Lactobacillus paracasei, microbial inoculant and application thereof

Abstract

The application belongs to the field of polycyclic aromatic hydrocarbon degrading microorganisms, and relates to a lactobacillus paracasei, a bacterial agent and application thereof. The lactobacillus paracasei has a preservation number of CGMCC No. 24463. The lactobacillus paracasei is stable in nature, can grow rapidly in soil or underground water with polycyclic aromatic hydrocarbon as a sole carbon source, has excellent polycyclic aromatic hydrocarbon degrading performance, and can be widely applied to soil or underground water containing polycyclic aromatic hydrocarbon repair.

Description

Technical Field

[0001] This invention belongs to the field of microorganisms that degrade polycyclic aromatic hydrocarbons (PAHs). Specifically, it relates to a Lactobacillus paracasei, an agent containing the Lactobacillus paracasei, and the application of the Lactobacillus paracasei and the agent in the degradation of PAHs. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are aromatic hydrocarbons containing two or more benzene rings. Based on the different ways the benzene rings are linked, they can be mainly divided into two categories. One category is non-fused-ring PAHs, including biphenyls, polyphenylene oxides, and polyphenylene aliphatic hydrocarbons. Compounds formed by benzene rings linked by σ bonds are called biphenyls. Biphenyl derivatives and polyphenylene oxides are named using biphenyl as the parent compound. Polyphenylene aliphatic hydrocarbons are compounds formed by replacing hydrogen atoms in aliphatic hydrocarbons with several benzene rings. These compounds are named using phenyl as a substituent and aliphatic hydrocarbon as the parent compound. The structure and properties of these non-fused-ring PAHs, such as biphenyls, polyphenylene oxides, and polyphenylene aliphatic hydrocarbons, are similar to those of monocyclic aromatic hydrocarbons. The other category is fused-ring PAHs, where two carbon atoms are shared by two benzene rings. For example, naphthalene has the molecular formula C6H ... 10 H8 is formed by the fusion of two benzene rings sharing two adjacent carbon atoms. Naphthalene is a white, flaky crystal with a melting point of 80°C and a boiling point of 218°C. It is insoluble in water but readily soluble in hot alcohols, ethers, and other organic solvents. It is volatile, sublimes easily, and has a characteristic odor. Naphthalene is the most abundant compound in coal tar, comprising approximately 10% in high-temperature coal tar. Anthracene has the molecular formula C6H2O. 14 H 10 Anthracene is composed of three fused benzene rings. It is found in coal tar, comprising approximately 0.25% of the coal. Phenanthrene is also found in coal tar and is an isomer of anthracene.

[0003] Polycyclic aromatic hydrocarbons (PAHs) can be categorized into natural and anthropogenic sources. Natural sources primarily originate from the biosynthetic processes of terrestrial and aquatic plants and microorganisms. Additionally, PAHs are found in natural forest and grassland fires, volcanic eruptions, fossil fuels, lignin, and sediment. Anthropogenic sources are mainly formed through the incomplete combustion or pyrolysis of various mineral fuels (such as coal, oil, and natural gas), wood, paper, and other hydrocarbon-containing materials under reducing conditions.

[0004] Unlike other persistent organic pollutants (PAHs) that are globally banned from production and use, most PAHs are primarily released from sources closely related to human activities. Therefore, the presence of PAHs in the environment is currently a major concern. PAHs are ubiquitous in various environments, primarily entering surface waters through atmospheric deposition, river runoff, urban and industrial wastewater, and leakage during transportation. Water is a crucial medium for PAH migration in the environment; surface runoff, atmospheric deposition, soil-sediment exchange, and air-water interface inputs and exchanges are all effective pathways for their diffusion. These pollutants have extremely low solubility in water and exhibit strong affinity and biochemical persistence, demonstrating a strong adsorption effect on marine sediments and often remaining stable in various natural media. However, when sediments are subjected to external forces such as waves, tides and ocean currents, trawling, shipping traffic, and hydraulic engineering operations, pollutants are released from the sediments, exacerbating pollution.

