example 1 on various tea garden pesticides. (1) Culture medium: Based on inorganic salt culture medium, its composition is: 1.5 g / L K2HPO4 + 0.5 g / L KH2PO4 + 1.0 g / L NH4NO3 + 0.2 g / L MgSO4·7H2O + 0.5 g / L NaCl, pH=7.0±0.2, autoclaved at 121℃ for 20 min before use, and a single pesticide is added as the sole carbon / nitrogen source; Pesticides: Imidacloprid, acetamiprid, and diflubenzuron analytical grade standards were prepared into single and mixed stock solutions of 1000 mg / L using methanol.

[0036] (2) Experimental design: Single pesticide pollution system, namely IM, ACE and DIF; Composite pollution simulation system, IM+ACE+DIF mixed pesticide group; Microbial treatment: Two levels of biological treatment are set up under each pesticide system: ① Control (CK): no microbial inoculation; ② Microbial treatment (CM): inoculation with compound microbial agent.

[0037] (3) Experimental methods: ① Establishment of the culture system: Add 100 mL of MSM medium to a 250 mL Erlenmeyer flask. Add single or mixed pesticide stock solutions to each flask to ensure an initial pesticide concentration of 10 mg / L.

[0038] ② Inoculation with bacterial agent: Except for the CK group, all other groups were inoculated with the corresponding bacterial agent at a rate of 2%. An equal volume of sterile water was added to the CK group.

[0039] ③Cultivation conditions: Place the Erlenmeyer flask in a constant temperature shaker at 30°C and 180 rpm and cultivate in the dark.

[0040] ④ Sample collection and testing: At days 0, 1, 3, 7, and 14 of culture, 5 mL of culture medium was aseptically collected, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm organic filter membrane, and the concentration of each pesticide residue was determined by HPLC.

[0041] ⑤Chromatographic conditions: mobile phase acetonitrile / water (60:40, v / v), flow rate 1.0 mL / min, column temperature 30°C, detection wavelength IM: 270 nm, ACE: 245 nm, DIF: 260 nm. Quantification was performed using the external standard method.

[0042] (4) Results Analysis: After 14 days of cultivation, the degradation rates of the three single pesticides in the CM (inoculated with compound bacteria) treatment group were: IM: 47.2%; ACE: 78.6%; DIF: 62.1%, all significantly higher than the control group (CK) at the same time point (p < 0.05). In the simulated system of multiple pesticide coexistence, the CM group maintained a simultaneous degradation efficiency of over 43.25% for the three pesticides. In the CM group, the residual concentrations of each pesticide showed an exponential decreasing trend with cultivation time, consistent with the first-order degradation kinetic model, and the half-life (t) was [not specified]. 1 / 2 The time was shortened by more than 50% compared to the control group.

[0043] IV. The optimal concentration of the compound microbial agent from Example 1 for applying to PE-mediated Cd contamination (PE+Cd) is shown in Table 3. Table 3

[0044] One-year-old Longjing 43 tea seedlings were transplanted into contaminated soil. Sixty days after transplanting, compound microbial agent suspension was applied by root irrigation according to Table 3. After the microbial agent was applied, the seedlings were cultured for another 60 days. Then, the contents of PE and Cd in the rhizosphere soil and tea seedling samples were collected. Three samples were taken from each group and the average value was taken. The results are shown in Table 4.

[0045] Table 4

[0046] Note: In the PE-Cd contaminated control group (PE+Cd) without bacterial treatment, the PE content was 1.66 mg / kg dry soil and the DTPA-Cd content was 0.55 mg / kg dry soil.

[0047] As shown in Table 4, the concentration of plant-available Cd (DTPA-Cd) in the soil decreased significantly with increasing concentration of the compound microbial inoculant. Compared with the untreated control group (0.55 mg / kg), the concentration of the inoculant at 2.5 × 10⁻⁶ mg / kg significantly decreased. 7 At a concentration of CFU / g, the Cd content decreased by 21.82%; at a concentration of 5.0 × 10⁻⁶ CFU / g, the Cd content decreased by 21.82%. 7 At a concentration of CFU / g, the decrease was 30.91%; at a concentration of 7.5 × 10⁻⁶. 7 At a concentration of CFU / g, the decrease was 36.36%; at a concentration of 1.0×10 8 At CFU / g, the concentration decreased by 41.82%. This indicates that the inactivation effect of the microbial agent on Cd exhibits a clear dose-dependent effect. High-concentration treatment with the microbial agent can significantly reduce the bioavailability of Cd in the soil, thereby reducing the risk of Cd absorption by tea plants.

