An anaerobic degradation strain SRB-2 and its application

Abstract

The present invention discloses an anaerobic degradation strain SRB-2 and its application, wherein the strain is Trichococcus sp.SRB‑2 is a facultative anaerobic Gram-positive bacterium, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC No.40302. Under anaerobic or microaerobic conditions, the strain has an anaerobic degradation half-life of 2.0 d for 20 mg / L of isopropylamine, and also exhibits a good bioremediation effect on isopropylamine-contaminated industrial wastewater. The strain of the present invention enriches the resource library of pesticide-degrading bacteria, has broad prospects in the application of bioremediation of isopropylamine pollution or preparation of isopropylamine anaerobic degradation bacterial agents, and has good application value in environmental protection.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial remediation of organic pollutants and relates to an anaerobic degradation strain SRB-2 and application thereof. Background Art

[0002] Isopropylamine [2-methyl-6-ethyl- N -(1-methyl-2-methoxyethyl)- N -Chloroacetanilide, Metolachlor] is a systemic, selective, pre-emergence chloroacetamide herbicide. It is widely used in wheat, rice, corn, peanut and other crop fields to control annual grass weeds and some broadleaf weeds. Isopropylamine has the advantages of higher safety and wider application of crops. Its global market share and scope of use are expanding year by year. In 2018, its usage reached 59,300 tons, making it the sixth largest herbicide in the world (the top five are glyphosate, glufosinate, mesotrione, 2,4-D and atrazine), and its usage is still increasing rapidly.

[0003] Metolachlor has high water solubility (480 mg / L at 25°C and pH 7.3) and low Kow value (octanol-water partition coefficient), so it has high fluidity and is easy to pollute surface water, groundwater and soil through surface runoff after application. Metolachlor residues in soil and water bodies can cause serious crop damage and threaten ecological and environmental safety. In recent years, crop damage caused by improper use of metolachlor has occurred from time to time, causing serious losses to agricultural production, especially in low-lying areas, sandy soils, rainy days or drip irrigation, where the damage is particularly serious. Studies have found that metolachlor is highly toxic to aquatic organisms such as fish, water fleas and algae. It was also found that metolachlor has a strong inhibitory effect on the number and activity of soil microorganisms, and significantly reduces the abundance and diversity of soil microorganisms.

[0004] Isopropylamine can be eliminated from the environment through photolysis, chemical degradation and microbial degradation, among which microorganisms are the main driving force for eliminating isopropylamine residual pollution in the environment. Compared with traditional physical and chemical methods, microbial degradation and remediation technology has the advantages of high efficiency, rapidity, no residue, and no secondary pollution. It is suitable for the degradation and remediation of large-scale non-point source pollution of toxic and harmful organic pollutants, and the remediation cost is low (only about 10% of the cost of traditional chemical and physical remediation). Microbial degradation is divided into aerobic microbial degradation and anaerobic microbial degradation according to the different needs of microorganisms for oxygen. Anaerobic bioremediation has the advantages of low energy consumption, high sludge load, low nutrient requirements, high economic benefits and a wide range of applications. It has become the main method for remediation of environmental pollutants. At present, the aerobic degradation strains of isopropylamine isolated and screened at home and abroad are Phanerochaete chrysosporium, Streptomoyces sp., Paracoccus sp. FLY-8, etc., which have low degradation efficiency for isopropylamine, such as strain Paracoccus sp. FLY-8 had a degradation rate of only 22% for 100 mg / L isopropylamine within 5 days. Since isopropylamine is highly water-soluble, it is easy to enter micro-aerobic or anaerobic environments such as groundwater and deep soil during use. However, only one strain has been reported to be able to anaerobically degrade isopropylamine. Proteiniclasticum sediminis BAD-10 T The degradation half-life of 100 μM (28 mg / L) metolachlor was as long as 30.6 days. Therefore, isolating and purifying the highly efficient anaerobic degradation strain of metolachlor and developing the bacterial agent for the remediation of metolachlor pollution are of great significance for enriching the resources of metolachlor degradation strains and eliminating the residual pollution of metolachlor quickly and at low cost. Summary of the invention

