Pseudomonas kamiae, bacterial agent, and application and method for degrading polycyclic aromatic hydrocarbons

Abstract

The application belongs to the technical field of soil and groundwater pollution remediation and bioengineering, and relates to a Pseudomonas koreensis, a bacterial agent, and a method for degrading polycyclic aromatic hydrocarbons. The preservation number of the Pseudomonas koreensis is CGMCC No.22735. The Pseudomonas koreensis or the bacterial agent has high polycyclic aromatic hydrocarbon degradation capacity, can be used for remediation of polycyclic aromatic hydrocarbon contaminated soil or groundwater, and has a good application prospect in green and sustainable remediation of polycyclic aromatic hydrocarbon contaminated sites.

Description

Technical Field

[0001] This invention belongs to the fields of soil and groundwater pollution remediation technology and bioengineering technology. Specifically, it relates to a Pseudomonas geniculateus, an agent containing the Pseudomonas geniculateus, the application of the Pseudomonas geniculateus and the agent in degrading polycyclic aromatic hydrocarbons (PAHs) and remediating PAH-contaminated soil or PAH-contaminated groundwater, and a method for degrading PAHs. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are widespread organic pollutants in the environment, possessing carcinogenic, teratogenic, and mutagenic effects. They easily accumulate in the environment, posing long-term risks to the ecological environment and human health. Currently, commonly used PAH remediation methods mainly include thermal desorption, chemical oxidation, and biodegradation. Compared to physical and chemical remediation, bioremediation has advantages such as low cost, simple operation, and minimal secondary pollution, making it a green and sustainable environmental governance technology.

[0003] CN113755338B discloses a polycyclic aromatic hydrocarbon (PAH) degrading bacterium, *Pyronema domesticum* LJD-1, its inoculum agent, and its applications. This strain was domesticated and isolated from soil samples from an oil-contaminated site and is capable of utilizing phenanthrene as a carbon source. After culturing for 7 days in an inorganic salt culture medium with an initial phenanthrene concentration of 50 mg / L, the phenanthrene degradation rate reached 79.1%; when prepared as an inoculum agent, the phenanthrene degradation rate was 61.0%.

[0004] CN104004684B discloses a polycyclic aromatic hydrocarbon (PAH) degrading bacterium, Bacillus sp. s8-t8-L9, and its applications. This strain can utilize PAHs such as naphthalene, phenanthrene, and pyrene, as well as diesel oil, as its sole energy and carbon source for growth and reproduction. Within 20 days, it can degrade naphthalene, phenanthrene, pyrene, and diesel oil in an inorganic salt culture medium by 58.46%, 52.14%, 23.14%, and 47.54%, respectively.

[0005] CN106434470B discloses a polycyclic aromatic hydrocarbon (PAH) degrading bacterium, Rhodococcus sp. YE-WL-2, and its applications. This strain can grow in solutions with fluorescein concentrations up to 300 mg / L. When the initial fluorescein concentration is 50 mg / L, the strain can degrade more than 40% of the fluorescein in the culture medium within 7 days, achieve a degradation rate of more than 50% after 14 days of cultivation, and achieve a degradation rate of more than 60% after 21 days of cultivation.

[0006] In existing technologies, most strains achieve a degradation rate of less than 80% for polycyclic aromatic hydrocarbons (PAHs), and this low remediation efficiency has become a bottleneck in the application of microbial remediation technology. Therefore, identifying highly efficient PAH-degrading bacteria is of great significance for promoting the application of microbial remediation. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of low degradation rate of polycyclic aromatic hydrocarbons (PAHs) and low remediation efficiency of PAH-contaminated soil or groundwater in existing technologies. This invention provides a *Pseudomonas geniculates* bacterium, a bacterial agent, a method for degrading PAHs, and its applications. Using the *Pseudomonas geniculates* bacterium or bacterial agent of this invention for PAH degradation results in high degradation efficiency.

[0008] To achieve the above objectives, the first aspect of the present invention provides a *Pseudomonas geniculata*, the preservation number of which is CGMCC No. 22735.

[0009] A second aspect of the present invention provides a bacterial agent containing *Pseudomonas geniculateus* as described above.

[0010] A third aspect of the present invention provides the use of the above-described Pseudomonas geniculateus or the above-described bacterial agent in the degradation of polycyclic aromatic hydrocarbons.

[0011] The fourth aspect of the present invention provides the application of the above-described Pseudomonas geniculates or the above-described bacterial agents in the remediation of polycyclic aromatic hydrocarbon (PAH) contaminated soil or PAH contaminated groundwater.

[0012] The fifth aspect of the present invention provides a method for degrading polycyclic aromatic hydrocarbons, the method comprising: contacting the above-described Pseudomonas geniculateus or the above-described bacterial agent with a pollutant containing polycyclic aromatic hydrocarbons.

[0013] The *Pseudomonas geniculates* or its agent described in this invention exhibits a high degradation rate of polycyclic aromatic hydrocarbons (PAHs). Specifically, in an inorganic salt liquid culture medium with a phenanthrene content of 100 mg / L, at a temperature of 30.0 ± 2.0 °C and a pH of 7.0 ± 0.2, the *Pseudomonas geniculates* achieves a phenanthrene degradation rate of over 80% in 3 days and over 95% in 5 days. In contaminated soil with a phenanthrene content of 100 mg / kg, at a temperature of 30.0 ± 2.0 °C and a pH of 7.0 ± 0.2, the phenanthrene degradation rate reaches over 90% in 14 days.

