Phthalate degrading strain and application thereof

Abstract

The invention discloses a phthalate degrading strain and application thereof. The strain LB2 is identified as Alicycliphilus sp., the strain LB2 is preserved in the China Center for Type Culture Collection on December 6, 2024, and the preservation number of the strain is CCTCC NO: M 20242749. The degradation strain LB2 can effectively degrade PAEs, the degradation rates of dibutyl phthalate, diamyl phthalate, diisobutyl phthalate and butyl benzyl phthalate in a water body all exceed 80%, PAEs residual pollution in environments such as soil and the water body can be effectively solved, and the ecological environment is protected.

Description

Technical Field

[0001] The present invention relates to a phthalate-degrading strain and its application, belonging to the field of high biotechnology. It uses microorganisms to efficiently degrade the residues of phthalates, and is applicable to the removal of phthalate residues in soil and water bodies to protect the environment. Background Art

[0002] Phthalate esters (PAEs), as a class of plasticizers widely used in industrial production and daily life, play a key role in enhancing the flexibility and processing performance of materials. In the field of plastic products, it is often used as a plasticizer in polyvinyl chloride (PVC) products, such as agricultural films, toys, packaging materials, etc.; in cosmetics and personal care products, PAEs can stabilize fragrances and extend the shelf life, for example, products such as nail polish and perfume; in building materials and coatings, PAEs act as solvents or stabilizers to enhance the durability and adhesion of materials. In the agricultural field, agricultural film covering is the main source of PAEs pollution, and its release amount increases with the duration of film covering. At present, the annual global production of PAEs is approaching 300 million tons, and it is predicted that this figure will jump to 500 million tons by 2050. It is worth noting that China has already become the country with the largest consumption of plasticizers in the world. In 2017 alone, its consumption accounted for as high as 42% of the total global consumption. This widespread use situation has also brought serious environmental risks. Due to the stable chemical properties and easy migration of PAEs, they have been widely detected in various environmental media. In water bodies, the concentrations of PAEs in rivers, lakes and coastal waters vary greatly, mainly due to industrial wastewater, urban sewage and the dumping of plastic waste. The pollution in some basins in China is showing an aggravating trend, and the concentrations in some areas have exceeded the ecological safety thresholds. In terms of soil, the residues of PAEs in the soil of protected agriculture are serious, and the use of agricultural films and livestock manure organic fertilizers are the main reasons. The content of PAEs in the soil of protected vegetable fields is closely related to the film covering time and the type of agricultural film. In organisms, PAEs can be transmitted through the food chain and accumulate in fish, vegetables and even the human body. PAEs have persistence, bioaccumulation and toxicity, and cause significant harm to the ecological environment. In aquatic ecosystems, acute toxicity can cause fish death, and chronic exposure can lead to growth inhibition and reproductive disorders, and can also affect organisms at higher trophic levels of the food chain through bioaccumulation. In terrestrial ecosystems, the growth of plants is hindered after absorbing PAEs, the safety of agricultural products is threatened, the structure of the soil microbial community changes, and the nutrient cycle and soil health are damaged. For human health, PAEs have endocrine disrupting effects, interfere with sex hormone metabolism, and lead to a decline in male sperm quality and abnormalities in the female reproductive system. Some PAEs such as di(2-ethylhexyl) phthalate are listed as potential carcinogens, and prenatal exposure may cause abnormal fetal development. In addition, dibutyl phthalate, as one of the most widely used PAEs, is an important environmental endocrine disruptor and carcinogenic, teratogenic and mutagenic substance. The US Environmental Protection Agency (EPA), the China National Environmental Monitoring Center and the European Union have all listed it as a priority control pollutant. Microbial degradation is an important way to treat PAEs pollution. Specific strains can use PAEs as a carbon source, significantly reduce the pollution concentration within a few weeks, and most of the metabolites are low-toxic or non-toxic, reducing secondary pollution. Combining microbial degradation with physical adsorption technology can also improve the repair efficiency. The contradiction between the wide application of PAEs and their environmental toxicity is prominent, and pollution control needs to combine source control and end-of-pipe repair technologies. Although the potential of microbial remediation is huge, the resources of PAEs-degrading strains still need to be further explored, and their stability and economic feasibility in practical applications also need to be further studied. Summary of the Invention

[0003] The present invention aims at the actual problems and needs of environmental remediation and provides a pure culture strain of microorganisms for degrading PAEs.

