Preparation method and application of immobilized microbial agent

Abstract

The invention provides a preparation method and application of an immobilized microbial agent, belongs to the technical field of microorganisms, can effectively reduce the content of PAHs in soil, can also increase the content of organic carbon in soil by immobilizing CO2, and is safe, effective and low in treatment cost. The method comprises the following steps: preparing a bacterial liquid of a functional flora; and the functional flora contains two strains of new bacteria, namely, Kocuria sp. BJ05 and Staphylococcus sp. BJ06, and the functional flora contains two strains of new bacteria, namely, Kocuria sp. BJ05 and Staphylococcus sp. BJ06. And immobilizing the bacterial liquid of the functional flora on the biochar to obtain the immobilized bacterial agent.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a preparation method of an immobilized bacterial agent and application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are organic compounds composed of two or more benzene rings arranged in a linear, angular or clustered manner. They are mainly derived from the incomplete combustion of organic matter. PAHs are widely present in the environment and can be spread through the soil, thereby affecting human health and the ecosystem.

[0003] Microbial technology is often used in the prior art to repair soil contaminated by PAHs. For example, free microorganisms are immobilized on a carrier (such as biochar) to obtain an immobilized bacterial agent, and the PAHs in the soil are removed by applying the immobilized bacterial agent to the soil. However, the existing microbial technology is still not effective in removing PAHs from the soil, and in the process of removing PAHs, it is not possible to simultaneously fix carbon dioxide in the soil. Summary of the invention

[0004] The present invention provides a preparation method of an immobilized bacterial agent and application thereof. The immobilized bacterial agent prepared by the present invention can effectively remove polycyclic aromatic hydrocarbons in the soil and simultaneously fix carbon dioxide in the soil.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing an immobilized bacterial agent, comprising: Prepare a bacterial solution of functional bacteria; the functional bacteria include Coxiella Kocuria sp. )BJ05, Staphylococcus aureus ( Staphylococcus sp. )BJ06, Pseudomonas putida ( Pseudomonas putida. )CICC23685, Sphingobacterium ( Sphingobium sp. ) RS2 and Acetobacter xylinum ( Acetobacter xylinum. )ATCC23767; among them, Coxiella ( Kocuria sp. )BJ05 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit number CGMCC No.33674; Staphylococcus aureus ( Staphylococcus sp. )BJ06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit number CGMCC No.33675; The bacterial liquid of the functional bacterial community was fixed on the biochar at a ratio of 5-50 mL: 1 g to obtain an immobilized bacterial agent.

[0006] In another aspect of the present invention, a bacterial solution of a functional bacterial flora is prepared, comprising: Each bacterium in the functional bacterial flora is activated and cultured separately until each bacterium in the functional bacterial flora reaches the logarithmic growth phase, and the cells of each bacterium are collected, and then the OD of each bacterium is adjusted with inorganic salt culture medium. 600 = 1.0, and obtained bacterial liquids of Coxiella BJ05, Staphylococcus BJ06, Pseudomonas putida CICC23685, Sphingobacterium RS2, and Acetobacter xylinum ATCC23767, respectively; The bacterial liquid of Coxsackievirus BJ05, the bacterial liquid of Staphylococcus aureus BJ06, the bacterial liquid of Pseudomonas putida CICC23685, the bacterial liquid of Sphingobacterium RS2 and the bacterial liquid of Acetobacter xylinum ATCC23767 were mixed in a volume ratio of 0.95~1.05:0.95~1.05:0.95~1.05:0.95~1.05 to obtain a bacterial liquid of functional flora.

[0007] In another aspect of the present invention, the fixing method is: The bacterial liquid of the functional bacterial community and the biochar were mixed in a ratio of 5-50 mL: 1 g to obtain a mixture, and the mixture was cultured at a constant temperature of 20-45 °C and 140-160 rpm for 0.5-4 days to obtain a culture solution, and then the culture solution was centrifuged at a speed of 11000-13000 rpm for 10-22 min and the supernatant was discarded. The culture solution was placed in an oven at 25-30 °C for drying for 6-10 h to obtain an immobilized bacterial agent.

[0008] The present invention also provides the use of the immobilized bacterial agent prepared by the above preparation method in simultaneously removing polycyclic aromatic hydrocarbons in soil and fixing carbon dioxide in soil.

[0009] In another aspect of the present invention, the polycyclic aromatic hydrocarbons include any one or more of naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzanthracene, chrysene and benzopyrene.