[0014] The *Pseudomonas geniculata* or its agent of the present invention has a highly efficient ability to degrade polycyclic aromatic hydrocarbons (PAHs) and can be used for the remediation of PAH-contaminated soil or groundwater. It has good application prospects for the green and sustainable remediation of PAH-contaminated sites.

[0015] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0016] Exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0017] Figure 1The colony morphology of the geniculate pseudomonas NAP-1 of the present invention on an agar solid medium plate is shown.

[0018] Biological Preservation Instructions

[0019] The *Pseudomonas geniculata* NAP-1 of this invention was deposited on June 21, 2021, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) (CGMCC), with accession number CGMCCNo. 22735. Detailed Implementation

[0020] 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.

[0021] The first aspect of the present invention provides a Pseudomonas geniculata, the preservation number of which is CGMCC No. 22735.

[0022] The *Pseudomonas geniculata* strain of this invention was screened from soil contaminated with polycyclic aromatic hydrocarbons in the Tianjin area.

[0023] The screening method includes: collecting contaminated soil, adding it to sterilized deionized water to prepare an inoculum; inoculating the inoculum into an inorganic salt culture medium with phenanthrene as the sole carbon source, and subculturing it 3-5 times; serially diluting the above culture medium and streaking it continuously until a single colony is isolated; scaling up the above single colony using LB medium, centrifuging to obtain bacterial cells, which are the strains to be identified. Resuspending the obtained bacterial cells in sterilized physiological saline to obtain a bacterial suspension, mixing the bacterial suspension in 70% glycerol at a 1:1 volume ratio, and storing it at -80°C.

[0024] The bacterial strains to be identified exhibited regular colony morphology on agar solid medium plates, appearing as yellow, round, bead-like protrusions. Individual colonies were relatively large, with smooth, moist, glossy, and opaque surfaces. Figure 1 As shown. The species of this strain was identified by 16S rDNA sequencing, and it was determined to be *Pseudomonas geniculateus*, named NAP-1.

[0025] The *Pseudomonas geniculateus* NAP-1 was deposited at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) with accession number CGMCC No. 22735 and deposit date of June 21, 2021.

[0026] A second aspect of the present invention provides a bacterial agent containing *Pseudomonas geniculateus* as described above.

[0027] In some embodiments of the present invention, the bacterial agent is a liquid bacterial agent and / or a solid bacterial agent. In the bacterial agent of the present invention, the concentration of *Pseudomonas geniculateus* is not particularly limited and can be selected according to specific circumstances. In some embodiments of the present invention, the viable count in the liquid bacterial agent is 10-1. 9 CFU / mL or higher. In some embodiments of the present invention, the viable count in the solid bacterial agent is 10-1. 9 CFU / g or higher.

[0028] In some embodiments of the present invention, the bacterial agent may also contain excipients. The excipients may be commonly used excipients in the art that will not affect the activity of *Pseudomonas geniculateus*.

[0029] Furthermore, the microbial agent provided by this invention can be prepared into different dosage forms, such as powders, ointments, and liquids, depending on the intended use.

[0030] The preparation method of the bacterial agent can be a conventional preparation method in the art. Preferably, the preparation method includes: activating the *Pseudomonas geniculates* 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 to achieve a viable count of 102. 9 A bacterial agent is obtained by obtaining a concentration of cfu / mL or higher. The culture medium can be a conventional medium used in the art for culturing *Pseudomonas geniculateus*, such as LB medium. The inventors have discovered that the *Pseudomonas geniculateus* of this invention can be cultured in LB medium. Preferably, for reasons of production cost and ease of operation, the culture medium is LB medium.

[0031] In this invention, the culture conditions can be those commonly known in the art for the culture of Pseudomonas geniculateus, and this invention does not limit them.

[0032] A third aspect of the present invention provides the use of the above-described Pseudomonas geniculateus or the above-described bacterial agent in the degradation of polycyclic aromatic hydrocarbons.

[0033] The fourth aspect of the present invention provides the application of the above-described Pseudomonas geniculates or the above-described bacterial agents in the remediation of polycyclic aromatic hydrocarbon (PAH) contaminated soil or PAH contaminated groundwater.

[0034] The fifth aspect of the present invention provides a method for degrading polycyclic aromatic hydrocarbons, the method comprising: contacting the above-described Pseudomonas geniculateus or the above-described bacterial agent with a pollutant containing polycyclic aromatic hydrocarbons.

[0035] In this invention, the polycyclic aromatic hydrocarbon refers to at least one of the aromatic hydrocarbons containing at least two benzene rings, including both fused-ring polycyclic aromatic hydrocarbons and non-fused-ring polycyclic aromatic hydrocarbons.

[0036] Specifically, the polycyclic aromatic hydrocarbons include, but are not limited to, naphthalene, anthracene, phenanthrene, pyrene, etc. At least one of benzo[a]pyrene, benzo[a]anthracene, benzo[a]fluoranthracene, ind[a]pyrene, biphenyl (i.e., diphenyl), biphenyls, and polyphenylalanine hydrocarbons. 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. The biphenyls may be terphenyl, etc., and the polyphenylalanine hydrocarbons may be diphenylmethane, diphenylethane, etc.

[0037] In some embodiments of the present invention, the pollutant containing polycyclic aromatic hydrocarbons is soil containing polycyclic aromatic hydrocarbons or groundwater containing polycyclic aromatic hydrocarbons.

[0038] 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.

