Formulation for reducing pesticide residues and application of scytonema in reducing pesticide residues

Abstract

The application belongs to the technical field of land pesticide residue degradation, and particularly relates to a preparation for reducing pesticide residues and application of cladophora in reducing pesticide residues. Scenedesmus obliquus It is found that the cladophora can effectively degrade four typical pesticides of atrazine, thiamethoxam, cypermethrin and nicosulfuron, has wide applicable temperature (such as 5 DEG C to 30 DEG C), short action time, high safety and low cost.

Description

Technical Field

[0001] This invention belongs to the field of soil pesticide residue degradation technology, specifically involving formulations for reducing pesticide residues and the application of Scenedesmus obliquus in reducing pesticide residues. Background Technology

[0002] Black soil, a globally scarce high-fertility soil resource, is a core strategic resource for ensuring national food security in Northeast my country's main grain-producing areas. However, with the development of intensive agriculture, pesticides such as atrazine (a corn herbicide), thiamethoxam (a neonicotinoid insecticide), cypermethrin (a pyrethroid insecticide), and nicosulfuron (a sulfonylurea herbicide) are widely used. Because these pesticides are chemically stable and have long half-lives (e.g., atrazine's half-life can reach 60–150 days), they easily form complex residual pollution in black soil and accumulate through the food chain.

[0003] Current pesticide pollution control technologies have significant limitations: (1) When four types of pesticides coexist, they may produce synergistic toxicity (e.g., the combined use of nicosulfuron and atrazine can increase the toxicity to aquatic organisms), exacerbating ecological risks; (2) The organic matter content of black soil is as high as 5% to 10%, and the strong adsorption effect on pesticide molecules significantly inhibits the degradation efficiency of traditional microbial remediation technologies; (3) The reported degradation strains (e.g., Acinetobacter and Pseudomonas) generally have problems such as single target (can only degrade 1-2 kinds of pesticides) and poor environmental adaptability (the activity of black soil drops sharply during the low temperature period), which makes it difficult to meet the treatment needs of complex pollution scenarios.

[0004] With the deepening of environmental protection and sustainable development concepts, green, efficient, and low-cost bioremediation technologies have become a research hotspot. Microalgae, as a type of photosynthetic autotrophic lower plant, possess advantages such as rapid growth, strong environmental adaptability, and the ability to simultaneously degrade pesticide residues, demonstrating enormous potential in pollution control. However, currently, broad-spectrum, low-temperature-resistant microalgae degradation technologies targeting typical compound pesticide pollution in black soil remain lacking. Developing microalgae remediation technologies with high degradation performance and environmental adaptability is of great significance for solving the problem of pesticide pollution in black soil, ensuring food security, and promoting ecological sustainable development. Summary of the Invention

[0005] Based on this, the present invention discovered *Scenedesmus obliquus* (… Scenedesmus obliquus It can effectively degrade four typical pesticides: atrazine, thiamethoxam, cypermethrin, and nicosulfuron. It has a wide applicable temperature range (e.g., 5℃~30℃), short action time, high safety, and low cost.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present application provides a pesticide residue-reducing preparation, which comprises a Scenedesmus obliquus having a preservation number of CGMCC No. 6567.

[0008] In another aspect, the present application provides an application of the Scenedesmus obliquus in reducing pesticide residue, the pesticide being one or a combination of atrazine, thiamethoxam, cypermethrin or nicosulfuron.

[0009] Preferably, the application comprises an application of the Scenedesmus obliquus in reducing pesticide residue in soil.

[0010] Preferably, in the above application, the Scenedesmus obliquus has a preservation number of CGMCC No. 6567.

[0011] Preferably, in the above application, the application is applicable at a temperature of 5-30℃.

[0012] In still another aspect, the present application provides a method for reducing pesticide residue in soil, which comprises inoculating the Scenedesmus obliquus into the soil, the pesticide being one or a combination of atrazine, thiamethoxam, cypermethrin or nicosulfuron.

[0013] Preferably, in the above method, the Scenedesmus obliquus has a preservation number of CGMCC No. 6567.

[0014] Preferably, in the above method, the Scenedesmus obliquus is inoculated at an amount of (0.8-1.2)×10 8 cells / g of soil.

[0015] Preferably, in the above method, nitrogen and phosphorus elements are further added to the soil.

