PAHs (polycyclic aromatic hydrocarbons) degrading strain as well as fungicide and application thereof

Abstract

The invention belongs to the technical field of biological agents and bioremediation, and particularly relates to a PAHs degrading strain as well as a fungicide and application thereof. The invention provides a strain for degrading PAHs (polycyclic aromatic hydrocarbons), the strain is Brucella anthrapi M1, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) M 2024922. The strain M1 can grow and breed by taking benzo (a) pyrene (BaP), pyrene or phenol as the unique carbon source and energy, and can quickly degrade PAHs; the strain is cultured in an inorganic salt culture medium with BaP, pyrene and phenol initial concentration of 5 mg / L for 5 days, and the degradation efficiency reaches 81%, 37% and 28% respectively. The bacterial strain is prepared into a microbial inoculum through crosslinking of sodium alginate and calcium chloride, and the degradation efficiencies of the microbial inoculum to BaP, pyrene or phenol respectively reach 46%, 37% and 80%. After the bacterial strain is prepared into a bacterial agent, the BaP degradation efficiency of the bacterial agent can reach 80%. Therefore, the strain and the fungicide thereof have a good application prospect in the bioremediation of PAHs (especially BaP) polluted soil or water.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparations and bioremediation, and specifically relates to a polycyclic aromatic hydrocarbons (PAHs) degrading strain, a bacterial agent and an application thereof, and in particular relates to a Brucella anthorpi M1 strain having PAHs degradation function, a bacterial agent prepared by using the degradation strain and an application thereof in degrading polycyclic aromatic hydrocarbons. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) generally refer to a class of compounds formed by hydrocarbons containing benzene rings arranged in linear, angular or clustered shapes. Currently, there are more than 200 known PAHs, 16 of which were listed as priority environmental pollutants by the United States Environmental Protection Agency (USEPA) in the 1980s. As the number of PAHs rings increases, the melting and boiling points and octanol-water partition coefficients of PAHs also increase significantly, making them more stable and difficult to remove in the environment, posing a serious threat to environmental ecological safety and human health.

[0003] With the continuous improvement of people's living standards and the rapid development of the economy, the demand for sewage irrigation, fertilizer and pesticide application, burning of crop straw, petrochemical fuels and other production and living raw materials is increasing day by day, and the emission of PAHs is also showing a rapid growth trend. PAHs generated during human activities will enter the soil directly on the one hand, and enter the soil through other means such as rainfall, snowfall and dustfall on the other hand, making the soil the main reservoir of PAHs in the environment. Therefore, the development of economical, efficient and environmentally friendly PAHs pollution remediation technology is still an urgent problem to be solved in current environmental science research.

[0004] Bioremediation has become a pollution remediation method favored by researchers in recent years due to its advantages of low investment, high efficiency and no secondary pollution. Microorganisms (including bacteria and fungi) can degrade PAHs into non-toxic substances and can even completely metabolize them into CO2 and water. At present, researchers have isolated many functional microorganisms that can degrade PAHs from different environmental media, such as strains of Pseudomonas, Mycobacterium, Sphingomonas, Stenotrophomonas and other genera.

[0005] After high molecular weight PAHs enter the environment, they are more likely to show persistence and difficulty in degradation due to their stable chemical properties and strong toxicity, and are mostly degraded by cometabolism. Abiotic effects have potential significance for the reduction of low molecular weight PAHs (2-ring and 3-ring), but have little effect on the reduction of high molecular weight PAHs with 4-6 rings. In addition, most strains reported so far have high activity in the degradation of PAHs with four rings or less, while there are few strains that can degrade PAHs with five rings or more, and Paracoccus aminovorans and others have been publicly reported. Bioremediation of high molecular weight PAHs-contaminated environments is the focus and difficulty of current research in the field of environmental remediation. Therefore, obtaining high molecular weight PAHs-degrading bacteria with strong environmental adaptability and high degradation activity and using them to prepare efficient microbial agents are essential prerequisites for bioremediation of PAH-contaminated environments. In this study, microbial strains with the function of degrading benzo(a)pyrene (BaP), pyrene or phenol were screened from soils that have been contaminated by PAHs for a long time, in order to provide biological resources and technical support for the bioremediation of PAHs. Summary of the invention

[0006] Based on the problems existing in the prior art, the first object of the present invention is to provide a strain of Brucella anthorpi M1 having the function of degrading polycyclic aromatic hydrocarbons (PAHs) (especially having the function of degrading benzo(a)pyrene (BaP), pyrene or phenol). The strain was isolated and identified from farmland soil contaminated by polycyclic aromatic hydrocarbons in 2023. There are no relevant reports on the degradation of PAHs by the strain at home and abroad.

