A bacterial agent for degrading isoproturon herbicide and preparation method thereof
By using bacteria agents with wheat straw biochar carrier and loading Sphinobiumsp.IPU-1 bacteria, the degradation problem of iseproximate residual pollution was solved, and efficient degradation and soil improvement effects were achieved.
Patent Information
- Application Number
- CN202211422710.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-15
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2042-11-15
AI Technical Summary
The prior art is difficult to effectively degrade residual contamination of isopropyl herbicides, and there are problems of secondary contamination and high cost in physical and chemical repair.
Using wheat straw biochar as a carrier, loading sphingosine monocytosine genus Sphingobiumsp.IPU-1 bacteria was prepared, and a bacterial agent was improved through the immobilization technology of biochar and bacterial bodies.
This fungus agent can quickly degrade isopropylene in the soil, with a degradation rate of 97.6%. At the same time, it improves soil acidity, improves the effectiveness of soil nutrients, and reduces the inhibitory effect of isopropylene on crop growth.
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Figure CN115747199B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial remediation of organic pollution, and in particular relates to a bacterial agent for degrading isoproturon herbicide and a preparation method thereof. Background Art
[0002] As a pre-emergence and post-emergence soil treatment agent, isoproturon is mainly used to control annual grass weeds and most broad-leaved weeds in wheat, barley, peanuts, potatoes, and tomato fields, such as foxtail millet, net grass, bluegrass, wild oats, matricaria, shepherd's purse, chickweed, and horsetail. It is one of the most widely used urea-substituted herbicides in the world. The national food safety standard stipulates that the maximum residue limit of isoproturon in brown rice and wheat is 0.05 mg / kg. With the need for weed control and pest control, the use of isoproturon is increasing. Isoproturon exists in soil, water environment, and related crops such as wheat and rice. Due to the occurrence of demethylation reactions, its metabolite demethylisoproturon will be generated, which remains in related cereal crops such as wheat and rice. At present, reports on the detection of isoproturon residues are mostly seen in food and water bodies. The pollution remediation of isoproturon mainly includes physical remediation, chemical remediation, and biological remediation. Physical remediation and chemical remediation are not widely used in practice due to their shortcomings such as incomplete remediation, easy to cause secondary pollution, and high remediation cost. Bioremediation technology has gradually become a mainstream technical means to repair environmental pollution due to its advantages such as low cost, thorough repair and no secondary pollution.
[0003] How to effectively use straw to solve problems, turn waste into treasure, make comprehensive use of it and solve straw pollution is an important strategic issue for sustainable agricultural development and has great practical significance. Biochar has a high specific surface area, many pores and good adsorption effect. It has unique natural advantages and huge development potential in microbial immobilization technology. Summary of the invention
[0004] Problem to be solved
[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a bacterial agent for degrading isoproturon herbicide and a preparation method thereof, so as to solve the problems of turning straw into treasure and isoproturon residue pollution.
[0006] Technical Solution
[0007] To solve the above problems, the present invention adopts the following technical solutions.
[0008] A bacterial agent for degrading the herbicide isoproturon, wherein the bacterial agent is a biochar carrier and loaded with Sphingomonas spp. Sphingobium sp. Made from IPU-1 bacteria;
[0009] The Sphingobium sp.The IPU-1 bacteria, numbered IPU-1, was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on September 27, 2022, with the accession number CCTCC M 20221514.
[0010] The above-mentioned bacterial agent for degrading the herbicide isoproturon,
[0011] The carrier in the bacterial agent is biochar prepared by anaerobic calcination of wheat straw, and the bacterial cell is a sphingomonas spp. Sphingobium sp. IPU-1 bacteria, wet bacterial content is 20g / L, biochar content is 20 mg / mL.
[0012] A method for preparing a bacterial agent for degrading the herbicide isoproturon comprises the following steps:
[0013] (1) Biochar preparation;
[0014] (2) Bacterial culture;
[0015] (3) Preparation of bacterial agents.