[0005] In view of the above-mentioned deficiencies of the prior art, the first object of the present invention is to provide an anaerobic degradation strain SRB-2, which is classified and named Trichococcus sp. SRB-2, deposited in the General Microbiology Center of China Microorganism Culture Collection on August 5, 2022, with the deposit number CGMCC No. 40302, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The second object of the present invention is to provide an application of an anaerobic degradation strain SRB-2 in degrading the residues of the herbicide isopropylamine.

[0007] The third object of the present invention is to provide an application of an anaerobic degradation strain SRB-2 in repairing a natural environment or wastewater treatment equipment contaminated by isopropylamine.

[0008] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0009] The anaerobic degradation strain SRB-2 was classified and named Trichococcus sp. SRB-2, deposited in the General Microbiology Center of China Microorganism Culture Collection on August 5, 2022, with the deposit number CGMCC No. 40302, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The present invention collects anaerobic sludge from a pesticide factory in Binzhou, Shandong Province, and obtains a strain capable of efficiently and rapidly anaerobically degrading isopropylamine after enrichment and domestication, separation and purification. The strain is identified by morphology and molecular biology as belonging to Trichococcus Genus, named Trichococcussp. SRB-2. This strain can rapidly degrade isopropylamine under anaerobic / microaerobic conditions. Under the same conditions, its degradation efficiency of isopropylamine is the highest among all strains reported so far. P. sediminis BAD-10 T The results showed that the anaerobic degradation strain SRB-2 can be used as a biodegradation bacterium for the bioremediation of isopropylamine-contaminated environment.

[0011] The anaerobic degradation strain SRB-2 is used for degrading herbicide residues, wherein the herbicide is metolachlor, and the degradation half-life of metolachlor at a concentration of 20 mg / L is 2.0 d.

[0012] The application of the anaerobic degradation strain SRB-2 in repairing the natural environment or wastewater treatment equipment contaminated by isopropylamine.

[0013] As an improvement, the natural environment is groundwater, surface water, flooded soil or wetland, and the wastewater treatment facility is an anaerobic reactor.

[0014] A highly efficient bacterial agent for anaerobic degradation of the herbicide metolachlor contains the anaerobic degradation strain SRB-2.

[0015] As an improvement, the number of anaerobic degradation bacteria SRB-2 in the bacterial agent is 1.0 × 10 5 –1.0 × 1.0 8 cfu / mL.

[0016] Beneficial effects:

[0017] Compared with the prior art, the present invention provides an anaerobic degradation strain SRB-2 and an application thereof, isolates and screens a high-efficiency anaerobic degradation strain of isopropylamine, identifies it, studies its degradation characteristics and bioremediation ability, and the results show that the present invention enriches the herbicide degradation bacteria resource library and provides a basis for developing new herbicide residue pollution remediation strategies.

[0018] (1) The present invention isolates and screens an anaerobic degradation strain SRB-2. Through testing, it is found that the strain is a Gram-negative bacterium that cannot move. Under strictly anaerobic or microaerobic conditions, the degradation half-life of the strain for 20 mg / L of isopropylamine is 2.0 days. The strain has a high anaerobic degradation efficiency for isopropylamine, thus enriching the seed resource library for anaerobic degradation of herbicides.

[0019] (2) The present invention found that under anaerobic conditions, the strain Trichococcus sp. SRB-2 was able to degrade isopropylamine rapidly and efficiently, indicating that strain SRB-2 had great potential for bioremediation of anaerobic environments contaminated by isopropylamine.