[0010] The preparation method provided by the present invention is to use two new strains of Coxsackie spp. Kocuria sp. ) BJ05 and Staphylococcus aureus ( Staphylococcus sp. ) The immobilized bacterial agent prepared by combining BJ06 with the other three bacteria can reduce the content of PAHs in the soil relatively efficiently, and can also increase the soil organic carbon (SOC) content by fixing CO2. It is safe, effective, and has a low treatment cost, making it suitable for wide promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a schematic diagram of the removal rate of 16 PAHs by Coxsackievirus BJ05; Figure 2 This is a schematic diagram of the removal rate of 16 PAHs by Staphylococcus aureus BJ06; Figure 3 It is a schematic diagram of the removal rate of 16 PAHs by Pseudomonas putida CICC23685; Figure 4 This is a schematic diagram of the removal rate of 16 PAHs by Sphingobacterium RS2; Figure 5 It is a schematic diagram of the removal rate of PAHs of different molecular weights and total PAHs by immobilized bacteria at different fixation times; Figure 6 The immobilized bacteria agent was applied to the soil at different fixed time. 13 C / 12 Schematic diagram of the percentage of C atoms (a), SOC content (b), the amount of CO2 assimilated in SOC (c), and carbon fixation rate (d); Figure 7 It is a schematic diagram of the removal rate of PAHs of different molecular weights and total PAHs obtained by the fixed ratio of bacterial liquid and biochar of different functional bacterial communities; Figure 8 It is the fixed ratio of bacterial liquid of different functional bacterial communities to biochar and is applied to the soil. 13 C / 12 Schematic diagram of the percentage of C atoms (a), SOC content (b), the amount of CO2 assimilated in SOC (c), and carbon fixation rate (d); Fig. 9 It is a schematic diagram of the removal rate of PAHs with different molecular weights and total PAHs by immobilized bacteria at different fixed temperatures; Fig.10 The immobilized bacteria were applied to the soil at different fixed temperatures. 13 C / 12 Schematic diagram of the percentage of C atoms (a), SOC content (b), the amount of CO2 assimilated in SOC (c) and carbon fixation rate (d). DETAILED DESCRIPTION

[0012] Example 1: This example describes a method for preparing an immobilized bacterial agent, including S1 to S3.

