Enrichment and culture method of acetochlor-degrading bacteria and its application

Abstract

The present invention discloses an enrichment culture method and application of an acetochlor-degrading bacterial system. The enrichment culture method comprises enriching and culturing a substrate-containing material in an enrichment medium; the enrichment medium comprises sodium salts, trace elements, potassium salts, ammonium salts, calcium and magnesium solutions, and vitamins; and the material is soil or activated sludge. Through the use of enrichment culture, the acetochlor-degrading bacterial system can significantly reduce acetochlor. Within this bacterial system, Sphingomonas accounts for a high proportion, and compared to existing enrichment culture methods, this bacterial system significantly improves the degradation rate and stability of acetochlor.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to an enrichment and cultivation method for an acetochlor-degrading bacterial system and an application thereof. Background Art

[0002] Corn is an important food crop in my country. Continuous corn cropping is common in Northeast China, and the application of sethoxydim may cause phytotoxicity, such as curling of the heart leaves and slow growth of the plants. In severe cases, it may even lead to the death of seedlings.

[0003] Soybean is an important food and oil crop and one of the most widely cultivated crops in the world. Its ability to biologically fix nitrogen through symbiosis with rhizobia plays a vital role in the sustainable development of modern agriculture.

[0004] Acetochlor is widely used in soybean fields. Acetochlor's main effects on soybeans include reduced nodule count, slower growth, lower chlorophyll content, and decreased root activity. Severe damage can also cause shoot dieback, terminal bud atrophy, abnormal plant growth, premature bud ripening, and premature bud ripening. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to quickly and stably obtain a bacterial system that can efficiently degrade acetochlor in soil and water. A method for enriching and culturing an acetochlor-degrading bacterial system and its application are provided. Through a specific enrichment and culturing method, a highly efficient acetochlor-degrading bacterial system stably containing Sphingomonas can be quickly obtained. The enrichment and culturing rate is fast and the stability is significantly improved compared with existing enrichment and culturing methods. In addition, this bacterial system has a fast acetochlor degradation rate and good passage stability. In the future, it can be used to reduce acetochlor in soil and water, thereby achieving the purpose of improving soil health and soil quality.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first aspect of the present invention provides a method for enriching and culturing an acetochlor-degrading bacterial strain, wherein the substrate used in the enrichment culture is acetochlor; preferably 92% (920 g / L) acetochlor technical;

[0008] The enrichment culture method comprises enriching and culturing the material containing the substrate in an enrichment medium;

[0009] The enrichment medium includes 0.008-0.08 mol / L sodium salt, 4-5 mg / L trace elements, 0.006-0.008 mol / L potassium salt, 0.006-0.008 mol / L ammonium salt, 40-60 mg / L calcium magnesium solution and 1.0-1.25 mg / L vitamins;

[0010] The material is soil or activated sludge.

[0011] In the present invention, the activated sludge refers to flocs that have the ability to absorb and biologically oxidize organic matter in water, and the flocs are composed of particulate matter, dissolved matter, microorganisms, and microbial secretions in the water.

[0012] In some embodiments, the enrichment culture method includes using acetochlor to contaminate soil and performing enrichment culture by gradiently increasing the acetochlor stress concentration in the enrichment culture medium, and the substrate used in the enrichment culture is 92% acetochlor technical.

[0013] In some embodiments, the enrichment culture is performed in a light-proof environment, and the temperature of the enrichment culture is 25-30°C or 30-35°C.

[0014] In some specific embodiments, the enrichment culture temperature is 30° C. This temperature is suitable for the growth of acetochlor-degrading bacteria in the bacterial strain.

[0015] In some embodiments, the enriched culture medium may be one of inorganic salt culture media.

[0016] In some embodiments, the sodium salt comprises NaNO3 and Na2HPO4.

[0017] In some embodiments, the concentration of the sodium salt is 0.008-0.04 mol / L.

[0018] In some specific embodiments, the sodium salt concentration is 0.04 mol / L; for example, each liter contains 0.5 g NaNO3 and 2.5 g Na2HPO4.

[0019] In some embodiments, the potassium salt comprises KH2PO4.

[0020] In some embodiments, the concentration of the potassium salt is 0.006-0.0073 mol / L.

[0021] In some specific embodiments, the concentration of the potassium salt is 0.0073 mol / L, for example, 1.0 g KH2PO4 per liter.

[0022] In some embodiments, the ammonium salt comprises (NH4)2SO4.

[0023] In some embodiments, the concentration of the ammonium salt is 0.006-0.0076 mol / L.

[0024] In some specific embodiments, the concentration of the ammonium salt is 0.0076 mol / L, for example, 1.0 g (NH 4 ) 2 SO 4 per liter.

[0025] In some embodiments, the calcium-magnesium solution comprises calcium chloride and magnesium chloride.

[0026] In some embodiments, the concentration of the calcium and magnesium solution is 40-50 mg / L.

[0027] In some specific embodiments, the concentration of the calcium-magnesium solution is 50 mg / L, for example, containing 30 mg CaCl2 and 20 mg MgCl2 per liter.

[0028] In some embodiments, the trace element is selected from the group consisting of aluminum, cobalt, copper, iron, bromine, boron, iodine, lithium, manganese, molybdenum, tungsten, nickel, tin, and zinc.

[0029] In some embodiments, the trace elements contain 25-100 μg / L of Na 2 WO 4 ·2H 2 O; for example, preferably 25-50 μg / L.

[0030] In some embodiments, the trace elements contain 10-25 μg / L or 25-50 μg / L SnCl2·2H2O.

[0031] In some specific embodiments, the trace elements contain 25 μg / L Na2WO4·2H2O and 25 μg / L SnCl2·2H2O.

[0032] In some embodiments, the vitamin is selected from para-aminobenzoic acid (VB10), biotin (VB7), folic acid (VB9), niacin (VB3), calcium pantothenate (VB5), pyridoxine hydrochloride (VB6), riboflavin (VB2), thiamine (VB1) and cobalamin (VB12).

[0033] In some embodiments, the folic acid (VB9) content is 100-200 μg / L or 200-400 μg / L.

[0034] In some embodiments, the biotin (VB7) content is 100-200 μg / L or 200-400 μg / L.

[0035] In some embodiments, the folic acid (VB9) content is 200-400 μg / L.

[0036] In some embodiments, the biotin (VB7) content is 100-200 μg / L.

[0037] In some specific embodiments, the folic acid (VB9) content is 200 μg / L.

[0038] In some specific embodiments, the biotin (VB7) content is 200 μg / L.