[0020] (3) The anaerobic degradation strain SRB-2 isolated by the present invention can be applied in natural environments or wastewater treatment equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The anaerobic degradation effect of the anaerobic degradation strain SRB-2 on isopropylamine; A is the liquid phase diagram of isopropylamine at different culture times; B is the anaerobic degradation dynamics of isopropylamine at different culture times;

[0022] Figure 2 A is the colony of anaerobic degradation strain SRB-2;

[0023] Figure 2 B is the bacterial morphology of the anaerobic degradation strain SRB-2;

[0024] Figure 3 This is the phylogenetic analysis of the 16S rRNA gene of the anaerobic degradation strain SRB-2;

[0025] Figure 4 A is the effect of temperature on the degradation of metolachlor by the anaerobic degradation strain SRB-2;

[0026] Figure 4 B is the effect of pH on the degradation of metolachlor by the anaerobic degradation strain SRB-2;

[0027] Figure 4 C is the effect of NaCl concentration on the degradation of metolachlor by the anaerobic degradation strain SRB-2;

[0028] Figure 5 Anaerobic degradation and remediation of isopropylamine in industrial wastewater by anaerobic degradation strain SRB-2, A is 5 mg / L; B is 20 mg / L. DETAILED DESCRIPTION

[0029] The present invention is further described below by way of examples in conjunction with the accompanying drawings; however, the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] The culture medium formula described in the following examples is as follows:

[0031] Anaerobic enrichment acclimatization medium (MSM, g / L): K2HPO4·3H2O, 0.35 g; KH2PO4, 0.27 g; MgCl2·6H2O, 0.15 g; NH4Cl, 0.53 g; CaCl2·2H2O, 0.073 g; sodium pyruvate, 0.1 g; sodium citrate, 0.1 g; FeSO4·7H2O, 0.60 g (added before boiling and aliquoting); ascorbic acid, 0.1 g (added before boiling and aliquoting); cysteine ​​hydrochloride, 0.02 g (added before boiling and aliquoting); resazurin, 1.0 mg; trace element complex solution, 1.0 mL; vitamin complex solution, 1.0 mL (added after sterilization); pH 7.0, pure water dilute to 1.0 L

[0032] Anaerobic isolation and culture medium (MPM, g / L): NH4Cl, 1.0 g; NaCl, 1.0 g; K2HPO4·3H2O, 0.5 g; MgCl2·6H2O, 0.05 g; Na2SO4, 1.1 g; CaCl2·2H2O, 0.05 g; sodium lactate, 2.0 g; ascorbic acid, 0.10 g; cysteine ​​hydrochloride, 0.2 g; yeast extract, 1.0 g; resazurin, 1.0 mg; trace element complex solution, 1.0 mL; vitamin complex solution, 1.0 mL; pH was adjusted to 7.2 with phosphate buffer and pure water was added to 1.0 L. 1.7% agar was added to the solid medium.

[0033] Deoxygenation of culture medium: Boil the prepared culture medium, and while it is still hot, dispense 30 mL of the culture medium into 50 mL serum bottles, plug the bottles with rubber stoppers and secure them with aluminum caps, fill them with nitrogen for 30 minutes to deoxygenate, and sterilize them with high-pressure steam at 115°C for 30 minutes. The culture medium should remain colorless after sterilization. If the color changes to slightly red or red, it means that oxygen has not been completely removed.

[0034] Detection conditions of isopropylamine: Ultra-high performance liquid chromatography (U3000, Thermo Fisher Scientific, USA); Thermo Fisher Syncronis C 18 (5 μm, 4.6 × 250 mm); mobile phase: acetonitrile: water = 80:10 (V / V); detection wavelength: 222 nm; flow rate: 1.0 mL / min; column temperature: 30 ℃; injection volume: 20 μL.

