A method for degrading organochlorine pesticides using a microbial agent comprising Phanerochaete chrysosporium and Ochrobacterium intermedia

Abstract

The present invention relates to a microbial agent comprising Phanerochaete chrysosporium and Ochrobacterium intermedia and a method for degrading organochlorine pesticides. The microbial agent comprises Phanerochaete chrysosporium and Ochrobacterium intermedia; the classification name of Phanerochaete chrysosporium is Phanerochaete chrysosporium. Phanerochaete chrysosporium , the deposit number is CGMCC No. 41170; the classification of the intermedia Ochrobacterium is named as Intermedia Ochrobacterium Ochrobactrum intermedium , deposit number is CGMCC No. 33645. The microbial agent disclosed in the present invention can efficiently degrade organochlorine pesticides.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a microbial agent comprising Phanerochaete chrysosporium and Ochrobacterium intermedia and a method for degrading organochlorine pesticides. Background Art

[0002] Organochlorine pesticides are a class of chlorine-containing organic synthetic pesticides, primarily including DDT, hexachloride (HCH), chlordane, aldrin, and dieldrin. Since the 1940s, these pesticides have been mass-produced and widely used worldwide in agriculture, forestry, and health sectors due to their highly effective insecticidal and fungicidal properties, relatively simple production processes, and low costs. They are used to control crop pests, forest pests, and sanitary pests, playing a vital role in ensuring agricultural production and controlling the spread of diseases such as malaria. However, over time, it has been discovered that organochlorine pesticides are highly stable and lipid-soluble, making them difficult to degrade in the environment. They can persist for long periods and accumulate in organisms and humans through bioaccumulation and biomagnification in the food chain, posing a serious threat to the ecological environment and human health. For example, high concentrations of organochlorine pesticides in many birds can lead to thinning of their eggshells and reduced reproductive capacity. Long-term exposure to organochlorine pesticide residues in humans can also increase the risk of cancer, disrupt the endocrine system, and affect reproductive and immune function.

[0003] Currently, while traditional physical and chemical treatment methods (such as incineration, adsorption, and chemical oxidation) can remove organochlorine pesticides, they have certain limitations. These include the high cost of energy and chemical reagents required for the treatment process; the potential for the production of toxic byproducts, which can cause secondary pollution; and the difficulty of large-scale application in soil and water remediation, resulting in a limited scope of application. Microbial degradation has become a research hotspot due to its advantages of high efficiency, environmental friendliness, and low cost. Many microorganisms (such as bacteria and fungi) have the ability to degrade organochlorine pesticides. Several highly efficient degrading strains have been isolated, including Pseudomonas, Sphingomonas, and white-rot fungi. However, the reported degrading strains (such as white-rot fungi and some Pseudomonas) lack tolerance to high concentrations of organochlorine pesticides and have limited dechlorination capabilities for complex chlorinated compounds, resulting in low degradation efficiency and a narrow degradation spectrum. Therefore, the development of efficient and environmentally friendly composite microbial degradation systems is urgently needed. Summary of the Invention

[0004] The purpose of the present disclosure is to provide a microbial agent comprising Phanerochaete chrysosporium and Ochrobacterium intermedia and a method for degrading organochlorine pesticides. The microbial agent of the present disclosure can efficiently degrade organochlorine pesticides.

[0005] In order to achieve the above-mentioned object, the present disclosure provides a microbial agent for the degradation of organochlorine pesticides in a first aspect, wherein the microbial agent comprises Phanerochaete chrysosporium and Ochrobacterium intermedia;

[0006] The classification of the fungus is named Phanerochaete chrysosporium Phanerochaete chrysosporium , the deposit number is CGMCC No. 41170; the classification of the intermedia Ochrobacterium is named as Intermedia Ochrobacterium Ochrobactrum intermedium , the deposit number is CGMCC No. 33645.

[0007] Optionally, the dosage form of the microbial agent includes a liquid agent and / or a solid agent.

