Fungicide for degrading sulfonylurea herbicides as well as preparation method and application of fungicide

Abstract

The invention discloses a microbial inoculum for degrading sulfonylurea herbicides and a preparation method and application thereof, and relates to the technical field of pesticide biodegradation, the microbial inoculum takes weak positive electrochemical modified biochar as a carrier to load a composite flora, the composite flora is a composite flora composed of Ancylobacter aquaticus CGMCC1.2804, Aspergillus niger CGMCC3.15663 and Bacillus subtilis CGMCC1.821, and the microbial inoculum has the advantages that the microbial inoculum is used for degrading sulfonylurea herbicides, the microbial inoculum is used for degrading the sulfonylurea herbicides, the microbial inoculum is used for degrading the sulfonylurea herbicides, the microbial inoculum is used for degrading the sulfonylurea herbicides, and the microbial inoculum is used for degrading the sulfonylurea herbicides; the biochar is subjected to positive electrochemical modification, so that the removal effect of the complex microbial inoculant on a nicosulfuron herbicide is enhanced; under the condition of low natural organic matter nutrition of 10mg / L, the removal effects on tribenuron-methyl, nicosulfuron and halosulfuron-methyl which are lower than 10mu g / L can reach 78.14%, 80.28% and 92.38% respectively.

Description

Technical Field

[0001] This application relates to the field of pesticide biodegradation technology, specifically a microbial agent for degrading sulfonylurea herbicides, its preparation method, and its application. Background Technology

[0002] Sulfonylurea herbicides are a class of herbicides that inhibit the activity of acetolactate synthase (ALS) in plants. With their high biological activity and selectivity, unique mechanism of action, long residual effect and broad spectrum of weed control, they are widely used to control grasses and some broadleaf weeds in fields of major grain and oil crops such as wheat, rice, corn and soybean.

[0003] Studies have shown that, like most agricultural chemicals, sulfonylurea herbicides can remain in the environment for weeks to months, with some, such as metsulfuron-methyl, persisting for over a year. This prolonged persistence allows them to enter nearby rivers and lakes with rainfall and farmland runoff, posing potential ecological risks to the surrounding aquatic environment. Recent reports indicate that the sulfonylurea herbicide bensulfuron-methyl is highly toxic to some submerged plants; even a few micrograms per liter can pose a high ecological risk to Elodea. Therefore, effectively reducing sulfonylurea herbicides in farmland drainage to prevent their entry into environmental water bodies is crucial for maintaining local aquatic ecosystem health.

[0004] However, there are currently few practical technologies for the efficient reduction of agricultural herbicides. On the one hand, the environmental infrastructure in agricultural production is weak, making it difficult for conventional water treatment technologies to achieve both high efficiency and affordability. On the other hand, agricultural non-point source herbicides are often present in trace concentrations in wastewater, which also contain a certain proportion of natural organic matter and other pollutants, resulting in complex water quality conditions and significant fluctuations in wastewater discharge cycles and volumes, making it difficult for conventional technologies to operate stably and continuously.

[0005] From the perspective of solving practical problems, this invention considers the abundant resources of degrading microorganisms, the low technical threshold for screening and application, high environmental compatibility, and minimal risk of secondary pollution, making it one of the most promising means to cut off the agricultural source of herbicides. However, despite numerous reports on microorganisms that efficiently degrade sulfonylurea herbicides, these reported microorganisms are still far from practical application. First, trace concentrations of herbicides are insufficient to serve as the primary carbon and nitrogen sources for microorganisms; therefore, previously reported microorganisms that primarily use herbicides as carbon and nitrogen sources show poor degradation efficiency under these conditions. Second, existing degrading microorganisms have not been reported to efficiently degrade sulfonylurea herbicides under conditions where low concentrations of natural organic matter serve as the primary carbon and nitrogen sources. Third, due to differences in chemical structure, sulfonylurea herbicides also vary, and existing highly efficient degrading bacteria often show poor degradation efficiency for some sulfonylurea herbicides due to these differences in chemical structure.

[0006] Based on the above background, this invention constructs a sulfonylurea herbicide-degrading microbial community by combining the characteristics of various microorganisms. By optimizing their composition and structure, harmonious symbiosis among various microorganisms is achieved. Simultaneously, considering the problems often encountered in practical applications, such as high microbial loss rates due to a lack of stable growth environments, replacement by native bacteria due to insufficient competitiveness, and loss of synergistic effects due to imbalances within the microbial community, the biochar derived from the sintering of agricultural waste such as straw is appropriately modified to provide a suitable carrier for the degrading microbial community. This invention provides a practical technology for the degradation of agricultural non-point source sulfonylurea herbicides. Summary of the Invention

