A composite bacterial agent for efficiently degrading polycyclic aromatic hydrocarbons, an immobilized bacterial agent material, and a preparation method and application thereof

Abstract

The present invention provides a composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons, an immobilized bacterium agent material, a preparation method thereof and an application thereof. The composite bacterium agent is composed of Aeromonas hydrophila with a preservation number of CCTCC M 2022117 at 2×10<supgt;7< / supgt> - 6×10<supgt;9< / supgt> cfu / mL, Staphyococcus at 1×10<supgt;6< / supgt> - 1×10<supgt;9< / supgt> cfu / mL, Sphingobacterium at 3×10<supgt;6< / supgt> - 6×10<supgt;8< / supgt> cfu / mL, Pseudomonas aeruginosa at 4×10<supgt;6< / supgt> - 3×10<supgt;8< / supgt> cfu / mL and Bacillus subtilis at 2×10<supgt;7< / supgt> - 4×10<supgt;9< / supgt> cfu / mL, which are mixed in a volume ratio of (30 - 80):(5 - 30):(5 - 30):(5 - 30):(10 - 30). The composite bacterium agent of the present invention has a high degradation effect on polycyclic aromatic hydrocarbons. For low-ring PAHs (four rings and below) contaminated soil with 400 mg / kg, the degradation effect can reach 94.5% in 7 days; for high-ring PAHs (four rings and above) contaminated soil with 80 mg / kg, the degradation effect can reach 90.6% in 14 days.

Description

Technical Field

[0001] The present invention relates to the technical fields of microbiology, biochemistry and fermentation engineering, and particularly relates to a composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons, an immobilized bacterium agent material, and a preparation method and application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are toxic, genotoxic, mutagenic and carcinogenic, and can cause various harms to the human body, such as damage to the respiratory system, circulatory system and nervous system, and damage to the liver and kidneys. They are recognized as the main organic pollutants affecting human health. Polycyclic aromatic hydrocarbons in the environment can enter the human body through various channels, thereby endangering human health. Therefore, the treatment and remediation of polycyclic aromatic hydrocarbon pollution have received widespread attention.

[0003] The main methods for the remediation of polycyclic aromatic hydrocarbons are physical, chemical and biological remediation. Among them, traditional physical and chemical remediation methods are prone to cause secondary pollution and have high treatment costs, which limit their application. The biological remediation method has the characteristics of small project volume, low energy consumption, low cost, environmental friendliness, etc., and has thus been steadily developed and widely applied in the field of polycyclic aromatic hydrocarbon pollution remediation. Microbial remediation refers to the process of degrading pollutants in soil and water under the metabolic action of microorganisms by stimulating indigenous bacteria or adding exogenous degrading bacterium agents to render the pollutants harmless.

[0004] However, due to the stable nature of polycyclic aromatic hydrocarbons and the lack of efficient degrading bacterium agents, there are generally problems such as low efficiency and long cycle in the microbial remediation of polycyclic aromatic hydrocarbons. Therefore, it is necessary to develop a bacterium agent for polycyclic aromatic hydrocarbons that has fast growth, strong adaptability and good effects in degrading PAHs. Summary of the Invention

[0005] The purpose of the present invention is to provide a composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons, an immobilized bacterium agent material, and a preparation method and application thereof. Under the synergistic action of these five bacteria, the dissolution of PAHs can be accelerated, the bioavailability can be increased, and the biodegradation of PAHs can be effectively promoted.

[0006] In a first aspect of the present invention, there is provided a composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons, and the composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons is composed of Aeromonas with a preservation number of CCTCC M 2022117 at 2×10 7 ~6×10 9 cfu / mL, Staphisagria at 1×10 6 ~1×10 9 cfu / mL, Sphingobacterium at 3×10 6 ~6×10 8 cfu / mL, Sphingomonas at 4×10 6 ~3×10 8Pseudomonas aeruginosa at cfu / mL and 2×10 7 ~4×10 9 Bacillus subtilis at cfu / mL are mixed in a volume ratio of (30 - 80):(5 - 30):(5 - 30):(5 - 30):(10 - 30).

[0007] The deposit number of the Stappia sp. is CCTCC AB 208228; the deposit number of the Sphingomonas sp. is CCTCC AB 2010361; the deposit number of the Pseudomonas aeruginosa is CCTCC AB 93066; the deposit number of the Bacillus subtilis is CCTCC AB 90008.

[0008] In the second aspect of the present invention, there is provided an immobilized bactericide material for efficiently degrading polycyclic aromatic hydrocarbons, which is composed of a carrier and a composite bactericide for efficiently degrading polycyclic aromatic hydrocarbons adsorbed on the surface of the carrier.

[0009] Furthermore, the carrier includes: one of a biochar carrier, chitosan, alginate, polyurethane, diatomite, bentonite, vermiculite, slag, porous ceramics.

[0010] The biochar carrier includes one or a combination of straw biochar, rice husk biochar, and pinecone biochar. Preparation of the biochar carrier: After crushing and drying straw, rice husks or pinecones, place them in a rotary electric furnace for heating. When the temperature rises to 105°C, introduce steam, keep the steam flow rate stable, heat at a rate of 10°C per minute to 600 - 800°C, keep warm for 3 - 4 hours and then stop heating, continue to introduce steam until the temperature is lower than 100°C. After the temperature drops to room temperature, grind and sieve, soak with inorganic strong acid for 5 - 6 hours, wash off the ash, rinse with deionized water until the pH value is constant, and dry at 105°C to obtain the biochar.