[0039] In this invention, the amount of *Pseudomonas geniculateus* or the inoculant 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 *Pseudomonas geniculateus* or the inoculant is such that the viable count in the soil containing polycyclic aromatic hydrocarbons is 10. 6 -10 8 cfu / g. The soil weight is on a dry basis.

[0040] In some embodiments of the present invention, the contact conditions include: a temperature of 20-40°C and a pH value of 5-10. The temperature can be any one of 20°C, 23°C, 26°C, 28°C, 30°C, 33°C, 35°C, 38°C, or 40°C, or a value within the range of any two of these values. The pH can be any one of 5.0, 5.3, 5.5, 5.7, 6.0, 6.3, 6.6, 6.8, 7.0, 7.2, 7.5, 7.8, 8.0, 8.3, 8.6, 8.8, 9.0, 9.2, 9.4, 9.8, or 10.0, or a value within the range of any two of these values. Preferably, to further improve the degradation rate of polycyclic aromatic hydrocarbons, the contact conditions are: a temperature of 25-38°C and a pH of 5.8-9.2. The inventors discovered that when the contact conditions are within the preferred range described above, the degradation rate of polycyclic aromatic hydrocarbons can be further improved.

[0041] 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.

[0042] In some embodiments of the present invention, when the pollutant containing polycyclic aromatic hydrocarbons is groundwater containing polycyclic aromatic hydrocarbons, the amount of *Pseudomonas geniculates* or the bacterial agent used is such that the viable count in the groundwater containing polycyclic aromatic hydrocarbons is 10. 6 -10 8 cfu / mL.

[0043] The present invention will be described in detail below through examples. Unless otherwise specified, all reagents and raw materials used in the following examples are commercially available, and all methods used are conventional methods in the art.

[0044] In the following embodiments:

[0045] LB medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, deionized water 1 L.

[0046] Inorganic salt culture medium: NH4Cl 0.67g / L, NaNO3 1.06g / L, MgSO4·7H2O 0.1g / L, CaCl2 0.1g / L, K2HPO4·3H2O 1.5g / L, KH2PO4 0.5g / L, FeCl3·6H2O 0.1g / L, deionized water 1L.

[0047] The product was purchased from Beijing Innocare Technology Co., Ltd.

[0048] Naphthalene was purchased from Beijing Innocare Technology Co., Ltd.

[0049] Purchased from Beijing Innocare Technology Co., Ltd.

[0050] Benzo[a]anthracene was purchased from Beijing Innocare Technology Co., Ltd.

[0051] Benzo[b]fluoranthene was purchased from Beijing Innocare Technology Co., Ltd.

[0052] Benzo[a]pyrene was purchased from Beijing Innocare Technology Co., Ltd.

[0053] Dibenzo[a,h]anthracene was purchased from Beijing Innocare Technology Co., Ltd.

[0054] Indo[1,2,3-cd]pyrene was purchased from Beijing Innocare Technology Co., Ltd.

[0055] The strain Pyronema domesticum LJD-1 was purchased from the Guangdong Provincial Microbial Culture Collection Center.

[0056] The strain Bacillus sp.s8-t8-L9 was purchased from the China General Microbiological Culture Collection Center.

[0057] Pseudomonas geniculata NAP-1 was deposited on June 21, 2021, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) (CGMCC accession number CGMCC No. 22735).

[0058] The detection method for polycyclic aromatic hydrocarbons (PAHs) in contaminated soil was based on the standard HJ 834-2017, "Determination of Semi-volatile Organic Compounds in Soils and Sediments by Gas Chromatography-Mass Spectrometry". Specifically, pressurized fluid extraction of PAHs was performed using a dichloromethane-acetone mixed solution. With a final volume of 1.0 mL and an injection volume of 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] The detection method for polycyclic aromatic hydrocarbon concentration in inorganic salt culture medium was carried out in accordance with the "Determination of Polycyclic Aromatic Hydrocarbons in Water by Liquid-Liquid Extraction and Solid-Phase Extraction by High Performance Liquid Chromatography" (HJ 478-2009).

[0060] The degradation rate (%) of polycyclic aromatic hydrocarbons (PAHs) in inorganic salt culture medium = (initial PAH concentration - remaining PAH concentration) / initial PAH concentration. Wherein, the initial PAH concentration refers to the PAH concentration in the inorganic salt culture medium without inoculated bacterial suspension.

[0061] The degradation rate (%) of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil = (initial PAH content - remaining PAH content) / initial PAH content. Wherein, the initial PAH content refers to the PAH content in the contaminated soil without inoculation with bacterial suspension.

[0062] Example 1

[0063] This example illustrates the degradation effect of Pseudomonas geniculatus NAP-1 on phenanthrene.

[0064] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated groundwater: A phenanthrene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane had completely evaporated, it was added to a sterile inorganic salt culture medium to make the phenanthrene concentration 100 mg / L, thus obtaining an inorganic salt culture medium with a phenanthrene concentration of 100 mg / L.

[0065] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0066] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9CFU was inoculated into an inorganic salt medium with a phenanthrene concentration of 100 mg / L. Temperature and pH were used as influencing factors to set up experimental and control groups (the control group was not inoculated with *Pseudomonas geniculates* NAP-1). The residual phenanthrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for phenanthrene in the inorganic salt medium by *P. geniculates* NAP-1 were determined, as well as the degradation effect after 5 days under the optimal conditions. The results showed that the optimal degradation conditions for *P. geniculates* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The phenanthrene degradation effects of *P. geniculates* NAP-1 under the optimal conditions (temperature 30.0 ± 2.0℃, pH 7.0 ± 0.2) after 3 and 5 days are shown in Table 1.

