Bacillus amyloliquefaciens for degrading n-methyl pyrrolidone as well as culture method and application of bacillus amyloliquefaciens

Abstract

The invention provides bacillus amyloliquefaciens for degrading n-methyl pyrrolidone as well as a culture method and application of the bacillus amyloliquefaciens. The preservation number of the bacillus amyloliquefaciens for degrading the n-methyl pyrrolidone is CGMCC (China General Microbiological Culture Collection Center) No.32488. According to the method, the degrading bacteria capable of degrading the n-methyl pyrrolidone in the air are collected by adopting a natural sedimentation method, the operation is simple, the removal effect is good, and the method is suitable for being popularized and applied to treatment of low-concentration NMP wastewater. The degrading bacterium can grow in an LB culture medium containing n-methyl pyrrolidone, and shows good NMP (N-Methyl Pyrrolidone) tolerance. After the bacillus amyloliquefaciens is inoculated into wastewater containing 100 mg / L of n-methyl pyrrolidone according to the inoculum size of 1%, shaking culture is carried out for 3 days under the conditions of 30 DEG C and 180 r / min, complete degradation of 100 mg / L of NMP can be achieved, and a good degradation effect is shown.

Description

Technical Field

[0001] The invention is used in the field of biotechnology and mainly relates to Bacillus amyloliquefaciens for degrading n-methylpyrrolidone and a culture method and application thereof. Background Art

[0002] N-methylpyrrolidone (NMP) is an excellent organic solvent with low volatility, high polarity, non-corrosiveness and good water solubility. It is widely used in many fields. In the chemical industry, NMP is often used for dissolving and cleaning polymer materials, resins, coatings and coatings because it can dissolve a variety of organic and inorganic substances; in the oil and gas industry, NMP is widely used in aromatic separation, solvent deasphalting, sulfur compound removal and oil field development. Its excellent solubility, high selectivity and low volatility make it a key solvent in petroleum refining, chemical processing and oil field exploitation; in the pharmaceutical industry, NMP is used as a solvent for drugs for extraction, separation and dissolution of drugs. For example, in the extraction process of traditional Chinese medicine, NMP can be used as a solvent to extract active compounds in plants, such as flavonoids, steroid compounds, etc., to ensure extraction efficiency. In the electronics industry, NMP is used as an electronic component cleaning agent, especially in the printed circuit board (PCB) and semiconductor manufacturing process, and can efficiently remove dissolved impurities and residues. In battery manufacturing, especially lithium battery production, NMP is one of the key solvents for preparing electrodes and conductive materials.

[0003] It can be seen that NMP is widely used as a solvent in most industrial production processes. Due to its good water solubility, it is easily discharged into the environment with wastewater. However, n-methylpyrrolidone has stable chemical properties and poor biodegradability, and is difficult to remove in the environment. Its physiological toxicity is significant. Once contaminated, it may cause long-term ecological damage and health problems. Therefore, treating NMP-containing wastewater and ensuring that it meets emission standards has become a major environmental problem that needs to be solved urgently. The main methods for treating NMP wastewater at home and abroad include physical and chemical methods, such as activated carbon adsorption, reverse osmosis ultrafiltration technology, Fenton oxidation, ozone oxidation, hydrogen peroxide oxidation and photocatalytic oxidation. Although these methods have a good removal effect on NMP wastewater, their process conditions are complex, the treatment cost is high, and secondary pollution is easily generated. Therefore, it is urgent to explore simple, economical and efficient treatment methods.

[0004] At present, biochemical methods have significant advantages in water treatment processes due to their environmental protection, high efficiency, economy, sustainability and strong adaptability. Studies have shown that microorganisms can effectively degrade organic pollutants in wastewater, and biochemical methods have a good effect on the degradation of NMP, but the strains that have been screened for the degradation of n-methylpyrrolidone are still relatively limited. Therefore, isolating and screening strains that can efficiently degrade NMP is of great significance for the treatment of wastewater containing NMP. Summary of the invention

[0005] In order to solve the above problems, the present invention discloses a Bacillus amyloliquefaciens for degrading n-methylpyrrolidone and a culture method and application thereof.