[0011] In the third aspect of the present invention, there is provided a preparation method of the immobilized bactericide material for efficiently degrading polycyclic aromatic hydrocarbons, and the method includes:

[0012] Respectively expand and culture the Aeromonas sp., Stappia sp., Sphingomonas sp., Pseudomonas aeruginosa and Bacillus subtilis. When OD600 = 1.0 ± 0.05, mix the bacterial solutions in the above ratio, and then add 1 - 5% (w / v) of the biochar carrier for adsorption culture;

[0013] After the adsorption culture is completed, centrifuge under sterile conditions to remove the upper suspension to obtain a solid product;

[0014] Freeze - dry the solid product to obtain the immobilized bactericide material for efficiently degrading polycyclic aromatic hydrocarbons.

[0015] In the fourth aspect of the present invention, there is provided an application of the above-mentioned composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons in degrading polycyclic aromatic hydrocarbons.

[0016] The method of the above application includes:

[0017] For water bodies polluted by polycyclic aromatic hydrocarbons: Take the above-mentioned composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons and add it to the water body polluted by polycyclic aromatic hydrocarbons at a volume ratio of 5-15% for biodegradation, ensuring that the dissolved oxygen in the water body is 2.0-6.0 mg / L;

[0018] For soil or sediment polluted by polycyclic aromatic hydrocarbons: Take the above-mentioned composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons and add it to the culture medium at a volume ratio of 5-15% to prepare a bacterial solution, and then mix the bacterial solution with the polluted soil or sediment evenly at a mass ratio of 1:1-3:1 for biodegradation, ensuring that the dissolved oxygen in the water body is 2.0-6.0 mg / L, stirring and adding 0.1-1.5% by volume of surfactant to promote the dissolution of polycyclic aromatic hydrocarbons.

[0019] It should be noted that when preparing the above-mentioned composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons, the culture systems of each bacterium are different. Among them, Aeromonas is independently cultured in a liquid medium with PAHs pyrene (Byrene) as the sole carbon source (the components of 1 L of this medium are as follows: 100 mg Byrene, 1.3-2.0 g KH2PO4, 4.5-6.5 g K2HPO3·3H2O, 0.5-2.4 g NH4Cl, 0.4-1.0 g NaCl, 100-500 mg MgSO4, 0-100 mg MnSO4·H2O, 0-100 mg FeSO4·7H2O, 0-100 mg CaCl2, 10 ml Tween80, pH = 7.0-7.2, autoclaved at 121 °C for 20 min, and the culture conditions are: 30 °C, rotation speed 200 rpm);

[0020] Stappia is cultured in 2216E liquid medium (the components of 1 L of this medium are as follows: 5 g tryptone, 0.1 g ferric citrate, 19.45 g NaCl, 5.9 g MgCl2, 0.55 g KCl, 3.24 g Na2SO4, 1.8 g CaCl2, 0.16 g Na2CO3, 0.08 g KBr, 34 mg SrCl2, 22 mg H3BO3, 4 mg NaSiO3, 2.4 mg NaF, 1.6 mg NH4NO3, 8 mg Na2HPO4, pH = 7.6, and the culture conditions are: 30 °C, rotation speed 200 rpm);

[0021] Sphingomonas, Pseudomonas aeruginosa, and Bacillus subtilis were expanded in LB liquid medium (the components of 1 L of this medium are as follows: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, pH = 7.0 - 7.2, autoclaved at 121 °C for 20 min, and the culture conditions are: 28 °C (Sphingomonas), 37 °C (Pseudomonas aeruginosa and Bacillus subtilis), rotation speed 200 rpm).

[0022] Unless otherwise specified, the drugs were purchased from Sinopharm Group Co., Ltd.

[0023] In the fifth aspect of the present invention, there is provided the application of the immobilized bacterium agent material for highly efficient degradation of polycyclic aromatic hydrocarbons in the degradation of polycyclic aromatic hydrocarbons.

[0024] The method of the application includes:[[]]END]]

[0025] First, the immobilized bacterium agent material was added to the culture solution at a ratio of 4 - 6 g / L to obtain an activated bacterial solution.

[0026] For polycyclic aromatic hydrocarbon - polluted water: The activated bacterial solution was added to the sewage containing polycyclic aromatic hydrocarbon pollutants at a ratio of 5 - 15% (v / v) for biodegradation, ensuring that the dissolved oxygen in the water body was 4.0 - 7.0 mg / L.

[0027] For polycyclic aromatic hydrocarbon - polluted soil or sediment: The activated bacterial solution was added to the culture solution at a ratio of 5 - 15% (v / v) to obtain a diluted bacterial solution, and then the polluted soil (sediment) was mixed evenly with the diluted bacterial solution at a ratio of 1:1 - 1:3 (w / v) for biodegradation, ensuring that the dissolved oxygen in the water body was 4.0 - 7.0 mg / L, and stirring and supplementing 0.1 - 1.5% by volume of surfactant to promote the dissolution of polycyclic aromatic hydrocarbons.