[0039] In some embodiments, the enrichment medium comprises: 0.008-0.04 mol / L sodium salt, wherein the sodium salt comprises NaNO3 and Na2HPO4; 0.006-0.008 mol / L potassium salt, wherein the potassium salt comprises KH2PO4; 0.006-0.008 mol / L ammonium salt, wherein the ammonium salt comprises (NH4)2SO4; and trace elements, wherein the trace elements preferably comprise 108-110 μg / L Al2(SO4)3·18H2O, 56-58 μg / L CoSO4·7H2O, 56-60 μg / L CuSO4·5H2O, 2.5-3.0 mg / L FeSO4·7H2O, 600-611 μg / L H3BO3, 25-28 μg / L KBr, 56-60 μg / L KI, 25-28 μg / L LiCl, 360-389 μg / L MnCl2·4H2O, 25-27 μg / L Na2MoO4·2H2O, 25-27 μg / L Na2WO4·2H2O, 58-64 μg / L NiCl2·6H2O, 25-28 μg / L SnCl2·2H2O, 34-36 μg / L ZnSO4·H2O; 50 mg / L calcium magnesium solution, the calcium magnesium solution containing 30 mg / LCaCl2 and 20 mg / L MgCl2; vitamins, preferably comprising 200-250 μg / L p-aminobenzoic acid (VB10), 200-250 μg / L biotin (VB7), 200-250 μg / L folic acid (VB9), 200-250 μg / L niacin (VB3), 100-120 μg / L calcium pantothenate (VB5), 100-120 μg / L pyridoxine hydrochloride (VB6), 100-120 μg / L riboflavin (VB2), 100-120 μg / L thiamine (VB1) and 1-1.2 μg / L cobalamin (VB12).

[0040] In some specific embodiments, the enrichment medium comprises: 0.04 mol / L sodium salt, which comprises 0.5 g / L NaNO3 and 2.5 g / L Na2HPO4; 0.0073 mol / L potassium salt, which comprises 1.0 g / L KH2PO4; 0.0076 mol / L ammonium salt, which comprises 1.0 g / L (NH4)2SO4; trace elements, which comprise 108 μg / LAl2(SO4)3·18H2O, 56 μg / L CoSO4·7H2O, 56 μg / L CuSO4·5H2O, 3.0 mg / L FeSO4·7H2O, 611 μg / L H3BO3, 28 μg / L KBr, 56 μg / L KI, 28 μg / L LiCl, 389 μg / L MnCl2·4H2O, 25 μg / L Na2MoO4·2H2O, 25μg / L Na2WO4·2H2O, 58μg / L NiCl2·6H2O, 25μg / L SnCl2·2H2O, 34μg / L ZnSO4·H2O; 50mg / L calcium magnesium solution, the calcium magnesium solution containing 30mg / L CaCl2 and 20mg / L MgCl2; vitamins, the vitamins containing 200μg / L p-aminobenzoic acid (VB10), 200μg / L biotin (VB7), 200μg / L folic acid (VB9), 200μg / L niacin (VB3), 100μg / L calcium pantothenate (VB5), 100μg / L pyridoxine hydrochloride (VB6), 100μg / L riboflavin (VB2), 100μg / L thiamine (VB1) and 1μg / L cobalamin (VB12).

[0041] In some embodiments, the enrichment culture further comprises: enriching and culturing the substrate for 6-7 days, and culturing the solution OD 600 When the value is 0.1-0.15, the concentration of acetochlor technical detected by high performance liquid chromatography is lower than 50%, 45% or 40% of the initial concentration.

[0042] In some embodiments, the enrichment culture further comprises: the abundance of Sphingomonas detected by 16S high-throughput sequencing is not less than 60%, preferably not less than 70%.

[0043] The second aspect of the present invention provides an acetochlor-degrading bacterial strain obtained according to the enrichment and cultivation method as described in the first aspect.

[0044] A third aspect of the present invention provides an acetochlor-degrading bacterial system, comprising Sphingomonas, Diaphorobacter, Shinella, Sphingobium, Brucella, Pseudoxanthomonas, Hyphomicrobium, Achromobacter, Bordetella, Dokdonella, Rhodopseudomonas, Bosea, Ramlibacter, Luteimonas, Pseudomonas, and Rhodanobacter.

[0045] The abundance of Sphingomonas in the acetochlor-degrading bacterial system is 40%-70%.

[0046] In some embodiments, the abundance of the genus Diaphorobacter is 3%-12%.

[0047] In some embodiments, the abundance of Shinella is 9%-26%.

[0048] In some embodiments, the abundance of Sphingobium is 0.01%-0.6%.

[0049] In some embodiments, the abundance of Brucella is 0.8%-4.5%.

[0050] In some embodiments, the abundance of Pseudoxanthomonas is 0.5%-0.7%.

[0051] In some embodiments, the abundance of Hyphomicrobium is 1%-2.2%.

[0052] In some embodiments, the abundance of Achromobacter is 4.5%-7.5%.

[0053] In some embodiments, the abundance of Bordetella is 0.1%-0.35%.

[0054] In some embodiments, the abundance of Dokdonella is 0.8%-4.5%.

[0055] In some embodiments, the abundance of Rhodopseudomonas is 0.1%-1.8%.

[0056] In some embodiments, the abundance of the genus Bosea is 0.2%-2.5%.

[0057] In some embodiments, the abundance of Ramlibacter is 0.1%-0.4%.

[0058] In some embodiments, the abundance of Luteimonas is 0.01%-0.1%.

[0059] In some embodiments, the abundance of Pseudomonas is 0.01%-0.7%.

[0060] In some embodiments, the abundance of the genus Rhodanobacter is 0.05%-1.6%.

[0061] In some specific embodiments, the abundance of each bacterial genus in the acetochlor-degrading bacterial system is shown in Table 12.

[0062] In the present invention, the "abundance" refers to the ratio of the number of a certain bacteria to the number of all bacteria in the acetochlor-degrading bacterial system. The bacteria are at the genus level.

[0063] The fourth aspect of the present invention provides a composition comprising the acetochlor-degrading bacterial system as described in the second aspect or the third aspect.

[0064] The fifth aspect of the present invention provides an acetochlor-degrading bacterial system according to the second aspect or the third aspect, or the use of the composition according to the fourth aspect for reducing acetochlor in soil and water.

[0065] In the present invention, the application can be used, for example, to degrade acetochlor in different concentrations and forms (acetochlor technical, emulsifiable concentrate, microemulsion, emulsion in water and wettable powder, etc.), promote the promotion of the pollutant-remediation bacterial agent (acetochlor-acetochlor-degrading bacterial system) model in farmland, and provide a basis for eliminating non-point source pollution of herbicides in farmland, improving soil health, and increasing crop yield and quality.

[0066] In some embodiments, the degradation of acetochlor is degradation of acetochlor at a concentration of 50-1000 mg / L.

[0067] In some embodiments, the degradation of acetochlor is degradation of acetochlor at a concentration of 50-800 mg / L.

[0068] In some embodiments, the degradation of acetochlor is the degradation of acetochlor at a concentration of 50-200 mg / L.

[0069] In some embodiments, the acetochlor is the technical, emulsifiable concentrate, microemulsion, aqueous emulsion or wettable powder of acetochlor.

[0070] The sixth aspect of the present invention provides a culture medium for enrichment culture of acetochlor-degrading bacteria, the culture medium comprising 0.008-0.08 mol / L sodium salt, 4-5 mg / L trace elements, 0.006-0.008 mol / L potassium salt, 0.006-0.008 mol / L ammonium salt, 40-60 mg / L calcium magnesium solution and 1.0-1.25 mg / L vitamins.