[0005] Most polycyclic aromatic hydrocarbons (PAHs) exhibit strong toxic side effects on organisms, including immunotoxicity and carcinogenicity. Benzo[a]pyrene has been classified as Group 1 by the International Atomic Energy Agency (IAEA) and is carcinogenic to humans. Other PAHs are classified into Groups 2A and 2B. Furthermore, PAHs have a high bioaccumulation rate, which exacerbates their adverse effects. Even trace amounts can lead to various diseases and cause serious harm to the human body. Increasing evidence suggests that long-term exposure to PAHs not only causes certain cancers but also other diseases such as obesity and type 2 diabetes (T2D).

[0006] Traditionally, PAH pollution remediation employs physical and chemical methods. Conventional methods utilizing temperature, electrodynamics, and redox reactions offer advantages such as short cycles and rapid results; however, their high energy consumption and significant damage to the original site cannot be ignored. Microbial degradation, due to its lower cost and ease of operation compared to other degradation methods, is gradually becoming the main research direction for PAH degradation.

[0007] In the natural environment, the decomposition and transformation mechanism of organic matter by microorganisms is the primary method for removing organic pollutants under non-human intervention conditions. This degradation and transformation mechanism is the core idea behind the use of microbial methods to treat organic pollutants. When the pollutants at a site are chain-like petroleum hydrocarbons or organic pesticides, microbial methods often have good treatment effects. However, when encountering PAHs with high hydrophobicity, the effectiveness of microbial methods is often unsatisfactory. To achieve better remediation results, it is not only necessary to improve the remediation methods but also to clarify the characteristics of microbial degradation and screen and develop microorganisms that match the structural characteristics and physicochemical properties of the target pollutants to achieve better degradation effects. Summary of the Invention

[0008] The purpose of this invention is to provide a Lactobacillus paracasei with excellent polycyclic aromatic hydrocarbon degradation performance, as well as its inoculum and applications.

[0009] The first aspect of the present invention provides a Lactobacillus paracasei ( Lactobacillus paracasei The Lactobacillus paracasei has the accession number CGMCC No. 24463.

[0010] A second aspect of the present invention provides a microbial agent containing the aforementioned Lactobacillus paracasei.

[0011] A third aspect of the invention provides the use of the aforementioned Lactobacillus paracasei or the aforementioned bacterial agent in the degradation of polycyclic aromatic hydrocarbons.

[0012] Furthermore, the application method includes contacting the Lactobacillus paracasei or the bacterial agent with a pollutant containing polycyclic aromatic hydrocarbons.

[0013] Furthermore, the polycyclic aromatic hydrocarbon is a fused-ring polycyclic aromatic hydrocarbon or a non-fused-ring polycyclic aromatic hydrocarbon.

[0014] Furthermore, the polycyclic aromatic hydrocarbon is selected from at least one of naphthalene, anthracene, phenanthrene, pyrene, β-pyrene, benzo[a]pyrene, benzo[a]anthracene, benzo[a]fluoranthracene, and ind[a]pyrene.

[0015] Furthermore, the non-fused-ring polycyclic aromatic hydrocarbon is selected from at least one of biphenyl, biphenylene, and polyphenylene aliphatic hydrocarbons.

[0016] Furthermore, the polyphenylene is biphenylene; the polyphenylene aliphatic hydrocarbon is at least one of diphenylmethane and diphenylethane.

[0017] Furthermore, the pollutant containing polycyclic aromatic hydrocarbons is soil containing polycyclic aromatic hydrocarbons or groundwater containing polycyclic aromatic hydrocarbons.

[0018] Furthermore, in the soil containing polycyclic aromatic hydrocarbons, the content of polycyclic aromatic hydrocarbons is 2-1000 mg / kg soil; in the groundwater containing polycyclic aromatic hydrocarbons, the content of polycyclic aromatic hydrocarbons is 2-1000 mg / L groundwater.

[0019] Furthermore, when the pollutant containing polycyclic aromatic hydrocarbons is soil containing polycyclic aromatic hydrocarbons, the amount of *Lactobacillus paracasei* or the inoculant used is such that the viable count in the soil containing polycyclic aromatic hydrocarbons is 10. 7 -10 9 When the pollutant containing polycyclic aromatic hydrocarbons is groundwater containing polycyclic aromatic hydrocarbons, the dosage of *Lactobacillus paracasei* or the bacterial agent is such that the viable count in the groundwater containing polycyclic aromatic hydrocarbons is 10cfu / g. 7 -10 9 cfu / mL.

