Broad-spectrum compound bacterium system for harmlessly and efficiently degrading neonicotinoid pesticides and application of broad-spectrum compound bacterium system

Abstract

The invention provides a broad-spectrum compound bacterium system for harmlessly and efficiently degrading neonicotinoid pesticides and application of the broad-spectrum compound bacterium system. The broad-spectrum compound bacterial system provided by the invention comprises a bacterial strain Pantoea agglomerans, a bacterial strain Enterobacter hormaechei and a bacterial strain Raoultella ornitinolytica, and the broad-spectrum compound bacterial system provided by the invention comprises the following components: a bacterial strain Pantoea agglomerans, a bacterial strain Enterobacter hormaechei and a bacterial strain Raoultella ornitinolytica. A compound bacterial system formed by compounding the three strains can harmlessly and efficiently degrade different types of NNIs (acetamiprid, imidacloprid and thiamethoxam), and the compound bacterial system is fixed on porous coating sponge with a large specific surface area, so that efficient, stable and repeated utilization can be realized. The broad-spectrum compound bacterial system provided by the invention is simple in component and good in stability, has broad-spectrum characteristics, and can realize harmless degradation of different types of NNIs. By optimizing the assembly mode of the strains, the removal rate of the NNIs can be remarkably increased, and deep mineralization of the NNIs can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of ecological environment restoration, and in particular to a broad-spectrum composite bacterial system for harmlessly and efficiently degrading neonicotinoid pesticides and applications thereof. Background Art

[0002] Neonicotinoids (NNIs) are the most widely used insecticide class worldwide due to their low toxicity, high efficacy, and good systemic absorption. Although NNIs effectively kill a wide range of target organisms, only approximately 10% is absorbed by crops, with the remainder remaining on crop surfaces and in the soil. Compared to other pesticides, NNIs are highly water-soluble, less volatile, and difficult to degrade. They can easily enter aquatic ecosystems through rainwater washoff, surface runoff, and soil leaching, causing pollution in various water bodies.

[0003] Currently, the main degradation technologies for NNIs, both domestically and internationally, include chemical oxidation, Fenton oxidation, photocatalysis, electrocatalysis, adsorption, and biological methods. Physical and chemical methods can effectively degrade NNIs, but they are costly and prone to secondary pollution. Bioremediation technologies, with their advantages of safety, effectiveness, low cost, no secondary pollution, ease of in-situ operation, and suitability for non-point source pollution control, have attracted considerable attention in the field of environmental remediation. Microorganisms are crucial pollutant decomposers in nature and play a crucial role in the degradation and transformation of pesticides. Therefore, microbial-based environmental remediation technologies hold broad application prospects in the ecological restoration of agricultural soils and water environments.

[0004] In recent years, researchers at home and abroad have identified numerous NNI-degrading strains or bacterial communities from contaminated environments. However, these NNI-dominant strains are typically only capable of degrading a single pesticide. Furthermore, most microorganisms can only decompose pesticides into a series of intermediate transformation products (TPs), failing to completely mineralize them. Furthermore, some TPs are even more environmentally hazardous than their parent pesticides. Treatment technologies that focus solely on the parent pesticides while ignoring toxic TPs are bound to lead to secondary pollution. Therefore, utilizing microbial remediation technologies to achieve harmless, efficient, and even complete mineralization of NNIs has become a pressing scientific challenge in the field of environmental remediation. Summary of the Invention

[0005] The purpose of the present invention is to provide a broad-spectrum composite bacterial system and its application for harmlessly and efficiently degrading neonicotinoid pesticides, so as to solve the problem that the current NNIs dominant strains can usually only degrade a single type of pesticide and easily produce toxic intermediates during the degradation of neonicotinoid pesticides.

[0006] The present invention is achieved in that:

[0007] The present invention screened out three dominant bacterial strains that can degrade different types of NNIs (acetamiprid, imidacloprid, and thiamethoxam). By combining the strains, a new broad-spectrum composite bacterial system with simple components that can harmlessly and efficiently degrade NNIs was constructed. The system was then fixed on a porous, large-surface-area coating sponge to achieve efficient, stable, and reusable use.