[0004] Another object of the present invention is to provide a microbial agent prepared from the degrading strain.

[0005] Still another object of the present invention is to provide a preparation method and application of the degrading microbial agent.

[0006] The object of the present invention can be achieved by the following technical solutions: The present invention provides a phthalate-degrading strain LB2, which is characterized in that its taxonomic name isAlicycliphilus sp., preserved at the China Center for Type Culture Collection on December 6, 2024, with the strain deposit number CCTCC NO: M 20242749.

[0007] Under laboratory shake flask culture conditions, strain LB2 can completely degrade 50 mg / L of dibutyl phthalate within 7 days without the addition of an external carbon source. -1 This strain can be produced using general fermentation equipment in the fermentation industry.

[0008] The degradation strain LB2 described in the present invention is applied in the degradation of PAEs, preferably in the degradation of PAEs in soil and water bodies. The PAEs are selected from dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate. Further preferably dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate.

[0009] The degradation strain LB2 described in the present invention is applied in the preparation of PAEs-degrading microbial agents. The PAEs are selected from dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate. Further preferably dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate.

[0010] A PAEs residue-degrading microbial agent produced with the degradation strain described in the present invention, prepared by fermenting the degradation strain described in the present invention.

[0011] The PAEs residue-degrading microbial agent described in the present invention is preferably produced by the following method: (1) Inoculate the test tube culture of the PAEs-degrading strain LB2 (CCTCC NO: M 20242749) into a shake flask of LB medium and shake culture until the logarithmic phase; (2) Inoculate the above-cultured strain into a seed tank at an inoculation amount of 10%, and culture until the logarithmic growth phase. The medium formula used in the seed tank is: glucose 8.0 g / L, -1 yeast extract 5.0 g / L, -1 K2HPO4 1.0 g / L, -1 NaCl 5.0 g / L, -1 CaCO3 2.0 g / L, -1 MgSO4 0.2 g / L, -1 soybean oil 0.1% (v / v), pH value 7.2 - 7.5; (3) Inoculate the seed liquid into the production tank for cultivation at an inoculation amount of 10%, and the culture medium used in the production tank is the same as that in the seed tank; (4) During the cultivation process in the seed tank and the production tank, the ventilation volume of sterile air is 1: 0.6 - 1.2, the stirring speed is 180 - 240 rpm, the cultivation temperature is 30 - 35 °C, the whole process cultivation time is 72 - 84 h, and the number of bacteria reaches 1 billion per mL after fermentation ends. -1 After that, the fermented broth is directly filled into liquid dosage forms with plastic packaging barrels or packaging bottles or filled into solid bacterial agent dosage forms with peat adsorption and packaging bags.

[0012] Application of the described bacterial agent in degrading phthalic acid esters, and the phthalic acid esters are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate. Further preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate.

[0013] The described bacterial agent is preferably applied to degrade phthalic acid esters in soil and water bodies.

[0014] Beneficial effects:

[0015] The present invention provides a bacterium LB2 that can effectively degrade PAEs. The degradation strain LB2 can completely degrade 50 mg / L of dibutyl phthalate within 7 days without adding an external carbon source, and has broad application potential and value. The degradation bacterial agent produced using this bacterium has the advantages of low production and use costs, convenient use, and good removal effects. It is suitable for large-scale popularization and use in areas of agricultural production. The present invention is of great significance for protecting the ecological environment and the physical health of people. -1 The present invention effectively solves the problem of excessive PAEs residues in agricultural land, neither affecting the normal use of agricultural films nor effectively treating the soil and water environment polluted by PAEs released from agricultural films, and protecting the ecological environment.

