Bacillus amyloliquefaciens for degrading n-methylpyrrolidone, and its cultivation method and application

Abstract

The present invention provides a kind of Bacillus amyloliquefaciens that degrades n-methyl pyrrolidone and its culture method and application, and the deposit number of the Bacillus amyloliquefaciens that degrades n-methyl pyrrolidone is CGMCC No.32488. The present invention adopts natural sedimentation method to collect degrading bacteria that can degrade n-methyl pyrrolidone in the air, which is simple to operate and has good removal effect, and is suitable for promotion and application in treating low-concentration NMP wastewater. The degrading bacteria can grow in LB culture medium containing n-methyl pyrrolidone and show good NMP tolerance. After the Bacillus amyloliquefaciens is accessed into the wastewater containing 100mg / Ln-methyl pyrrolidone with an inoculum size of 1%, it is shaken and cultured for 3 days at 30°C and 180r / min, and the complete degradation of 100mg / L NMP can be achieved, showing good degradation effect.

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-corrosive properties, and good water solubility, making it widely used in various fields. In the chemical industry, NMP is commonly used for dissolving and cleaning polymer materials, resins, paints, and coatings due to its ability to dissolve a wide range of organic and inorganic substances. In the oil and gas industry, NMP is widely used in aromatics separation, solvent deasphalting, sulfur compound removal, and oilfield development. Its excellent solubility, high selectivity, and low volatility make it a key solvent in petroleum refining, chemical processing, and oilfield extraction. In the pharmaceutical industry, NMP is used as a solvent for the extraction, separation, and dissolution of pharmaceuticals. For example, in the extraction of traditional Chinese medicine, NMP can be used as a solvent to extract active compounds from plants, such as flavonoids and steroids, ensuring efficient extraction. In the electronics industry, NMP is used as a cleaning agent for electronic components, particularly in printed circuit board (PCB) and semiconductor manufacturing processes, effectively removing dissolved impurities and residues. In battery manufacturing, especially lithium battery production, NMP is a key solvent for the preparation of 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, making it difficult to remove from 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 issue that needs to be addressed 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 good removal effects on NMP wastewater, their process conditions are complex, the treatment cost is high, and they are prone to secondary pollution. Therefore, there is an urgent need to explore simple, economical and efficient treatment methods.

[0004] Currently, biochemical methods hold significant advantages in water treatment due to their environmental friendliness, high efficiency, cost-effectiveness, sustainability, and adaptability. Research has shown that microorganisms can effectively degrade organic pollutants in wastewater, and biochemical methods are particularly effective for NMP degradation. However, the number of strains currently identified for the degradation of n-methylpyrrolidone is relatively limited. Therefore, isolating and screening strains that are highly effective in degrading NMP is crucial for treating NMP-containing wastewater. Summary of the Invention

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

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

[0007] A Bacillus amyloliquefaciens strain capable of degrading n-methylpyrrolidone is disclosed. The preservation number of the Bacillus amyloliquefaciens strain capable of degrading n-methylpyrrolidone is CGMCC No. 32488.

[0008] In a further improvement, the 16S rDNA sequence of the Bacillus amyloliquefaciens is shown as 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%; and 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 time is 12 h to 72 h.

[0013] A further improvement is that 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: Expose a culture dish containing LB liquid medium containing 2% n-methylpyrrolidone to air to collect airborne bacteria and fungi, and then close 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 from the culture dish in step 2 and add it to 100 ml of inorganic salt liquid culture medium containing 200 mg / L N-methylpyrrolidone. Shake and culture at 180 rpm and 30°C. Transfer 5% of the inoculum to fresh inorganic salt liquid culture medium every 24 hours for 5 times.

