Klebsiella variicola with benzoic acid tolerance and application thereof

Abstract

The present application belongs to the technical field of bioremediation, and particularly relates to a kind of variable klebsiella KVB40a with benzoic acid tolerance and application thereof, the variable klebsiella KVB40a is preserved in China General Microbiological Culture Collection Center, and the preservation time is April 13, 2026, and the preservation number is CGMCC No.7.719.The variable klebsiella KVB40a in the present application can grow in LB culture medium containing 1.0g / L benzoic acid, the tolerance of evolved strain to benzoic acid can be stably inherited, and the benzoic acid decomposition ability is maintained.The evolved strain recovers growth in 6-12h under acidic conditions, and the lag phase is significantly shorter than that of the original strain;The original strain is completely inhibited when the mass percentage of NaCl is 5%, but KVB40a can grow normally, which indicates that the tolerance of KVB40a to acid and salt is better than that of the original strain.

Description

Technical Field

[0001] This invention belongs to the field of biological soil remediation technology, specifically relating to a benzoic acid-tolerant Klebsiella pneumoniae KVB40a and its applications. Background Technology

[0002] Benzoic acid is a widely used organic compound with strong application potential. However, its use has made it a common environmental pollutant, impacting not only soil but also contributing significantly to recalcitrant industrial wastewater from the chemical, food, dye, and pharmaceutical industries. It is a typical recalcitrant organic pollutant, and its large accumulation in water bodies leads to serious environmental pollution problems. Current methods for treating benzoic acid in the environment mainly include adsorption, extraction, biological methods, catalytic oxidation, and combined technologies. The remediation of benzoic acid in contaminated soil primarily employs biodegradation methods. Numerous studies on the biodegradation of benzoic acid have revealed its degradation mechanisms from multiple perspectives, yielding in-depth results. Research indicates that under aerobic conditions, the benzene ring structure of benzoic acid requires the catalytic action of specific oxidases to complete hydroxylation and cleave. Therefore, benzene ring cleavage is considered a key step in the microbial decomposition of benzoic acid. Previous studies, through in-depth analysis of the mechanisms of microbial degradation of benzoic acid and its derivatives, have clarified that benzoic acid compounds possess multiple different degradation pathways. Current research focuses on screening microbial resources capable of degrading benzoic acid compounds, deeply analyzing the complex and diverse metabolic pathways of these compounds, and systematically conducting targeted optimization studies on core functional genes. Most current research on benzoic acid-degrading microorganisms concentrates on bacteria, but in addition to bacteria capable of degrading autotoxic substances, some fungi also exist in the natural environment that can degrade these substances.

[0003] Klebsiella variegata ( Klebsiella variicola Klebsiella pneumoniae (Klebsiella pneumoniae) is a Gram-negative, facultative anaerobic, non-spore-forming bacterium. In systematic taxonomy, Klebsiella pneumoniae is classified into the kingdom Bacteria, phylum Proteobacteria, class Gamma-Proteobacteria, order Enterobacteriaceae, and family Enterobacteriaceae. Klebsiella pneumoniae is the type species of the genus. Current research on the environmental adaptation of Klebsiella pneumoniae mainly focuses on its tolerance to salt stress. Studies have shown that Klebsiella pneumoniae alleviates salt-alkali stress by regulating its antioxidant system, including increasing the activity of antioxidant enzymes and the content of non-enzymatic antioxidants. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a benzoic acid-tolerant Klebsiella pneumoniae strain KVB40a and its application.

[0005] The first aspect of the present invention provides a benzoic acid-tolerant Klebsiella variegata KVB40a, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 13, 2026, with accession number CGMCC No. 7.719.

[0006] This invention isolates Klebsiella variegata KV1 from pesticide-contaminated soil. High-throughput genome sequencing was used to obtain its complete genome map and annotate its gene functions, confirming the strain as Klebsiella variegata. KV1 was evolved into KVB40a, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 7.719. The entry 1757B in the accession certificate is named KVB40a in this invention, and its classification number is [missing information]. Klebsiella sp. Deposit date: April 13, 2026; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The KVB40a of this invention can grow in LB medium containing 1.0 g / L benzoic acid and retains its benzoic acid decomposition ability. KVB40a recovers its growth under acidic conditions within 6-12 hours, and can grow normally when the NaCl mass percentage is 5%-10%. This indicates that KVB40a has good growth characteristics under different environments, and therefore can be used to decompose benzoic acid in various wastewaters and polluted soils.

[0007] The second aspect of this invention provides a benzoic acid decomposing agent, with the aforementioned Klebsiella variegata KVB40a as the sole active ingredient; The *Klebsiella variegata* KVB40a described herein decomposes benzoic acid in an environment with a benzoic acid concentration of 0.5 g / L to 1 g / L. The *Klebsiella variegata* KVB40a of this invention can still grow normally in an environment with a benzoic acid concentration of 0.5 g / L to 1 g / L and retain its ability to decompose benzoic acid, indicating that the *Klebsiella variegata* KVB40a of this invention has good tolerance to benzoic acid, and therefore can be used to decompose benzoic acid in environments with severe benzoic acid contamination.

[0008] In another preferred embodiment, the benzoic acid decomposing agent is Klebsiella variegata KVB40a bacterial powder or Klebsiella variegata KVB40a fermentation broth.

[0009] In another preferred embodiment, the benzoic acid decomposing agent is applied to the object to be treated to decompose benzoic acid.

[0010] In another preferred embodiment, the object of treatment includes pesticide-contaminated soil or industrial wastewater. Specifically, the industrial wastewater includes wastewater generated in the chemical, food, dye, and pharmaceutical industries.

[0011] In another preferred embodiment, the benzoic acid decomposing agent is used for pesticide-contaminated soil or industrial wastewater with a pH of 5 to 9.

[0012] The third aspect of the present invention provides the application of the aforementioned Klebsiella variegata KVB40a in the decomposition of benzoic acid under salt stress.