[0016] The present invention effectively solves the problem of excessive PAEs residues in agricultural land, neither affecting the normal use of agricultural films nor effectively treating the soil and water environment polluted by PAEs released from agricultural films, and protecting the ecological environment. Brief description of the drawings

[0017] Figure 1 Colony morphology (a) of the strain LB2 of the present invention on LB medium and transmission electron microscope picture (b) of its bacterial cells.

[0018] Figure 2 16S rRNA phylogenetic tree of the strain LB2 of the present invention.

[0019] Figure 3 Genomic phylogenetic tree of the strain LB2 of the present invention.

[0020] Figure 4 UV detection chart of the degradation of dibutyl phthalate by the strain LB2 of the present invention.

[0021] Figure 5 HPLC detection chart of the degradation of dibutyl phthalate by the strain LB2 of the present invention.

[0022] Figure 6 Degradation curve of dibutyl phthalate by the strain LB2 of the present invention.

[0023] Figure 7 GC-MS identification chart of the metabolites of dibutyl phthalate degraded by the strain LB2 of the present invention.

[0024] Figure 8 Influence of temperature on the degradation of dibutyl phthalate by the strain LB2 of the present invention.

[0025] Figure 9 Influence of initial pH on the degradation of dibutyl phthalate by the strain LB2 of the present invention.

[0026] Figure 10 Influence of salt concentration on the degradation of dibutyl phthalate by the strain LB2 of the present invention.

[0027] Figure 11 Influence of metal ions on the degradation of dibutyl phthalate by the strain LB2 of the present invention.

[0028] Figure 12 Comparison of the degradation efficiency of different PAEs by the strain LB2 of the present invention. Biological material preservation information

[0029] LB2, classified and named as Alicycliphilus sp. LB2, deposited in the China Center for Type Culture Collection, the accession number of the strain is CCTCC NO: M 20242749, the deposition date is December 6, 2024, and the deposition address is Wuhan University, Wuhan, China. Detailed implementation mode

[0030] Example 1

[0031] The strain LB2 of the present invention was isolated from the soil of a farmland in Huaibei, Anhui. The specific isolation and screening method of the strain is as follows: Take 10.0 g of soil sample and add it to 100 mL containing 30 mg L -1Dibutyl phthalate liquid inorganic salt medium (hereinafter referred to as MSM), cultured in a shaker at 30 °C and 180 rpm for 7 d, transferred to fresh same medium with an inoculation amount of 15% (v / v), and continuously enriched and passaged four times. Scanned within the range of 200 - 350 nm using an ultraviolet spectrophotometer to detect the degradation effect of the fifth-generation enriched solution. The effective enriched solution was diluted and spread on an LB solid medium containing dibutyl phthalate, cultured at 30 °C for 5 d, and single colonies on the plate were picked into 3 mL of liquid LB test tube medium, then stored and transferred to 20 mL of MSM containing 30 mg L -1 Dibutyl phthalate, cultured at 30 °C for 7 d. Subsequently, extracted with an equal volume of dichloromethane, and the effect was detected by an ultraviolet spectrophotometer. Finally, the dibutyl phthalate-degrading strain LB2 was obtained. -1 The strain LB2 was inoculated into liquid LB medium, cultured in a shaker at 30 °C and 180 rpm for 24 h, the cells were collected by centrifugation, and the cells of the strain LB2 were sent to Shanghai Ling'en Biotechnology Co., Ltd. by dry ice for genome framework map scanning. The genomic information of the strain LB2 was obtained.