[0008] The three dominant strains screened out in the present invention are Pantoea agglomerans (P-1 for short), Enterobacter hormaechei (E-2 for short) and Raoultella ornithinolytica (R-3 for short).

[0009] In the above scheme, preferably, the composite bacterial system is prepared by mixing P-1, E-2, and R-3 in different proportions, and the bacterial solution (OD 600 =1.0) The combination ratio is x:y:z (x=1~2, y=1~2, z=1~2).

[0010] In the above scheme, preferably, the composite bacterial system is firmly "anchored" on the porous coating sponge through the adhesion effect of PDA to form an immobilized composite bacterial system, and its 24-hour leakage rate is less than 7%.

[0011] In the above scheme, preferably, by adjusting the dosage of DA (0.01-5.0 g / 20 mL Tris-HCl buffer), water bath temperature (5-80° C.), and reaction time (0.5-24 h), immobilized carriers with different adhesion forces can be obtained.

[0012] In the above scheme, preferably, by controlling the composite bacterial suspension (0.1-5.0 mL, OD 600 =1), the immobilized composite bacterial system with different loading amounts can be obtained.

[0013] The broad-spectrum composite bacterial system provided by the present invention can be used to harmlessly and efficiently degrade neonicotinoid pesticides, including acetamiprid, imidacloprid, and thiamethoxam.

[0014] Preferably, the composite bacterial system can degrade one or more of acetamiprid, imidacloprid and thiamethoxam in water or soil with a pH of 3 to 11 and a temperature of -5 to 40°C; the concentration of acetamiprid, imidacloprid and thiamethoxam in the water or soil is 0.01 to 1000 mg / L.

[0015] Preferably, the composite bacterial system can simultaneously degrade one or more of acetamiprid, imidacloprid, and thiamethoxam.

[0016] Preferably, after the composite bacterial system is fixed on the porous coating sponge to form an immobilized composite bacterial system, it can be reused efficiently and stably, and the number of reuses is 2 to 100 times.

[0017] Preferably, by adjusting the composition ratio of the composite bacterial system, the composite bacterial system can simultaneously degrade one or more of acetamiprid, imidacloprid and thiamethoxam in the contaminated soil, and the mineralization degree is higher than 80%.

[0018] More preferably, by optimizing the composition ratio of the composite bacterial system, the composite bacterial system can be used to degrade one or more of acetamiprid, imidacloprid, and thiamethoxam in polluted water, with a mineralization degree higher than 90% and a corresponding TOC removal rate higher than 80%.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) The broad-spectrum composite bacterial system provided by the present invention can efficiently degrade different types of NNIs, breaking through the limitation that most current microorganisms can only decompose a single pesticide.

[0021] (2) Compared with a single bacterial community, the broad-spectrum composite bacterial system provided by the present invention has stronger environmental tolerance and stability, stable propagation, high degradation efficiency, greatly shortened degradation time, and can utilize the synergistic effect of multiple microorganisms to efficiently degrade multiple pesticides. It also contains a rich degradation enzyme system, which can achieve complete degradation of intermediate metabolites.

[0022] (3) The broad-spectrum composite bacterial system provided by the present invention has simple components, good stability, and broad-spectrum properties, enabling harmless degradation of different types of NNIs. By optimizing the combination of strains, the removal rate of NNIs can be significantly improved, and deep mineralization can be achieved.

[0023] (4) By immobilizing the free composite bacterial system on a porous coated sponge carrier, the present invention obtains a low-cost, high-efficiency, stable, and reusable immobilized composite bacterial system. This composite bacterial system can be used for in-situ remediation of actual water bodies and soils, has broad market prospects and development potential, and is of great practical significance for promoting the application of microbial remediation technology in the environmental field. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are optical microscope and SEM images of three broad-spectrum dominant strains; among them, a corresponds to the first strain Pantoea agglomerans (abbreviated as P-1), b corresponds to the second strain Enterobacter hormaechei (abbreviated as E-2), and c corresponds to the third strain Raoultella ornithinolytica (abbreviated as R-3).