[0007] Technical Problem Solved: This application provides a microbial agent for degrading sulfonylurea herbicides, its preparation method, and its application. By using modified biochar loading, it achieves efficient degradation of trace concentrations of sulfonylurea herbicides under low-concentration natural organic matter conditions. This solves the problem in existing technologies where sulfonylurea herbicides can remain in the environment for weeks to months, with some herbicides, such as metsulfuron-methyl, even for over a year. This long residual time allows them to enter nearby rivers and lakes with rainfall and farmland runoff, posing potential ecological risks to the surrounding aquatic environment. Furthermore, the sulfonylurea herbicide bensulfuron-methyl is highly toxic to some submerged plants; even a few micrograms per liter can cause damage to Elodea. High ecological risks, complex water quality, and large fluctuations in drainage cycles and water volume make it difficult for conventional technologies to operate stably and continuously. Microorganisms that use herbicides as their main carbon and nitrogen sources are very ineffective in degrading them under these conditions. Existing degrading microorganisms have not been reported to have the ability to efficiently degrade sulfonylurea herbicides under conditions where natural organic matter is the main carbon and nitrogen source. Existing highly efficient degrading bacteria often have poor degradation effects on some sulfonylurea herbicides due to differences in their chemical structures. In practical applications, technical problems such as the lack of a stable growth environment leading to high microbial loss rates, insufficient competitiveness resulting in replacement by native bacteria, and imbalance in the proportion of bacteria within the microbial community leading to the loss of synergistic effects are often encountered.

[0008] The specific technical solution of this invention is as follows:

[0009] A microbial agent for degrading sulfonylurea herbicides, wherein the microbial agent uses weakly positively charged modified biochar as a carrier and loads a complex microbial community, wherein the complex microbial community consists of Ancylobacter aquaticus CGMCC1.2804, Aspergillus niger CGMCC3.15663, and Bacillus subtilis CGMCC1.821.

[0010] Furthermore, the complex microbial community comprises 15%-30% Ancylobacter aquaticus, 15%-30% Aspergillus niger, and 40%-70% Bacillus subtilis.

[0011] Furthermore, the weakly positively charged modified biochar carrier is prepared by calcining corn cobs at 400°C in an oxygen-free environment to obtain biochar, and then by positively charging it with amino groups as the main functional groups.

[0012] This application also discloses a method for preparing a microbial agent for degrading sulfonylurea herbicides, comprising the following steps:

[0013] Step 1: Preparation and modification of biochar: The collected corn cobs were rinsed with water to remove particulate matter, air-dried, crushed with a pulverizer, and calcined in a tube furnace under nitrogen protection. The heating rate was 3℃ / min, and the calcination time was 400℃ for 4 hours. After cooling to room temperature, the biochar was removed and passed through a 100-mesh sieve, with a particle size of 100-300μm. The obtained biochar BC was stirred with water for 12 hours and allowed to settle naturally to remove floating carbon powder and bottom ash. The obtained biochar material was placed in a mixture of sulfuric acid and nitric acid and mechanically stirred at 300 rpm for 8 hours. Two volumes of water were added to the mixed acid and stirred for 30 minutes. The supernatant was discarded, and ammonia and sodium thiosulfate were added until the pH reached 7. The volume ratio of ammonia to sodium thiosulfate was 1:1 (V:m). The mixture was mechanically stirred at 300 rpm for 5 hours. The supernatant was filtered, washed three times with water, dried, and stored to obtain modified biochar.

[0014] The second step is the cultivation of bacterial suspension: the compound bacterial suspension is inoculated into the culture medium to obtain a bacterial suspension;

[0015] The third step is the preparation of the bacterial agent: 0.1g-0.5g of modified biochar is added to 100mL of inorganic salt culture medium and sterilized in an autoclave at 121℃ for 20min. After cooling to room temperature, the bacterial suspension prepared in the second step is inoculated with inorganic salt culture medium: bacterial suspension at a volume ratio of 1:10. The suspension is fixed in a shaker at 200 rpm for 12h, then removed and centrifuged at 4000 rpm to remove the supernatant. The suspension is washed with 0.9% physiological saline, centrifuged again, and washed twice to obtain the bacterial agent loaded with modified biochar. After drying at 30℃, the suspension is stored in a refrigerator at 4℃.

[0016] Furthermore, in the second step, the culture medium consists of 2 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L sodium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate heptahydrate, 0.02 g / L calcium chloride, 1 mL Staub's trace element solution, and 5 mL sterile nutrient yeast extract. It is cultured in a shaker at 30°C for 24 h, and is ready for use when the OD600 value increases to 0.8 ± 0.1.

[0017] Furthermore, in the third step, the inorganic salt culture medium consists of 2 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L sodium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate heptahydrate, 0.02 g / L calcium chloride, and 1 mL Staub's trace element solution.

[0018] This invention also discloses the application of a microbial agent for degrading sulfonylurea herbicides in the enhanced reduction of sulfonylurea herbicides in farmland drainage ditches.

[0019] Explanation of the principle: To reduce the biotoxicity, especially the herbicidal activity, of sulfonylurea herbicides, molecular docking theory—a docking calculation based on the herbicide substrate and its target protein for herbicidal activity—was used to verify the results. It was found that the removal of sulfonylurea bridges and halogen atoms from some halogenated herbicides is key to reducing herbicidal activity. *Ancylobacter* bacteria have a strong ability to remove halogens, while *Aspergillus* bacteria have a strong ability to break sulfonylurea bridges. The combined effect of *Ancylobacter* and *Aspergillus* bacteria is highly likely to have a good degradation effect on various sulfonylurea herbicides.