[0008] Optionally, the microbial agent is a solid agent, and the number of viable bacteria of Phanerochaete chrysosporium in the microbial agent is (2-9)×10 8 CFU / g.

[0009] Optionally, the microbial agent is a liquid agent, and the number of viable bacteria of Phanerochaete chrysosporium in the microbial agent is (2-9)×10 8 CFU / mL.

[0010] Optionally, in the microbial agent, the ratio of the number of viable bacteria of the Phanerochaete chrysosporium to the number of viable bacteria of the Ochrobacterium intermedia is (1-3):1.

[0011] The second aspect of the present disclosure provides a method for degrading organochlorine pesticides, wherein the degradation agent used includes the microbial agent described in the first aspect of the present disclosure.

[0012] Optionally, the organochlorine pesticide includes one or more of bis(p-chlorophenyl)trichloroethane, 2,4-dichlorophenol and γ-hexachlorocyclohexane.

[0013] Optionally, the degradation is carried out in an air atmosphere under the following conditions: pH value of 5.0-8.5, temperature of 25-30° C., and time of 168-336 h.

[0014] Optionally, the method includes applying the microbial agent to a water body.

[0015] Optionally, the application amount of the microbial agent is (0.8-1.6)×10 8 CFU.

[0016] Through the above technical solution, the present invention compounds specific Phanerochaete chrysosporium and Ochrobacterium intermedia to form a microbial agent, which has a synergistic effect, so that the microbial agent has a high efficiency in degrading a variety of organochlorine pesticides and pesticide intermediates. It can reduce the residual amount of organochlorine pesticides in the water and soil environment in a short period of time, reduce the harm of organochlorine pesticides to the environment and organisms, improve the utilization efficiency of soil and water bodies, and reduce production costs. The method disclosed in the present invention is environmentally friendly, avoids the secondary pollution problems caused by traditional physical degradation and chemical degradation methods, produces good economic and ecological benefits, and has good application prospects.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description.

[0018] Biomaterial deposit information

[0019] Ochrobacterium intermedia of the present disclosure Ochrobactrum intermedium The strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms (CGMCC), the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is: February 24, 2025, the strain deposit number is: CGMCC No. 33645. DETAILED DESCRIPTION

[0020] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0021] A first aspect of the present disclosure provides a microbial agent for degrading organochlorine pesticides, wherein the microbial agent comprises Phanerochaete chrysosporium and Ochrobacterium intermedia;

[0022] The classification of the fungus is named Phanerochaete chrysosporium Phanerochaete chrysosporium , the deposit number is CGMCC No. 41170; the classification of the intermedia Ochrobacterium is named as Intermedia Ochrobacterium Ochrobactrum intermedium , the deposit number is CGMCC No. 33645.

[0023] The inventors of the present disclosure isolated Phanerochaete chrysosporium and Ochrobacterium intermedia and compounded them into a microbial agent. The two agents exhibit synergistic effects. Compared to single agents, the compound agent has highly efficient degradation capabilities against a variety of organochlorine pesticides and pesticide intermediates, broadening the degradation spectrum, providing high tolerance to high-concentration organochlorine pesticides, and exhibiting good dechlorination capabilities for organochlorine pesticides with complex structures. The microbial agent disclosed herein can efficiently and environmentally reduce the content of organochlorine pesticides in water and soil environments, reducing the harm of organochlorine pesticides to the environment and organisms, and avoiding the problems of physical and chemical degradation methods that are prone to secondary pollution and have limited scope of application.

[0024] According to one embodiment of the present disclosure, the microbial agent is used to degrade organochlorine pesticides in soil and / or water.

[0025] According to one embodiment of the present disclosure, the microbial agent consists of Phanerochaete chrysosporium, Ochrobacterium intermedia, optional sterile water and optional buffer.

