Preparation method and application of an immobilized microbial agent

Abstract

The present invention provides a preparation method and application of an immobilized bacterial agent, which belongs to the field of microbial technology and can reduce the content of PAHs in soil relatively efficiently, and can also increase the content of soil organic carbon by fixing CO2, which is safe, effective and has low processing cost. The method comprises: preparing a bacterial solution of a functional bacterial community. The functional bacterial community contains two new strains of Coxsackievirus ( Kocuria sp. ) BJ05 and Staphylococcus aureus ( Staphylococcus sp. )BJ06. The bacterial liquid of the functional bacterial community is fixed on the biochar to obtain an immobilized bacterial agent.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a preparation method and application of an immobilized bacterium agent. Background Art

[0002] Polycyclic Aromatic Hydrocarbons (PAHs) are a class of organic compounds composed of two or more benzene rings arranged linearly, angularly or in clusters, and their main source is the incomplete combustion of organic substances. PAHs are widely present in the environment and can be transmitted through the soil, thereby affecting human health and the ecosystem.

[0003] In the prior art, microbial technology is often used to repair soil contaminated with PAHs. For example, free microorganisms are immobilized on a carrier (such as biochar) to obtain an immobilized bacterium agent, and the PAHs in the soil are removed by applying the immobilized bacterium agent to the soil. However, the existing microbial technology still has poor removal effect on PAHs in the soil, and during the process of removing PAHs, carbon dioxide in the soil cannot be fixed simultaneously. Summary of the Invention

[0004] The present invention provides a preparation method and application of an immobilized bacterium agent. The immobilized bacterium agent prepared by the present invention can preferably remove polycyclic aromatic hydrocarbons in the soil, and at the same time fix carbon dioxide in the soil.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a preparation method of an immobilized bacterium agent, including:

[0007] Preparing a bacterial solution of a functional bacterial community; the functional bacterial community includes Kocuria sp. Kocuria Staphylococcus sp. BJ05, Staphylococcus Pseudomonas putida. BJ06, Pseudomonas putida Sphingobium sp. CICC23685, Sphingomonas Acetobacter xylinum. RS2, and Acetobacter xylinum Kocuria sp. ATCC23767; among them, Kocuria Staphylococcus sp. BJ05 was deposited on February 27, 2025 at the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 33674; Staphylococcus

[0008] The bacterial suspension of the functional bacterial community is immobilized on biochar at a ratio of 5 - 50 mL:1 g to obtain the immobilized microbial agent.

[0009] On the other hand, the present invention prepares the bacterial suspension of the functional bacterial community, including:

[0010] Each bacterium in the functional bacterial community is separately activated and cultured until each bacterium in the functional bacterial community reaches the logarithmic growth phase. The cells of each bacterium are collected, and then the OD of the cells of each bacterium is adjusted with an inorganic salt medium 600 = 1.0 to obtain the bacterial suspension of Kocuria sp. BJ05, the bacterial suspension of Staphylococcus sp. BJ06, the bacterial suspension of Pseudomonas putida CICC23685, the bacterial suspension of Sphingobium sp. RS2, and the bacterial suspension of Acetobacter xylinum ATCC23767 respectively;

[0011] According to the volume ratio of 0.95 - 1.05:0.95 - 1.05:0.95 - 1.05:0.95 - 1.05:0.95 - 1.05, the bacterial suspension of Kocuria sp. BJ05, the bacterial suspension of Staphylococcus sp. BJ06, the bacterial suspension of Pseudomonas putida CICC23685, the bacterial suspension of Sphingobium sp. RS2, and the bacterial suspension of Acetobacter xylinum ATCC23767 are mixed to obtain the bacterial suspension of the functional bacterial community.

[0012] On the other hand, the immobilization method is as follows:

[0013] The bacterial suspension of the functional bacterial community and biochar are mixed at a ratio of 5 - 50 mL:1 g to obtain a mixture. The mixture is incubated at a constant temperature of 20 - 45 °C and 140 - 160 rpm for 0.5 - 4 days to obtain a culture solution. Then the culture solution is centrifuged at a speed of 11000 - 13000 rpm for 10 - 22 min and the supernatant is discarded, and then placed in an oven at 25 - 30 °C for drying for 6 - 10 h to obtain the immobilized microbial agent.

[0014] The present invention also provides the application of the immobilized microbial agent prepared by the above preparation method in simultaneously removing polycyclic aromatic hydrocarbons in soil and fixing carbon dioxide in soil.

[0015] On the other hand, the polycyclic aromatic hydrocarbons include any one or more of naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, and benzo[a]pyrene.