[0020] As is well known, when microorganisms degrade pollutants, microorganisms from the same genus often exhibit different carbon and nitrogen source requirements due to their specific species. In other words, to achieve a synergistic effect, specific species within these two genera must first be selected. This invention, after multiple rounds of comparison, preferentially selected *Ancylobacter aquaticus* (CGMCC1.2804) and *Aspergillus niger* (CGMCC3.15663). Secondly, considering their nutritional requirements, another species capable of efficiently transforming environmental substrates was selected to provide stable carbon and nitrogen sources for *Ancylobacter aquaticus* and *Aspergillus niger*. This invention repeatedly compared several common genera and species with good degradation and transformation effects on natural organic matter (primarily considering the needs of practical applications; the laboratory used one or more of humic acid, bovine serum albumin, and sodium alginate for simulation). It was found that most species either inhibit *Ancylobacter aquaticus* and / or *Aspergillus niger*. The growth of Niger bacteria either causes them to lose their degradation ability (presumably due to a mismatch in carbon sources, etc., which prevents the expression of degradative enzymes); thus, the harmonious symbiotic relationship among the three is not only reflected in the number of species (proportion), but also in other more microscopic scales; through optimization, this invention found that Bacillus subtilis (CGMCC1.821) and Ancylobacter aquaticus have a good symbiotic relationship with Aspergillus niger, and under varying nutrient conditions (mainly referring to the simulated concentration and component ratio of natural organic matter), especially under oligotrophic (natural organic matter) environmental conditions, they can still have good removal effects on various sulfonylurea herbicides at low concentration levels.

[0021] The beneficial effects of this invention are:

[0022] 1. The compound microbial agent described in this invention patent has a good symbiotic relationship under natural organic matter conditions below 10 mg / L, and can achieve efficient degradation of various trace concentrations of sulfonylurea herbicides;

[0023] 2. The microbial agent described in this invention mainly contains Ancylobacter aquaticus (15%-30%), Aspergillus niger (15%-30%), and Bacillus subtilis (40%-70%), which have a good symbiotic relationship and synergistic effect, and have a good degradation effect on a variety of sulfonylurea herbicides.

[0024] 3. The microbial agent described in this invention, through appropriate positive electrochemical modification, enhances the removal efficiency of the compound microbial agent against nicosulfuron herbicide by up to 41.62%;

[0025] 4. The microbial agent described in this invention can achieve removal rates of 78.14%, 80.28%, and 92.38% for benzsulfuron, nicosulfuron, and clopyralid at concentrations below 10 μg / L under low natural organic matter nutrient conditions of 10 mg / L. Attached Figure Description

[0026] Figure 1 These are scanning electron microscope images of the microbial agent and its biochar material used to degrade sulfonylurea herbicides according to the present invention, wherein a is a surface morphology image of the composite microbial agent and b is a surface morphology image of the modified biochar.

[0027] Figure 2 This is a diagram showing the water contact angle and charge density of the modified biochar used in the microbial agent for degrading sulfonylurea herbicides according to the present invention, where a is the water contact angle diagram of the modified biochar under different modification conditions, and b is the charge density diagram of the modified biochar under different modification conditions.

[0028] Figure 3 This is a comparative graph showing the degradation effects of the microbial agent and its microbial raw materials for degrading sulfonylurea herbicides according to the present invention on various sulfonylurea herbicides. In the graph, a is the degradation of 10 mg / L high concentration nicosulfuron by the microbial agent and its microbial raw materials, and b is the degradation of 10 μg / L low concentration nicosulfuron by the microbial agent and its microbial raw materials.

[0029] Figure 4 This is a graph showing the degradation effect of the microbial agent described in this invention for degrading sulfonylurea herbicides on three sulfonylurea herbicides at a concentration of 3 μg / L under oligonutrient conditions. Detailed Implementation

[0030] To make the objectives and advantages of this invention clearer, the invention will be specifically described below with reference to embodiments. It should be understood that the following text is merely used to describe one or more specific embodiments of the invention and does not strictly limit the scope of protection specifically claimed by the invention.

[0031] The following strains of *Ancylobacter aquaticus* CGMCC1.2804 are disclosed in this application: https: / / cgmcc.net / directory / data?number=&genus=Ancylobacter+aquaticus&species=&yiming=&page=1; *Aspergillus niger* CGMCC3.15663 ...