[0020] Furthermore, the contact conditions include a temperature of 15-40°C and a pH value of 5-10.

[0021] The Lactobacillus paracasei of this invention is stable and can grow rapidly in soil or groundwater where polycyclic aromatic hydrocarbons (PAHs) are the sole carbon source. It exhibits excellent PAH degradation performance and can be widely applied to the remediation of soil or groundwater containing PAHs.

[0022] Other features and advantages of the present invention will be described in detail in the following detailed description section.

[0023] Biological Preservation Instructions

[0024] The biomaterial of this invention, *Lactobacillus paracasei* F1, is classified and named *Lactobacillus paracasei* (… Lactobacillus paracasei The specimen was deposited on March 2, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO.24463. Detailed Implementation

[0025] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0026] This invention provides a Lactobacillus paracasei ( Lactobacillus paracasei The *Lactobacillus paracasei* described herein has the accession number CGMCC No. 24463. The *Lactobacillus paracasei* (accession number CGMCC No. 24463) provided by this invention is a facultative anaerobic bacterium, Gram-positive, and was isolated from polycyclic aromatic hydrocarbon contaminated soil in Tianjin.

[0027] The specific screening and separation steps are as follows: (1) Domestication Add 3-5g of soil to a sterilized inorganic salt culture medium (150mL / 250mL Erlenmeyer flask), add phenanthrene as the sole carbon source, shake well, and incubate at 30℃ and 150rpm in a shaker. After 5 days, transfer 1mL of the culture solution to the culture flask, repeating at least 3 times.

[0028] (2) Separation

[0029] After diluting the acclimatized bacterial culture to a suitable concentration, the culture was separated by streak plating. Three replicates were prepared for each concentration gradient. After streaking, the culture dishes were placed in an incubator and incubated at 30°C for 3 days.

[0030] (3) Purification

[0031] Observe the various colonies growing on the agar plate surface, noting differences in size, shape, edge, surface structure, transparency, color, and other characteristics, and take photos for preservation. Repeatedly purify colonies that do not form single bacteria until the colonies on the same plate are uniform in size and morphology.

[0032] The purified *Lactobacillus paracasei* (named F1) was sent to Beijing Meiji Sanger Biomedical Technology Co., Ltd. for 16S rDNA strain identification. The sequence of the spliced ​​strain was compared with that obtained from NCBI analysis, showing that this strain is similar to *Lactobacillus paracasei* (…). Lactobacillus paracasei strain The gene sequence of the isolate showed 100% homology, and strain F1 was identified as *Lactobacillus paracasei*. Lactobacillus paracasei ).

[0033] The Lactobacillus paracasei F1 colonies provided by this invention are milky white, round, mostly with a diameter of more than 1.0 mm, with a smooth, raised surface and neat edges.

[0034] The inventors of this invention have discovered that *Lactobacillus paracasei* (CGMCC No. 24463) has good degradation activity against various types of polycyclic aromatic hydrocarbons (PAHs). Adding a bacterial agent containing *Lactobacillus paracasei* (CGMCC No. 24463) to soil or groundwater contaminated with PAHs can remediate PAH pollution in soil or groundwater.

[0035] The bacterial agent may also contain excipients. The excipients may be commonly used excipients in the art that will not affect the activity of Lactobacillus paracasei.

[0036] The preparation method of the bacterial agent can be a conventional preparation method in the art. Preferably, the preparation method includes: activating *Lactobacillus paracasei* as described above and culturing it in a culture medium to obtain a bacterial stock solution; centrifuging the bacterial stock solution to obtain bacterial cells and preparing a bacterial suspension with sterile water (the viable count in the bacterial suspension is typically 10^6). 9 The bacterial agent is obtained by using a concentration of cfu / mL or higher. The culture medium can be any medium commonly used in the art for culturing Lactobacillus paracasei.