[0016] The preparation method provided by the present invention uses two new bacteria, Kocuria sp. ( Kocuria sp. ), BJ05 and Staphylococcus sp. ( Staphylococcus sp.The immobilized microbial agent prepared by combining BJ06 with the other three kinds of bacteria can efficiently reduce the content of PAHs in the soil, and can also increase the content of soil organic carbon (SOC) by fixing CO2. It is safe and effective with low treatment cost and is suitable for wide promotion. Brief Description of the Drawings

[0017] Figure 1 It is a schematic diagram of the removal rate of 16 kinds of PAHs by Kocuria sp. BJ05;

[0018] Figure 2 It is a schematic diagram of the removal rate of 16 kinds of PAHs by Staphylococcus sp. BJ06;

[0019] Figure 3 It is a schematic diagram of the removal rate of 16 kinds of PAHs by Pseudomonas putida CICC23685;

[0020] Figure 4 It is a schematic diagram of the removal rate of 16 kinds of PAHs by Sphingobacterium sp. RS2;

[0021] Figure 5 It is a schematic diagram of the removal rate of immobilized microbial agent on different molecular weights and total PAHs at different immobilization times;

[0022] Figure 6 It is the immobilized microbial agent applied to the soil at different immobilization times 13 C / 12 Schematic diagram of the percentage of C atoms (a), SOC content (b), the amount of assimilated CO2 in SOC (c), and the carbon fixation rate (d);

[0023] Figure 7 It is a schematic diagram of the removal rate of immobilized microbial agent obtained by the immobilization ratio of the bacterial solution of different functional microbial communities and biochar on different molecular weights and total PAHs;

[0024] Figure 8 It is the immobilized microbial agent applied to the soil obtained by the immobilization ratio of the bacterial solution of different functional microbial communities and biochar 13 C / 12 Schematic diagram of the percentage of C atoms (a), SOC content (b), the amount of assimilated CO2 in SOC (c), and the carbon fixation rate (d);

[0025] Figure 9 It is a schematic diagram of the removal rate of immobilized microbial agent on different molecular weights and total PAHs at different immobilization temperatures;

[0026] Figure 10 It is the immobilized microbial agent applied to the soil at different immobilization temperatures 13 C / 12Schematic diagram of the percentage of C atoms (a), SOC content (b), the amount of assimilated CO2 in SOC (c), and carbon sequestration rate (d). Detailed implementation method

[0027] Example 1: This example describes a preparation method of an immobilized microbial agent, including S1 - S3.

[0028] S1. Prepare the bacterial solution of the functional microbial community:

[0029] Among them, the functional microbial community includes Kocuria sp. Kocuria Staphylococcus sp. ) BJ05, Staphylococcus Pseudomonas putida. ) BJ06, Pseudomonas putida Sphingobium sp. ) CICC23685, Sphingomonas Acetobacter xylinum. ) RS2, and Acetobacter xylinum

[0030] The above Kocuria Kocuria sp. ) BJ05 and Staphylococcus Staphylococcus sp. ) BJ06 are two newly obtained strains in this application;

[0031] The above Kocuria Kocuria sp. ) BJ05 was deposited on February 27, 2025, at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33674;

[0032] The above Staphylococcus Staphylococcus sp. ) BJ06 was deposited on February 27, 2025, at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33675;

[0033] The above Pseudomonas putida CICC23685, Sphingomonas RS2, and Acetobacter xylinum ATCC23767 are existing strains; specifically, the above Pseudomonas putida CICC23685 was purchased from the China National Center for Industrial Culture Collection of Microorganisms, the above Sphingomonas RS2 was purchased from the China General Microbiological Culture Collection Center, and the above Acetobacter xylinum ATCC23767 was purchased from Beijing BioWin Biotechnology Co., Ltd.;

[0034] It should be noted that the above-mentioned Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, and Sphingobium sp. RS2 are polycyclic aromatic hydrocarbon (PAHs) - degrading bacteria, and the above-mentioned Acetobacter xylinum ATCC23767 is a carbon - fixing bacterium; the above - mentioned PAHs - degrading bacteria are used to remove PAHs from the soil, and the above - mentioned carbon - fixing bacterium is used to fix carbon dioxide in the soil;

[0035] The enrichment culture process of the above - mentioned Kocuria sp. BJ05 and Staphylococcus sp. BJ06 will be described in detail below;

[0036] Three healthy - growing plant samples (Alopecurus aequalis, Trifolium pratense L., Conyza canadensis) were selected from a sewage outlet of a PAHs - polluted factory area in Nanjing (the PAHs concentration in the soil was 89.45 - 178.35 mg·kg -1 and the pyrene concentration was 0.95 - 2.32 mg·kg -1 ) for the isolation, screening, and identification of pyrene - degrading functional endophytic bacteria (pyrene is a polycyclic aromatic hydrocarbon);

[0037] The specific process of the above - mentioned isolation, screening, and identification of pyrene - degrading functional endophytic bacteria is as follows;

[0038] After rinsing the surface of the plant samples with deionized water (Deionized water, 18.2 MΩ - cm), 75% ethanol and 0.1% NaClO solution were added in sequence for rinsing for 2 - 3 min; the surface - sterilized plant samples were thoroughly washed 3 times with sterile water to remove the disinfectant solution, placed in a fresh LB solid medium, and incubated at 30 °C for 72 h to check whether the surface sterilization of the plants was complete;

