Pseudomonas having n-methylpyrrolidone hydrolytic acidification ability and use thereof

Abstract

This invention discloses a single-celled bacterium capable of hydrolyzing and acidifying N-methylpyrrolidone (N-methylpyrrolidone) and its applications. Using activated sludge, long-term material for treating industrial wastewater, as the bacterial source, and an inorganic salt culture medium with N-methylpyrrolidone as the sole carbon source as the directional screening medium, a single-celled bacterium, NJUST59, capable of efficiently and stably degrading N-methylpyrrolidone under microaerobic hydrolysis and acidification conditions was obtained through enrichment, domestication, separation, purification, and re-screening of degradation performance. The strain is accession number CCTCC NO: M 20261554. The N-methylpyrrolidone strain NJUST59 of this invention can utilize N-methylpyrrolidone as both the sole carbon source and electron donor, achieving the hydrolysis and acidification of N-methylpyrrolidone in water without the addition of an external organic carbon source. It is suitable for the removal and treatment of recalcitrant compounds in wastewater from industries such as lithium batteries, fine chemicals, and electronic materials.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology for degrading organic pollutants, specifically relating to a single-celled bacterium with N-methylpyrrolidone hydrolysis and acidification capabilities and its applications. Background Technology

[0002] N-methylpyrrolidone (NMP) is a nitrogen-containing five-membered heterocyclic compound with strong polarity, high water solubility, high chemical stability, and low volatility. It is widely used in lithium-ion battery electrode manufacturing, electronic chemicals, pharmaceutical intermediates, pesticide synthesis, engineering plastics, coatings, and polymer processing. With the rapid development of the new energy battery, electronic materials, and fine chemical industries, the production and use of NMP are expanding. It easily enters industrial wastewater systems during equipment cleaning, solvent recovery, mother liquor discharge, and production wastewater collection. Because this substance is highly miscible with water, it is difficult to remove from the aquatic environment through simple phase separation. Insufficient wastewater pretreatment or biochemical treatment capacity can easily increase the organic and nitrogen loads of subsequent treatment units, posing potential risks to receiving water bodies and the surrounding environment.

[0003] Existing treatment methods for N-methylpyrrolidone (NMP)-containing wastewater typically include recovery and separation, chemical oxidation, and biodegradation. For high-concentration NMP wastewater, distillation, extraction, and membrane separation can achieve solvent recovery; however, these methods have high requirements for equipment conditions, energy consumption, and operation management, and their adaptability to low-concentration, complex-composition, or highly variable mixed industrial wastewater is limited. Advanced oxidation, electrochemical oxidation, and photocatalytic oxidation technologies can disrupt some stable organic structures, but they usually suffer from high reagent consumption, high operating costs, and difficulty in controlling intermediate products. In contrast, biological treatment methods have advantages such as lower operating costs, less secondary pollution, and high potential for deep pollutant transformation, making them more suitable as the main or enhanced treatment stage in integrated wastewater treatment systems.

[0004] Hydrolysis acidification is a crucial front-end unit in the biochemical treatment of industrial wastewater, typically placed before aerobic or advanced treatment. It buffers water quality fluctuations and reduces the load of some organic pollutants. Through facultative or anaerobic microorganisms, it converts some recalcitrant organic matter into smaller organic acids, alcohols, amines, or other intermediates that are more readily utilized by subsequent units. For nitrogen-containing heterocyclic pollutants, the hydrolysis acidification system can create a different microbial niche than aerobic tanks under lower dissolved oxygen conditions, which is beneficial for enriching functional microbial communities adapted to reducing, low-oxygen, or micro-oxygen environments. However, N-methylpyrrolidone has a stable structure, and its degradation process may involve the release of organic nitrogen, the generation of amine intermediates, and electron donor-acceptor conversion. This places higher demands on the substrate tolerance, environmental adaptability, and metabolic continuity of the functional strains in the hydrolysis acidification unit. If the system lacks dominant microorganisms capable of effectively initiating N-methylpyrrolidone conversion, problems such as long start-up periods, low pollutant removal rates, intermediate product accumulation, and increased subsequent biochemical loads can easily occur. Therefore, obtaining functional strains that can efficiently degrade N-methylpyrrolidone under micro-oxygen hydrolysis and acidification conditions and have strong environmental adaptability is key to improving the biological treatment efficiency of N-methylpyrrolidone-containing wastewater.