[0007] The second object of the present invention is to provide the application of Brucella anthorpi M1 in the degradation of PAHs.

[0008] The third object of the present invention is to provide a PAHs-degrading bacterial agent, the main biologically active components of which include the Brucella anthorpi M1.

[0009] The fourth object of the present invention is to provide application of the PAHs-degrading bacterial agent in PAHs degradation.

[0010] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0011] A polycyclic aromatic hydrocarbons (PAHs) degrading strain, which is Brucella anthorpi, is coded and named Brucella anthorpi M1 by the inventor for the convenience of recording and management. The morphological characteristics of the strain are as follows: the colonies are light yellow, the surface is smooth and moist, flat, the edges are flat, and the diameter is about 3-4 mm; Gram staining is negative, it is a facultative anaerobe, and the methyl red reaction is negative.

[0012] The strain preservation number is: CCTCC M 2024922, the preservation unit is: China Center for Type Culture Collection (CCTCC); the preservation unit address is: Wuhan University, Wuhan City, Hubei Province (the postal code of the preservation unit address is: 430072); the submission date for preservation is: May 14, 2024, and the survival test date is May 21, 2024.

[0013] Furthermore, the present invention also provides the use of the Brucella anthorpi in the bioremediation of a PAHs-contaminated environment.

[0014] Specifically, the PAHs-contaminated environment is water or soil contaminated by PAHs.

[0015] Furthermore, the present invention also provides the use of the Brucella anthorpi in degrading polycyclic aromatic hydrocarbons (PAHs).

[0016] Specifically, the polycyclic aromatic hydrocarbons (PAHs) are one or more of benzo(a)pyrene, pyrene and phenol.

[0017] Specifically, during application, a Brucella anthorpi M1 bacterial solution stored at -80°C is taken, thawed at room temperature, cultured in a culture medium, and then the bacteria are collected by centrifugation and dissolved in sterile water to obtain a bacterial suspension, which is then added to water, soil or culture medium containing polycyclic aromatic hydrocarbons (PAHs), thereby achieving the degradation of polycyclic aromatic hydrocarbons (PAHs).

[0018] Specifically, the amount of bacterial suspension added is 2-8% (v / v or v / w), preferably 5% (v / v or v / w), of the water, soil or culture medium containing polycyclic aromatic hydrocarbons (PAHs); OD 600 About 1.0.

[0019] Specifically, the concentration of polycyclic aromatic hydrocarbons (PAHs) is 2 to 10 mg·L -1 , preferably 5 mg·L -1 .

[0020] Specifically, the Brucella anthorpi can have a high degradation efficiency for polycyclic aromatic hydrocarbons (PAHs) at 25°C, 28°C, 30°C, 35°C or 37°C.

[0021] Specifically, the Brucella anthorpi can have a high degradation efficiency for polycyclic aromatic hydrocarbons (PAHs) under pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5.

[0022] Furthermore, the present invention also provides the use of the Brucella anthorpi M1 in the preparation of a PAHs-degrading bacterial agent.

[0023] Furthermore, the present invention also provides a PAHs-degrading bacterial agent, the main biologically active components of which include the Brucella anthorpi M1.