[0039] Weighed 0.5 g of the surface - sterilized plant sample and placed it in a sterilized mortar, then immediately added 10 mL of sterile water and ground it evenly with a pestle; 5 mL of the grinding liquid was aspirated with a sterile pipette tip and inoculated into 100 mL of a PAHs - degrading medium containing pyrene (hereinafter referred to as PDM) (pyrene concentration was 50 mg·L -1 ) and placed in a shaking incubator (Shaking Incubator, HZ - X100) at 30 °C and 150 r·min -1 for 7 days to obtain an enriched culture solution; among them, the formula of the above - mentioned PDM was: adding 50 mg·L -1 of pyrene to the inorganic salt medium (i.e., MSM medium) to obtain the above - mentioned PDM;

[0040] Optionally, the formulation of the above inorganic salt medium can be: potassium dihydrogen phosphate 0.8 g·L -1 , dipotassium 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 formulation of the 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 inorganic salt medium can also be prepared from other formulations, which are not limited in this example;

[0041] Take 5 mL of the enrichment culture solution, re-add it to the PDM, and continuously enrich and culture it 4 times; use the gradient dilution and plate coating method to isolate and screen functional endophytic bacteria with pyrene degradation characteristics (Tametal., 2002); coat the dilution solution on a solid PDM plate containing pyrene, and statically culture it in a constant temperature incubator (GNP-9050BS-III, CIMO) at 30 °C for 3 - 7 d to screen out functional endophytic colonies with pyrene degradation characteristics and repeatedly purify to obtain single colonies; the above solid PDM plate is prepared from 1.6 - 1.8 g of agar: 100 mL of the above PDM;

[0042] Perform 16S rRNA sequence homology analysis on the single colonies, and the specific steps are as follows;

[0043] Obtain the genomic DNA of the single colonies using a DNA extraction kit (Axygen Company, USA) and perform PCR amplification;

[0044] The forward primer for the above PCR amplification was 16S rDNA-27F, and the reverse primer was 16S rDNA-1492R (Invitrogen, Shanghai, China) (Byers et al., 1998); among them, the nucleotide sequence of the above 16S rDNA-27F was shown as SEQ ID NO: 1 (5’-AGAGTTTGATCCTGGCTCAG-3’), and the nucleotide sequence of the above 16S rDNA-1492R was shown as SEQ ID NO: 2 (5’-TACCTTGTTACGACTT-3’);

[0045] The PCR reaction system (25 μL) for the above PCR amplification: Premix 12.5 μL, template DNA 1 μL, primers 16S-27F and 16S-1492R each 0.5 μL, double-distilled water 10.5 μL;

[0046] The amplification program for the above PCR amplification was: (a) pre-denaturation: 94°C, 4 min; (b) denaturation: 94°C, 30 s; (c) annealing: 55°C, 30 s; (d) extension: 72°C, 30 s; (e) cycle the above (b), (c) and (d) 30 times; (f) final extension: 72°C, 10 min; (g) incubation: 10°C, 10 min;

[0047] After the above PCR amplification products were verified by agarose gel electrophoresis, Nanjing GenScript Biotech Co., Ltd. performed 16S rRNA gene sequencing on them; using the NCBI Blast program, the sequencing results were compared and analyzed in the GenBank database (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and phylogenetic trees were constructed using Clustalx1.83 and MEGA 5.05 software to obtain strain species information, and they were named Kocuria ( Kocuria sp. ) BJ05 and Staphylococcus ( Staphylococcus sp. ) BJ06;

[0048] Next, the degradation abilities of Kocuria BJ05, Staphylococcus BJ06, Pseudomonas putida CICC23685, and Sphingobacterium sp. RS2 for PAHs were verified;

[0049] Specifically, the degradation capabilities of the above-mentioned Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, and Sphingomonas sp. RS2 for 11 PAHs were verified through experiments; the above 11 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 benzo[a]pyrene (BbF);

[0050] For any one of Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, and Sphingomonas sp. RS2, the above experimental process is as follows;

[0051] Add 11 PAHs to 19 mL of PDM so that the concentration of each PAH in the 11 PAHs is 3 mg / L; add 1 mL of the bacterial solution of the above bacteria (the OD of the bacterial solution 600nm is 1) for cultivation; and add 20 mL of methanol to the PDM on the 5th day and ultrasonicate for 1 h to obtain a solution; after filtering the solution through a 2-μm organic filter membrane, use high-performance liquid chromatography (hereinafter referred to as HPLC) to measure the concentration of 11 PAHs and calculate the removal rate of PAHs; the calculation formula for the removal rate of PAHs is as follows;

[0052] RE = (PC0 - PC1) / PC0

[0053] where RE is the removal rate of PAHs, PC0 is the concentration of PAHs in the soil on the 0th day, and PC1 is the concentration of PAHs in the soil after treatment with the immobilized bacterial agent;

[0054] The final experimental results are shown in Table 1;

[0055] Table 1: Removal rate table of PAH-degrading bacteria for PAHs

[0056] ;

[0057] As can be seen from Table 1, the above-mentioned Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, and Sphingomonas sp. RS2 all have good degradation effects on the above 11 PAHs;

