Application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water

Abstract

The application discloses application of nitrogen-fixing cyanobacteria in degradation of thiamethoxam in water bodies, and comprises the following steps: inoculating nitrogen-fixing cyanobacteria Nostoc sp.PCC7120 into liquid culture medium to carry out pre-culturing to logarithmic growth phase, and obtaining first algal liquid; inoculating the first algal liquid into new liquid culture medium to obtain second algal liquid; adding the second algal liquid into thiamethoxam contaminated water bodies to carry out degradation, so as to remove thiamethoxam. The nitrogen-fixing cyanobacteria Nostoc sp.PCC7120 can efficiently degrade and convert thiamethoxam in water bodies, and is not easy to cause secondary pollution; as environmental beneficial nitrogen-fixing cyanobacteria, the Nostoc sp.PCC7120 can improve nitrogen source in the polluted environment through photosynthesis and nitrogen fixation while degrading thiamethoxam pollutants; and the Nostoc sp.PCC7120 is easy to obtain, has high growth and reproduction speed, and is low in cost, so is suitable for popularization and application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial water pollution remediation, and particularly relates to application of nitrogen-fixing blue-green algae in degradation of thiamethoxam in water. BACKGROUND

[0002] With the vigorous development of modern agriculture, pesticides are still a key element of agricultural practice. Thiamethoxam (THX), chemical name 3-(2-chloro-5-thiazolylmethyl)-5-methyl-N-nitro-4H-1,3,5-tetrahydrooxadiazine-4-imine, is a thionicotinoid insecticide in the second generation of neonicotinoid pesticides (NNIs). It has the characteristics of high prevention effect, long effective period, low drug dosage, etc., and is a good variety to replace carbamate and organofluorine insecticides and gradually becomes one of the most widely used neonicotinoid insecticides. However, in the agricultural ecosystem, about 70% of NNIs cannot be effectively absorbed or metabolized by environmental substrates and organisms. These unused NNIs are relatively stable in chemical properties and have high water solubility, and can migrate to the ecosystem in various ways, causing potential impact on the environment. According to research reports, bees, fish, frogs, birds and other animal populations that prey on these vertebrates directly decrease under the exposure of NNIs. In addition, NNIs and their metabolites have been frequently detected in the human body through dietary and water intake pathways, including serum, hair, milk and saliva, which may bring serious health risks such as neurotoxicity and diabetes. These problems have attracted people's high attention to the environmental fate of NNIs and their metabolites.

[0003] NNIs can persist and accumulate in water environment. Therefore, the development of effective strategies to remove NNIs from water bodies has become an important international concern. At present, various 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 application is often subject to some limitations, such as secondary pollution of by-products, adverse environmental conditions, cost issues and long processing cycle. 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 minimal risk of secondary pollution, and is a safe remediation technology. However, there is currently a lack of efficient degradation of neonicotinoid pesticides in water environment, so it is of great significance to seek a biological resource that can efficiently degrade neonicotinoid pesticides in water. SUMMARY

[0004] The technical problem solved by the present application is to overcome the shortcomings of the prior art and provide an application of nitrogen-fixing cyanobacteria in degrading water thiabendazole, which is good in effect and green in environmental protection.

[0005] To solve the above technical problems, the present application adopts the following technical scheme: an application of nitrogen-fixing cyanobacteria in degrading water thiabendazole, in which nitrogen-fixing cyanobacteria Nostoc sp.PCC7120 is used to degrade thiabendazole in water.

[0006] The application of nitrogen-fixing cyanobacteria in degrading water thiabendazole preferably comprises the following steps:

[0007] (1) The nitrogen-fixing cyanobacteria is resuscitated, inoculated into a liquid culture medium and pre-cultured to the logarithmic growth phase to obtain a first algal liquid;

[0008] (2) The first algal liquid is taken, the supernatant is removed, the nitrogen-fixing cyanobacteria after washing with the liquid culture medium is transferred into a new liquid culture medium to obtain a second algal liquid;

[0009] (3) The second algal liquid is added into a thiabendazole-polluted water body for degradation to remove thiabendazole.

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

[0011] In the application of nitrogen-fixing cyanobacteria in degrading water thiabendazole, the pre-culturing temperature is preferably 20-35℃, the pre-culturing time is preferably 10-15 days, light and dark are alternated every 12-14 hours, the light intensity is 2000-3000 lux, and the algal liquid is shaken every 6-8 hours.