[0032] On December 6, 2024, it was deposited in the China Center for Type Culture Collection, and the strain preservation number was CCTCC NO: M20242749. The strain LB2 was light yellow, round, and convex on the LB medium. The colony diameter was 1 - 2 mm (

[0033] a). Its main biological characteristics were G Figure 1 , the cells were rod-shaped, about 0.8 μm wide and 2.0 μm long, without flagella ( － b), aerobic; positive for catalase, oxidase, and indole reaction; negative for V.P. reaction; unable to hydrolyze starch and solidify litmus milk. The 16S rRNA gene sequence of the strain LB2 was compared and analyzed in the database EzBioCloud, and a 16S rRNA phylogenetic tree was constructed by the Neighbor-joining method. The results showed that the strain LB2 was closely related to Figure 1 genus ( Alicycliphilus ). According to the genomic sequencing results of the strain LB2, the genome framework map of the strain LB2 contained 122 Scaffolds, with a size of about 3.81 Mb. The average G + C content was 67.5%. The genome of the strain LB2 contained 3574 genes and 52 tRNAs. A genomic phylogenetic tree was constructed between the genome of the strain LB2 and the genome of the closest related type strain. The results showed that the strain LB2 and the strain Figure 2 clustered together ( Alicycliphilus denitrificans K601 T ). Figure 3). Combining the colony morphological characteristics, physiological and biochemical characteristics of the strain, and the alignment analysis of the 16S rRNA gene and genomic phylogenetic tree, the strain LB2 was finally identified as Alicycliphilus genus. Example 2

[0034] 2.1 Seed liquor preparation The strain LB2 was inoculated into 100 mL of LB medium and cultured on a shaker at 30 °C and 180 rpm. After 48 h, the cells were collected by centrifugation at 6,000 rpm, washed twice with sterilized MSM, and finally resuspended in 40 mL of sterilized MSM as the seed liquor for standby.

[0035] 2.2 Detection of PAEs and identification of metabolites Ultraviolet scanning method: Take 3 mL of the sample to be tested, place it in a 10 mL centrifuge tube, add 3 mL of dichloromethane, and vortex for 1 min. After the mixture stands and separates, discard the upper aqueous phase, and add an excessive amount of anhydrous sodium sulfate to remove the water in the organic phase. The processed sample was detected using a U-T6 ultraviolet-visible spectrophotometer in the wavelength range of 200 - 350 nm, and the degradation of PAEs was judged by analyzing the changes in the ultraviolet absorption peaks of PAEs.

[0036] High performance liquid chromatography (HPLC) detection method: Take 3 mL of the sample to be tested, place it in a 10 mL centrifuge tube, add 10 µL of 25% hydrochloric acid to adjust the sample solution to acidic, then add 3 mL of ethyl acetate for extraction, vortex and mix evenly for 1 min, and then let it stand until the organic phase and the aqueous phase are completely separated. After the separation is completed, carefully aspirate the upper organic phase and transfer it to a new 5 mL centrifuge tube, add an excessive amount of anhydrous sodium sulfate and shake well to remove the water in the organic phase. Aspirate 2 mL of the processed organic phase and place it in a fume hood until the ethyl acetate has completely evaporated. After the ethyl acetate has evaporated, add 350 µL of methanol and shake for 3 - 5 min to ensure that the sample is completely dissolved. After passing the sample through a 0.22 µm organic phase filter, it was detected and analyzed using a high performance liquid chromatograph (Dionex UltiMate 3000). Liquid phase detection conditions: The mobile phase is methanol: water: glacial acetic acid = 80:20:0.5 (v / v / v), the column temperature is 30 °C, an Agilent 5 HC-C18 HPLC column (250×4.6 mm), the flow rate is set at 0.8 mL min -1 , the injection volume is 20 µL, the ultraviolet detection wavelength is set at 235 nm, and the detection time for a single sample is 15 min.