[0058] On this basis, the embodiments of the present application also used the above experimental method to measure the degradation effects of the above-mentioned Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, and Sphingomonas sp. 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;

[0059] The above experimental results are as Figures 1 to 4 shown, where Figure 1 is a schematic diagram of the removal rate of 16 PAHs by Kocuria sp. BJ05, Figure 2 is a schematic diagram of the removal rate of 16 PAHs by Staphylococcus sp. BJ06, Figure 3 is a schematic diagram of the removal rate of 16 PAHs by Pseudomonas putida CICC23685, Figure 4 is a schematic diagram of the removal rate of 16 PAHs by Sphingomonas sp. RS2. As Figures 1 to 4 can be seen, the above-mentioned Pseudomonas putida CICC23685, Sphingomonas sp. RS2, Kocuria sp. BJ05, and Staphylococcus sp. BJ06 all have good degradation effects on the above 16 PAHs;

[0060] In this embodiment, the bacterial solutions for preparing the functional flora include S11 - S12:

[0061] S11. Each bacterium in the functional flora is separately activated and cultured until each bacterium in the functional flora reaches the logarithmic growth phase. The bacterial cells of each bacterium are collected, and then the OD of the bacterial cells of each bacterium is adjusted with an inorganic salt medium 600 = 1.0 to obtain the bacterial solution of Kocuria sp. BJ05, the bacterial solution of Staphylococcus sp. BJ06, the bacterial solution of Pseudomonas putida CICC23685, the bacterial solution of Sphingomonas sp. RS2, and the bacterial solution of Acetobacter xylinum ATCC23767 respectively;

[0062] Among them, the above OD 600 = 1.0 means that the optical density of the bacterial solution of each bacterium in the functional flora at a wavelength of 600 nm is 1.0;

[0063] In this embodiment, for each bacterium in the functional flora, the process of separately activating and culturing it is as follows:

[0064] Step 1: Respectively place Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, and Sphingomonas sp. RS2 in LB medium for cultivation; place Gluconacetobacter xylinus ATCC23767 in acetic acid bacteria medium for cultivation, and cultivate each strain at a temperature of 30 °C with a constant shaking speed of 150 rpm for 12 h; then, at a temperature of 4 °C, centrifuge at a speed of 8000 rpm for 5 min and discard the supernatant.

[0065] The formula of the above LB medium is: Tryptone 10.0 g·L -1 , Yeast extract 5.0 g·L -1 , NaCl 10.0 g·L -1 , and the pH value of the LB medium is 7.0;

[0066] The formula of the above acetic acid bacteria 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 this acetic acid bacteria medium is 6.6 - 7.0;

[0067] Step 2: Resuspend the cells of each strain with inorganic salt medium, and then centrifuge the cells of each strain at a speed of 8000 rpm for 5 min.

[0068] Step 3: Repeat Step 2, resuspend the cells of each strain with inorganic salt medium and vortex to mix evenly. Finally, adjust the OD of the obtained cell suspension 600 to 1.0 to obtain the cell suspension of each strain, namely the cell suspension of Kocuria sp. BJ05, the cell suspension of Staphylococcus sp. BJ06, the cell suspension of Pseudomonas putida CICC23685, the cell suspension of Sphingomonas sp. RS2, and the cell suspension of Gluconacetobacter xylinus ATCC23767;

[0069] S12: According to the volume ratio of 1:1:1:1:1, mix the cell suspension of Kocuria sp. BJ05, the cell suspension of Staphylococcus sp. BJ06, the cell suspension of Pseudomonas putida CICC23685, the cell suspension of Sphingomonas sp. RS2, and the cell suspension of Gluconacetobacter xylinus ATCC23767 to obtain the cell suspension of the functional flora, and place it at a temperature of 4 °C for standby;

[0070] S2: Prepare biochar:

[0071] In this embodiment, the plant material for preparing biochar is corn straw. The specific process of S2 is as follows:

[0072] After cleaning, the corn straw is placed in an oven and blanched at 105 °C for 30 min, then baked at 55 °C for 24 h. After drying, it is crushed by a crusher. Then, the crushed corn straw is heated at a heating rate of 5 °C / min under a nitrogen atmosphere. After reaching 800 °C (i.e., the pyrolysis temperature is 800 °C), it is fired at a constant temperature for 2 h. After firing and cooling, it is ground through a 60-mesh sieve to obtain a carbon material. The obtained carbon material is washed with 0.1% HCl solution, rinsed with ultrapure water multiple times after removing tar and ash, and finally dried to obtain biochar;

[0073] It can be understood that the biochar used in this application can also be commercially available biochar, and the source of biochar in the embodiments of this application is not limited;

[0074] S3. Fix the bacterial solution of the functional bacterial community on the biochar at a ratio of 5 - 50 mL:1 g to obtain an immobilized bactericide:

[0075] Weigh the biochar and place it in a conical flask. After sterilization, it is cooled to room temperature. The bacterial solution of the functional bacterial community is mixed with the biochar at a ratio of 10 mL:1 g to obtain a mixture. The mixture is placed in a constant temperature shaking incubator and cultured at 30 °C (i.e., the fixed temperature is 30 °C) and 150 rpm for 1 day (i.e., the fixed time is 1 day), then taken out and transferred to a centrifuge tube and centrifuged at 12000 rpm for 20 min. After discarding the supernatant, it is placed in an oven with an internal temperature of 28 °C and dried for 8 h to obtain an immobilized bactericide.