[0037] The polycyclic aromatic hydrocarbons refer to aromatic hydrocarbons containing two or more benzene rings, including both fused-ring polycyclic aromatic hydrocarbons and non-fused-ring polycyclic aromatic hydrocarbons.

[0038] The fused-ring polycyclic aromatic hydrocarbons include, but are not limited to, at least one of naphthalene, anthracene, phenanthrene, pyrene, α, benzo[a]pyrene, benzo[a]anthracene, benzo[a]fluoranthracene, and ind[a]pyrene. The benzo[a]pyrene includes, for example, benzo[a]pyrene and benzo[e]pyrene. The benzo[a]anthracene includes, for example, benzo[a]anthracene and dibenzo[a,h]anthracene. The ind[a]pyrene includes, for example, ind[1,2,3-cd]pyrene.

[0039] The non-fused-ring polycyclic aromatic hydrocarbons include, but are not limited to, at least one of biphenyl (i.e., biphenyl), biphenylene oxide, and polyphenylene aliphatic hydrocarbons. The biphenylene oxide can be terphenyl, etc., and the polyphenylene aliphatic hydrocarbons can be diphenylmethane, diphenylethane, etc.

[0040] The Lactobacillus paracasei and its agent of the present invention are applicable to various pollutants containing polycyclic aromatic hydrocarbons, including but not limited to soil or groundwater containing polycyclic aromatic hydrocarbons.

[0041] In some embodiments of the present invention, when the pollutant containing polycyclic aromatic hydrocarbons (PAHs) is soil containing PAHs, the PAH content, calculated as PAHs, is 2-1000 mg / kg soil. For example, it can be any one of the following values, or a value within a range of any two of the following: 2 mg / kg soil, 5 mg / kg soil, 10 mg / kg soil, 50 mg / kg soil, 100 mg / kg soil, 200 mg / kg soil, 300 mg / kg soil, 500 mg / kg soil, 700 mg / kg soil, and 1000 mg / kg soil. The soil weight is on a dry basis.

[0042] In this invention, the amount of *Lactobacillus paracasei* F1 or the bacterial agent used can be selected within a wide range. In some embodiments of this invention, when the pollutant containing polycyclic aromatic hydrocarbons is soil containing polycyclic aromatic hydrocarbons, the amount of *Lactobacillus paracasei* F1 or the bacterial agent used is such that the viable count in the soil containing polycyclic aromatic hydrocarbons is 10. 7 -10 9 cfu / g. The soil weight is on a dry basis.

[0043] In some embodiments of the present invention, when the pollutant containing polycyclic aromatic hydrocarbons is groundwater containing polycyclic aromatic hydrocarbons, the concentration of polycyclic aromatic hydrocarbons is 2-1000 mg / L of groundwater, for example, it can be any one of the following values: 2 mg / L, 5 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 500 mg / L, 700 mg / L, or 1000 mg / L of groundwater, or a value within the range of any two of the above values.

[0044] In some embodiments of the present invention, when the pollutant containing polycyclic aromatic hydrocarbons is groundwater containing polycyclic aromatic hydrocarbons, the amount of *Lactobacillus paracasei* F1 or the bacterial agent used is such that the viable bacteria count in the groundwater containing polycyclic aromatic hydrocarbons is 10. 7 -10 9 cfu / mL.

[0045] The conditions under which Lactobacillus paracasei F1 or its inoculum comes into contact with pollutants containing polycyclic aromatic hydrocarbons are typically natural environmental conditions, such as a temperature of 15-40°C. The strains of this invention can degrade polycyclic aromatic hydrocarbons over a wide pH range of 5-10.

[0046] The present invention will be further described below with reference to the embodiments, but the scope of the present invention is not limited to these embodiments.

[0047] Soil model evaluation method for bioremediation of petroleum hydrocarbon contaminated soil

[0048] The effectiveness of bioremediation methods for petroleum hydrocarbon pollution in soil is ultimately evaluated by the degradation effect of petroleum hydrocarbons in the soil. Therefore, a soil model evaluation method for bioremediation of petroleum hydrocarbon-contaminated soil was established. The model evaluation method is as follows: ① Enriching the bacterial agent in an inorganic salt culture medium with the target pollutant as the sole carbon source; ② Centrifuging to obtain the enriched bacterial agent; ③ Introducing the bacterial agent into the soil along with water-retaining agents, soil conditioners, and nutrients; ④ Adding the target pollutant to the soil; ⑤ Regularly monitoring the petroleum hydrocarbon content and total number of bacteria in the soil to calculate the degradation effect of petroleum hydrocarbons.

