A water microbe for degrading N-methylpyrrolidone and its application

Abstract

The present invention relates to the field of microorganisms and sewage treatment technology, and provides a water microbacterium for degrading N-methylpyrrolidone and its application. The strain is a water microbacterium (Aquamicrobium sp.) HZ-F-003, and the deposit number is CGMCC No.32176. The water microbacterium HZ-F-003 provided by the present invention can adapt to an environment of pH 5-9, can degrade 200-1000mg / L of N-methylpyrrolidone wastewater, and the 24h degradation rate is close to 100%. It can tolerate a salt concentration of 1wt%-4wt%, and has great application potential in the field of treating high-salt and high-concentration N-methylpyrrolidone wastewater.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and sewage treatment, and in particular to a water microbe that degrades N-methylpyrrolidone and an application thereof. Background Art

[0002] N-Methyl pyrrolidone (NMP) has good water miscibility, is soluble in most organic solvents such as ether, acetone, and can dissolve most organic compounds, inorganic compounds, polar gases, natural and synthetic polymer compounds. Because of its strong selectivity, good chemical stability, excellent thermal stability, high polarity, low corrosiveness and other characteristics, it is widely used in industries such as liquid crystal, semiconductor, circuit board, carbon nanotube, aramid fiber, PPS, ultrafiltration membrane, OLED panel photoresist etching, printing ink, medicine, medicine, veterinary drugs, pharmaceutical intermediates. Because of the good water-soluble characteristics of NMP, it is very easy to enter the environment along with the discharge of sewage, pollute water bodies, vegetation, soil etc., and then cause irreparable environmental problems, and simultaneously it has biological toxicity and teratogenicity, may have adverse effects on human reproductive system, respiratory tract, liver and kidney. Therefore, how to efficiently and quickly degrade N-Methyl pyrrolidone, eliminate the pollution of N-Methyl pyrrolidone to the environment, has become one of the hot issues in the field of environmental governance.

[0003] Currently, the main methods for treating N-methylpyrrolidone in wastewater include adsorption, membrane treatment, photocatalysis, iron-carbon microelectrolysis, and combustion treatment. However, these methods have disadvantages such as high cost, high energy consumption, and the tendency to cause secondary pollution. In contrast, biological treatment technology offers the advantages of low cost, low energy consumption, and high removal rates. It can achieve non-toxic and harmless environmental treatment without causing secondary pollution, making it the most widely used wastewater treatment technology. However, due to the toxicity and difficult-to-degrade nature of N-methylpyrrolidone, it is necessary to find bacterial strains that can tolerate its toxicity and degrade it.

[0004] Most existing strains capable of degrading N-methylpyrrolidone not only have slow degradation rates and long degradation cycles, but also require the addition of an additional carbon source, which increases the operating costs and time of sewage treatment plants in practical applications. Furthermore, given that some N-methylpyrrolidone-containing wastewaters have high salinity, high-salinity environments often have a strong inhibitory effect on microorganisms, resulting in their inability to degrade N-methylpyrrolidone and causing problems such as excessive effluent levels. Therefore, it is of great significance to obtain a strain that does not require the addition of an additional carbon source and can degrade N-methylpyrrolidone in high-salinity environments. Summary of the Invention

[0005] In light of this, the present invention has purified and screened a strain of water microbes that can efficiently degrade N-methylpyrrolidone in wastewater. This strain does not require the addition of an additional carbon source during the degradation process. The strain has excellent environmental adaptability and can tolerate high-salt environments, making it suitable for treating high-salt wastewater. By optimizing fermentation conditions, the present invention achieves high-density cultivation of the water microbes, which is then prepared into a bioinfective agent, achieving the goal of low-cost, high-efficiency, and pollution-free degradation of N-methylpyrrolidone in field applications.

[0006] The technical solution of the present invention is implemented as follows: In the first aspect, the present invention provides a water microbacterium for degrading N-methylpyrrolidone, wherein the strain is water microbacterium (Aquamicrobium sp.) HZ-F-003, which was deposited on October 14, 2024 at the General Microbiology Center of China Culture Collection Administration Committee, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101; Deposit Number CGMCC No. 32176.

[0007] In a second aspect, the present invention provides an application of water microbes for degrading N-methylpyrrolidone in treating N-methylpyrrolidone wastewater.

[0008] On the basis of the above technical solution, preferably, Aquamicrobium sp. HZ-F-003 is inoculated into wastewater containing N-methylpyrrolidone to degrade N-methylpyrrolidone.

