Application of nitrogen-fixing cyanobacteria in degradation of thiamethoxam in water body

Abstract

The invention discloses an application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water, which comprises the following steps: inoculating nitrogen-fixing cyanobacteria Nostoc sp.PCC7120 into a liquid culture medium, and pre-culturing to a logarithmic phase to obtain a first cyanobacteria solution; inoculating the first algae solution into a new liquid culture medium to obtain a second algae solution; and adding the second algae liquid into the thiamethoxam polluted water body for degradation so as to remove thiamethoxam. The nitrogen-fixing cyanobacteria Nostoc sp.PCC7120 can efficiently degrade and convert thiamethoxam in a water body, secondary pollution is not prone to being generated, the Nostoc sp.PCC7120 serves as the nitrogen-fixing cyanobacteria beneficial to the environment, thiamethoxam pollutants are degraded, meanwhile, a nitrogen source in the polluted environment can be improved through photosynthesis and nitrogen fixation, and the Nostoc sp.PCC7120 is easy to obtain, high in growth and reproduction speed, low in cost and suitable for application and popularization.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial water pollution remediation, and particularly relates to the application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies. Background Art

[0002] With the vigorous development of modern agriculture, pesticides remain a key element in agricultural practices. Thiamethoxam (THX), with the chemical name of 3-(2-chloro-5-thiazolylmethyl)-5-methyl-N-nitro-4H-1,3,5-trihydrooxadiazine-4-imine, is a thionictin-type insecticide among the second-generation neonicotinoid pesticides (NNIs). It has the characteristics of high efficacy, long residual period, and low dosage, and has gradually become one of the most widely used neonicotinoid pesticides as a better variety to replace carbamate and organofluorine insecticides. However, in the agricultural ecosystem, approximately 70% of NNIs cannot be effectively absorbed or metabolized by environmental matrices and organisms. These unutilized NNIs are relatively stable in chemical properties and have high water solubility, and can migrate into the ecosystem in various ways, causing potential impacts on the environment. According to research reports, the population numbers of bees, fish, frogs, birds, and other animals that prey on these vertebrates directly decline when exposed to the NNI environment. In addition, NNIs and their metabolites have been frequently detected in the human body through dietary and water intake routes, including in serum, hair, milk, and saliva, which may pose serious health risks such as neurotoxicity and diabetes. These problems have attracted great attention to the environmental fate of NNIs and their metabolites.

[0003] NNIs can persist and accumulate in the water environment. Therefore, the development of strategies for effectively removing NNIs from water bodies has become an important international concern. Currently, a variety of innovative technologies have been adopted to remove NNIs, including advanced oxidation processes, adsorption methods, membrane treatment technologies, and various biological methods. Although progress has been made in these technologies, their applications are often restricted by some factors, such as secondary pollution of by-products, harsh environmental conditions, cost issues, and long treatment cycles. Therefore, there is an urgent need to develop efficient, environmentally friendly, and sustainable methods to remove NNIs from the environment, especially in aquatic systems. Bioremediation utilizes the basic metabolic activities of microorganisms such as bacteria, fungi, and algae to eliminate pollutants. This method is environmentally friendly, cost-effective, and has a minimum risk of secondary pollution, and is a safe remediation technology. However, there is a lack of efficient degradation biological resources for neonicotinoid pesticide pollution in the water environment at present. Therefore, it is of great significance to seek a biological resource that can efficiently degrade neonicotinoid pesticides in water bodies. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide an application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies, which has good effects and is green and environmentally friendly.

[0005] To solve the above technical problem, the present invention adopts the following technical solution: an application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies, using nitrogen-fixing cyanobacteria Nostoc sp. PCC7120 to degrade thiamethoxam in water bodies.