[0067] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was taken, pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of phenanthrene was weighed and dissolved in n-hexane solution, and sprayed evenly onto the soil sample to make the phenanthrene content 100 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a phenanthrene content of 100 mg / kg was obtained.

[0068] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into contaminated soil with a phenanthrene content of 100 mg / kg. Temperature and pH were used as influencing factors. Experimental and control groups were established (the control group was not inoculated with *Pseudomonas geniculata* NAP-1). The remaining phenanthrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for phenanthrene in contaminated soil by *P. geniculata* NAP-1 were determined, as well as the degradation effect after 20 days under these optimal conditions. The results showed that the optimal degradation conditions for *P. geniculata* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The phenanthrene degradation effects of *P. geniculata* NAP-1 after 14 and 20 days under the optimal conditions (temperature 30.0 ± 2.0℃, pH 7.0 ± 0.2) are shown in Table 1.

[0069] Example 2

[0070] This example illustrates the degradation effect of Pseudomonas geniculateus NAP-1 on naphthalene.

[0071] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated groundwater: A naphthalene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane had completely evaporated, it was added to a sterile inorganic salt culture medium to make the naphthalene concentration 100 mg / L, thus obtaining an inorganic salt culture medium with a naphthalene concentration of 100 mg / L.

[0072] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0073] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into an inorganic salt medium with a naphthalene concentration of 100 mg / L. Temperature and pH were considered as influencing factors. An experimental group and a control group (the control group was not inoculated with *Pseudomonas geniculates* NAP-1) were established. The remaining naphthalene content was periodically monitored, and the degradation rate of polycyclic aromatic hydrocarbons (PAHs) was calculated. The optimal degradation conditions for naphthalene in the inorganic salt medium by *Pseudomonas geniculates* NAP-1 were determined, as well as the degradation effect after 5 days under these optimal conditions. The results showed that the optimal degradation conditions for *Pseudomonas geniculates* NAP-1 were 30.0 ± 2.0 °C and pH 7.0 ± 0.2. The naphthalene degradation effects of *Pseudomonas geniculates* NAP-1 under the optimal conditions (30.0 ± 2.0 °C, pH 7.0 ± 0.2) after 3 and 5 days are shown in Table 1.

[0074] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of naphthalene was weighed and dissolved in n-hexane solution, and then evenly sprayed onto the soil sample to make the naphthalene content 100 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a naphthalene content of 100 mg / kg was obtained.

[0075] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into contaminated soil with a naphthalene content of 100 mg / kg. Temperature and pH were used as influencing factors. An experimental group and a control group (the control group was not inoculated with *Pseudomonas geniculata* NAP-1) were established. The remaining naphthalene content was periodically monitored, and the degradation rate of polycyclic aromatic hydrocarbons (PAHs) was calculated. The optimal degradation conditions for naphthalene in contaminated soil by *Pseudomonas geniculata* NAP-1 and the degradation effect after 20 days under the optimal conditions were determined. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The naphthalene degradation effects of *Pseudomonas geniculata* NAP-1 after 14 and 20 days under the optimal conditions (temperature 30.0 ± 2.0℃, pH 7.0 ± 0.2) are shown in Table 1.

[0076] Example 3

[0077] This example illustrates the effect of Pseudomonas geniculate nucleus NAP-1 on... The degradation effect.

[0078] Preparation of simulated polycyclic aromatic hydrocarbon-contaminated groundwater: using n-hexane The solution was filtered through a sterile membrane and added to a sterile conical flask. After the hexane had completely evaporated, it was added to a sterile inorganic salt culture medium. The concentration was 100 mg / L, and the result was... Inorganic salt culture medium with a concentration of 100 mg / L.

[0079] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0080] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was administered to the above-mentioned In an inorganic salt culture medium with a concentration of 100 mg / L, with temperature and pH as influencing factors, experimental and control groups were set up (the control group was not inoculated with Pseudomonas geniculates NAP-1). Residual bacteria were periodically monitored. Content, calculation of polycyclic aromatic hydrocarbon degradation rate, and determination of the effect of Pseudomonas geniculate nucleus NAP-1 on inorganic salt culture medium. The optimal degradation conditions and degradation effect after 5 days under optimal conditions were investigated. The results showed that the optimal degradation conditions for *Pseudomonas geniculateus* NAP-1 were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of *Pseudomonas geniculateus* NAP-1 under optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) was observed after 5 days. The degradation effect is shown in Table 1.

[0081] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then air-dried. A certain amount was weighed out. Dissolved in n-hexane solution, and evenly sprayed onto the above soil sample. The content is 100 mg / kg (wherein, the weight of the soil is on a dry basis), obtained after the hexane has completely volatilized. Contaminated soil with a content of 100 mg / kg.

[0082] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was administered to the above-mentioned In contaminated soil with a concentration of 100 mg / kg, experimental and control groups were set up, with temperature and pH as influencing factors (the control group was not inoculated with Pseudomonas geniculates NAP-1). Residual levels were periodically monitored. Content, calculation of polycyclic aromatic hydrocarbon degradation rate, and determination of the effect of Pseudomonas geniculates NAP-1 on contaminated soil The optimal degradation conditions and degradation effect after 20 days under these optimal conditions were investigated. The results showed that the optimal degradation conditions for *Pseudomonas geniculateus* NAP-1 were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of *Pseudomonas geniculateus* NAP-1 under these optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) for 20 days was determined. The degradation effect is shown in Table 1.