[0016] More preferably, in the above method, the ratio of the nitrogen and phosphorus elements is (9-11):1, and the phosphorus element is added at an amount of (4-6) mg / kg of soil.

[0017] The present application has the following beneficial effects:

[0018] (1) The present application uses the Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron, and a single treatment can reduce the pesticide residue in soil to below the limit in GB 2763-2021, thereby ensuring the safe reuse of arable land.

[0019] (2) The present application uses the Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron, and the pesticide degradation flux per unit area of black soil reaches 35.2 mg / (kg·d), which is 305% higher than that of the traditional method (8.7 mg / (kg·d)).

[0020] (3) The application uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron, and the algal liquid of Scenedesmus obliquus can be expanded on site (cost 0.8 yuan per liter), and the dosage per mu needs 375 liters, and the comprehensive cost can be reduced by about 85% compared with the prior art.

[0021] (4) The application uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron, and the temperature range is wide, which can be applied to the temperature range of 5-30 DEG C in the northeast black soil area, covering 92% of the annual agricultural time window.

[0022] (5) The application uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron, and the degradation rate of the pesticide can reach more than 88% at room temperature for 28 days, and the degradation rate of the pesticide can reach more than 66% at 10 DEG C low temperature for 45 days, the action time is short, and the effect is quick.

[0023] (6) The application uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron in soil, and will not generate metabolic toxins, and will not damage the soil structure, and is high in safety.

[0024] (7) The application uses Scenedesmus obliquus to degrade atrazine, thiamethoxam, cypermethrin and nicosulfuron, which is more spectral than the prior art (such as traditional microbial remediation and chemical oxidation method). DETAILED DESCRIPTION

[0025] The examples are used to better illustrate the application, but are not the content of the application, and are only limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above application, which still belong to the protection scope of the application.

[0026] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless otherwise apparent in context, the singular form of expression includes the plural form of expression. As used herein, it should be understood that terms such as "include", "have", "contain", etc. are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the application are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations may exist or can be added. As used herein, " / " can be interpreted as "and" or "or" depending on the circumstances.

[0027] In a first aspect, the embodiments of the application provide a preparation for reducing pesticide residues, which comprises Scenedesmus obliquus, and the preservation number of the Scenedesmus obliquus is CGMCC No. 6567.

[0028] In a second aspect, the application provides an application of the Scenedesmus obliquus in reducing pesticide residues, the pesticide being one or more combinations of atrazine, thiamethoxam, cypermethrin or nicosulfuron.

[0029] It should be noted that the application finds that the Scenedesmus obliquus can effectively degrade four typical pesticides of atrazine, thiamethoxam, cypermethrin and nicosulfuron, and has a wide applicable temperature (such as 5-30℃), a short action time, high safety and low cost. Scenedesmus obliquus In addition, the application finds that the Scenedesmus obliquus has a cross-class synergistic degradation capability, and has a synchronous high-efficiency degradation path for four types of pesticides with different chemical structures (including chloro-triazine, thio-neonicotinoid, pyrethroid and sulfonylurea), and the degradation rate needs to reach >85% (28 days, initial concentration of 10 mg / kg); in addition, the application can activate the "multi-enzyme cascade system" in the Scenedesmus obliquus cells (such as dehalogenase to decompose atrazine, cytochrome P450 to oxidize thiamethoxam, esterase to hydrolyze cypermethrin, and sulfonylurea hydrolyase to cut nicosulfuron), thereby solving the problem of single enzyme system of traditional strains.

[0030] In some specific examples, the application includes an application of the Scenedesmus obliquus in reducing pesticide residues in soil.

[0031] It should be noted that the Scenedesmus obliquus in the application is particularly suitable for reducing pesticide residues in soil, can not damage the soil structure, and has high safety without producing toxic metabolites.

[0032] In some specific examples, in the above application, the preservation number of the Scenedesmus obliquus is CGMCC No. 6567.

[0033] It should be noted that the Scenedesmus obliquus in the application is preferably collected from the surface layer (0-10 cm) of the northeast black soil, and the Scenedesmus obliquus obtained by separation has a preservation number of CGMCC No. 6567, and the information of the Scenedesmus obliquus with the preservation number of CGMCC No. 6567 is recorded in the public patent CN102943046A.

[0034] In some specific examples, in the above application, the applicable temperature of the application is 5-30℃.