[0006] The above application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies preferably includes the following steps:

[0007] (1) Resuscitate the nitrogen-fixing cyanobacteria and inoculate them into a liquid medium for pre-culturing until the logarithmic growth phase to obtain a first algal solution;

[0008] (2) Take the first algal solution, remove the supernatant, wash it with a liquid medium, and transfer the washed nitrogen-fixing cyanobacteria into a new liquid medium to obtain a second algal solution;

[0009] (3) Add the second algal solution to the thiamethoxam-polluted water body for degradation to remove thiamethoxam.

[0010] The above application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies preferably, in step (1), the pre-culturing is carried out under constant temperature conditions with light and dark cycle culture.

[0011] The above application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies preferably, the temperature of the pre-culturing is 20°C to 35°C, the time of the pre-culturing is 10 days to 15 days, light and dark alternation is carried out every 12h to 14h, the intensity of the light is 2000 lux to 3000 lux, and the algal solution is shaken every 6h to 8h.

[0012] The above application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies preferably, in step (2), the initial optical density of the second algal solution at 680 nm is 1.5 to 1.6, and the corresponding algal cell number is 0.99×10 7 cell / mL to 1.05×10 7 cell / mL.

[0013] The above application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies preferably, in step (3), the volume of the second algal solution accounts for 5% to 10% of the volume of the degrading water body, the degrading water body is the thiamethoxam-polluted water body after adding the second algal solution, and the mass concentration of thiamethoxam in the thiamethoxam-polluted water body is 1 ppm to 100 ppm.

[0014] The application of the above-mentioned nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies. Preferably, in step (3), the degradation is carried out under constant temperature conditions with light and dark cycles. The constant temperature is 20°C to 35°C, the degradation time is 6 days to 8 days, the light and dark are alternated every 12h to 14h, and the light intensity is 2000lux to 3000lux.

[0015] The application of the above-mentioned nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies. Preferably, the liquid medium is BG11 liquid medium with a mass concentration of 0.15% to 0.20% and a pH of 7.00 to 7.20.

[0016] The application of the above-mentioned nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies. Preferably, in step (3), after the degradation, the following treatment is further included: taking the degraded mixed solution, centrifuging and separating, and taking the supernatant for quantitative analysis of the remaining amount of thiamethoxam.

[0017] The application of the above-mentioned nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies. Preferably, the dosage of the mixed solution is 5mL to 10mL; the rotation speed of the centrifugal separation is 8000r / min to 10000r / min, and the centrifugal separation time is 15min to 20min; the dosage of the supernatant is 1mL to 1.5mL.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] The application of the nitrogen-fixing cyanobacteria of the present invention in degrading thiamethoxam in water bodies uses the nitrogen-fixing cyanobacteria Nostoc sp. PCC7120 to degrade thiamethoxam in water bodies. The degradation efficiency of thiamethoxam with a mass concentration of 1ppm to 100ppm can reach 100% within 6 to 8 days. Its degradation efficiency is significantly better than other solutions of the prior art, and no secondary pollution is generated, having the advantages of environmental protection. The nitrogen-fixing cyanobacteria Nostoc sp. PCC7120 of the present invention grows and reproduces rapidly, and its growth and reproduction speed is better in an environment containing thiamethoxam, and is also better than other types of nitrogen-fixing cyanobacteria, reducing the culture cost of microorganisms to a certain extent and laying a foundation for future industrial applications; at the same time, as an environmentally beneficial nitrogen-fixing cyanobacteria, Nostoc sp. PCC7120 can improve the nitrogen source in the polluted environment through photosynthesis and nitrogen fixation while degrading thiamethoxam pollutants. The present invention uses nitrogen-fixing cyanobacteria to degrade and transform the neonicotinoid pesticide thiamethoxam, which can prevent or solve the accumulation of thiamethoxam in water environment and prevent the pollution of thiamethoxam to water environment. The response speed of the nitrogen-fixing cyanobacteria Nostoc sp. PCC7120 of the present invention in an environment containing thiamethoxam is significantly better than other types of nitrogen-fixing cyanobacteria. Description of the Drawings

[0020] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention.