[0032] The modified biochar used in the following examples was prepared by rinsing collected corn cobs with water to remove particulate matter, air-drying them naturally, crushing them with a pulverizer, and calcining them in a tube furnace under nitrogen protection. The heating rate was 3℃ / min, and the calcination time was 400℃ for 4 hours. After cooling to room temperature, the biochar was removed and passed through a 100-mesh sieve, with a particle size of 100-300μm. The obtained biochar BC was stirred with water for 12 hours and allowed to settle naturally to remove floating carbon powder on the surface and ash deposited at the bottom. The obtained biochar material was placed in a mixture of sulfuric acid and nitric acid, wherein the mass-to-volume ratio of biochar to mixed acid was 1:5 to 1:1, and the volume ratio of nitric acid to sulfuric acid was 5:1 to 1:1. The mixture was mechanically stirred at 300 rpm for 8 hours. Add twice the volume of water to the mixed acid mixture and stir for 30 minutes. Discard the supernatant, then add ammonia and sodium thiosulfate until the pH reaches 7. The volume-to-mass ratio of ammonia and sodium thiosulfate is 1:1, with the mass ratio of sodium thiosulfate to nitric acid being 10:1 (in excess). Stir mechanically at 300 rpm for 5 hours. Filter the supernatant, wash it three times with water, and dry it to obtain modified biochar, which is then stored.

[0033] The bacterial cultures used in the following examples were as follows: *Ancylobacter aquaticus* CGMCC1.2804, *Aspergillus niger* CGMCC3.15663, and *Bacillus subtilis* CGMCC1.821 were inoculated into the following culture media, respectively. The *Ancylobacter aquaticus* comprised 15%-30% of the complex bacterial flora, while *Aspergillus niger* comprised 10%-30%. The culture medium consists of 15%-30% Niger bacteria and 40%-70% Bacillus subtilis bacteria. The culture medium is composed of 2 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L sodium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate heptahydrate, 0.02 g / L calcium chloride, 1 mL Staub's trace element solution, and 5 mL sterile nutrient yeast extract. It is cultured in a shaker at 30°C for 24 h. It is ready for use when the OD600 value increases to 0.80.

[0034] The preparation method of the biochar-degrading microbial composite inoculant used in the following examples is as follows: Take 0.1g-0.5g of modified biochar and add it to 100mL of inorganic salt culture medium. Sterilize in an autoclave at 121℃ for 20min. Take 100mL (total volume of the three bacterial suspensions with an OD600 value of 0.80) according to the bacterial suspension volume ratio of Ancylobacter aquaticus:Aspergillus niger:Ancylobacter aquaticus of 10:10:80 to 40:40:20. Centrifuge and discard the supernatant. Redissolve the precipitated bacterial strains in the same volume of inorganic salt culture medium and mix. Then, according to the set biochar quality and bacterial density, the volume ratio of biochar suspension and mixed bacterial suspension was adjusted for inoculation. After cooling to room temperature, the bacterial suspension prepared in the second step was inoculated with an inorganic salt medium: bacterial suspension volume ratio of 1:10. The suspension was placed in a shaker at 200 rpm for 12 h for fixation, then removed and centrifuged at 4000 rpm to remove the supernatant. The suspension was washed with 0.9% physiological saline, centrifuged again, and washed twice to obtain the modified biochar-loaded bacterial agent. After drying at 30°C, the suspension was stored in a refrigerator at 4°C.

[0035] Example 1

[0036] The biochar modification conditions were: BC:mixed acid ratio of 1:5 (m:V); nitric acid:sulfuric acid ratio of 5:1 (V:V);

[0037] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0038] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0039] Other carbon sources: None added;

[0040] Initial temperature and pH: 25℃, pH=7;

[0041] Test method and cycle: Static test, 24 hours;

[0042] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0043] The modified biochar exhibited a water contact angle of 45.6° and a charge density of 4.12 meq / g. Liquid chromatography analysis of the nicosulfuron concentration before and after the experiment showed that the removal rate of nicosulfuron in the control group was 11.86%, while the removal rate in the experimental group was 57.45%, with 42.31% of the removal rate attributed to microbial contribution.

[0044] Example 2

[0045] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0046] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0047] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0048] Other carbon sources: None added;

[0049] Initial temperature and pH: 25℃, pH=7;

[0050] Test method and cycle: Static test, 24 hours;

[0051] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0052] The modified biochar exhibited a water contact angle of 51.42° and a charge density of 3.53 meq / g. Liquid chromatography analysis of the nicosulfuron concentration before and after the experiment revealed a removal rate of 10.21% in the control group and 64.36% in the experimental group, with 54.15% of the removal rate attributed to microbial contribution.

[0053] Example 3

[0054] The biochar modification conditions were: BC:mixed acid ratio of 1:1 (m:V); nitric acid:sulfuric acid ratio of 1:1 (V:V);

[0055] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0056] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0057] Other carbon sources: None added;

[0058] Initial temperature and pH: 25℃, pH=7;

[0059] Test method and cycle: Static test, 24 hours;

[0060] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0061] The modified biochar exhibited a water contact angle of 60.89° and a charge density of 2.64 meq / g, as determined by water contact angle testing. Liquid chromatography analysis revealed that the removal rate of nicosulfuron before and after the experiment was 7.71% in the control group and 40.12% in the experimental group, with 32.41% of the removal rate attributed to microbial contribution.