[0013] S1. Prepare the bacterial solution of functional bacteria: Among them, the functional flora includes Coxiella Kocuria sp. )BJ05, Staphylococcus aureus ( Staphylococcus sp. )BJ06, Pseudomonas putida ( Pseudomonas putida. )CICC23685, Sphingobacterium ( Sphingobium sp. ) RS2 and Acetobacter xylinum ( Acetobacter xylinum.)ATCC23767; The above-mentioned Coxsackie bacteria ( Kocuria sp. ) BJ05 and Staphylococcus aureus ( Staphylococcus sp. ) BJ06 are two new strains obtained in this application; The above-mentioned Coxsackie bacteria ( Kocuria sp. )BJ05 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33674; The above Staphylococci ( Staphylococcus sp. ) BJ06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33675; The above-mentioned Pseudomonas putida CICC23685, Sphingobacterium RS2 and Acetobacter xylinum ATCC23767 are existing bacteria; specifically, the above-mentioned Pseudomonas putida CICC23685 was purchased from the China Industrial Microbiological Culture Collection Administration Center, the above-mentioned Sphingobacterium RS2 was purchased from the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the above-mentioned Acetobacter xylinum ATCC23767 was purchased from Beijing Biobo Biotechnology Co., Ltd.; It should be noted that the above-mentioned Coxiella BJ05, Staphylococcus BJ06, Pseudomonas putida CICC23685 and Sphingobacterium RS2 are polycyclic aromatic hydrocarbons (hereinafter referred to as PAHs) degrading bacteria, and the above-mentioned Acetobacter xylinum ATCC23767 is a carbon-fixing bacteria; the above-mentioned PAHs-degrading bacteria are used to remove PAHs in the soil, and the above-mentioned carbon-fixing bacteria are used to fix carbon dioxide in the soil; The enrichment culture process of the above-mentioned Coxsackie BJ05 and Staphylococcus aureus BJ06 is described in detail below; From the sewage outlet of a PAHs-contaminated factory in Nanjing (PAHs concentration in soil was 89.45~178.35 mg·kg -1 , where the concentration of pyrene is 0.95~2.32mg·kg -1 ) Three healthy plant samples (Alopecurus aequalis, Trifolium pratense L., and Conyza canadensis) were selected to isolate, screen, and identify endophytic bacteria with pyrene (pyrene is a polycyclic aromatic hydrocarbon) degradation function; The specific process of the above-mentioned pyrene-degrading functional endophytic bacteria isolation, screening and identification is as follows; After the surface of the plant sample was rinsed with deionized water (18.2 MΩ-cm), 75% ethanol and 0.1% NaClO solution were added successively for 2-3 min. The surface-sterilized plant sample was fully washed with sterile water for 3 times, the disinfectant was removed, and the sample was placed in a fresh LB solid culture medium and cultured at 30°C for 72 h to check whether the plant surface was completely sterilized. Weigh 0.5 g of surface-sterilized plant sample and place it in a sterile mortar. Then add 10 mL of sterile water and grind it evenly with a mortar pestle. Use a sterile pipette to draw 5 mL of the grinding solution and inoculate it into 100 mL of PAHs degradation medium (hereinafter referred to as PDM) containing pyrene (pyrene concentration is 50 mg·L -1 ), placed at 30℃, 150r·min -1 The cells were cultured in a constant temperature shaking incubator (HZ-X100) for 7 days to obtain an enriched culture fluid; wherein the formula of the PDM is as follows: 50 mg·L -1 pyrene, and the above-mentioned PDM is obtained; Optionally, the formula of the inorganic salt culture medium can be: potassium dihydrogen phosphate 0.8 g·L -1 , potassium hydrogen phosphate 0.2 g·L -1 , ammonium sulfate 1.0 g·L -1 , magnesium sulfate 0.2 g·L -1 , calcium chloride 0.01 g·L -1 , sodium chloride 0.1 g·L -1 , trace element solution: 1 mL·L -1 ; The formula of trace element solution is: ferrous sulfate 0.5 g·L -1 , zinc sulfate 0.4 g·L -1 , manganese sulfate 0.02 g·L -1 , copper sulfate 0.01 g·L -1 , boric acid 0.01 g·L -1 , sodium molybdate 0.01 g·L -1 ; The above-mentioned inorganic salt culture medium can also be prepared by other formulas, which is not limited in this embodiment; Take 5 mL of enriched culture solution and add it back into PDM, and continue enrichment culture for 4 times; use gradient dilution and plate spreading method to separate and screen functional endophytic bacteria with pyrene degradation characteristics (Tametal., 2002); spread the diluted solution on solid PDM plate containing pyrene, and culture it in a 30℃ constant temperature incubator (GNP-9050BS-III, CIMO) for 3-7 days, screen functional endophytic colonies with pyrene degradation characteristics and repeatedly purify to obtain single colonies; the above solid PDM plate is prepared by 1.6-1.8 g agar: 100 mL of the above PDM; Single colonies were subjected to 16S rRNA sequence homology analysis, and the specific steps were as follows; Genomic DNA of single colonies was obtained using a DNA extraction kit (Axygen Company, USA) and amplified by PCR; The forward primer for the PCR amplification is 16S rDNA-27F, and the reverse primer is 16S rDNA-1492R (Invitrogen, Shanghai, China) (Byers et al., 1998); wherein the nucleotide sequence of the 16S rDNA-27F is shown in SEQ ID NO: 1 (5'-AGAGTTTGATCCTGGCTCAG-3'), and the nucleotide sequence of the 16S rDNA-1492R is shown in SEQ ID NO: 2 (5'-TACCTTGTTACGACTT-3'); The PCR reaction system (25 μL) for the above PCR amplification: 12.5 μL of Premix, 1 μL of template DNA, 0.5 μL each of primers 16S-27F and 16S-1492R, and 10.5 μL of redistilled water; The amplification program of the above PCR amplification is as follows: (a) preliminary denaturation: 94°C, 4 min; (b) denaturation: 94°C, 30 s; (c) annealing: 55°C, 30 s; (d) extension: 72°C, 30 s; (e) 30 cycles of (b), (c) and (d); (f) final extension: 72°C, 10 min; (g) insulation: 10°C, 10 min; The PCR amplification products were verified by agarose gel electrophoresis and then sequenced by Nanjing GenScript Biotechnology Co., Ltd. The sequencing results were compared and analyzed in the GenBank database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using the NCBI Blast program. The phylogenetic tree was constructed using Clustalx1.83 and MEGA 5.05 software to obtain the species information of the strain, which was named Coxiella ( Kocuria sp. ) BJ05 and Staphylococcus aureus ( Staphylococcus sp. )BJ06; Next, the degradation ability of Coxiella BJ05, Staphylococcus aureus BJ06, Pseudomonas putida CICC23685 and Sphingobacterium RS2 on PAHs was verified; Specifically, the degradation ability of the above-mentioned Coxiella BJ05, Staphylococcus BJ06, Pseudomonas putida CICC23685 and Sphingobacterium RS2 for 11 kinds of PAHs was verified by experiments; the above-mentioned 11 kinds of PAHs are naphthalene (NAP), acenaphthylene (ANA), acenaphthene (ANY), fluorene (FLU), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLT), pyrene (PYR), benzo(a)anthracene (BaA), chrysene (CHR) and BaP-benzo(a)pyrene (BbF); For any of Coxiella BJ05, Staphylococcus aureus BJ06, Pseudomonas putida