[0021] Figure 1 This is the effect diagram of the removal of different concentrations of thiamethoxam by nitrogen-fixing cyanobacteria in Example 1 of the present invention.

[0022] Figure 2 This is the effect diagram of the removal of thiamethoxam by different types of nitrogen-fixing cyanobacteria under the same conditions. Detailed implementation manners

[0023] The following further describes the present invention with reference to the accompanying drawings of the specification and specific preferred embodiments, but does not limit the protection scope of the present invention thereby. The materials and instruments used in the following embodiments are commercially available unless otherwise specified.

[0024] Example 1

[0025] An application of a nitrogen-fixing cyanobacterium of the present invention in degrading thiamethoxam in water bodies, using the nitrogen-fixing cyanobacterium Nostoc sp. PCC7120 to degrade thiamethoxam in water bodies, includes the following steps:

[0026] (1) Select the nitrogen-fixing cyanobacterium Nostoc sp. PCC7120 for resuscitation, and inoculate it into a BG11 liquid medium with a mass concentration of 0.17% and a pH of 7.10 ± 0.1. Cultivate it in a constant temperature incubator at 25 ± 1 °C, and perform an enlarged pre-culture for 15 days to the logarithmic growth phase under the condition of 12 h light / 12 h dark cycle with a light intensity of 3000 lux (lux) to obtain a first algal solution. During this period, shake the algal solution every 8 hours, and randomly change the light position to simulate the light change in the real environment.

[0027] Among them, the nitrogen-fixing cyanobacterium Nostoc sp. PCC7120 was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, and the algal strain number is: FACHB-418.

[0028] The composition of the BG11 liquid medium is as follows: per liter, it includes 1.5 grams of sodium nitrate, 20 milligrams of sodium carbonate, 6 milligrams of citric acid, 0.39 milligrams of sodium molybdate, 6 milligrams of ammonium iron citrate, 0.08 milligrams of copper sulfate, 0.075 grams of magnesium sulfate, 0.05 milligrams of cobalt nitrate, 0.036 grams of calcium chloride, 2.86 milligrams of boric acid, 0.001 grams of disodium ethylenediaminetetraacetate, 1.81 milligrams of manganese chloride, 0.04 grams of potassium dihydrogen phosphate, and 0.22 milligrams of zinc sulfate. The manufacturer of the BG11 medium is Haibo Biotechnology Co., Ltd., and the product number is HB8793.

[0029] (2) Take a certain amount of the first algal solution. After removing the supernatant, wash it 3 times with BG11 liquid medium with a mass concentration of 0.17%, and then transfer the washed nitrogen-fixing cyanobacteria into a new BG11 liquid medium with a mass concentration of 0.17% to make the initial optical density of Nostoc sp. PCC7120 at 680 nm be 1.5. At this time, the number of nitrogen-fixing cyanobacteria cells is 0.99×10 7 cell / mL to obtain the second algal solution.

[0030] (3) Take three equal volumes of the second algal solution and add them to three polluted waters with the mass concentrations of thiamethoxam (THX) being 1 ppm, 10 ppm, and 80 ppm respectively for degradation to simulate the removal of thiamethoxam in actual water bodies. The volume of the second algal solution accounts for 8% of the volume of the degraded water body. The degraded water body is the thiamethoxam-polluted water body after adding the second algal solution. The degradation is carried out under constant temperature conditions with light and dark cycles. The constant temperature is 25±1 °C, and the degradation time is 8 days. Light and dark are alternated every 12 h, and the light intensity is 3000 lux. Take three BG11 media with a mass concentration of 0.17% without inoculating Nostoc sp. PCC7120 and add them to three polluted waters with the mass concentrations of thiamethoxam being 1 ppm, 10 ppm, and 80 ppm respectively for degradation under the same conditions as a blank control group;

[0031] (4) After the degradation is completed, take 10 mL of the degraded mixture and transfer it to a 10 ml centrifuge tube. Centrifuge it at 8000 r / min for 15 min. After centrifugation, take 1.5 mL of the supernatant and use a high-performance liquid chromatograph for quantitative analysis to obtain the remaining amount of thiamethoxam.