[0024] Furthermore, the present invention also provides a method for preparing the PAHs-degrading bacterial agent, comprising the following steps:

[0025] (1) taking a Brucella anthorpi M1 bacterial solution stored at -80°C, thawing it at room temperature, culturing it in a shaking platform in a culture medium until the logarithmic phase, centrifuging the bacterial solution, collecting the bacterial cells, and dissolving the bacterial cells in sterile water to obtain a bacterial suspension;

[0026] (2) adding the embedding agent into deionized water at 55-65° C. (preferably 60° C.), stirring to dissolve it, sterilizing it under high temperature and high pressure, and cooling it to obtain an embedding agent solution; dissolving the crosslinking agent into deionized water at room temperature (25±5° C.), sterilizing it under high temperature and high pressure, and cooling it to obtain a crosslinking agent solution;

[0027] (3) adding the bacterial suspension of step (1) to the cooled embedding agent solution and mixing to obtain a mixed solution, adding the mixed solution dropwise to the crosslinking agent solution, crosslinking at 0 to 5° C. (preferably 4° C.) for 2 to 8 hours (preferably 6 hours), and then washing the surface to obtain the PAHs-degrading bacterial agent.

[0028] Specifically, the concentration of the bacterial suspension in step (1) is about 10 9 CFU / mL.

[0029] Specifically, the embedding agent in step (2) is any one of sodium alginate, chitosan and polyvinyl alcohol.

[0030] Specifically, in step (2), the cross-linking agent is anhydrous calcium chloride or glyoxal.

[0031] Specifically, the concentration of the embedding agent solution in step (2) is (0.01-0.03) g / mL.

[0032] Specifically, the concentration of the cross-linking agent solution in step (2) is (0.01-0.05) g / mL.

[0033] Specifically, in step (3), the volume ratio of the bacterial suspension to the embedding agent solution is 1:(5-10), preferably 1:9.

[0034] Specifically, in step (3), the volume ratio of the mixed solution to the cross-linking agent solution is 1:2 to 1:3.

[0035] Furthermore, the present invention also provides the use of the PAHs-degrading bacterial agent in the bioremediation of a PAHs-contaminated environment.

[0036] Specifically, the PAHs-contaminated environment is water or soil contaminated by PAHs.

[0037] Furthermore, the present invention also provides the use of the PAHs-degrading bacterial agent in degrading polycyclic aromatic hydrocarbons (PAHs).

[0038] Specifically, the polycyclic aromatic hydrocarbons (PAHs) are one or more of benzo(a)pyrene, pyrene and phenol.

[0039] Specifically, when used, the PAHs-degrading bacterial agent is added to water, soil or culture medium containing polycyclic aromatic hydrocarbons (PAHs), so as to achieve the degradation of polycyclic aromatic hydrocarbons (PAHs).

[0040] Specifically, the addition amount of the PAHs-degrading bacterial agent is 0.4%-0.5% (w / v or w / w) of the water, soil or culture medium containing polycyclic aromatic hydrocarbons (PAHs).

[0041] Specifically, the concentration of polycyclic aromatic hydrocarbons (PAHs) is 2 to 10 mg·L -1 , preferably 5 mg·L -1 .

[0042] Furthermore, the present invention also provides a method for degrading PAHs, which utilizes the Brucella anthorpi M1 or the PAHs-degrading bacterial agent to be applied to a PAHs-contaminated environment to degrade PAHs.

[0043] Specifically, the PAHs-contaminated environment is water or soil contaminated by PAHs.

[0044] Specifically, the application amount of the Brucella anthorpi M1 is 2-8% (v / v or v / w) of the water, soil or culture medium containing polycyclic aromatic hydrocarbons (PAHs), preferably 5% (v / v or v / w);

[0045] The application amount of the PAHs degrading bacterial agent is 0.4%-0.5% (w / v or w / w) of the water, soil or culture medium containing polycyclic aromatic hydrocarbons (PAHs).

[0046] Specifically, the concentration of polycyclic aromatic hydrocarbons (PAHs) is 2 to 10 mg·L -1 , preferably 5 mg·L -1 .

[0047] Compared with the prior art, the beneficial effects of the present invention are:

[0048] The invention adopts enrichment, separation and culture techniques to separate a highly efficient degradation strain M1 from PAHs-contaminated soil, which can use benzo(a)pyrene (BaP), pyrene or phenol as the sole carbon source. According to the strain morphology, physiological and biochemical characteristics, Gram reaction and 16S rDNA gene sequence analysis and phylogenetic analysis, the 16S rDNA gene sequencing results of the strain show that the similarity with Brucella anthorpi DP5 is as high as 99.7%, and the strain is identified as Brucella anthorpi, belonging to the genus Brucella.