[0062] Example 4

[0063] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0064] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 15:15:70 (volume ratio);

[0065] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0066] Other carbon sources: None added;

[0067] Initial temperature and pH: 25℃, pH=7;

[0068] Test method and cycle: Static test, 24 hours;

[0069] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0070] The modified biochar exhibited a water contact angle of 51.42° and a charge density of 3.53 meq / g. Liquid chromatography analysis revealed that the removal rate of nicosulfuron before and after the experiment was 11.35% in the control group and 50.68% in the experimental group, with 39.33% of the removal attributed to microbial contribution.

[0071] Example 5

[0072] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0073] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 30:30:40 (volume ratio);

[0074] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0075] Other carbon sources: None added;

[0076] Initial temperature and pH: 25℃, pH=7;

[0077] Test method and cycle: Static test, 24 hours;

[0078] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0079] The modified biochar showed a water contact angle of 51.42° and a charge density of 3.53 meq / g after water contact angle testing. Liquid chromatography analysis of the concentration of nicosulfuron before and after the experiment showed a removal rate of 9.68% in the control group and 48.34% in the experimental group, with 38.66% of the removal rate attributed to microbial contribution.

[0080] Example 6

[0081] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0082] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0083] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0084] Other carbon sources: 10 mg / L humic acid;

[0085] Initial temperature and pH: 25℃, pH=7;

[0086] Test method and cycle: Static test, 24 hours;

[0087] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0088] The modified biochar exhibited a water contact angle of 51.42° and a charge density of 3.53 meq / g. Liquid chromatography analysis of the nicosulfuron concentration before and after the experiment showed that the removal rate of nicosulfuron in the control group was 15.15%, while the removal rate in the experimental group was 92.39%, with 77.24% of the removal attributed to microbial contribution.

[0089] Example 7

[0090] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0091] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0092] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0093] Other carbon sources: 10 mg / L bovine serum albumin;

[0094] Initial temperature and pH: 25℃, pH=7;

[0095] Test method and cycle: Static test, 24 hours;

[0096] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0097] The modified biochar exhibited a water contact angle of 51.42° and a charge density of 3.53 meq / g. Liquid chromatography analysis of the nicosulfuron concentration before and after the experiment showed that the removal rate of nicosulfuron in the control group was 12.35%, while the removal rate in the experimental group was 80.69%, with 68.34% of the removal considered to be due to microbial contribution.

[0098] Example 8

[0099] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0100] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0101] Treatment target: 10 mg / L nicosulfuron aqueous solution;

[0102] Other carbon sources: 10 mg / L sodium alginate;

[0103] Initial temperature and pH: 25℃, pH=7;

[0104] Test method and cycle: Static test, 24 hours;

[0105] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0106] The modified biochar exhibited a water contact angle of 51.42° and a charge density of 3.53 meq / g, as determined by water contact angle testing. Liquid chromatography analysis revealed that the removal rate of nicosulfuron before and after the experiment was 14.22% in the control group and 85.66% in the experimental group, with 71.44% of the removal attributed to microbial contribution.

[0107] Example 9

[0108] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0109] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0110] Treatment target: 10 μg / L benzsulfuron-methyl aqueous solution;

[0111] Other carbon sources: 10 mg / L humic acid;

[0112] Initial temperature and pH: 25℃, pH=7;

[0113] Test method and cycle: Static test, 24 hours;

[0114] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of bensulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0115] The modified biochar showed a water contact angle of 51.42° and a charge density of 3.53 meq / g. Liquid chromatography analysis of the concentrations of benzsulfuron before and after the experiment revealed a removal rate of 10.23% for benzsulfuron in the control group and 78.14% for nicosulfuron in the experimental group. Of this, 67.91% was attributed to microbial contribution.

[0116] Example 10

[0117] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0118] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0119] Treatment target: 10 μg / L nicosulfuron aqueous solution;

[0120] Other carbon sources: 10 mg / L humic acid;

[0121] Initial temperature and pH: 25℃, pH=7;

[0122] Test method and cycle: Static test, 24 hours;

[0123] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of nicosulfuron-methyl inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0124] The modified biochar exhibited a water contact angle of 51.42° and a charge density of 3.53 meq / g. Liquid chromatography analysis of the nicosulfuron concentration before and after the experiment showed that the removal rate of nicosulfuron in the control group was 12.89%, while the removal rate in the experimental group was 80.28%, with 67.39% of the removal attributed to microbial contribution.

[0125] Example 11

[0126] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0127] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0128] Treatment target: 10 μg / L clopyralid aqueous solution;

[0129] Other carbon sources: 10 mg / L humic acid;

[0130] Initial temperature and pH: 25℃, pH=7;

[0131] Test method and cycle: Static test, 24 hours;

[0132] The bacterial agent was added at a dosage of 1 / 1000 (mass ratio) of the treated water volume. 0.1 g of the bacterial agent was added to 100 mL of chlorpyrifos inorganic salt medium. The medium was incubated for 24 h at 200 rpm in a shaker at pH 7 and 25℃. The experiment was repeated three times, and the average value was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0133] The modified biochar exhibited a water contact angle of 51.42° and a charge density of 3.53 meq / g. Liquid chromatography analysis of the concentration of chlorpyrifos before and after the experiment showed that the removal rate of chlorpyrifos in the control group was 8.89%, while the removal rate in the experimental group was 92.38%, with 83.49% of the removal rate attributed to microbial contribution.