[0016] The preparation method of the above-mentioned bacterial agent for degrading the herbicide isoproturon,
[0017] The method for preparing biochar in step (1) is as follows:
[0018] The collected wheat straw was cut into small segments of 3-5 cm, and rinsed repeatedly with tap water to remove surface dust and debris. After washing, it was washed with pure water until neutral, dried naturally in a ventilated place, and then transferred to an oven for drying overnight; the dried straw was put into a grinder, crushed and sieved through an 80-mesh sieve, and then the straw powder was evenly loaded into a quartz boat, filled and compacted tightly, and placed in a muffle furnace. Nitrogen was passed through the muffle furnace for 5 min. After the air was exhausted, a tube was connected to the exhaust port and placed in a NaOH solution to form a liquid seal. The temperature was raised to the set temperature at a rate of 2°C / min, kept constant for 2 h, and taken out after cooling to room temperature. It was sieved through a 100-mesh sieve to obtain biochar with a particle size of 200 μm, and prepared in batches multiple times. Finally, the wheat straw biochar prepared from different batches was mixed evenly, put into a ziplock bag, and placed in a dry environment for refrigeration.
[0019] The preparation method of the above-mentioned bacterial agent for degrading the herbicide isoproturon,
[0020] The method for culturing the bacterial solution in step (2) is as follows:
[0021] Isoproturon-degrading bacteria Sphingobium sp.IPU-1 was inoculated into LB liquid culture medium, and after culturing to the logarithmic growth phase, the culture fluid was collected, the supernatant was discarded by centrifugation, and the cells were washed three times with physiological saline. After the cells were collected, the cell concentration was adjusted to OD600nm of 1.0 with a buffer solution and set aside.
[0022] The preparation method of the above-mentioned bacterial agent for degrading the herbicide isoproturon,
[0023] The method for preparing the bacterial agent in step (3) is as follows:
[0024] A certain amount of wheat straw biochar was added to 50 mL of inorganic salt liquid culture medium, sterilized at 121°C in a high-pressure steam sterilizer for 20 min, cooled to room temperature, and inoculated with the above-prepared bacterial suspension at a 10% inoculation amount (v / v) to obtain a mixed immobilized culture medium of biochar and bacterial liquid, fixed in a shaker, taken out, centrifuged and discarded the supernatant; washed with 0.9% saline, centrifuged for 5 min and discarded the supernatant, washed the lower sediment with sterile saline, repeated this process twice, and finally obtained Sphingobium sp. IPU-1 and biochar immobilized bacteria were dried at 30°C and stored in a refrigerator at 4°C.
[0025] Beneficial Effects
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] (1) The bacterial agent of the present invention can better retain the genus Sphingomonas ( Sphingobium sp. )The degradation activity of IPU-1 bacteria. Sphingomonas spp. Sphingobium sp. )The IPU-1 bacteria are adsorbed in the bacterial agent, which can avoid the loss of bacteria caused by the flow of water and reduce the damage to the bacteria caused by mechanical friction. The production cost is low and it can be used directly by putting it into the soil, which has broad application prospects.
[0028] (2) The bacterial agent of the present invention can quickly degrade isoproturon residual in the soil. The half-life of natural degradation of 20 mg / L isoproturon in the soil is 39.42 days, while it is only 8.67 days after adding the bacterial agent. When the potted plant experiment was carried out for 25 days, the degradation rate reached 97.6%.
[0029] (3) The bacterial agent of the present invention can improve the acidity of soil contaminated by isoproturon, thereby increasing the effectiveness of soil nutrients and improving the soil ecological environment.
[0030] (4) The bacterial agent of the present invention can effectively alleviate the inhibitory effect of isoproturon on various physiological indicators (seedling weight, root length, plant height) of wheat and corn plants in contaminated soil, and reduce the impact of isoproturon residues on the growth of wheat and corn plants in contaminated soil. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 The physical shape diagram and scanning electron microscope diagram of the bacterial agent of the present invention;
[0032] Figure 2 This is a diagram showing the degradation effect of the bacterial agent of the present invention on isoproturon in soil;
[0033] Figure 3 This is a graph showing the effect of the bacterial agent of the present invention on the pH of soil contaminated by isoproturon;
[0034] Figure 4 This is a graph showing the effect of the bacterial agent of the present invention on the growth of wheat plants in the soil;
[0035] Figure 5 This is a graph showing the effect of the bacterial agent of the present invention on the growth of corn plants in the soil;
[0036] Figure 6 This is a data graph showing the degradation rate of isoproturon by the bacterial agent of the present invention. DETAILED DESCRIPTION
[0037] The present invention is further described below in conjunction with specific embodiments.