[0049] Experiments have shown that the strain M1 can grow and reproduce with benzo(a)pyrene (BaP), pyrene or phenol as the sole carbon source and energy source, and can degrade PAHs relatively quickly; after being cultured in an inorganic salt medium with an initial concentration of 5 mg / L of BaP, pyrene and phenol for 5 days, its degradation efficiency reached 81%, 37% and 28% respectively. The strain was cross-linked with sodium alginate and calcium chloride to make a bacterial agent, and the degradation efficiency of the three (pyrene, phenol or BaP) reached 46%, 37% and 80% respectively. In addition, the strain has a wide range of growth adaptation, and its optimal growth environment is pH 7.5 and temperature is 30°C, at which time the strain has the highest degradation efficiency for BaP. After the strain M1 is prepared into a bacterial agent, its degradation efficiency for BaP can reach up to 80%. Therefore, the strain and its bacterial agent have good application prospects in the bioremediation of soil or water contaminated by PAHs (especially BaP). BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is the residual concentration of benzo(a)pyrene in the enrichment circulation device in Example 1 of the present invention;

[0051] Figure 2 Figure 1 shows the colony growth morphology (a) and topography (b) of strain M1 in Example 1 of the present invention;

[0052] Figure 3 is the phylogenetic tree of strain M1 in Example 1 of the present invention;

[0053] Figure 4 The degradation kinetics of benzo(a)pyrene by strain M1 in Example 2 of the present invention;

[0054] Figure 5 The degradation kinetics of pyrene and phenol by strain M1 in Example 2 of the present invention;

[0055] Figure 6 This is a morphological diagram of the bacterial agent prepared using strain M1 in Example 3 of the present invention;

[0056] Figure 7 This is a graph showing the degradation of different pollutants by the bacterial agent prepared by strain M1 in Example 3 of the present invention;

[0057] Figure 8 This is a graph showing the degradation of benzo(a)pyrene by strain M1 in Example 4 of the present invention at different temperatures (a) and different pH values ​​(b). DETAILED DESCRIPTION

[0058] In order to better understand the present invention, the present invention is further described below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments, and any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0059] Example 1 Isolation and Identification of Brucella anthorpi

[0060] Embodiment 1 provides the screening method of Brucella anthorpi, and the specific steps are as follows:

[0061] 1. Preparation of culture medium, soil and raw materials

[0062] In 2023, farmland soil contaminated by polycyclic aromatic hydrocarbons was collected downwind of a steel plant in Nanjing. The PAHs content was about 2.6 mg / kg. Stones and plant residues were selected and discarded, and the soil was stored at 4°C for future use.

[0063] Preparation of inorganic salt medium (MSM) (1L): K2HPO4 6.0g, KH2PO4 5.5g, Na2SO4 2.0g, KCl2.0g, NH4NO3 2.0g, MgSO4 0.2g, trace metal solution 1mL, distilled water 1L. Trace element nutrient solution: EDTA·2Na 500mg, ZnSO4·7H2O 10mg, MnSO4·H2O 5mg, H3BO3 30mg, CoSO4·7H2O24mg, CuSO4·5H2O 5mg, Na2MoO4·2H2O 5mg, Ca(OH)2 50mg per liter of water, pH 7.2. The medium was sterilized at high temperature and high pressure (121℃, 110kPa) for 20min before use.

[0064] Prepare 5000 mg·L of dimethylformamide (DMF) -1 The benzo(a)pyrene (BaP) stock solution was sterilized by filtration using a 0.22 μm hydrophobic polyvinylidene fluoride (PVDF) sterile filter membrane, 200 μL of the BaP stock solution was added to 200 mL of sterilized MSM to obtain a BaP-MSM liquid culture medium, and agar powder was added in an amount of 1.8% (w / v) to obtain a BaP-MSM solid culture medium.

[0065] LB medium (1 L): NaCl 10.0 g, yeast extract 5.0 g, peptone 10.0 g, agar 20.0 g, pH 7.0, sterilized at 121 °C for 20 min.