[0013] In another preferred embodiment, the Klebsiella variegata KVB40a is used to decompose benzoic acid in an environment with a mass percentage of 5%~10% NaCl.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention isolated Klebsiella variegata KV1 from pesticide-contaminated soil. Through adaptive evolution experiments, the concentration of benzoic acid was continuously increased, ultimately yielding three evolved strains: KVB40a, KVB40b, and KVB40c. These strains exhibited significantly enhanced tolerance to benzoic acid. KVB40a, with its strong benzoic acid tolerance, was selected for further research. Experimental results showed that after 40 rounds of adaptive evolution, KVB40a could grow in LB medium containing 1.0 g / L benzoic acid. The benzoic acid tolerance of KVB40a was stably inherited, and it retained its ability to decompose benzoic acid. Under acidic conditions, the evolved strains recovered growth within 6-12 hours, with a significantly shorter lag phase than the original strain. At a NaCl concentration of 5%, the growth of KV1 was completely inhibited, while KVB40a grew normally, indicating that KVB40a's tolerance to both acid and salt was superior to that of KV1. This invention achieves significantly enhanced benzoic acid tolerance through adaptive evolution of KV1. This study reveals significant changes in KVB40a in gene expression, metabolic regulation, and physiological adaptability, laying a theoretical foundation for subsequent research on the tolerance mechanisms of other phenolic compounds in KVB40a. It also provides a powerful tool for the remediation and bioremediation of benzoic acid pollution and promotes the application of microbial degradation technology in environmental protection. Attached Figure Description

[0015] Figure 1 Figure 1 shows the growth of KV1 under different benzoic acid concentrations.

[0016] Figure 2 This is a growth curve of KV1 in different concentrations of benzoic acid.

[0017] Figure 3 The growth curves are for KVB40a, KVB40b, and KVB40c.

[0018] Figure 4 The figure shows the results of the KVB40a stability test against benzoic acid.

[0019] Figure 5 The figures show the growth curves of KV1 and KVB40a. A represents the growth curves of the two strains without benzoic acid inhibitors; B represents the growth curves of the two strains cultured in LB liquid medium with a benzoic acid concentration of 0.7 g / L; and C represents the growth curves of the two strains cultured in LB liquid medium with a benzoic acid concentration of 1.0 g / L.

[0020] Figure 6 The results of KV1 and KVB40a on solid culture media with different benzoic acid concentrations are shown in the figure.

[0021] Figure 7 The images show the staining results of KV1 and KVB40a cells; A shows the observation of stained cells under a microscope, with PMB representing phosphoric acid-methylene blue staining solution; B shows the lethality at different time points in benzoic acid-free medium; C shows the lethality at different time points in medium containing 0.7 g / L benzoic acid; D shows the lethality at different time points in medium containing 1.0 g / L benzoic acid. Six fields of view were selected for observation each time, and the lethality was calculated. The error bars in the figures represent the standard error (SEM). A t-test was used to analyze the significance of the data for each group of samples; ns: no significant difference between samples; *: statistically significant difference between samples, p < 0.05; **: statistically significant difference between samples, p < 0.01; ***: highly significant statistical difference between samples, p < 0.001.

[0022] Figure 8 The growth curves of KV1 and KVB40a in MSM with different benzoic acid concentrations are shown.

[0023] Figure 9 The growth curves of KV1 and KVB40a under different pH conditions are shown in Figure A, where KV1 and KVB40a grow at pH=9; Figure B, where KV1 and KVB40a grow at pH=7; and Figure C, where KV1 and KVB40a grow at pH=5. Both strains were replicated in triplicate at different pH values.

[0024] Figure 10C shows the growth curves of KV1 and KVB40a under different salt concentrations; D shows the growth curves of KV1 and KVB40a with 3% NaCl by mass; E shows the growth curves of KV1 and KVB40a with 5% NaCl by mass; and E shows the growth curves of KV1 and KVB40a with 10% NaCl by mass. Three replicates were set up for both KV1 and KVB40a under different salt concentrations.

[0025] Figure 11 This is a graph showing the length of the KV1 gene.

[0026] Figure 12 This is the KV1 whole genome map.

[0027] Figure 13 This is a phylogenetic tree diagram of KV1.

[0028] Figure 14 A functional classification diagram for KV1 gene function annotation; where A represents biological processes, B represents cellular components, and C represents molecular functions.

[0029] Figure 15 KEGG metabolic pathway classification diagram for functional annotation of variable KV1 genes.

[0030] Figure 16 A classification diagram of COG function annotations for KV1.

[0031] Figure 17 A graph showing the functional classification of CAZy in KV1 and the corresponding gene count.

[0032] Figure 18 The diagram shows the non-synonymous SNV mutation VENN of three evolved strains.

[0033] Figure 19 Pearson correlation plots for transcriptome samples of KV1 and KVB40a.

[0034] Figure 20 Volcano plots were used to remove background from KV1 and KVB40a; where A represents differentially expressed genes of KV1 before and after 6 h of benzoic acid treatment; and B represents differentially expressed genes of KVB40a before and after 6 h of benzoic acid treatment.

[0035] Figure 21 The graph shows the gene expression analysis of KV1 and KVB40a. In the graph, A represents the upregulated gene expression of the two strains, and B represents the downregulated gene expression of the two strains.

[0036] Figure 22Pearson correlation analysis plots for KV1 and KVB40a metabolites; where A represents the Pearson correlation analysis for positively charged metabolites and B represents the Pearson correlation analysis for negatively charged metabolites.

[0037] Figure 23 The diagrams show the VENN diagrams of KV1 and KVB40a metabolites; where A is the VENN diagram of the specifically upregulated metabolites after 6 h of culture under benzoic acid conditions; and B is the VENN diagram of the specifically downregulated metabolites.

[0038] Figure 24 The diagram shows the enrichment pathways of differentially expressed metabolites of KVB40a. A represents the enrichment pathway analysis results of metabolites that are specifically upregulated after 6 hours of culture under benzoic acid inhibition; B represents the enrichment pathway analysis results of metabolites that are specifically downregulated after 6 hours of culture under benzoic acid inhibition.

[0039] Figure 25 This is a diagram showing the combined analysis of the arginine and proline metabolic pathways in KV1 and KVB40a.

[0040] Figure 26 This diagram illustrates the pathways related to the synthesis of coenzymes for energy metabolism in KV1 and KVB40a. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0042] 1. Isolation, culture and identification of Klebsiella variegata.