[0026] According to one embodiment of the present disclosure, the dosage form of the microbial agent includes a liquid agent and / or a solid agent. When the microbial agent is a solid agent, it can be directly administered or configured as a liquid agent for administration; the liquid agent can be directly administered after being cultured in a liquid culture medium, or it can be configured as a liquid agent for administration after being cultured.

[0027] According to one embodiment of the present disclosure, the microbial agent is a solid agent, in which the number of viable bacteria of Phanerochaete chrysosporium is (2-9)×10 8 CFU / g, including but not limited to 2×10 8 CFU / g, 2.5×10 8 CFU / g, 3×10 8 CFU / g, 3.5×10 8 CFU / g, 4×10 8 CFU / g, 4.5×10 8 CFU / g, 5×10 8 CFU / g, 5.5×10 8 CFU / g, 6×10 8 CFU / g, 6.5×10 8 CFU / g, 7×10 8 CFU / g, 7.5×10 8 CFU / g, 8×10 8 CFU / g, 8.5×10 8 CFU / g, or a range consisting of any two thereof; microbial agents with the above-mentioned viable bacterial counts have better organochlorine pesticide degradation effects.

[0028] According to one embodiment of the present disclosure, the microbial agent is a liquid agent, in which the number of viable bacteria of Phanerochaete chrysosporium is (2-9)×10 8 CFU / mL, including but not limited to 2×10 8 CFU / mL, 2.5×10 8 CFU / mL, 3×10 8 CFU / mL, 3.5×10 8 CFU / mL, 4×10 8 CFU / mL, 4.5×10 8 CFU / mL, 5×10 8 CFU / mL, 5.5×10 8 CFU / mL, 6×10 8 CFU / mL, 6.5×10 8 CFU / mL, 7×10 8 CFU / mL, 7.5×10 8 CFU / mL, 8×10 8 CFU / mL, 8.5×10 8 CFU / mL, or a range consisting of any two thereof; microbial agents with the above viable bacterial counts have better organochlorine pesticide degradation effects.

[0029] According to one embodiment of the present disclosure, in the microbial agent, the ratio of the number of viable bacteria of the Phanerochaete chrysosporium to the number of viable bacteria of the Ochrobacterium intermedia is (1-3):1, preferably (2-2.5):1, including but not limited to 1:1, 1.2:1, 1.5:1, 1.8:1, 2:1, 2.2:1, 2.5:1, 2.75:1, 2.9:1, 3:1, or a range consisting of any two thereof. In the microbial agent with the above ratio, Phanerochaete chrysosporium and Ochrobacterium intermedia have good synergistic effect, which can further improve the degradation rate of organochlorine pesticides.

[0030] According to one embodiment of the present disclosure, the microbial agent is a liquid agent, and the method for preparing the microbial agent includes: configuring Phanerochaete chrysosporium and Ochrobacterium intermedia to form a bacterial suspension; specifically, mixing the bacterial suspension of Phanerochaete chrysosporium with the bacterial suspension of Ochrobacterium intermedia; centrifuging or filtering the cultured Phanerochaete chrysosporium and Ochrobacterium intermedia separately, collecting the bacterial bodies, and resuspending them with sterile water or buffer respectively to obtain a bacterial suspension of Phanerochaete chrysosporium and a bacterial suspension of Ochrobacterium intermedia, and then mixing the two; or, directly resuspending the cultured bacterial bodies of Phanerochaete chrysosporium and Ochrobacterium intermedia with sterile water or buffer to form a bacterial suspension; the type of buffer is conventional in the art.

[0031] According to one embodiment of the present disclosure, the microbial agent is a solid agent, and the method for preparing the microbial agent includes: mixing and stirring a composite bacterial solution, peat soil, diatomaceous earth, and peptone in a weight ratio of 2:0.3:0.5:0.3, and then freeze-drying to prepare a solid composite bacterial agent.