[0066] 2. Isolation of BaP-degrading strains

[0067] Weigh 3.0g of soil sample contaminated by PAHs and mix it with several activated carbon particles with a diameter of 1-3cm so that microorganisms can attach to the surface of carbon particles. Place it in an enrichment circulation device at 28°C, and circulate the BaP-MSM liquid culture medium through the mixture of soil and activated carbon particles continuously for continuous enrichment culture. Replace the new culture medium every week and measure the residual concentration of BaP in the enrichment circulation device to calculate the degradation efficiency of BaP. Repeat the cycle in this way to enrich the microorganisms on the activated carbon particles. During the enrichment culture process, an air peristaltic pump is used to allow air to enter the enrichment circulation device, and the ventilation volume is adjusted to allow the culture solution to complete the cycle.

[0068] After the degradation of BaP in the circulation device is stabilized, the enriched circulating liquid and activated carbon particles are separated, the activated carbon particles are taken out and crushed, the enriched circulating liquid is shaken and diluted 100-1000 times with sterile water, 100uL is applied to the BaP-MSM solid culture medium, placed in a bacterial incubator, and cultured at 30°C for 2 days. Bacteria with different colony morphology are selected from the plate and separated by streaking. The purified single colony is picked and cultured in an inorganic salt culture medium to the logarithmic phase (the obtained strain is numbered as M1), the bacterial solution obtained after culture is mixed with sterile glycerol and dispensed into sterile 2mL cryopreservation tubes (glycerol concentration is 20% by mass), and stored at -80°C for a long time.

[0069] 3. Identification of BaP-degrading strains

[0070] 3.1 Extraction and detection of BaP: 20 mL of ethyl acetate was added to 20 mL of the enriched circulating liquid collected in step 2, and the BaP in the aqueous phase was dissolved in the organic phase ethyl acetate by oscillation extraction using a vortexer to complete the extraction; then 2 mL of the extracted organic phase was blown dry under a nitrogen blower, dissolved and diluted with acetonitrile, and then determined using a Shimadzu high performance liquid chromatography (HPLC) system in Japan, with a mobile phase of water and acetonitrile (1:9, v:v), and the flow rate was set to 1 mL min -1 The detector was a fluorescence detector (RF-20A) equipped with a C18 reverse phase column (VP-ODS250×4.6mm), the column temperature was 35°C, and the excitation wavelength of the detector (λ Ex ) and emission wavelength (λ Em ) are set to 290nm and 410nm respectively.

[0071] The results showed that the residual BaP concentration in the enrichment circulation device was less than 1 mg·L after 11 weeks of continuous circulation culture. -1 ( Figure 1 ), and its degradation efficiency is stable at about 90%, which indicates that with the extension of enrichment culture time, the degradation effect of microorganisms on BaP is significantly improved, indicating that the activity of BaP-degrading bacteria in the enrichment circulation device is continuously improved, and a large number of microorganisms capable of degrading BaP are enriched in the enrichment circulation device.

[0072] 3.2 Identification of screening strains: The pure bacteria isolated in step 2 were inoculated into LB liquid culture medium and incubated at 30°C for 150 rpm. -1 The strains were cultured on a shaker for 24 hours, and their colony morphology was observed, and their morphological characteristics were observed using a scanning electron microscope (SEM). Gram staining, methyl red test, aerobic test, citrate utilization test, and nitrate reduction test were also performed to identify their physiological and biochemical characteristics.

[0073] The bacterial genomic DNA rapid extraction kit (spin column type, Shanghai Jierui Bioengineering Co., Ltd.) was used to extract bacterial DNA according to the instructions, and the bacterial 16S rDNA was amplified by PCR using F27 (5'-CAGCGGTACCAGAGTTT GATCCTGGCTCAG) and R1492 (5'-CTCTCTGCAGTACGGCTACCTTGTTACGACTT) as primers.

[0074] The PCR reaction system is 50 μL, including 25 μL Premix Taq TM (a mixture of DNA polymerase, buffer, and dNTP at twice the concentration), 1 μL of 10 mol·L -1 Forward primer and reverse primer, template DNA 1μL, sterile double distilled water 22μL. PCR reaction conditions are 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, 30 cycles, and finally 72℃ extension for 10min. PCR products were sequenced by Sangon Biotechnology, and the sequencing results were compared for homology in the NCBI database. At the same time, high-throughput sequences were downloaded, and multiple sequence alignments were performed using MEGA software to establish a phylogenetic tree.