[0043] 1.1 Isolation and culture of Klebsiella variegata Klebsiella variegata KV1 was isolated from heavy metal-contaminated soil in Shifang City, Sichuan Province. Hereinafter, KV1 will be referred to simply as KV1. The complete genome of KV1 was obtained through high-throughput genome sequencing, and its gene functions were annotated, confirming that this strain is Klebsiella variegata. KV1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 7.719. The entry 1757B in the accession certificate is named KVB40a in this invention, and its classification number is [not specified in the original text]. Klebsiella sp. Deposit date: April 13, 2026; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Culture medium composition: Each liter of LB medium contains the following ingredients by weight percentage: 1% sodium chloride, 0.5% yeast extract, 1% peptone, pH=7.0. For solid media, add 1.5%-2% agar. Each liter of benzoic acid LB medium contains the following ingredients by weight percentage: benzoic acid, 1% sodium chloride, 0.5% yeast extract, 1% peptone. For solid media, add 1.5%-2% agar. The temperature should not exceed 100℃ when adding benzoic acid to LB medium. Each liter of inorganic salt medium (MSM) contains the following ingredients at the following concentrations: 1.0 g / L ammonium sulfate, 0.5 g / L potassium dihydrogen phosphate, 1.5 g / L dipotassium hydrogen phosphate, 1.0 g / L sodium chloride, and 0.1 g / L magnesium sulfate heptahydrate, with suitable benzoic acid, and pH adjusted to 7.2.

[0045] 1.2 Experimental Methods 1) Confirmation of initial inhibitor concentration: Step 1: Inoculate the Klebsiella variegata strain into LB solid medium and incubate at 37°C for 48 hours until colonies grow on the medium. Pick a single colony and place it in LB liquid medium, then activate it at 220 rpm and 37°C for 24 hours.

[0046] Step 2: Mix Klebsiella variegata KV1 cells with an OD600 concentration of 0.1 at a ratio of 10... -1 Perform serial dilutions, and then spot the diluted bacterial solutions in 5 μL onto LB solid medium with benzoic acid concentrations ranging from 0.0 g / L to 1.0 g / L (with a concentration point every 0.1 g / L). Incubate at 37°C for 24 to 36 hours and observe the growth of the strains on the medium.

[0047] Step 3: Seed cells at an OD600 concentration of 0.1 in LB liquid medium containing inhibitors (add different concentrations of the inhibitor benzoic acid to the medium and set a concentration gradient based on the results of spotting on solid medium), and culture at 220 r / min and 37℃. Perform 3 replicates, with medium without inhibitors as the control group.

[0048] Step 4: Take samples at 0h, 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h, and 108h respectively, and measure the absorbance of the bacterial solution at a wavelength of 600nm using a zeroed microplate reader, and plot the growth curve as required.

[0049] Step 5: Based on the growth curve results, select the benzoic acid concentration that has a significant inhibitory effect within 24 hours as the initial benzoic acid concentration for this adaptive evolution experiment.

[0050] 2) Adaptive evolution experiment Step 1: Inoculate Klebsiella variegata cells into LB liquid medium and culture at 220 r / min and 37℃ for 24 h, i.e., culture to the exponential growth phase of the cells.

[0051] Step 2: Inoculate the activated Klebsiella variegata into LB liquid medium containing benzoic acid inhibitor at a cell concentration of OD600=0.1, and continue to culture at 220 r / min and 37℃ until the cells grow to the exponential growth phase.

[0052] Step 3: Collect the mutated cells for the next round of adaptive evolution. Repeat step 2 for 5 rounds. Dilute the bacterial culture obtained in the 5th round and spread it on LB solid medium containing benzoic acid. Pick single colonies for preservation.

[0053] Step 4: Repeat steps 2 and 3, continuously increasing the concentration of the inhibitor in the culture medium at a concentration gradient of 0.1 g / L. Finally, after 40 cycles, the benzoic acid concentration in the LB medium was increased to 1.0 g / L, yielding an evolved strain of *Klebsiella variegata* with a certain degree of tolerance to benzoic acid.

[0054] 3) Verification of benzoic acid tolerance in evolved strains Step 1: Inoculate the Klebsiella variegata evolutionary strain obtained through adaptive evolution into LB liquid medium, set the shaker environment to 220 r / min and 37℃, and culture until the exponential growth phase of the cells.

[0055] Step 2: The activated Klebsiella variegata evolutionary strain was inoculated again into LB liquid medium at a cell concentration of OD600=0.1 and cultured at 220 r / min and 37℃ until the exponential growth phase.

[0056] Step 3: Repeat step 2 for 5 rounds.

[0057] Step 4: After five rounds of repeated culture in LB liquid medium, the strain was inoculated into LB liquid medium with 1.0 g / L benzoic acid at a cell concentration of OD600=0.1. The strain was cultured at a shaking speed of 220 r / min and a temperature of 37℃. The growth status of the strain was observed and a growth curve was plotted.

[0058] 4) Growth curve plotting: To accurately evaluate the growth characteristics of Klebsiella variegata under different benzoic acid concentrations, this experiment measured its growth status using a microplate reader and plotted growth curves. The experiment was conducted in triplicate to ensure data reliability and reproducibility. The specific steps are as follows: Step 1: Inoculate the Klebsiella variegata strain into LB liquid medium and incubate at 220 r / min and 37℃ for 24 h to activate it.

[0059] Step 2: The activated cells were transferred to LB liquid medium containing different concentrations of benzoic acid, and the OD600 value was adjusted to 1×(1×10⁻⁶). 7 The culture was carried out at 37℃ with constant temperature shaking at 220 r / min. Samples were taken at 0h, 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h, and 108h. The microplate reader was zeroed with sterile blank culture medium, and the absorbance value of the bacterial solution was measured at a wavelength of 600 nm.

[0060] Step 3: Draw the growth curve as required. The growth curve can intuitively show the growth dynamics of the strain under different benzoic acid concentrations, including the characteristics of different growth stages such as the lag phase, logarithmic growth phase and stationary phase. By comparing the growth curves under different concentrations, the effect of benzoic acid on the growth of Klebsiella variegata can be analyzed.