[0006] In order to achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] A bacillus amyloliquefaciens capable of degrading n-methylpyrrolidone, wherein the preservation number of the bacillus amyloliquefaciens capable of degrading n-methylpyrrolidone is CGMCC No.32488.

[0008] In a further improvement, the 16S rDNA sequence of the Bacillus amyloliquefaciens is as shown in SEQ ID NO.1.

[0009] A use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone. The Bacillus amyloliquefaciens is as described above, and the Bacillus amyloliquefaciens is used for degrading n-methylpyrrolidone.

[0010] As a further improvement, when the Bacillus amyloliquefaciens is used to degrade n-methylpyrrolidone, the inoculation amount is 0.1-5%; the concentration of n-methylpyrrolidone is 100 mg / L-1000 mg / L.

[0011] As a further improvement, when the concentration of n-methylpyrrolidone is 100 mg / L, the inoculation amount of Bacillus amyloliquefaciens is 1%.

[0012] As a further improvement, when the Bacillus amyloliquefaciens is used to degrade n-methylpyrrolidone, the temperature is 27.5° C. to 37.5° C. and the duration is 12 h to 72 h.

[0013] In a further improvement, the temperature is 30°C to 32.5°C.

[0014] A method for culturing Bacillus amyloliquefaciens for degrading n-methylpyrrolidone comprises the following steps:

[0015] Step 1: exposing a culture dish containing LB liquid culture medium containing 2% n-methylpyrrolidone to air to collect airborne bacteria and fungi, and then closing the culture dish;

[0016] Step 2: Transfer the culture dish to a constant temperature incubator at 30°C and culture for 48 hours;

[0017] Step 3: Take the bacterial solution in the culture dish of step 2, add it to 100 ml of inorganic salt liquid culture medium with N-methylpyrrolidone concentration of 200 mg / L, shake culture at 180 r / min and 30°C, transfer 5% of the inoculum to fresh inorganic salt culture medium every 24 hours, and transfer it 5 times continuously;

[0018] Step 4: Take 1.0 ml of the enriched bacterial solution obtained in step 3, add it to 9.0 ml of sterile water to prepare enriched solution 10-1, then take 1.0 ml of enriched solution 10-1, add it to 9.0 ml of sterile water, mix well, and prepare enriched solution 10-2. Similarly, the enriched solution is gradiently diluted to form enriched solution 10-1, enriched solution 10-2, enriched solution 10-3 and enriched solution 10-4 in sequence;

[0019] Step 5: 0.1 ml of each of the enriched solution 10-1, the enriched solution 10-2, the enriched solution 10-3 and the enriched solution 10-4 was taken and spread on LB solid medium containing 300 mg / L N-methylpyrrolidone, and cultured in a constant temperature incubator at 30° C. for 24 hours;

[0020] Step 6: Select a single colony with obvious differences on the LB solid medium in step 5, purify and culture it by streaking separation, and obtain a purified strain after 3 consecutive purifications;

[0021] Step 7: Transfer the purified strain to an inorganic salt culture medium containing a preset concentration of N-methylpyrrolidone, and culture it in a constant temperature oscillator at 30°C and 180rpm for 72 hours. Analyze the degradation effect of each strain on N-methylpyrrole by HPLC, and select the strain with the highest degradation efficiency for identification and preservation.

[0022] As a further improvement, the deposit number of the bacterial strain with the highest degradation efficiency is CGMCC No.32488.

[0023] As a further improvement, the LB liquid culture medium has a formula of: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 10.0 g / L sodium chloride, and the rest is water, and then is sterilized in a high temperature autoclave at 121° C. for 20 minutes;

[0024] The LB solid culture medium is formulated as follows: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 10.0 g / L sodium chloride, 22.0 g / L agar, and the remainder is water, and then sterilized in a 121° C. high-temperature autoclave for 20 minutes.

[0025] The formula of the inorganic salt culture medium is: K2HPO4 1.5 g / L, NaCl 1.0 g / L, KH2PO4 0.5 g / L, MgSO4 0.2 g / L, and the rest is water. It is prepared by sterilization in a high-temperature sterilizer at 121°C for 20 minutes.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] 1. The present invention adopts a natural sedimentation method to collect degrading bacteria that can degrade n-methylpyrrolidone in the air. The operation is simple and the removal effect is good. It is suitable for promotion and application in treating low-concentration NMP wastewater.