[0134] Example 12

[0135] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0136] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0137] Treatment targets: A mixed aqueous solution of benzsulfuron, nicosulfuron, and clopyralid, all at 3 μg / L;

[0138] Other carbon sources: a mixed aqueous solution of humic acid, bovine serum albumin, and sodium alginate, all at 3 mg / L;

[0139] Initial temperature and pH: 25℃, pH=7;

[0140] Test method and cycle: continuous flow dynamic test, hydraulic residence time: 6h;

[0141] The bacterial agent was packed into the column bed, and the above-mentioned treatment objects and other carbon source conditions were scaled up 20 times for a pre-run for one week. Then, the treatment objects and other carbon source conditions were adjusted to the above conditions and run for another 3 days to ensure stable operation of the device. The formal experiment then began, using two sets of devices as parallel experiments, with five samples taken from each parallel, and the average value of the 10 results was taken. The bacterial agent inactivated by autoclaving at 121℃ was used as a blank control in this experiment.

[0142] The concentrations of the three sulfonylurea herbicides before and after the experiment were detected by liquid chromatography-mass spectrometry. The average removal rate of bensulfuron in the control group was 60.87%, the removal rate of nicosulfuron was 85.36%, and the removal rate of chlorpyrifos was 78.69%. All of these were considered to be the effect of microbial degradation.

[0143] Example 13

[0144] The biochar modification conditions were: BC:mixed acid ratio of 1:2 (m:V); nitric acid:sulfuric acid ratio of 2:1 (V:V);

[0145] Microbial strains: Ancylobacter aquaticus: Aspergillus niger: Bacillus subtilis = 20:20:60 (volume ratio);

[0146] Treatment targets: A mixed aqueous solution of benzsulfuron, nicosulfuron, and clopyralid, all at 3 μg / L;

[0147] Other carbon sources: Drainage water from a farmland, total organic carbon content 8.74 mg / L, pH = 6.72;

[0148] Initial temperature: 25℃;

[0149] Test method and cycle: continuous flow dynamic test, hydraulic residence time: 6h;

[0150] The bacterial agent was filled into the column bed, and the treatment target was scaled up 20 times for a pre-run for one week. Then, the treatment conditions were adjusted to the above conditions and run for another 3 days to ensure stable operation of the device. The formal experiment then commenced, using two parallel sets of devices, with five samples taken from each parallel set. The average value of the 10 results was taken. The bacterial agent inactivated at 121℃ in an autoclave served as a blank control in this experiment.

[0151] The concentrations of the three sulfonylurea herbicides before and after the experiment were detected by liquid chromatography-mass spectrometry. The average removal rate of bensulfuron in the control group was 65.38%, the removal rate of nicosulfuron was 82.67%, and the removal rate of chlorpyrifos was 83.18%. These were all considered to be the effect of microbial degradation.

[0152] Comparative Example 1

[0153] Ancylobacter aquaticus was cultured to an OD600 of 0.80, then centrifuged from a medium containing nutrient yeast extract, reconstituted into an inorganic salt medium, and its OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used, without any other external carbon source, to treat 10 mg / L and 10 μg / L nicosulfuron and clopyralid aqueous solutions, respectively. A static test method was used for 24 h, with an initial temperature of 25℃ and pH of 7. All experiments were performed in triplicate, and the average degradation results were taken.

[0154] The initial concentration of herbicides at 10 mg / L was detected by liquid chromatography. After 24 h, the degradation rates of nicosulfuron and clopyralid at 10 mg / L by *Ancylobactera quaticus* were 17.52% and 22.58%, respectively.

[0155] Comparative Example 2

[0156] Aspergillus niger was cultured to an OD600 of 0.80, then centrifuged from a medium containing nutrient yeast extract, reconstituted into an inorganic salt medium, and its OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used, without any other external carbon source, to treat 10 mg / L nicosulfuron and clopyralid aqueous solutions, respectively. A static test method was used for 24 h, with an initial temperature of 25℃ and pH of 7. All experiments were performed in triplicate, and the average degradation results were taken.

[0157] The initial concentration of herbicides at 10 mg / L was detected by liquid chromatography. After 24 h, the degradation rates of nicosulfuron and clopyralid at 10 mg / L by Aspergillus niger were 22.76% and 20.81%, respectively.

[0158] Comparative Example 3

[0159] Bacillus subtilis was cultured to an OD600 of 0.80, then centrifuged from a medium containing nutrient yeast extract, reconstituted into an inorganic salt medium, and its OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used, without any other external carbon source, to treat 10 mg / L nicosulfuron and clopyralid aqueous solutions, respectively. A static test method was used for 24 h, with an initial temperature of 25℃ and pH of 7. All experiments were performed in triplicate, and the average degradation results were taken.