[0048] Regarding PE content, all microbial agent treatment groups showed a slight decrease compared to the control group (1.66 mg / kg), but the decrease was small (maximum decrease of 9.04%). This is because the microbial agent mainly physically encapsulates PE particles by secreting extracellular polymeric substances (EPS), blocking their Cd adsorption sites, rather than directly degrading PE. Therefore, the total residual amount of PE in the soil did not change significantly. This result confirms the core mechanism of this invention: the microbial agent, through a dual-effect synergy of "physical encapsulation and chemical passivation," blocks PE-mediated Cd migration at the source, rather than simply removing PE itself.

[0049] Considering both soil Cd passivation efficiency and inoculant usage cost, the recommended optimal field application concentration for this invention is 5.0 × 10⁻⁶. 7 ~1.0×10 8 CFU / g, of which 1.0×10 8 The CFU / g treatment group showed the largest reduction in the bioavailable Cd content (41.82%), and can be considered the preferred concentration to ensure the best remediation effect.

[0050] V. The composite microbial agents prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were subjected to a concentration of 1.0 × 10⁻⁶. 8 The concentration of CFU / g of the microbial agent was used to test the remediation of tea soil containing PE-mediated Cd, imidacloprid, Pb, and As contamination. One-year-old Longjing 43 tea seedlings were transplanted into the contaminated soil. Sixty days after transplanting, the compound microbial agent suspension was applied by root irrigation. After the microbial agent was applied, the soil was cultured for another 60 days. Then, the contents of PE and Cd in the rhizosphere soil and tea seedling samples were collected. Three samples were taken from each group and the average value was taken. The results are shown in Table 5.

[0051] Table 5

[0052] Note: The initial concentration is the measured value of the control group without bacterial treatment of PE-Cd-IM-Pb-As composite pollution; PE content is the total residue in the soil; Cd, Pb, and As contents are the contents of available forms in the soil (DTPA extraction method); IM content is the amount of imidacloprid residue in the soil.

[0053] As shown in Table 5, under the condition of multiple complex pollution of PE-Cd-IM-Pb-As, there are significant differences in the remediation effects of each test case on the pollutants.

[0054] 1. The restorative effects of Examples 1-3 (the microbial agents of this invention) Example 1 (live bacteria ratio 1:1, containing 10% pesticide-degrading bacteria) and Example 2 (live bacteria ratio 1:2, containing 10% pesticide-degrading bacteria) showed the best overall remediation effect. Compared with the initial concentration, after treatment in Example 1, the PE content decreased by 8.43%, the available Cd content decreased by 43.64%, the IM residue degradation rate reached 47.00%, the available Pb content decreased by 28.40%, and the available As content decreased by 26.60%.

[0055] The results of Example 2 were basically the same as those of Example 1, indicating that efficient synergistic repair can be achieved in the range of live bacteria ratio of 1:1 to 1:2.

[0056] Example 3 (without pesticide-degrading bacteria) was significantly inferior to Examples 1 and 2 in terms of IM degradation, with IM residue decreasing by only 11.00%, indicating that the addition of pesticide-degrading bacteria (Alcaligenes f. alkali-producing bacteria and Fusarium) is key to achieving efficient degradation of organic pesticides. However, the passivation effect of Example 3 on heavy metals (Cd, Pb, As) was basically consistent with that of Examples 1 and 2, proving that the core bacterial group (Bacillus subtilis and Rhodopseudomonas palustris) is sufficient to undertake the heavy metal remediation function, and the addition of pesticide-degrading bacteria does not affect the heavy metal passivation ability of the core bacterial group.

[0057] 2. Repair effects of comparison ratios 1-2 (where the ratio of bacterial strains exceeds 1:1 to 1:2). The combined remediation effects of Comparative Examples 1 (viable bacteria ratio 1:0.5) and 2 (viable bacteria ratio 1:2.5) were significantly worse than those of the Example 1. Compared with Example 1, the Cd passivation efficiency of Comparative Example 1 decreased by 35.42%, Pb passivation efficiency decreased by 23.18%, and As passivation efficiency decreased by 20.43%; the effect of Comparative Example 2 was basically the same as that of Comparative Example 1. This indicates that when the viable bacteria ratio of Bacillus subtilis to Rhodopseudomonas palustris exceeds the range of 1:1 to 1:2, the synergistic metabolic effect of the two strains weakens, the secretion of EPS decreases, resulting in a reduction in the physical encapsulation and chemical complexation ability of heavy metals. At the same time, the IM degradation effect in Comparative Examples 1 and 2 was also significantly lower than that in Example 1 (the residual amount decreased by only 9% to 10%), which may be related to the weakened metabolic activity of the core bacterial community affecting the growth microenvironment of pesticide-degrading bacteria.

