Application of Klebsiella pneumoniae M1

Abstract

The application discloses Klebsiella pneumoniae M1 and an application thereof, wherein the Klebsiella pneumoniae M1 has a preservation number of CCTCC NO: M20251762 and is preserved in the China Center for Type Culture Collection on August 4, 2025. The Klebsiella pneumoniae M1 in the application is derived from natural soil, has strong environmental adaptability and metabolic capacity, can effectively utilize metribuzin as a carbon and nitrogen source to grow and degrade, and adds diversified candidate bacterial resources to the field of microbial remediation.

Description

Technical Field

[0001] This invention belongs to the fields of environmental microbiology and bioremediation technology, specifically relating to the application of Klebsiella pneumoniae M1. Background Technology

[0002] Metazon is a highly effective systemic triazine herbicide that is primarily absorbed through plant roots and leaves. It interferes with electron transport in photosystem II, thereby inhibiting photosynthesis and causing weed death. Its chemical name is 4-amino-6-tert-butyl-3-methylthio-1,2,4-triazine-5(4H)-one, with the molecular formula C8H10. 14 N4OS is commonly used in fields of soybeans, potatoes, and tomatoes to control annual broadleaf weeds and some grassy weeds. However, due to its high water solubility, strong chemical stability, and long half-life, metribuzin degrades slowly in the natural environment, is easily adsorbed by soil, and remains for a long time, leading to increasingly serious soil and water pollution problems. In recent years, metribuzin has been detected frequently in surface water and soil samples from multiple regions, and its residual concentration has reached environmental risk levels. This pollutant is considered an endocrine disruptor; once it enters the body, it may damage the nervous, immune, and reproductive systems, and long-term exposure may threaten human health.

[0003] Traditional methods for addressing metribuzin pollution include physical adsorption, chemical oxidation, and photocatalytic degradation. However, these methods often involve high costs, complex operations, and the risk of potential secondary pollution. In contrast, microbial remediation technology, with its economic efficiency, environmental friendliness, and sustainability, has become an ideal choice for treating pesticide residues. This technology utilizes the metabolic capabilities of microorganisms to convert organic pollutants into non-toxic or low-toxic substances and has been applied in various environmental pollution control projects. Currently, for similar triazine herbicides such as atrazine, simazine, and promethazine, various degrading strains, including bacteria from genera such as Pseudomonas and Bacillus, have been isolated from soil. These strains degrade the herbicides by producing specific enzyme systems, such as hydrolyzing side chains or cleaving triazine rings. However, research on the microbial degradation of metribuzin remains relatively insufficient, with a limited number of reported degrading strains and varying degradation performance.

[0004] In existing technologies, some studies have used enrichment culture methods to screen Burkholderia strains, such as *Burkholderia cepacia*, from farmland soil, enabling the degradation of metribuzin under laboratory conditions, but requiring a long culture period. Other reports indicate that Bacillus strains, such as *Bacillus* sp., exhibit degradation activity at neutral pH and suitable temperatures. Furthermore, *Paracoccus* sp. and *Rhodococcus* sp. have also been shown to have metribuzin degradation potential, and these strains can enhance degradation in the rhizosphere soil environment. Although these strains provide preliminary resources for the bioremediation of metribuzin, the problem of a lack of highly efficient strains remains. Existing strains have a narrow range of adaptability and limited tolerance to temperature, pH, and pollutant concentrations, leading to unstable degradation efficiency in real-world complex environments. Moreover, most studies are limited to single-factor experimental optimization, neglecting the interactions between multiple factors and failing to comprehensively improve the efficiency of the degradation process.

[0005] Klebsiella, an important member of the Enterobacteriaceae family, is widely distributed in natural environments such as soil, water bodies, and plant rhizospheres, and has been found to have the potential to degrade a variety of pollutants. For example, some Klebsiella strains can effectively degrade pesticides such as polycyclic aromatic hydrocarbons and chlorimuron-methyl, as well as inorganic pollutants such as ammonia nitrogen, demonstrating strong metabolic diversity and environmental adaptability. However, existing literature has not reported the use of Klebsiella for the degradation of metribuzin, reflecting the need to expand the diversity of strain resources in the field of metribuzin microbial degradation. Especially in the soil environment of hot and humid southern regions, screening for heat- and acid- and alkali-tolerant strains is of significant practical importance to adapt to local climate and soil conditions. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies of the prior art and provide a Klebsiella pneumoniae M1 strain.