[0035] Example 1 Isolation, screening and identification of strain SRB-2

[0036] (1) Enrichment, domestication, isolation and screening of anaerobic metolachlor-degrading bacteria

[0037] Anaerobic activated sludge collected from a long-term production plant of isopropyl metolachlor was inoculated into a serum bottle containing 20 mg / L isopropyl metolachlor and 30 mL MSM, and cultured in a shaker at 30 °C and 150 rpm for 7 days. The enriched solution of the previous generation was transferred to 30 mL of fresh MSM containing 20 mg / L isopropyl metolachlor at 5% content. The concentration of isopropyl metolachlor in the bottle was measured each time the transfer was made until the isopropyl metolachlor was degraded by more than 90%, and the transfer was continued to the fourth generation. The enriched solution of the fifth generation was taken, and the diluted enriched solution was spread on the MPM solid medium containing 100 mg / L isopropyl metolachlor in an anaerobic operation box by the dilution coating method. The plate was inverted and placed in an anaerobic tank, and cultured at 30 °C for 7 days. After a single colony grew on the plate, a single colony was picked and streaked for purification multiple times, and its degradation effect on isopropyl metolachlor was verified.

[0038] Using isopropylamine as the selection pressure, an anaerobic degrading bacterium was isolated and purified, numbered SRB-2. Trichococcus sp. SRB-2, deposited in the General Microbiology Center of China Microorganism Culture Collection on August 5, 2022, with the deposit number CGMCC No. 40302, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0039] As the incubation time increased, the absorption peak of isopropylamine in the HPLC liquid chromatogram gradually decreased ( Figure 1 A); Within 8 days of cultivation, the anaerobic degradation rate of strain SRB-2 for 20 mg / L of isopropylamine reached more than 95% ( Figure 1 B).

[0040] (2) Morphological and molecular biological identification of anaerobic degradation strain SRB-2

[0041] In the anaerobic operation box, the anaerobic degradation strain SRB-2 was inoculated on the MPM solid plate and cultured at 30 ℃ for 7 days. The colony morphology during the observation period was round in shape, with a diameter of 0.5-3.0 mm, and a moist, irregular, light yellow surface ( Figure 2 A); The bacteria are spherical or nearly elliptical, without flagella, non-motile, and 0.6-1.0 μm × 0.8-1.2 μm in size ( Figure 2 B). The strain is Gram-positive and grows facultatively anaerobic; it can grow at a temperature of 0-42 °C, with an optimum growth temperature of 25-30 °C; it can grow at a pH of 5.0-10.0, with an optimum growth pH of 7.0-8.0; it can tolerate an oxygen concentration of 2.0%, and it can reduce sulfate to produce hydrogen sulfide.

[0042] The total DNA of the anaerobic degradation strain SRB-2 was extracted using the high salt method, and then the universal primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) for bacterial identification were designed. The extracted total DNA of strain SRB-2 was used as a template to amplify the 16S rRNA gene sequence of strain SRB-2. The product after PCR amplification was entrusted to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The 16S rRNA gene sequence of strain SRB-2 obtained by sequencing was analyzed for homology with the 16S rRNA gene of the reported model strain in the EzBioCloud database (www.ezbiocloud.net). The model strain-related sequences with higher homology were selected, and the phylogenetic tree of the resistant strain was constructed by the neighbor-joining method using MEGA 6.0 software. Figure 3 As shown, the 16S rRNA gene sequence of the strain SRB-2 isolated and obtained in the present invention is Trichococcus collinsii 37AN3* T The highest homology is 99.9%.

[0043] Therefore, based on the morphological and molecular biological identification of the anaerobic degradation strain SRB-2, the anaerobic degradation strain SRB-2 was identified as Trichococcus Genus, named Trichococcus sp. SRB-2. The strain SRB-2 isolated in the present invention belongs to Trichococcus genus, and the currently reported Proteiniclasticum The different taxonomic status enriches the seed resource library of isopropylamine-degrading bacteria. The strain was deposited in the General Microbiological Center of China Microbiological Culture Collection on August 5, 2022, with the deposit number CGMCC No. 40302, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0044] Example 2 Study on the anaerobic degradation characteristics of metolachlor by anaerobic degradation strain SRB-2

[0045] (1) Preparation of liquid bacterial agent of anaerobic degradation strain SRB-2

[0046] In an anaerobic operation box, the strain SRB-2 grown on the MPM solid plate was inoculated into a 250 mL vial containing 100 mL of deoxygenated MPM liquid medium and cultured at 30 °C and 150 rpm until the logarithmic phase (4 d). The bacteria were collected by centrifugation at 3380 × g for 5 min, washed twice with MSM and resuspended in MSM. The bacterial solution concentration OD was adjusted. 600 1.0 (~1.2 × 10 8cfu / mL), which is liquid bacterial agent.