[0035] It should be noted that the expression amount of the anti-freezing protein (AFPs) of the Scenedesmus obliquus in the application increases at low temperature, and the cell membrane fluidity does not change significantly, that is, the temperature applicable range of the Scenedesmus obliquus in the application for degrading pesticide residues is wide, for example, 5-30℃, such as 5℃, 10℃, 15℃, 20℃ or 25℃, etc.; that is, it is more suitable for the temperature change of the northeast black soil.

[0036] Thirdly, embodiments of the present invention provide a method for reducing pesticide residues in soil, the method comprising: inoculating *Scenedesmus obliquus* into the soil, wherein the pesticide is one or more combinations of atrazine, thiamethoxam, cypermethrin, or nicosulfuron.

[0037] In some specific examples, the preservation number of *Scenedesmus obliquus* in the above method is CGMCC No. 6567.

[0038] In some specific examples, the inoculum size of *Scenedesmus obliquus* in the above method is (0.8–1.2) × 10⁻⁶. 8 cells / g soil.

[0039] It should be noted that, when using *Scenedesmus obliquus* to degrade pesticide residues in soil, the inoculation amount can be (0.8–1.2) × 10⁻⁶ per gram of soil. 8 cells are Scenedesmus oblique.

[0040] In some specific examples, nitrogen and phosphorus elements are also added to the soil in the above method.

[0041] It should be noted that nitrogen and phosphorus elements can be added to promote the degradation of pesticide residues in the soil. Nitrogen and phosphorus elements are well known in the field, for example, in a mixture of NH4Cl and K2HPO4.

[0042] In some specific examples, the ratio of nitrogen to phosphorus in the above method is (9-11):1, and the amount of phosphorus added is (4mg-6mg) / kg soil.

[0043] It should be noted that the ratio of nitrogen to phosphorus in this invention is (9-11):1 (e.g., 9.5:1, 10:1 or 10.5:1, etc.), and the amount of phosphorus added is 4mg to 6mg per kg of soil, such as 4.5mg, 5mg or 5.5mg, etc.

[0044] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0045] In the following examples, Scenedesmus obliquus ( Scenedesmus obliquus The sample was collected from the surface layer (0-10cm) of black soil in Northeast China, isolated, and verified as Scenedesmus obliquus by 18S rRNA sequencing. Its preservation number is CGMCC No. 6567.

[0046] In the following examples, BG-11 medium (pH 7.0) includes: NaNO31.5 g / L, K2HPO40.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, ferric citrate 0.006 g / L, EDTA 0.001 g / L, trace element solution 1 mL / L (1 L trace element solution contains H3BO3(boric acid): 2.86 g, MnCl2·4H2O (manganese chloride tetrahydrate): 1.81 g, ZnSO4·7H2O (zinc sulfate heptahydrate): 0.222 g, Na2MoO4·2H2O (sodium molybdate dihydrate): 0.39 g, CuSO4·5H2O (copper sulfate pentahydrate): 0.079 g, Co (NO3)2·6H2O (cobalt nitrate hexahydrate): 0.0494 g).

[0047] In the following examples, the soil used is typical black soil from Gongzhuling, Jilin Province (organic matter 6.8%, pH 6.5), which is sieved through a 2 mm sieve and sterilized at 121°C for 1 h.

[0048] In the following examples, the method for culturing Scenedesmus obliquus to the logarithmic growth phase (OD680=1.2±0.1, about 7 days) is as follows: Scenedesmus obliquus is inoculated into BG-11 medium (pH 7.0) and cultured in a shaker (for about 7 days) to the logarithmic phase; the culture temperature is 25±1°C or 10±1°C; the light conditions are 12 h of light (white light LED, 3000 lux) followed by 12 h of darkness; the shaker speed is 120 rpm.

[0049] I. Contaminated soil simulation

[0050] The pesticides atrazine, thiamethoxam, cypermethrin and nicosulfuron are dissolved in solvents to prepare different pesticide solutions, and the pesticide solutions are uniformly sprayed on the soil (dry soil). After 48 h of ventilation in the dark, the solvents are evaporated to obtain different pesticide-contaminated soils; the solvents and amounts of each pesticide added are shown in Table 1.

[0051] Table 1 Solvents and amounts of each pesticide used

[0052]

[0053] Note: 10 mL of the pesticide solution (containing 10 mg of pesticide) is added to 1 kg of dry soil, and the concentration after evaporation of the solvent is 10 mg / kg.