[0028] The formula of the culture solution is as follows:[[]]END]]

[0029] The components in 1 L of the culture solution are as follows: 1.3 - 2.0 g of KH2PO4, 4.5 - 6.5 g of K2HPO3·3H2O, 0.5 - 2.4 g of NH4Cl, 0.4 - 1.0 g of NaCl, 100 - 500 mg of MgSO4, 0 - 100 mg of MnSO4·H2O, 0 - 100 mg of FeSO4·7H2O, 0 - 100 mg of CaCl2, such that pH = 7.0 - 7.2, and the salinity is maintained within the range of 0.7 - 1.2%.

[0030] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:[[]]END]]

[0031] An efficient polycyclic aromatic hydrocarbon-degrading composite bacterium agent, an immobilized bacterium agent material, a preparation method and an application thereof provided by the present invention. The core degrading strain Aeromonas in the composite bacterium agent of the present invention is screened from the activated sludge in the secondary sedimentation tank of a coking wastewater treatment plant by means of acclimation culture and enrichment separation, etc., and has the advantages of fast growth, strong adaptability and good PAHs degradation effect. In addition to Aeromonas, the composite bacterium agent of the present invention is also compounded with four strains of Pseudomonas aeruginosa, Stappia sp., Sphingomonas sp. and Bacillus subtilis. Among them, both Stappia sp. and Sphingomonas sp. have good PAHs degradation ability; while Pseudomonas aeruginosa and Bacillus subtilis have the ability to produce biosurfactants such as rhamnolipids. Under the synergistic action of the five bacteria, the dissolution of PAHs can be accelerated, the bioavailability can be increased, and the biodegradation of PAHs can be effectively promoted. The composite bacterium agent of the present invention has a high degradation effect on polycyclic aromatic hydrocarbons. For the soil contaminated with low-ring PAHs (four rings and below) at 400 mg / kg, a degradation effect of 94.5% can be achieved in 7 days; for the soil contaminated with high-ring PAHs (four rings and above) at 80 mg / kg, a degradation effect of 90.6% can be achieved in 14 days (as shown in Figure 1 and Figure 2 ).

[0032] The preservation date of the Aeromonas of the present invention is February 14, 2022, and the preservation number is CCTCC NO: M2022117. Its taxonomic name is Aeromonas sp. BCP-3, the name of the preservation unit is China Center for Type Culture Collection, the address is Wuhan University, Wuhan, Hubei, China, and the postal code is 430072. Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0034] Figure 1 It is the degradation effect of the composite bacterium agent of the present invention on soil low-ring PAHs;

[0035] Figure 2 It is the degradation effect of the composite bacterium agent of the present invention on soil high-ring PAHs;

[0036] Figure 3 It is the SEM image of the immobilized polycyclic aromatic hydrocarbon-degrading bacterium agent material of the present invention;

[0037] Figure 4 It is the degradation effect of the immobilized bacterium agent material of the present invention on PAHs in actual sewage;

[0038] Figure 5 For the degradation effect of the immobilized bacterium agent material of the present invention on PAHs in actual soil. Specific embodiments

[0039] The following will specifically describe the present invention in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0040] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of conflict, this specification shall prevail.

[0041] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or by existing methods.

[0042] The preparation method of the Aeromonas of the present invention is as follows:

[0043] The inventors of the present application screened out a strain of microorganism from the activated sludge collected from the secondary sedimentation tank of a coking wastewater treatment plant in Wuhan through means such as acclimation culture and enrichment separation, and found that this strain has the ability to efficiently degrade polycyclic aromatic hydrocarbons. After analyzing the colony morphology, biochemistry, and 16S rRNA sequencing of this bacterium, the homology of this strain with multiple strains of the genus Pseudomonas is above 96%. Combining the physiological and biochemical characteristics, it is preliminarily determined that this strain belongs to the genus Pseudomonas, and it is named Aeromonas sp. BCP-3.

[0044] Take this Aeromonas, and expand the culture in LB liquid medium. Take the fresh bacterial liquid and extract its genome using a bacterial genome extraction kit. Use the universal primers 27F and 1492R to perform PCR amplification on the extracted genome. The PCR system is: 25 μL of 2x Taq Plus PCR Master Mix, 19 μL of ddH2O, 2 μL of universal primer 27F, 2 μL of universal primer 1492R, and 2 μL of template DNA. Take 10 μL of the product obtained by PCR amplification and perform electrophoresis detection in 1.5% agarose gel. After confirming that there are clear and bright bands, send the remaining PCR product to a sequencing company for sequencing, and the sequencing result is as described in SEQ ID NO:1.

[0045] The sequencing results were compared and analyzed with the gene sequences in the database (NCBI) by the Blast search program, and a phylogenetic tree was constructed ( Figure 2 as shown). The homology between the current strain and Aeromonas reached 96%. Combining physiological and biochemical characteristics, the strain was determined to be a member of the genus Aeromonas and named Aeromonas sp. BCP-3. It was deposited at the China Center for Type Culture Collection on February 14, 2022, with the deposit number CCTCC NO: M 2022117.

[0046] Compared with ordinary Aeromonas, the Aeromonas of the present invention has a high degradation effect on polycyclic aromatic hydrocarbons. For a simulated polycyclic aromatic hydrocarbon solution of 305 mg / L, a degradation effect of 90% can be achieved in 7 days; for an actual polycyclic aromatic hydrocarbon sewage of 389.81 mg / L, a degradation effect of 92% can be achieved in 14 days; for an actual polycyclic aromatic hydrocarbon-contaminated soil of 135.53 mg / kg, a degradation effect of 74% can be achieved in 21 days.