[0007] Another object of the present invention is to provide the application of the above-mentioned Klebsiella pneumoniae M1.

[0008] The technical solution of the present invention is as follows:

[0009] A strain of Klebsiella pneumoniae M1, with accession number CCTCC NO: M20251762, was deposited on August 4, 2025, at the China Center for Type Culture Collection (Wuhan University, Wuhan, China).

[0010] The application of Klebsiella pneumoniae M1 in the degradation of metribuzin.

[0011] In a preferred embodiment of the present invention, the Klebsiella pneumoniae M1 is inoculated into a culture medium or environment containing metribuzin for degradation.

[0012] More preferably, the degradation is carried out at a temperature of 24-36℃, a pH of 4-10, an initial concentration of 20-150 mg / L of metribuzin, and an inoculum size of 1%-10%.

[0013] More preferably, the degradation is carried out at a temperature of 30.39°C, a pH of 7.28, an initial concentration of 40.46 mg / L of metribuzin, and an inoculum concentration of 6.96%.

[0014] A method for degrading metribuzin involves inoculating Klebsiella pneumoniae M1 into a culture medium or environment containing metribuzin for degradation.

[0015] In a preferred embodiment of the present invention, the degradation is carried out at a temperature of 24-36°C, a pH of 4-10, an initial concentration of 20-150 mg / L of metribuzin, and an inoculum size of 1%-10%.

[0016] More preferably, the degradation is carried out at a temperature of 30.39°C, a pH of 7.28, an initial concentration of 40.46 mg / L of metribuzin, and an inoculum concentration of 6.96%.

[0017] A cypermethrin degradation composition comprising, as an active ingredient, live cells of Klebsiella pneumoniae M1 or its metabolites.

[0018] In a preferred embodiment of the invention, a carrier or auxiliary agent is also included.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention provides a novel methamidophos-degrading strain, Klebsiella pneumoniae M1, which is derived from natural soil and has strong environmental adaptability and metabolic capacity. It can effectively utilize methamidophos as a carbon and nitrogen source for growth and degradation, adding a diverse range of candidate bacterial resources to the field of microbial remediation.

[0021] 2. This invention reveals the influence of environmental factors such as temperature, pH, initial concentration and inoculum amount on the growth and degradation performance of the strain, and provides targeted optimization guidance, which helps to improve degradation efficiency and stability under different soil and water conditions.

[0022] 3. This invention employs response surface methodology to comprehensively evaluate the interactions of multiple factors, thereby achieving precise control of the degradation process, improving the predictability and practicality of the overall remediation effect, and reducing experimental costs.

[0023] 4. The Klebsiella pneumoniae M1 strain of the present invention is well adapted to neutral to slightly alkaline environments and high-humidity soils, making it particularly suitable for farmland pollution remediation in hot and humid southern regions. This reduces the environmental risks associated with chemical remediation methods and promotes the sustainable development of green agriculture.

[0024] 5. This invention expands the application potential of Klebsiella spp. in the field of herbicide degradation, provides new ideas, strain libraries and methodological references for the biodegradation of related triazine pollutants, and enhances the universality of microbial technology. Attached Figure Description

[0025] Figure 1 The colony characteristics and Gram staining morphology of strain M1 in Example 1 of this invention are shown. Among them: (a) Colony morphology of strain M1 on LB medium; (b) Colony morphology of strain M1 on MSM medium containing 200 mg / L metribuzin; (c) Gram staining results of strain M1, magnified 100×.

[0026] Figure 2 This shows a phylogenetic tree (NJ method) constructed based on the 16S rRNA gene sequence of strain M1 in Example 1 of the present invention.

[0027] Figure 3 The growth curve (a) and the degradation curve (b) of strain M1 in Example 1 of the present invention are shown.

[0028] Figure 4 This demonstrates the effects of different environmental conditions on the growth of strain M1 and the degradation of metribuzin (72 h) in Example 1 of the present invention.

[0029] Figure 5 This presents one of the response surface analyses of the effect of the interaction of various factors on the degradation rate of metribuzin in Example 1 of the present invention.

[0030] Figure 6 This is the second response surface analysis of the effect of the interaction of various factors on the degradation rate of metribuzin in Example 1 of the present invention. Detailed Implementation

[0031] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0032] Example 1

[0033] 1. Materials and Methods

[0034] 1.1 Test Materials

[0035] 1.1.1 Test soil: Soil samples were collected in the vegetable growing area of ​​Xiamen City, Fujian Province (24°41'43" N, 118°4'15" E). Surface impurities such as dead branches and fallen leaves were removed, and a 5-10 cm layer of soil was taken, air-dried, crushed, and sieved for later use.