[0075] Results and Conclusions:

[0076] 1) The colony of strain M1 is about 3-4 mm in diameter, light yellow in color, smooth, moist, flat, with smooth edges. Under SEM, the strain is observed to be rod-shaped ( Figure 2 ). Strain M1 is Gram-negative, facultative anaerobic, and has a negative methyl red reaction. The strain reduces glucose to produce less acid, or the produced acid is further converted into other substances (such as alcohol, ketone, aldehyde, gas, and water, etc.). The nitrate reduction test is positive, and the strain can reduce nitrate to nitrite. The citrate utilization test is positive, and the strain can use sodium citrate as a carbon source and produce carbonate after decomposing citrate.

[0077] 2) The 16S rRNA gene sequence of strain M1 was obtained by PCR amplification and gene sequencing (the sequence is shown in SEQ ID NO.1). The 16S rRNA gene sequence of strain M1 and some 16S rRNA gene sequences with high similarity were used to construct a phylogenetic tree, thereby obtaining the homology results between their 16S rRNA genes. The constructed phylogenetic tree is shown in Figure 3 As shown. Figure 3It can be seen that strain M1 and Brucella anthorpi DP5 are on the same branch, indicating that the strain has a high homology (99.76%) with Brucella anthorpi DP5. Therefore, the functional strain M1 isolated in this experiment belongs to the species Brucella anthorpi, and is named Brucella anthorpi M1. The inventor deposited this bacterium in the China Center for Type Culture Collection (CCTCC) on May 14, 2024, and the survival detection date was May 21, 2024. The address of the depository is: Wuhan University, Wuhan City, Hubei Province (the postal code of the depository address is: 430072), and the deposit number is CCTCC M 2024922.

[0078] Example 2 Degradation of BaP, pyrene and phenol by Brucella anthorpi

[0079] (I) Degradation of BaP

[0080] The isolated Brucella anthorpi M1 was inoculated into LB liquid medium and cultured at 30°C for 18 h. The cells were collected by centrifugation, washed with sterile water, and resuspended to adjust the OD 600 =1.0, and obtain a bacterial suspension for later use. Inoculate the bacterial suspension of M1 into a triangular flask containing BaP-MSM liquid culture medium (BaP concentration is 5 mg·L) at a 5% (v / v) inoculation volume. -1 ), which is the experimental group M1; at the same time, sterile water was added to the Erlenmeyer flask containing BaP-MSM liquid culture medium instead of bacterial suspension (BaP concentration was 5 mg·L -1 ) was used as the blank control group CK; 5% (v / v) sterilized bacterial suspension was used as the sterilized control group SCK. Each group was repeated 3 times at 30°C, 150 r·min -1 The samples were shaken and cultured in a constant temperature incubator for 5 days. The concentration of BaP in the samples was extracted and determined by shaking extraction method at 0, 1, 3, and 5 days of culture, and the degradation kinetic curve of BaP was drawn.

[0081] The results are as follows Figure 4As shown in the figure, the concentration of BaP in the blank control group CK and the control group SCK inoculated with inactivated bacteria hardly changed with the increase of culture time, which indicates that the non-biological degradation of BaP under culture conditions is almost negligible. In the experimental group M1 inoculated with bacterial suspension, the concentration of BaP in the culture medium gradually decreased with the extension of culture time. The concentration of BaP in the culture medium dropped to 3.6 mg / L at 1 day, and then to 1.8 mg / L at 3 days. At this time, the degradation efficiency of BaP was about 62%. At 5 days of culture, the concentration of BaP dropped to 0.9 mg / L, and its degradation efficiency reached 81%.

[0082] (II) Degradation of pyrene and phenol

[0083] According to the same method as in Part (I) above, the bacterial suspension of M1 was inoculated into MSM liquid medium with pyrene or phenol as the sole carbon source (the concentration of pyrene or phenol was 5 mg·L -1 ) as experimental group M1, at 30℃, 150r min -1 The pollutant degradation effect was measured after shaking culture in a constant temperature incubator for 5 days.