[0047] Next, a composite bactericide, an immobilized bactericide material, and their preparation methods and applications for efficiently degrading polycyclic aromatic hydrocarbons of the present application will be described in detail in combination with examples and experimental data.

[0048] Example 1: Composite Bactericide for Efficient Degradation of Polycyclic Aromatic Hydrocarbons and Its Preparation Method and Application

[0049] Preparation of the composite bactericide: Five kinds of bacteria were respectively expanded in the above independent culture systems. Among them, Aeromonas was independently expanded in a culture solution with pyrene (Byrene) as the only PAHs carbon source; Stappia was expanded in 2216E liquid medium; Sphingomonas, Pseudomonas aeruginosa, and Bacillus subtilis were expanded in LB liquid medium (the components and culture conditions of each medium are shown in the above specification). When the OD600 of the five kinds of bacterial solutions reached 1.0, Aeromonas, Pseudomonas aeruginosa, Stappia, Sphingomonas, and Bacillus subtilis were mixed in a ratio of 55%, 10%, 10%, 10%, and 15%. The total viable bacteria count was measured to be 4.3×10 8 cfu / ml. That is, a composite bactericide capable of degrading PAHs was obtained and stored for later use.

[0050] Preparation of low-ring polycyclic aromatic hydrocarbon (PAH)-simulated contaminated soil: Weigh appropriate amounts of 5 low-ring PAHs (including 150 mg of Nap, 100 mg of Ace, 50 mg of Phe, 50 mg of Ant, and 50 mg of Fla), dissolve them in 300 ml of acetone solution, immerse this solution in 1 kg of simulated soil, and age it in the dark for 7 days. After the acetone has evaporated, contaminated soil containing 5 low-ring PAHs can be obtained (with the content of each pollutant being: 150 mg / kg of Nap, 100 mg / kg of Ace, 50 mg / kg of Phe, 50 mg / kg of Ant, and 50 mg / kg of Fla), and it can be stored for future use.

[0051] Experimental and testing conditions: Take appropriate amounts of PAH-simulated contaminated soil and culture solution (for the detailed composition of the culture solution, see the above-mentioned specification) at a mass ratio of 1:2 and place them in a 250-ml conical flask. Set up a control group (adding 10% of the total volume of the culture solution) and an experimental group (adding 10% of the total volume of the complex bacterial agent), with three replicates in each group. Under the conditions of 30 °C and 200 rpm, culture them with shaking in the dark for 21 days, and intermittently aerate to maintain the dissolved oxygen within the range of 2.0 - 6.0 mg / L. Samples are taken on days 0, 1, 3, 7, 14, and 21 respectively to measure the concentration of residual PAHs in the system.

[0052] Experimental results: The degradation effects of each PAH component are as shown in the appendix Figure 1 . To facilitate the comparison of the degradation effects between the experimental group and the control group, the degradation rates of each PAH component on day 7 are summarized in Table 1 below.

[0053] Table 1 Comparison of the 7-day degradation effects of the bacterial agent of the present invention on low-ring PAH-simulated soil (mg / kg)

[0054]

[0055] From Figure 1 and Table 1, it can be seen that for low-ring PAHs at 400 mg / kg, the bacterial agent of the present invention has a significant degradation effect, with a degradation rate of 94.5% achieved in 7 days, and the degradation rates on days 14 and 21 are 99.0% and 99.5% respectively.

[0056] Example 2. Complex bacterial agent for highly efficient degradation of polycyclic aromatic hydrocarbons, its preparation method and application

[0057] Preparation of compound bacterium agent: Five kinds of bacteria were respectively amplified in the above independent culture systems. Among them, Aeromonas was independently amplified in a culture solution with pyrene (Byrene) as the only PAHs carbon source; Stappia was amplified in 2216E liquid medium; Sphingomonas, Pseudomonas aeruginosa and Bacillus subtilis were amplified in LB liquid medium (the components and culture conditions of each medium are shown in the above specification). When the OD600 of the five kinds of bacterial solutions reached 1.0, Aeromonas, Pseudomonas aeruginosa, Stappia, Sphingomonas and Bacillus subtilis were mixed according to the proportion of 50%, 12.5%, 12.5%, 12.5% and 12.5%, and the total viable count was measured to be 2×10 9 cfu / ml. That is, a compound bacterium agent capable of degrading PAHs was obtained and stored for later use.

[0058] Preparation of high-ring polycyclic aromatic hydrocarbon simulated contaminated soil: Take corresponding masses of 5 kinds of high-ring PAHs (including 25 mg Byr, 25 mg BaA, 25 mg BbF, 2.5 mg Bap and 2.5 mg DBahA) and dissolve them in 300 ml of acetone solution. Immerse this solution in 1 kg of simulated soil, and after aging in the dark for 7 days until the acetone volatilizes, 5 kinds of high-ring PAHs contaminated soil can be obtained (the content of each pollutant is: 25 mg / kg Byr, 25 mg / kg BaA, 25 mg / kg BbF, 2.5 mg / kg Bap and 2.5 mg / kg DBahA), and store it for later use.