[0054] II. Soil grouping

[0055] ​The different pesticide-contaminated soils obtained in the above "one" are divided into an experimental group and a pesticide control group, and an uncontaminated soil is used as a blank control group; the experimental group, the pesticide control group and the blank control group are treated according to the treatment modes shown in Table 2 below; wherein, the preparation of the algal liquid is: the algae mud collected by centrifugation (4000 rpm, 10 min) of the oblique grid algae grown to the logarithmic phase is resuspended with sterile water to 10 7 cells / mL to obtain the algal liquid; in addition, an equal amount of NH4Cl-K2HPO4 mixed solution (N 50 mg / kg, P 5 mg / kg) with an N / P ratio of 10:1 is also added to the soil in each treatment group (NH4Cl: N content 26.17% (N accounts for the proportion of the molecular weight of NH4Cl), required NH4Cl concentration = 5.0 mg / mL ÷ 26.17% ≈ 19.1 mg / mL (i.e. 19.1 g / L); K2HPO4: P content 17.78% (P accounts for the proportion of the molecular weight of K2HPO4), required K2HPO4 concentration = 0.5 mg / mL ÷ 17.78% ≈ 2.81 mg / mL (i.e. 2.81 g / L)), and the water content of the soil is adjusted to 60% of the maximum water holding capacity (first measure the maximum water holding capacity of the soil, then calculate the target water content, add the corresponding water amount by weighing method, and verify after balancing).

[0056] Table 2 Treatment modes of soil in each group

[0057] ,

[0058] III. Sampling and detection

[0059] In the following detection, the detection method for pesticides in the soil is: 5 g of soil sample treated in each treatment group is dissolved in 20 mL of acetonitrile, ultrasonicated for 30 min, centrifuged and filtered through a 0.22 μm filter to obtain a sample to be detected; then the sample to be detected is subjected to HPLC-MS / MS (Agilent 1290-6470) detection; wherein, the HPLC-MS / MS detection conditions of each pesticide are shown in Table 3 below (other conditions are carried out according to the instrument manual).

[0060] Table 3 HPLC-MS / MS detection conditions of each pesticide

[0061] ,

[0062] (I) Degradation rate detection

[0063] (1) Degradation rate detection of normal temperature group

[0064] The pesticide content in the soil treated in each group in the normal temperature group was detected by the above-mentioned pesticide detection method in the soil at 7th day, 14th day and 28th day of the culture, and the degradation rate was calculated, degradation rate (%) = (Ct / C0) x 100 (Ct: concentration at t time, C0: initial concentration); the calculation results are shown in Table 4.

[0065] Table 4 Degradation rate of pesticides in soil treated in each group in normal temperature group at different culture times

[0066] ,

[0067] From Table 4 above, it can be seen that when degraded for 28 days, the degradation rate of the four types of pesticides in the Scenedesmus obliquus in the application is all > 85%, meeting the requirements of the crop rotation window period (≤ 30 days); especially for the degradation of cypermethrin, it can reach more than 90% within 28 days.

[0068] (2) Degradation rate detection in low temperature group

[0069] The pesticide content in the soil treated in each group in the low temperature group was detected by the above-mentioned pesticide detection method in the soil at 28th day and 45th day of the culture, and the degradation rate was calculated, degradation rate (%) = (Ct / C0) x 100 (Ct: concentration at t time, C0: initial concentration); the calculation results are shown in Table 5.

[0070] Table 5 Degradation rate of pesticides in soil treated in each group in low temperature group at different culture times

[0071] ,

[0072] From Table 5 above, it can be seen that when degraded for 45 days at low temperature, the degradation rate of the pesticides in the Scenedesmus obliquus in the application can all reach more than 65%, especially the degradation rate of cypermethrin can reach more than 70%.

[0073] (2) Toxic intermediate detection

[0074] The screening method for the following toxic intermediate detection includes:

[0075] (1) Sample pretreatment process refinement

[0076] QuEChERS method optimization steps:

[0077] (1-1) Soil crushing: 10 g of air-dried black soil is weighed, 10 g of anhydrous sodium sulfate is added and ground into powder (passing through a 2 mm sieve) to improve extraction efficiency;

[0078] (1-2) Extraction and salting-out: 15 mL acetonitrile (containing 1% glacial acetic acid) was added and shaken vigorously for 5 min, then 4 g of anhydrous MgSO4 and 1 g of NaCl were added, shaken for 3 min, and centrifuged (4000 rpm, 10 min), and the supernatant was taken;