[0028] 2. The Bacillus amyloliquefaciens in the present invention can grow in LB medium containing n-methylpyrrolidone and show good NMP tolerance. After the Bacillus amyloliquefaciens was inoculated with 1% of the wastewater containing 100 mg / L n-methylpyrrolidone, it was shaken and cultured for 3 days at 30°C and 180r / min, and the complete degradation of 100 mg / L NMP was achieved, showing a good degradation effect. The degradation bacteria of the present invention have important scientific significance for the research on biochemical treatment of NMP. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Schematic diagram of Bacillus amyloliquefaciens colony;

[0030] Figure 2 :Schematic diagram of the evaluation of the NMP degradation effect of 4 bacterial strains;

[0031] Figure 3 :Schematic diagram of the effect of culture time on the degradation effect of Bacillus amyloliquefaciens;

[0032] Figure 4 : Schematic diagram of the effect of NMP concentration on the degradation effect of Bacillus amyloliquefaciens;

[0033] Figure 5 :Schematic diagram of the effect of inoculation amount on the degradation effect of Bacillus amyloliquefaciens;

[0034] Figure 6 : Schematic diagram of the effect of culture temperature on the degradation effect of Bacillus amyloliquefaciens.

[0035] Specific implementation method

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] Cultivation, identification and preservation of Bacillus amyloliquefaciens

[0043] (1) Enrichment and domestication of strains

[0044] Place a 200ml LB liquid culture medium dish containing 2% n-methylpyrrolidone in the laboratory on the 4th floor of Building 1, Guizhou Science City, Baiyun District, Guiyang City, Guizhou Province, 1.5 meters above the ground, and expose it to the air for 24 hours to collect bacteria and fungi in the air. Subsequently, close the culture dish and transfer it to a constant temperature incubator at 30°C for 48 hours. Take 5ml of bacterial liquid from the above culture dish, add it to 100ml of inorganic salt liquid culture medium containing 200mg / L N-methylpyrrolidone, and shake and culture at 180r / min and 30°C. Transfer to fresh inorganic salt culture medium at a 5% inoculum every 24 hours, and transfer it 5 times in a row.

[0045] The formula of LB liquid medium is: peptone 10.0g / L, yeast extract powder 5.0g / L, sodium chloride 10.0g / L, which is sterilized in a 121℃ high temperature autoclave for 20 minutes. The formula of inorganic salt medium is: K2HPO4 1.5g / L, NaCl 1.0g / L, KH2PO4 0.5g / L, MgSO4 0.2g / L, which is sterilized in a 121℃ high temperature autoclave for 20 minutes.

[0046] (2) Isolation and screening of strains

[0047] Take 1.0 ml of the enriched bacterial solution obtained in (1) and add it to 9.0 ml of sterile water to prepare a 10-1 enriched solution. Then, take 1.0 ml of the prepared 10-1 enriched solution and add it to 9.0 ml of sterile water, mix thoroughly, and prepare a 10-2 enriched solution. Similarly, the enriched solution is gradient diluted to four different gradients from 10-1 to 10-4. Take 0.1 ml of the dilution of each gradient and spread it on LB solid culture medium containing 300 mg / L N-methylpyrrolidone, and culture it in a constant temperature incubator at 30°C for 24 hours. Select single colonies with obvious differences on the culture medium, purify and culture them by streaking separation method, and obtain 4 purified strains after 3 consecutive purifications. Add the purified strains to 5.0 ml of inorganic salt culture medium for enrichment culture for 24 hours. Next, centrifuge at 12000r / min for 5 minutes, then pour off the supernatant, add 5.0ml of 0.9% sodium chloride solution and shake well to make a uniform bacterial suspension, then centrifuge at 12000r / min for 5 minutes again, then pour off the supernatant. After washing twice according to this method, add 0.9% sodium chloride solution, shake for 1 minute and resuspend the bacteria. Finally, draw 1.0ml of the resuspended bacterial solution and add it to 100ml of inorganic salt liquid culture medium containing N-methylpyrrolidone at a concentration of 200mg / L, and culture it at 30°C and 180rpm in a constant temperature oscillator for 72 hours. The degradation effect of different strains on N-methylpyrrolidone is analyzed by high performance liquid chromatography, and the strain with the highest degradation efficiency is selected for identification, preservation and subsequent experiments.