[0032] Use a high-performance liquid chromatograph to quantitatively analyze thiamethoxam, and calculate the removal rate according to the formula: v=(C 0 -C n ) / C 0 *100%, where C n is the concentration of thiamethoxam on the nth day, and C 0 is the concentration of thiamethoxam at day 0. The removal effects of nitrogen-fixing cyanobacteria Nostoc sp. PCC7120 on thiamethoxam with concentrations of 1 ppm, 10 ppm, and 80 ppm are as Figure 1As shown in the figure, in the blank group of Example 1, the photo- and hydrolysis rates of thiamethoxam with mass concentrations of 1 ppm, 10 ppm, and 80 ppm on the 6th day were 0.73%, 1.19%, and 1.63% respectively, indicating that thiamethoxam was difficult to decompose under the culture system of the blank group; the degradation rates of thiamethoxam with mass concentrations of 1 ppm, 10 ppm, and 80 ppm in the experimental group on the 6th day of culture were 100%, 97.48%, and 90.96% respectively; the degradation rates of thiamethoxam at 1 ppm, 10 ppm, and 80 ppm in the experimental group on the 8th day of culture were all 100%.

[0033] Investigate the application of different species of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water

[0034] Using the nitrogen-fixing cyanobacteria Nostoc sp. PCC7120, Nostoc linckia FACHB-104, Tolypothrix tenuis FACHB-129, and Anabaena azotica FACHB-888 to degrade thiamethoxam in water respectively, including the following steps:

[0035] (1) Resuscitate the above four species of nitrogen-fixing cyanobacteria respectively, and inoculate them into four portions of BG11 liquid medium with a mass concentration of 0.17% and a pH of 7.10 ± 0.1. Cultivate them in a constant temperature incubator at 25 ± 1°C, and perform an expanded pre-culture for 15 days to the logarithmic growth phase under the condition of 12 h light / 12 h dark cycle with a light intensity of 3000 lux (lux). Obtain the first algal solutions of the four species of nitrogen-fixing cyanobacteria respectively. During this period, shake the algal solution once every 8 hours and randomly change the light position to simulate the light change in the real environment.

[0036] (2) Take out a certain amount of the first algal solutions of the four species of nitrogen-fixing cyanobacteria respectively. After removing the supernatant, wash them 3 times with BG11 liquid medium with a mass concentration of 0.17%, and then transfer the washed nitrogen-fixing cyanobacteria into a new BG11 liquid medium with a mass concentration of 0.17% to make the initial optical density of the nitrogen-fixing cyanobacteria at 680 nm be 1.5. At this time, the number of nitrogen-fixing cyanobacteria cells is 0.99×10 7 cell / mL, and obtain the second algal solutions of the four species of nitrogen-fixing cyanobacteria respectively.

[0037] (3) Add equal amounts of the second algal solutions of the four species of nitrogen-fixing cyanobacteria into four portions of polluted water bodies with a thiamethoxam mass concentration of 1 ppm respectively for degradation. Among them, the volume of the second algal solution accounts for 8% of the volume of the degraded water body. The degraded water body is the thiamethoxam-polluted water body after adding the second algal solution. The degradation is carried out under the condition of light and dark cycle at a constant temperature. The constant temperature is 25 ± 1°C, the degradation time is 8 days, and the light and dark are alternated every 12 h. The light intensity is 3000 lux.

[0038] (4) After the degradation is completed, 10 mL of each of the four degraded mixed solutions is taken and transferred to four 10 mL centrifuge tubes respectively. Centrifuge at 8000 r / min for 15 min. After centrifugation, take 1.5 mL of the supernatant from each and perform quantitative analysis using a high performance liquid chromatograph to obtain the remaining amount of thiamethoxam.