[0025] Figure 2 is the phylogenetic tree of strain Pantoea agglomerans.

[0026] Figure 3 is the phylogenetic tree of strain Enterobacter hormaechei.

[0027] Figure 4 is the phylogenetic tree of strain Raoultella ornithinolytica.

[0028] Figure 5 is the degradation efficiency of the three dominant bacteria on different initial concentrations of acetamiprid; among them, a corresponds to strain P-1, b corresponds to strain E-2, and c corresponds to strain R-3.

[0029] Figure 6 is the degradation efficiency of acetamiprid (C0=20 mg / L) by the three dominant bacteria at different pH values; wherein a corresponds to strain P-1, b corresponds to strain E-2, and c corresponds to strain R-3.

[0030] Figure 7 is the degradation efficiency of acetamiprid (C0=20 mg / L) by three dominant bacteria at different temperatures; among them, a corresponds to strain P-1, b corresponds to strain E-2, and c corresponds to strain R-3.

[0031] Figure 8 The graphs are the residual amount and rate constant of acetamiprid under different composition ratios of the composite bacteria system; among them, a is the residual amount graph under different composition ratios, and b is the reaction rate constant graph under different composition ratios.

[0032] Figure 9 It is the degradation efficiency of the composite bacterial system on acetamiprid and the TOC removal rate under the optimal combination ratio.

[0033] Figure 10 1 are SEM images of the porous coating sponge and the composite bacterial system fixed on the porous coating sponge; wherein, a is the SEM image of the porous coating sponge, and b is the SEM image of the composite bacterial system fixed on the porous coating sponge.

[0034] Figure 11 It is the remediation effect of free composite bacterial system on soil contaminated by NNIs (acetamiprid, imidacloprid, thiamethoxam).

[0035] Figure 12 are the degradation rates of typical NNIs (acetamiprid, imidacloprid, and thiamethoxam) in actual surface water samples by the immobilized composite bacterial system and the TOC removal rates; where a is the degradation rate and b is the TOC removal rate.

[0036] Figure 13 It is the reusability of the immobilized composite bacterial system. DETAILED DESCRIPTION

[0037] The present invention constructs a broad-spectrum composite bacterial system with simple components, good stability, and the ability to harmlessly and efficiently degrade NNIs. This bacterial system is capable of efficiently degrading different types of NNIs (acetamiprid, imidacloprid, and thiamethoxam), and has the advantages of being environmentally friendly, low-cost, high-efficiency, simple to operate, stable, reusable, and having a wide pH range of applicability. More importantly, it can achieve a high degree of mineralization of NNIs, providing a new and efficient treatment approach for the in-situ remediation of their contaminated soil and water environments, which is of great practical significance for ensuring the sustainable development of the ecological environment and human health.

[0038] The present invention is described in detail below through specific embodiments.

[0039] 1. Isolation and purification of monoclonal dominant strains and their degradation characteristics

[0040] Acclimated soil samples were mixed with sterilized mineral salt medium (MSM). Aqueous dispersants of acetamiprid, imidacloprid, and thiamethoxam were added to three different plots, and the cultures were shaken at 200 rpm and 30°C. After multiple rounds of induced acclimation and continuous enrichment using soil-water mixtures and liquid culture media, single colonies were isolated and purified using a gradient dilution method. Single colonies with good growth and stable passages were selected for expansion and identification using 16S rDNA sequencing. Three dominant strains of NNIs were identified through testing of their degradation capacity.

[0041] The morphology of the three dominant strains is as follows Figure 1 The phylogenetic tree is shown in Figure 2 、 Figure 3 、 Figure 4 The first is Pantoea agglomerans (P-1), a Gram-positive strain with a regular 5μm rod-shaped electron microscopic morphology. The second is Enterobacter hormaechei (E-2), a Gram-negative strain with a 7μm rod-shaped electron microscopic morphology. The third is Raoultella ornithinolytica (R-3), a Gram-positive strain with a 4μm rod-shaped electron microscopic morphology.