[0049] The specific operating steps are as follows: 1. Take 1.5 mL of bacterial solution into an Erlenmeyer flask containing 150 mL of inorganic salt culture medium, then add the target contaminant, shake at 150 r / min for 5-10 days (depending on the amount of bacteria) to obtain the enriched bacterial solution.

[0050] 2. Centrifuge the obtained enriched bacterial solution at 8000 r / min to separate the solid and liquid layers, discard the upper culture medium solution, add 100 mL of inorganic salt culture medium, shake thoroughly, and centrifuge again at 8000 r / min to separate the solid and liquid layers, discard the upper solution, and keep the bottom solid for later use.

[0051] 3. Add water-retaining agent, soil conditioner, nutrients and bottom solids to the soil, mix thoroughly, and add deionized water at the same time to maintain the soil moisture content within a certain range.

[0052] 4. Add the target pollutant to the soil and mix well.

[0053] 5. Test the content of target pollutants in the soil and the total number of bacteria in the soil weekly.

[0054] 6. Water every two days to keep the soil moisture content within a certain range, and turn the soil 1-3 times a week.

[0055] Inorganic salt culture medium: NH4Cl 0.67g, NaNO3 1.06g, MgSO4·7H2O 0.1g, CaCl2 0.1g, K2HPO4·3H2O 1.5g, KH2PO4 0.5g, FeCl3·6H2O 0.1g, deionized water 1L, pH 7.0~7.5.

[0056] Analytical methods

[0057] The method for determining heterotrophic bacteria refers to the plate counting method in GB / T14643.1-1993 standard "Analysis Methods for Industrial Circulating Cooling Water".

[0058] The method for determining polycyclic aromatic hydrocarbon (PAH) content in soil was based on the standard "Determination of Semi-volatile Organic Compounds in Soil and Sediments by Gas Chromatography-Mass Spectrometry" (HJ 834-2017). Specifically, pressurized fluid extraction of PAHs was performed using a dichloromethane-acetone mixed solution. When the final volume was 1.0 mL and the injection volume was 1.0 μL, the method detection limit for PAHs was 0.06–0.3 mg / kg, and the lower limit of quantification was 0.24–1.20 mg / kg.

[0059] Methods for determining total soil bacteria count

[0060] The total number of bacteria in the soil was determined by plate culture colony counting, and the culture medium for soil bacteria was meat extract peptone medium.

[0061] The specific measurement steps are as follows: Weigh 10g of soil sample and place it in an Erlenmeyer flask containing 90mL of sterile deionized water. Place the flask on a shaker and shake at 160 times / min at room temperature for 4 hours to thoroughly mix the soil sample with the water, thus fully dispersing and separating the microbial cells from the soil. This is 10g. -1 Soil suspension: Take 1 mL of this soil suspension and add it to 9 mL of sterile water containing 8.5 g / L NaCl. Mix thoroughly by pipetting three times using a sterile pipette to prepare 10... -2 Soil suspension. Repeat this process to prepare 10... -3 10 -4 10 -5 10 -6 10 -7 10 -8 Soil suspensions at different dilutions. After selecting an appropriate dilution, the total bacterial count of the soil suspensions was determined using the mixed plate count method.

[0062] The total number of bacteria in the soil, x (cFU / g), is calculated using the following formula.

[0063]

[0064] In the formula: x — Total number of soil bacteria, cells / g; e – Total number of bacteria in the soil suspension, CFU / mL; V – Volume of soil suspension, mL; m — Sample size, g; w —Soil moisture content,%.

[0065] The composition of the meat extract peptone medium is as follows: NaCl 10g, peptone 10g, beef extract 5g, agar 18g, deionized water 1L, pH 7.2~7.4.