[0038] Example 1
[0039] The steps for preparing the bacterial agent for degrading isoproturon are as follows:
[0040] (1) Preparation of wheat straw biochar:
[0041] The collected wheat straw was cut into small sections of 3-5 cm, and rinsed repeatedly with tap water to remove dust and debris on the surface. After washing, it was washed with pure water until neutral, dried naturally in a ventilated place, and then transferred to an oven for drying overnight. Take an appropriate amount of dry straw and put it into a grinder (25000 rpm). After crushing, it was sieved through an 80-mesh sieve. Then the straw powder was evenly loaded into a quartz boat, filled and compacted tightly, and placed in a muffle furnace. Nitrogen was passed through the muffle furnace for 5 min. After the air was exhausted, a tube was connected to the exhaust port and placed in a NaOH solution to form a liquid seal. The temperature was raised to the set temperature at a rate of 2℃ / min, kept constant for 2 h, and taken out after cooling to room temperature. It was sieved through a 100-mesh sieve to obtain biochar with a particle size of 200μm. Because the amount of biochar required was large and the capacity of the muffle furnace was limited, it was prepared in batches for multiple times. Finally, the wheat straw biochar prepared from different batches was mixed evenly, put into a ziplock bag, and placed in a dry environment for refrigeration.
[0042] (2) Sphingobium sp. Preparation of IPU-1 bacteria
[0043] Isoproturon-degrading bacteria Sphingobium sp.IPU-1 was inoculated into LB liquid culture medium, and after culturing to the logarithmic growth phase, the culture fluid was collected, the supernatant was discarded by centrifugation, and the cells were washed three times with physiological saline. After the cells were collected, the cell concentration was adjusted to OD600nm of 1.0 with a buffer solution and set aside.
[0044] (3) Preparation Sphingobium sp. IPU-1 and biochar immobilization bacteria
[0045] Take a certain amount of wheat straw biochar and add it to 50 mL of inorganic salt liquid culture medium, sterilize it in a high-pressure steam sterilizer at 121℃ for 20 min, cool it to room temperature, and inoculate the above-prepared bacterial suspension at a 10% inoculation amount (v / v) to obtain a mixed immobilized culture medium of biochar and bacterial liquid, place it in a shaker for a certain period of time, take it out, centrifuge and discard the supernatant. Wash it with 0.9% saline, centrifuge it for 5 min, discard the supernatant, wash the lower sediment with sterile saline, repeat this process twice, and the final result is Sphingobium sp. IPU-1 and biochar immobilized bacteria were dried at 30°C and stored in a refrigerator at 4°C.
[0046] Mineral Salt Medium (MSM): MgSO 4 7H 2 O 0.4 g / L, K 2 HPO 4 0.2 g / L, (NH 4 ) 2 SO 4 0.2 g / L, CaSO 4 0.08 g / L, FeSO 4 7H 2 O 0.002 g / L, and the pH was adjusted to 7.4.
[0047] LB medium (Luria-Bertani Medium): 10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract powder, pH adjusted to 7.0. LB solid medium: Add 1.8% (m / m) agar powder to LB liquid medium.
[0048] The above culture media were sterilized by high-temperature steam sterilization at 121°C for 20 min.
[0049] Depend on Figure 1 It can be seen that the bacterial agent is in the form of black powder with a rough surface, many pores, good adsorption effect and good permeability.
[0050] Example 2 Remediation of soil contaminated with isoproturon by degradation bacteria
[0051] (1) Degradation of isoproturon in contaminated soil
[0052] First, accurately weigh isoproturon and mix it evenly into the soil to make its concentration reach 10.0 mg / kg and 20.0 mg / kg respectively; then, the experiment was carried out according to the experimental design in Table 1; finally, 10 g of soil was randomly extracted from each experimental group on days 0, 1, 3, 5, 7, 10, 15, 20, and 25 for isoproturon residue determination. In the experiment, the following treatments were performed: 1) Regularly add appropriate amount of water to make the soil moisture content reach 60% of the optimal water holding capacity; 2) keep the bacterial amount of the experimental group equal, and repeat each experimental level three times; 3) All treatments were incubated at constant temperature (30℃) and dark conditions for 24 h, in order to make isoproturon fully adsorbed in the soil.