[0018] Step 4: Take 1.0 ml of the enriched bacterial solution obtained in step 3 and 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 and add it to 9.0 ml of sterile water. Mix thoroughly to prepare enriched solution 10-2. Repeat this process to prepare 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 enrichment solutions 10-1, 10-2, 10-3, and 10-4 were taken and spread onto 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 streak 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 180 rpm for 72 hours. Analyze the degradation effect of each strain on N-methylpyrrole by high performance liquid chromatography, 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] A further improvement is that 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 remainder is water, and then 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 water, and then sterilized in a high-temperature autoclave at 121° C. 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 sterilizing 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 the natural sedimentation method to collect the degrading bacteria of n-methylpyrrolidone in the air, which is simple to operate and has a good removal effect, and is suitable for promotion and application in treating low-concentration NMP wastewater.

[0028] 2. The Bacillus amyloliquefaciens bacteria of the present invention can grow in LB medium containing n-methylpyrrolidone and exhibits good NMP tolerance. When inoculated with a 1% inoculum of this bacteria into wastewater containing 100 mg / L n-methylpyrrolidone and cultured at 30°C and 180 rpm for three days, the bacteria completely degraded 100 mg / L of NMP, demonstrating excellent degradation results. The degrading bacteria of the present invention have important scientific significance for 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 four 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 influence of culture temperature on the degradation effect of Bacillus amyloliquefaciens.

[0035] Specific implementation methods

[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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be 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] Culture, identification and preservation of Bacillus amyloliquefaciens

[0043] (1) Enrichment and domestication of strains

[0044] A 200ml LB liquid culture medium dish containing 2% n-methylpyrrolidone was placed 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 exposed to the air for 24 hours to collect bacteria and fungi in the air. Subsequently, the culture dish was closed and transferred to a constant temperature incubator at 30°C for 48 hours. 5ml of bacterial liquid was taken from the above culture dish and added to 100ml of inorganic salt liquid culture medium containing 200mg / L N-methylpyrrolidone. The culture was shaken at 180r / min and 30°C. Every 24 hours, 5% of the inoculum was transferred to fresh inorganic salt culture medium, and the transfer was repeated 5 times.

[0045] The LB liquid medium was prepared by autoclaving at 121°C for 20 minutes using the following formula: 10.0 g / L peptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride. The inorganic salt medium was prepared by autoclaving at 121°C for 20 minutes using the following formula: 1.5 g / L K₂HPO₄, 1.0 g / L NaCl, 0.5 g / L KH₂PO₄, and 0.2 g / L MgSO₄.

[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 it 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 and purify and culture them by streaking separation method. After three consecutive purifications, four purified strains are obtained. The purified strains are added to 5.0 ml of inorganic salt culture medium for enrichment culture for 24 hours. Next, centrifuge at 12,000 r / min for 5 minutes, discard the supernatant, add 5.0 ml of 0.9% sodium chloride solution and shake to make a uniform bacterial suspension, then centrifuge again at 12,000 r / min for 5 minutes, and discard the supernatant. After washing twice in this way, add 0.9% sodium chloride solution and resuspend the bacteria after shaking for 1 minute. Finally, draw 1.0 ml of the resuspended bacterial solution and add it to 100 ml of inorganic salt liquid culture medium containing 200 mg / L of N-methylpyrrolidone. Incubate in a constant temperature oscillator at 30°C and 180 rpm 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 a slant culture medium, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. Molecular biological identification methods were used, and 16S rDNA universal primer 1492r was used for PCR amplification of 16S rDNA. PCR reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 57°C annealing for 30 seconds, 72°C extension for 90 seconds, and 72°C extension for 10 minutes after 30 cycles. The PCR product was subjected to 1.5% agarose gel electrophoresis. After agarose gel electrophoresis, the PCR product was recovered, purified, and sequenced. Based on the obtained 16S rDNA sequence, a homologous sequence was searched in GenBank by Blast and homologous sequence analysis and comparison were performed. The 16S rDNA sequence of Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0050] The slant culture medium formula is: 10.0 g / L NaCl, 10.0 g / L peptone, 5.0 g / L yeast extract powder, and 22.0 g / L agar. Weigh these ingredients proportionally and dissolve them in 100 mL of sterile water. Sterilize the mixture in an autoclave at 121°C for 20 minutes. When the temperature drops to 60°C, pour the culture medium into a sterilized test tube to the 1 / 3rd of its height. Seal the tube with a rubber stopper and place it on a 1 cm thick wooden strip. Allow to cool to obtain the slant culture medium.