[0047] (2) Study on the degradation characteristics of anaerobic degradation strain SRB-2

[0048] Add 1.0 mL of liquid inoculum of strain SRB-2 (OD 600 = 0.6) and a certain amount of isopropylamine to make the final concentration of isopropylamine 20 mg / L, and then cultured in a shaker at 30 ℃ and 150 rpm. After 6 days, samples were taken to determine the concentration of isopropylamine. The blank group did not add strain SRB-2, and each group was repeated three times. According to the above operation, the effects of different temperatures (10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃ and 42 ℃), different pH (5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), and different NaCl concentrations (0%, 1.0%, 2.0%, 3.0%, 4.0% and 5.0%, w / v) on the degradation of isopropylamine by the anaerobic degradation strain SRB-2 were studied.

[0049] Depend on Figure 4 It can be seen that the anaerobic degradation rate of anaerobic degradation strain SRB-2 for isopropylamine is greater than 50% at a temperature of 15-42 °C; the anaerobic degradation rate of anaerobic degradation strain SRB-2 for isopropylamine is greater than 70% at 20-37 °C, and the optimal temperature for anaerobic degradation of isopropylamine is 25-30 °C ( Figure 4 A); At pH 6.0-10.0, the anaerobic degradation rate of isopropylamine by the anaerobic degradation strain SRB-2 was greater than 60%, and the optimal pH for anaerobic degradation of isopropylamine was 7.0-8.0 ( Figure 4 B); When the NaCl concentration is 0%-4.0%, the anaerobic degradation rate of isopropylamine by the anaerobic degradation strain SRB-2 is greater than 60%, and the optimal NaCl concentration for anaerobic degradation of isopropylamine is 0%-3.0% ( Figure 4 C).

[0050] The above results show that the anaerobic degradation strain SRB-2 has good environmental adaptability under a wide range of temperature (15-42 ℃), pH (6.0-10.0) and NaCl concentration (0%-3.0%) conditions, and can efficiently degrade isopropylamine.

[0051] Example 3 Determination of the anaerobic degradation ability of the anaerobic degradation strain SRB-2 on metolachlor

[0052] 1.0 mL of liquid inoculum (OD 600= 0.6) was added to 30 mL of deoxygenated MSM at pH 7.0, and then a certain amount of isopropylamine was added to make the final concentrations of 5 mg / L, 20 mg / L and 50 mg / L, and then placed in a shaker at 30 ℃ and 150 rpm for cultivation. Samples were taken at different time points to determine the concentration of isopropylamine, and the anaerobic degradation dynamics of isopropylamine were fitted to calculate its half-life. The blank group was without the anaerobic degradation strain SRB-2, and each group was repeated three times.

[0053] Table 1 The first-order kinetic parameters of anaerobic degradation of metolachlor at different concentrations by strain SRB-2

[0054]

[0055] The experimental results are shown in Table 1. The degradation dynamics of isopropylamine by strain SRB-2 conforms to the law of the first-order kinetic equation. With time as the horizontal axis and the residual concentration of isopropylamine as the vertical axis, the first-order kinetic equation ( C = C 0e -kt ) fitting, the correlation coefficient of the fitting equation (0.9294-0.9984) shows that the anaerobic degradation dynamics of anaerobic degradation strain SRB-2 on isopropylamine conforms to the law of first-order kinetics. The degradation half-life of anaerobic degradation strain SRB-2 on isopropylamine at concentrations of 5 mg / L, 20 mg / L, 28 mg / L and 50 mg / L ( T 1 / 2 = ln2 / k) were 0.53 d, 2.04 d, 2.95 d and 5.18 d respectively. The anaerobic degradation capacity of strain SRB-2 for isopropylamine (28 mg / L) was the highest among strains. P. sediminis BAD-10 T The anaerobic degradation strain SRB-2 screened and obtained by the present invention has a high degradation efficiency for isopropylamine and has a good application potential for the remediation of isopropylamine pollution.