[0009] On the basis of the above technical solution, preferably, the inoculation amount of Aquamicrobium sp. HZ-F-003 is 1% to 4%.

[0010] Based on the above technical solution, preferably, the pH value of the wastewater is 5-9.

[0011] On the basis of the above technical solution, preferably, the salt concentration of the wastewater is 1 wt%-4 wt%.

[0012] On the basis of the above technical solution, preferably, the concentration of N-methylpyrrolidone in the wastewater is 200-1000 mg / L.

[0013] In a third aspect, the present invention provides a bacterial agent for degrading N-methylpyrrolidone, comprising Aquamicrobium sp. HZ-F-003.

[0014] The water microbe for degrading N-methylpyrrolidone and its application in the present invention have the following beneficial effects compared with the prior art:

[0015] Currently known strains that can degrade N-methylpyrrolidone mainly include Paracoccus, Bacillus, Ochrobacter, etc., and there are no literature reports on water microbes that can degrade N-methylpyrrolidone.

[0016] The water microbe (Aquamicrobium sp.) HZ-F-003 provided by the present invention can adapt to an environment with a pH of 5-9, can degrade 200-1000 mg / L of N-methylpyrrolidone wastewater, and has a degradation rate of nearly 100% within 24 hours. It can also tolerate a salt concentration of 1wt%-4wt%, and has great application potential in the field of treating high-salt and high-concentration N-methylpyrrolidone wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a scanning electron microscope photo of water microbe HZ-F-003;

[0019] Figure 2 is the effect of initial pH value on strain HZ-F-003;

[0020] Figure 3 is the effect of inoculum size on strain HZ-F-003;

[0021] Figure 4 is the effect of initial N-methylpyrrolidone concentration on strain HZ-F-003;

[0022] Figure 5 is the effect of sodium chloride concentration on strain HZ-F-003;

[0023] Figure 6 This is the degradation curve of strain HZ-F-003 for N-methylpyrrolidone. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Example 1 Strain enrichment, separation and purification

[0026] Strain enrichment: 20 g of activated sludge from long-term N-methylpyrrolidone wastewater treatment was mixed with 300 mL of enrichment medium and placed in a 500 mL conical flask for enrichment. A sufficient amount of N-methylpyrrolidone (1000-2000 mg / L) was added and cultured in a shaking incubator at 30°C and 150 rpm. The N-methylpyrrolidone content in the supernatant was tested every two days. If the N-methylpyrrolidone had been completely degraded, the supernatant was replaced with fresh enrichment medium and the enrichment culture continued. This process was repeated 5-7 times, and the mixed enrichment was then isolated and purified.

[0027] Strain separation and purification: Take 1 mL of the mixed enrichment and perform gradient dilution. The dilution solution is the sterilized enrichment medium. The enrichment is diluted to 10 -1 ~10 -6 Six gradients were prepared. 100 μL of each gradient was spread onto solid enrichment medium containing 500 mg / L N-methylpyrrolidone. Five replicates were prepared for each gradient. Cultures were maintained at 30°C under microaerobic conditions for approximately 72 hours. Colonies that emerged from the plates were selected for streak purification. After four to five rounds of streak purification, strain HZ-F-003 was obtained. Pure colonies were inoculated onto slant medium and stored in a refrigerator at 4°C.

[0028] Activated medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, initial pH 7.2. Solid medium: add 2 wt% agar powder to the formula.

[0029] Enrichment culture medium (inorganic salt culture medium): KH2PO40.5g / L, K2HPO40.6g / L, MgSO40.06g / L, NaCl10g / L, CaCl20.08g / L, FeSO40.008g / L, MnSO40.008g / L, NH4Cl4g / L, initial pH 7.2.

[0030] After the culture medium is prepared, it must be sterilized in a high-pressure steam autoclave at 121°C for 30 minutes. After sterilization, N-methylpyrrolidone is added, and the amount added depends on the specific needs of the experiment.

[0031] Example 2 Strain Identification

[0032] (1) Strain morphology: The strain HZ-F-003 was streaked on a solid activated culture medium and cultured at 30°C under microaerobic conditions for 24-48 hours. The colony diameter was 0.5-1.5 mm, the colony was round, white, with a raised, moist, shiny surface, opaque, odorless, and with a complete and smooth edge. The strain was a Gram-negative bacterium. The microscopic morphology of the strain was observed by scanning electron microscopy, indicating that the strain was a bacillus, 4-6 μm long (see Figure 1 ).