[0059] Experimental and test conditions: Take appropriate amounts of polycyclic aromatic hydrocarbon simulated contaminated soil and culture solution (the components of the culture solution are detailed in the above specification) according to a mass ratio of 1:3 and place them in a 250 ml conical flask. Set a control group (adding 10% of the total volume of the culture solution) and an experimental group (adding 10% of the total volume of the compound bacterium agent), with three parallels in each group. Under the conditions of 30 °C and 200 rpm, culture with shaking in the dark for 21 days, and intermittently aerate to keep the dissolved oxygen within the range of 2.0 - 6.0 mg / L. Samples were taken on days 0, 1, 3, 7, 14, and 21 respectively to test the concentration of residual PAHs in the system.

[0060] Experimental results: The degradation effects of each PAHs component are shown in the appendix Figure 2 . In order to facilitate the comparison of the degradation effects between the experimental group and the control group, the degradation rates of each PAHs component on the 14th day are summarized in Table 2 below.

[0061] Table 2 Comparison of the 14-day degradation effects of the bacterium agent of the present invention on high-ring PAHs simulated soil (mg / kg)

[0062]

[0063] From Figure 2As can be seen from Table 2, for the high-ring PAHs at 80 mg / kg, the degradation effect of the bacterial agent of the present invention is significant. The degradation rate can reach 90.6% in 14 days, and the degradation rate at 21 days slightly increases to 94.1%.

[0064] Example 3: Composite Bacterial Agent for Highly Degrading Polycyclic Aromatic Hydrocarbons, Its Preparation Method and Application

[0065] Preparation of the composite bacterial agent: The five bacteria are respectively expanded in the above independent culture systems. Among them, Aeromonas is independently expanded in a culture solution with pyrene as the only PAHs carbon source; Stappia is expanded in 2216E liquid medium; Sphingomonas, Pseudomonas aeruginosa and Bacillus subtilis are expanded in LB liquid medium (the components and culture conditions of each medium are shown in the above specification). When the OD600 of the five bacterial solutions reaches 1.0, Aeromonas, Pseudomonas aeruginosa, Stappia, Sphingomonas and Bacillus subtilis are mixed according to the proportions of 60%, 10%, 10%, 8% and 12%. The total viable count is measured to be 9×10 8 cfu / ml. That is, a composite bacterial agent capable of degrading PAHs is obtained and stored for later use.

[0066] Actual contaminated soil: An appropriate amount of contaminated soil is taken from a coking contaminated site in Hangzhou, naturally air-dried in a cool place, ground, passed through a 10-mesh sieve, and impurities such as branches are removed, and then stored for later use. Tests by the national standard method (HJ 805-2016) show that the initial total amount of 16 PAHs in this soil is 294.20 mg / kg, and the content of each PAHs component is shown in Table 3 below.

[0067] Experimental and test conditions: An appropriate amount of polycyclic aromatic hydrocarbon-simulated contaminated soil and culture solution (the components of the culture solution are detailed in the above specification) are taken in a mass ratio of 1:3 and placed in a 250 ml conical flask. A control group (adding 10% of the total volume of the culture solution) and an experimental group (adding 10% of the total volume of the composite bacterial agent) are set up, with three parallels in each group. Under the conditions of 30 °C and 200 rpm, the mixture is cultured in the dark with shaking for 21 days, and intermittent aeration is carried out to keep the dissolved oxygen within the range of 2.0 - 6.0 mg / L. Samples are taken at 0, 7, and 14 days respectively to test the concentration of residual PAHs in the system.

[0068] Experimental results: The degradation rates of each PAHs component are summarized in Table 3 below.

[0069] Table 3 Comparison of the 14-day degradation effect of the bacterial agent of the present invention on actual PAHs-contaminated soil (mg / kg)

[0070]

[0071] As shown in Table 3, the degradation effect of each PAHs component is obvious. For the actual PAHs-contaminated soil with a total amount of 294.2 mg / kg, the degradation rate can reach 97.1% in 14 days.

[0072] Example 4: Immobilized bacterial agent material for efficient degradation of polycyclic aromatic hydrocarbons and its preparation method and application

[0073] 1. Preparation of immobilized PAHs degradation bacterial agent materials

[0074] Preparation of biochar carrier: The straw and rice husk are crushed and dried in a ratio of 1:1 (w / w), and then placed in a rotary electric furnace for heating. When the temperature reaches 105°C, water vapor is introduced, and the water vapor flow rate is kept stable. The temperature is increased by 10°C per minute and heated to 600-800°C. After keeping warm for 3-4 hours, the heating is stopped and water vapor is continued to be introduced until the temperature is lower than 100°C. After the temperature drops to room temperature, the mixture is ground and sieved, and the ash is washed off after being soaked in a strong inorganic acid for 5-6 hours. The mixture is rinsed with deionized water until the pH value is constant, and then dried at 105°C to obtain the biochar carrier.

[0075] Preparation of immobilized bacterial agent materials: First, the three bacteria were cultured in the above-mentioned independent culture systems, among which Aeromonas was cultured independently in a culture medium with pyrene as the only PAHs carbon source; Pseudomonas aeruginosa and Bacillus subtilis were cultured in LB liquid culture medium (the components of each culture medium and the culture conditions are shown in the above instructions); secondly, when the OD600 of the three bacterial solutions was 1.0, Aeromonas, Pseudomonas aeruginosa, Stappella, Sphingobacterium and Bacillus subtilis were mixed at a ratio of 55%, 15%, 5%, 10% and 15%, and the total number of viable bacteria was measured to be 4.3×10 8 cfu / ml; then, 5% (w / v) biochar carrier was added to the mixed bacterial solution and adsorbed and cultured for 12 hours; finally, after the adsorption culture was completed, centrifuged at 5000rpm for 10 minutes under sterile conditions, the upper suspension was removed, and the solid product at the bottom was freeze-dried to obtain the immobilized PAHs-degrading bacterial agent material.

