Pseudomonas putida for aerobic degradation of n-methylpyrrolidone and its application

Abstract

The application discloses an aerobic degrading N-methyl pyrrolidone Pseudomonas putida and application thereof. The application takes activated sludge for long-term treatment of heterocyclic compounds as a bacterial source, and takes inorganic salt culture medium with N-methyl pyrrolidone as a sole carbon source as a screening culture medium, and a Pseudomonas putida NJUST57 capable of rapidly degrading and mineralizing N-methyl pyrrolidone under aerobic conditions is separated and purified, and the preservation number is CCTCC NO: M 20261552. The Pseudomonas putida can utilize oxygen as an electron acceptor and N-methyl pyrrolidone as a sole electron donor, realizes rapid mineralization and conversion of N-methyl pyrrolidone in water, and is suitable for treatment of wastewater of lithium batteries, fine chemical industry, medicine, electronic materials and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology for degrading organic pollutants, specifically relating to a strain of Pseudomonas putida that aerobically degrades N-methylpyrrolidone and its applications. Background Technology

[0002] N-methylpyrrolidone (NMP), also known as 1-methyl-2-pyrrolidone, is a typical nitrogen-containing heterocyclic organic solvent. It is characterized by good water solubility, low volatility, high polarity, high chemical stability, and low corrosivity, and is widely used in industrial production processes such as lithium-ion batteries, pharmaceuticals, pesticides, engineering plastics, coatings, dyes, synthetic fibers, and electronic materials. Due to the large quantities of NMP used in industrial systems and its easy entry into the environment via industrial wastewater, its migration risk in water bodies and soil is high. NMP has a stable structure and poor biodegradability; conventional hydrolysis, photolysis, and natural degradation are insufficient for its efficient removal. Furthermore, NMP exhibits biotoxicity and developmental toxicity, potentially posing risks to aquatic ecosystems and human health. Therefore, developing efficient, stable, and environmentally friendly biological treatment technologies for industrial wastewater containing NMP has become a crucial issue urgently needing to be addressed in the field of organic solvent pollution control.

[0003] Currently, the main methods for treating N-methylpyrrolidone-containing wastewater include physicochemical and biological treatment methods. Physicochemical methods include adsorption, extraction, membrane separation, ozone oxidation, photocatalytic oxidation, and electrochemical oxidation. While these methods can separate or oxidize N-methylpyrrolidone, they typically suffer from high costs, high energy consumption, and the potential for secondary pollution. Biological treatment methods offer advantages such as low cost, wide applicability, environmental friendliness, and a well-developed engineering foundation, making them an important technology for treating organic solvent-based wastewater. Aerobic biological treatment, in particular, utilizes the oxidative metabolism of microorganisms to convert and degrade N-methylpyrrolidone, demonstrating significant practical application potential. However, N-methylpyrrolidone is structurally stable, has poor biodegradability, and exhibits certain biotoxicity. Existing microbial systems often suffer from long adaptation periods, low degradation efficiency, weak tolerance, and insufficient operational stability in high-concentration N-methylpyrrolidone wastewater, making it difficult to meet the demands of stable treatment of actual industrial wastewater. Therefore, obtaining functional strains that can efficiently degrade N-methylpyrrolidone under aerobic conditions and have strong environmental adaptability is key to improving the biological treatment efficiency of N-methylpyrrolidone-containing wastewater.

[0004] A search revealed that Chinese patent application CN119931895B discloses an aqueous microbacterium, Aquamicrobium sp. HZ-F-003, which degrades N-methylpyrrolidone. This strain is capable of processing 200-1000 mg·L⁻¹ of N-methylpyrrolidone.-1 N-methylpyrrolidone. However, this strain still requires approximately 24 hours to complete efficient degradation, and its 24-hour degradation time is [not specified] for 1000 mg·L [amount not specified]. -1 The degradation rate of N-methylpyrrolidone was 99.89%, at 200 mg·L⁻¹. -1 The degradation rate of N-methylpyrrolidone was 85%, with a relatively long degradation cycle. Furthermore, this strain belongs to the genus *Aquatic Microbe*, and there is still room for improvement regarding its rapid start-up capability, short-cycle degradation ability, and adaptability to different aerobic wastewater systems in conventional aerobic environments. Therefore, obtaining a strain that can significantly shorten the pollutant removal cycle and improve the response speed of aerobic biological treatment units to N-methylpyrrolidone shock loads has significant application value for the efficient bio-enhanced treatment of N-methylpyrrolidone-containing wastewater. Summary of the Invention