CICC23685, and Sphingobacterium RS2, the above experimental process is as follows; Eleven PAHs were added to 19 mL PDM, so that the concentration of each of the 11 PAHs was 3 mg / L. 1 ml of the bacterial solution of the above bacteria was added to PDM (OD 600nm 1) was used for cultivation; and on the 5th day, 20 mL of methanol was added to the PDM, and ultrasonicated for 1 hour to obtain a solution; after filtering the solution with a 2 μm organic filter membrane, the concentrations of 11 PAHs were determined by high performance liquid chromatography (hereinafter referred to as HPLC), and the removal rate of PAHs was calculated; the calculation formula for the removal rate of PAHs is as follows; RE = (PC0-PC1) / PC0 Among them, RE is the removal rate of PAHs, PC0 is the concentration of PAHs in the soil on day 0, and PC1 is the concentration of PAHs in the soil after treatment with immobilized bacterial agents; The final experimental results are shown in Table 1; Table 1: Removal rate of PAHs by PAHs-degrading bacteria ; As shown in Table 1 , the above-mentioned Coxiella BJ05, Staphylococcus BJ06, Pseudomonas putida CICC23685 and Sphingobacterium RS2 all have good degradation effects on the above-mentioned 11 PAHs; On this basis, the examples of the present application also use the above experimental method to determine the degradation effects of the above-mentioned Coxsackievirus BJ05, Staphylococcus aureus BJ06, Pseudomonas putida CICC23685 and Sphingobacterium RS2 on 16 PAHs; the 16 PAHs include NAP-naphthalene, ANA-acenaphthene, ANY-acenaphthylene, FLU-fluorene, PHE-phenanthrene, ANT-anthracene, FLT-fluoranthene, PYR-pyrene, BaA-benzo(a)anthracene, CHR-chrysene, BbF-benzo(b)fluoranthene, BkF-benzo(k)fluoranthene, BaP-benzo(a)pyrene, DBA-dibenzo(ah)anthracene, BPE-benzo(ghi)perylene and IPY-indeno(1,2,3-cd)pyrene; The above experimental results are as follows Figures 1 to 4 As shown, Figure 1 This is a schematic diagram of the removal rate of 16 PAHs by Coxsackievirus BJ05. Figure 2 This is a schematic diagram of the removal rate of 16 PAHs by Staphylococcus aureus BJ06. Figure 3 This is a schematic diagram of the removal rate of 16 PAHs by Pseudomonas putida CICC23685. Figure 4 The removal rate of 16 PAHs by Sphingobacterium RS2 is shown in Figure 2. Figures 1 to 4 It can be seen that the above-mentioned Pseudomonas putida CICC23685, Sphingobacterium RS2, Coxsackie BJ05 and Staphylococcus aureus BJ06 all have good degradation effects on the above-mentioned 16 PAHs; In this embodiment, the preparation of the bacterial solution of the functional bacterial flora includes S11-S12: S11. Each bacterium in the functional bacterial flora is activated and cultured separately until each bacterium in the functional bacterial flora reaches the logarithmic growth phase, and the cells of each bacterium are collected, and then the OD of each bacterium is adjusted using an inorganic salt culture medium. 600 = 1.0, and obtained bacterial liquids of Coxiella BJ05, Staphylococcus BJ06, Pseudomonas putida CICC23685, Sphingobacterium RS2, and Acetobacter xylinum ATCC23767, respectively; Among them, the above OD 600 =1.0 means that the optical density of the bacterial solution of each bacteria in the functional bacterial community at a wavelength of 600 nanometers is 1.0; In this embodiment, for each type of bacteria in the functional flora, the process of activating and culturing it separately is as follows: Step 1, culture Coxiella BJ05, Staphylococcus aureus BJ06, Pseudomonas putida CICC23685 and Sphingobacterium RS2 in LB medium respectively; culture Acetobacter xylinum ATCC23767 in acetic acid bacteria medium, and culture each bacterium at 30°C with constant temperature shaking at 150 rpm for 12 h; then centrifuge at 8000 rpm for 5 min at 4°C and discard the supernatant; The formula of the LB medium is: tryptone 10.0 g·L -1 , yeast powder 5.0 g·L -1 , NaCl 10.0 g·L -1 , the pH value of LB medium was 7.0; The formula of the above acetic acid bacteria culture medium is: polypeptone 5.0 g·L -1 , yeast extract 5.0 g·L -1 , glucose 5.0 g·L -1 , mannitol 5.0 g·L -1 , MgSO4·7H2O 1.0 g·L -1 , ethanol 5.0 mL·L -1 ; The pH of the acetic acid bacteria culture medium is 6.6-7.0; Step 2: resuspend the cells of each kind of bacteria in an inorganic salt medium, and then centrifuge the cells of each kind of bacteria at 8000 rpm for 5 min; Step 3: Repeat step 2 and resuspend each bacterial cell in inorganic salt medium and vortex to mix. Finally, calculate the OD value of the bacterial suspension. 600 Adjust to 1.0 to obtain bacterial solutions of each type of bacteria, namely, bacterial solution of Coxiella BJ05, bacterial solution of Staphylococcus aureus BJ06, bacterial solution of Pseudomonas putida CICC23685, bacterial solution of Sphingobacterium RS2, and bacterial solution of Acetobacter xylinum ATCC23767; S12, mixing the bacterial solution of Coxiella BJ05, the bacterial solution of Staphylococcus aureus BJ06, the bacterial solution of Pseudomonas putida CICC23685, the bacterial solution of Sphingobacterium RS2 and the bacterial solution of Acetobacter xylinum ATCC23767 in a volume ratio of 1:1:1:1:1 to obtain a bacterial solution of functional bacteria, and placing it at 4°C for later use; S2. Preparation of biochar: In this embodiment, the plant material for preparing biochar is corn stalks, and the specific process of S2 is as follows: The corn stalks were washed and placed in an oven, fixed at 105 °C for 30 min, then baked at 55 °C for 24 h, and then crushed with a pulverizer after drying; the crushed corn stalks were heated at a heating rate of 5 °C / min under a nitrogen atmosphere, and after reaching 800 °C (i.e., the pyrolysis temperature was 800 °C), they were calcined at a constant temperature for 2 h, and after cooling, they were ground through a 60-mesh sieve to obtain a carbon material; the obtained carbon material was washed with 0.1% HCl solution, and after removing tar and ash, it was rinsed with ultrapure water for several times, and finally dried to obtain biochar; It is understandable that the biochar used in this application may also be commercially available biochar, and the examples of this application do not limit the source of the biochar; S3. Fix the bacterial liquid of the functional bacterial community on the biochar at a ratio of 5-50 mL: 1 g to obtain an immobilized bacterial agent: Weigh the biochar and place it in a conical flask, sterilize it and cool it to room temperature; mix the bacterial liquid of the functional bacteria and the biochar in a ratio of 10 mL: 1 g to obtain a mixture; place the mixture in a constant temperature shaking incubator, culture it at 30 ℃ (i.e., the fixed temperature is 30 ℃) and 150 rpm for 1 day (i.e., the fixed time is 1 day), then take it out and transfer it to a centrifuge tube and centrifuge it at 12000 rpm for 20 min, discard the supernatant and place it in an oven with an internal temperature of 28 ℃ for drying for 8 h to obtain an immobilized bacterial agent.