[0061] 5) Cell viability assay: The original and evolved strains were activated separately in LB liquid medium. The activated original and evolved strains were then inoculated into LB liquid medium with benzoic acid concentrations of 0.0 g / L, 0.7 g / L, and 1.0 g / L, respectively, at an OD600 of 0.1 and cultured with shaking at 200 r / min and 37℃. Samples were collected at 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h. The samples were diluted with deionized water to ensure a cell count of approximately 80 cells per field of view. 1 mL of the diluted sample was mixed with 1 mL of 0.1% phosphate-methylene blue staining solution and stained for 1 min. 2 μL of the mixture was added to a glass slide, covered with a coverslip, and observed using a 400x optical microscope (40 × 10). Approximately 10 fields of view were randomly selected, and the total number of cells and the number of stained cells in each field of view were counted. Data analysis was performed based on the total number of cells and the number of stained cells obtained from the obtained fields of view. The viability curves of Klebsiella variegata were plotted with culture time on the x-axis and lethality corresponding to each culture time on the y-axis.

[0062] 6) Effects of pH and salt concentration on the adaptability of bacterial strains Plotting growth curves of the strain in culture media with different pH values: (1) The original Klebsiella variegata and the evolved Klebsiella variegata were activated and cultured in LB liquid medium at 200 r / min and 37 ℃ for 24 h.

[0063] (2) The pH of LB liquid medium was adjusted by using inorganic salts potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4).

[0064] (3) The activated Klebsiella variegata original strain and evolved strain were inoculated into LB liquid medium containing 0.7 g / L benzoic acid at pH 5, 7 and 9 respectively at a cell concentration of OD600=0.1 and cultured by shaking at 200 r / min and 37℃.

[0065] (4) Samples were taken at 0h, 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 96h, and 108h, and the absorbance of the bacterial solution was measured at a wavelength of 600nm using a zeroed microplate reader.

[0066] Plotting growth curves of bacterial strains in media with different salt concentrations: Since sodium chloride (NaCl) is the most common salt in production and daily life, and is widely present in the natural environment and industrial applications, the growth status of bacterial strains and their salt tolerance at different salt concentrations were verified by increasing the NaCl concentration in the liquid culture medium. NaCl was added at mass percentages of 0%, 1%, 3%, 5%, and 10% to LB liquid culture medium containing 0.7 g / L benzoic acid. The original and evolved *Klebsiella variegata* strains were activated and cultured in LB liquid culture medium at 200 r / min and 37℃ for 24 h. The activated strains were then inoculated into the above-mentioned liquid culture media with different salt concentrations at a cell concentration of OD600 = 0.1. The cultures were shaken and cultured at 200 r / min and 37℃, and samples were taken at different time points. The absorbance of the bacterial solution was measured at a wavelength of 600 nm using a zeroed microplate reader. Growth curves were plotted as required.

[0067] 7) Benzoic acid decomposition ability test (1) The original Klebsiella variegata and the evolved Klebsiella variegata were activated in LB liquid medium at 220 r / min and 37 ℃ for 24 h.

[0068] (2) Prepare MSM liquid culture medium with benzoic acid concentrations of 0 g / L, 0.5 g / L and 0.7 g / L, and adjust the pH value to a suitable range after sterilization for subsequent experiments.

[0069] (3) Centrifuge the activated original strain and the evolved strain at a cell concentration of OD600=0.1 (centrifuge at 10000r / min for 2min), remove the supernatant, and rinse with distilled water 1-2 times to remove residual LB medium components.

[0070] (4) The obtained strain was added to the prepared MSM liquid medium containing benzoic acid and cultured under shaking conditions at 220 r / min and 37℃. The photometric value of the bacterial solution was measured at a wavelength of 600 nm using a zeroed microplate reader.

[0071] (5) Draw the growth curve as required.

[0072] 8) Gene sequencing, transcriptome and metabolome analysis: KV1 genome sequencing, evolutionary strain genome resequencing, Klebsiella variegata transcriptome analysis and metabolome analysis were all provided by Novogene Biotechnology Co., Ltd.

[0073] 2. Results 2.1 Confirmation of the initial inhibitor benzoic acid concentration Before conducting adaptive evolution experiments on KV1, it was necessary to first determine the initial concentration of benzoic acid, the inhibitor used for adaptive evolution, to ensure that the strain's growth would be inhibited at this concentration, specifically manifested as a prolonged lag phase. The study employed a spot inoculation method, inoculating KV1 onto solid LB medium with different concentrations of benzoic acid. Figure 1 To investigate the growth of KV1 on solid culture plates with benzoic acid concentrations of 0 g / L, 0.6 g / L, 0.7 g / L, and 0.8 g / L, KV1 grew normally and showed good growth on the control culture plate with a benzoic acid concentration of 0 g / L. At a benzoic acid concentration of 0.6 g / L, the growth was similar to that of the control group. At a benzoic acid concentration of 0.7 g / L, KV1 could grow, but its growth was inhibited compared to the control group. When the concentration was increased to 0.8 g / L, KV1 did not grow on the solid culture plate.

[0074] Based on the TLC results, KV1 was cultured in LB liquid medium containing 0 g / L, 0.6 g / L, and 0.7 g / L benzoic acid, with 0 g / L LB medium as a control. Growth curves were plotted. Figure 2 The growth curve results showed that when the benzoic acid concentration was 0.7 g / L, the growth of KV1 was significantly inhibited. It was observed that KV1 was inhibited throughout the 0–24 h period, with a significantly prolonged lag phase and a decreased growth rate. However, KV1 gradually recovered its growth activity after 24 h. Compared to the culture medium with a lower benzoic acid concentration, KV1 reached a plateau phase at approximately 72 h at this benzoic acid concentration. This indicates that this concentration effectively screens for tolerant strains after adaptive evolution without completely inhibiting strain growth. Therefore, 0.7 g / L benzoic acid was chosen as the initial concentration for adaptive evolution in this experiment.