[0042] The growth factors of the three dominant strains were optimized to improve the degradation efficiency. Detailed steps: The suspension of the three dominant strains in the logarithmic growth phase was inoculated into 20mL MSM culture medium (C0=10mg / L), and cultured at 30℃ and 120rpm for 5 days. Then, all the above bacterial solutions were centrifuged at 2500rpm for 3min, repeated 3 times, and resuspended with 2mL PBS to obtain an acclimated bacterial suspension. 2mL of the acclimated bacterial suspension was inoculated into 20mL MSM culture medium (C0=10mg / L). The pH (5, 6, 7, 8, 9), temperature (25℃, 30℃, 35℃), inoculation amount (0.5% (0.1mL), 2% (0.4mL), 5% (1mL), 10% (2mL), 15% (3mL), 20% (4mL), bacterial solution OD 600 =1), additional carbon and nitrogen sources (0.05-2.0 g / L), and initial acetamiprid concentrations (C0, 20 mg / L, 40 mg / L, 100 mg / L, 200 mg / L) were optimized to determine the optimal conditions. The growth factors that significantly affected the degradation rate of acetamiprid were the initial concentration of NNIs, pH value, and temperature. The effects of these three growth factors on the degradation rate of acetamiprid are shown in Figure 2. Figure 5 、 Figure 6 、 Figure 7 .Depend on Figure 5 It can be seen that too high an initial concentration of acetamiprid will inhibit the degradation rate of the strain, and the degradation rate is higher when the initial concentration is 20 mg / L and 40 mg / L. Figure 6 It can be seen that the three broad-spectrum dominant strains can maintain high catalytic degradation activity in a wide pH range. Figure 7 It can be seen that at the temperature of 25℃~35℃, the three broad-spectrum dominant strains all have good catalytic degradation activity.

[0043] 2. Construction and immobilization of composite bacterial system

[0044] The bacterial suspensions of P-1, E-2 and R-3 in logarithmic growth phase (OD 600 =1.0) were inoculated into MSM culture medium containing acetamiprid (C0=20 mg / L) according to different group volume ratios (1:1:1, 1:1:2, 2:1:1, 2:1:2, 2:2:1, 1:2:1, 1:2:2), cultured at pH=7, 30°C, and 120rpm with shaking, sampled regularly, filtered through a 0.22μm water filter membrane, and the residual amount of acetamiprid was detected by HPLC-MS / MS. At the same time, the removal rate of total organic carbon (TOC) was tested. By optimizing the inoculation ratio, the degradation efficiency of the composite bacterial system can be significantly improved. The effect of different group ratios on the degradation efficiency of the composite bacterial system is shown in Figure 2. Figure 8 As shown, it can be seen that the ratio of P-1, E-2, and R-3 is 1:1:1, which is the best group ratio.

[0045] The degradation efficiency of NNIs (taking acetamiprid as an example, C0 = 20 mg / L, degradation system is MSM medium (pH = 7)) and TOC removal rate of the composite bacterial system constructed under the optimal combination ratio (inoculation amount of each strain is 5%:5%:5%) are shown as follows: Figure 9 The results showed that the composite bacteria system could harmlessly degrade more than 95% of NNIs (C0 = 20 mg / L) within 3 days, with a mineralization degree higher than 78.6%.

[0046] Based on the adhesion effect of polydopamine (PDA), a composite microbial system constructed under optimal conditions was immobilized on a porous coating sponge using a one-pot method. 0.1 g of dopamine hydrochloride (DA) was dissolved in 20 mL of Tris-HCl buffer (10 mM, pH = 8.5), 10 mL of bacterial solution was added, and a piece of PDA-modified sponge (PDA / MOF@Sponge) was immersed in the bacterial solution. The mixture was stirred at 150 rpm in a water bath at 20-60 ° C for 2-6 hours and washed three times with DI water to obtain an immobilized composite microbial system (Microbe-PDA / MOF@Sponge). Its SEM morphology is shown in Figure 2. Figure 10 The results showed that the three bacteria were clearly attached to the MOF@Sponge skeleton. The outstanding advantage of this immobilized composite bacterial system is that it can be recycled and reused.

