Preparation method and application of carboxymethyl cellulose coated phosphorus-doped biochar composite material loaded phosphate solubilizing bacteria functionalized microbial agent

Abstract

The invention discloses a preparation method and application of a carboxymethylcellulose-coated phosphorus-doped biochar composite material loaded phosphate solubilizing bacteria functionalized microbial agent, and relates to the technical field of contaminated soil remediation functional materials. The invention aims to solve the problem of low passivation efficiency caused by serious inhibition of biological activity of phosphate solubilizing bacteria by high-concentration heavy metals when the soil lead / cadmium pollution is repaired by adopting a bioremediation method. The method comprises the following steps: mixing a phosphate solubilizing bacteria culture solution with phosphorus-modified biochar, putting the mixture into a constant-temperature oscillator for full oscillation, and performing full centrifugation and freeze drying on the mixture to obtain the biochar-based biological agent loaded with the phosphate solubilizing bacteria; and adding the charcoal-based biological agent loaded with the phosphate solubilizing bacteria into a sodium carboxymethyl cellulose solution, uniformly stirring, and freeze-drying to obtain the phosphate solubilizing bacteria functionalized agent loaded with the carboxymethylcellulose-coated phosphorus-doped charcoal composite material. The preparation method and the application of the phosphate solubilizing bacteria functionalized microbial agent loaded on the phosphorus-doped charcoal composite material coated with the carboxymethyl cellulose can be obtained by adopting the preparation method and the application of the phosphate solubilizing bacteria functionalized microbial agent.

Description

Technical Field

[0001] The present invention relates to the technical field of functional materials for remediating contaminated soil, and in particular to a preparation method and application of a functionalized bacterial agent of phosphate-solubilizing bacteria loaded on a carboxymethyl cellulose-coated phosphorus-doped biochar composite material. Background Art

[0002] Lead (Pb) and cadmium (Cd) are among the most frequently detected heavy metals in soil. They can accumulate in soil through pesticide application, solid waste disposal, atmospheric deposition, and wastewater irrigation. Cadmium and lead can accumulate and migrate in soil over long periods of time. Therefore, they pose a high risk of entering the food chain, and their impacts on organisms within the food web are of particular concern.

[0003] Currently, common technologies for treating heavy metal contaminated soil include bioremediation, phytoremediation, electrodynamic remediation, and chemical stabilization. Among them, bioremediation is considered a promising solution due to its low cost and feasible operation. Phosphate-solubilizing bacteria (PSB), as a functional strain, can convert insoluble phosphates into soluble phosphates by producing organic acids and acid phosphatases. These soluble phosphates can further react with heavy metals (such as lead and cadmium) to form stable lead / cadmium phosphate compounds (such as Pb5(PO4)3OH and Cd5(PO4)3OH), thereby achieving the passivation of heavy metals. However, high concentrations of lead and cadmium may severely inhibit the biological activity of PSB, resulting in a decrease in its passivation efficiency.

[0004] To address this issue, microbial immobilization technology (MIT) has been widely used. This technology immobilizes functional microorganisms on a carrier material, protecting them from harmful substances and thus maintaining high microbial activity and stability. Biochar (BC) is considered an ideal carrier material due to its porous structure and high biocompatibility. However, raw BC has limited capacity to immobilize lead and cadmium, making it unable to effectively mitigate the toxicity of high-concentration heavy metals to PSB.

[0005] Phosphorus-modified biochar (PBC) has garnered significant attention in recent years. By introducing more functional groups, PBC enhances its passivation properties against heavy metals, making it an ideal sanctuary for PSBs. Furthermore, the phosphorus in PBC serves as an additional phosphorus source for PSBs, promoting their growth and activity. However, in complex soil environments, the performance of PBC can be disrupted by factors such as pH fluctuations and temperature changes, leading to degradation of its functionality. Summary of the Invention

[0006] The purpose of the present invention is to solve the problem that when using bioremediation methods to repair soil lead / cadmium pollution, high concentrations of heavy metals will seriously inhibit the biological activity of phosphate-solubilizing bacteria (PSB), thereby resulting in low passivation efficiency. The present invention provides a preparation method and application of a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a functionalized bacterial agent of phosphate-solubilizing bacteria.