[0014] Optionally, the ambient temperature of the mixture in the constant temperature shaking incubator may also be 20°C or 45°C, and the shaking speed may also be 140 rpm or 160 rpm. The rotation speed of the culture solution may also be 11000 rpm or 13000 rpm, and the centrifugation time may also be 10 min or 22 min. The drying temperature may also be 25°C or 30°C, and the drying time may also be 6 h or 10 h. The above conditions may be determined as appropriate, and are not limited in the embodiments of the present application.

[0015] Example 2: The difference between this example and example 1 is that in S2, the pyrolysis temperature is 400°C.

[0016] Example 3: The difference between this example and example 1 is that in S2, the pyrolysis temperature is 600°C.

[0017] Embodiment 4: This embodiment differs from Embodiment 1 in that: in S3, the fixed time is 0.5 days.

[0018] Embodiment 5: The difference between this embodiment and embodiment 1 is that in S3, the fixed time is 2 days.

[0019] Embodiment 6: The difference between this embodiment and embodiment 1 is that in S3, the fixed time is 3 days.

[0020] Embodiment 7: This embodiment differs from Embodiment 1 in that: in S3, the fixed time is 4 days.

[0021] Example 8: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional bacteria to the biochar is 5 ml: 1 g.

[0022] Example 9: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional bacteria to the biochar is 20 ml: 1 g.

[0023] Example 10: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional bacteria to the biochar is 30 ml: 1 g.

[0024] Example 11: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional bacteria to the biochar is 40 ml: 1 g.

[0025] Example 12: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional bacteria to the biochar is 50 ml: 1 g.

[0026] Example 13: The difference between this example and Example 1 is that in S3, the fixed temperature is 20°C.

[0027] Embodiment 14: The difference between this embodiment and embodiment 1 is that in S3, the fixed temperature is 25°C.

[0028] Example 15: The difference between this example and Example 1 is that in S3, the fixed temperature is 35°C.

[0029] Example 16: The difference between this example and Example 1 is that in S3, the fixed temperature is 40°C.

[0030] Example 17: The difference between this example and Example 1 is that in S3, the fixed temperature is 45°C.

[0031] Example 18: The difference between this example and Example 1 is that in S2, the plant material is wheat straw.

[0032] Example 19: The difference between this example and Example 1 is that in S2, the plant material is rice straw.

[0033] Example 20: The difference between this example and Example 1 is that in S12, the volume ratio is 0.95:1.05:1.05:1.05:1.05.

[0034] Example 21: The difference between this example and Example 1 is that in S12, the volume ratio is 1.05:0.95:0.95:0.95:0.95.

[0035] Example 22: This example provides an application of an immobilized bacterial agent, which is based on the application of the immobilized bacterial agent prepared by the preparation method provided in Example 1 to simultaneously remove polycyclic aromatic hydrocarbons in the soil and fix carbon dioxide in the soil.

[0036] In this embodiment, the application method of the immobilized bacterial agent is: putting the immobilized bacterial agent into the soil (i.e., soil contaminated by polycyclic aromatic hydrocarbons), and mixing the immobilized bacterial agent with the soil at a mass ratio of 0.95%:1 to remove polycyclic aromatic hydrocarbons in the soil and fix carbon dioxide (hereinafter referred to as CO2) in the soil. The above-mentioned polycyclic aromatic hydrocarbons (hereinafter referred to as PAHs) include naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzanthracene, chrysene and benzopyrene.

[0037] After 20 days of mixing the immobilized bacteria with the soil, the residual concentration of PAHs in the soil was measured and the removal rate of PAHs was calculated to evaluate the removal capacity of the immobilized bacteria on PAHs in the soil. 13 C value and soil organic carbon (SOC) content and calculate 13 C / 12 The percentage of C atoms, the amount of CO2 assimilated in SOC and the carbon fixation rate are used to evaluate the ability of the experimental subjects to fix CO2.

[0038] The results of the determination of the removal of PAHs in the soil and the fixation of CO2 in the soil by the above-mentioned immobilized bacterial agent are shown in Table 2 below.

[0039] Table 2: Results of the removal of PAHs and fixation of CO2 in soil by immobilized bacteria ; As shown in Table 2, the total removal rate of PAHs by the immobilized bacterial agent prepared by the preparation method provided in Example 1 was 80.693% on the 20th day, which shows that the above-mentioned immobilized bacterial agent can effectively remove PAHs in the soil. The assimilated CO2 amount in SOC reached 48.751 mg / Kg, and the carbon fixation rate reached 2.438 mg / (Kg·d), which shows that the above-mentioned immobilized bacterial agent also has a good effect in fixing CO2 in the soil. It can be seen that the above-mentioned immobilized bacterial agent can simultaneously remove PAHs in the soil and fix CO2 in the soil.