[0005] In recent years, research on the biological treatment of N-methylpyrrolidone wastewater has gradually shifted from simple pollutant removal to screening functional bacterial communities, utilizing electron donors, and co-converting nitrogen. Zhu et al. found that comamonas testosteroni can accelerate the monooxygenation conversion of nitrogen-containing heterocyclic pollutants through bioaugmentation and promote subsequent mineralization processes, indicating that this bacterium has good functional potential in the biotransformation of nitrogen-containing heterocyclic pollutants (Zhu, G., Zhang, YM, Chen, SY, et al. Howbioaugmentation with Comamonas testosteroni accelerates pyridine mono-oxygenation and mineralization. Environ. Res. 2021, 193, 110553.). However, existing research mainly focuses on the conversion and utilization of N-methylpyrrolidone in denitrification or aerobic systems, and there is still a lack of clear technical solutions for comamonas that can stably adapt to and convert N-methylpyrrolidone under microaerobic hydrolysis and acidification conditions. Therefore, screening for monocytogenes that can adapt to low-oxygen or micro-oxygen environments and have stable hydrolytic acidification capabilities for N-methylpyrrolidone is of great application value for enhancing the front-end biological treatment of industrial wastewater containing N-methylpyrrolidone, reducing the load on subsequent treatments, and improving the overall process stability. Summary of the Invention

[0006] The purpose of this invention is to provide a single-celled bacterium with the ability to hydrolyze and acidify N-methylpyrrolidone and its applications. This strain can efficiently convert N-methylpyrrolidone into volatile fatty acids using N-methylpyrrolidone as the sole carbon source and electron donor under microaerobic hydrolysis and acidification conditions.

[0007] The inventors used activated sludge, which has been used for the long-term degradation of heterocyclic compounds, as the bacterial source. Using a screening medium with N-methylpyrrolidone as the sole carbon source, they purified and isolated the strain, obtaining a strain capable of using N-methylpyrrolidone as an electron donor for microaerobic hydrolysis and acidification. Molecular biological identification confirmed it to be a Commamonas testosteroni, and it was named Commamonas testosteroni NJUST59. This strain was deposited on July 6, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261554.

[0008] The present invention provides a method for culturing the above-mentioned monoclonal bacterium NJUST59, specifically: inoculating monoclonal bacterium NJUST59 into a culture medium with a pH of 7.0 and a culture temperature of 30 ± 0.5℃, and culturing under microaerobic conditions.

[0009] Furthermore, the microaerobic conditions are: dissolved oxygen ≤ 0.2 mg / L. -1 .

[0010] The present invention also provides the application of the above-mentioned monoclonal bacterium NJUST59 in the treatment of wastewater containing N-methylpyrrolidone.

[0011] Furthermore, the specific application method is as follows: the seed culture of NJUST59 monoclonal bacteria is inoculated into wastewater containing N-methylpyrrolidone, and the N-methylpyrrolidone is degraded under anaerobic conditions.

[0012] The present invention also provides the application of the above-mentioned monoclonal bacterium NJUST59 in the micro-aerobic hydrolysis acidification treatment of wastewater containing N-methylpyrrolidone.

[0013] Furthermore, the micro-aerobic hydrolysis acidification treatment method is as follows: The seed culture of *N. jute* strain NJUST59 is inoculated into wastewater containing N-methylpyrrolidone, and the dissolved oxygen is kept at ≤0.2 mg / L. -1 Microaerobic conditions promote the hydrolysis and acidification of N-methylpyrrolidone into volatile fatty acids.

[0014] The monoclonal bacterium NJUST59 of this invention can utilize N-methylpyrrolidone as the sole carbon source and electron donor to achieve the hydrolysis and acidification of N-methylpyrrolidone in water without the addition of an external organic carbon source. It has the advantages of short biotransformation cycle, strong substrate specificity, high adaptability to hydrolysis and acidification, and high potential for bio-enhanced applications. It is suitable for the removal and treatment of recalcitrant compounds in wastewater from industries such as lithium batteries, fine chemicals, and electronic materials. Attached Figure Description

[0015] Figure 1 This is a scanning electron microscope image of the monoclonal bacterium NJUST59.

[0016] Figure 2 This is a graph showing the degradation effect of N-methylpyrrolidone by the monoclonal bacterium NJUST59 in a liquid medium with an initial concentration of 5.0 mM.

[0017] Figure 3 This describes the generation of volatile fatty acids from the hydrolysis products of NJUST59 monoclonal bacteria in simulated wastewater with an initial N-methylpyrrolidone concentration of 5.0 mM. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0019] The reagents and culture media used in the following examples have the following compositions:

[0020] LB medium: 25 g L -1 LB medium.

[0021] Inorganic salt culture medium: 1.53 g / L -1 NaHPO4·12H2O, 0.38 g L -1 KH2PO4, 0.1 g L -1 MgSO4, 0.05 g / L -1 CaCl2, 10 mL of trace element solution SL-4, and the concentration of N-methylpyrrolidone are added according to experimental needs.