[0079] (1-3) Purification: 5 mL of the supernatant was taken into a centrifuge tube containing 150 mg of PSA and 900 mg of MgSO4, vortexed for 2 min, and the supernatant was taken after centrifugation and filtered through a 0.22 μm filter membrane for LC-MS / MS analysis;

[0080] (1-4) Derivatization (for polar intermediates): If aniline intermediates (such as atrazine desethyl derivatives) need to be detected, dansyl chloride (D6 labeled) can be added for derivatization to improve mass spectrometric response;

[0081] (2) Instrument parameters and scanning strategy

[0082] LC-MS / MS full scan mode:

[0083] Chromatographic column: ACQUITY UPLC BEH C18 (1.7 μm, 2.1 x 100 mm), column temperature 40℃; mobile phase: A phase 0.1% formic acid water, B phase 0.1% formic acid acetonitrile, gradient elution (0-5 min, 5%-30% B; 5-10 min, 30%-80% B; 10-12 min, 80% B);

[0084] Mass spectrometry parameters: ion source: electrospray ionization (ESI+), capillary voltage 3.5 kV, ion source temperature 150℃, desolvation gas temperature 500℃, flow rate 1000 L / h; scan range: m / z 50-600, scan rate 2000 Da / s, resolution 70,000 (FWHM), mass deviation ≤5 ppm; data-dependent secondary scan (ddMS 2 ): automatic triggering of secondary fragmentation, collision energy 15-40 eV, for structure analysis of unknown intermediates.

[0085] (3) APGC-HRMS supplementary detection

[0086] Chromatographic column: TG-5MS capillary column (30 m x 0.25 mm x 0.25 μm), programmed temperature (40℃ for 2 min, increased to 320℃ at 15℃ / min, held for 5 min);

[0087] Ion source: atmospheric pressure gas phase ionization (APGC), transfer line temperature 280℃, ion source temperature 150℃, using soft ionization mode (proton transfer), generating molecular ion peaks (such as thiamethoxam [M+H]+m / z 292.098);

[0088] Mass spectrometry parameters: scan range m / z 50-600, resolution 20,000 (FWHM), mass deviation ≤5 ppm, used to verify LC-MS / MS results and reduce matrix interference.

[0089] (4) Data processing and standard comparison

[0090] (4-1) Database construction: Establish a standard database containing target pesticides (atrazine, thiamethoxam, etc.) and their known metabolites (such as thiazolamine, de-ethyl atrazine), store retention time, accurate mass-to-charge ratio, characteristic fragment ions and abundance ratio.

[0091] Example: [M+H]+ m / z 169.043 of thiazolamine, characteristic fragment ion m / z 125.022 (loss of SO2); [M+H]+ m / z 186.087 of de-ethyl atrazine, fragment ion m / z 158.061 (loss of CO2).

[0092] (4-2) Screening process: Target screening: Use MassLynx software to extract ion chromatogram (EIC), set mass extraction window ±5 ppm, compare standard retention time and fragment ions, if matching degree ≥85% and signal-to-noise ratio (S / N) ≥3, then confirm as positive; Non-target screening: Peak extraction is performed on full scan data, compared with MassBank and HMDB databases through Compound Discoverer software, combined with element composition prediction (such as C 10 H 14 ClN5O2S accurate mass m / z 305.068), infer potential intermediate structure; False positive exclusion: standard addition verification is performed on suspected peaks, if retention time and fragment ions are consistent, then confirm as target intermediate; If not consistent, then exclude matrix interference through high resolution mass spectrometry isotope distribution pattern (such as Cl element M+2 peak).

[0093] The detection method for pesticide detection in soil described above (full scan mode (m / z 50-600) + standard comparison) was used to screen the toxic intermediates (atrazine toxic intermediates: desisopropyl atrazine, hydroxyatrazine, cyanuric acid; thiamethoxam toxic intermediates: N-nitroguanidine, thiamethoxam; cypermethrin toxic intermediates: 3-phenoxybenzoic acid, dichlorophenyl ether chrysanthemum acid; nicosulfuron toxic intermediates: 2-chloro-N,N-dimethyl nicotinamide, sulfonamide formate) in the soil of each experimental group of the normal temperature group at 28 days of culture and the toxic intermediates (thiamethoxam-thiazolamine and atrazine-deethyl derivative) in the soil of each experimental group of the low temperature group at 45 days of culture, and the results showed that no known toxic intermediates (thiazolamine and atrazine-deethyl derivative) were detected in each experimental group of the normal temperature group and the low temperature group; the final products were all mineralized into CO2, H2O, Cl - , SO2 2- and NO3 - and other inorganic substances.