[0048] (3) Identification of strains

[0049] After the strain obtained in (2) was inoculated into the slant culture medium, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. The molecular biological identification method was used, and the 16S rDNA universal primer 1492r was used for PCR amplification of 16S rDNA. PCR reaction conditions: 95°C pre-denaturation for 5min; 94°C denaturation for 30s, 57°C annealing for 30s, 72°C extension for 90s, and 72°C extension for 10min after 30 cycles. The PCR product was subjected to 1.5% agarose gel electrophoresis, and the PCR product was recovered, purified and sequenced after agarose gel electrophoresis. Based on the obtained 16S rDNA sequence, a homologous sequence was searched in GenBank by Blast and a homologous sequence analysis and comparison were performed. The 16S rDNA sequence of Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0050] The formula of slant culture medium is: 10.0g / L NaCl, 10.0g / L peptone, 5.0g / L yeast extract powder, 22.0g / L agar. Weigh these raw materials in proportion and mix and dissolve with 100mL sterile water. Sterilize the mixture in an autoclave at 121℃ for 20 minutes; when the temperature drops to 60℃, pour the culture medium into the sterilized test tube at 1 / 3 of the height, then seal it with a rubber stopper and place it on a 1 cm thick wooden strip. After cooling, the slant culture medium is obtained.

[0051] The 16S rDNA gene sequence comparison analysis showed that the strain belonged to the genus Bacillus, and the sequence alignment identity (Per.Ident) with Bacillus amyloliquefaciens reached 100.0%.

[0052] (4) Preservation of strains

[0053] After the obtained strain was inoculated into the slant culture medium, it was sent to the General Microbiology Center of China Microorganism Culture Collection Administration for preservation, with the preservation number CGMCC No.32488, classification name: Bacillus amyloliquefaciens, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation time is November 4, 2024.

[0054] like Figure 1 As shown, the strain obtained in Example 1 is rod-shaped on LB solid medium, and the colonies are opaque and round with neat edges and light yellow. The strain has oval spores, the spores are located from the mesogenetic to the subterminal, and the spore capsules are not swollen.

[0055] Example 2 Effect of different strains on NMP degradation

[0056] The four strains in Example 1 were inoculated into an inorganic salt liquid culture medium containing 200 mg / L NMP at an inoculum of 1%. After culturing in a constant temperature oscillator at 30°C and 150 r / min for 72 hours, the residual NMP concentration in the culture medium was measured and the degradation rate was calculated. Degradation rate = (initial NMP mass concentration - NMP mass concentration after reaction) / initial NMP mass concentration × 100%. The results are shown in Figure 2 All the strains screened were able to degrade NMP after 72 hours. Strain 1 performed best, with a degradation efficiency of 93.4% under aerobic conditions, followed by strain 3, with a degradation efficiency of 82.3%. The other two strains had lower NMP degradation efficiencies. Strain 1 with the highest degradation efficiency was selected for identification, preservation and further research.

[0057] Example 3 Effect of culture time on the degradation effect of Bacillus amyloliquefaciens

[0058] The amyloliquefaciens strain in Example 2 was inoculated into an inorganic salt liquid medium containing 100 mg / L NMP at a 1% inoculum. The culture was carried out in a constant temperature oscillator at 30°C and 150 r / min for 72 hours. Samples were taken every 12 hours and measured. The results are shown in Table 1. Figure 3 . The Bacillus amyloliquefaciens can completely degrade 100 mg / L of NMP within 72 hours. Microorganisms need a certain adaptation period when they initially come into contact with organic solvents. Therefore, within the first 24 hours, the degradation efficiency of the strain is low and the NMP content does not change much. After a period of adaptation, the microorganisms in the strain enter the logarithmic growth phase by changing metabolic pathways and regulating enzyme expression. During this stage, the microorganisms grow and divide rapidly, the synthesis and metabolic activities of enzymes reach a peak, and the degradation ability is significantly improved. Therefore, after 24 hours, the degradation efficiency of the strain is improved, and the NMP content quickly drops to zero. The experimental results show that Bacillus amyloliquefaciens requires a longer period of culture to gradually enhance its degradation ability, and a culture time of 72 hours can enable the strain to achieve the best degradation performance.