[0071] In some embodiments, the culture medium includes 0.008-0.04 mol / L sodium salt, which includes NaNO3 and Na2HPO4; 0.006-0.008 mol / L potassium salt, which is KH2PO4; 0.006-0.008 mol / L ammonium salt, which is (NH4)2SO4; and trace elements, which preferably include 108-110 μg / L Al2(SO4)3·18H2O, 56-58 μg / L CoSO4·7H2O, 56-60 μg / L CuSO4·5H2O, 2.5-3.0 mg / L FeSO4·7H2O, 600-611 μg / L H3BO3, 25-28 μg / L KBr, 56-60 μg / L KI, 25-28 μg / L LiCl, 360-389 μg / L MnCl2·4H2O, 25-27μg / LNa2MoO4·2H2O, 25-27μg / L Na2WO4·2H2O, 58-64μg / L NiCl2·6H2O, 25-28μg / L SnCl2·2H2O, 34-36μg / L ZnSO4·H2O; 50mg / L calcium magnesium solution, the calcium magnesium solution contains 30mg / L CaCl2 and 20mg / LMgCl2; vitamins, preferably comprising 200-250μg / L p-aminobenzoic acid (VB10), 200-250μg / L biotin (VB7), 200-250μg / L folic acid (VB9), 200-250μg / L niacin (VB3), 100-120μg / L calcium pantothenate (VB5), 100-120μg / L pyridoxine hydrochloride (VB6), 100-120μg / L riboflavin (VB2), 100-120μg / L thiamine (VB1) and 1-1.2μg / L cobalamin (VB12).

[0072] In some specific embodiments, the culture medium includes 0.04 mol / L sodium salt, which includes 0.5 g / L NaNO3 and 2.5 g / L Na2HPO4; 0.0073 mol / L potassium salt, which is 1.0 g / L KH2PO4; 0.0076 mol / L ammonium salt, which is 1.0 g / L (NH4)2SO4; and trace elements, which include 108 μg / L Al2(SO4)3·18H2O, 56 μg / L CoSO4·7H2O, 56 μg / L CuSO4·5H2O, 3.0 mg / L FeSO4·7H2O, 611 μg / L H3BO3, 28 μg / L KBr, 56 μg / L KI, 28 μg / L LiCl, 389 μg / L MnCl2·4H2O, 25 μg / L Na2MoO4·2H2O, 25μg / L Na2WO4·2H2O, 58μg / L NiCl2·6H2O, 25μg / L SnCl2·2H2O, 34μg / L ZnSO4·H2O; 50mg / L calcium magnesium solution, the calcium magnesium solution containing 30mg / L CaCl2 and 20mg / L MgCl2; vitamins, the vitamins containing 200μg / L p-aminobenzoic acid (VB10), 200μg / L biotin (VB7), 200μg / L folic acid (VB9), 200μg / L niacin (VB3), 100μg / L calcium pantothenate (VB5), 100μg / L pyridoxine hydrochloride (VB6), 100μg / L riboflavin (VB2), 100μg / L thiamine (VB1) and 1μg / L cobalamin (VB12).

[0073] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0074] The reagents and raw materials used in the present invention are commercially available.

[0075] The positive progress effect of the present invention is:

[0076] The present invention has discovered and confirmed that an acetochlor-degrading bacterial strain is suitable for cultivation in an enrichment medium with acetochlor as the sole carbon source. The present invention provides an enrichment cultivation method for an acetochlor-degrading bacterial strain, specifically using 92% acetochlor technical as a substrate and cultivating it in an enrichment medium containing sodium salt, potassium salt, ammonium salt, vitamins, trace elements, and calcium and magnesium solution. In one example, the bacterial strain can degrade 65% of acetochlor six days after inoculation, with Sphingomonas accounting for 60%; and degrade 85% of acetochlor nine days after inoculation, with Sphingomonas accounting for 65%. The enrichment cultivation method of the present invention significantly improves the degradation rate and stability of acetochlor by the bacterial strain compared to existing enrichment cultivation methods; it has efficient and stable acetochlor degradation capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The figure shows the effect of different temperatures on the enrichment of acetochlor-degrading bacteria.

[0078] Figure 2 This is the standard curve of low concentration acetochlor.

[0079] Figure 3 This is the standard curve of high concentration acetochlor.

[0080] Figure 4 The effect of different concentrations of sodium salt on the enrichment of acetochlor-degrading bacteria.

[0081] Figure 5 This is the effect of trace element content on the enrichment of acetochlor-degrading bacteria.

[0082] Figure 6 Effects of different concentrations of W and Sn on the enrichment of acetochlor-degrading bacteria.

[0083] Figure 7 The effect of vitamin content on the enrichment of acetochlor-degrading bacteria.

[0084] Figure 8 The effect of different concentrations of folic acid and biotin on the enrichment of acetylcholine-degrading bacteria.

[0085] Figure 9 It is the stability of the acetochlor-degrading bacterial line.

[0086] Figure 10 The residual amount of acetochlor at different concentrations degraded by the bacterial system.

[0087] Figure 11 is the degradation rate of acetochlor-degrading bacteria on different concentrations of acetochlor.

[0088] Figure 12 The degradation ability of acetochlor-degrading bacteria on different acetochlor preparations.

[0089] Figure 13The composition of different bacterial communities is at the genus level.

[0090] Figure 14 The phylum level represents the composition of different bacterial communities.

[0091] Figure 15 This is the residue of acetochlor in the soil of the pot test.

[0092] Figure 16 This is the residue of acetochlor in the soil of the field plot test. DETAILED DESCRIPTION

[0093] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0094] The reagents and raw materials used in the examples are commercially available.

[0095] Example 1: Effect of different culture temperatures on the enrichment of acetochlor-degrading bacteria

[0096] In order to find a test sample that can tolerate acetochlor and contains a large number of bacteria with potential degradation ability, thereby obtaining a better screening effect; in this embodiment, soil from the workshop of the acetochlor production plant was selected as the sampling sample. Since the concentration of acetochlor at this location is relatively high, the microorganisms surviving in this environment may tolerate or degrade and utilize acetochlor. Therefore, taking the soil from the acetochlor workshop as a sample for screening and enriching acetochlor-degrading bacteria is more purposeful and more operational, which is conducive to further screening and enrichment.