[0083] Example 4

[0084] This example illustrates the degradation effect of Pseudomonas geniculateus NAP-1 on benzo[a]anthracene.

[0085] Preparation of simulated polycyclic aromatic hydrocarbon contaminated groundwater: A benzo[a]anthracene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane was completely evaporated, it was added to a sterile inorganic salt culture medium to make the benzo[a]anthracene concentration 100 mg / L, thus obtaining an inorganic salt culture medium with a benzo[a]anthracene concentration of 100 mg / L.

[0086] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0087] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into an inorganic salt medium containing 100 mg / L of benzo[a]anthracene. Temperature and pH were used as influencing factors. An experimental group and a control group (the control group was not inoculated with *Pseudomonas geniculata* NAP-1) were established. The remaining benzo[a]anthracene content was periodically measured, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for benzo[a]anthracene in the inorganic salt medium by *Pseudomonas geniculata* NAP-1 were determined, as well as the degradation effect after 5 days under these optimal conditions. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on benzo[a]anthracene under the optimal conditions (temperature 30.0 ± 2.0℃, pH 7.0 ± 0.2) for 5 days is shown in Table 1.

[0088] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of benzo[a]anthracene was weighed and dissolved in n-hexane solution, and sprayed evenly onto the soil sample to make the benzo[a]anthracene content 100 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a benzo[a]anthracene content of 100 mg / kg was obtained.

[0089] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into contaminated soil containing 100 mg / kg of benzo[a]anthracene. Temperature and pH were used as influencing factors. Experimental and control groups were established (the control group was not inoculated with *Pseudomonas geniculata* NAP-1). The remaining benzo[a]anthracene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for benzo[a]anthracene in contaminated soil by *Pseudomonas geniculata* NAP-1 and its degradation effect after 20 days under these optimal conditions were determined. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on benzo[a]anthracene under the optimal conditions (30.0±2.0℃, pH 7.0±0.2) for 20 days is shown in Table 1.

[0090] Example 5

[0091] This example illustrates the degradation effect of Pseudomonas geniculateus NAP-1 on benzo[b]fluoranthene.

[0092] Preparation of simulated polycyclic aromatic hydrocarbon contaminated groundwater: A benzo[b]fluoranthene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane was completely evaporated, it was added to a sterile inorganic salt culture medium to make the benzo[b]fluoranthene concentration 70 mg / L, thus obtaining an inorganic salt culture medium with a benzo[b]fluoranthene concentration of 70 mg / L.

[0093] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0094] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into an inorganic salt medium containing 70 mg / L benzo[b]fluoranthene. Temperature and pH were used as influencing factors. An experimental group and a control group (the control group was not inoculated with *Pseudomonas geniculata* NAP-1) were established. The remaining benzo[b]fluoranthene content was periodically measured, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for benzo[b]fluoranthene in the inorganic salt medium by *Pseudomonas geniculata* NAP-1 were determined, as well as the degradation effect after 5 days under these optimal conditions. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on benzo[b]fluoranthene under the optimal conditions (30.0 ± 2.0℃, pH 7.0 ± 0.2) for 5 days is shown in Table 1.

[0095] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of benzo[b]fluoranthene was weighed and dissolved in n-hexane solution, and then evenly sprayed onto the soil sample to make the benzo[b]fluoranthene content 70 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a benzo[b]fluoranthene content of 70 mg / kg was obtained.

[0096] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into contaminated soil containing 70 mg / kg of benzo[b]fluoranthene. Temperature and pH were used as influencing factors. Experimental and control groups were established (the control group was not inoculated with *Pseudomonas geniculata* NAP-1). The remaining benzo[b]fluoranthene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for benzo[b]fluoranthene in contaminated soil by *Pseudomonas geniculata* NAP-1 and the degradation effect after 20 days under these optimal conditions were determined. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on benzo[b]fluoranthene under the optimal conditions (30.0±2.0℃, pH 7.0±0.2) for 20 days is shown in Table 1.

[0097] Example 6

[0098] This example illustrates the degradation effect of Pseudomonas geniculateus NAP-1 on benzo[a]pyrene.

[0099] Preparation of simulated polycyclic aromatic hydrocarbon contaminated groundwater: A benzo[a]pyrene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane was completely evaporated, it was added to a sterile inorganic salt culture medium to make the benzo[a]pyrene concentration 30 mg / L, thus obtaining an inorganic salt culture medium with a benzo[a]pyrene concentration of 30 mg / L.

[0100] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0101] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9CFU was inoculated into an inorganic salt medium containing 30 mg / L benzo[a]pyrene. Temperature and pH were considered as influencing factors. An experimental group and a control group (the control group was not inoculated with *Pseudomonas geniculates* NAP-1) were established. The remaining benzo[a]pyrene content was periodically measured, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for benzo[a]pyrene in the inorganic salt medium by *Pseudomonas geniculates* NAP-1 were determined, as well as the degradation effect after 5 days under these optimal conditions. The results showed that the optimal degradation conditions for *Pseudomonas geniculates* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The degradation effect of *Pseudomonas geniculates* NAP-1 on benzo[a]pyrene under the optimal conditions (30.0 ± 2.0℃, pH 7.0 ± 0.2) for 5 days is shown in Table 1.

[0102] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of benzo[a]pyrene was weighed and dissolved in n-hexane solution, and sprayed evenly onto the soil sample to make the benzo[a]pyrene content 30 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a benzo[a]pyrene content of 30 mg / kg was obtained.