[0076] The scanning electron microscopy image of the immobilized PAHs-degrading bacterial agent prepared under these conditions is shown in Figure 3 As shown. Figure 3 It can be seen that the degradation bacteria are evenly dispersed on the surface and pores of the biochar carrier and are well fixed.

[0077] 2. Degradation effect of immobilized PAHs-degrading bacterial agent on 16 PAHs in actual sewage

[0078] Immobilized bacterial agent material: Prepare the immobilized polycyclic aromatic hydrocarbons degradation bacterial agent material according to the method of Example 1

[0079] Actual sewage: The source of the sewage is the water in the regulating tank of a coking wastewater treatment plant in Wuhan. Through testing and analysis by the national standard method (HJ 478 - 2009), it is found that the total amount of the initial 16 PAHs in this sewage is 399.32 mg / L. The content of each PAHs component is shown in Table 4 below.

[0080] Experimental conditions: The experiment was carried out in 250 - ml conical flasks, with a total reaction system of 200 ml. A blank control group (adding 20 ml of culture medium and 180 ml of actual sewage), a free bacterial agent experimental group (adding 20 ml of activated free bacterial agent and 180 ml of actual sewage), and an immobilized bacterial agent experimental group (adding 20 ml of activated immobilized bacterial agent and 180 ml of actual sewage) were set up. There were three parallels in each group, placed in a shaker, and cultured under the conditions of 30 °C and 200 rpm in the dark with intermittent aeration to keep the dissolved oxygen within the range of 4.0 - 7.0 mg / L. Samples were taken on days 0, 1, 3, 7, 14, and 21 respectively, and the concentration of residual PAHs in the system was tested by the national standard method (HJ 478 - 2009). (Among them, the free bacterial agent refers to the dispersed bacterial agent not loaded on the biochar carrier. Its preparation method is that each strain is first separately amplified in an independent culture system. When OD600 = 1.0 ± 0.05, the bacterial solutions are mixed in a certain proportion, centrifuged under sterile conditions, and the upper suspension is removed to obtain a solid product; the solid product is freeze - dried for later use and needs to be activated before use. The activation method is the same as that of the immobilized bacterial agent; the formula of the culture medium and the activation of the immobilized bacterial agent refer to the specific implementation plan)

[0081] Experimental results: The degradation effect of PAHs in the actual sewage is as attached Figure 4 , and in order to facilitate the comparison of the degradation effects between the experimental group and the control group, the content of each PAHs component on the 14th day in the system is summarized in Table 4 below.

[0082] Table 4 Comparison of the 14 - day degradation effect of the immobilized bacterial agent material of the present invention on 16 PAHs in sewage (mg / L)

[0083]

[0084]

[0085] From Figure 4 and Table 4, it can be seen that both the free bacterial agent experimental group and the immobilized bacterial agent experimental group have obvious degradation effects on PAHs in the sewage. However, the immobilized bacterial agent experimental group has obvious advantages, with a faster degradation rate. It has tended to be stable on the 14th day of degradation and reached a degradation rate of 98%.

[0086] 3. Degradation effect of the immobilized polycyclic aromatic hydrocarbon - degrading bacterial agent material on 16 PAHs in actual soil

[0087] Immobilized bacterium agent material: Prepare the immobilized polycyclic aromatic hydrocarbon degrading bacterium agent material in the manner of Example 1

[0088] Actual soil: The soil was sourced from a disused coking plant site in Xuzhou, air-dried naturally in a shaded place, ground, passed through a 10-mesh sieve, and impurities such as branches were removed before storage for later use. Through testing and analysis by the national standard method (HJ 805-2016), it was found that the initial total amount of 16 PAHs in this soil was 154.68 mg / kg, and the content of each PAH component is shown in Table 5 below.

[0089] Experimental conditions: The experiment was carried out in 250-ml conical flasks. The contaminated soil and the culture solution (diluted bacterial solution) were mixed at a ratio of 1:3 (w / v). A blank control group (adding 50 g of polycyclic aromatic hydrocarbon-actually contaminated soil and 150 ml of culture solution), a free bacterium agent experimental group (adding 50 g of polycyclic aromatic hydrocarbon-actually contaminated soil, 15 ml of activated free bacterium agent, and 135 ml of culture solution), and an immobilized bacterium agent experimental group (adding 50 g of polycyclic aromatic hydrocarbon-actually contaminated soil, 15 ml of activated immobilized bacterium agent, and 135 ml of culture solution) were set up. Each group had three parallels, placed in a shaker, and cultured under the conditions of 30 °C and 200 rpm in the dark with intermittent aeration to maintain the dissolved oxygen within the range of 4.0 - 7.0 mg / L. Samples were taken on days 0, 1, 3, 7, 14, and 21 respectively, and the concentration of residual PAHs in the system was tested by the national standard method (HJ805-2016). (Among them, the preparation and activation of the free bacterium agent refer to Specific Example 2; the formula of the culture solution and the activation of the immobilized bacterium agent refer to the specific implementation plan)

[0090] Experimental results: The degradation effect of PAHs in the actual soil is as shown in the appendix Figure 5 For the convenience of comparing the degradation effects of the experimental group and the control group, the content of each PAH component on the 21st day in the system is summarized in Table 5 below.