[0040] Specifically, the specific process of determining the residual concentration of PAHs in soil is as follows: Step 1, drying the soil, grinding and mixing the dried soil; mixing n-hexane and dichloromethane in a ratio of 1:1 to obtain a mixed solution; weighing 2 g of silica gel and 2 g of anhydrous sodium sulfate and placing them in a chromatography column; Step 2: Activate the chromatography column twice with the mixed solution, then take 2 g of soil and add 10 mL of the mixed solution to obtain a test sample; Step 3, vortex mix the sample and extract it by ultrasonic for 30 min; then centrifuge the sample at 2000 rpm for 5 min, collect the supernatant of the sample and pour it into the chromatography column, and continue to use it as the sample; Step 4. Repeat step 3 twice; Step 5: Add 10 mL of the mixed solution to the chromatography column to elute the sample to obtain an extract; after the extract is completely evaporated in a rotary evaporator, elute with 2 mL of methanol to obtain an eluate; filter the eluate through a 2 μm organic filter membrane, and use HPLC to determine the concentrations of 11 PAHs to obtain the residual concentration of PAHs.

[0041] The calculation formula for calculating the removal rate of PAHs is referred to the relevant description in Example 1. 13 The C value and SOC content are measured by an isotope ratio mass spectrometer and an element analyzer. Since the measurement method of the isotope ratio mass spectrometer and the element analyzer belongs to the commonly used technical means in the technical field, this embodiment will not be described in detail.

[0042] Specifically, in the determination of δ 13 After calculating the C value and SOC content, the calculation formula for the amount of CO2 assimilated by microorganisms in the above SOC is as follows; ; in, 13 CC soc is the amount of CO2 assimilated by microorganisms in SOC, in mg kg -1 ; SOC is organic carbon content, unit is g·kg -1 ; AT %( labeled ) is the treated soil 13 C / 12 C atomic percentage; AT %( unlabeled ) is the untreated soil 13 C / 12 C atomic percentage.

[0043] The calculation formula for the above carbon fixation rate is as follows; CFR = ( 13 C - C soc ) / T Wherein, CFR is the carbon fixation rate, T is the culture time, and the unit is day, for example, 20 days.

[0044] Example 23: The difference between this example and Example 21 is that the mass ratio of the immobilized bacterial agent to the soil is 1%:1.

[0045] Example 24: The difference between this example and Example 21 is that the mass ratio of the immobilized bacterial agent to the soil is 1.05%:1.

[0046] Example 25: The difference between this example and Example 21 is that the PAHs is pyrene.

[0047] Example 26: The difference between this example and Example 21 is that the PAHs include naphthalene and pyrene.

[0048] Example 27: The difference between this example and Example 21 is that the PAHs include acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene.

[0049] Experimental Example 1: This experimental example is based on the biochar-based bacterial agent prepared by the preparation method provided in Examples 1 to 3, and explores the effect of different pyrolysis temperatures on the effect of the prepared immobilized bacterial agent on removing PAHs and fixing CO2 in the soil.

[0050] Four experimental groups were set up. The experimental object of the first experimental group was the immobilized bacterial agent prepared in Example 1, the experimental object of the second experimental group was the immobilized bacterial agent prepared in Example 2, the experimental object of the third experimental group was the immobilized bacterial agent prepared in Example 3, and the experimental object of the fourth experimental group was the bacterial liquid (i.e., free bacterial population) of the functional bacterial community prepared by S1 in Example 1.

[0051] For each experimental group, the experimental device was a serum bottle (capacity of 100 mL) containing 10 g of soil (farmland soil was used). The soil contained 11 PAHs, and the concentration of each PAH was 3 mg / kg. The 11 PAHs were naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzanthracene, chrysene and benzopyrene.

[0052] Furthermore, the serum bottle 13 The CO2 content was 5%. Specifically, after 90% of the CO2 in the serum bottle was consumed, the top of the serum bottle was flushed with pressurized synthetic air 20% O2, 80% N2 for 1 min to maintain aerobic conditions, and then 5 mL 13 CO2.

[0053] During the experiment, sterile water was first added to the soil in the serum bottle until the soil moisture content was 60%, and then 0.1g of the experimental object was added to the soil. The serum bottle was then placed at 28°C for incubation, and the gas in the serum bottle was renewed every 5 days to make the serum bottle 13 The CO2 content was 5%. On the 20th day, the soil in the serum bottle was collected for freeze-drying, and the dried soil was stored at -20 °C. Finally, the PAHs removal capacity of the experimental subjects was evaluated by measuring the residual concentration of PAHs in the soil and calculating the removal rate of PAHs. 13 C value and SOC content and calculate 13 C / 12 The percentage of C atoms, the amount of CO2 assimilated in SOC and the carbon fixation rate are used to evaluate the ability of the experimental subjects to fix CO2.