[0012] In the application of nitrogen-fixing cyanobacteria in degrading water thiabendazole, the initial optical density of the second algal liquid at 680 nm is preferably 1.5-1.6, and the corresponding number of algal cells is preferably 0.99x10 7 cell / mL-1.05x10 7 cell / mL.

[0013] In the application of nitrogen-fixing cyanobacteria in degrading water thiabendazole, the volume of the second algal liquid accounts for 5-10% of the volume of the degradation water body in step (3), the degradation water body is a thiabendazole-polluted water body to which the second algal liquid is added, and the mass concentration of thiabendazole in the thiabendazole-polluted water body is 1-100 ppm.

[0014] Preferably, in the step (3), the degradation is carried out under constant temperature condition with light and dark cycle, the constant temperature is 20-35 DEG C, the degradation time is 6-8 days, the light and dark cycle is carried out once every 12-14 hours, and the light intensity is 2000-3000 lux.

[0015] Preferably, the liquid culture medium is BG11 liquid culture medium with mass concentration of 0.15-0.20% and pH of 7.00-7.20.

[0016] Preferably, in the step (3), after the degradation, the following treatment is further included: taking the mixed solution after degradation, centrifugal separation, and taking the supernatant to quantitatively analyze the residual amount of thiamethoxam.

[0017] Preferably, the taking amount of the mixed solution is 5-10 mL; the centrifugal separation speed is 8000-10000 r / min, and the centrifugal separation time is 15-20 min; and the taking amount of the supernatant is 1-1.5 mL.

[0018] Compared with the prior art, the application has the following advantages:

[0019] The application of the nitrogen-fixing blue algae in degrading thiamethoxam in water has the following advantages: the nitrogen-fixing blue algae Nostoc sp.PCC7120 can degrade thiamethoxam in water with mass concentration of 1-100 ppm in 6-8 days, and the degradation efficiency is 100%, which is significantly better than that of other prior art solutions, and no secondary pollution is caused, and the application has the advantages of green environmental protection; the nitrogen-fixing blue algae Nostoc sp.PCC7120 grows and reproduces fast, and its growth and reproduction speed is better in the environment with thiamethoxam than other types of nitrogen-fixing blue algae, and the culture cost of the microorganism is reduced to some extent, which lays a foundation for future industrial application; meanwhile, the nitrogen-fixing blue algae Nostoc sp.PCC7120 can improve the nitrogen source in the polluted environment through photosynthesis and nitrogen fixation while degrading thiamethoxam pollutants; the application can prevent or solve the accumulation of thiamethoxam in water environment and prevent the pollution of thiamethoxam to water environment by degrading and converting thiamethoxam with the nitrogen-fixing blue algae; and the response speed of the nitrogen-fixing blue algae Nostoc sp.PCC7120 in the environment with thiamethoxam is significantly better than that of other types of nitrogen-fixing blue algae. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application.

[0021] Figure 1 Figure for removal effect of nitrogen-fixing cyanobacteria in Embodiment 1 of the present application on thiamethoxam of different concentrations.

[0022] Figure 2 Figure for removal effect of thiamethoxam by different types of nitrogen-fixing cyanobacteria under the same conditions. DETAILED DESCRIPTION

[0023] The present application will be further described below in combination with the drawings of the specification and specific preferred embodiments, but the protection scope of the present application is not limited therefore. The materials and instruments used in the following embodiments are commercially available unless otherwise specified.

[0024] Embodiment 1

[0025] The present application discloses an application of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water, which degrades thiamethoxam in water by using nitrogen-fixing cyanobacteria Nostoc sp. PCC7120, and includes the following steps:

[0026] (1) Resuscitate nitrogen-fixing cyanobacteria Nostoc sp. PCC7120, inoculate into BG11 liquid medium with a mass concentration of 0.17% and pH of 7.10±0.1, cultivate in a constant temperature incubator at 25±1℃, and expand pre-culture under 12h light / 12h dark cycle conditions with a light intensity of 3000 lux for 15 days to logarithmic growth phase, to obtain first algae liquid, shake the algae liquid every 8 hours during the period, and randomly change the light position to simulate the light change in real environment.

[0027] The nitrogen-fixing cyanobacteria Nostoc sp. PCC7120 is purchased from the Freshwater Algae Culture Collection of China Academy of Sciences, and the algae strain number is FACHB-418.

[0028] The composition of the BG11 liquid medium is as follows: 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 per liter. 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, remove the supernatant, and wash three times with 0.17% BG11 liquid medium. Then transfer the washed nitrogen-fixing cyanobacteria into a new 0.17% BG11 liquid medium, so that the initial optical density of Nostoc sp. PCC7120 at 680 nm is 1.5, and the number of nitrogen-fixing cyanobacteria cells is 0.99 × 10⁻⁶. 7 The second algal solution was obtained at a cell / mL.