[0076] Optionally, the environmental temperature of the above mixture placed in the constant temperature shaking incubator can also be 20 °C or 45 °C, and the shaking speed can also be 140 rpm or 160 rpm. The rotation speed of the above culture solution can also be 11000 rpm or 13000 rpm, and the centrifugation time can also be 10 min or 22 min. The drying temperature can also be 25 °C or 30 °C, and the drying time can also be 6 h or 10 h. The above conditions can be determined according to the situation, and the embodiments of this application do not limit them.

[0077] Example 2: The difference between this example and Example 1 is that in S2, the pyrolysis temperature is 400 °C.

[0078] Example 3: The difference between this example and Example 1 is that in S2, the pyrolysis temperature is 600 °C.

[0079] Example 4: The difference between this example and Example 1 is that in S3, the fixed time is 0.5 days.

[0080] Example 5: The difference between this example and Example 1 is that in S3, the fixed time is 2 days.

[0081] Example 6: The difference between this example and Example 1 is that in S3, the fixed time is 3 days.

[0082] Example 7: The difference between this example and Example 1 is that in S3, the fixed time is 4 days.

[0083] Example 8: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional flora to the biochar is 5 ml: 1 g.

[0084] Example 9: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional flora to the biochar is 20 ml: 1 g.

[0085] Example 10: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional flora to the biochar is 30 ml: 1 g.

[0086] Example 11: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional flora to the biochar is 40 ml: 1 g.

[0087] Example 12: The difference between this example and Example 1 is that in S3, the ratio of the bacterial liquid of the functional flora to the biochar is 50 ml: 1 g.

[0088] Example 13: The difference between this example and Example 1 is that in S3, the fixed temperature is 20 °C.

[0089] Example 14: The difference between this example and Example 1 is that in S3, the fixed temperature is 25 °C.

[0090] Example 15: The difference between this example and Example 1 is that in S3, the fixed temperature is 35 °C.

[0091] Example 16: The difference between this example and Example 1 is that in S3, the fixed temperature is 40 °C.

[0092] Example 17: The difference between this example and Example 1 is that in S3, the fixed temperature is 45 °C.

[0093] Example 18: The difference between this example and Example 1 is that in S2, the plant material is wheat straw.

[0094] Example 19: The difference between this example and Example 1 is that in S2, the plant material is rice straw.

[0095] 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.

[0096] 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.

[0097] Example 22: This example provides an application of the immobilized bacterium agent, which is the application of the immobilized bacterium agent prepared based on the preparation method provided in Example 1 in simultaneously removing polycyclic aromatic hydrocarbons in soil and fixing carbon dioxide in soil.

[0098] In this example, the application method of the immobilized bacterium agent is as follows: Put the immobilized bacterium agent into the soil (i.e., the soil contaminated by polycyclic aromatic hydrocarbons), and mix the immobilized bacterium agent and the soil according to the mass ratio of 0.95%:1 to remove polycyclic aromatic hydrocarbons in the soil and fix carbon dioxide (hereinafter simply referred to as CO2) in the soil. Among them, the above polycyclic aromatic hydrocarbons (hereinafter simply referred to as PAHs) include naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, and benzo[a]pyrene.

[0099] Twenty days after the immobilized bacterium agent is mixed with the soil, measure the residual concentration of PAHs in the soil and calculate the removal rate of PAHs to evaluate the removal ability of the immobilized bacterium agent to PAHs in the soil; by measuring the δ 13 C value and the content of soil organic carbon (hereinafter simply referred to as SOC) in the soil and calculating 13 C / 12 the percentage of C atoms, the amount of assimilated CO2 in SOC, and the carbon fixation rate, evaluate the ability of the experimental object to fix CO2.

[0100] The measurement results of the above immobilized bacterium agent for removing PAHs in the soil and fixing CO2 in the soil are shown in Table 2 below.

[0101] Table 2: Measurement results of the immobilized bacterium agent for removing PAHs in the soil and fixing CO2 in the soil

[0102] ;

[0103] As can be seen from Table 2, on the 20th day, the total removal rate of PAHs by the immobilized bacterial agent prepared based on the preparation method provided in Example 1 was 80.693%. It can be seen that the above immobilized bacterial agent can better remove PAHs in the soil. The amount of assimilated CO2 in SOC reached 48.751 mg / Kg, and the carbon fixation rate reached 2.438 mg / (Kg·d). It can be seen that the above immobilized bacterial agent also has a good effect on fixing CO2 in the soil. Thus, it can be known that the above immobilized bacterial agent can simultaneously remove PAHs in the soil and fix CO2 in the soil.