[0054] It should be noted that three repeated experimental groups were set for each treatment in the above experimental process, and the average value of the results of the three repeated experimental groups was taken as the final result of each treatment.

[0055] The above method for determining the residual concentration of PAHs in soil and calculating the removal rate of PAHs is used to calculate 13 C / 12 The methods for the percentage of C atoms, the amount of CO2 assimilated in SOC and the carbon fixation rate refer to the relevant description in Example 21 and are not repeated in this experimental example.

[0056] The experimental results of the above four experimental groups are shown in Table 3 below.

[0057] Table 3: Comparison of the effects of four experimental groups on PAHs removal and CO2 fixation ; As shown in Table 3, from the perspective of total PAHs removal rate, the total PAHs removal rates of the first experimental group (pyrolysis temperature of 800 °C), the second experimental group (pyrolysis temperature of 400 °C) and the third experimental group (pyrolysis temperature of 600 °C) were all higher than those of the fourth experimental group (free bacterial colony). 13 C / 12In terms of the percentage of C atoms, SOC content, the amount of CO2 assimilated in SOC and the carbon fixation rate, the CO2 fixed by the first, second and third experimental groups is also better than that of the fourth experimental group. Comparing the effects of the first, second and third experimental groups on PAHs removal and CO2 fixation, it can be seen that the immobilized bacterial agent of the first experimental group has the best effect. It can be seen that in the preparation process of the immobilized bacterial agent, when the pyrolysis temperature is 800 ℃, the immobilized bacterial agent has the best effect on PAHs removal and CO2 fixation, so 800 ℃ is a better pyrolysis temperature.

[0058] Experimental Example 2: This experimental example is based on the biochar-based bacterial agent prepared by the preparation method provided in Example 1, Example 4, Example 5, Example 6 and Example 7, and explores the effect of different fixation times on the effect of the prepared immobilized bacterial agent on removing PAHs and fixing CO2 in the soil.

[0059] Five experimental groups were set up. The experimental object of the first experimental group was the immobilized bacterial agent prepared in Example 1, the experimental object of the second experimental group was the immobilized bacterial agent prepared in Example 4, the experimental object of the third experimental group was the immobilized bacterial agent prepared in Example 5, the experimental object of the fourth experimental group was the immobilized bacterial agent prepared in Example 6, and the experimental object of the fifth experimental group was the immobilized bacterial agent prepared in Example 7.

[0060] The above experimental process and the determination method required for the effect of PAHs removal and CO2 fixation refer to the relevant description in the above experimental example 1, and this experimental example will not be repeated here.

[0061] The experimental results of the above five experimental groups are as follows Figure 5 and Figure 6 As shown. Figure 5 It can be seen that from the perspective of PAHs removal rate, the experimental data obtained from the third experimental group (fixed time is 2 days) showed that the removal rates of low, medium and high molecular weight and total PAHs were significantly higher than those of other fixed time periods. Figure 6 It can be seen that from the comprehensive perspective of SOC content, assimilated CO2 in SOC and carbon fixation rate under different fixation times, the third experimental group (fixation time of 2 days) has the most significant carbon fixation effect. It can be seen that in the preparation process of immobilized bacterial agents, when the fixation time is 2 days, the immobilized bacterial agent has the best effect on PAHs removal and CO2 fixation, so 2 days is the optimal fixation time.

[0062] Specifically, low, medium and high molecular weight refers to low molecular weight PAHs among 11 PAHs, medium molecular weight PAHs among 11 PAHs and high molecular weight PAHs among 11 PAHs. In this experimental example, low molecular weight PAHs among 11 PAHs include naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene and anthracene; medium molecular weight PAHs among 11 PAHs include fluoranthene, pyrene, benzanthracene and chrysene; high molecular weight PAH among 11 PAHs include benzopyrene.

[0063] Experimental Example 3: This experimental example is based on the biochar-based bacterial agent prepared by the preparation method provided in Example 1, Example 8, Example 9, Example 10, Example 11 and Example 12, and explores the effect of the fixed ratio of bacterial liquid and biochar of different functional bacterial communities on the effect of the prepared immobilized bacterial agent in removing PAHs and fixing CO2 in the soil.

[0064] Six experimental groups were set up. The experimental object of the first experimental group was the immobilized bacterial agent prepared in Example 1, the experimental object of the second experimental group was the immobilized bacterial agent prepared in Example 8, the experimental object of the third experimental group was the immobilized bacterial agent prepared in Example 9, the experimental object of the fourth experimental group was the immobilized bacterial agent prepared in Example 10, the experimental object of the fifth experimental group was the immobilized bacterial agent prepared in Example 11, and the experimental object of the sixth experimental group was the immobilized bacterial agent prepared in Example 12.

[0065] The above experimental process and the determination method required for the effect of PAHs removal and CO2 fixation refer to the relevant description in the above experimental example 1, and this experimental example will not be repeated here.