[0059] Example 4 Effect of NMP concentration on the degradation effect of Bacillus amyloliquefaciens

[0060] The amyloliquefaciens strain in Example 2 was inoculated at 1% in an inorganic salt liquid culture medium containing 100 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L and 1000 mg / L NMP. The culture was carried out in a constant temperature oscillator at 30°C and 150 r / min for 72 hours, the residual NMP concentration in the culture medium was measured, and the degradation rate was calculated. The results are shown in Figure 4 , Bacillus amyloliquefaciens has a significant degradation effect when the NMP mass concentration does not exceed 600mg / L, and the degradation rate can reach more than 90%. When the NMP concentration is 100mg / L, Bacillus amyloliquefaciens can completely degrade NMP. When the NMP mass concentration exceeds 600mg / L, the effect of Bacillus amyloliquefaciens on degrading NMP is significantly reduced, and when the NMP concentration reaches 1000mg / L, the degradation rate is only 59.4%. An appropriate amount of NMP contributes to the growth and activity of Bacillus amyloliquefaciens. Within a certain concentration range, Bacillus amyloliquefaciens can effectively utilize these organic substrates for metabolism and produce active intermediates, which sometimes promote the degradation process. However, if the NMP concentration is too high, the growth of Bacillus amyloliquefaciens may be restricted, the growth rate may slow down, or even enter an inhibitory state, thereby reducing its degradation ability.

[0061] Example 5 Effect of inoculum size on the degradation effect of Bacillus amyloliquefaciens

[0062] The amyloliquefaciens strain in Example 2 was inoculated into an inorganic salt liquid culture medium containing 100 mg / L NMP at an inoculum of 0.1%, 0.5%, 1%, 2%, 3%, 4%, and 5%. The culture was carried out in a constant temperature oscillator at 30°C and 150 r / min for 72 hours, and the residual NMP concentration in the culture medium was measured and the degradation rate was calculated. The results are shown in Figure 5 When the inoculation size is less than 1%, the degradation efficiency of NMP by Bacillus amyloliquefaciens increases with the increase of inoculation size. When the inoculation size is 1%, Bacillus amyloliquefaciens can completely degrade 100 mg / L of NMP. As the inoculation size increases to 5%, the removal effect of NMP by Bacillus amyloliquefaciens decreases slightly. When the inoculation size is low, the number of strains is small and the ability to degrade NMP is limited, resulting in low degradation efficiency. The degradation efficiency is low. After increasing the inoculation size, the number of strains increases, and the degradation efficiency of NMP increases accordingly. As the inoculation size increases further, due to limited nutrition in the culture medium, competition occurs between strains, which inhibits the growth of the strains, resulting in a decrease in their ability to degrade NMP.

[0063] Example 6 Effect of culture temperature on the degradation effect of Bacillus amyloliquefaciens

[0064] The Bacillus amyloliquefaciens in Example 2 was inoculated into an inorganic salt liquid culture medium containing 100 mg / L NMP at an inoculum of 1%, and cultured in a constant temperature oscillator at 27.5-37.5°C and 150 r / min for 72 hours. The residual NMP concentration in the culture medium was measured and the degradation efficiency was calculated. The results are shown in Figure 6 In the culture temperature range of 27.5-30℃, the degradation efficiency of Bacillus amyloliquefaciens on NMP increases with the increase of temperature, indicating that the temperature rise in this temperature range is conducive to the growth of the strain, thereby accelerating the degradation of NMP; when the temperature reaches 30℃, the degradation efficiency reaches the highest, and NMP can be completely degraded, indicating that 30℃ is the optimal growth temperature of Bacillus amyloliquefaciens; at the culture temperature of 30-32.5℃, the strain still has a good degradation effect on NMP; but when the temperature rises to 35-37.5℃, the degradation rate drops sharply, indicating that this temperature range causes the strain to inactivate, which is not conducive to its growth and degradation of NMP. The experimental results show that 27.5-32.5℃ is a temperature range suitable for the cultivation of Bacillus amyloliquefaciens, and the strain has good degradation performance within this range, among which 30℃ is the optimal culture temperature.