[0097] 5 g of acetochlor-contaminated soil was added to a solution containing 0.04 mol / L sodium salt (0.5 g NaNO3 and 2.5 g Na2HPO4 per liter), 0.0073 mol / L potassium salt (1.0 g KH2PO4 per liter), 0.0076 mol / L ammonium salt (1.0 g (NH4)2SO4 per liter), trace elements (108 μg Al2(SO4)3·18H2O, 56 μg CoSO4·7H2O, 56 μg CuSO4·5H2O, 3.0 mg FeSO4·7H2O, 611 μg H3BO3, 28 μg KBr, 56 μg KI, 28 μg LiCl, 389 μg MnCl2·4H2O, 25 μg Na2MoO4·2H2O, 25 μg Na2WO4·2H2O, 58 μg NiCl2·6H2O, 25μg SnCl2·2H2O, 34μg ZnSO4·H2O), calcium magnesium solution (30mg CaCl2, 20mg In a 100 ml inorganic salt culture medium containing 200 μg p-aminobenzoic acid (VB10), 200 μg biotin (VB7), 200 μg folic acid (VB9), 200 μg nicotinic acid (VB3), 100 μg calcium pantothenate (VB5), 100 μg pyridoxine hydrochloride (VB6), 100 μg riboflavin (VB2), 100 μg thiamine (VB1), and 1 μg cobalamin (VB12) per liter, 92% acetochlor technical was added as the sole carbon source, and the acetochlor stress concentration was increased sequentially to an acetochlor effective concentration of 50, 200, 400, and 600 mg / L. The culture was transferred every 15 days at an inoculum size of 1 / 20. 600 mg / L acetochlor was added to the inorganic salt medium, and the enriched bacterial strain was inoculated at an inoculum size of 1 / 20, and cultured on a shaker at 160 rpm at different temperatures of 10, 15, 20, 25, 30, 35, and 40°C, with three replicates per group. The acetochlor residues in each group were detected by high performance liquid chromatography, and the optimal culture temperature for enrichment of the acetochlor-degrading bacterial strain was determined to be 30°C (Table 1, Figure 1 ).

[0098] Table 1 Effects of different temperatures on the enrichment of acetochlor-degrading bacteria

[0099] Temperature (℃) 10 15 20 25 30 35 40 Degradation rate 1 1.56％ 10.43％ 31.65％ 61.55％ 98.56％ 62.76％ 13.56％ Degradation rate 2 3.43％ 11.56％ 24.94％ 63.76％ 100.00％ 85.52％ 21.45％ Degradation rate 3 5.28％ 15.58％ 31.85％ 76.91％ 96.43％ 68.04％ 16.42％

[0100] Among them, the standard curve of low concentration acetochlor is:

[0101] y=6.3846x-15.729R 2 =0.9992 (Table 2, Figure 2 );

[0102] Table 2 Liquid phase data of low concentration acetochlor standard curve

[0103] Concentration (mg / L) Peak time Peak area 10 2.671 55.93319 25 2.685 146.02919 50 2.712 282.38382 75 2.708 469.02808 100 2.709 619.46014 150 2.71 959.83917 200 2.713 1251.80493

[0104] The standard curve of high concentration acetochlor is:

[0105] y=5.0039x+109.11R 2 =0.9997 (Table 3, Figure 3 );

[0106] Table 3 High concentration acetochlor standard curve liquid phase data

[0107] Concentration (mg / L) Peak time Peak area 100 2.721 507.94565 250 2.722 1372.43445 500 2.724 2668.85742 750 2.723 3931.26025 1000 2.724 5121.68604 1500 2.724 7593.54883 2000 2.722 1.01E+04

[0108] The standard curves of high and low concentrations of acetochlor were fitted to y = 5.0141x + 101.93R 2 =0.9994, the results show that the linear relationship is good.

[0109] Example 2: Effects of different sodium salt concentrations on the enrichment of acetochlor-degrading bacteria

[0110] 5 g of acetochlor-contaminated soil was added to a solution containing 0.0073 mol / L potassium salt (1.0 g KH2PO4 per liter), 0.0076 mol / L ammonium salt (1.0 g (NH4)2SO4 per liter), trace elements (108 μg Al2(SO4)3·18H2O, 56 μg CoSO4·7H2O, 56 μg CuSO4·5H2O, 3.0 mg FeSO4·7H2O, 611 μg H3BO3, 28 μg KBr, 56 μg KI, 28 μg LiCl, 389 μg MnCl2·4H2O, 25 μg Na2MoO4·2H2O, 25 μg Na2WO4·2H2O, 58 μg NiCl2·6H2O, 25 μg SnCl2·2H2O, 34 μg To a 100 ml inorganic salt culture medium containing 2% ZnSO4·H2O), a calcium magnesium solution (containing 30 mg CaCl2 and 20 mg MgCl2 per liter), and vitamins (containing 200 μg p-aminobenzoic acid (VB10), 200 μg biotin (VB7), 200 μg folic acid (VB9), 200 μg nicotinic acid (VB3), 100 μg calcium pantothenate (VB5), 100 μg pyridoxine hydrochloride (VB6), 100 μg riboflavin (VB2), 100 μg thiamine (VB1), and 1 μg cobalamin (VB12) per liter), 92% acetochlor technical was added as the sole carbon source, and the acetochlor stress concentration was increased sequentially to an acetochlor effective concentration of 50, 200, 400, and 600 mg / L. The culture was transferred every 15 days at an inoculum size of 1 / 20. 600 mg / L acetochlor was added to the inorganic salt medium, and the enriched bacterial strain was inoculated at an inoculum size of 1 / 20. The culture was carried out at 30 ° C and 160 rpm in a shaking table at different sodium salt concentrations of 0.008, 0.04, 0.08, and 0.16 mol / L, respectively. Three replicates were used in each group. The acetochlor residue in each group was detected by high performance liquid chromatography. The results showed that when the sodium salt concentration of the acetochlor-degrading bacterial strain in the inorganic salt medium was about 0.04 mol / L, a bacterial strain that efficiently degraded acetochlor could be enriched (Table 4, Figure 4 ).

[0111] Table 4 Effects of different sodium salt concentrations on the enrichment of acetochlor-degrading bacteria

[0112]

[0113] Example 3: Effect of trace element content on the enrichment of acetochlor-degrading bacteria

[0114] Take 5g of acetochlor-contaminated soil and add it to a solution containing 0.04mol / L sodium salt (0.5g NaNO 3，All trace elements (108 μg / L each) were added to 100 ml of inorganic salt culture medium containing 2.5 g Na2HPO4), 0.0073 mol / L potassium salt (1.0 g KH2PO4 per liter), 0.0076 mol / L ammonium salt (1.0 g (NH4)2SO4 per liter), calcium magnesium solution (30 mg CaCl2, 20 mg MgCl2 per liter) and vitamins (200 μg p-aminobenzoic acid (VB10), 200 μg biotin (VB7), 200 μg folic acid (VB9), 200 μg nicotinic acid (VB3), 100 μg calcium pantothenate (VB5), 100 μg pyridoxine hydrochloride (VB6), 100 μg riboflavin (VB2), 100 μg thiamine (VB1), and 1 μg cobalamin (VB12) per liter). Al2(SO4)3·18H2O, 56μg CoSO4·7H2O, 56μg CuSO4·5H2O, 3.0mg FeSO4·7H2O, 611μg H3BO3, 28μg KBr, 56μg KI, 28μg LiCl, 389μg MnCl2·4H2O, 25μg Na2MoO4·2H2O, 25μg Na2WO4·2H2O, 58μg NiCl2·6H2O, 25μg SnCl2·2H2O, 34μg ZnSO4·H2O), no trace elements added, all trace elements except Na2WO4·2H2O (each liter contains 108μg Al2(SO4)3·18H2O, 56μg CoSO4·7H2O, 56μg CuSO4·5H2O, 3.0mg 4H2O), all trace elements except SnCl2 (each liter contains 108μg Al2(SO4)3·18H2O, 56μg CoSO4·7H2O, 56μg CuSO4·5H2O, 3.0mg FeSO4·7H2O, 611μg H3BO3, 28μg KBr, 56μg KI, 28μg LiCl, 389μg MnCl2·4H2O, 25μg Na2MoO4·2H2O, 25μg NiCl2·6H2O, 25μg SnCl2·2H2O, 34μg ZnSO4·H2O), and 1.5mg FeSO4·7H2O. Na2WO4·2H2O, 58μg NiCl2·6H2O, 34μg ZnSO4·H2O). 92% acetochlor technical was added as the sole carbon source. The acetochlor stress concentration was increased sequentially to an effective acetochlor concentration of 50, 200, 400, and 600 mg / L. The inoculum size was 1 / 20, and the cells were switched every 15 days.600 mg / L acetochlor was added to an inorganic salt medium, and the enriched strain was inoculated at a 1 / 20 inoculum size. The culture was shaken at 30°C and 160 rpm, with three replicates per group. The acetochlor residue in each group was determined by HPLC. Removal of W or Sn significantly reduced the acetochlor degradation ability of the enriched acetochlor-degrading strain (Table 5). Figure 5 ), and the optimal concentration for enrichment was 25 μg / L (Table 6, Figure 6 ).