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

[0052] (4) Preservation of strains

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

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

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

[0056] The four strains in Example 1 were inoculated at a 1% inoculum into an inorganic salt liquid culture medium containing 200 mg / L NMP. After 72 hours of culture in a constant temperature shaker at 30°C and 150 rpm, the residual NMP concentration in the culture medium was measured and the degradation rate was calculated. Degradation rate = (initial NMP mass concentration - post-reaction NMP mass concentration) / initial NMP mass concentration × 100%. The results are shown in Figure 2 All screened strains were able to degrade NMP after 72 hours. Strain 1 performed best, achieving an NMP degradation efficiency of 93.4% under aerobic conditions; strain 3 came in second with an efficiency of 82.3%. The remaining two strains had lower NMP degradation efficiencies. Strain 1, which showed 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 Bacillus amyloliquefaciens strain from Example 2 was inoculated at 1% in an inorganic salt liquid culture medium containing 100 mg / L NMP. The culture was carried out in a constant temperature shaker at 30°C and 150 rpm 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 require 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 culture period 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 Bacillus amyloliquefaciens from Example 2 was inoculated at a 1% inoculum into inorganic salt liquid culture media containing 100 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, and 1000 mg / L NMP, respectively. The cultures were incubated in a constant temperature shaker at 30°C and 150 rpm for 72 hours. The residual NMP concentration in the culture medium was measured, and the degradation rate was calculated. The results are shown in Table 2. Figure 4 When the NMP mass concentration does not exceed 600mg / L, Bacillus amyloliquefaciens has a significant degradation effect, with a degradation rate of 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 NMP degradation 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 also promote the degradation process. However, if the NMP concentration is too high, it may cause the growth of Bacillus amyloliquefaciens to be restricted, the growth rate to 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 Bacillus amyloliquefaciens strain from Example 2 was inoculated into an inorganic salt liquid culture medium containing 100 mg / L NMP at inoculum concentrations of 0.1%, 0.5%, 1%, 2%, 3%, 4%, and 5%. The culture was incubated in a constant temperature shaker at 30°C and 150 rpm for 72 hours. The residual NMP concentration in the culture medium was measured and the degradation rate was calculated. The results are shown in Table 1. 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 nutrients 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 strain from Example 2 was inoculated at 1% inoculum into an inorganic salt liquid culture medium containing 100 mg / L NMP. The culture was incubated in a constant temperature shaker at 27.5-37.5°C and 150 rpm for 72 hours. The residual NMP concentration in the culture medium was measured and the degradation efficiency was calculated. Figure 6 Within the culture temperature range of 27.5-30°C, the NMP degradation efficiency of Bacillus amyloliquefaciens increased with increasing temperature, indicating that warming in this temperature range is beneficial for strain growth, thereby accelerating NMP degradation. At 30°C, the degradation efficiency reached its highest level, with NMP completely degraded, indicating that 30°C is the optimal growth temperature for Bacillus amyloliquefaciens. The strain's NMP degradation efficiency remained good at a culture temperature of 30-32.5°C. However, the degradation rate dropped sharply when the temperature rose to 35-37.5°C, indicating that this temperature range inactivates the strain and is not conducive to its growth and NMP degradation. The experimental results show that 27.5-32.5°C is the ideal temperature range for the cultivation of Bacillus amyloliquefaciens, and the strain exhibits excellent degradation performance within this range, with 30°C being the optimal culture temperature.

[0065] The above is only a specific guide implementation method 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 strain 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, wherein 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 is used for degrading n-methylpyrrolidone.

4. The use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 3, wherein 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 100 mg / L-1000 mg / L.

5. The use of Bacillus amyloliquefaciens for degrading n-methylpyrrolidone according to claim 4, characterized in that: When the concentration of n-methylpyrrolidone is 100 mg / L, the inoculum size of Bacillus amyloliquefaciens is 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 time 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~32.5°C.

Images