[0104] Specifically, the specific process for measuring the residual concentration of PAHs in the soil is as follows:

[0105] Step 1: Dry the soil, grind and mix the dried soil evenly; mix n-hexane and dichloromethane in a ratio of 1:1 to obtain a mixed solution; weigh 2 g of silica gel and 2 g of anhydrous sodium sulfate and place them in a chromatography column.

[0106] Step 2: Activate the chromatography column twice with the mixed solution, and then add 2 g of soil to 10 mL of the mixed solution to obtain a measurement sample.

[0107] Step 3: Vortex and mix the measurement sample and ultrasonically extract for 30 min; then centrifuge the measurement sample at a speed of 2000 rpm for 5 min, collect the supernatant of the measurement sample and pour it into the chromatography column, and continue as the measurement sample.

[0108] Step 4: Repeat Step 3 twice.

[0109] Step 5: Add 10 mL of the mixed solution to the chromatography column to elute the measurement sample to obtain an extract; after completely evaporating the extract in a rotary evaporator, elute it with 2 mL of methanol to obtain an eluate; the eluate is filtered through a 2 μm organic filter membrane, and the concentrations of 11 PAHs are measured by HPLC to obtain the residual concentration of PAHs.

[0110] The calculation formula for the removal rate of PAHs refers to the relevant description in Example 1. The δ 13 C value and SOC content in the above soil are measured by an isotope ratio mass spectrometer and an elemental analyzer. Since the measurement methods using an isotope ratio mass spectrometer and an elemental analyzer belong to common technical means in the technical field, they will not be elaborated in this example.

[0111] Specifically, after measuring the δ 13 C value and SOC content in the soil, the calculation formula for the amount of CO2 assimilated by microorganisms in the above SOC is as follows;

[0112] ;

[0113] Among them, 13C-C soc is the amount of CO₂ assimilated by microorganisms in SOC, with the unit of mg·kg -1 ; SOC is the organic carbon content, with the unit of g·kg -1 ; AT %( labeled ) is the percentage of 13 C / 12 C atomic number in the treated soil; AT %( unlabeled ) is the percentage of 13 C / 12 C atomic number in the untreated soil.

[0114] The calculation formula for the carbon sequestration rate is as follows;

[0115] CFR = ( 13 C - C soc ) / T

[0116] where CFR is the carbon sequestration rate, T is the incubation time, with the unit of days, such as 20 days.

[0117] Example 23: The difference between this example and Example 21 is that the mass ratio of the immobilized microbial agent to the soil is: 1%:1.

[0118] Example 24: The difference between this example and Example 21 is that the mass ratio of the immobilized microbial agent to the soil is: 1.05%:1.

[0119] Example 25: The difference between this example and Example 21 is that the PAHs is pyrene.

[0120] Example 26: The difference between this example and Example 21 is that the PAHs include naphthalene and pyrene.

[0121] Example 27: The difference between this example and Example 21 is that the PAHs include acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene.

[0122] Experimental Example 1: Based on the biochar-based microbial agent prepared by the preparation methods provided in Examples 1 to 3, this experimental example explores the influence of different pyrolysis temperatures on the effects of the prepared immobilized microbial agent in removing PAHs and fixing CO₂ in the soil.

[0123] Four experimental groups are set up. The experimental object of the first experimental group is the immobilized microbial agent prepared in Example 1, the experimental object of the second experimental group is the immobilized microbial agent prepared in Example 2, the experimental object of the third experimental group is the immobilized microbial agent prepared in Example 3, and the experimental object of the fourth experimental group is the bacterial liquid of the functional microbial community prepared from S1 in Example 1 (i.e., the free microbial community).

[0124] For each experimental group, the experimental device was a serum bottle (with a capacity of 100 mL) containing 10 g of soil (farmland soil). 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, benz[a]anthracene, chrysene, and benzo[a]pyrene.

[0125] Furthermore, inside the above-mentioned serum bottle 13 the CO2 content was 5%. Specifically, after 90% of the CO2 in the serum bottle was consumed, first, the top of the serum bottle was rinsed with pressurized synthetic air (20% O2, 80% N2) for 1 min to maintain aerobic conditions, and then 5 mL 13 CO2 was added.

[0126] During the experiment, first, sterile water was added to the soil in the serum bottle until the soil moisture content reached 60%. Then, 0.1 g of the experimental object was added to the soil. Next, the serum bottle was placed in an incubator at 28 °C, and the gas inside the serum bottle was updated every 5 days to make the 13 CO2 content 5%. On the 20th day, the soil in the serum bottle was collected for freeze-drying, and the dried soil was stored in an environment at -20 °C. Finally, by measuring the residual concentration of PAHs in the soil and calculating the removal rate of PAHs, the removal ability of the experimental object for PAHs in the soil was evaluated; by measuring the δ 13 C value and SOC content in the soil and calculating the 13 C / 12 C atomic number percentage, the amount of assimilated CO2 in SOC, and the carbon sequestration rate, the ability of the experimental object to fix CO2 was evaluated.

[0127] It should be noted that each treatment in the above experimental process was set up with three replicate experimental groups, and the average value of the results of the three replicate experimental groups was used as the final result of each treatment.