[0066] The experimental results of the above six experimental groups are as follows Figure 7 and Figure 8 As shown. Figure 7 It can be seen that from the perspective of PAHs removal rate, when the fixed ratio of functional bacterial flora to biochar is 20mL:1g (corresponding to the third experimental group), the removal rates of low, medium and high molecular weight and total PAHs are significantly higher than those of other addition amounts. Figure 8 It can be seen that from the comprehensive perspective of SOC content, the amount of CO2 assimilated in SOC and the carbon fixation rate, the carbon fixation capacity is most significant when the fixed ratio of the functional bacterial community's bacterial solution to biochar is 20mL:1g (corresponding to the third experimental group). It can be seen that in the preparation process of the immobilized bacterial agent, when the fixed ratio of the functional bacterial community's bacterial solution to biochar is 20mL:1g, the immobilized bacterial agent has the best effect on PAHs removal and CO2 fixation, so 20mL:1g is a better fixed ratio of the functional bacterial community's bacterial solution to biochar.

[0067] Experimental Example 4: This experimental example is based on the biochar-based bacterial agent prepared by the methods provided in Examples 1, 13, 14, 15, 16 and 17, and explores the effects of different fixation temperatures on the effects of the prepared immobilized bacterial agent on removing PAHs and fixing CO2 in the soil.

[0068] Six experimental groups were set up. The experimental object of the first experimental group was the immobilized bacterial agent prepared in Example 1, the experimental object of the second experimental group was the immobilized bacterial agent prepared in Example 13, the experimental object of the third experimental group was the immobilized bacterial agent prepared in Example 14, the experimental object of the fourth experimental group was the immobilized bacterial agent prepared in Example 15, the experimental object of the fifth experimental group was the immobilized bacterial agent prepared in Example 16, and the experimental object of the sixth experimental group was the immobilized bacterial agent prepared in Example 17.

[0069] The above experimental process and the determination method required for the effect of PAHs removal and CO2 fixation refer to the relevant description in the above experimental example 1, and this experimental example will not be repeated here.

[0070] The experimental results of the above six experimental groups are as follows Fig. 9 and Fig.10 As shown. Fig. 9 It can be seen that from the perspective of PAHs removal rate, when the fixed temperature is 35 ℃ (corresponding to the fourth experimental group), the removal rates of low, medium and high molecular weight and total PAHs are significantly higher than those at other fixed temperatures. Fig.10 It can be seen that from the comprehensive perspective of SOC content, CO2 assimilated in SOC and carbon fixation rate, the carbon fixation capacity is most significant when the fixed temperature is 35°C. It can be seen that in the preparation process of the immobilized bacterial agent, when the fixed temperature is 35°C, the immobilized bacterial agent has the best effect on PAHs removal and CO2 fixation, so 35°C is the optimal fixed temperature.

Claims

1. A method for preparing an immobilized bacterial agent, characterized in that: include: Prepare bacterial solution of functional bacteria; The functional flora includes Coxsackie bacteria ( Kocuria sp. )BJ05, Staphylococcus aureus ( Staphylococcus sp. )BJ06, Pseudomonas putida ( Pseudomonas putida. )CICC23685, Sphingobacterium ( Sphingobium sp. ) RS2 and Acetobacter xylinum ( Acetobacter xylinum. ) ATCC23767; wherein the Coxsackie syndrome ( Kocuria sp. ) BJ05 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit number CGMCC No.33674; the Staphylococcus aureus ( Staphylococcus sp. )BJ06 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 27, 2025, with the deposit number CGMCC No.33675; The bacterial liquid of the functional bacterial community is fixed on the biochar at a ratio of 5-50 mL: 1 g to obtain an immobilized bacterial agent.

2. The method according to claim 1, characterized in that The bacterial liquid for preparing the functional bacterial flora comprises: Each bacterium in the functional bacterial group is activated and cultured separately until each bacterium in the functional bacterial group reaches a logarithmic growth phase, the cells of each bacterium are collected, and the OD of each bacterium is adjusted using an inorganic salt culture medium. 600 = 1.0, and obtained bacterial liquids of Coxiella BJ05, Staphylococcus BJ06, Pseudomonas putida CICC23685, Sphingobacterium RS2, and Acetobacter xylinum ATCC23767, respectively; The bacterial solution of Coxsackievirus BJ05, the bacterial solution of Staphylococcus aureus BJ06, the bacterial solution of Pseudomonas putida CICC23685, the bacterial solution of Sphingobacterium RS2 and the bacterial solution of Acetobacter xylinum ATCC23767 are mixed in a volume ratio of 0.95~1.05:0.95~1.05:0.95~1.05:0.95~1.05 to obtain the bacterial solution of the functional flora.

3. The method according to claim 1, characterized in that The fixing method is: The bacterial liquid of the functional bacterial community and the biochar are mixed in a ratio of 5-50 mL: 1 g to obtain a mixture, and the mixture is cultured at a constant temperature of 20-45°C and 140-160 rpm for 0.5-4 days to obtain a culture solution, and then the culture solution is centrifuged at a speed of 11000-13000 rpm for 10-22 min and the supernatant is discarded, and the culture solution is placed in an oven at 25-30°C for drying for 6-10 h to obtain an immobilized bacterial agent.

4. Use of the immobilized bacterial agent prepared by the method according to any one of claims 1 to 3 in simultaneously removing polycyclic aromatic hydrocarbons in soil and fixing carbon dioxide in soil.

5. The use according to claim 4, characterized in that The polycyclic aromatic hydrocarbons include any one or more of naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzanthracene, chrysene and benzopyrene.

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