[0115] Table 5 Effects of trace element content on the enrichment of acetochlor-degrading bacteria

[0116] Trace element content ( / L) Degradation rate 1 Degradation rate 2 Degradation rate 3 All trace elements 98.07％ 99.49％ 100.00％ No added trace elements 12.45％ 9.67％ 10.23％ <![CDATA[All trace elements removed from Na2WO4·2H2O]]> 36.23％ 25.23％ 28.23％ <![CDATA[All trace elements removed from SnCl2·2H2O]]> 23.56％ 26.23％ 35.26％

[0117] Table 6 Effects of different concentrations of W and Sn on the enrichment of acetochlor-degrading bacteria

[0118] Trace element content ( / L) Degradation rate 1 Degradation rate 2 Degradation rate 3 <![CDATA[0μg Na2WO4·2H2O]]> 10.22％ 15.23％ 19.56％ <![CDATA[10μg Na2WO4·2H2O]]> 47.56％ 58.97％ 48.98％ <![CDATA[25μg Na2WO4·2H2O]]> 98.89％ 100.00％ 100.00％ <![CDATA[50μg Na2WO4·2H2O]]> 90.00％ 91.56％ 85.81％ <![CDATA[100μg Na2WO4·2H2O]]> 87.00％ 83.21％ 79.23％ <![CDATA[0μg SnCl2·2H2O]]> 19.56％ 6.87％ 14.92％ <![CDATA[10μg SnCl2·2H2O]]> 84.56％ 76.89％ 68.27％ <![CDATA[25μg SnCl2·2H2O]]> 97.87％ 96.89％ 100.00％ <![CDATA[50μg SnCl2·2H2O]]> 75.65％ 80.05％ 84.41％ <![CDATA[100μgSnCl2·2H2O]]> 47.67％ 35.36％ 30.20％

[0119] Example 4: Effect of vitamin content on the enrichment of acetochlor-degrading bacteria

[0120] Take 5g of acetochlor-contaminated soil and add it to a solution containing 0.04mol / L sodium salt (0.5g NaNO 3，2.5gNa2HPO4), 0.0073mol / L potassium salt (containing 1.0g KH2PO4 per liter), 0.0076mol / L ammonium salt (containing 1.0g (NH4)2SO4 per liter), trace elements (containing 108μg Al2(SO4)3·18H2O, 56μg CoSO4·7H2O, 56μg CuSO4·5H2O, 3.0mg FeSO4·7H2O, 611μg H3BO3, 28μg KBr, 56μg KI, 28μg LiCl, 389μg MnCl2·4H2O, 25μgNa2MoO4·2H2O, 25μg Na2WO4·2H2O, 58μg NiCl2·6H2O, 25μg SnCl2·2H2O, 34μg ZnSO4·H2O), calcium magnesium solution (containing 30 mg CaCl2 and 20 mg MgCl2 per liter), all vitamins (containing 200 μg p-aminobenzoic acid (VB10), 200 μg biotin (VB7), 200 μg folic acid (VB9), 200 μg niacin (VB3), 100 μg calcium pantothenate (VB5), 100 μg pyridoxine hydrochloride (VB6), 100 μg riboflavin (VB2), 100 μg thiamine (VB1), and 1 μg cobalamin (VB12) per liter), no vitamins, and all vitamins except folic acid (containing 200 μg p-aminobenzoic acid (VB10), 200 μg biotin (VB7), 200μg niacin (VB3), 100μg calcium pantothenate (VB5), 100μg pyridoxine hydrochloride (VB6), 100μg riboflavin (VB2), 100μg thiamine (VB1), 1μg cobalamin (VB12)), all vitamins except biotin (each liter contains 200μg para-aminobenzoic acid (VB10), 200μg folic acid (VB9), 200μg niacin (VB3), 100μg calcium pantothenate (VB5), 100μg pyridoxine hydrochloride (VB6), 100μg riboflavin (VB2), 100μg thiamine (VB1), 1μg cobalamin (VB12)). 92% acetochlor technical was added as the sole carbon source, and the acetochlor stress concentration was increased to an effective acetochlor concentration of 50, 200, 400, and 600 mg / L, with an inoculum size of 1 / 20, and the culture was transferred every 15 days. 600 mg / L acetochlor was added to the inorganic salt medium, and the enriched bacterial strain was inoculated at an inoculum size of 1 / 20. The culture was shaken at 30°C and 160 rpm, with three replicates per group. The acetochlor residue in each group was determined by high-performance liquid chromatography. The removal of folic acid (VB9) or biotin (VB7) significantly reduced the degradation capacity of the acetochlor-degrading bacterial strain (Table 7, Figure 7 ), and the optimal concentration was 200 μg / L (Table 8, Figure 8 ).