[0128] The method for measuring the residual concentration of PAHs in the soil and calculating the removal rate of PAHs, calculating the 13 C / 12 C atomic number percentage, the amount of assimilated CO2 in SOC, and the carbon sequestration rate refer to the relevant descriptions in Example 21, and will not be elaborated in this experimental example.

[0129] The experimental results of the above four experimental groups are shown in Table 3 below.

[0130] Table 3: Comparison table of the effects of four experimental groups on PAHs removal and CO2 fixation

[0131] ;

[0132] As can be seen from Table 3, in terms of the total removal rate of PAHs, 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) are all higher than those of the fourth experimental group (free bacteria). From 13 C / 12 In terms of the percentage of C atoms, SOC content, the amount of assimilated CO2 in SOC, and the carbon sequestration rate, the fixed CO2 of the first experimental group, the second experimental group, and the third experimental group is also better than that of the fourth experimental group. Comparing the effects of the first experimental group, the second experimental group, and the third experimental group on PAHs removal and CO2 fixation, it can be seen that the immobilized bacteria agent of the first experimental group has the best effect. Thus, it can be known that during the preparation of the immobilized bacteria agent, when the pyrolysis temperature is 800 °C, the immobilized bacteria agent has the best effect on PAHs removal and CO2 fixation. Therefore, 800 °C is the preferred pyrolysis temperature.

[0133] Experimental Example 2: Based on the biochar-based bacteria agents prepared by the preparation methods provided in Example 1, Example 4, Example 5, Example 6, and Example 7, this experimental example explores the influence of different immobilization times on the effects of the prepared immobilized bacteria agents on removing PAHs and fixing CO2 in the soil.

[0134] Five experimental groups are set up. The experimental object of the first experimental group is the immobilized bacteria agent prepared in Example 1, the experimental object of the second experimental group is the immobilized bacteria agent prepared in Example 4, the experimental object of the third experimental group is the immobilized bacteria agent prepared in Example 5, the experimental object of the fourth experimental group is the immobilized bacteria agent prepared in Example 6, and the experimental object of the fifth experimental group is the immobilized bacteria agent prepared in Example 7.

[0135] The above experimental process and the measurement methods for the effects of PAHs removal and CO2 fixation refer to the relevant descriptions in Experimental Example 1 above, and will not be elaborated here in this experimental example.

[0136] The experimental results of the above five experimental groups are as Figure 5 and Figure 6 shown. As Figure 5 can be seen, in terms of the PAHs removal rate, among the experimental data obtained from the third experimental group (immobilization time of 2 days), the removal rates of low, medium, and high molecular weight and total PAHs are all significantly higher than those of other immobilization times. As Figure 6It can be seen that, considering the SOC content, the amount of assimilated CO2 in SOC, and the carbon fixation rate at different fixed times comprehensively, the carbon fixation effect of the third experimental group (with a fixed time of 2 days) is the most significant. It can be known that during the preparation process of the immobilized inoculant, when the fixed time is 2 days, the immobilized inoculant has the best effect on PAHs removal and CO2 fixation. Therefore, 2 days is the preferred fixed time.

[0137] Specifically, low, medium, and high molecular weights refer to the low molecular weight PAHs among the 11 PAHs, the medium molecular weight PAHs among the 11 PAHs, and the high molecular weight PAHs among the 11 PAHs. In this experimental example, the low molecular weight PAHs among the 11 PAHs include naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, and anthracene; the medium molecular weight PAHs among the 11 PAHs include fluoranthene, pyrene, benz[a]anthracene, and chrysene; the high molecular weight PAH among the 11 PAHs includes benzo[a]pyrene.

[0138] Experimental Example 3: Based on the biochar-based inoculant prepared by the preparation methods provided in Example 1, Example 8, Example 9, Example 10, Example 11, and Example 12, this experimental example explores the influence of the immobilization ratio of the bacterial liquid of different functional flora to biochar on the effects of the prepared immobilized inoculant in removing PAHs and fixing CO2 in soil.

[0139] Six experimental groups are set up. The experimental object of the first experimental group is the immobilized inoculant prepared in Example 1, the experimental object of the second experimental group is the immobilized inoculant prepared in Example 8, the experimental object of the third experimental group is the immobilized inoculant prepared in Example 9, the experimental object of the fourth experimental group is the immobilized inoculant prepared in Example 10, the experimental object of the fifth experimental group is the immobilized inoculant prepared in Example 11, and the experimental object of the sixth experimental group is the immobilized inoculant prepared in Example 12.

[0140] The above experimental process and the measurement methods required for the effects of PAHs removal and CO2 fixation refer to the relevant descriptions in the above Experimental Example 1, and this experimental example will not elaborate here.