[0091] Table 5 Comparison of the 21-day degradation effects of the immobilized bacterium agent material of the present invention on 16 PAHs in soil (mg / kg)

[0092]

[0093] From Figure 5 and Table 5, it can be seen that for the PAH-actually contaminated soil with a total amount of 154.68 mg / kg, the immobilized bacterium agent material of the present invention has a significant degradation effect, and the degradation rate reaches 93.2% at 21 days, and the degradation effect has been significantly improved compared with the free bacterium agent experimental group.

[0094] Experimental Example 1. Comparison of the abilities of 5 kinds of bacteria to degrade simulated polycyclic aromatic hydrocarbons alone

[0095] To demonstrate the synergistic effect of the composite bacterial agent of the present invention, the degradation capabilities of five bacteria on actual polycyclic aromatic hydrocarbon (PAH) wastewater and actual PAH-contaminated soil are compared as follows:

[0096] (1) Comparison of degradation capabilities for actual wastewater

[0097] Sample source: The actual PAH wastewater is the same as the actual wastewater in Example 4. The total amount of the initial 16 PAHs is 399.32 mg / L, and the content of each PAH component is shown in Table 4.

[0098] Bacterial strain preparation: The five bacteria, namely Aeromonas, Stappia, Sphingomonas, Pseudomonas aeruginosa, and Bacillus subtilis, are respectively expanded in independent culture systems (the components and culture conditions of each culture medium are as described in the above specification). When the biomass of the five bacteria reaches 2×10 7 ~6×10 9 cfu / mL, 1×10 6 ~1×10 9 cfu / mL, 3×10 6 ~6×10 8 cfu / mL, 4×10 6 ~3×10 8 cfu / mL, and 2×10 7 ~4×10 9 cfu / mL respectively, they are stored for standby.

[0099] Experimental conditions: The experiment is carried out in 250 ml conical flasks, with a total reaction system of 200 ml. An Aeromonas experimental group, a Stappia experimental group, a Sphingomonas experimental group, a Pseudomonas aeruginosa experimental group, and a Bacillus subtilis experimental group are set up (20 ml of bacterial solution and 180 ml of actual wastewater are added to each experimental group). Each group has three parallels, which are placed in a shaker and cultured under the conditions of 30 °C and 200 rpm in the dark with intermittent aeration to maintain the dissolved oxygen within the range of 4.0 - 7.0 mg / L. Samples are taken on the 14th day, and the concentration of residual PAHs in the system is tested by the national standard method (HJ 478 - 2009).

[0100] Experimental results: The summary of the individual degradation capabilities of the 5 bacteria on actual wastewater is shown in Table 6.

[0101] (2) Comparison of degradation capabilities for actual soil

[0102] The actual PAH soil is the same as the actual soil in Example 3. The total amount of the initial 16 PAHs is 154.68 mg / kg, and the content of each PAH component is shown in Table 3.

[0103] Bacterial strain preparation: The same as the bacterial strain preparation in Comparative Example (1).

[0104] Experimental conditions:

[0105] Testing by the national standard method (HJ 805-2016) shows that the initial total amount of 16 PAHs in this soil is 294.20 mg / kg. The content of each PAH component is shown in Table 6 below.

[0106] Experimental and testing conditions: Take appropriate amounts of polycyclic aromatic hydrocarbon-simulated contaminated soil and bacterial solution according to a mass ratio of 1:3 and place them in a 250 ml conical flask. Set up Aeromonas hydrophila experimental group, Stapylococcus experimental group, Sphingomonas experimental group, Pseudomonas aeruginosa experimental group and Bacillus subtilis experimental group (15 ml of bacterial solution, 135 ml of culture medium (the composition of the culture medium is detailed in the above specification) and 50 g of actual soil are added to each experimental group) (10% of the total volume of the bacterial agent is added), and there are three parallels in each group. Under the conditions of 30 °C and 200 rpm, culture with shaking in the dark for 21 days, and aerate intermittently to keep the dissolved oxygen within the range of 2.0 - 6.0 mg / L. And sample on the 14th day, and test the concentration of residual PAHs in the system by the national standard method (HJ 805-2016).

[0107] Experimental results: The individual degradation capabilities of 5 kinds of bacteria for actual sewage are summarized in Table 6.

[0108] Table 6 Comparison of the degradation capabilities of 5 kinds of bacteria for actual sewage and actual contaminated soil

[0109]

[0110]

[0111] It can be seen from the data in Table 6 that, compared with the blank control group, Aeromonas hydrophila has obvious degradation capabilities for both actual sewage and soil; Stapylococcus and Sphingomonas have weak degradation capabilities; Pseudomonas aeruginosa and Bacillus subtilis basically do not have degradation capabilities. And the composite bacterial agent formed by these 5 kinds of bacteria has very excellent degradation capabilities, and the degradation effects on both actual sewage and actual contaminated soil reach more than 95%, achieving the synergistic degradation purpose of "1 + 1 > 2".