[0121] Table 7 Effect of vitamin content on the enrichment of acetochlor-degrading bacteria

[0122] Vitamin content ( / L) Degradation rate 1 Degradation rate 2 Degradation rate 3 All vitamins 95.62％ 100.00％ 95.36％ No added vitamins 18.64％ 7.30％ 12.01％ All vitamins except folic acid (VB9) 24.36％ 18.47％ 12.75％ All vitamins minus biotin (VB7) 36.23％ 14.79％ 28.56％

[0123] Table 8 Effects of different concentrations of folic acid and biotin on the enrichment of acetylcholine-degrading bacteria

[0124]

[0125]

[0126] Example 5: Stability of acetochlor-degrading bacterial strains

[0127] According to the optimal acetochlor enrichment culture method, 100 ml of inorganic salt culture medium (containing 0.04 mol / L sodium salt (0.5 g NaNO per liter) was added. 3， 2.5g Na2HPO4), 0.0073mol / L potassium salt (containing 1.0g KH2PO4 per liter), 0.0076mol / L ammonium salt (containing 1.0g (NH4)2SO4 per liter), trace elements (containing 108μg Al2(SO4)3·18H2O, 56μg CoSO4·7H2O, 56μgCuSO4·5H2O, 3.0mg FeSO4·7H2O, 611μg H3BO3, 28μg KBr, 56μg KI, 28μg LiCl, 389μgMnCl2·4H2O, 25μg Na2MoO4·2H2O, 25μg Na2WO4·2H2O, 58μg NiCl2·6H2O, 25μg SnCl2·2H2O, 34μg ZnSO4·H2O), calcium magnesium solution (containing 30 mg CaCl2 and 20 mg MgCl2 per liter), and vitamins (containing 200 μg para-aminobenzoic acid (VB10), 200 μg biotin (VB7), 200 μg folic acid (VB9), 200 μg niacin (VB3), 100 μg calcium pantothenate (VB5), 100 μg pyridoxine hydrochloride (VB6), 100 μg riboflavin (VB2), 100 μg thiamine (VB1), and 1 μg cobalamin (VB12) per liter) 92% acetochlor technical was added as the sole carbon source, and the acetochlor stress concentration was increased to 50, 200, 400, and 600 mg / L, respectively. The inoculum size was 1 / 20, and the culture was transferred every 15 days (from the fifth generation, the effective concentration of acetochlor was 600 mg / L). The culture temperature was 30°C, the inoculum size was 1 / 20, and the acetochlor-degrading bacteria system was continuously cultured for 14 generations and could stably degrade acetochlor. Figure 9 ).

[0128] Example 6: Degradation rate of acetochlor at different concentrations by acetochlor-degrading bacteria

[0129] When the acetochlor-degrading bacteria were subcultured to the fourth generation, the bacteria were cultured in an inorganic salt medium (containing 0.04 mol / L sodium salt (0.5 g NaNO 3， 2.5g Na2HPO4), 0.0073mol / L potassium salt (containing 1.0g KH2PO4 per liter), 0.0076mol / L ammonium salt (containing 1.0g (NH4)2SO4 per liter), trace elements (containing 108μg Al2(SO4)3·18H2O, 56μg CoSO4·7H2O, 56μgCuSO4·5H2O, 3.0mg FeSO4·7H2O, 611μg H3BO3, 28μg KBr, 56μg KI, 28μg LiCl, 389μgMnCl2·4H2O, 25μg Na2MoO4·2H2O, 25μg Na2WO4·2H2O, 58μg NiCl2·6H2O, 25μg SnCl2·2H2O, 34μg ZnSO4·H2O), calcium magnesium solution (containing 30 mg CaCl2 and 20 mg MgCl2 per liter), and vitamins (containing 200 μg p-aminobenzoic acid (VB10), 200 μg biotin (VB7), 200 μg folic acid (VB9), 200 μg niacin (VB3), 100 μg calcium pantothenate (VB5), 100 μg pyridoxine hydrochloride (VB6), 100 μg riboflavin (VB2), 100 μg thiamine (VB1), and 1 μg cobalamin (VB12) per liter)) were added with different concentrations of 92% acetochlor technical to an effective concentration of acetochlor of 200, 800, and 1000 mg / L, and the OD was adjusted. 600 =0.3 bacterial suspension was inoculated into the above-mentioned acetochlor inorganic salt culture medium according to an inoculum size of 1 / 20, and the uninoculated sample was used as the blank control treatment group. The culture time was 18 days at 30°C and 160rpm shaking. The acetochlor-degrading bacteria system degraded 200mg / L acetochlor by more than 85%, 800mg / L acetochlor by more than 65%, and 1000mg / L acetochlor by more than 44% on the 6th day after inoculation; on the 9th day after inoculation, it could degrade 200mg / L acetochlor by more than 94%, 800mg / L acetochlor by more than 85%, and 1000mg / L acetochlor by more than 94%; on the 12th day after inoculation, it could degrade three concentrations of acetochlor by more than 95%. The acetochlor-degrading bacteria system can effectively degrade acetochlor in a large range (Table 9, Table 10, Figure 10 , Figure 11 ).

[0130] Table 9 Degradation of acetochlor residues at different concentrations by bacterial systems

[0131]

[0132] Table 10 Degradation rate of acetochlor degrading bacteria on different concentrations of acetochlor

[0133]

[0134]

[0135] Example 7: Degradation ability of acetochlor-degrading bacteria on different acetochlor pesticide formulations

[0136] When the acetochlor-degrading bacteria were subcultured to the fourth generation, the bacteria were cultured in an inorganic salt medium (containing 0.04 mol / L sodium salt (0.5 g NaNO 3， 2.5g Na2HPO4), 0.0073mol / L potassium salt (containing 1.0g KH2PO4 per liter), 0.0076mol / L ammonium salt (containing 1.0g (NH4)2SO4 per liter), trace elements (containing 108μg Al2(SO4)3·18H2O, 56μg CoSO4·7H2O, 56μgCuSO4·5H2O, 3.0mg FeSO4·7H2O, 611μg H3BO3, 28μg KBr, 56μg KI, 28μg LiCl, 389μgMnCl2·4H2O, 25μg Na2MoO4·2H2O, 25μg Na2WO4·2H2O, 58μg NiCl2·6H2O, 25μg SnCl2·2H2O, 34μg ZnSO4·H2O), calcium magnesium solution (containing 30mg CaCl2 and 20mg MgCl2 per liter) and vitamins (containing 200μg p-aminobenzoic acid (VB10), 200μg biotin (VB7), 200μg folic acid (VB9), 200μg nicotinic acid (VB3), 100μg calcium pantothenate (VB5), 100μg pyridoxine hydrochloride (VB6), 100μg riboflavin (VB2), 100μg thiamine (VB1), and 1μg cobalamin (VB12) per liter) were added with different acetochlor pesticide preparations: 50% emulsifiable concentrate, 900g / L emulsifiable concentrate, 990g / L emulsifiable concentrate, 50% microemulsion, 40% emulsion in water, 48% emulsion in water, 50% emulsion in water, 20% wettable powder, and 40% wettable powder. The OD 600 = 0.3 was inoculated into the above-mentioned acetochlor inorganic salt medium at an inoculum size of 1 / 20. Uninoculated samples were used as blank control treatment groups. The cultures were shaken at 30°C and 160 rpm for 7 and 15 days. The acetochlor-degrading bacteria could degrade different types of pesticide formulations by more than 90% within 15 days (Table 11, Figure 12 ).