[0047] 3. Practical application of composite bacterial systems

[0048] The composite bacterial system was applied to the degradation of NNIs in soil media. Three 20 mL suspensions of the composite bacterial system with the best composition ratio (OD 600 = 1.0) were added to 500 g of soil containing acetamiprid, imidacloprid, and thiamethoxam (C0 of each NNI = 50 mg / kg, soil water holding capacity 60%), mixed evenly, and cultured in an artificial climate incubator at 28°C for 15 days. Figure 11 As shown in the figure, under the action of microorganisms, the removal rates of acetamiprid, imidacloprid and thiamethoxam are 98.1%, 91.4% and 85.3% respectively.

[0049] Figure 12This is the removal effect of the immobilized composite bacterial system on typical NNIs (acetamiprid, imidacloprid, thiamethoxam, C0 = 20 mg / L) in actual surface water samples. When the immobilized composite bacterial system (sponge 10cm×10cm×10cm) was used to treat 1L of water sample, the degradation rates of acetamiprid, imidacloprid and thiamethoxam reached 99.3%, 95.2% and 90.7% respectively within 7 days, and the corresponding TOC removal rates were 87.9%, 85.2% and 80.3% respectively, indicating that the immobilized composite bacterial system can efficiently degrade different types of NNIs and deeply mineralize them. The present invention evaluated the microbial leakage rate of the immobilized composite bacterial system in 100mL of deionized water under mild oscillation conditions of 100rpm through a static leakage test. The results showed that the cumulative leakage rates after immersion for 6h, 12h, and 24h were 3.40%, 5.20%, and 6.30%, respectively. The lower leakage rates indicated that the composite bacteria system was firmly "anchored" on the porous coating sponge through the adhesion effect of PDA.

[0050] The present invention further verifies the cyclic stability of the immobilized composite bacterial system. Figure 13 When the immobilized composite bacterial system (sponge 10cm×10cm×10cm) was used to treat 1L of water sample containing acetamiprid (C0=10mg / L) for 48 hours each time, the degradation efficiency still reached 82.3% after five consecutive uses, indicating that the prepared immobilized composite bacterial system can be reused efficiently and stably.

Claims

1. A broad-spectrum composite bacterial system that can harmlessly and efficiently degrade neonicotinoid pesticides, characterized by: These include strains Pantoea agglomerans, Enterobacter hormaechei, and Raoultella ornithinolytica.

2. The broad-spectrum composite bacterial system for harmless and efficient degradation of neonicotinoid pesticides according to claim 1 is characterized in that: The composite bacterial system is prepared by compounding bacterial liquids of three bacterial strains in logarithmic growth phase according to the ratio of x:y:z, wherein x=1-2, y=1-2, and z=1-2.

3. The broad-spectrum composite bacterial system for harmless and efficient degradation of neonicotinoid pesticides according to claim 2 is characterized in that: x=1, y=1, z=1.

4. The broad-spectrum composite bacterial system for harmless and efficient degradation of neonicotinoid pesticides according to claim 1 is characterized in that: The composite bacterial system is fixed on the porous coating sponge.

5. Use of the broad-spectrum composite bacterial system for harmlessly and efficiently degrading neonicotinoid pesticides according to any one of claims 1 to 4 in degrading neonicotinoid pesticides.

6. The use according to claim 5, characterized in that: The neonicotinoid pesticides include acetamiprid, imidacloprid and thiamethoxam.

7. The use according to claim 6, characterized in that: The composite bacterial system can degrade one or more of acetamiprid, imidacloprid and thiamethoxam in water or soil with a pH of 3 to 11 and a temperature of -5 to 40°C.

8. The use according to claim 7, characterized in that: The concentrations of acetamiprid, imidacloprid and thiamethoxam in water or soil are 0.01-1000 mg / L.

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