[0032] According to one embodiment of the present disclosure, Phanerochaete chrysosporium can be cultured in a culture medium. The culture medium type and culture method are not particularly limited. For example, it can be a PDB liquid culture medium, cultured at 28°C and 180 rpm for 72 to 120 hours, until the viable cell count reaches 10 6 to 10 7 CFU / mL, the specific formula of the culture medium is not particularly limited.

[0033] According to one embodiment of the present disclosure, Ochrobacter intermedia can be cultured in a culture medium. The type of culture medium and the culture method are not particularly limited. For example, it can be a nutrient broth medium (NB) and cultured at 32°C and 180 rpm for 48 to 60 hours until the number of viable bacteria reaches 10 8 to 10 9 CFU / mL, the specific formula of the culture medium is not particularly limited.

[0034] The second aspect of the present disclosure provides a method for degrading organochlorine pesticides, wherein the degradation agent used includes the microbial agent described in the first aspect of the present disclosure.

[0035] According to one embodiment of the present disclosure, the organochlorine pesticide includes one or more of dichlorodiphenyltrichloroethane (commonly known as DDT), 2,4-dichlorophenol and γ-hexachlorocyclohexane (commonly known as lindane).

[0036] According to one embodiment of the present disclosure, the degradation is carried out in an air atmosphere under the following conditions: pH value of 5.0-8.5, temperature of 25-30°C, and time of 168-336 hours. The above conditions are conducive to the degradation of organochlorine pesticides and improve the degradation rate.

[0037] According to one embodiment of the present disclosure, the method comprises: applying the microbial agent to soil, wherein the concentration of the organochlorine pesticide in the soil is 50-150 mg / kg; optionally, the application amount of the microbial agent is (0.8-1.6)×10 8 CFU, including but not limited to 0.8×10 8 CFU, 0.9×10 8 CFU, 1.0×10 8 CFU, 1.1×10 8 CFU, 1.2×10 8 CFU, 1.3×108 CFU, 1.4×10 8 CFU, 1.5×10 8 CFU, 1.6×10 8 CFU, or a range consisting of any two thereof; the above dosage can reduce the application amount of the bacterial agent while maintaining a high degradation rate, thereby reducing costs.

[0038] According to one embodiment of the present disclosure, the method comprises: applying the microbial agent to a water body, wherein the concentration of the organochlorine pesticide in the water body is 50-150 mg / L; optionally, the application amount of the microbial agent is (0.8-1.6)×10 8 CFU, including but not limited to 0.8×10 8 CFU, 0.9×10 8 CFU, 1.0×10 8 CFU, 1.1×10 8 CFU, 1.2×10 8 CFU, 1.3×10 8 CFU, 1.4×10 8 CFU, 1.5×10 8 CFU, 1.6×10 8 CFU, or a range consisting of any two thereof; the above dosage can reduce the application amount of the bacterial agent while maintaining a high degradation rate, thereby reducing costs.

[0039] The present invention is further described in detail below with reference to the examples, but the scope of the present invention is not limited to the following examples.

[0040] Unless otherwise specified, all culture media used were commercially available products.

[0041] Test method for the content of organochlorine pesticides in soil: carried out according to the method described in the standard "Gas Chromatography Method for the Determination of Organochlorine Pesticides in Soil and Sediments" (HJ 921-2017).

[0042] Test method for the content of organochlorine pesticides in water: carried out according to the method described in the standard "Determination of organochlorine pesticides and polychlorinated biphenyls in water by gas chromatography" (SL 497-2010).

[0043] Example 1

[0044] 1. Strain culture

[0045] (1) Inoculate Phanerochaete chrysosporium CGMCC No. 41170 into PDB liquid culture medium and shake culture at 28℃ and 180 r / min for 48 hours to obtain seed liquid. Then inoculate the seed liquid into fresh PDB liquid culture medium at a 5% inoculum volume and shake culture for 5 days until the bacterial concentration reaches 10 6 to 10 7 CFU / mL.