[0075] 2.2 Growth phenotypes of the original strain and the evolved strain After 40 rounds of adaptive evolution of KV1 using an adaptive evolutionary experimental procedure, the concentration of the inhibitor benzoic acid in LB liquid medium was increased from 0.7 g / L to 1.0 g / L, resulting in the evolved strains: KVB40a, KVB40b, and KVB40c. It was found that among the three strains, KVB40a showed the best growth in the medium containing 1.0 g / L benzoic acid, exhibiting superior growth compared to the other two strains between 12 and 24 hours. Figure 3 Therefore, KVB40a will be used as the main evolved strain, and further research will be conducted on this strain. KVB40a can survive in an environment of 1.0 g / L benzoic acid, and its tolerance to benzoic acid is significantly improved compared with KV1. KVB40a was inoculated into LB medium and cultured at 220 r / min and 37℃ for 24 h, and then inoculated into LB medium at an OD600 concentration of 0.1 for a total of 5 rounds of culture. After 5 rounds of activation culture, KVB40a5 and KVB40a were inoculated into 15 mL of LB liquid medium with a benzoic acid concentration of 1.0 g / L, and the OD600 value was measured at regular intervals to plot the growth curve. Figure 4 The study found that KVB40a5, after five rounds of stable activation and culture, showed no significant difference in benzoic acid tolerance compared to Klebsiella variegata KVB40a. This indicates that the tolerance of evolved strains to benzoic acid is not a stress response at the transcriptional expression level, but rather a stable increase in tolerance. KV1 and KVB40a were activated by inoculating 20 mL of LB liquid medium and cultured at 220 rpm and 37°C for 24 h. Then, cells were inoculated at OD600 = 0.1 into 15 mL of LB liquid medium with benzoic acid concentrations of 0 g / L, 0.7 g / L, and 1.0 g / L, respectively. Growth curves were plotted by periodically measuring OD600 values. Figure 5 The growth curves revealed that the two strains exhibited similar growth patterns in the absence of benzoic acid. In a medium with 0.7 g / L benzoic acid, KV1 showed a significantly longer lag phase than KVB40a. In a medium with 1.0 g / L benzoic acid, KV1 growth was completely inhibited, showing no signs of recovery even after 72 hours. KVB40a, on the other hand, resumed growth between 24 and 36 hours, reaching a stable state after 96 hours. This confirms that KVB40a, obtained through adaptive evolutionary selection, demonstrates improved tolerance to benzoic acid compared to KV1, exhibiting a shorter lag phase at initial inhibitor concentrations and the ability to resume normal growth even after significantly increasing the inhibitor concentration in the medium.

[0076] To further verify the tolerance-enhancing effect of KVB40a, a spot test was used to compare and analyze KV1 and KVB40a. The cell concentration of the activated bacterial solution was adjusted to OD600=0.1, and the bacterial solution was serially diluted and inoculated onto solid culture media containing different concentrations of benzoic acid to obtain the growth status of KV1 and KVB40a on solid culture media under different benzoic acid concentrations. Figure 6 It was observed that both KV1 and KVB40a could grow normally on LB medium without benzoic acid; on solid culture plates containing 0.7 g / L benzoic acid, it was observed that the strains grew at an OD600 of 10... -4 and 10 -5 At the desired dilution, KVB40a showed significantly better growth than KV1, indicating that KVB40a possesses stronger adaptability under the initial benzoic acid inhibitor concentration. When the benzoic acid concentration in the solid culture plate reached 1.0 g / L, KV1 ceased to grow on the plate, but KVB40a continued to grow normally. The results of the TLC further confirm that after 40 rounds of selection through adaptive evolution experiments, the obtained KVB40a exhibited significantly improved tolerance to the benzoic acid inhibitor. Figure 7 The images show the different states of KV1 and KVB40a before and after staining. Multiple different microscopic fields were randomly selected. The figures show that in benzoic acid-free medium, there was no significant difference in lethality rates between KV1 and KVB40a at 0h, 6h, and 12h, indicating that the growth of the two strains is similar in general medium without inhibition. In mediums containing 0.7g / L and 1.0g / L benzoic acid, there was no significant difference in lethality rates at 0h, but the difference became highly significant at 6h. At 12h, there was a statistically significant difference between KV1 and KVB40a in medium containing 0.7g / L benzoic acid, and a highly significant statistical difference when the benzoic acid concentration was increased to 1.0g / L. This result shows that in the early stage of cell growth (6h), 0.7g / L and 1.0g / L benzoic acid... Benzoic acid exhibited a more significant inhibitory effect on KV1, leading to an increased cell mortality rate. However, when the culture time was extended to 12 hours, the inhibitory effect of 0.7 g / L benzoic acid on KV1 weakened, while the inhibitory effect of 1.0 g / L benzoic acid on KV1 remained, resulting in an increased cell mortality rate. For KVB40a, although both inhibitor concentrations showed some inhibitory effect, the mortality rate of KVB40a decreased after 12 hours of culture, and growth began to resume. Therefore, cell staining and mortality rate analysis confirm that, compared to KV1, KVB40a, obtained after 40 rounds of adaptive evolution, exhibits improved tolerance to benzoic acid.

[0077] 2.3 Verification of the strain's ability to decompose benzoic acid Benzoic acid was added to MSM medium as the sole carbon source for the growth of KV1 and KVB40a. In this validation study, the benzoic acid concentration was 0.5 g / L, and the initial inhibitory concentration was 0.7 g / L. MSM without benzoic acid was used as a control group. Growth curves of the two strains cultured in MSM medium with different concentrations of benzoic acid are shown below. Figure 8 The growth curves show that neither KV1 nor KVB40a grows in benzoic acid-free MSM medium. Both strains can grow in environments containing 0.5 g / L and 0.7 g / L benzoic acid, but the lag phase is noticeably shorter at lower benzoic acid concentrations, indicating that benzoic acid concentration affects strain growth. In the 0.7 g / L benzoic acid environment, KV1 begins to grow around 12 hours, with a longer lag phase than the evolved strain. While the lag phase is less pronounced in the 0.5 g / L benzoic acid environment, a longer lag phase is still observed in KV1. This indicates that both KV1 and KVB40a possess benzoic acid decomposition capabilities, with KVB40a showing significantly improved benzoic acid tolerance compared to KV1. The decrease in OD values ​​during the later stages of culture is likely due to the depletion of benzoic acid, the carbon source in the MSM medium, leading to nutrient deficiency and strain death.