[0094] (Three) Bioavailability test

[0095] The Tenax-TA continuous extraction method was used to test the proportion of bioavailable pesticides and the amount of humic acid-pesticide combination in the soil of the experimental group (test pesticide: atrazine) and the pesticide control group of the normal temperature group at 28 days of culture, and the specific method is as follows:

[0096] I) Detection of bioavailable pesticide proportion

[0097] (1) Tenax-TA resin (particle size 60-80 mesh, purchased from Sigma-Aldrich Company, USA), soaked in methanol for 24h before use, washed with distilled water to neutral, dried at 60°C for standby; the extraction container is a 50mL centrifuge tube (with a polytetrafluoroethylene inner cover);

[0098] (2) Take 10.0g of soil sample passing through 2mm sieve in the centrifuge tube, add 0.5g of Tenax-TA resin and 20mL of 0.01mol / L CaCl2 solution (as extraction medium), place in a constant temperature oscillator, oscillate at 25°C and 200r / min. Take out the centrifuge tube at 1h, 2h, 4h, 8h, 12h, 24h, 48h and 72h, centrifuge at 3000r / min for 10min, collect the supernatant; at the same time, supplement with equal amount of fresh CaCl2 solution and continue to oscillate extraction;

[0099] (3) The pesticide concentration in the supernatant at each time point was determined by gas chromatography-mass spectrometry (GC-MS, model 7890A-5975C, Agilent, USA), and the total amount of extracted bioavailable pesticides was calculated. The bioavailable pesticide proportion calculation formula was: (bioavailable pesticide amount / total pesticide residue amount in soil) x 100%. The determination of total pesticide residue in soil was performed in accordance with "Gas Chromatographic Method for Determination of Hexachlorocyclohexane and DDT in Soil" (GB / T 14550-2003).

[0100] II) Humic acid-pesticide binding amount test method

[0101] The determination of humic acid-pesticide binding amount was performed by alkali dissolution-centrifugal separation method combined with high performance liquid chromatography (HPLC) detection, and the specific steps were as follows:

[0102] (1) 5.0 g of soil sample was weighed, 50 mL of 0.1 mol / L NaOH solution was added, and extraction was performed at 25°C for 1 h, followed by centrifugation at 4000 r / min for 15 min to collect the supernatant (containing humic acid), and the extraction was repeated twice, and the supernatants were combined;

[0103] (2) 1 mol / L HCl was added to the supernatant to adjust the pH to 1.0, and the humic acid was allowed to precipitate for 24 h, followed by centrifugation at 3000 r / min for 10 min, and the supernatant was discarded; the precipitate was washed with 0.01 mol / L HCl for 3 times, 20 mL of methanol was added for ultrasonic extraction for 30 min (power 300 W), followed by centrifugation at 4000 r / min for 10 min, and the extract was collected, and the extraction was repeated twice, and the extract was combined and diluted to 50 mL;

[0104] (3) HPLC (model 1260 Infinity, Agilent, USA) was used to determine the pesticide concentration in the extract, the chromatographic column was C18 column (250 mm x 4.6 mm, 5 μm), the mobile phase was methanol-water (volume ratio 70:30), the flow rate was 1.0 mL / min, and the detection wavelength was set according to the characteristics of the target pesticide (such as 210 nm for organophosphorus pesticides). The humic acid-pesticide binding amount calculation formula was: (total mass of pesticides in the extract / dry weight of soil sample), and the result was expressed in "mg / g".

[0105] The above test results are shown in Table 6.

[0106] Table 6 Bioavailability and humic acid-pesticide binding amount of soil in each treatment group

[0107] ,

[0108] From the above table 6, it can be seen that the proportion of bioavailable pesticides in the treated soil in the experimental group is significantly higher than that in the pesticide control group without using Chlorella sloanei, which is increased by about 108%, and the humic acid-pesticide combination amount in the treated soil in the experimental group is significantly lower than that in the pesticide control group without using Chlorella sloanei, which is reduced by about 63%.