[0103] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into contaminated soil containing 30 mg / kg of benzo[a]pyrene. Temperature and pH were used as influencing factors. Experimental and control groups were established (the control group was not inoculated with *Pseudomonas geniculata* NAP-1). The residual benzo[a]pyrene content was periodically monitored, and the degradation rate of polycyclic aromatic hydrocarbons (PAHs) was calculated. The optimal degradation conditions for benzo[a]pyrene in contaminated soil by *Pseudomonas geniculata* NAP-1 and its degradation effect after 20 days under these optimal conditions were determined. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on benzo[a]pyrene after 20 days under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) is shown in Table 1.

[0104] Example 7

[0105] This example illustrates the degradation effect of Pseudomonas geniculateus NAP-1 on dibenzo[a,h]anthracene.

[0106] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated groundwater: A dibenzo[a,h]anthracene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane had completely evaporated, it was added to a sterile inorganic salt culture medium to make the dibenzo[a,h]anthracene concentration 30 mg / L, thus obtaining an inorganic salt culture medium with a dibenzo[a,h]anthracene concentration of 30 mg / L.

[0107] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0108] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into an inorganic salt medium containing 30 mg / L dibenzo[a,h]anthracene. Temperature and pH were used as influencing factors. An experimental group and a control group (the control group was not inoculated with *Pseudomonas geniculata* NAP-1) were established. The remaining dibenzo[a,h]anthracene content was periodically measured, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for dibenzo[a,h]anthracene in the inorganic salt medium by *Pseudomonas geniculata* NAP-1 were determined, as well as the degradation effect after 5 days under these optimal conditions. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on dibenzo[a,h]anthracene after 5 days under the optimal conditions (temperature 30.0 ± 2.0℃, pH 7.0 ± 0.2) is shown in Table 1.

[0109] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of dibenzo[a,h]anthracene was weighed and dissolved in n-hexane solution, and sprayed evenly onto the soil sample to make the dibenzo[a,h]anthracene content 30 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a dibenzo[a,h]anthracene content of 30 mg / kg was obtained.

[0110] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into contaminated soil containing 30 mg / kg of dibenzo[a,h]anthracene. Temperature and pH were used as influencing factors. An experimental group and a control group (the control group was not inoculated with *Pseudomonas geniculata* NAP-1) were established. The remaining dibenzo[a,h]anthracene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for dibenzo[a,h]anthracene in contaminated soil by *Pseudomonas geniculata* NAP-1 and the degradation effect under these optimal conditions for 20 days were determined. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on dibenzo[a,h]anthracene under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) for 20 days is shown in Table 1.

[0111] Example 8

[0112] This example illustrates the degradation effect of Pseudomonas geniculateus NAP-1 on indo[1,2,3-cd]pyrene.

[0113] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated groundwater: Indo[1,2,3-cd]pyrene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane had completely evaporated, it was added to a sterile inorganic salt culture medium to achieve an indo[1,2,3-cd]pyrene concentration of 30 mg / L, thus obtaining an inorganic salt culture medium with an indo[1,2,3-cd]pyrene concentration of 30 mg / L.

[0114] After activating the preserved Pseudomonas geniculateae NAP-1 cells, the bacterial stock solution was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10⁻⁶. 9 cfu / mL.

[0115] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into an inorganic salt medium containing 30 mg / L indo[1,2,3-cd]pyrene. Temperature and pH were used as influencing factors. Experimental and control groups were established (the control group was not inoculated with *Pseudomonas geniculata* NAP-1). The remaining indo[1,2,3-cd]pyrene content was periodically measured, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for indo[1,2,3-cd]pyrene in the inorganic salt medium by *Pseudomonas geniculata* NAP-1 were determined, as well as the degradation effect after 5 days under these optimal conditions. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0 ± 2.0℃ and pH 7.0 ± 0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on indo[1,2,3-cd]pyrene after 5 days under the optimal conditions (30.0 ± 2.0℃, pH 7.0 ± 0.2) is shown in Table 1.

[0116] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of indene[1,2,3-cd]pyrene was weighed and dissolved in n-hexane solution, and then evenly sprayed onto the soil sample to achieve an indene[1,2,3-cd]pyrene content of 30 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with an indene[1,2,3-cd]pyrene content of 30 mg / kg was obtained.

[0117] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9CFU was inoculated into contaminated soil containing 30 mg / kg of indo[1,2,3-cd]pyrene. Temperature and pH were used as influencing factors. Experimental and control groups were established (the control group was not inoculated with *Pseudomonas geniculata* NAP-1). The remaining indo[1,2,3-cd]pyrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for indo[1,2,3-cd]pyrene in contaminated soil by *Pseudomonas geniculata* NAP-1 were determined, as well as the degradation effect after 20 days under these optimal conditions. The results showed that the optimal degradation conditions for *Pseudomonas geniculata* NAP-1 were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of *Pseudomonas geniculata* NAP-1 on indo[1,2,3-cd]pyrene after 20 days under the optimal conditions (30.0±2.0℃, pH 7.0±0.2) is shown in Table 1.

[0118] Comparative Example 1

[0119] This comparative example is used to compare the degradation effect of Pyronema domesticum LJD-1 (GDMCC No. 61797) on phenanthrene.

[0120] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated groundwater: A phenanthrene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane had completely evaporated, it was added to a sterile inorganic salt culture medium to make the phenanthrene concentration 100 mg / L, thus obtaining an inorganic salt culture medium with a phenanthrene concentration of 100 mg / L.