[0022] Trace element SL-4: 0.5 g L -1 EDTA, 0.2 g / L -1 FeSO4·7H2O, 100 mL L -1 Trace element SL-6.

[0023] Trace element SL-6: 0.01 g L -1 ZnSO4·7H2O, 0.03 g L -1 MnCl2·4H2O, 0.3 g L-1 H3BO4, 0.2 g L -1 CoCl2·6H2O, 0.01 g L -1 CuCl2·2H2O, 0.02 g L -1 NiCl2·6H2O, 0.03 g L -1 Na2MoO4·2H2O.

[0024] Inorganic salt agar solid medium plates: Add 30 g / L to the inorganic salt medium. -1 The agar was sterilized in a high-pressure steam autoclave at 121°C for 20 minutes, then poured into sterile petri dishes and cooled to room temperature to obtain inorganic salt agar solid culture medium plates.

[0025] Example 1

[0026] Screening, isolation, and identification of NJUST59 monoclonal bacteria:

[0027] 1. Screening and isolation of strains

[0028] A 5 g sample was taken from activated sludge that has been used for the long-term degradation of heterocyclic compounds and added to 100 mL of sterile physiological saline (0.85% sodium chloride solution). After stirring evenly, the mixture was allowed to stand for 2 hours. 1 mL of the supernatant was added to an inorganic salt liquid culture medium that had been sterilized at 121℃ for 20 minutes. The medium was then incubated on a shaker at 180 rpm for three days. After three consecutive enrichment cycles, the culture medium was serially diluted with sterile physiological saline to a final concentration of 10. -4 -10 -10 To prepare inorganic salt agar solid medium plates, 20 μL of the diluted culture solution was spread onto the plates and incubated at 30–35 °C for three days. Single colonies showing significant differences were selected from the plates and purified using the streak plate method. After five consecutive purifications, a single strain was obtained and preserved as an agar slant.

[0029] Prepare 100 mL of inorganic salt liquid culture medium containing N-methylpyrrolidone and transfer it to a 120 mL serum bottle. Aerate the system with pure helium to maintain dissolved oxygen at approximately 0.2 mg / L. -1 After sterilization at 121℃ for 20 minutes, the purified strain was inoculated and cultured in a constant temperature shaking incubator at 180 rpm and 30 ± 0.5℃, and the concentration change of N-methylpyrrolidone was monitored. A strain that effectively removed N-methylpyrrolidone from the culture medium was selected and named NJUST59, which was then preserved as a slant culture and stored at -80℃.

[0030] 2. Identification of strains

[0031] Morphological and physiological / biochemical tests were performed on the strain. The 16S rRNA gene sequence of the strain was determined, and the 16S rRNA gene sequence of the strain was compared with known sequences in the NCBI database using BLAST. The species of the bacterium was determined at the molecular biological level.

[0032] (1) Morphological characteristics: On solid culture medium, NJUST59 colonies are nearly round with relatively neat edges, light pink in color, and relatively regular in shape. In liquid culture medium, they exhibit diffuse turbidity. The cells of this strain are rod-shaped or short rod-shaped with blunt ends, and the cells are relatively dispersed, with local cell aggregation visible. The bacterial morphology is relatively regular, the cell outline is clear, and the surface shows slight roughness, wrinkles, or attachment structures. The bacterial size is approximately 0.3~0.5 μm × 1.0~2.2 μm. Figure 1 This is a scanning electron microscope image of the monoclonal bacterium NJUST59.

[0033] (2) Physiological and biochemical characteristics: Gram-negative bacteria, nitrate reductase positive.

[0034] (3) Molecular biological identification: Using the nuclear DNA of strain NJUST59 as a template, PCR amplification was performed using universal primers for bacterial amplification to determine the gene sequence of strain NJUST59. The 16S rRNA gene sequence of the strain was compared with known sequences in the NCBI database using BLAST. The results showed that the sequence similarity between strain NJUST59 and Commamonas testosteroni MH064250.1 was over 99%.

[0035] Based on morphological, physiological and biochemical tests and molecular biological analysis, strain NJUST59 was identified as a monocystic bacterium and named NJUST59.