[0046] 200g potatoes (peeled, cut into small pieces, boiled for 30 minutes, filtered through gauze to collect the filtrate), 20g glucose, 1000mL distilled water, natural pH value (no additional adjustment required).

[0047] (2) Inoculate Ochrobacterium intermedia CGMCC No. 33645 into NB liquid culture medium and culture at 32°C and 180 rpm for 48 h until the viable count reaches 10 8 to 10 9 CFU / mL.

[0048] NB liquid culture medium formula: tryptone (10 g / L), beef extract powder (5 g / L), NaCl (5 g / L).

[0049] 2. Bacterial liquid preparation

[0050] The cultured bacterial liquids of Phanerochaete chrysosporium CGMCC No. 41170 and Ochrobacter intermedia CGMCC No. 33645 were centrifuged separately, the bacterial bodies were collected, and resuspended in sterile water to prepare bacterial suspensions.

[0051] 3. Preparation of microbial agents

[0052] The bacterial suspension of Phanerochaete chrysosporium CGMCC No. 41170 and the bacterial suspension of Ochrobacter intermedia CGMCC No. 33645 were mixed in proportion to obtain microbial agent A1. The parameters are listed in Table 1.

[0053] Examples 2 to 5

[0054] Microbial agents A2 to A5 were prepared using the method of Example 1, except that the viable counts and ratios of Phanerochaete chrysosporium and Ochrobacterium intermedia in the microbial agents were different from those in Example 1. The parameters are listed in Table 1.

[0055] Comparative Example 1

[0056] The microbial agent D1 was prepared by the method of Example 1, except that the Ochrobactrum intermedia suspension was not added. The parameters are listed in Table 1.

[0057] Comparative Example 2

[0058] The microbial agent D2 was prepared by the method of Example 1, except that the Phanerochaete chrysosporium suspension was not added. The parameters are listed in Table 1.

[0059] Comparative Example 3

[0060] The microbial agent D3 was prepared by the method of Example 1, except that the bacterial suspension of Phanerochaete chrysosporium CGMCC No. 41170 was replaced with a bacterial suspension of Phanerochaete chrysosporium CGMCC 5.0776 (purchased from the General Microbiology Center of China Culture Collection Administration). The parameters are listed in Table 1.

[0061] Comparative Example 4

[0062] The microbial agent D4 was prepared by the method of Example 1, except that the bacterial suspension of Ochrobacterium intermedia CGMCC No. 33645 was replaced with a commercially available bacterial suspension of Ochrobacterium intermedia HZB220764 (Huicao Biotechnology Co., Ltd.). The parameters are listed in Table 1.

[0063] Table 1

[0064]

[0065] Test Example 1 Degradation of organochlorine pesticides in soil

[0066] A pot experiment was conducted to determine the degradation rate of organochlorine pesticides by microorganisms. Dichlorodiphenyltrichloroethane (DDT), γ-hexachlorocyclohexane (lindane), or 2,4-dichlorophenol was uniformly mixed into the test soil at a concentration of 150 mg / kg. The test inoculum was then added to the soil and stirred thoroughly. Each pot contained 5 kg of soil at a pH of 7.0 and placed in an incubator set to 28°C. Eight days after application of the inoculum, the soil levels of DDT, lindane, and 2,4-dichlorobenzene were measured. This was repeated three times. The results are shown in Table 2, and the data are the average of these three tests.

[0067] In Table 2, degradation rate = (pesticide content in soil before degradation treatment - pesticide content in soil after degradation treatment) / pesticide content in soil before degradation treatment × 100%.

[0068] Table 2

[0069]

[0070] The above data demonstrate that the microbial agent disclosed herein has a strong degradation effect on organochlorine pesticides in soil environments. A comparison of Test Example 5 with Test Examples 1-4 demonstrates that, under the same conditions, the degradation effect is enhanced when the ratio of viable Phanerochaete chrysosporium to viable Ochrobacterium intermedia is within the range of (1-3):1. Furthermore, a comparison of Test Examples 4 and 5 with Test Examples 3 and 6 demonstrates that the degradation effect is further enhanced when the ratio of viable Phanerochaete chrysosporium to viable Ochrobacterium intermedia is within the preferred range of (2.0-2.5):1.