[0053] Table 1 Grouping of potted plant experiments
[0054] Experimental Grouping Experimental treatment CK Soil + MSM P Soil + Isoproturon + MSM BC Soil + Biochar BCP Soil + isoproturon + biochar PY Soil + isoproturon + bacterial solution BCPY Soil + isoproturon + immobilized bacteria
[0055] Extraction method of isoproturon in soil: accurately weigh 10 g of dry weight of soil sample and place it in a 50 mL centrifuge tube, add 5 g NaCl, 15 mL acetonitrile, and 5 mL deionized water, mix and place on a shaker for vigorous shaking for 6 h, centrifuge and take 5 mL of supernatant, blow dry with a nitrogen blower, and then make up to volume with methanol in a 1:1 ratio, ultrasonicate for 5 min, and filter through a 0.22 um filter membrane for use.
[0056] The experimental results are as follows Figure 2 It can be seen that the half-life of natural degradation of isoproturon in soil is 39.42 days, and it is only 8.67 days after adding the bacterial agent; when the pot experiment was carried out for 25 days, the degradation rate reached 97.6%, which was basically completely degraded.
[0057] (2) After 25 days of the potted experiment, soil samples were taken for pH measurement: the instrument was calibrated with two buffer solutions of different pH values to ensure that both buffer checks were normal and the allowable deviation was within 0.02 pH. 10 g of air-dried soil sample that passed through a 1 mm sieve was weighed and placed in a 25 mL beaker. 25 mL of distilled water was added to mix and allowed to stand for 30 min. The pH value of the suspension was measured with a calibrated pH meter. The experimental results are shown in the figure. Figure 3 It can be seen that the application of microbial agents will change the soil pH and affect the biochemical process of the soil.
[0058] Effects of isoproturon-degrading bacteria on the growth of corn and wheat plants after remediation of contaminated soil
[0059] After 25 days of bacterial treatment, sow in the experimental soil of each treatment. Select corn and wheat seeds with full grains and no diseases, soak them for 24 hours, spread them on gauze and place them in a dark incubator for germination. After the seeds germinate, select young shoots with consistent growth and transplant them into the experimental soil of each treatment, cover them with a layer of topsoil, and culture the potted plants in a greenhouse with a daylighting time of 14 h / d and a temperature of 25°C. After the seedlings grow out, observe their growth every day. After one week, remove the wheat and corn from the soil, wash them with deionized water, wipe them dry with absorbent paper, and store them. Measure the root length, plant height, and seedling weight of wheat and corn, and draw a chart for analysis. The experimental results are as follows: Figure 4 , 5 It can be seen that the application of fungal agents can alleviate the inhibitory effect of isoproturon on various physiological indicators of plants.
[0060] In addition, the supplementary test contents are as follows:
[0061] Comparison of adsorption and embedding methods
[0062] Preparation of embedded bacterial agent:
[0063] ① Weigh 14 g polyvinyl alcohol, 1 g sodium alginate, and 2 g biochar and place them in 100 mL ultrapure water (in a 250 ml conical flask) and sterilize at 121°C for 20 min. Cool to room temperature;
[0064] ② When the temperature drops to 30℃, add 2 g of wet bacteria and mix well.
[0065] ③ Use a sterilized 10 mL syringe with an inner diameter of 0.4 mm to add the above mixture dropwise to the cross-linking solution. Place the cross-linking solution on a magnetic stirrer and keep it rotating so that the dripping balls will not stick together. After dripping, place it in a 4℃ refrigerator for cross-linking and fixation for 24 hours;
[0066] ④ Wash the prepared bacterial agent twice with physiological saline and store it in a 4℃ refrigerator for later use.