[0033] (2) Molecular biological identification: 16S rRNA identification of the strain

[0034]

[0035] The 16S rRNA sequence was subjected to BLAST comparison, and the comparison results showed that the strain HZ-F-003 was Aquamicrobium defluvii.

[0036] (3) Physiological and biochemical testing of strains

[0037] Physiological and biochemical identification of the strain revealed a Gram-negative bacterium. It was oxidase and catalase positive, unable to liquefy gelatin, but able to utilize glucose, nitrate, malonic acid, and salicylic acid. It was unable to utilize lysine and arginine, but could utilize a small amount of carbohydrates as a carbon source. The indole production test was negative.

[0038] (4) Strain preservation

[0039] Strain HZ-F-003 is designated Aquamicrobium sp. HZ-F-003. It was deposited on October 14, 2024, at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, China; accession number: CGMCC No. 32176. The strain was found to be viable on October 14, 2024, as determined by the collection.

[0040] Example 3 Optimization of culture conditions for strain HZ-F-003

[0041] (1) Optimization of initial pH value

[0042] Activation of strain HZ-F-003: Streak strain HZ-F-003 onto a solid plate containing activation medium containing 500 mg / L N-methylpyrrolidone and incubate inverted at 30°C for 24 hours. Inoculate a single colony into enrichment medium and incubate at 30°C at 150 rpm for 48 hours.

[0043] The initial pH value of the inorganic salt culture medium was adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0 respectively, 500 mg / L N-methylpyrrolidone was added, and the above activated bacteria were inoculated into the culture medium at a ratio of 1%. The culture was placed in a shaker and cultured at 30°C and 150 r / min. Samples were taken after 24 hours and the OD was measured. 600 and N-methylpyrrolidone concentration, calculate the degradation rate of N-methylpyrrolidone, the result is as follows Figure 2 shown.

[0044] analyze Figure 2The data show that the strain showed good N-methylpyrrolidone degradation ability and growth in the initial pH range of 7.0-9.0. Among them, when the initial pH value was 8.0, the strain grew best and had the highest N-methylpyrrolidone degradation rate, indicating that 8.0 is the optimal pH value for the strain to degrade N-methylpyrrolidone. Figure 2 It can also be seen that the degradation of N-methylpyrrolidone and the growth OD of the strain 600 There is a positive correlation between the two groups, which indicates that the strain can utilize N-methylpyrrolidone for its own growth and reproduction.

[0045] (2) Optimization of inoculation volume

[0046] The activated strain HZ-F-003 was inoculated into an inorganic salt medium with an initial pH of 8.0 at a ratio of 0.5%, 1%, 2%, 3%, and 4%, and 500 mg / L of N-methylpyrrolidone was added. The culture was placed in a shaker at 30°C and 150 rpm, and samples were taken after 24 hours to measure the OD 600 and N-methylpyrrolidone concentration, calculate the degradation rate of N-methylpyrrolidone, the result is as follows Figure 3 shown.

[0047] analyze Figure 3 The data show that the degradation rate of N-methylpyrrolidone is close to 100% when the inoculation amount is 2%, 3% and 4%, among which the degradation rate is the highest when the inoculation amount is 3%. 600 It is also the highest, indicating that 3% is the optimal inoculum amount of the strain.

[0048] (3) Optimization of initial N-methylpyrrolidone concentration

[0049] The activated strain HZ-F-003 was inoculated into an inorganic salt medium with an initial pH of 8.0 at a ratio of 3%, and 200 mg / L, 500 mg / L, 800 mg / L, and 1000 mg / L of N-methylpyrrolidone were added respectively. The culture was placed in a shaker at 30°C and 150 r / min, and samples were taken after 24 hours to measure the OD 600 and N-methylpyrrolidone concentration, calculate the degradation rate of N-methylpyrrolidone, the result is as follows Figure 4 shown.

[0050] from Figure 4 As can be seen from the figure, with the increase of the initial concentration of N-methylpyrrolidone, the DO of the strain 600 The degradation rates of N-methylpyrrolidone and strain OD increased gradually. When the initial N-methylpyrrolidone concentration was 1000 mg / L, the 600 It can reach 1.73, which is significantly higher than other experimental groups. When the initial N-methylpyrrolidone concentration is 200 mg / L, the DO of the strain600 The degradation rates of N-methylpyrrolidone and pyrrolidone were inhibited to a certain extent. This may be because the growth and reproduction of the strain requires N-methylpyrrolidone to provide it with sufficient carbon and nitrogen sources to achieve a higher OD 600 and N-methylpyrrolidone degradation rates.