[0078] 2.4 Effects of environmental factors on strain growth 1) Effects of different pH values ​​on the growth of the two strains To investigate the growth characteristics of KV1 and KVB40a under different pH conditions, KV1 and KVB40a were cultured in LB liquid medium containing 0.7 g / L benzoic acid at pH 5, 7, and 9. The absorbance was measured at different time points, and growth curves were plotted. Figure 9 Comparing the growth of KV1 and KVB40a under different pH values, it was found that in neutral and slightly alkaline environments (pH=7 and 9), the overall growth of KV1 was not significantly different from that of KVB40a within 3-12 hours, and their growth lag periods were similar, indicating that the inhibitory effect of benzoic acid on the two strains was not significantly different under these conditions. However, under slightly acidic conditions (pH=5), KVB40a gradually resumed growth after 6 hours, and its lag period was significantly shorter than that of KV1. Furthermore, KVB40a's growth was significantly better than KV1's between 6 and 36 hours. Therefore, it is believed that both KV1 and KVB40a have a certain adaptability to benzoic acid in slightly alkaline and neutral environments, and there is no significant difference in their adaptability. Compared with KV1, KVB40a, obtained through 40 rounds of adaptive evolution, has a stronger adaptability to benzoic acid in slightly acidic environments, further reflecting the enhanced acid tolerance of KVB40a.

[0079] 2) Effects of different salt concentrations on the growth of the two strains LB liquid medium containing 0.7 g / L benzoic acid was supplemented with NaCl at concentrations of 0%, 1%, 3%, 5%, and 10% (w / w), with 0% salt as the control. KV1 and KVB40a strains were cultured, and growth curves of the same strain at different salt concentrations were plotted based on OD600 values ​​measured at different time points. Figure 10 According to the growth curves, KVB40a showed significantly better salt tolerance than KV1. KV1 was completely inhibited when the NaCl mass percentage reached 3%, while KVB40a could grow in liquid medium with a mass percentage of 5%. The growth curves of the evolved strains under different salt concentrations showed that the lag phase of the strains was prolonged with increasing salt concentration, and the growth of KVB40a was only completely inhibited at a salt concentration of 10%.

[0080] 2.5 Multi-omics study of Klebsiella variegata 1) KV1 genome Starting with rigorously quality-controlled data, the genome of *Klebsiella variegata* was assembled using Unicycler software. Data from second- and third-generation sequencing were integrated, and chromosome and plasmid sequences were screened. The chromosome sequences were assembled into a circular genome structure, and coding gene prediction was performed on the newly sequenced genome. The total length of the KV1 genome was determined to be 5723812 bp. Predictive analysis yielded 5457 coding genes with a total length of 4964022 bp. Calculations showed that coding regions accounted for 86.73% of the total genome length. The gene length distribution is shown in the figure below. Figure 11 As shown. Based on the genome sequences assembled from the sequencing samples, and considering the predictive analysis results of coding genes, Circos software was used to systematically process and integrate the sample genome data to construct a whole-genome map of KV1. This map clearly displays the structure and characteristic distribution of the KV1 genome, providing an intuitive reference for subsequent genome analysis. Figure 12 The obtained KV1 whole genome was compared with other strains of the Klebsiella genus, including Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae ), Klebsiella acidogenum ( Klebsiella oxytoca Klebsiella Michigani ( Klebsiella michiganensis ), Klebsiella pneumoniae ( Klebsiella quasipneumoniae ), Klebsiella wilbachia ( Klebsiella huaxiensi ) and Klebsiella variegata ( Klebsiella variicola Perform genome comparison analysis and draw a phylogenetic tree. Figure 13Based on the phylogenetic tree, KV1 is most closely related to *Klebsiella variegata*, thus further confirming at the genetic level that KV1 obtained from pesticide-contaminated soil does indeed belong to *Klebsiella variegata*. Through sequence repetition prediction, the KV1 genome contains 23 SINEs with a total length of 1521 bp; 19 LINEs with a total length of 1828 bp; 96 microsatellite DNA sequences with a total length of 7430 bp; and 3 microsatellite DNA sequences with a total length of 137 bp (Table 1).

[0081] Table 1. Statistics of Repeated Sequences By processing the gene data related to Klebsiella pneumoniae KV1 using the GO database, these genes can be annotated according to three main categories: cellular component, molecular function, and biological process. Figure 14 According to the annotation results, among the genes annotated in the KV1 genome that play a role in biological processes, those related to metabolic processes are the most numerous, totaling 1871; followed by genes related to cellular processes, with 1731 genes. Among the genes classified by molecular function, the two most numerous categories are those related to catalytic activity and binding. The KV1 genome was annotated using the KEGG database. Figure 15 The study revealed that the most densely enriched pathway was carbohydrate metabolism (423 genes), followed by membrane transport (411 genes). In addition to these two pathways, amino acid metabolism and the metabolism of cofactors and vitamins also showed enrichment of over 200 genes each. Overall, KEGG database analysis showed that the genes of bacterium KV1 were concentrated in metabolism-related pathways. The COG database, belonging to the protein database category, allows for the accurate annotation of specific protein sequences into corresponding COG categories using alignment techniques. Each COG cluster is composed of orthologous protein sequences, and this compositional characteristic allows for further reasonable inferences about the function of the protein sequence. Figure 16As shown, 611 genes related to carbohydrate transport and metabolism and 546 genes related to amino acid transport and metabolism were annotated in the KV1 genome, which is similar to the results obtained from the KEGG database analysis. The CAZy database is a specialized database in the field of carbohydrate-active enzymes, including five major enzyme categories: carbohydrate esterases (CEs), glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), and oxidoreductases (AAs). The CAZy functional classification and corresponding gene counts of KV1 are shown below. Figure 17 As shown, according to the statistical results, the KV1 genome has the most genes encoding glycoside hydrolases, totaling 149, followed by genes encoding glycosyltransferases, totaling 107. In addition, the KV1 genome also has 42 genes encoding carbohydrate-related components and 15 genes encoding glycoside hydrolases.

[0082] There is a certain correlation between benzoic acid degradation capacity and enzyme-encoding genes annotated in the CAZy database, particularly regarding oxidoreductases and carbohydrate esterases. Glycoside hydrolases may be involved in the hydrolysis of benzoic acid ester bonds when it combines with other sugars; polysaccharide lyases may participate in the cleavage of polysaccharides when benzoic acid combines with other polysaccharides; carbohydrate esterases directly participate in the hydrolysis of ester bonds and may play a role in the degradation of benzoic acid esters; oxidoreductases participate in redox reactions and may play a key role in the oxidation of benzoic acid. However, core enzymes involved in benzoic acid degradation, such as benzoic acid-coenzyme A ligase, are not directly annotated in the CAZy database. Therefore, it is preliminarily concluded that KV1 has a strong carbohydrate degradation capacity and the potential to degrade benzoic acid.