[0036] 1.1.2 Main reagents and instruments: Mefenoxam solution standard (100 μg·mL) -1 Purchased from the National Center for Standard Materials. Mefenoxam technical grade (97% purity) purchased from Shanghai Yuanye Biotechnology Co., Ltd. Ultraviolet spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.); High-performance liquid chromatograph (Waters).

[0037] 1.1.3 Culture Medium: Minimal Salt Medium (MSM): 2.00 g glucose, 2.00 g K₂HPO₄, 0.45 g KH₂PO₄, 0.10 g MgSO₄, 0.40 g NaCl, 1 mL trace element stock solution, and distilled water to a final volume of 1 L. The agar powder addition to the solid culture medium is 15 g·L⁻¹. -1 LB solid medium: 10 g peptone, 10 g NaCl, 5 g yeast extract, 15 g agar, dissolved in 1 L distilled water, pH 7.0. Autoclave at 121℃ for 30 min.

[0038] 1.2 Methods

[0039] 1.2.1 Isolation and purification of degrading bacteria: 5.0 g of soil sample was added to MSM medium containing 50 mg / L metribuzin and incubated at 30 ℃ and 180 r·min. -1 After 7 days of shaking culture under controlled conditions, 10 mL of the culture medium was added to MSM medium with a concentration of 100 mg / L metribuzin, and the culture was continued at 30℃ and 180 r / min in the dark with shaking for another 7 days. Following this method, the herbicide concentration was increased by 100 mg / L every 7 days until it reached 300 mg / L, at which point the culture was terminated (i.e., the herbicide concentration was added in a gradient of 50, 100, 200, and 300 mg / L). The culture medium for the last cycle was diluted to 10... –8 The concentration gradient was spread onto MSM solid plates containing 200 mg / L metribuzin for further culture and purification, and finally a single colony strain was obtained, numbered M1, and stored at 4°C.

[0040] 1.2.2 Morphological observation and phylogenetic tree construction: The purified bacterial strains were transferred to LB agar plates and cultured at 30℃ for 2-3 days. The morphological appearance of the strains was observed. Simultaneously, the purified bacterial culture was stored in centrifuge tubes and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing using universal bacterial primers 27F (5'-agtttgatcmtggctcag-3', SEQ ID NO.01) and 1492R (5'-ggttaccttgttacgactt-3', SEQ ID NO.02). The sequencing results were compared for BLAST homology on the NCBI website. Strains with similar homology were selected, and a phylogenetic tree was constructed using the MEGA X nearest neighbor method.

[0041] 1.2.3 Preparation of bacterial suspension: Prepare LB liquid medium, activate the isolated and purified bacterial strain, and add 1 mL of bacterial suspension to 100 mL of LB liquid medium. Incubate at 30℃ and 180 r / min in a shaker for 12 h. Centrifuge at 6000 r / min for 5 min, discard the supernatant, and resuspend the bacteria by pipetting with sterile water. Centrifuge again at 6000 r / min for 5 min. Repeat the above operation 3 times. Adjust the bacterial concentration with sterile water and then measure the OD value using a spectrophotometer. 600 The value is around 1.0, which will be used for subsequent experiments.

[0042] 1.2.4 Determination of the growth curve and degradation curve of the strain against metribuzin: 2 mL of the above bacterial culture was inoculated into MSM containing 100 mg / L metribuzin and incubated at 30 ℃ and 180 r·min. -1 Cultured under the specified conditions, OD was measured within 96 hours. 600 The growth curve of the strain on medium containing metribuzin was plotted, and the content of metribuzin in the culture medium was determined by high performance liquid chromatography to plot the degradation curve of metribuzin by the strain.

[0043] 1.2.5 Effects of Environmental Conditions on the Growth and Degradation of Mefenoxam: The effects of environmental conditions on the growth of strain M1 and its degradation of mefenoxam were investigated by varying temperature, pH, inoculum size, and mefenoxam concentration. MSM liquid medium containing 50 mg / L mefenoxam was prepared, and 2 mL of inoculum was added. Five treatments were set up at different temperatures (24, 27, 30, 33, and 36℃), pH 7.0, and shaking culture at 180 r / min; five treatments were set up at different pH values ​​(4.0, 6.0, 7.0, 8.0, and 10.0), and shaking culture at 30℃ and 180 r / min; five treatments were set up at different inoculum sizes (1%, 2%, 5%, 8%, and 10%, volume ratio), and shaking culture at 30℃ and 180 r / min. Prepare MSM by adjusting the concentration of metribuzin to 20, 50, 80, 100, and 150 mg / L, add 2 mL of inoculum, and incubate at pH 7.0, 30 ℃, and 180 r·min. -1 Culture. The control group used culture medium without bacterial suspension. Each treatment was performed in triplicate. OD was measured at 48 h and 72 h after culture. 600 Value and residue of metribuzin.