[0005] The purpose of this invention is to provide an aerobic *Pseudomonas putida* strain that degrades N-methylpyrrolidone and its applications. This strain can rapidly and efficiently degrade N-methylpyrrolidone under aerobic conditions, using N-methylpyrrolidone as an electron donor and oxygen as an electron acceptor.

[0006] The inventors used activated sludge, which has long been used for the treatment 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 aerobic degradation using N-methylpyrrolidone as an electron donor. Molecular biological identification confirmed it as *Pseudomonas putida*, and it was named *Pseudomonas putida* NJUST57. 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 20261552.

[0007] The present invention provides a method for culturing the above-mentioned Pseudomonas putida NJUST57, the specific steps of which are: inoculating Pseudomonas putida NJUST57 into LB medium, the pH of the medium is 7.0, and the culture temperature is 30 ± 0.5℃.

[0008] The present invention also provides the application of the above-mentioned *Pseudomonas putida* NJUST57 in the treatment of wastewater containing N-methylpyrrolidone.

[0009] Furthermore, the specific application method is as follows: inoculate the seed culture of Pseudomonas putidae NJUST57 into wastewater containing N-methylpyrrolidone, and degrade N-methylpyrrolidone under aerobic conditions.

[0010] The *Pseudomonas putida* strain of this invention can utilize oxygen as an electron acceptor and N-methylpyrrolidone as the sole electron donor to achieve the mineralization and transformation of N-methylpyrrolidone in water, at a concentration of 200 mg·L⁻¹. -1 N-methylpyrrolidone can be completely degraded within 6 hours, with a fast degradation rate, making it suitable for the treatment of lithium batteries, fine chemicals, pharmaceuticals, electronic materials, and other materials containing recalcitrant heterocyclic compounds. Attached Figure Description

[0011] Figure 1 This is a scanning electron microscope image of Pseudomonas putida NJUST57.

[0012] Figure 2 It is *Pseudomonas putida* NJUST57 at an initial N-methylpyrrolidone concentration of 200 mg / L. -1 The degradation effect of N-methylpyrrolidone in simulated wastewater is shown in the figure.

[0013] Figure 3 It is *Pseudomonas putida* NJUST57 at an initial N-methylpyrrolidone concentration of 200 mg / L. -1 The diagram shows the effect of total organic carbon removal in simulated wastewater. Detailed Implementation

[0014] The technical solution of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0015] The reagents and culture media used in the following examples have the following compositions:

[0016] LB medium: 25 g L -1 LB medium.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] Example 1

[0022] Screening, isolation, and identification of *Pseudomonas putida* NJUST57:

[0023] 1. Screening and isolation of strains

[0024] 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 two 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 (0.85% sodium chloride solution) 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.

[0025] 100 mL of simulated wastewater containing N-methylpyrrolidone was prepared and placed in a 250 mL Erlenmeyer flask. After sterilization at 121 °C for 20 minutes, the purified strain was inoculated and cultured in a constant temperature shaking incubator at 180 rpm and 30 ± 0.5 °C. The concentration change of N-methylpyrrolidone was monitored. The strain that could effectively remove N-methylpyrrolidone from the culture medium was selected and named NJUST57. It was then preserved as a slant culture and stored at -80 °C.

[0026] 2. Identification of strains

[0027] Morphological and physiological / biochemical tests were performed on the strain. The 16S rRNA gene sequence (SEQ ID No. 1) 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.

[0028] (1) Morphological characteristics: On solid culture medium, NJUST57 colonies are nearly round with relatively neat edges, pale flesh-colored to light pink, and relatively regular in shape. In liquid culture medium, they diffuse and cause turbidity. The cells of this strain are rod-shaped with blunt ends, relatively uniform in size, and have slight wrinkles or rough structures on the surface, with a size of approximately 0.4~0.6 μm × 1.0~2.2 μm. Figure 1 Scanning electron microscope image of Pseudomonas putida NJUST57.