[0071] Test Example 2 Degradation of organochlorine pesticides in water

[0072] An experiment on the degradation of organochlorine pesticides in water was conducted using an inorganic salt culture medium. The formula of the inorganic salt culture medium is: 1.0 g / L NH4NO3, 1.0 g / L NaCl, 1.5 g / L KH2PO4, 0.5 g / L K2HPO4, 0.1 g / L MgSO4, and the pH was adjusted to 5.5. DDT, lindane, or 2,4-dichlorophenol was added to the inorganic salt culture medium at a concentration of 150 mg / L. The test bacteria were inoculated into the inorganic salt culture medium. 200 mL of the inorganic salt culture medium was placed in a shake flask and cultured at 28°C and 200 r / min for 8 days. The DDT, lindane, and 2,4-dichlorobenzene contents in the culture medium were then determined. The test was repeated three times. The test results are listed in Table 3. The data in the table are the average values ​​of the three tests.

[0073] In Table 3, degradation rate = (pesticide content in the solution before degradation treatment - pesticide content in the solution after degradation treatment) / pesticide content in the solution before degradation treatment × 100%.

[0074] Table 3

[0075]

[0076] Based on the above data, the microbial agent disclosed herein has a good degradation effect on organochlorine pesticides in aquatic environments, effectively reducing the content of organochlorine pesticides in water. A comparison of Test Example 20 with Test Examples 16-19 shows that, under the same conditions, when the ratio of the viable count of Phanerochaete chrysosporium to the viable count of Ochrobacterium intermedia is within the range of (1-3):1, the degradation effect can be improved. A comparison of Test Examples 17 and 18 with Test Examples 16 and 19 shows that when the ratio of the viable count of Phanerochaete chrysosporium to the viable count of Ochrobacterium intermedia is within the preferred range of (2.0-2.5):1, the degradation effect can be further improved, reducing the content of organochlorine pesticides in water.

[0077] The preferred embodiments of the present disclosure are described in detail above. However, the present disclosure is not limited to the specific details of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.

[0078] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0079] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A method for degrading organochlorine pesticides, wherein the degradation agent used includes a microbial agent, characterized in that: The microbial agents include Phanerochaete chrysosporium and Ochrobacterium intermedia; The classification of the fungus is named Phanerochaete chrysosporium Phanerochaete chrysosporium , the deposit number is CGMCC No. 41170; the classification of the intermedia Ochrobacterium is named as Intermedia Ochrobacterium Ochrobactrum intermedium , deposit number is CGMCC No. 33645; In the microbial agent, the ratio of the number of viable bacteria of Phanerochaete chrysosporium to the number of viable bacteria of Ochrobacterium intermedia is (1-3):1; The organochlorine pesticides include one or more of di-p-chlorophenyl trichloroethane, 2,4-dichlorophenol and gamma-hexachlorocyclohexane.

2. The method according to claim 1, wherein The dosage form of the microbial agent includes liquid and / or solid dosage forms.

3. The method according to claim 2, wherein: The microbial agent is a solid agent, in which the number of viable bacteria of Phanerochaete chrysosporium is (2-9)×10 8 CFU / g.

4. The method according to claim 2, wherein: The microbial agent is a liquid agent, in which the number of viable bacteria of Phanerochaete chrysosporium is (2-9)×10 8 CFU / mL.

5. The method according to claim 1, wherein The degradation is carried out in an air atmosphere under the following conditions: pH value of 5.0-8.5, temperature of 25-30° C., and time of 168-336 hours.

6. The method according to claim 1, wherein The method comprises: applying the microbial agent to a water body.

7. The method according to claim 6, wherein: The application amount of the microbial agent is (0.8-1.6)×10 8 CFU.

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