[0051] (4) Tolerance of strains to salt

[0052] The activated strains were inoculated into an inorganic salt medium with an initial pH of 8.0 at a ratio of 3%, and 1000 mg / L N-methylpyrrolidone was added. Different experimental groups were added with 0%, 1%, 2%, 3% and 4% sodium chloride, respectively. The culture was placed in a shaker at 30°C and 150 rpm. Samples were taken after 24 hours to measure the OD 600 and N-methylpyrrolidone concentration, calculate the degradation rate of N-methylpyrrolidone, the result is as follows Figure 5 shown.

[0053] from Figure 5 As can be seen from the figure, the strain showed good growth and N-methylpyrrolidone degradation rate in the sodium chloride concentration range of 1% to 3%. This shows that the strain can tolerate salt concentrations of 1% to 3% and can be used to treat high-salinity wastewater when used on site.

[0054] (5) Degradation curve of N-methylpyrrolidone

[0055] Based on the optimized initial concentration of N-methylpyrrolidone (1000 mg / L), the optimal pH value (8.0), and the optimal inoculum size (3%), the activated strain was inoculated into an inorganic salt medium. Cultured at 30°C, 150 rpm, and sampled after 24 hours to measure OD 600 and N-methylpyrrolidone concentrations, the results are as follows Figure 6 shown.

[0056] observe Figure 6 The data show that strain HZ-F-003 entered the logarithmic growth phase 8 hours after inoculation, with rapid bacterial growth and a significant increase in the N-methylpyrrolidone degradation rate. After 20 hours of culture, the strain gradually entered the stationary phase, and the N-methylpyrrolidone degradation rate stabilized. After 24 hours of culture, the N-methylpyrrolidone concentration dropped to 0 mg / L.

[0057] Example 4 Application of strains in treating high-salt and high-N-methylpyrrolidone wastewater

[0058] A high-salt and high-N-methylpyrrolidone wastewater from a sewage treatment plant was used for a simulation experiment on treating high-salt and high-N-methylpyrrolidone wastewater with the HZ-F-003 strain. The salt concentration in the wastewater from the sewage treatment plant reached 2.8%, and the N-methylpyrrolidone concentration reached 830.41 mg / L, which seriously exceeded the COD standard for sewage discharge.

[0059] In the experimental group, the activated strain HZ-F-003 was inoculated into the wastewater at an inoculum size of 3%, and cultured at 30°C and 150 r / min with shaking.

[0060] The control group did not add bacteria and was cultured at 30℃ and 150r / min.

[0061] Samples were taken at 0h and 24h to detect the N-methylpyrrolidone concentration and OD 600 and pH value changes, and the results are shown in Table 1.

[0062] Table 1 Application of strains in treating high-salt and high-N-methylpyrrolidone wastewater

[0063]

[0064] Analysis of the data in Table 1 indicates that after inoculating wastewater with a 3% concentration of the N-methylpyrrolidone-degrading strain HZ-F-003 and incubating with shaking for 24 hours, the N-methylpyrrolidone concentration in the wastewater dropped to 0.92 mg / L. The N-methylpyrrolidone degradation rate reached 99.89% within 24 hours. This indicates that the N-methylpyrrolidone-degrading strain HZ-F-003 can rapidly degrade high concentrations of N-methylpyrrolidone in high-salinity wastewater without the need for additional carbon and nitrogen sources. This demonstrates the significant potential of this strain in treating high-salinity, high-concentration N-methylpyrrolidone wastewater.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A water microbe for degrading N-methylpyrrolidone, characterized in that: The strain is Aquamicrobium sp. HZ-F-003, and its preservation number is CGMCC No.32176.

2. Use of a water microbe for degrading N-methylpyrrolidone according to claim 1 in treating N-methylpyrrolidone wastewater, characterized in that: Aquamicrobium sp. HZ-F-003 was inoculated into wastewater containing N-methylpyrrolidone to degrade N-methylpyrrolidone.

3. The use according to claim 2, characterized in that: The inoculation amount of the water microbacterium (Aquamicrobium sp.) HZ-F-003 is 1% to 4%.

4. The use according to claim 2, wherein: The pH value of the wastewater is 5-9.

5. The use according to claim 2, characterized in that: The salt concentration of the wastewater is 1 wt% to 4 wt%.

6. The use according to claim 2, characterized in that: The concentration of N-methylpyrrolidone in the wastewater is 200-1000 mg / L.

7. A bacterial agent for degrading N-methylpyrrolidone, characterized in that: The invention comprises the water microbacterium (Aquamicrobium sp.) HZ-F-003 described in claim 1.

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