[0083] 2) Resequencing of the genome of Klebsiella variegata The three evolved strains KVB40a, KVB40b, and KVB40c were used as samples for genome resequencing to remove noise and identify key genes related to benzoic acid tolerance in the evolved strains. After activation, the three strains were sampled and named JYZ401, JTZ402, and JYZ403. According to Table 2, the raw data yield of each sample ranged from 1232.886M to 2236.227M, with high sequencing quality (Q20 > 96.73%, Q30 > 90.75%) and GC content ranging from 48.68% to 57.13%. In summary, all samples had sufficient data, met sequencing quality standards, and had a reasonable GC content distribution, making them suitable for library construction, sequencing, and further analysis.

[0084] Table 2 Genome resequencing data The three parallel evolutionary strains KVB40a, KVB40b, and KVB40c were all resequencing analyzed. Non-synonymous SNV mutation genes in the three strains were selected for analysis. Figure 18 The comparison results are as follows. Based on the comparative analysis, we obtained nine genes with common nonsynonymous SNV mutations in the three strains, including tufA, fimA, fimC, fimD, trmD, oadA1, valS, lysR, and one gene encoding a conserved protein of the DUF2345 family that has not yet been characterized. We further analyzed these genes to understand the variation of each gene and the protein encoded by the gene, and summarized the specific mutation information of these genes in Table 3 below.

[0085] Table 3 Mutant genes in evolutionary strains Note: - indicates that the item is not included.

[0086] Gene tufa The gene encodes Elongation Factor Tu (EF-Tu), a GTPase that plays a key role in the translational elongation process of protein synthesis. fimA The gene primarily encodes the major subunit protein of type I fimbriae (FimA), which is widely present in a variety of bacteria, especially in Gram-negative bacteria; fimC The gene encodes a molecular chaperone protein (FimC), which plays an important role in the assembly of bacterial type I pili; fimD The gene encodes an outer membrane chaperone protein (FimD), which is a key protein in the assembly and transport of type I pili; trmD It is a gene that encodes tRNA methyltransferase, which is responsible for catalyzing the methylation of guanine at position 37 in the tRNA molecule; oadA1 The gene encodes pyruvate / oxaloacetate carboxyltransferase; valS The enzyme encoding valine-tRNA synthetase is an amino acid activator that covalently links valine residues to their corresponding tRNA molecules, forming Val-tRNA. Since valine-tRNA is an essential substrate for protein synthesis via ribosomes, this process plays a crucial role in protein synthesis. lysR It encodes a DNA-binding transcriptional regulator belonging to the LysR family, which is widely found in bacteria and participates in the regulation of a variety of physiological functions.

[0087] 3) Transcriptomic sequencing of two types of Klebsiella variegata The KV1 samples collected at 0h were named BA001, BA002, and BA003, and the KVB40a samples collected at the same time were named BA0401, BA0402, and BA0403. The KV1 samples collected at 6h were named BA601, BA602, and BA603, and the KVB40a samples collected at the same time were named BA6401, BA6402, and BA6403. As shown in Table 4, the sequencing clean bases for almost all samples were greater than 1.0G. Saccharomyces cerevisiae has 5457 genes, each approximately 910bp, and a transcript set is approximately 5 × 10⁻⁶. 6 The bp result indicates that the sequencing depth was greater than 200×. Generally, a sequencing depth of 100× is sufficient for analysis, but this sequencing depth exceeded 100×, resulting in ample data. Both Q20 and Q30 were greater than 90%, indicating a very low error rate and very high data quality. In summary, the sequencing data was plentiful and of very high quality, suitable for analysis. Figure 19 The results of the correlation analysis between samples are shown. As can be seen from the figure, the correlation between the biological replicate samples is high, which indicates that the data of these replicate samples are reliable and consistent and can be used for subsequent research analysis.

[0088] Table 4. Correlation among transcriptome sequencing data Background removal analysis was performed on the sequencing results of KV1 and KVB40a. By comparing the sequencing results before and after treatment, differentially expressed genes of the strain under benzoic acid (BA) stress were obtained. The selection criteria for differentially expressed genes were |log2(FoldChange)|>0 ​​and padj≤0.05.

[0089] After 6 hours of BA treatment, the total number of genes differentially expressed by KV1 was 2085, including 996 upregulated genes and 1089 downregulated genes. Figure 20 A); In contrast, bacterial KVB40a differentially expressed a total of 1725 genes, including 820 upregulated genes and 905 downregulated genes, such as... Figure 20 As shown in B.

[0090] The identification of these differentially expressed genes provides a foundation for further functional analysis and helps to reveal the key gene expression changes that occur in KVB40a during adaptation to benzoic acid stress and their potential biological significance.

[0091] By comparing the upregulated genes among differentially expressed genes of KV1 and KVB40a, based on... Figure 21A revealed 737 genes unique to KV1 and 561 genes unique to KVB40a. Enrichment analysis of the 561 upregulated genes unique to KVB40a using the KEGG database yielded the results shown in Table 5. The analysis revealed that these genes were primarily enriched in the ABC transporters, arginine and proline metabolism, quorum sensing, and lysine degradation pathways.

[0092] Simultaneously, the downregulated genes in the differentially expressed genes of the two strains were compared, based on... Figure 21 As shown in B, among these genes, KV1 has a total of 725 unique genes, while KVB40a has 541 unique genes. Gene enrichment analysis of the 541 unique KVB40a genes revealed that the downregulated genes unique to KVB40a are mainly enriched in the ribosome, carbon metabolism, and methane metabolism pathways (Table 5). Transcriptome analysis revealed that some specifically upregulated genes in KVB40a are enriched in the ABC transporter pathway, which not only promotes the production of cold shock proteins (such as cspABCD2) but is also closely related to phosphate uptake. These functions are related to the acid tolerance of the strain. Specifically, this is reflected in the genes... rbsA , dppA , pstA , pstS and pstC All strains showed more than 2-fold specific upregulation.