[0037] Determination of dibutyl phthalate and its intermediate metabolites by gas chromatography-mass spectrometry (GC-MS): Take 3 mL of the sample to be tested and perform freeze-drying. After drying, add 350 μL of methanol to the centrifuge tube, shake well, filter through a 0.22 μm organic phase filter, and then detect using gas chromatography-mass spectrometry. The chromatograph is a Finngen Trace DSQ gas chromatography-mass spectrometer. The chromatographic conditions are as follows: The initial column temperature is 50 °C, maintained for 0.5 min, then heated at a rate of 8 °C min -1 to 280 °C and maintained for 5.0 min. The transfer line temperature is 280 °C, and the injection port temperature is 280 °C. The flow rate of the carrier gas (high-purity nitrogen) is 1.0 mL min -1 . The mass spectrometry conditions are as follows: EI injector, ion source temperature 200 °C, scanning range ( m / z ) 30 - 350.

[0038] 2.3 Degradation of dibutyl phthalate by strain LB2 Inoculate strain LB2 into 100 mL of MSM containing 50 mg L -1 dibutyl phthalate at an inoculation amount of 3% (v / v), and culture it on a shaker at 30 °C and 180 rpm for 5 days. Then, according to the method in Example 1, use an ultraviolet spectrophotometer to detect the degradation of dibutyl phthalate. The results are as Figure 4 shown. Strain LB2 can effectively degrade dibutyl phthalate. The detection results by high performance liquid chromatography (HPLC) are as Figure 5 shown. Dibutyl phthalate (retention time: 10.100 min) was almost completely degraded by strain LB2, and new metabolites were produced.

[0039] 2.4 Degradation curve of dibutyl phthalate by strain LB2 and identification of metabolites Inoculate strain LB2 into 100 mL of MSM containing 50 mg L -1 dibutyl phthalate at an inoculation amount of 3% (v / v), and culture it on a shaker at 30 °C and 180 rpm. Take 3 mL of samples every 24 h until the 7th day. Detect the residual amount of dibutyl phthalate, calculate the degradation rate, and plot the time-degradation curve of strain LB2 for dibutyl phthalate. As Figure 6 shown, without an external carbon source, strain LB2 can completely degrade dibutyl phthalate within 7 days.

[0040] Perform GC-MS identification on the samples taken at regular intervals. The results are as Figure 7 shown. A total of three substances were detected by GC-MS. From the corresponding molecular weights, they are known to be dibutyl phthalate (Figure 7 a), monobutyl phthalate ( Figure 7 b) and phthalic acid ( Figure 7 c). In addition, when phthalic acid was used as the sole carbon source, the degradation experiment showed that strain LB2 could not degrade phthalic acid. It can be seen from this that strain LB2 could not completely mineralize dibutyl phthalate, but first degraded it into monobutyl phthalate, and then further degraded it into the final product phthalic acid ( Figure 7 d).

[0041] 2.5 Effect of temperature on the degradation of dibutyl phthalate by strain LB2 In the MSM supplemented with dibutyl phthalate at a final concentration of 50 mg / L, the seed liquid of strain LB2 was inoculated at an inoculation amount of 3% (v / v). The cultures were incubated on a shaker at 180 rpm at 16, 28, 30, 37 and 42 °C. After 4 days, samples were taken to detect the residual amount of dibutyl phthalate, and the degradation rate was calculated to determine the effect of temperature on the degradation of dibutyl phthalate by strain LB2. The results are as -1 shown. The degradation rate of dibutyl phthalate by strain LB2 was the highest at 30 °C. When the temperature was higher than 37 °C, its degradation efficiency decreased significantly. Figure 8 shown, the degradation rate of dibutyl phthalate by strain LB2 was the highest at 30 °C. When the temperature was higher than 37 °C, its degradation efficiency decreased significantly.

[0042] 2.6 Effect of initial pH on the degradation of dibutyl phthalate by strain LB2 In the MSM with initial pH values of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, 50 mg / L -1 of dibutyl phthalate was added. The seed liquid of strain LB2 was inoculated at an inoculation amount of 3% (v / v). The cultures were incubated on a shaker at 30 °C and 180 rpm. After 4 days, samples were taken to detect the residual amount of dibutyl phthalate, and the degradation rate was calculated to determine the effect of pH on the degradation of dibutyl phthalate by strain LB2. The non-inoculated degradation strain was used as a control. As Figure 9 shown, the degradation effect of dibutyl phthalate by strain LB2 was the best at pH 7.0; it could degrade dibutyl phthalate well in the range of pH 5.0 - 7.0; while when the pH was less than 5.0 or greater than 7.0, its degradation ability decreased significantly.