[0160] The initial concentration of herbicides at 10 mg / L was detected by liquid chromatography. After 24 h, the degradation rates of nicosulfuron and chlorpyrifos by Bacillus subtilis at 10 mg / L were 6.54% and 7.89%, respectively.

[0161] Comparative Example 4

[0162] Ancylobacter aquaticus was cultured to an OD600 of 0.80, then centrifuged from a medium containing nutrient yeast extract, reconstituted into an inorganic salt medium, and its OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used to treat 10 μg / L nicosulfuron and clopyralid aqueous solutions without any other external carbon source. A static test method was used for 24 h, with an initial temperature of 25℃ and pH of 7. All experiments were performed in triplicate, and the degradation results were averaged.

[0163] The initial concentration of herbicides at 10 μg / L was detected using triple quadrupole liquid chromatography-mass spectrometry. After 24 h, the degradation rates of 10 μg / L nicosulfuron and clopyralid by *Ancylobacter aquaticus* were 12.38% and 14.75%, respectively.

[0164] Comparative Example 5

[0165] Aspergillus niger was cultured to an OD600 of 0.80, then centrifuged from a medium containing nutrient yeast extract, reconstituted into an inorganic salt medium, and its OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used to treat 10 μg / L nicosulfuron and clopyralid aqueous solutions without any other external carbon source. A static treatment method was used for 24 h at an initial temperature of 25℃ and pH 7. All experiments were performed in triplicate, and the average degradation results were taken.

[0166] The initial concentration of herbicides at 10 μg / L was detected using triple quadrupole liquid chromatography-mass spectrometry. After 24 h, the degradation rates of nicosulfuron and clopyralid at 10 μg / L by Aspergillus niger were 18.38% and 16.66%, respectively.

[0167] Comparative Example 6

[0168] Bacillus subtilis was cultured to an OD600 of 0.80, then centrifuged from a medium containing nutrient yeast extract, reconstituted into an inorganic salt medium, and its OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used to treat 10 μg / L nicosulfuron and clopyralid aqueous solutions without any other external carbon source. A static test method was used for 24 h, with an initial temperature of 25℃ and pH of 7. All experiments were performed in triplicate, and the average degradation results were taken.

[0169] The initial concentration of herbicides at 10 μg / L was detected using triple quadrupole liquid chromatography-mass spectrometry. After 24 h, the degradation rates of 10 μg / L nicosulfuron and clopyralid by Bacillus subtilis were 3.68% and 5.58%, respectively.

[0170] Comparative Example 7

[0171] Ancylobacter aquaticus, Aspergillus niger, and Bacillus subtilis were cultured to an OD600 of 0.80. They were then separated from a culture medium containing nutrient-rich yeast extract in a 2:2:6 volume ratio by centrifugation, reconstituted into an inorganic salt medium, and their OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used, without any other external carbon source, to treat 10 mg / L nicosulfuron and clopyrazosulfuron aqueous solutions, respectively. A static test method was used for 24 h, with an initial temperature of 25℃ and pH of 7. All experiments were performed in triplicate, and the average degradation results were taken.

[0172] The initial concentration of herbicides at 10 mg / L was detected by liquid chromatography. After 24 h, the degradation rates of nicosulfuron and clopyralid at 10 mg / L by the compound bacteria were 20.35% and 29.68%, respectively.

[0173] Comparative Example 8

[0174] *Ancylobacter aquaticus*, *Aspergillus niger*, and *Bacillus subtilis* were cultured to an OD600 of 0.80. They were then separated from a nutrient-rich yeast extract medium by centrifugation in a 2:2:6 volume ratio, reconstituted in an inorganic salt medium, and their OD600 was adjusted to be not less than 0.75 for degradation experiments. 10 mL of the bacterial culture was used, without any other external carbon source, to treat 10 μg / L nicosulfuron and clopyrazosulfuron aqueous solutions, respectively. A static test method was used for 24 h, with an initial temperature of 25℃ and pH of 7. All experiments were performed in triplicate, and the average degradation results were taken.

[0175] The initial concentration of herbicides at 10 μg / L was analyzed using triple quadrupole liquid chromatography-mass spectrometry. After 24 h, the degradation rates of 10 μg / L nicosulfuron and clopyralid were 25.68% and 31.57%, respectively.

[0176] Figure 1 It can be observed that porosity is an important characteristic of the modified biochar, which is beneficial for the adsorption of micro pollutants. The microbial agent contains a large number of microbial cells with intact morphology, indicating that its structure has not been destroyed, which is an important prerequisite for it to exert its degradation ability.

[0177] Figure 2 The results show that the water contact angle of the modified biochar gradually increases as the amount of nitric acid in the mixed acid decreases, while its hydrophilicity gradually decreases. Conversely, the charge density gradually decreases as the amount of nitric acid in the mixed acid decreases. By appropriately adjusting the ratio of nitric acid, modified biochar materials with suitable water contact angles and charge densities can be obtained. Based on the examples, a BC:mixed acid ratio of 1:2 (m:V) and a nitric acid:sulfuric acid ratio of 2:1 (V:V) are more suitable.