[0067] Bacterial preparation by embedding method
[0068] Study on the degradation activity of bacterial agents in MSM
[0069] To contain 100.0 mg L -1 Add the degradation bacteria to 20 mL of MSM containing isoproturon and incubate at 37 °C and 150 rmin -1 After culturing on a shaker for 0, 2, 4, 6, 8, 9, 10, 11, and 12 hours, the residual concentration of isoproturon was extracted and detected. At the same time, the embedding and biochar agents without degradation bacteria were set as controls, and 3 replicates were set for both the control and the treatment.
[0070] Experimental Results
[0071] like Figure 6 As shown in the figure, the degradation rate of isoproturon by the adsorbed bacteria reached 95% at 8 hours, and was basically completely degraded after 10 hours. The degradation rate of the embedded bacteria was 74.5% at 8 hours, and was completely degraded after 12 hours.
[0072] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope determined by the claims submitted for the present invention.
Claims
1. A bacterial agent for degrading the herbicide isoproturon, characterized in that: The bacterial agent is a biochar carrier, loaded with Sphingomonas spp. Sphingobium sp. Made from IPU-1 bacteria; The Sphingobium sp. The IPU-1 bacteria, numbered IPU-1, was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on September 27, 2022, with the accession number CCTCC M 20221514.
2. The bacterial agent for degrading the herbicide isoproturon according to claim 1, characterized in that: The carrier in the bacterial agent is biochar prepared by anaerobic calcination of wheat straw, and the bacterial cell is a sphingomonas spp. Sphingobium sp. IPU-1 bacteria, wet bacterial content is 20g / L, biochar content is 20 mg / mL.
3. A method for preparing a bacterial agent for degrading the herbicide isoproturon as claimed in claim 1, comprising the following steps: (1) Biochar preparation; (2) Bacterial culture; (3) Preparation of bacterial agents.
4. The method for preparing the bacterial agent for degrading the herbicide isoproturon according to claim 3, characterized in that: The method for preparing biochar in step (1) is as follows: The collected wheat straw was cut into small segments of 3-5 cm, and rinsed repeatedly with tap water to remove surface dust and debris. After washing, it was washed with pure water until neutral, dried naturally in a ventilated place, and then transferred to an oven for drying overnight; the dried straw was put into a grinder, crushed and sieved through an 80-mesh sieve, and then the straw powder was evenly loaded into a quartz boat, filled and compacted tightly, and placed in a muffle furnace. Nitrogen was passed through the muffle furnace for 5 min. After the air was exhausted, a tube was connected to the exhaust port and placed in a NaOH solution to form a liquid seal. The temperature was raised to the set temperature at a rate of 2°C / min, kept constant for 2 h, and taken out after cooling to room temperature. It was sieved through a 100-mesh sieve to obtain biochar with a particle size of 200 μm, and prepared in batches multiple times. Finally, the wheat straw biochar prepared from different batches was mixed evenly, put into a ziplock bag, and placed in a dry environment for refrigeration.
5. The method for preparing the bacterial agent for degrading the herbicide isoproturon according to claim 3, characterized in that: The method for culturing the bacterial solution in step (2) is as follows: Isoproturon-degrading bacteria Sphingobium sp. IPU-1 was inoculated into LB liquid culture medium, and after culturing to the logarithmic growth phase, the culture fluid was collected, the supernatant was discarded by centrifugation, and the cells were washed three times with physiological saline. After the cells were collected, the cell concentration was adjusted to OD600nm of 1.0 with a buffer solution and set aside.
6. The method for preparing the bacterial agent for degrading the herbicide isoproturon according to claim 3, characterized in that: The method for preparing the bacterial agent in step (3) is as follows: A certain amount of wheat straw biochar was added to 50 mL of inorganic salt liquid culture medium, sterilized at 121°C in a high-pressure steam sterilizer for 20 min, cooled to room temperature, and inoculated with the above-prepared bacterial suspension at a volume percentage of 10% to obtain a mixed immobilized culture medium of biochar and bacterial liquid. After being fixed in a shaker, it was taken out and centrifuged to discard the supernatant; it was washed with 0.9% saline, centrifuged for 5 min, and then the supernatant was discarded. The lower sediment was washed with sterile saline, and this process was repeated twice. Sphingobium sp. IPU-1 and biochar immobilized bacteria were dried at 30°C and stored in a refrigerator at 4°C.