[0043] 2.7 Effect of inoculation amount on the degradation of dibutyl phthalate by strain LB2 The inoculation amounts of 0.5%, 1%, 2.5%, 5% and 8% (v / v) were respectively inoculated into the MSM containing 50 mg / L -1 of dibutyl phthalate. The cultures were incubated on a shaker at 30 °C and 180 rpm. The content of dibutyl phthalate was measured at 4 days. As Figure 10As shown, the inoculum size has a direct relationship with the degradation efficiency of dibutyl phthalate. The larger the inoculum size, the higher the degradation efficiency of dibutyl phthalate.

[0044] 2.8 Effect of Metal Ions on the Degradation of Dibutyl Phthalate by Strain LB2 Add 50 mg L -1 concentration of dibutyl phthalate into 100 mL of MSM with a pH of 7.0, and add metal ions (Al 3+ , Cr 3+ , Cu 2+ , Fe 3+ , Mg 2+ and Ni 2+ ) with a final concentration of 1 mM respectively. Inoculate the seed solution of strain LB2 at an inoculum size of 3% (v / v). After inoculation, place the samples in a shaker at 30 °C and 180 rpm for 4 days. After the culture is completed, take samples and perform HPLC detection and analysis to determine the degradation rate under the influence of different metal ions. As Figure 11 can be seen, Cu 2+ and Fe 3+ have a slight inhibitory effect on the degradation of dibutyl phthalate by strain LB2, while Cr 3+ and Mg 2+ have a slight promoting effect on the degradation, and other metal ions have no significant effect on the degradation effect.

[0045] 2.9 Comparison of the Degradation Efficiency of Strain LB2 on Different PAEs Add different PAEs with a final concentration of 50 mg L -1 , including dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), dipentyl phthalate (DNPP), dioctyl phthalate (DOP), diisobutyl phthalate (DIBP) and benzyl butyl phthalate (BBP) into 100 mL of MSM with a pH of 7.0 respectively, and inoculate the seed solution of LB2 at an inoculum size of 3% (v / v). After inoculation, place the samples in a shaker at 30 °C and 180 rpm for 4 days. After the culture is completed, take samples and perform HPLC detection and analysis to determine the degradation ability of strain LB2 on different PAEs. As Figure 12 can be seen, the strain has the degradation ability for all the tested substrates, and the degradation effects on dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate and benzyl butyl phthalate are the best. Example 3

[0046] The original strain of the dibutyl phthalate-degrading strain LB2 of the present invention was activated on a test tube slant, and its degradation performance was measured, and then it was inoculated on the test tube slant for standby. The test tube strain was inoculated into a 1,000 mL shake flask containing 200 mL of LB medium (LB medium formula: peptone 10.0 g, yeast extract 5.0 g, sodium chloride 5.0 g, water 1 L, pH 7.4), and cultured at a constant temperature with shaking until the logarithmic phase to prepare for inoculation into the first-stage seed tank. The first-stage seed tank is 50 L, the feeding amount is 40 L, and the medium formula is: glucose 8.0 g / L -1 , yeast extract 5.0 g / L -1 , K2HPO4 1.0 g / L -1 , NaCl 5.0 g / L -1 , CaCO3 2.0 g / L -1 , MgSO4 0.2 g / L -1 , soybean oil 0.1% (v / v), pH value 7.2 - 7.5.