[0137] Table 11 Degradation rates of acetochlor-degrading bacteria on different acetochlor pesticide formulations

[0138]

[0139] Example 8: Stability of acetochlor-degrading bacterial community composition

[0140] Acetochlor-contaminated soil was subjected to different treatments and named AT1, AT2, AS, and ABT1, respectively. The specific treatments were as follows: AT1 was enriched and cultured according to the method described in Example 5 of the present invention; AT2 was enriched and cultured according to the method described in Example 5 of the present invention, except that the culture temperature was 22°C; AS was enriched and cultured according to the method described in Example 5 of the present invention, except that Na2WO4·2H2O and SnCl2·2H2O were not added to the inorganic salt culture medium; and ABT1 was enriched and cultured according to the method described in Example 5 of the present invention, except that folic acid (VB9) and biotin (VB7) were not added to the inorganic salt culture medium. The culture was carried out on a shaker at 160 rpm for 15 days. AT1 (the preferred acetochlor-degrading bacterial strain enriched by the enrichment method of the present invention) degraded more than 50% of the acetochlor within 3 days after inoculation and more than 60% within 6 days after inoculation, at a steady rate. The results of 16S high-throughput sequencing showed that Sphingomonas accounted for 60% of AT1 on the 6th day and 65% on the 9th day, and Sphingomonas was able to degrade a variety of aromatic compounds. The above shows that the AT1 strain has high stability in degradation ability and composition ( Figure 13 , Figure 14 ), the abundance of bacteria in the AT1 strain is shown in Table 12.

[0141] Table 12 Abundance of bacteria in acetochlor-degrading bacterial strain AT1

[0142]

[0143]

[0144] Example 9: Application of acetochlor-degrading bacteria in indoor pot plant experiments

[0145] In the indoor potted plant experiment, different treatments were set up, namely, a control group (no acetochlor removal and no bacterial agent), a bacterial agent group (with bacterial agent added), a low-concentration group (3 mg / kg acetochlor applied), a low-concentration repair group (3 mg / kg acetochlor applied and bacterial agent added), a high-concentration group (15 mg / kg acetochlor applied), and a high-concentration repair group (15 mg / kg acetochlor applied and bacterial agent added). The specific operation was as follows: acetochlor was sprayed into 2 mm air-dried soil at a ratio of 3 or 15 mg / kg soil; when the acetochlor-degrading bacterial system enriched by the enrichment method described in Example 5 of the present invention was passaged to the 8th generation, the bacteria were collected, and the bacterial solution concentration was adjusted to OD600 = 1, sprayed at 1 ml / 100 g of soil into 2 mm of air-dried soil. After spraying the soil with acetochlor and the degrading agent, the soil moisture content of each group was adjusted to 20%. The soil was then distributed into pots and incubated at 25°C. Soil samples were collected on days 0, 3, 7, 14, 28, and 49 and analyzed for acetochlor content by liquid chromatography-mass spectrometry. The results showed that starting from the third day after treatment, the addition of the agent significantly reduced the acetochlor content in the soil compared to the group without the agent. This demonstrates that the acetochlor degradation and enrichment method of the present invention can successfully enrich strains of bacteria that efficiently degrade acetochlor and can be used in potted plant experiments (Table 13, Figure 15 ).

[0146] Table 13 Acetochlor residues in potted soil

[0147]

[0148]

[0149] Example 10: Application of acetochlor-degrading bacteria in field plot trials

[0150] Different treatments were set up in the field plot test, namely the control group (no acetochlor application and no bacterial agent addition), the bacterial agent group (microbial agent addition), the low-concentration group (1500 g / hectare acetochlor application), the low-concentration remediation group (1500 g / hectare acetochlor application and microbial agent addition), the high-concentration group (7500 g / hectare acetochlor application), and the high-concentration remediation group (7500 g / hectare acetochlor application and microbial agent addition). The specific operation is as follows: acetochlor was sprayed on the soil at a ratio of 1500 g / hectare and 7500 g / hectare. When the acetochlor-degrading bacterial system enriched by the enrichment method described in this patent was subcultured to the 9th generation, the bacteria were collected, and the bacterial solution concentration was adjusted to OD 600 =1, according to 4L / 20m 2 The soil was sprayed with the agent; soil samples were collected at 0, 10, 20, and 40 days and analyzed for acetochlor content by liquid chromatography-mass spectrometry. The results showed that the addition of the agent significantly reduced the acetochlor content in the soil compared to the control group without the agent, indicating that the acetochlor degradation and enrichment method of the present invention can successfully enrich a strain of bacteria that efficiently degrades acetochlor and can be used in field plot trials (Table 14, Figure 16 ).

[0151] Table 14 Acetochlor residues in field plot test soil

[0152]

[0153]

[0154] The foregoing description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein and should not be construed as excluding other embodiments. The present invention is adaptable to various other combinations, modifications, and variations, and is capable of modification within the scope of the concepts described herein, through the teachings above, or through techniques or knowledge in the relevant field. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention are intended to be within the scope of the appended claims.

Claims

1. A method for enriching and culturing an acetochlor-degrading bacterial strain, characterized in that: The substrate used in the enrichment culture is acetochlor; the enrichment culture method comprises enriching and culturing a material containing the substrate in an enrichment culture medium; The enrichment medium includes 0.008-0.04 mol / L sodium salt, 4-5 mg / L trace elements, 0.006-0.008 mol / L potassium salt, 0.006-0.008 mol / L ammonium salt, 40-60 mg / L calcium magnesium solution and 1.0-1.25 mg / L vitamins; the trace elements include aluminum, cobalt, copper, iron, bromine, boron, iodine, lithium, manganese, molybdenum, tungsten, nickel, tin and zinc, and the trace elements contain 25-100 μg / L Na2WO4·2H2O and 25-50 μg / L SnCl2·2H2O; the vitamins include p-aminobenzoic acid, biotin, folic acid, niacin, calcium pantothenate, pyridoxine hydrochloride, riboflavin, thiamine and cobalamin, wherein the content of folic acid is 200-400 μg / L. μg / L, and the content of biotin is 100-200 μg / L; The material is soil or activated sludge; the temperature of the enrichment culture is 25-30°C.

2. The enrichment culture method according to claim 1, characterized in that The enrichment culture is carried out in a light-proof environment.

3. The enrichment culture method according to claim 1, characterized in that The calcium-magnesium solution comprises calcium chloride and magnesium chloride; and / or, the concentration of the calcium and magnesium solution is 40-50 mg / L; and / or, the sodium salt comprises NaNO3 and Na2HPO4; and / or, the potassium salt comprises KH2PO4; and / or, the concentration of the potassium salt is 0.006-0.0073 mol / L; and / or, the ammonium salt comprises (NH4)2SO4; And / or, the concentration of the ammonium salt is 0.006-0.0076 mol / L.

4. The enrichment culture method according to claim 1, characterized in that The trace elements contain 25-50 μg / L of Na2WO4·2H2O.

5. The enrichment culture method according to claim 1, characterized in that: The enrichment culture medium includes: the trace elements; the vitamins; 0.008-0.04 mol / L sodium salt, wherein the sodium salt comprises NaNO3 and Na2HPO4; 0.006-0.008 mol / L potassium salt, wherein the potassium salt comprises KH2PO4; 0.006-0.008 mol / L ammonium salt, wherein the ammonium salt comprises (NH4)2SO4; and 50 mg / L calcium magnesium solution, wherein the calcium magnesium solution comprises 30 mg / L CaCl2 and 20 mg / L MgCl2.