[0056] Example 4 Application of anaerobic degradation strain SRB-2 to bioremediation of isopropylamine contaminated in industrial wastewater

[0057] Take sewage from the anaerobic treatment station of a sewage treatment plant in Nanjing, store it in a sealed plastic bottle and place it in a cool place. Take 1.0 L of sewage, add 0.6 g of FeSO4·7H2O and 1.0 mg of resazurin, and divide it into 50 mL vials, 30 mL in each bottle. Add a certain amount of isopropylamine to the vials containing 30 mL of sewage to make the concentrations reach 5 mg / L and 20 mg / L respectively, then seal it with a butyl rubber stopper and fix it with an aluminum cap, fill it with high-purity nitrogen for 30 minutes to remove all dissolved oxygen in the bottle. In the anaerobic operation box, add 1.0 mL of liquid inoculant (OD 2.0) of the anaerobic degradation strain SRB-2 to each bottle of sample. 600 = 0.6), the sewage samples were cultured in a shaker at 30 ℃ and 150 rpm, and the concentration of isopropylamine in the bottles was measured at different time points. The control group was without bacterial solution, and each group was repeated three times.

[0058] The results of bioremediation of isopropylamine in industrial wastewater by anaerobic degradation strain SRB-2 are as follows: Figure 5 As shown. In the blank group with a concentration of 5 mg / L of metolachlor, the anaerobic degradation rate of metolachlor was less than 8% during the experimental period (0-10 d); the treatment group with the addition of strain SRB-2 was able to completely degrade metolachlor within 10 d, and its anaerobic degradation half-life was 1.7 d. In the blank group with a concentration of 20 mg / L of metolachlor, the anaerobic degradation rate of metolachlor was less than 13.7% during the experimental period (0-14 d); the treatment group with the addition of strain SRB-2 was able to completely degrade metolachlor within 14 d, and its anaerobic degradation half-life was 3.2 d.

[0059] The above results show that the anaerobic degradation strain SRB-2 showed a good remediation effect on isopropylamine in sewage and has great application potential.

Claims

1. A strain SRB-2 for anaerobic degradation of the herbicide metolachlor, characterized in that: The strain SRB-2 is classified and named Trichococcus sp., deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 5, 2022, with the deposit number CGMCC No. 40302, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of the strain SRB-2 for anaerobic degradation of the herbicide metolachlor according to claim 1 in degradation of herbicide residues, characterized in that: The herbicide is isopropylamine.

3. Use of the strain SRB-2 for anaerobic degradation of the herbicide metolachlor according to claim 1 in repairing a natural environment contaminated by metolachlor.

4. The use according to claim 3, characterized in that: The natural environment is groundwater, surface water, flooded soil or wetland.

5. Use of the strain SRB-2 for anaerobic degradation of the herbicide metolachlor according to claim 1 in repairing wastewater treatment equipment contaminated by metolachlor.

6. The use according to claim 5, characterized in that: The wastewater treatment equipment is an anaerobic reactor.

7. A highly efficient anaerobic bacterial agent for degrading the herbicide isopropylamine, characterized in that: A strain SRB-2 containing the anaerobic herbicide degrading metolachlor as claimed in claim 1.

8. The bacterial agent according to claim 7, characterized in that The number of strain SRB-2 in the bacterial agent is 1.0 × 10 5 –1.0 ×10 8 cfu / mL.

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