[0030] (3) Take three equal portions of the second algal solution and add them to three portions of polluted water with thiamethoxam (THX) concentrations of 1 ppm, 10 ppm, and 80 ppm, respectively, for degradation to simulate the removal of thiamethoxam from actual water bodies. The volume of the second algal solution accounts for 8% of the volume of the water body to be degraded. The water body to be degraded is the thiamethoxam-polluted water body after the addition of the second algal solution. Degradation is carried out under constant temperature conditions with alternating light and dark cycles. The constant temperature is 25±1℃, and the degradation time is 8 days. Light and dark cycles are performed every 12 hours, and the light intensity is 3000 lux. Take three portions of 0.17% BG11 culture medium without Nostoc sp. PCC7120 inoculation and add them to three portions of polluted water with thiamethoxam concentrations of 1 ppm, 10 ppm, and 80 ppm, respectively, for degradation under the same conditions, as blank control groups.

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

[0032] Thiamethoxam was quantitatively analyzed using high performance liquid chromatography (HPLC), and the removal rate was calculated using the formula: v = (C0 - C...) n ) / C0*100%, where C n Cn represents the concentration of thiamethoxam on day n, and C0 represents the concentration of thiamethoxam on day 0. The removal efficiency of the nitrogen-fixing cyanobacterium Nostoc sp. PCC7120 for thiamethoxam concentrations of 1 ppm, 10 ppm, and 80 ppm is shown in the figure. Figure 1 As shown, in Example 1, the light and water degradation rates of 1 ppm, 10 ppm, and 80 ppm thiamethoxam in the blank group on day 6 were 0.73%, 1.19%, and 1.63%, respectively, indicating that thiamethoxam was difficult to decompose in the culture system of the blank group. In the experimental group, the degradation rates of 1 ppm, 10 ppm, and 80 ppm thiamethoxam on day 6 were 100%, 97.48%, and 90.96%, respectively. In the experimental group, the degradation rates of 1 ppm, 10 ppm, and 80 ppm thiamethoxam were all 100% on day 8.

[0033] Investigation on the application of different kinds of nitrogen-fixing cyanobacteria in degrading thiabendazole in water

[0034] Thiabendazole in water was degraded by nitrogen-fixing cyanobacteria Nostoc sp. PCC7120, nitrogen-fixing cyanobacteria Nostoc linckia FACHB-104, nitrogen-fixing cyanobacteria Tolypothrix tenuis FACHB-129 and nitrogen-fixing cyanobacteria Anabaena azotica FACHB-888, respectively, including the following steps:

[0035] (1) The above four kinds of nitrogen-fixing cyanobacteria were recovered and inoculated into four BG11 liquid culture media with a mass concentration of 0.17% and a pH of 7.10±0.1, and cultured in a constant temperature incubator at 25±1℃, with a light intensity of 3000 lux for 12h light / 12h dark cycle for 15 days to the logarithmic growth phase, to obtain the first algae liquid of the four kinds of nitrogen-fixing cyanobacteria, and the algae liquid was shaken every 8 hours and the light position was randomly changed to simulate the light change in the real environment.

[0036] (2) A certain amount of the first algae liquid of the four kinds of nitrogen-fixing cyanobacteria was taken out, washed with 0.17% BG11 liquid medium three times after removing the supernatant, and then transferred into new 0.17% BG11 liquid medium, so that the initial optical density of the nitrogen-fixing cyanobacteria at 680nm was 1.5, at this time the number of nitrogen-fixing cyanobacteria cells was 0.99×10 7 cell / mL, to obtain the second algae liquid of the four kinds of nitrogen-fixing cyanobacteria.

[0037] (3) Equal amounts of the second algae liquid of the four kinds of nitrogen-fixing cyanobacteria were added to four thiabendazole-contaminated water bodies with a mass concentration of 1 ppm for degradation, wherein the volume of the second algae liquid accounted for 8% of the volume of the degradation water body, the degradation water body was the thiabendazole-contaminated water body after adding the second algae liquid, the degradation was carried out under constant temperature conditions with light and dark cycles, the constant temperature was 25±1℃, and the degradation time was 8 days, with light and dark alternation every 12h, and the light intensity was 3000 lux.

[0038] (4) After degradation, 10mL of each of the four degraded mixed liquids was taken and transferred into four 10ml centrifuge tubes, centrifuged at 8000r / min for 15min, and 1.5mL of supernatant was taken after centrifugation, and high performance liquid chromatography was used for quantitative analysis to obtain the residual amount of thiabendazole.