[0066] The following examples and comparative examples all adopted the following experimental methods: The soil used was dry soil sterilized at 250℃ for 24 hours. Sawdust was added to improve soil aeration; polyacrylic acid was added to improve soil water retention; and urea was added to supplement the soil's nitrogen source. All were mixed thoroughly. The solid portion of the bacterial solution was then fully mixed with deionized water and added to the soil. Finally, the target pollutant, polycyclic aromatic hydrocarbons (PAHs), was dissolved in 3 mL of acetone and added to the soil. The soil ambient temperature range was 18-23℃.

[0067] Turn the soil once a week and add deionized water until the soil moisture content is at its initial value. No other intervention measures are taken.

[0068] The method for obtaining the solid portion of the bacterial solution includes: (1) taking 1.5 mL of bacterial solution into an Erlenmeyer flask containing 150 mL of inorganic salt culture medium, adding the target pollutant as the sole carbon source, and placing it in a shaker at 30°C and 150 r / min for 5-10 days to obtain OD. 600 The enriched bacterial solution is 1.0. (2) Centrifuge the obtained enriched bacterial solution at 8000 r / min to separate the solid and liquid components, discard the upper culture medium solution, add 100 mL of inorganic salt culture medium, shake thoroughly, and centrifuge again at 8000 r / min to separate the solid and liquid components, discard the upper solution, and obtain the solid part of the bacterial solution.

[0069] The inorganic salt culture medium consists of: 0.67g NH4Cl, 1.06g NaNO3, 0.1g MgSO4·7H2O, 0.1g CaCl2, 1.5g K2HPO4·3H2O, 0.5g KH2PO4, 0.1g FeCl3·6H2O, and 1L deionized water, with a pH of 7.0~7.5.

[0070] In the following examples and comparative examples: Biphenyl was purchased from Beijing Innocare Technology Co., Ltd.

[0071] 1,4-Bis(triphenyl) was purchased from Beijing Innocare Technology Co., Ltd.

[0072] The diphenylmethane was purchased from Beijing Innocare Technology Co., Ltd.

[0073] Naphthalene was purchased from Beijing Innocare Technology Co., Ltd.

[0074] Anthracene was purchased from Beijing Innocare Technology Co., Ltd.

[0075] The product was purchased from Beijing Innocare Technology Co., Ltd.

[0076] The product was purchased from Beijing Innocare Technology Co., Ltd.

[0077] Benzo[a]pyrene was purchased from Beijing Innocare Technology Co., Ltd.

[0078] Oligotrophomonas was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC1.6393; Acinetobacter was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC1.10395. T ; Dietrichia coli was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC1.6332. T .

[0079] Example 1

[0080] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of biphenyl to the well-mixed soil.

[0081] Example 2

[0082] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of 1,4-benzene to the well-mixed soil.

[0083] Example 3

[0084] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of diphenylmethane to the well-mixed soil.

[0085] Example 4

[0086] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of naphthalene to the well-mixed soil.

[0087] Example 5

[0088] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of anthracene to the well-mixed soil.

[0089] Example 6

[0090] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of phenanthrene to the well-mixed soil.

[0091] Example 7

[0092] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of pyrene to the well-mixed soil.

[0093] Example 8

[0094] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Lactobacillus paracasei with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of benzo[a]pyrene to the well-mixed soil.

[0095] Comparative Example 1

[0096] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix oligotrophic bacteria with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of biphenyl to the well-mixed soil.

[0097] Comparative Example 2

[0098] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Acinetobacter bacillus with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of diphenylmethane to the well-mixed soil.

[0099] Comparative Example 3

[0100] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Dietariformis bacteria with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of phenanthrene to the well-mixed soil.

[0101] Comparative Example 4

[0102] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Mix Dietrichia coli with 300g of deionized water to form aqueous solution 3. Weigh 45g of sawdust and mix it with 2400g of soil. Then add aqueous solutions 1, 2, and 3 and mix thoroughly. Add 150mg of benzo[a]pyrene to the well-mixed soil.

