Aquamicrobe for degrading N-methyl pyrrolidone and application thereof

Abstract

The invention relates to the technical field of microorganisms and sewage treatment, and provides an aquatic microbacterium for degrading N-methyl pyrrolidone and application of the aquatic microbacterium. The bacterial strain is Aquamicrobe sp. HZ-F-003, and the preservation number of the bacterial strain is CGMCC (China General Microbiological Culture Collection Center) No. 32176. The aquatic microbe HZ-F-003 provided by the invention can adapt to an environment with the pH value of 5-9, can degrade 200-1000mg / L of N-methyl pyrrolidone wastewater, has the 24h degradation rate close to 100%, can tolerate the salt concentration of 1-4wt%, and has great application potential in the field of treatment of high-salt and high-concentration N-methyl pyrrolidone wastewater.

Description

Technical Field

[0001] The 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-methylpyrrolidone (NMP) has good water miscibility, is soluble in most organic solvents such as ether and 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, semiconductors, circuit boards, carbon nanotubes, aramid fibers, PPS, ultrafiltration membranes, OLED panel photoresist etching, inks, medicine, medicine, veterinary drugs, and pharmaceutical intermediates. Because of the good water solubility of NMP, it is very easy to enter the environment with the discharge of sewage, causing pollution to water bodies, vegetation, soil, etc., and then causing irreparable environmental problems. At the same time, it has biological toxicity and teratogenicity, and may have adverse effects on the reproductive system, respiratory tract, liver and kidneys of the human body. Therefore, how to efficiently and quickly degrade N-methylpyrrolidone and eliminate the pollution of N-methylpyrrolidone to the environment has become one of the hot issues in the field of environmental governance.

[0003] At present, the main methods for treating N-methylpyrrolidone in wastewater include adsorption, membrane treatment, photocatalysis, iron-carbon micro-electrolysis, combustion treatment, etc., but they have the disadvantages of high cost, high energy consumption, and easy to cause secondary pollution. In contrast, biological treatment technology has the advantages of low cost, low energy consumption, high removal rate, etc. It can achieve non-toxic and harmless environmental governance and will not cause secondary pollution. It is currently the most widely used wastewater treatment technology. However, due to the toxicity and difficult-to-degrade characteristics of N-methylpyrrolidone, it is necessary to find strains that can tolerate the toxicity of N-methylpyrrolidone and can degrade it.

[0004] Most of the existing strains that can degrade N-methylpyrrolidone not only have a slow degradation rate and a long cycle, but also require additional carbon sources, which increases the operating cost and time of sewage treatment plants in actual application. At the same time, considering that some N-methylpyrrolidone wastewater contains high salinity, high-salt environments usually have a strong inhibitory effect on microorganisms, resulting in the inability of microorganisms to degrade N-methylpyrrolidone, causing problems such as excessive effluent content. Therefore, it is of great significance to obtain a strain that does not require additional carbon sources and can degrade N-methylpyrrolidone in a high-salt environment. Summary of the invention

[0005] In view of this, the present invention purifies and screens a water microbe that can efficiently degrade N-methylpyrrolidone in sewage, and no additional carbon source needs to be added during the degradation process. The strain has good environmental adaptability, can tolerate high-salt environments, and is suitable for the treatment of high-salt wastewater. The present invention optimizes fermentation conditions to achieve high-density cultivation of water microbes, and then prepares a biological bacterial agent to achieve the goal of being able to degrade N-methylpyrrolidone at low cost, high efficiency, and pollution-free when applied on site.

[0006] The technical solution of the present invention is implemented as follows: In the first aspect, the present invention provides an aquamicrobium for degrading N-methylpyrrolidone, wherein the strain is Aquamicrobium sp. HZ-F-003, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 14, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101; Collection 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 scheme, preferably, water microbes (Aquamicrobium sp.) HZ-F-003 are 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 1wt%-4wt%.

[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, including Aquamicrobium sp. HZ-F-003.

[0014] The water microbes for degrading N-methylpyrrolidone and the application thereof of 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, Ochrobacterium, etc. There is no literature report on water microorganisms that can degrade N-methylpyrrolidone.

[0016] The water microbe (Aquamicrobium sp.) HZ-F-003 provided by the present invention can adapt to an environment of pH 5-9, can degrade 200-1000 mg / L of N-methylpyrrolidone wastewater, the 24h degradation rate is close to 100%, and 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. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

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

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

[0020] Figure 3 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 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 described embodiments 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 creative work are within the scope of protection of the present invention.

[0025] Example 1 Strain enrichment, separation and purification

[0026] Strain enrichment: Take 20g of activated sludge from long-term treatment of N-methylpyrrolidone wastewater, mix it with 300mL of enrichment medium, and put it into a 500mL conical flask for enrichment. In addition, add sufficient amount of N-methylpyrrolidone (1000-2000mg / L), and culture it in a shaking table at 30℃ and 150r / min. Detect the content of N-methylpyrrolidone in the supernatant every 2 days. If it has been completely degraded, replace the supernatant with fresh enrichment medium and continue enrichment culture. After repeating this 5-7 times, take the mixed enrichment for separation and purification.