[0044] 1.3 Data Statistics and Analysis

[0045] SPSS Statistics 26 software was used for statistical significance analysis of the data (P<0.05), and Origin 9.0 was used for plotting.

[0046] 2 Results and Analysis

[0047] 2.1 Isolation and Identification of Acetaminophen-Degrading Bacteria

[0048] A strain of metribuzin-degrading bacteria, designated M1, was screened, isolated, and purified from soil in vegetable-growing areas of Xiamen City using a domestication and enrichment culture method. This strain can grow in a medium with metribuzin as the sole carbon and nitrogen source. Figure 1 As shown, M1 colonies are round with neat edges; white in color; smooth and glossy surface; moist texture with slight stickiness, easily picked up; after 3 days of culture, the colony size is approximately 2 mm. After Gram staining and observation under an optical microscope, the strain is coccobacillus-shaped and stained red, belonging to Gram-negative cocci.

[0049] BLASTn homology alignment results are as follows Figure 2As shown, strain M1 exhibits high 16S rRNA gene sequence similarity (99.86%) to multiple strains of *Klebsiella pneumoniae*, indicating a very close phylogenetic relationship. Neighbor-joining (NJ) phylogenetic trees were constructed using MEGA 12 software for homologous strains. The results showed that strain M1 clustered with *Klebsiella pneumoniae* subsp. *pneumoniae* strain S4 on the same monophyletic branch, with a support rate as high as 98%. Based on morphological observation, sequence homology, and phylogenetic tree construction, strain M1 was identified as *Klebsiella pneumoniae*. The 16S rRNA gene sequence obtained from the sequencing of strain M1 was submitted to NCBI's GeneBank, obtaining accession number PV658896.

[0050] 2.2 Growth characteristics of strain M1 and its degradation characteristics of metribuzin

[0051] The growth curve of strain M1 after culturing for 96 h at 30℃, pH 7, inoculum size of 2%, and methamidophos concentration of 100 mg / L ( Figure 3 a) and its degradation curve of metribuzin ( Figure 3 b) As shown in the figure. The growth of strain M1 follows an S-shaped curve. From 0 to 10 h, strain M1 grows slowly; from 10 to 72 h, the OD600 value rises rapidly, and the strain enters a rapid growth phase; as the culture time increases (72 to 96 h), the growth rate of the OD600 value gradually slows down and tends to stabilize, and the growth rate of the strain gradually decreases.

[0052] In the initial stage of cultivation (0-20 h), the concentration of metribuzin rapidly decreased from 100 mg / L to approximately 85 mg / L, while the degradation rate rapidly increased to approximately 17%. This rapid degradation corresponds to the rapid growth of the degrading bacteria, indicating that after adapting to the environment, the bacteria can efficiently utilize metribuzin as a nitrogen source for growth and metabolism. As the cultivation time increased (20-40 h), the metribuzin concentration continued to decrease, but the rate of decrease slowed slightly, and the upward trend in the degradation rate also slowed down. In the later stage of cultivation (40-96 h), the metribuzin concentration finally dropped to approximately 70 mg / L, with a degradation rate of approximately 30%, and the degradation rate still showed an increasing trend over time.

[0053] 2.3 Study on the degradation of metribuzin by strain M1 under different environmental conditions

[0054] Within the temperature range of 24℃-36℃, the growth trend of strain M1 first increased and then decreased with increasing temperature, and the biomass of strain M1 was highest at 30℃. Figure 4 -a1), the degradation rate of metribuzin was 30.7% ( Figure 4 -a2). The biomass and degradation rate of strain M1 at 27℃ were both lower than at 37℃. Growth and degradation ability of strain M1 were inhibited at temperatures above 30℃ or below 27℃. The results indicate that the optimal temperature range for atrazine degradation by strain M1 is 27℃-30℃, with 30℃ being the optimal culture temperature for this strain.