[0121] After activating Pyronema domesticum LJD-1 cells, a stock culture was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, containing approximately 10% viable cells. 9 cfu / mL.

[0122] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into an inorganic salt medium with a phenanthrene concentration of 100 mg / L. Temperature and pH were considered as influencing factors. An experimental group and a control group (the control group was not inoculated with Pyronema domesticesticum LJD-1) were established. The residual phenanthrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for phenanthrene in the inorganic salt medium by Pyronema domesticesticum LJD-1 were determined, as well as the degradation effect after 5 days under the optimal conditions. The results showed that the optimal degradation conditions for Pyronema domesticesticum LJD-1 were 28.0 ± 2.0℃ and pH 6.0 ± 0.2. The phenanthrene degradation effects of Pyronema domesticesticum LJD-1 after 3 and 5 days under the optimal conditions (temperature 28.0 ± 2.0℃, pH 6.0 ± 0.2) are shown in Table 1.

[0123] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was taken, pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of phenanthrene was weighed and dissolved in n-hexane solution, and sprayed evenly onto the soil sample to make the phenanthrene content 100 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a phenanthrene content of 100 mg / kg was obtained.

[0124] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into contaminated soil with a phenanthrene content of 100 mg / kg. Temperature and pH were used as influencing factors. Experimental and control groups were established (the control group was not inoculated with Pyronema domesticum LJD-1). The remaining phenanthrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for phenanthrene in contaminated soil by Pyronema domesticum LJD-1 and its degradation effect after 20 days under optimal conditions were determined. The results showed that the optimal degradation conditions for Pyronema domesticum LJD-1 were 28.0±2.0℃ and pH 6.0±0.2. The phenanthrene degradation effects of Pyronema domesticum LJD-1 after 14 and 20 days under optimal conditions (temperature 28.0±2.0℃, pH 6.0±0.2) are shown in Table 1.

[0125] Comparative Example 2

[0126] This comparative example is used to compare the degradation effect of Pyronema domesticum LJD-1 (GDMCC No. 61797) on benzo[a]pyrene.

[0127] Preparation of simulated polycyclic aromatic hydrocarbon contaminated groundwater: A benzo[a]pyrene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane was completely evaporated, it was added to a sterile inorganic salt culture medium to make the benzo[a]pyrene concentration 30 mg / L, thus obtaining an inorganic salt culture medium with a benzo[a]pyrene concentration of 30 mg / L.

[0128] After activating Pyronema domesticum LJD-1 cells, a stock culture was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, containing approximately 10% viable cells. 9 cfu / mL.

[0129] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9CFU was inoculated into an inorganic salt medium containing 30 mg / L benzo[a]pyrene. Temperature and pH were considered as influencing factors. An experimental group and a control group (the control group was not inoculated with Pyronema domesticesticum LJD-1) were established. The remaining benzo[a]pyrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for benzo[a]pyrene in the inorganic salt medium by Pyronema domesticesticum LJD-1 and the degradation effect after 5 days under the optimal conditions were determined. The results showed that the optimal degradation conditions for Pyronema domesticesticum LJD-1 were 28.0 ± 2.0℃ and pH 6.0 ± 0.2. The degradation effect of Pyronema domesticesticum LJD-1 on benzo[a]pyrene after 5 days under the optimal conditions (temperature 28.0 ± 2.0℃, pH 6.0 ± 0.2) is shown in Table 1.

[0130] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of benzo[a]pyrene was weighed and dissolved in n-hexane solution, and sprayed evenly onto the soil sample to make the benzo[a]pyrene content 30 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a benzo[a]pyrene content of 30 mg / kg was obtained.

[0131] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 Pyronema domesticum LJD-1 was inoculated into contaminated soil containing 30 mg / kg of benzo[a]pyrene. Experimental and control groups (without inoculation of Pyronema domesticum LJD-1) were established, with temperature and pH as influencing factors. The residual benzo[a]pyrene content was periodically monitored, and the degradation rate of polycyclic aromatic hydrocarbons (PAHs) was calculated. The optimal degradation conditions for benzo[a]pyrene in contaminated soil by Pyronema domesticum LJD-1 and its degradation effect after 20 days under these optimal conditions were determined. The results showed that the optimal degradation conditions for Pyronema domesticum LJD-1 were 28.0±2.0℃ and pH 6.0±0.2. The degradation effect of Pyronema domesticum LJD-1 on benzo[a]pyrene after 20 days under the optimal conditions (temperature 28.0±2.0℃, pH 6.0±0.2) is shown in Table 1.

[0132] Comparative Example 3

[0133] This comparative example is used to compare the degradation effect of Bacillus sp.s8-t8-L9 (CGMCC No. 9032) on phenanthrene.

[0134] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated groundwater: A phenanthrene solution was prepared with n-hexane, filtered through a sterile filter membrane, and added to a sterile conical flask. After the n-hexane had completely evaporated, it was added to a sterile inorganic salt culture medium to make the phenanthrene concentration 100 mg / L, thus obtaining an inorganic salt culture medium with a phenanthrene concentration of 100 mg / L.

[0135] Bacillus sp. s8-t8-L9 cells were activated and cultured in LB medium to obtain a stock solution. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, containing approximately 10% viable cells. 9 cfu / mL.