[0039] In Example 1, the blank group showed a photochemical and water decomposition rate of 0.73% for 1 ppm thiamethoxam on day 6, indicating that thiamethoxam is difficult to decompose in a culture system without nitrogen-fixing cyanobacteria. Quantitative analysis of thiamethoxam was performed using high-performance liquid chromatography (HPLC), and the remaining amount was calculated using the formula: v = 1 - (C0 - C...). n ) / C0*100%, where C n C is the concentration of thiamethoxam on day n, and C0 is the concentration of thiamethoxam on day 0. Figure 2 The graph shows the removal effect of different species of nitrogen-fixing cyanobacteria on a 1 ppm concentration of thiamethoxam. Figure 2 As shown, Nostoc sp. PCC7120 had a 0% residual amount of 1 ppm thiamethoxam after degradation on day 6, with a degradation rate of 100%. Under the same conditions, three nitrogen-fixing cyanobacteria, Nostoc linckia FACHB-104, Tolypothrix tenuis FACHB-129, and Anabaena azotica FACHB-888, still had relatively high residual amounts of 1 ppm thiamethoxam after degradation on day 8, at 44.9%, 39.6%, and 50.0%, respectively. This demonstrates the recalcitrant degradation characteristics of thiamethoxam and further proves the excellent degradation effect of the nitrogen-fixing cyanobacterium Nostoc sp. PCC7120 used in this invention on thiamethoxam.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. The use of nitrogen-fixing cyanobacteria in degrading thiamethoxam in water bodies, characterized in that, Nitrogen-fixing cyanobacteria Nostoc sp. PCC7120 degrade thiamethoxam in water bodies.

2. Use of the nitrogen-fixing cyanobacterium according to claim 1 for degrading thiamethoxam in a water body, characterized in that, comprising the following steps: (1) resuscitating the nitrogen-fixing cyanobacteria, inoculating into liquid medium to pre-culture to logarithmic growth phase to obtain a first algal liquid; (2) taking the first algal liquid, removing supernatant, washing with liquid medium, and transferring the washed nitrogen-fixing cyanobacteria into a new liquid medium to obtain a second algal liquid; (3) adding the second algal liquid into a thiamethoxam contaminated water body for degradation to remove thiamethoxam.

3. Use of the nitrogen-fixing cyanobacterium according to claim 2 for degrading thiamethoxam in a water body, characterized in that, In step (1), the pre-culture is carried out under constant temperature conditions with light and dark cycle culture.

4. Use of the nitrogen-fixing cyanobacterium according to claim 3 for degrading thiamethoxam in a water body, characterized in that, The pre-culture temperature is 20-35℃, the pre-culture time is 10-15 days, light and dark are alternated every 12-14 hours, the light intensity is 2000-3000 lux, and the algal liquid is shaken every 6-8 hours.

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

6. Use of the nitrogen-fixing cyanobacterium according to claim 2 for degrading thiamethoxam in a water body, characterized in that, In step (3), the volume of the second algal liquid accounts for 5-10% of the volume of the degradation water body, and the mass concentration of thiamethoxam in the thiamethoxam contaminated water body is 1-100 ppm.

7. Use of nitrogen-fixing cyanobacteria according to claim 6 for degrading thiamethoxam in a water body, characterized in that, In step (3), the degradation is carried out under constant temperature conditions with light and dark cycle, the constant temperature is 20-35℃, the degradation time is 6-8 days, light and dark are alternated every 12-14 hours, and the light intensity is 2000-3000 lux.

8. Use of the nitrogen-fixing cyanobacteria according to any one of claims 2 to 7 for the degradation of thiamethoxam in a water body, characterized in that, The liquid medium is BG11 liquid medium with a mass concentration of 0.15-0.20% and a pH of 7.00-7.

20.

9. Use of the nitrogen-fixing cyanobacterium according to any one of claims 2 to 7 for the degradation of thiamethoxam in a water body, characterized in that, In step (3), after the degradation is completed, the following treatment is further included: taking the mixed liquid after degradation, centrifuging, taking supernatant, and quantitatively analyzing the residual amount of thiamethoxam.

10. Use of the nitrogen-fixing cyanobacterium according to claim 9 for degrading thiamethoxam in a water body, characterized in that, The amount of the mixed liquid taken is 5-10 mL; the centrifugation speed is 8000-10000 r / min, the centrifugation time is 15-20 min; and the amount of the supernatant taken is 1-1.5 mL.

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