[0109] (Four) Low-temperature fluidity test

[0110] The DPH fluorescence polarization test was used to test the expression amount of antifreeze proteins (AFPs) of Chlorella sloanei at normal temperature and low temperature (using

[0111] The antifreeze protein ELISA detection kit (model: AFP-ELISA-Kit, purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.) was used for detection) and cell membrane fluidity (Chlorella sloanei cells in the logarithmic growth phase were washed 3 times with phosphate buffered saline (PBS, pH 7.4), and the cell concentration was adjusted to 1×10 6 / mL; 10 μmol / L of DPH fluorescent probe was added to the cell suspension, and the cell suspension was incubated at 37°C in the dark for 30 min; a fluorescence spectrophotometer (model: F-7000, purchased from Hitachi, Japan) was used to measure the fluorescence polarization degree (P value), the excitation wavelength was 360 nm, the emission wavelength was 430 nm, the fluorescence intensity in the parallel direction and the perpendicular direction was measured respectively, and the P value was calculated according to the formula (P= (I∥-GI⊥) / (I∥+GI⊥), wherein G is the correction factor).

[0112] The detection results are shown in the following table 7.

[0113] Table 7 Antifreeze protein (AFPs) expression amount and cell membrane fluidity of Chlorella sloanei at normal temperature and low temperature

[0114] ,

[0115] Note: The lower the P value, the stronger the membrane fluidity (0.15-0.25 is the ideal metabolic activity interval); in addition, the cell membrane is a single-layer membrane structure wrapped outside the algal cell protoplast, mainly composed of lipids and proteins, which is not only a barrier for material exchange and signal transmission between cells and the external environment, but also directly affects the material transport, energy conversion and metabolic reaction efficiency of the cells, especially in a low-temperature environment, the stability of the protoplast membrane fluidity is the key to maintaining the normal physiological function of the algal cells.

[0116] From the above table 7, it can be seen that the Chlorella sloanei in the application can express antifreeze proteins at 10°C, and the cell membrane fluidity is in the ideal metabolic activity range.

[0117] (Five) Soil micro-ecological balance test

[0118] In the following tests, the microbial Shannon diversity test method is:

[0119] (1) Soil microbial total DNA extraction: The soil genomic DNA extraction kit, model: FastDNA®Spin Kit for Soil, is purchased from the United States MP Biomedicals Company, and the operation is strictly in accordance with the kit instructions;

[0120] (2) PCR amplification: In addition to the above primers and amplification system, the PCR reaction conditions refer to the parameters specified in "Soil Microbial Diversity Determination High-throughput Sequencing Method" (NY / T3425-2019): 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 45 s, a total of 35 cycles; 72°C final extension for 10 min.

[0121] (3) Sequencing and data analysis: The high-throughput sequencing platform operation process refers to the Illumina MiSeq System Standard Operating Procedures (Illumina official document, Document#15039740v01); the specific parameter settings of QIIME software (version 1.9.1) refer to its official user manual (http: / / qiime.org / install / index.html).

[0122] In the following tests, the relative abundance of Actinobacteria is tested by: OTU taxonomic annotation based on high-throughput sequencing data uses RDPClassifier (version 2.11), and the Silva 132 rRNA gene database (https: / / www.arb-silva.de / ) is selected as the database. The specific annotation method refers to the "Microbial Taxonomic Annotation Process" chapter in "High-throughput Sequencing Data Analysis Guide" (China Environmental Science Press, 2018, authors: Li Juan, etc.).

[0123] In the following examples, the earthworm survival rate test method is:

[0124] (1) Test substrate pretreatment: After the soil sample is collected, impurities are removed through a 2mm sieve, and the pH value (requirements 6.0-7.5) and organic matter content (requirements 2-10%) are adjusted according to Appendix A of "Chemical Pesticide Environmental Safety Evaluation Test Guidelines Part 11: Acute Toxicity Test of Bombyx mori" (GB / T31270.11-2014);

[0125] (2) Standard soil configuration: According to the provisions of GB / T31270.11-2014, it is mixed by quartz sand (20-50 mesh), kaolin and peat (volume ratio 7:2:1), and the organic matter content of peat is ≥80%, purchased from KSB AG Company in Germany.

[0126] The microbial Shannon diversity and relative abundance of Actinobacteria in the soil of each experimental group in the normal temperature group after 28 days of culture were tested according to the above method, and the fluctuation rate was calculated, and the survival rate of earthworms was observed, and the results are shown in Table 8 as follows.