[0084] The results are as follows Figure 5 As shown in the figure, after 5 days of cultivation, the pyrene concentration increased from 4.88 mg·L -1 Reduced to 3.03 mg·L -1 The degradation efficiency was about 37%; the phenol concentration increased from 4.96 mg·L -1 Down to 3.48 mg·L -1 , and its degradation efficiency is about 28%. Although the degradation efficiency of this strain for pyrene and phenol is lower than that of BaP, it can still slowly degrade a variety of pollutants and has good application potential in environmental pollution remediation.

[0085] Example 3 Preparation and Application of Brucella anthorpi Bacterial Agent

[0086] (i) The isolated Brucella anthorpi M1 was inoculated into LB medium and cultured on a shaker at 30°C and 150 rpm for 18 hours until the logarithmic phase. 100 mL of the bacterial solution (approximately 10 8 CFU / mL) was centrifuged at 10000 rpm for 5 min, the supernatant was discarded, and 10 mL of sterile water was added to obtain a bacterial suspension of Brucella anthorpi M1 (about 10 9 CFU / mL);

[0087] Weigh 2.5 g of sodium alginate and add it to 90 mL of deionized water heated to 60°C in advance, stir to dissolve it, then sterilize it at 121°C for 15 min, and cool it to obtain an embedding agent solution; weigh 6.0 g of anhydrous calcium chloride and dissolve it in 200 mL of deionized water at room temperature (25±5°C), stir to dissolve it, then sterilize it at 121°C for 15 min, and cool it to obtain a cross-linking agent solution;

[0088] 10 mL of bacterial suspension was added to 90 mL of the cooled embedding agent solution and mixed to obtain 100 mL of mixed solution. The mixed solution was added dropwise to 200 mL of the crosslinking agent solution using a 50 mL syringe (the inner diameter of the needle tip was 2 mm). After crosslinking at 4°C for 6 hours, the surface was rinsed with physiological saline to obtain the Brucella M1 inoculum ( Figure 6 ).

[0089] The plate counting method was used to count the strain M1 embedded in the microbial agent. The counting results showed that the number of live bacteria per gram of microbial agent was 2.52×10 9 CFU g -1 .

[0090] (II) Using the prepared bacterial agent with a concentration of 5 mg L -1 The degradation experiment was carried out with an aqueous solution of pyrene, phenol or BaP. The experimental method was the same as that in Part (I) of Example 2, and the addition amount of the M1 degradation bacterial agent was 0.4%-0.5% (w / v).

[0091] The experimental results showed that after 5 days of cultivation, the concentrations of the three pollutants were reduced to 2.46 mg·L -1 , 2.95mg·L -1 and 1.00mg·L -1 , and their degradation efficiencies were 46%, 37% and 80% respectively ( Figure 7 The degradation effect of M1 bacterial agent on BaP is better than that of free bacteria M1, and it has strong application potential in environmental remediation.

[0092] Example 4 Degradation of Benzo(a)pyrene by Brucella anthorpi under Different Conditions

[0093] (I) Degradation performance under different temperature conditions

[0094] The isolated Brucella anthorpi M1 was inoculated into LB liquid medium and cultured at 30°C for 18 h. The cells were collected by centrifugation, washed with sterile water, and resuspended to adjust the OD 600=1, obtain bacterial suspension, and set aside. Inoculate the bacterial suspension of M1 into a triangular flask containing BaP-MSM liquid culture medium at a 5% inoculation amount, and use sterile water instead of the bacterial suspension as a blank control group CK. The BaP concentration in all treatments is 5 mg / L, and each group is repeated 3 times; culture for 5 days at 25°C, 28°C, 30°C, 35°C or 37°C, and then extract and measure the BaP in the solution. Degradation efficiency = (BaP concentration in CK - BaP concentration in treatment) / BaP concentration in CK × 100%.

[0095] (II) Degradation performance under different pH conditions

[0096] The pH of the MSM liquid culture medium was adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5 with 1 M NaOH solution or 1 M HCl solution. The bacterial suspension was inoculated into 20 mL of MSM liquid culture medium with different pH values ​​according to the above method. The BaP concentration in all treatments was 5 mg / L, and each group was repeated 3 times. The culture was carried out at the optimal temperature for 5 days, and the degradation efficiency of BaP was extracted and determined.