[0055] The growth of strain M1 at different pH levels and the degradation of metribuzin are as follows: Figure 4 -b1 and Figure 4 As shown in Figure -b2, the growth ability of strain M1 is optimal at pH 7-8, with its OD... 600 The pH values ​​were 0.556 and 0.622, respectively. M1 showed the best degradation effect on metribuzin at pH 6-8, with degradation rates of 33.84%, 40.02%, and 37.87% after 72 h of culture. When the pH was strongly acidic (pH=4) or strongly alkaline (pH=10), M1 grew slowly, and the degradation rate of metribuzin was less than 25%. The results indicate that strain M1 has a wide pH tolerance range and is more adapted to neutral and slightly alkaline environments.

[0056] The effects of initial concentration of metribuzin on the growth and degradation of strain M1 are as follows: Figure 4 -c1、 Figure 4 As shown in -c2. Within the initial concentration range of 20-150 mg / L of metribuzin, the OD of the bacterial culture increased with increasing metribuzin concentration. 600 The OD value and degradation rate of metribuzin showed a trend of first increasing and then decreasing. At a concentration of 50 mg / L, the OD value was... 600 The bacterial culture exhibited the highest OD value and degradation rate, reaching 40.85% after 72 hours. At concentrations of 20 mg / L and 80 mg / L, the degradation rate of bromide by strain M1 was slightly lower than at 50 mg / L, at 35.04% and 34.44%, respectively. At a bromide concentration of 150 mg / L, the bacterial culture OD value... 600 It has the lowest value and degradation rate of cypermethrin.

[0057] Figure 4 -d1、 Figure 4-d2 represents the growth of M1 and its degradation rate of metribuzin under different inoculum sizes. 1%, 2%, 5%, 8%, and 10% bacterial suspension were added to an inorganic salt medium containing 100 mg / L metribuzin. The results showed that the OD600 value of the bacterial suspension and the degradation rate of metribuzin gradually increased with increasing inoculum size, reaching the highest degradation rate of 63.30% at an inoculum size of 8%. The degradation rate decreased after the inoculum size exceeded 8%. At inoculum sizes of 5% and 10%, the degradation rates were approximately 38.78% and 46.12%, respectively; while at inoculum sizes of 1% and 2%, the degradation rates were relatively low, at 11.54% and 18.51%, respectively.

[0058] 2.4 Results of Box-Behnken Response Surface Optimization

[0059] Based on the single-factor results above, a four-factor, three-level experiment was designed, involving temperature (A), pH (B), initial concentration of metribuzin (C), and inoculum size (D) (Table 1). The degradation rate of metribuzin was used as the response value, and the Box-Behnken response surface methodology was employed to optimize the culture conditions of strain M1, resulting in 27 experimental groups (Table 2). Strain M1 was cultured according to the combinations of temperature, pH, initial concentration of metribuzin, and inoculum size listed in Table 2. After 72 hours of culture, the residual amount of metribuzin was measured, and the degradation rate of metribuzin by strain M1 was calculated.

[0060] Based on the results in Table 2, four factors (temperature, pH, initial concentration of metribuzin, and inoculum size) were used as independent variables, and metribuzin degradation rate was used as the dependent variable. Data analysis was performed using Design-Expert 13 software, yielding a quadratic regression equation:

[0061]

[0062] As shown in Table 3, the model is statistically significant (P < 0.0001), and the lack-of-fit term is not significant. The regression equation is R0. 2 = 0.9820, R 2 adj = 0.9610, indicating that the model fits well and can effectively explain the relationship between variables. Therefore, using this model to optimize the culture conditions for the degradation of metribuzin by strain M1 is reliable. Analysis of variance shows that temperature, pH, initial metribuzin concentration, and inoculum size have significant effects on the model (P < 0.05). Based on the F-value, the initial metribuzin concentration has the largest F-value, and its impact on the model is greater than that of temperature, pH, and inoculum size.