[0029] (2) Physiological and biochemical characteristics: Gram-negative bacteria, catalase positive.

[0030] (3) Molecular biological identification: Using the nuclear DNA of strain NJUST57 as a template, PCR amplification was performed using universal primers for bacterial amplification to determine the gene sequence of strain NJUST57. 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 NJUST57 and Pseudomonas putida AY622320.1 was over 99%.

[0031] Based on morphological, physiological and biochemical tests and molecular biological analysis, strain NJUST57 was identified as *Pseudomonas putida* and named *Pseudomonas putida* NJUST57.

[0032] Example 2

[0033] Experiment on the rapid degradation of N-methylpyrrolidone by Pseudomonas putida NJUST57 under aerobic conditions:

[0034] Pseudomonas putida NJUST57 was inoculated into a solution containing 200 mg L. -1 In LB broth containing N-methylpyrrolidone, the bacteria were cultured on a shaker at 180 rpm at 30 ± 0.5 °C to enrich *Pseudomonas putida* NJUST57. After the strain entered the logarithmic growth phase (approximately 48 hours), the resulting bacterial suspension was centrifuged for 10 minutes (4 °C, 8000 rpm) using an ultra-low temperature centrifuge to obtain deposited bacterial cells. These cells were resuspended in sterile liquid inorganic salt medium, centrifuged, and washed three times. The bacterial cells were then resuspended in sterile liquid inorganic salt medium to obtain a seed culture with an OD600 of 1.5–2.0.

[0035] Add 100 mL of N-methylpyrrolidone to a 250 mL wide-mouth conical flask with an initial concentration of 200 mg / L. -1 Inorganic salt liquid culture medium was used as simulated wastewater. The wide-mouth conical flasks were then sealed with sterile, breathable sealing film and autoclaved at 121°C for 20 minutes, then allowed to cool to room temperature. The seed culture was inoculated into serum bottles at an inoculation volume of 5%, and cultured on a shaker at 30 ± 0.5°C and 180 rpm. The concentrations of N-methylpyrrolidone and total organic carbon in the simulated wastewater were monitored. The N-methylpyrrolidone concentration was determined by high-performance liquid chromatography (HPLC), and the total organic carbon concentration was determined by a total organic carbon analyzer. An experimental group without inoculation with *Pseudomonas putida* NJUST57 was established as a non-biological control system; the remaining procedures were the same as for the aerobic system.

[0036] Experimental results are as follows Figure 2 As shown, in an aerobic system inoculated with *Pseudomonas putida* NJUST57, N-methylpyrrolidone was completely degraded within 6 hours. In a non-biological control system not inoculated with *Pseudomonas putida* NJUST57, the concentration of N-methylpyrrolidone did not change significantly. Figure 3 As shown, the total organic carbon content in the aerobic system gradually decreased, reaching a mineralization rate of over 82% after 14 hours, while the total organic carbon content in the non-biological control system remained unchanged. These results demonstrate that the *Pseudomonas putida* NJUST57 of this invention is suitable for the aerobic biochemical treatment of N-methylpyrrolidone wastewater, achieving rapid removal of N-methylpyrrolidone from the wastewater.

Claims

1. *Pseudomonas putida* NJUST57, characterized by, The accession number is CCTCC NO: M20261552.

2. The method for culturing *Pseudomonas putida* NJUST57 according to claim 1, characterized in that, The specific steps are as follows: Inoculate Pseudomonas putida NJUST57 into LB medium with a pH of 7.0 and a culture temperature of 30 ± 0.5℃.

3. The application of the *Pseudomonas putida* NJUST57 as described in claim 1 in the treatment of wastewater containing N-methylpyrrolidone.

4. The application according to claim 3, characterized in that, The specific application method is as follows: inoculate the seed culture of Pseudomonas putidae NJUST57 into wastewater containing N-methylpyrrolidone, and degrade N-methylpyrrolidone under aerobic conditions.

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