[0039] In the blank group of Example 1, the photolysis and hydrolysis rate of 1 ppm thiamethoxam on the 6th day was 0.73%, indicating that thiamethoxam is difficult to decompose in a culture system without nitrogen-fixing cyanobacteria. Quantitative analysis of thiamethoxam was carried out using a high performance liquid chromatograph. According to the formula, the remaining amount of thiamethoxam was calculated, v = 1 - (C 0 - C n ) / C 0 * 100%, where C n is the concentration of thiamethoxam on the nth day, and C 0 is the concentration of thiamethoxam at day 0. Figure 2 is the removal effect diagram of different types of nitrogen-fixing cyanobacteria on 1 ppm thiamethoxam. As Figure 2 shown, the remaining amount of 1 ppm thiamethoxam degraded by Nostoc sp. PCC7120 on the 6th day was 0%, and its degradation rate was 100%. Under the same conditions, the remaining amounts of thiamethoxam with a mass concentration of 1 ppm degraded by three nitrogen-fixing cyanobacteria, Nostoc linckia FACHB-104, Tolypothrix tenuis FACHB-129, and Anabaena azotica FACHB-888, were still relatively high on the 8th day, being 44.9%, 39.6%, and 50.0% respectively. This shows the recalcitrant characteristics of thiamethoxam and further proves the excellent degradation effect of the nitrogen-fixing cyanobacterium Nostoc sp. PCC7120 used in the present invention.

[0040] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes, without departing from the spirit and technical solution of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. An application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water, characterized in that: Degradation of thiamethoxam in water by nitrogen-fixing cyanobacteria Nostocsp. PCC7120.

2. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to claim 1, characterized in that: The following steps are involved: (1) reviving the nitrogen-fixing cyanobacteria, inoculating them into a liquid culture medium and pre-culturing them until the logarithmic growth phase to obtain a first algae liquid; (2) taking the first algae liquid, removing the supernatant, washing with a liquid culture medium, and transferring the washed nitrogen-fixing cyanobacteria into a new liquid culture medium to obtain a second algae liquid; (3) adding the second algae liquid to the water body contaminated by thiamethoxam to degrade the water body so as to remove thiamethoxam.

3. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to claim 2, characterized in that: In step (1), the pre-culture is carried out under constant temperature conditions with light and dark cycles.

4. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to claim 3, characterized in that: The pre-culture temperature is 20°C to 35°C, the pre-culture time is 10 to 15 days, light and darkness are alternated every 12 to 14 hours, the light intensity is 2000 lux to 3000 lux, and the algae liquid is shaken every 6 to 8 hours.

5. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to claim 2, characterized in that: In step (2), the initial optical density of the second algae solution at 680 nm is 1.5-1.6, and the corresponding number of algae cells is 0.99×10 7 cell / mL~1.05×10 7 cell / mL.

6. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to claim 2, characterized in that: In step (3), the volume of the second algae liquid accounts for 5% to 10% of the volume of the degraded water body, and the mass concentration of thiamethoxam in the thiamethoxam-contaminated water body is 1 ppm to 100 ppm.

7. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to claim 6, characterized in that: In step (3), the degradation is carried out under constant temperature conditions with light and dark cycles, the constant temperature is 20°C to 35°C, the degradation time is 6 days to 8 days, light and dark are alternated every 12h to 14h, and the intensity of the light is 2000lux to 3000lux.

8. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to any one of claims 2 to 7, characterized in that: The liquid culture medium is a BG11 liquid culture medium with a mass concentration of 0.15% to 0.20% and a pH of 7.00 to 7.

20.

9. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to any one of claims 2 to 7, characterized in that: In step (3), after the degradation is completed, the following treatment is further included: taking the mixed solution after degradation, centrifuging it, and taking the supernatant to quantitatively analyze the remaining amount of thiamethoxam.

10. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water according to claim 9, characterized in that: The amount of the mixed solution is 5 mL to 10 mL; the rotation speed of the centrifugal separation is 8000 r / min to 10000 r / min, and the time of the centrifugal separation is 15 min to 20 min; the amount of the supernatant is 1 mL to 1.5 mL.

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