[0063] Table 1. Box-Behnken Experimental Factors and Levels

[0064]

[0065] Table 2 Response Surface Experiment Scheme and Results

[0066]

[0067] Table 3. Analysis of Variance Table of Response Surface Experiment Results

[0068] source sum of squares Degrees of freedom Mean Square F value p-value Significance Model 4128.43 14 294.89 46.8 <0.0001 ** A 46.05 1 46.05 7.31 0.0192 * B 686.4 1 686.4 108.95 <0.0001 ** C 1300.25 1 1300.25 206.38 <0.0001 ** D 491.9 1 491.9 78.08 <0.0001 ** AB 21.76 1 21.76 3.45 0.0878 AC 57.68 1 57.68 9.16 0.0105 * AD 24.01 1 24.01 3.81 0.0747 BC 126.11 1 126.11 20.02 0.0008 ** BD 94.48 1 94.48 15 0.0022 ** CD 123.54 1 123.54 19.61 0.0008 ** <![CDATA[A 2 ]]> 515.49 1 515.49 81.82 <0.0001 ** <![CDATA[B 2 ]]> 1086.8 1 1086.8 172.5 <0.0001 ** <![CDATA[C 2 ]]> 332.96 1 332.96 52.85 <0.0001 ** <![CDATA[D 2 ]]> 612.4 1 612.4 97.2 <0.0001 ** residual 75.6 12 6.3 Missing item 71.06 10 7.11 3.13 0.2666 Not significant Pure error 4.55 2 2.27 sum 4204.03 26

[0069] Note: * indicates significant (P < 0.05), ** indicates highly significant (P < 0.01)

[0070] The response surface plot is a three-dimensional surface plot of the response value of metribuzin degradation rate as a function of four factors: temperature, pH, initial metribuzin concentration, and inoculum size. Figure 5 and Figure 6 The response surface plots for temperature and pH, and temperature and inoculum size are circular (P > 0.05), while the remaining response surface plots are elliptical (P < 0.05). Combined with the ANOVA results in Table 3, the interactions between temperature and pH, and temperature and inoculum size are not significant, while the interactions between temperature and initial concentration of metribuzin, pH and initial concentration of metribuzin, pH and inoculum size, and initial concentration of metribuzin and inoculum size are significant. Analysis using Design-Expert 13 software revealed the optimal culture conditions for metribuzin-degrading bacteria M1 as follows: culture temperature 30.39℃, pH 7.28, initial metribuzin concentration 40.46 mg / L, and inoculum size 6.96%. Under these conditions, after culturing strain M1 for 72 hours, the metribuzin degradation rate reached 70.44%.

[0071] 2.5 Validation of Culture Conditions

[0072] To verify the accuracy of the model's predictions, based on the optimal conditions analyzed by the software, the metribuzin-degrading bacterium M1 was cultured at a temperature of 30℃, pH 7.3, an initial concentration of metribuzin of 40 mg / L, and an inoculum size of 7%. After 72 hours, the metribuzin degradation rates were 70.46%, 70.07%, and 69.97%, respectively, with a relative error of 0.27% compared to the theoretical value of 70.44%. Therefore, optimizing the culture conditions of strain M1 using the Box-Behnken response surface methodology is feasible, and the optimized culture conditions demonstrate good repeatability and practical application value.

[0073] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A type of Klebsiella pneumoniae M1, characterized in that: Its accession number is CCTCCNO: M20251762, and it was deposited at the China Center for Type Culture Collection on August 4, 2025.

2. The application of Klebsiella pneumoniae M1 as described in claim 1 in the degradation of metribuzin.

3. The application as described in claim 2, characterized in that: The Klebsiella pneumoniae M1 strain was inoculated into a culture medium or environment containing metribuzin for degradation.

4. The application as described in claim 3, characterized in that: The degradation was carried out at a temperature of 24-36℃, a pH of 4-10, an initial concentration of 20-150 mg / L of metribuzin, and an inoculum size of 1%-10%.

5. The application as described in claim 4, characterized in that: The degradation was carried out at a temperature of 30.39°C, pH 7.28, an initial concentration of 40.46 mg / L of metribuzin, and an inoculum concentration of 6.96%.

6. A method for degrading metribuzin, characterized in that: The Klebsiella pneumoniae M1 of claim 1 is inoculated into a culture medium or environment containing cypermethrin for degradation.

7. The method as described in claim 6, characterized in that: The degradation was carried out at a temperature of 24-36℃, a pH of 4-10, an initial concentration of 20-150 mg / L of metribuzin, and an inoculum size of 1%-10%.

8. The method as described in claim 7, characterized in that: The degradation was carried out at a temperature of 30.39°C, pH 7.28, an initial concentration of 40.46 mg / L of metribuzin, and an inoculum concentration of 6.96%.

9. A composition for the degradation of metribuzin, characterized in that: Its active ingredients include live cells of Klebsiella pneumoniae M1 as described in claim 1 or its metabolites.

10. The metribuzin degradation composition according to claim 9, characterized in that: It also includes carriers or auxiliary agents.

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