6. The enrichment culture method according to claim 5, wherein: The trace elements include 108-110 μg / LAl2(SO4)3·18H2O, 56-58 μg / L CoSO4·7H2O, 56-60 μg / L CuSO4·5H2O, 2.5-3.0 mg / LFeSO4·7H2O, 600-611 μg / L H3BO3, 25-28 μg / L KBr, 56-60 μg / L KI, 25-28 μg / L LiCl, 360-389 μg / L MnCl2·4H2O, 25-27 μg / L Na2MoO4·2H2O, 25-27 μg / L Na2WO4·2H2O, 58-64 μg / L NiCl2·6H2O, 25-28 μg / L SnCl2·2H2O, 34-36 μg / L ZnSO4·H2O.

7. The enrichment culture method according to claim 5, wherein: The vitamins include 200 μg / L p-aminobenzoic acid, 200 μg / L biotin, 200 μg / L folic acid, 200 μg / L niacin, 100 μg / L calcium pantothenate, 100 μg / L pyridoxine hydrochloride, 100 μg / L riboflavin, 100 μg / L thiamine, and 1 μg / L cobalamin.

8. The enrichment culture method according to claim 5, wherein: The enrichment medium comprises: 0.04 mol / L sodium salt, which comprises 0.5 g / L NaNO3 and 2.5 g / L Na2HPO4; 0.0073 mol / L potassium salt, which comprises 1.0 g / L KH2PO4; 0.0076 mol / L ammonium salt, which comprises 1.0 g / L (NH4)2SO4; and trace elements, which comprise 108 μg / L Al2(SO4)3·18H2O, 56 μg / L CoSO4·7H2O, 56 μg / L CuSO4·5H2O, 3.0 mg / L FeSO4·7H2O, 611 μg / L H3BO3, 28 μg / L KBr, 56 μg / L KI, 28 μg / L LiCl, 389 μg / L MnCl2·4H2O, 25 μg / L Na2MoO4·2H2O, 25 μg / L Na2WO4·2H2O, 58 μg / L NiCl2·6H2O, 25 μg / L SnCl2·2H2O, 34 μg / L ZnSO4·H2O; 50 mg / L calcium magnesium solution, the calcium magnesium solution contains 30 mg / LCaCl2 and 20 mg / L MgCl2; the vitamins contain 200 μg / L p-aminobenzoic acid, 200 μg / L biotin, 200 μg / L folic acid, 200 μg / L niacin, 100 μg / L calcium pantothenate, 100 μg / L pyridoxine hydrochloride, 100 μg / L riboflavin, 100 μg / L thiamine and 1 μg / L cobalamin.

9. The enrichment culture method according to any one of claims 1 to 8, characterized in that: The enrichment culture further comprises: enriching and culturing the substrate for 6-7 days, and culturing the solution OD 600 When the value is 0.1-0.15, the concentration of acetochlor technical detected by HPLC is lower than 50%, 45% or 40% of the initial concentration; And / or, the enrichment culture further comprises: the abundance of Sphingomonas detected by 16S high-throughput sequencing accounts for no less than 60%.

10. The enrichment culture method according to claim 9, wherein: The abundance of the genus Sphingomonas is not less than 70%.

11. A culture medium for enrichment culture of acetochlor-degrading bacteria, characterized in that: The culture medium includes 0.008-0.04 mol / L sodium salt, 4-5 mg / L trace elements, 0.006-0.008 mol / L potassium salt, 0.006-0.008 mol / L ammonium salt, 40-60 mg / L calcium magnesium solution and 1.0-1.25 mg / L vitamins; the trace elements include aluminum, cobalt, copper, iron, bromine, boron, iodine, lithium, manganese, molybdenum, tungsten, nickel, tin and zinc, and the trace elements contain 25-100 μg / L Na2WO4·2H2O and 25-50 μg / L SnCl2·2H2O; the vitamins include p-aminobenzoic acid, biotin, folic acid, niacin, calcium pantothenate, pyridoxine hydrochloride, riboflavin, thiamine and cobalamin, wherein the content of folic acid is 200-400 μg / L, and the content of biotin is 100-200 μg / L.

12. The culture medium according to claim 11, characterized in that The culture medium includes the trace elements; the vitamins; 0.008-0.04 mol / L sodium salt, wherein the sodium salt comprises NaNO3 and Na2HPO4; 0.006-0.008 mol / L potassium salt, wherein the potassium salt comprises KH2PO4; 0.006-0.008 mol / L ammonium salt, wherein the ammonium salt comprises (NH4)2SO4; and 50 mg / L calcium magnesium solution, wherein the calcium magnesium solution comprises 30 mg / L CaCl2 and 20 mg / L MgCl2.

13. The culture medium according to claim 12, characterized in that The trace elements include 108-110 μg / L Al2(SO4)3·18H2O, 56-58 μg / L CoSO4·7H2O, 56-60 μg / L CuSO4·5H2O, 2.5-3.0 mg / L FeSO4·7H2O, 600-611 μg / L H3BO3, 25-28 μg / L KBr, 56-60 μg / L KI, 25-28 μg / L LiCl, 360-389 μg / L MnCl2·4H2O, 25-27 μg / L Na2MoO4·2H2O, 25-27 μg / L Na2WO4·2H2O, 58-64 μg / LNiCl2·6H2O, 25-28 μg / L SnCl2·2H2O, 34-36 μg / L ZnSO4·H2O.

14. The culture medium according to claim 12, characterized in that The vitamins include 200 μg / L p-aminobenzoic acid, 200 μg / L biotin, 200 μg / L folic acid, 200 μg / L niacin, 100 μg / L calcium pantothenate, 100 μg / L pyridoxine hydrochloride, 100 μg / L riboflavin, 100 μg / L thiamine, and 1 μg / L cobalamin.

15. The culture medium according to any one of claims 11 to 14, characterized in that The culture medium includes 0.04 mol / L sodium salt, which contains 0.5 g / L NaNO3 and 2.5 g / L Na2HPO4; 0.0073 mol / L potassium salt, which contains 1.0 g / L KH2PO4; 0.0076 mol / L ammonium salt, which contains 1.0 g / L (NH4)2SO4; and trace elements, which contain 108 μg / L Al2(SO4)3·18H2O, 56 μg / L CoSO4·7H2O, 56 μg / L CuSO4·5H2O, 3.0 mg / L FeSO4·7H2O, 611 μg / L H3BO3, 28 μg / L KBr, 56 μg / L KI, 28 μg / L LiCl, 389 μg / L MnCl2·4H2O, 25 μg / L Na2MoO4·2H2O, 25 μg / L Na2WO4·2H2O, 58 μg / L NiCl2·6H2O, 25 μg / L SnCl2·2H2O, 34 μg / L ZnSO4·H2O; 50 mg / L calcium magnesium solution, the calcium magnesium solution contains 30 mg / LCaCl2 and 20 mg / L MgCl2; the vitamins contain 200 μg / L p-aminobenzoic acid, 200 μg / L biotin, 200 μg / L folic acid, 200 μg / L niacin, 100 μg / L calcium pantothenate, 100 μg / L pyridoxine hydrochloride, 100 μg / L riboflavin, 100 μg / L thiamine and 1 μg / L cobalamin.

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