[0127] Table 8 Microbial changes in the soil and earthworm survival of each experimental group after 28 days of culture

[0128] ,

[0129] From the above Table 8, it can be seen that the Scenedesmus obliquus in the application does not destroy the soil micro-ecological balance, which meets the standard of GB / T31270-2014.

[0130] Four, pesticide degradation flux testing per unit area of black soil

[0131] The pesticide degradation flux testing of black soil in the embodiments of the application is carried out according to the following method:

[0132] Experimental group: Scenedesmus obliquus (CGMCC No. 6567) is inoculated (inoculation amount is 8×10 6 cells / g of black soil) into black soil containing atrazine to simulate the actual farmland environment.

[0133] Control group: The black soil containing atrazine is treated by a traditional method (natural degradation treatment).

[0134] Sterile condition control

[0135] In order to exclude the interference of other microorganisms, the experiment is carried out in a sterile environment:

[0136] (1) The black soil sample is treated by high temperature sterilization (121℃, 30 minutes).

[0137] (2) Scenedesmus obliquus is pre-cultured in a sterile culture medium to the logarithmic growth phase to ensure pure culture.

[0138] Environmental parameter simulation, the experimental conditions strictly simulate the ecological environment of black soil:

[0139] (1) The temperature is controlled at 25±1℃, the humidity is 75%-85%, and the light cycle is 12 hours of light / 12 hours of darkness (simulating natural day-night rhythm).

[0140] (2) The soil moisture is adjusted to 60% of the saturated water holding capacity to maintain microbial activity.

[0141] High-efficiency detection technology

[0142] The following method is used to quantitatively analyze the pesticide residues in the soil:

[0143] (1) HPLC-MS / MS: detect polar pesticides such as atrazine, thiamethoxam, with a sensitivity of 0.01-0.05 mg / kg;

[0144] (2) GC-ECD: analyze pyrethroid pesticides such as cypermethrin, with a detection limit as low as 0.001 mg / kg;

[0145] (3) SPE: purify soil extracts through HLB columns or QuEChERS kits to reduce matrix interference.

[0146] Time gradient sampling

[0147] Periodic sampling during the experimental period (0, 3, 7, 14, 21 days), 3 parallel samples were collected each time, and the pesticide residue was determined. The concentration of atrazine in black soil was detected by HPLC-MS / MS.

[0148] Degradation rate formula

[0149] Degradation flux (mg / (kg·d)) is calculated by the following formula: ,

[0150] Where C0 is the initial pesticide concentration (mg / kg), C_t is the residual concentration after t days, and t is the time (days).

[0151] The improvement over the traditional method is calculated by the following formula:

[0152] ,

[0153] ,

[0154] The test results show that the degradation flux of black soil pesticide (atrazine) per unit area reaches 35.2 mg / (kg·d), which is 305% higher than the traditional method (8.7 mg / (kg·d)).

[0155] The performance indicators of the present application for degrading pesticides in soil by Chlorella oblonga are compared with those of the prior art, as shown in Table 9.

[0156] Table 9 Comparison of the present application with the prior art

[0157] ,

[0158] From Table 9 above, it can be seen that the performance indicators of the present application for degrading pesticides in soil using Chlorella oblonga are superior to those of the traditional microbial remediation and chemical oxidation method in the prior art.

[0159] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. Application of Scenedesmus obliquus in reducing pesticide residue in soil, the pesticide is A, or a combination of cypermethrin and A; A is one or more combination of diafenthiuron, thiamethoxam or nicosulfuron; the accession number of Scenedesmus obliquus is CGMCC No. 6567.

2. Use according to claim 1, characterized in that, The application temperature is 5-30℃.

3. A method of reducing pesticide residues in soil, characterized by, The method comprises: inoculating Scenedesmus obliquus into soil, the pesticide is A, or a combination of cypermethrin and A; A is one or more combination of diafenthiuron, thiamethoxam or nicosulfuron; the accession number of Scenedesmus obliquus is CGMCC No. 6567.

4. The method of claim 3, wherein, The inoculation amount of Scenedesmus obliquus was (0.8-1.2) x 10 8 cells / g soil.

5. The method according to claim 3 or 4, characterized in that, Nitrogen and phosphorus elements are also added to the soil.

6. The method of claim 5, wherein, The ratio of nitrogen and phosphorus elements is (9-11):1, and the addition amount of phosphorus element is (4-6) mg / kg soil.