[0036] Example 2

[0037] Rapid conversion of N-methylpyrrolidone by NJUST59 under microaerobic hydrolysis and acidification conditions:

[0038] 1. Inoculate NJUST59 monoclonal bacteria into LB liquid medium containing 5.0 mM N-methylpyrrolidone and culture in a shaker at 180 rpm at 30 ± 0.5 °C to enrich the NJUST59 bacterial strain. After the strain enters the logarithmic growth phase (approximately 48 hours), centrifuge the obtained bacterial suspension at 8000 rpm for 10 minutes at 4 °C using an ultra-low temperature centrifuge to obtain deposited bacterial cells. Resuspend the cells in sterile inorganic salt liquid medium, centrifuge, and repeat the washing process three times. Resuspend the bacterial cells in sterile liquid inorganic salt medium to obtain a seed culture with OD600 = 1.5~2.0.

[0039] 2. Add 100 mL of inorganic salt liquid culture medium containing an initial concentration of 5.0 mM N-methylpyrrolidone to a 120 mL serum bottle as simulated wastewater. Autoclave at 121°C for 20 minutes and allow to stand until room temperature. Inoculate the above seed culture into the serum bottle at an inoculum volume of 5%. Continuously purge with high-purity helium gas to maintain dissolved oxygen in the serum bottle and wastewater at approximately 0.2 mg / L. -1 A micro-aerobic hydrolysis acidification system was constructed. Serum bottles were sealed with butyl rubber stoppers and aluminum caps and cultured on a shaker at 30 ± 0.5℃ and 180 rpm. Changes in the concentrations of N-methylpyrrolidone and volatile fatty acids in the wastewater were monitored, and both concentrations were determined by high-performance liquid chromatography (HPLC). An experimental group without inoculation with NJUST59 was established as a non-biological control system, with the remaining procedures the same as the hydrolysis acidification system. High-purity helium gas was continuously purged, and dissolved oxygen was maintained at approximately 0 mg / L. -1 The system was used as an anaerobic control, and the remaining operations were the same as those for the microaerobic hydrolysis acidification system.

[0040] Experimental results are as follows Figure 2 As shown, in the microaerophilic hydrolysis acidification system inoculated with *N. cytomegalovirus* NJUST59, the concentration of N-methylpyrrolidone (N-methylpyrrolidone) at 5 mM decreased with culture time, reaching 2.43 ± 0.16 mM after 108 hours, significantly lower than the 3.40 ± 0.10 mM in the anaerobic control system. In the non-biological control system without inoculated strains, the N-methylpyrrolidone concentration remained stable throughout the culture process. Figure 3 As shown, the concentration of volatile fatty acids in the microaerobic hydrolysis acidification system gradually increased to 3.65 ± 0.10 mM with the culture time, which was significantly higher than that in the anaerobic control system, indicating that this strain can promote the generation and accumulation of volatile fatty acids during the conversion of N-methylpyrrolidone.

[0041] The above results indicate that the microbial bacterium NJUST59 can adapt to a microaerobic hydrolysis acidification environment and promote the biotransformation of N-methylpyrrolidone under these conditions. The removal of N-methylpyrrolidone in this microaerobic hydrolysis acidification system is mainly characterized by acidification transformation, accompanied by the generation of volatile fatty acids. This process helps to convert structurally stable and poorly biodegradable N-methylpyrrolidone into small-molecule organic acids that are easily utilized by subsequent biochemical units, reducing the load on subsequent treatments and improving the stability of the biological treatment system.

Claims

1. Commonas testosteroni NJUST59, characterized by, The accession number is CCTCC NO: M20261554.

2. The method for culturing NJUST59 monoclonal bacteria according to claim 1, characterized in that, Specifically, the monoclonal bacteria NJUST59 was inoculated into a culture medium with a pH of 7.0 and a culture temperature of 30 ± 0.5℃ under microaerobic conditions.

3. The cultivation method according to claim 2, characterized in that, Microaerobic conditions: dissolved oxygen ≤ 0.2 mg / L -1 .

4. The application of the monoclonal bacterium NJUST59 as described in claim 1 in the treatment of wastewater containing N-methylpyrrolidone.

5. The application according to claim 3, characterized in that, The specific application method is as follows: inoculate the seed culture of NJUST59 monoclonal bacteria into wastewater containing N-methylpyrrolidone, and degrade N-methylpyrrolidone under anaerobic conditions.

6. The application of the monoclonal bacterium NJUST59 as described in claim 1 in the micro-aerobic hydrolysis acidification treatment of wastewater containing N-methylpyrrolidone.

7. The application according to claim 6, characterized in that, The micro-aerobic hydrolysis acidification treatment method is as follows: Inoculate the seed culture of *N. jute* strain NJUST59 into wastewater containing N-methylpyrrolidone, and maintain dissolved oxygen ≤ 0.2 mg / L. -1 Microaerobic conditions promote the hydrolysis and acidification of N-methylpyrrolidone into volatile fatty acids.

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