[0103] Comparative Example 5

[0104] Weigh 6g of polyacrylic acid and dissolve it in 200g of deionized water to form aqueous solution 1. Weigh 0.96g of urea and dissolve it in 100g of deionized water to form aqueous solution 2. Weigh 45g of sawdust and mix it into 2400g of soil. Then add aqueous solutions 1 and 2 and 300g of deionized water and mix well. Add 150mg of benzo[a]pyrene to the well-mixed soil.

[0105] Test Example 1

[0106] Soil samples from the above examples and comparative examples were taken periodically to measure the residual polycyclic aromatic hydrocarbon (PAH) content in the soil samples and to investigate the PAH degradation. The results are shown in Table 1.

[0107] Table 1. Polycyclic aromatic hydrocarbon content in soil samples from the examples and comparisons

[0108] Note: The unit for polycyclic aromatic hydrocarbon content is mg / kg.

[0109] As can be seen from the results in Table 1, the *Lactobacillus paracasei* of the present invention has a significant degradation effect on various types of polycyclic aromatic hydrocarbons, and is superior to the comparative strain.

[0110] Test Example 2

[0111] Soil samples from the above examples and comparative examples were taken periodically to measure the total number of bacteria in the soil samples and to investigate the microbial growth. The results are shown in Table 2.

[0112] Table 2. Soil microbial growth in the examples and comparative examples

[0113] Note: Total bacterial count is measured in units of 10⁻⁶. 6 cfu / g soil.

[0114] As shown in Table 2, the growth of Lactobacillus paracasei in the soil reached its peak at 21 days and decreased to a level comparable to that at 7 days by 49 days. During this period, the content of polycyclic aromatic hydrocarbons (PAHs) in the soil samples continued to decrease, indicating that the decrease in PAH content in the soil was due to the degradation by Lactobacillus paracasei.

[0115] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

[0116] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

Claims

1. A type of Lactobacillus paracasei ( Lactobacillus paracasei ), characterized in that, The Lactobacillus paracasei has the accession number CGMCC No. 24463.

2. A microbial agent, characterized in that, The microbial agent contains Lactobacillus paracasei as described in claim 1.

3. The application of Lactobacillus paracasei according to claim 1 or the bacterial agent according to claim 2 in the degradation of polycyclic aromatic hydrocarbons, wherein the polycyclic aromatic hydrocarbons are fused-ring polycyclic aromatic hydrocarbons or non-fused-ring polycyclic aromatic hydrocarbons.

4. The application according to claim 3, wherein, The application method includes contacting the Lactobacillus paracasei of claim 1 or the bacterial agent of claim 2 with a pollutant containing polycyclic aromatic hydrocarbons.

5. The application according to claim 3, wherein, The fused-ring polycyclic aromatic hydrocarbon is selected from at least one of naphthalene, anthracene, phenanthrene, pyrene and benzo[a]pyrene.

6. The application according to claim 3, wherein, The non-fused-ring polycyclic aromatic hydrocarbon is selected from at least one of biphenyl, 1,4-triphenyl and diphenylmethane.

7. The application according to claim 4, wherein, The pollutants containing polycyclic aromatic hydrocarbons are soil or groundwater containing polycyclic aromatic hydrocarbons.

8. The application according to claim 7, wherein, In the soil containing polycyclic aromatic hydrocarbons, the content of polycyclic aromatic hydrocarbons is 2-1000 mg / kg soil, calculated as polycyclic aromatic hydrocarbons. The groundwater containing polycyclic aromatic hydrocarbons (PAHs) has a PAH content of 2-1000 mg / L.

9. The application according to claim 7, wherein, When the pollutant containing polycyclic aromatic hydrocarbons is soil containing polycyclic aromatic hydrocarbons, the amount of *Lactobacillus paracasei* or the inoculant used is such that the viable count in the soil containing polycyclic aromatic hydrocarbons is 10. 7 -10 9 cfu / g; When the pollutant containing polycyclic aromatic hydrocarbons is groundwater containing polycyclic aromatic hydrocarbons, the dosage of the *Lactobacillus paracasei* or the bacterial agent is such that the viable bacteria count in the groundwater containing polycyclic aromatic hydrocarbons is 10. 7 -10 9 cfu / mL.

10. The application according to claim 4, wherein, The contact conditions include a temperature of 15-40°C and a pH value of 5-10.

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