[0112] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

[0113] Finally, it should also be noted that the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0114] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0115] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A composite microbial agent for efficiently degrading polycyclic aromatic hydrocarbons, characterized in that, The composite bacterium agent for efficiently degrading polycyclic aromatic hydrocarbons consists of Aeromonas with a preservation number of CCTCC M 2022117 at 2×10 7 ~6×10 9 cfu / mL, Staphisococcus at 1×10 6 ~1×10 9 cfu / mL, Sphingobacterium at 3×10 6 ~6×10 8 cfu / mL, Pseudomonas aeruginosa at 4×10 6 ~3×10 8 cfu / mL, and Bacillus subtilis at 2×10 7 ~4×10 9 cfu / mL, which are mixed in a volume ratio of (30 - 80):(5 - 30):(5 - 30):(5 - 30):(10 - 30).

2. The composite microbial agent for efficiently degrading polycyclic aromatic hydrocarbons according to claim 1, wherein The preservation numbers of the Stappia bacteria are CCTCC AB 208228; the preservation numbers of the Sphingomonas bacteria are CCTCC AB 2010361; the preservation numbers of the Pseudomonas aeruginosa bacteria are CCTCC AB 93066; the preservation numbers of the Bacillus subtilis bacteria are CCTCC AB90008.

3. An immobilized bacterium agent material for efficiently degrading polycyclic aromatic hydrocarbons, characterized in that, The immobilized bacterium agent material consists of a carrier and the composite bacterium agent for highly degrading polycyclic aromatic hydrocarbons as claimed in claim 1 or 2 adsorbed on the surface of the carrier.

4. The immobilized bacterium agent material for efficiently degrading polycyclic aromatic hydrocarbons according to claim 3, wherein The carrier includes: one of a biochar carrier, chitosan, alginate, polyurethane, diatomite, bentonite, vermiculite, slag, and porous ceramics.

5. A method for preparing the immobilized bacterium agent material for efficiently degrading polycyclic aromatic hydrocarbons according to claim 3 or 4, characterized in that, The method includes: The Aeromonas bacteria, Stappia bacteria, Sphingomonas bacteria, Pseudomonas aeruginosa bacteria, and Bacillus subtilis bacteria are respectively expanded and cultured to obtain bacterial solutions. When OD600 = 1.0 ± 0.05, the bacterial solutions are mixed according to the ratio, and then 1-5% of the biochar carrier with a mass-volume ratio is added for adsorption culture. After the adsorption culture is completed, centrifugation is carried out under sterile conditions to remove the upper suspension to obtain a solid product. The solid product is freeze-dried to obtain an immobilized bacterium agent material for highly degrading polycyclic aromatic hydrocarbons.

6. Application of the composite bacterium agent for highly degrading polycyclic aromatic hydrocarbons as claimed in claim 1 or 2 in degrading polycyclic aromatic hydrocarbons.

7. The application according to claim 6, wherein The method of the application includes: For polycyclic aromatic hydrocarbon-polluted water: Take the composite bacterium agent for highly degrading polycyclic aromatic hydrocarbons as claimed in claim 1 or 2 and add it to the polycyclic aromatic hydrocarbon-polluted water at a volume ratio of 5-15% for biodegradation, ensuring that the dissolved oxygen in the water is 2.0-6.0 mg / L. For polycyclic aromatic hydrocarbon-polluted soil or sediment: Take the composite bacterium agent for highly degrading polycyclic aromatic hydrocarbons as claimed in claim 1 or 2 and add it to the culture solution at a volume ratio of 5-15% to prepare a bacterial solution. Then, the bacterial solution is mixed evenly with the polluted soil or sediment at a mass ratio of 1:1-3:1 for biodegradation, ensuring that the dissolved oxygen in the water is 2.0-6.0 mg / L, and stirring and supplementing 0.1-1.5% by volume of a surfactant to promote the dissolution of polycyclic aromatic hydrocarbons.

8. Application of the immobilized bacterium agent material for highly degrading polycyclic aromatic hydrocarbons as claimed in claim 3 or 4 in degrading polycyclic aromatic hydrocarbons.

9. The application according to claim 8, characterized in that, The method of the application includes: First, the immobilized bacterium agent material is added to the culture solution at a ratio of 4-6 g / L to obtain an activated bacterial solution. For polycyclic aromatic hydrocarbon-polluted water: The activated bacterial solution is added to the sewage containing polycyclic aromatic hydrocarbon pollutants at a volume ratio of 5-15% for biodegradation, ensuring that the dissolved oxygen in the water is 4.0-7.0 mg / L. For polycyclic aromatic hydrocarbon-polluted soil or sediment: The activated bacterial solution is added to the culture solution at a volume ratio of 5-15% to obtain a diluted bacterial solution. Then, the polluted soil is mixed evenly with the diluted bacterial solution at a mass-volume ratio of 1:1-1:3 for biodegradation, ensuring that the dissolved oxygen in the water is 4.0-7.0 mg / L, and stirring and supplementing 0.1-1.5% by volume of a surfactant to promote the dissolution of polycyclic aromatic hydrocarbons.

10. The application according to claim 7 or 9, characterized in that, The formula of the culture solution is: The components in 1 L of the culture solution are as follows: 1.3 - 2.0 g of KH2PO4, 4.5 - 6.5 g of K2HPO3·3H2O, 0.5 - 2.4 g of NH4Cl, 0.4 - 1.0 g of NaCl, 100 - 500 mg of MgSO4, 0 - 100 mg of MnSO4·H2O, 0 - 100 mg of FeSO4·7H2O, 0 - 100 mg of CaCl2, with the pH adjusted to 7.0 - 7.2 and the salinity maintained within the range of 0.7 - 1.2%.

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