[0058] 3. Repair effect of Comparative Example 3 (without targeted domestication) Comparative Example 3 used the original strain that had not been directionally acclimatized to PE-Cd combined pollution stress, and its overall remediation effect was inferior to that of Example 1. Compared with Example 1, the Cd passivation efficiency of Comparative Example 3 decreased by 26.09%, Pb passivation efficiency decreased by 17.58%, As passivation efficiency decreased by 14.17%, and IM degradation efficiency decreased by 8.52%. This indicates that directional acclimatization endowed the strain with specific adaptability to the combined pollution environment, enabling it to efficiently secrete EPS and maintain metabolic activity in the presence of PE, while the functional expression of the unacclimatized strain was limited under combined pollution stress.

[0059] 4. Overall Conclusion As can be seen from the comparison between the embodiments and comparative examples, the technical advantages of the present invention stem from the synergistic effect of the following key elements: (1) Specific live bacteria ratio range (1:1~1:2): Ensure that the synergistic metabolism of Bacillus subtilis and Rhodopseudomonas palustris reaches the optimal level, so as to achieve efficient secretion of EPS and passivation of heavy metals; (2) Targeted acclimatization treatment: to enable the strain to acquire specific adaptability to the PE-Cd compound pollution environment, and to ensure the functional stability of the bacterial agent under complex pollution conditions; (3) Modular microbial agent design: The core microbial community (Bs + Rp) ensures the heavy metal remediation function, and pesticide degradation microbial modules can be added as needed to expand the broad spectrum remediation capability, realizing a flexible application mode of "one microbial multi-effect and as-needed combination".

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics, characterized in that: The compound microbial agent includes Bacillus subtilis and Rhodopseudomonas palustris, which have been selectively domesticated.

2. The composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics according to claim 1, characterized in that: The weight ratio of Bacillus subtilis and Rhodopseudomonas palustris is 1:1~2.

3. The composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics according to claim 1, characterized in that: The compound microbial agent also includes alkaloid Pseudomonas aeruginosa and Fusarium, and the total amount of alkaloid Pseudomonas aeruginosa and Fusarium is 10-30% of the total amount of Bacillus subtilis and Rhodopseudomonas palustris.

4. The composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics according to claim 3, characterized in that: The mass ratio of alkalogenic Pseudomonas to Fusarium is 1:

1.

5. A method for preparing a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics, characterized in that: Includes the following steps: Step 1: Mix Bacillus subtilis and Rhodopseudomonas palustris in a certain ratio and inoculate them into a low-nutrient acclimatization medium. Use polyethylene microplastics and Cd composite contamination to create a target stress and set a concentration gradient. Perform stepwise subculturing at each concentration. When the strain grows stably at 2 times the concentration and the extracellular polymer secretion is ≥30% higher than before acclimatization, the acclimatization is complete. Step 2: After directional domestication, Bacillus subtilis and Rhodopseudomonas palustris were inoculated into LB liquid medium and photosynthetic cell medium, respectively, and cultured until the logarithmic growth phase to activate the strains; Step 3: Centrifuge the bacterial solution to collect the bacterial cells and resuspend them by weight. Step 4, the bacillus subtilis and rhodopseudomonas palustris obtained after step 3 are compounded, and the total bacterial concentration is adjusted to 1 x 10 9 CFU / mL.

6. The method for preparing a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics according to claim 5, characterized in that: The activation temperature of the strain is 30~35℃, the shaking speed is 180~220rpm, and the time is 12~18h.

7. The method for preparing a composite microbial agent for remediating cadmium pollution mediated by polyethylene microplastics according to claim 5, characterized in that: Alkalogenic Pseudomonas and Fusarium were also added during the compounding process.

8. The application of a composite microbial agent according to any one of claims 1 to 4 or a composite microbial agent prepared by any one of claims 5 to 7 in tea garden soil contaminated with cadmium mediated by polyethylene microplastics.

9. An application according to claim 8, characterized in that: The concentration of the complex microbial agent ranges from 2.5 x 10 7 1.0 x 10 8 CFU / g.

10. An application according to claim 8, characterized in that: The concentration range of the compound microbial agent is 5.0 × 10⁻⁶. 7 ~1.0×10 8 CFU / g.

Images