[0136] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into an inorganic salt medium with a phenanthrene concentration of 100 mg / L. Experimental and control groups (the control group was not inoculated with Bacillus sp. s8-t8-L9) were established, with temperature and pH as influencing factors. The residual phenanthrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for phenanthrene in the inorganic salt medium by Bacillus sp. s8-t8-L9 and the degradation effect after 5 days under the optimal conditions were determined. The results showed that the optimal degradation conditions for Bacillus sp. s8-t8-L9 were 25.0 ± 2.0℃ and pH 7.5 ± 0.2. The phenanthrene degradation effects of Bacillus sp. s8-t8-L9 after 3 and 5 days under the optimal conditions (temperature 25.0 ± 2.0℃, pH 7.5 ± 0.2) are shown in Table 1.

[0137] Preparation of simulated polycyclic aromatic hydrocarbon (PAH) contaminated soil: Soil uncontaminated with PAHs was taken, pretreated by sieving to remove impurities such as small stones and grass roots, and then air-dried. A certain amount of phenanthrene was weighed and dissolved in n-hexane solution, and sprayed evenly onto the soil sample to make the phenanthrene content 100 mg / kg (wherein, the weight of the soil is on a dry basis). After the n-hexane was completely volatilized, contaminated soil with a phenanthrene content of 100 mg / kg was obtained.

[0138] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9CFU was inoculated into contaminated soil with a phenanthrene content of 100 mg / kg. Temperature and pH were used as influencing factors. An experimental group and a control group (the control group was not inoculated with Bacillus sp. s8-t8-L9) were established. The residual phenanthrene content was periodically monitored, and the polycyclic aromatic hydrocarbon (PAH) degradation rate was calculated. The optimal degradation conditions for phenanthrene in contaminated soil by Bacillus sp. s8-t8-L9 and the degradation effect after 20 days under the optimal conditions were determined. The results showed that the optimal degradation conditions for Bacillus sp. s8-t8-L9 were 25.0±2.0℃ and pH 7.5±0.2. The phenanthrene degradation effects of Bacillus sp. s8-t8-L9 after 14 and 20 days under the optimal conditions (temperature 25.0±2.0℃, pH 7.5±0.2) are shown in Table 1.

[0139] Table 1. Degradation effect of polycyclic aromatic hydrocarbons by Pseudomonas geniculata NAP-1

[0140]

[0141] As shown in Table 1, the *Pseudomonas geniculates* NAP-1 of this invention exhibits excellent degradation activity against various polycyclic aromatic hydrocarbons (PAHs) in soil or groundwater. In an inorganic salt liquid culture medium with a phenanthrene content of 100 mg / L, at a temperature of 30.0 ± 2.0 °C and a pH of 7.0 ± 0.2, the phenanthrene degradation rate reached over 95% after 5 days. In contaminated soil with a phenanthrene content of 100 mg / kg, at a temperature of 30.0 ± 2.0 °C and a pH of 7.0 ± 0.2, the phenanthrene degradation rate reached over 95% after 20 days; the degradation rate is significantly better than that of the control strain.

[0142] 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.

[0143] 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 geniculate pseudomonad ( Pseudomonas geniculata ), characterized in that, The preservation number of the *Pseudomonas geniculateus* is CGMCC No. 22735.

2. A microbial agent, characterized in that, The bacterial agent contains the *Pseudomonas geniculateus* as described in claim 1.

3. The microbial agent according to claim 2, wherein, The microbial agent is a liquid microbial agent and / or a solid microbial agent; The liquid bacterial agent contains 10 live bacteria. 9 cfu / mL or higher; The number of viable bacteria in the solid bacterial agent is 10. 9 CFU / g or higher.

4. The application of the *Pseudomonas geniculateus* of claim 1 or the bacterial agent of claim 2 or 3 in the degradation of polycyclic aromatic hydrocarbons.

5. The application of the *Pseudomonas geniculata* of claim 1 or the bacterial agent of claim 2 or 3 in the remediation of polycyclic aromatic hydrocarbon (PAH) contaminated soil or PAH contaminated groundwater.

6. A method for degrading polycyclic aromatic hydrocarbons, characterized in that, The method includes contacting the *Pseudomonas geniculateus* of claim 1 or the bacterial agent of claim 2 or 3 with a contaminant containing polycyclic aromatic hydrocarbons.

7. The method according to claim 6, wherein, The polycyclic aromatic hydrocarbon is at least one of the aromatic hydrocarbons containing at least two benzene rings.

8. The method according to claim 7, wherein, The polycyclic aromatic hydrocarbon is selected from at least one of naphthalene, phenanthrene, benzo[a]pyrene, benzo[a]anthracene, benzo[a]fluoranthracene, and ind[a]pyrene.

9. The method according to any one of claims 6-8, wherein, The pollutants containing polycyclic aromatic hydrocarbons are soil or groundwater containing polycyclic aromatic hydrocarbons.

10. The method according to claim 9, 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.

11. The method according to claim 9, wherein, When the pollutant containing polycyclic aromatic hydrocarbons is soil containing polycyclic aromatic hydrocarbons, the amount of *Pseudomonas geniculates* or the inoculant used is such that the viable count in the soil containing polycyclic aromatic hydrocarbons is 10. 6 -10 8 cfu / g; When the pollutant containing polycyclic aromatic hydrocarbons is groundwater containing polycyclic aromatic hydrocarbons, the dosage of the *Pseudomonas geniculates* or the bacterial agent is such that the viable bacteria count in the groundwater containing polycyclic aromatic hydrocarbons is 10. 6 -10 8 cfu / mL.

12. The method according to any one of claims 6-8, wherein, The contact conditions include a temperature of 20-40°C and a pH value of 5-10.

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