[0027] Strain isolation 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 6 gradients, 100 μL of each gradient was spread on a solid enrichment medium containing 500 mg / L N-methylpyrrolidone, and 5 parallels were made for each gradient. The culture was carried out at 30°C under microaerobic conditions for about 72 hours, and different colonies grown on the plate were picked for streaking purification. After 4-5 rounds of streaking purification, a strain HZ-F-003 was finally obtained. The pure colonies were inoculated on the 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 is prepared by adding 2 wt% agar powder to the formula.

[0029] Enriched culture medium (inorganic salt culture medium): KH2PO4 0.5g / L, K2HPO4 0.6g / L, MgSO4 0.06g / L, NaCl 10g / L, CaCl2 0.08g / L, FeSO4 0.008g / L, MnSO4 0.008g / 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 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 a complete, smooth edge. The strain was a Gram-negative bacterium. The microscopic morphology of the strain was observed by scanning electron microscopy, and it was found that the bacterium was a bacillus, 4-6 μm long (see Figure 1 ).

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

[0034]

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

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

[0037] The physiological and biochemical identification results of the strain were Gram-negative bacteria. Oxidase and catalase were positive, it could not liquefy gelatin, but could utilize glucose, nitrate, malonic acid and salicylic acid, could not utilize lysine and arginine, but could utilize a small amount of carbohydrates as carbon source, and the indole production test was negative.

[0038] (4) Strain preservation

[0039] The strain HZ-F-003 was classified and named Aquamicrobium sp. HZ-F-003. It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee on October 14, 2024, 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. The collection center tested the strain as alive on October 14, 2024.

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

[0041] (1) Optimization of initial pH value

[0042] Activation of strain HZ-F-003: Streak strain HZ-F-003 on a solid plate of activation medium containing 500 mg / L N-methylpyrrolidone, and incubate at 30°C for 24 hours. Pick a single colony and inoculate it into enrichment medium at 30°C and 150 r / min shaking for 48 hours.

[0043] The initial pH value of the inorganic salt medium was adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0, 500 mg / L of N-methylpyrrolidone was added, and the above activated bacteria were inoculated into the medium at a ratio of 1%, and the medium was placed in a shaker and cultured at 30°C and 150 r / min. Samples were taken after 24 hours to detect OD 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, which indicates that the strain can utilize N-methylpyrrolidone for its own growth and reproduction.

[0045] (2) Optimization of inoculation amount

[0046] The activated strain HZ-F-003 was inoculated into an inorganic salt medium with an initial pH value of 8.0 at a ratio of 0.5%, 1%, 2%, 3% and 4%, and 500 mg / L of N-methylpyrrolidone was added. The medium was placed in a shaker and cultured at 30°C and 150 r / min. Samples were taken after 24 hours to detect 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 value 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 detect 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 initial concentration of N-methylpyrrolidone, the DO of the strain 600 The degradation rates of N-methylpyrrolidone and iodine gradually increased. When the initial N-methylpyrrolidone concentration was 1000 mg / L, the OD 600 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 rate.

[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 of 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 r / min, and samples were taken after 24 hours to detect 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 It can be seen that the strain showed good growth and N-methylpyrrolidone degradation rate in the sodium chloride concentration range of 1% to 3%, indicating that the strain tolerates 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 above optimized optimal N-methylpyrrolidone initial concentration (1000 mg / L), optimal pH value (8.0) and optimal inoculum size (3%), the activated strain was inoculated into the inorganic salt medium. The culture was shaken at 30°C and 150 r / min. Samples were taken after 24 hours to detect OD 600 and N-methylpyrrolidone concentration, the results are as follows Figure 6 shown.

[0056] observe Figure 6 From the data, we can see that strain HZ-F-003 entered the logarithmic growth phase 8h after inoculation, the bacterial growth rate became faster, and the degradation rate of N-methylpyrrolidone also increased significantly. After 20h of culture, the strain gradually entered the stable phase, and the degradation rate of N-methylpyrrolidone tended to be stable. After 24h of culture, the concentration of N-methylpyrrolidone dropped to 0mg / 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 a 3% inoculation rate 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 concentration of N-methylpyrrolidone and OD 600 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 shows that after 3% of the N-methylpyrrolidone-degrading strain HZ-F-003 was inoculated in the wastewater and shaken for 24 hours, the N-methylpyrrolidone concentration in the wastewater dropped to 0.92 mg / L. The degradation rate of N-methylpyrrolidone reached 99.89% at 24 hours. This indicates that the N-methylpyrrolidone-degrading strain HZ-F-003 can quickly degrade high-concentration N-methylpyrrolidone in high-salinity sewage without the need for additional carbon and nitrogen sources, which proves that the strain has great application potential in the field of treating high-salinity and 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 protection scope 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 deposit number is CGMCC No.32176.

2. Use of the water microbe for degrading N-methylpyrrolidone as claimed in claim 1 in treating N-methylpyrrolidone wastewater.

3. The use according to claim 2, characterized in that: Aquamicrobium sp. HZ-F-003 was inoculated into wastewater containing N-methylpyrrolidone to degrade N-methylpyrrolidone.

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

5. The use according to claim 2, characterized in that: The pH value of the wastewater is 5-9.

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

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

8. A bacterial agent for degrading N-methylpyrrolidone, characterized in that: It comprises the water microbacteria (Aquamicrobium sp.) HZ-F-003 described in claim 1.

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