[0065] The above is only a specific guiding implementation mode of the present invention, but the design concept of the present invention is not limited to this. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. A Bacillus amyloliquefaciens for degrading n-methylpyrrolidone, characterized in that: The deposit number of the Bacillus amyloliquefaciens that degrades n-methylpyrrolidone is CGMCC No.32488.

2. The Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 1, characterized in that: The 16S rDNA sequence of the Bacillus amyloliquefaciens is shown as SEQ ID NO.

1.

3. A use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone, characterized in that: The Bacillus amyloliquefaciens is as claimed in claim 1 or 2, and the Bacillus amyloliquefaciens is used for degrading n-methylpyrrolidone.

4. The use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 3, characterized in that: When the bacillus amyloliquefaciens is used to degrade n-methyl pyrrolidone, the inoculation amount is 0.1-5%; the concentration of n-methyl pyrrolidone is 100mg / L-1000mg / L.

5. The use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 4, characterized in that: When the concentration of n-methylpyrrolidone was 100 mg / L, the inoculation amount of Bacillus amyloliquefaciens was 1%.

6. The use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 3, characterized in that: When the Bacillus amyloliquefaciens is used to degrade n-methylpyrrolidone, the temperature is 27.5° C. to 37.5° C. and the duration is 12 h to 72 h.

7. The use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 6, characterized in that: The temperature is 30°C to 32.5°C.

8. A method for culturing Bacillus amyloliquefaciens for degrading n-methylpyrrolidone, characterized in that: The steps include: Step 1: exposing a culture dish containing LB liquid culture medium containing 2% n-methylpyrrolidone to air to collect airborne bacteria and fungi, and then closing the culture dish; Step 2: Transfer the culture dish to a constant temperature incubator at 30°C and culture for 48 hours; Step 3: Take the bacterial solution in the culture dish of step 2, add it to 100 ml of inorganic salt liquid culture medium with N-methylpyrrolidone concentration of 200 mg / L, shake culture at 180 r / min and 30°C, transfer 5% of the inoculum to fresh inorganic salt culture medium every 24 hours, and transfer it 5 times continuously; Step 4: Take 1.0 ml of the enriched bacterial solution obtained in step 3, add it to 9.0 ml of sterile water to prepare enriched solution 10-1, then take 1.0 ml of enriched solution 10-1, add it to 9.0 ml of sterile water, mix well, and prepare enriched solution 10-2. Similarly, the enriched solution is gradiently diluted to form enriched solution 10-1, enriched solution 10-2, enriched solution 10-3 and enriched solution 10-4 in sequence; Step 5: 0.1 ml of each of the enriched solution 10-1, the enriched solution 10-2, the enriched solution 10-3 and the enriched solution 10-4 was taken and spread on LB solid medium containing 300 mg / L N-methylpyrrolidone, and cultured in a constant temperature incubator at 30° C. for 24 hours; Step 6: Select the single colony with obvious differences on the LB solid medium in step 5, purify and culture it by streaking separation method, and obtain the purified strain after 3 consecutive purifications; Step 7: Transfer the purified strain to an inorganic salt culture medium containing a preset concentration of N-methylpyrrolidone, and culture it in a constant temperature oscillator at 30°C and 180rpm for 72 hours. Analyze the degradation effect of each strain on N-methylpyrrole by HPLC, and select the strain with the highest degradation efficiency for identification and preservation.

9. The method for culturing Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 8, characterized in that: The deposit number of the bacterial strain with the highest degradation efficiency is CGMCC No.32488.

10. The method for culturing Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 8, characterized in that: The LB liquid culture medium is formulated as follows: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 10.0 g / L sodium chloride, and the remainder water, and then sterilized in a 121° C. high-temperature autoclave for 20 minutes. The LB solid culture medium is formulated as follows: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 10.0 g / L sodium chloride, 22.0 g / L agar, and the remainder is water, and then sterilized in a 121° C. high-temperature autoclave for 20 minutes. The formula of the inorganic salt culture medium is: K2HPO4 1.5 g / L, NaCl 1.0 g / L, KH2PO4 0.5 g / L, MgSO4 0.2 g / L, and the rest is water. It is prepared by sterilization in a high-temperature sterilizer at 121°C for 20 minutes.

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