[0097] The results are as follows Figure 8 As shown, at a temperature of 25°C-30°C, the degradation efficiency of strain M1 on BaP was 70.6%-79.1%, and its degradation efficiency reached the maximum at 30°C.

[0098] At the optimum temperature of 30°C and acidic conditions (pH 5.5 and 6), the degradation efficiency of BaP was low, only 40.2% and 42.3%; at pH 6.5, 7, 7.5 and 8, the degradation efficiency of BaP was relatively stable, 74.8%, 75.3%, 80.1% and 72.6% respectively, of which the degradation efficiency reached the highest at pH 7.5; when pH was 8, the degradation efficiency of BaP was only 53%. It can be seen that Brucella anthorpi M1 has a wide range of adaptability, and has a good degradation effect on BaP in an environment of 25°C-30°C and pH 5.5-8.5, and its optimal environmental temperature is 30°C and the optimal pH is 7.5.

[0099] The above embodiments are illustrative of the implementation methods of the present invention. Although the present invention is illustrated and described using specific embodiments, it should be appreciated that the implementation methods of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A PAHs-degrading strain, characterized in that: The strain is Brucella ( Brucella anthorpi )M1, the strain preservation number is: CCTCC M 2024922.

2. Brucella described in claim 1 ( Brucella anthorpi ) in the bioremediation of PAHs-contaminated environments, characterized in that: The PAHs-polluted environment is water or soil contaminated by PAHs; the PAHs are one or more of benzo(a)pyrene, pyrene and phenol.

3. Brucella described in claim 1 ( Brucella anthorpi ) in the degradation of PAHs, characterized in that, The PAHs are one or more of benzo(a)pyrene, pyrene and phenol.

4. A PAHs-degrading bacterial agent, characterized in that: The main biologically active ingredients of the bacterial agent include the Brucella described in claim 1 ( Brucella anthorpi )M1.

5. The method for preparing the PAHs-degrading bacterial agent according to claim 4, characterized in that: The following steps are involved: (1) Take Brucella stored at -80℃ ( Brucella anthorpi ) M1 bacterial solution, thawed at room temperature, cultured in culture medium to logarithmic phase, centrifuged the bacterial solution, collected the bacterial cells, and dissolved the bacterial cells in sterile water to obtain a bacterial suspension; (2) Add the embedding agent into deionized water at 55-65°C, stir to dissolve it, sterilize it under high temperature and high pressure, and cool it to obtain an embedding agent solution; dissolve the cross-linking agent in deionized water at room temperature, sterilize it under high temperature and high pressure, and cool it to obtain a cross-linking agent solution; (3) Add the bacterial suspension of step (1) to the cooled embedding agent solution and mix well to obtain a mixed solution, add the mixed solution to a cross-linking agent solution, cross-link at 0-5° C. for 2-8 hours, and then wash the surface to obtain the PAHs-degrading bacterial agent.

6. The preparation method according to claim 5, characterized in that: The embedding agent in step (2) is any one of sodium alginate, chitosan and polyvinyl alcohol; the cross-linking agent in step (2) is anhydrous calcium chloride or glyoxal.

7. The preparation method according to claim 5, characterized in that: The concentration of the embedding agent solution in step (2) is (0.01-0.03) g / mL; the concentration of the cross-linking agent solution in step (2) is (0.01-0.05) g / mL.

8. The preparation method according to claim 5, characterized in that: The volume ratio of the bacterial suspension to the embedding agent solution in step (3) is 1:(5-10); the volume ratio of the mixed solution to the cross-linking agent solution in step (3) is 1:2-1:

3.

9. The use of the PAHs-degrading bacterial agent according to claim 4 in bioremediation of a PAHs-contaminated environment, characterized in that: The PAHs-polluted environment is water or soil contaminated by PAHs; the PAHs are one or more of benzo(a)pyrene, pyrene and phenol.

10. Use of the PAHs-degrading bacterial agent according to claim 4 in degrading PAHs, characterized in that: The PAHs are one or more of benzo(a)pyrene, pyrene and phenol.

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