[0178] Figure 3 The results showed that the microbial raw materials themselves had poor degradation effects on nicosulfuron, both at high concentrations (10 mg / L) and trace concentrations (10 μg / L). The mixture of the three microbial raw materials significantly improved the degradation efficiency (different letters in the figure indicate significant differences in degradation rates), indicating a good synergistic effect among the three. Furthermore, it was found that the improvement in degradation rate by the compound microbial agent was closely related to the modified biochar used as the loading agent; only by controlling the characteristics of the biochar could good results be achieved. In addition, the addition of an external carbon source helped improve the degradation of herbicides by the microbial agent.

[0179] Figure 4 The results indicate that the compound microbial agent has a good degradation effect on trace concentrations of various sulfonylureas, both under simulated natural organic conditions and under real farmland ditch drainage water quality conditions, demonstrating its promising application prospects for the degradation of sulfonylureas in farmland drainage ditches.

[0180] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained in this invention are implemented according to conventional methods in the art unless otherwise specified or limited.

Claims

1. A microbial agent for degrading sulfonylurea herbicides, characterized in that, The bacterial agent uses weakly positively charged modified biochar as a carrier to load a complex bacterial community, which consists of Ancylobacter aquaticus CGMCC1.2804, Aspergillus niger CGMCC3.15663, and Bacillus subtilis CGMCC1.

821.

2. The microbial agent for degrading sulfonylurea herbicides according to claim 1, characterized in that, The complex microbial community comprises 15%-30% Ancylobacter aquaticus, 15%-30% Aspergillus niger, and 40%-70% Bacillus subtilis.

3. The microbial agent for degrading sulfonylurea herbicides according to claim 1, characterized in that, The weakly positively charged modified biochar carrier is prepared by calcining corn cobs at 400°C in an oxygen-free environment, followed by positively charged modification with amino groups as the main functional groups.

4. A method for preparing a microbial agent for degrading sulfonylurea herbicides as described in any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Preparation and modification of biochar: The collected corn cobs were rinsed with water to remove particulate matter, air-dried, crushed with a pulverizer, and calcined in a tube furnace under nitrogen protection. The heating rate was 3℃ / min, and the calcination time was 400℃ for 4 hours. After cooling to room temperature, the biochar was removed and passed through a 100-mesh sieve, with a particle size of 100-300μm. The obtained biochar BC was stirred with water for 12 hours and allowed to settle naturally to remove floating carbon powder and bottom ash. The obtained biochar material was placed in a mixture of sulfuric acid and nitric acid and mechanically stirred at 300 rpm for 8 hours. Two volumes of water were added to the mixed acid and stirred for 30 minutes. The supernatant was discarded, and ammonia and sodium thiosulfate were added until the pH reached 7. The volume ratio of ammonia to sodium thiosulfate was 1:1 (V:m). The mixture was mechanically stirred at 300 rpm for 5 hours. The supernatant was filtered, washed three times with water, dried, and stored to obtain modified biochar. The second step is the cultivation of bacterial suspension: the compound bacterial suspension is inoculated into the culture medium to obtain a bacterial suspension; The third step is the preparation of the bacterial agent: 0.1g-0.5g of modified biochar is added to 100mL of inorganic salt culture medium and sterilized in an autoclave at 121℃ for 20min. After cooling to room temperature, the bacterial suspension prepared in the second step is inoculated with inorganic salt culture medium: bacterial suspension at a volume ratio of 1:

10. The suspension is fixed in a shaker at 200 rpm for 12h, then removed and centrifuged at 4000 rpm to remove the supernatant. The suspension is washed with 0.9% physiological saline, centrifuged again, and washed twice to obtain the bacterial agent loaded with modified biochar. After drying at 30℃, the suspension is stored in a refrigerator at 4℃.

5. The method for preparing the microbial agent for degrading sulfonylurea herbicides according to claim 4, characterized in that, In the second step, the culture medium consists of 2 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L sodium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate heptahydrate, 0.02 g / L calcium chloride, 1 mL Staub's trace element solution, and 5 mL sterile nutrient yeast extract solution. It is cultured in a shaker at 30°C for 24 h, and is ready for use when the OD600 value increases to 0.8 ± 0.

1.

6. The method for preparing the microbial agent for degrading sulfonylurea herbicides according to claim 4, characterized in that, The inorganic salt culture in the third step consists of 2 g / L disodium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L sodium nitrate, 0.5 g / L ammonium chloride, 0.1 g / L magnesium sulfate heptahydrate, 0.02 g / L calcium chloride, and 1 mL Staub's trace element solution.

7. The application of the microbial agent for degrading sulfonylurea herbicides as described in any one of claims 1-3 in the enhanced reduction of sulfonylurea herbicides in farmland drainage ditches.

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