[0093] In addition, genes specifically upregulated in evolved strains also include proX , opuUCC , bcsC as well as rpoS These genes were upregulated more than 1-fold specifically in the evolved strains. Since these genes are closely related to the structure and function of the strain's cell membrane, it suggests that the adaptively evolved KVB40a may enhance its tolerance to benzoic acid by influencing biomembrane formation and regulating osmosis, thereby improving biomembrane stability and the efficiency of intracellular and extracellular substance exchange. Some upregulated genes in KVB40a are also specifically enriched in quorum sensing pathways, such as... ddpB , ydcV and ydcZ Upregulation of genes can affect biofilm formation and intercellular synergy of KVB40a. The expression of these genes is shown in Table 6.

[0094] Table 5. KEGG enrichment pathways in the transcriptome Table 6. Gene expression of Klebsiella variegata 4) Non-targeted metabolomes of two types of Klebsiella variegata from Figure 22 As can be seen, there is a high correlation between the data from the replicate samples in this metabolomics assay, indicating that the data are reliable and can be used for further analysis. After culturing for 6 hours under benzoic acid conditions, differential expression analysis of metabolites in KV1 and KVB40a revealed that 332 metabolites were upregulated in KV1 and 272 metabolites were upregulated in KVB40a, of which 40 metabolites were specifically upregulated in KVB40a. A total of 127 metabolites were downregulated in KV1 and 114 metabolites were downregulated in KVB40a, with 39 metabolites showing specific downregulation in KVB40a. Figure 23 Enrichment analysis of metabolite-specific expression in KVB40a revealed that, after 6 hours of culture under benzoic acid inhibition, metabolites specifically upregulated in the riboflavin metabolism pathway were enriched. Figure 24 A), specifically downregulated metabolites are mainly enriched in phenylalanine metabolism, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and pantothenate and CoA biosynthesis. Figure 24B). Based on metabolomics analysis, the specific upregulated metabolites riboflavin and FAD (flavin adenine dinucleotide) enriched in KVB40a along the riboflavin metabolic pathway are associated with strain tolerance and play important roles in both the cellular respiratory chain and cell protection. Flavin mononucleotide (FMN) and riboflavin are precursors of flavin adenine dinucleotide (FAD). In KVB40a cultured for 6 h in a medium containing 0.7 g / L benzoic acid, only FAD was specifically upregulated; FMN showed an upregulated trend in both KV1 and KVB40a. Metabolite analysis of KV1 and KVB40a cultured for 6 h in LB liquid medium containing 0.7 g / L benzoic acid showed that agmatine, dephospho-CoA, and phenylacetaldehyde were specifically downregulated in KVB40a. Guanidine is associated with arginine degradation and spermidine formation. However, analysis showed that under benzoic acid inhibition, arginine levels were not significantly upregulated in either strain, while spermidine levels were upregulated by more than 1-fold. In the arginine and proline metabolic pathway, guanidine first degrades to putrescine, which then degrades to spermidine. Putrescine can also degrade to 4-aminobutyraldehyde dimethyl acetal (GABA). Although this metabolite did not show significant up- or down-regulation, the results showed a down-regulation trend in KV1 and an up-regulation trend in KVB40a. Figure 25 ).

[0095] Analyzing these three pathways together—the phenylalanine metabolic pathway, the biosynthetic pathways of phenylalanine, tyrosine, and tryptophan, and the biosynthetic pathway of pantothenic acid and coenzyme A—revealed that dephosphorylated coenzyme A kinase can be synthesized from pantothenic acid. While pantothenic acid was upregulated in both strains, the upregulation was more than 2-fold higher in KV1 than in KVB40a. Dephosphorylated coenzyme A kinase showed specific downregulation in KVB40a, while coenzyme A, produced downstream of pantothenic acid, was upregulated in both strains, with an upregulation of more than 7-fold in KVB40a, significantly greater than in KV1. Therefore, it can be preliminarily concluded that KVB40a, under benzoic acid stress, consumes pantothenic acid and dephosphorylated coenzyme A kinase to generate coenzyme A to resist benzoic acid damage. In this pathway, phenylacetaldehyde can be converted to phenylacetic acid, which can then undergo a series of reactions to ultimately produce acetyl-CoA and succinyl-CoA, both important metabolites involved in the tricarboxylic acid cycle (TCA). Analysis of the test results revealed that, apart from the specific downregulation of phenylacetaldehyde in the evolved strains, phenylacetic acid and the two coenzymes were not specifically expressed. Phenylacetic acid was downregulated in both strains, while acetyl-CoA was upregulated in both strains. However, acetyl-CoA was upregulated more than three times in the evolved strains, far exceeding the upregulation in the original strains. This suggests that the consumption of phenylacetaldehyde in the evolved strains may be used for the production of acetyl-CoA. Figure 26 ).

[0096] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Klebsiella pneumoniae KVB40a with benzoic acid tolerance, characterized in that, The Klebsiella variegata ( Klebsiella variicola KVB40a is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 13, 2026, with accession number CGMCC No. 7.

719.

2. A benzoic acid decomposing agent, characterized in that, The Klebsiella variegata KVB40a described in claim 1 is the sole active ingredient; The Klebsiella variegata KVB40a decomposes benzoic acid in an environment with a benzoic acid concentration of 0.5 g / L to 1 g / L.

3. The benzoic acid decomposing agent according to claim 2, characterized in that, The benzoic acid decomposing agent is Klebsiella variegata KVB40a bacterial powder or Klebsiella variegata KVB40a fermentation broth.

4. The benzoic acid decomposing agent according to claim 2, characterized in that, The benzoic acid decomposing agent is applied to the object to be treated to decompose benzoic acid.

5. The benzoic acid decomposing agent according to claim 4, characterized in that, The objects to be treated include pesticide-contaminated soil or industrial wastewater.

6. The benzoic acid decomposing agent according to claim 5, characterized in that, The benzoic acid decomposing agent is used for pesticide-contaminated soil or industrial wastewater with a pH value of 5-9.

7. The application of Klebsiella variegata KVB40a as described in claim 1 in the decomposition of benzoic acid under salt stress.

8. The application of Klebsiella pneumoniae KVB40a according to claim 7 in the decomposition of benzoic acid under salt stress, characterized in that, The Klebsiella variegata KVB40a was used to decompose benzoic acid in an environment with a mass percentage of 5%~10% NaCl.

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