[0141] The experimental results of the above six experimental groups are as Figure 7 and Figure 8 shown. It can be Figure 7 seen that from the perspective of the PAHs removal rate, when the immobilization ratio of the bacterial liquid of the functional flora to biochar is 20 mL:1 g (corresponding to the third experimental group), the removal rates of low, medium, high molecular weight, and total PAHs are all significantly higher than those of other addition amounts. It can be Figure 8It can be seen that considering the SOC content, the amount of assimilated CO2 in SOC, and the carbon sequestration rate comprehensively, the carbon sequestration ability is the most significant when the fixed ratio of the functional microbial community broth to biochar is 20 mL:1 g (corresponding to the third experimental group). It can be seen from this that during the preparation of the immobilized microbial agent, when the fixed ratio of the functional microbial community broth to biochar is 20 mL:1 g, the immobilized microbial agent has the best effect on PAHs removal and CO2 fixation. Therefore, 20 mL:1 g is the preferred fixed ratio of the functional microbial community broth to biochar.

[0142] Experimental Example 4: Based on the biochar-based microbial agent prepared by the methods provided in Example 1, Example 13, Example 14, Example 15, Example 16, and Example 17, this experimental example explores the influence of different immobilization temperatures on the effects of the prepared immobilized microbial agent in removing PAHs and fixing CO2 in soil.

[0143] Six experimental groups were set up. The experimental object of the first experimental group was the immobilized microbial agent prepared in Example 1, the experimental object of the second experimental group was the immobilized microbial agent prepared in Example 13, the experimental object of the third experimental group was the immobilized microbial agent prepared in Example 14, the experimental object of the fourth experimental group was the immobilized microbial agent prepared in Example 15, the experimental object of the fifth experimental group was the immobilized microbial agent prepared in Example 16, and the experimental object of the sixth experimental group was the immobilized microbial agent prepared in Example 17.

[0144] The above experimental process and the measurement methods required for the effects of PAHs removal and CO2 fixation refer to the relevant descriptions in Experimental Example 1 above, and will not be elaborated in this experimental example.

[0145] The experimental results of the above six experimental groups are as Figure 9 and Figure 10 shown. It can be seen from Figure 9 that from the perspective of the PAHs removal rate, when the immobilization temperature is 35 °C (corresponding to the fourth experimental group), the removal rates of low, medium, and high molecular weight and total PAHs are significantly higher than those of other immobilization temperatures. It can be seen from Figure 10 that considering the SOC content, the amount of assimilated CO2 in SOC, and the carbon sequestration rate comprehensively, the carbon sequestration ability is the most significant when the immobilization temperature is 35 °C. It can be seen from this that during the preparation of the immobilized microbial agent, when the immobilization temperature is 35 °C, the immobilized microbial agent has the best effect on PAHs removal and CO2 fixation. Therefore, 35 °C is the preferred immobilization temperature.

Claims

1. A preparation method of an immobilized microbial agent, characterized in that, Comprising: A bacterial solution for preparing a functional bacterial community; The functional flora includes Kocuria ( Kocuria sp. ), BJ05, Staphylococcus ( Staphylococcus sp. ), BJ06, Pseudomonas putida ( Pseudomonas putida. ), CICC23685, Sphingobium ( Sphingobium sp. ), RS2, and Acetobacter xylinum ( Acetobacter xylinum. ), ATCC23767; among them, the Kocuria ( Kocuria sp. ), BJ05 was deposited on February 27, 2025, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 33674; the Staphylococcus ( Staphylococcus sp. ), BJ06 was deposited on February 27, 2025, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 33675; including: Each bacterium in the functional flora is separately activated and cultured until each bacterium in the functional flora reaches the logarithmic growth phase. The bacterial cells of each bacterium are collected, and then the OD of the bacterial cells of each bacterium is adjusted with an inorganic salt medium 600 = 1.0 to obtain the bacterial solutions of Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, Sphingobium sp. RS2, and Acetobacter xylinum ATCC23767, respectively; Mix the bacterial solutions of Kocuria sp. BJ05, Staphylococcus sp. BJ06, Pseudomonas putida CICC23685, Sphingobium sp. RS2, and Acetobacter xylinum ATCC23767 according to the volume ratio of 0.95 - 1.05:0.95 - 1.05:0.95 - 1.05:0.95 - 1.05:0.95 - 1.05 to obtain the bacterial solution of the functional bacterial community; Fix the bacterial solution of the functional bacterial community on biochar at a ratio of 5 - 50 mL:1 g to obtain an immobilized microbial agent.

2. The method according to claim 1, wherein The immobilization method is as follows: Mix the bacterial solution of the functional bacterial community and the biochar at a ratio of 5 - 50 mL:1 g to obtain a mixture. Incubate the mixture at a constant temperature of 20 - 45 °C and 140 - 160 rpm for 0.5 - 4 days to obtain a culture solution. Then centrifuge the culture solution at a speed of 11000 - 13000 rpm for 10 - 22 min and discard the supernatant. Place it in an oven at 25 - 30 °C and dry for 6 - 10 h to obtain the immobilized microbial agent.

3. Application of the immobilized microbial agent prepared by the method according to any one of claims 1 - 2 in simultaneously removing polycyclic aromatic hydrocarbons from soil and fixing carbon dioxide in soil.

4. The application according to claim 3, wherein The polycyclic aromatic hydrocarbons include any one or more of naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, chrysene, and benzo[a]pyrene.

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