[0047] After the feeding was completed, it was sterilized by high-pressure moist heat at 121 °C. After cooling to 30 °C, the cultured shake flask strain was inoculated into the 50 L first-stage seed tank at an inoculation amount of 10%. It was cultured until the logarithmic growth phase (about 84 h), the stirring speed was 220 rpm, and the sterile air inlet volume was 1:0.8. The seed liquid reaching the logarithmic phase was inoculated into the second-stage seed tank at an inoculation amount of 10%. The second-stage seed tank is 500 L, the feeding amount is 400 L, and the medium formula and culture conditions are the same as those of the first-stage seed tank. The seed liquid reaching the logarithmic phase was inoculated into the production tank for culture, and the medium components of the production tank were the same as those of the seed tank medium. The production tank has a capacity of 5 tons and a feeding amount of 4.5 tons. The production tank after feeding was sterilized by high-pressure moist heat at 121 °C under a pressure of 1.1 kg / cm -2 . After sterilization, it was cooled to 30 °C, and sterile air was introduced to maintain a sterile state for standby. The temperature of the inoculated production tank was controlled at 30 - 35 °C. During the culture process of the production tank, the sterile air inlet volume was 1:1.0, and the stirring speed was 240 rpm. The total culture time of the entire process flow was 84 h. After fermentation, the number of bacteria reached more than 1 billion / mL -1 .

[0048] After fermentation was completed, the culture solution was directly discharged from the tank and sub-packed into liquid dosage forms using plastic packaging barrels or packaging bottles, or adsorbed with peat and sub-packed into solid microbial agent dosage forms using packaging bags.

Claims

1. A phthalate-degrading strain LB2, characterized in that, Taxonomic name is Alicycliphilus sp., which was deposited in the China Center for Type Culture Collection on December 6, 2024, with the accession number of CCTCC NO: M 20242749.

2. Use of the degrading strain LB2 as claimed in claim 1 in degrading phthalate esters; the phthalate esters are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate. Further preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate.

3. Use of the degrading strain LB2 as claimed in claim 1 in preparing a phthalate ester degrading microbial agent; the phthalate esters are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate. Further preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate.

4. A phthalate ester degrading microbial agent produced by the phthalate ester degrading strain LB2 as claimed in claim 1.

5. The preparation method of the phthalate-degrading bacterial agent according to claim 4, characterized in that, Comprising the following steps: 1) Inoculating the test tube culture of the phthalate ester degrading strain LB2 into a shake flask of LB medium and culturing with shaking until the logarithmic phase; 2) Inoculate the above-prepared bacterial strain into the seed tank at an inoculation amount of 10%, and culture it until the logarithmic growth phase. The culture medium formula used in the seed tank is as follows: glucose 8.0 g / L -1 , yeast extract 5.0 g / L -1 , K2HPO4 1.0 g / L -1 , NaCl 5.0 g / L -1 , CaCO3 2.0 g / L -1 , MgSO4 0.2 g / L -1 , soybean oil 0.1% (v / v), pH value 7.2 - 7.5; 3) Inoculating the seed liquid into the production tank for culture at an inoculation amount of 10%, and the medium used in the production tank is the same as that in the seed tank; 4) During the cultivation process in the seed tank and production tank, the aeration rate of sterile air is 1: 0.6 - 1.2, the stirring speed is 180 - 240 rpm, the cultivation temperature is 30 - 35 °C, the total process cultivation time is 72 - 84 h, and the number of bacteria reaches 1 billion per mL after fermentation ends. -1 Above, the fermented liquid is directly filled into liquid dosage forms using plastic packaging barrels or packaging bottles after leaving the tank, or solid microbial agent dosage forms are filled using peat adsorption and packaging bags.

6. Use of the microbial agent as claimed in claim 4 in degrading phthalate esters, the phthalate esters are selected from any one or more of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dipentyl phthalate, dioctyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate. Further preferably, any one or more of dibutyl phthalate, dipentyl phthalate, diisobutyl phthalate, and benzyl butyl phthalate.

7. The application according to claim 6, characterized in that Use of the microbial agent as claimed in claim 6 in degrading phthalate esters in soil and water bodies.

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