[0007] The preparation method of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent is carried out according to the following steps:

[0008] Step S1, preparing phosphorus-modified biochar:

[0009] The biomass powder and potassium dihydrogen phosphate are added into a tube furnace, heated to 470-500° C. under a nitrogen atmosphere, and kept at 470-500° C. for 110-120 minutes; after the holding period, the mixture is cooled, soaked, rinsed, and dried overnight to obtain phosphorus-modified biochar;

[0010] Step S2: preparing a BC-based biological agent loaded with PSB:

[0011] The phosphate-solubilizing bacteria culture solution is mixed with the phosphorus-modified biochar obtained in step S1, and then placed in a constant temperature oscillator and fully shaken. The mixture is then fully centrifuged and finally freeze-dried to obtain a biochar-based bioagent loaded with phosphate-solubilizing bacteria;

[0012] Step S3, preparing a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent:

[0013] The biochar-based biological agent loaded with phosphate-solubilizing bacteria obtained in step S2 is added to the sodium carboxymethyl cellulose solution, stirred evenly, and freeze-dried to obtain a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a functionalized agent of phosphate-solubilizing bacteria.

[0014] Application of carboxymethyl cellulose coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent, and application of the carboxymethyl cellulose coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent in passivating lead and cadmium in soil.

[0015] Beneficial effects of the present invention:

[0016] 1. The preparation method of the functionalized bacterial agent loaded with phosphate-solubilizing bacteria on the CMC-coated phosphorus-doped biochar composite material of the present invention adopts the coupling of chemical synthesis and biological fixation to realize the preparation of the functionalized bacterial agent. The main mechanism of action includes: (1) PSB can convert insoluble phosphate into soluble phosphate by generating organic acid and phosphate. The soluble phosphate can further interact with Pb(II) / Cd(II) to convert the mobile Pb / Cd into stable compounds (Pb5(PO4)3OH and Cd5(PO4)3OH). (2) The functionalized bacterial agent loaded with phosphate-solubilizing bacteria on the CMC-coated phosphorus-doped biochar composite material has good heavy metal immobilization performance and can form a complex with Pb / Cd through the rich functional groups (hydroxyl, carboxyl, etc.) on the surface. In addition, due to its rich pore structure, the CMC-coated phosphorus-doped biochar can serve as a supporting material to provide a shelter for PSB, thereby improving the tolerance of phosphate-solubilizing bacteria to harmful high Pb / Cd concentrations and providing nutrients (N, P, K, etc.) to PSB, thereby improving microbial activity and stability and improving the passivation effect. Therefore, the coupling of CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent and PSB not only enhances the biological function of PSB, but also enhances the passivation of Pb / Cd. The prepared functionalized bacterial agent has efficient passivation performance and good phosphorus-solubilizing characteristics, which promotes the development and application of heavy metal passivators in soil.

[0017] 2. The preparation process of the CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent of the present invention is fast and simple.

[0018] The invention can obtain a preparation method and application of a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A scanning electron microscope image showing the surface morphology of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1;

[0020] Figure 2 The Fourier transform infrared spectrum of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown;

[0021] Figure 3 The X-ray photoelectron spectrum of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown;

[0022] Figure 4 The surface carbon element distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent after passivation of Pb in Example 1 is shown;

[0023] Figure 5The surface phosphorus distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent after passivation of Pb in Example 1 is shown;

[0024] Figure 6 The distribution diagram of lead elements on the surface of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material after the Pb is passivated by the phosphate-solubilizing bacteria functionalized agent in Example 1;

[0025] Figure 7 The surface carbon element distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent after passivation of Pb in Example 1 is shown;

[0026] Figure 8 The surface phosphorus distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent after passivation of Pb in Example 1 is shown;

[0027] Figure 9 The surface Cd element distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent after passivation of Pb in Example 1 is shown;

[0028] Figure 10 The experimental diagram shows the solubility of soluble phosphate by the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized bacterial agent in Example 1. PSB represents phosphate-solubilizing bacteria, and CPB-S represents the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized bacterial agent prepared in Example 1.

[0029] Figure 11 The experimental graph shows the effect of pH on the adsorption of Pb(II) by the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1. ◆ represents 20 mg / L, ● represents 50 mg / L, and ▲ represents 100 mg / L.

[0030] Figure 12 The experimental graph shows the effect of pH on the adsorption of Cd(II) by the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1. ◆ represents 20 mg / L, ● represents 50 mg / L, and ▲ represents 100 mg / L.

[0031] Figure 13 The kinetic experimental diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown. 1 represents the kinetic Avrami model fitting Pb(II), 2 represents the kinetic second-order model fitting Pb(II), and 3 represents the kinetic first-order model fitting Pb(II);

[0032] Figure 14The kinetic experimental diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown. 1 represents the kinetic Avrami model fitting Cd(II), 2 represents the kinetic second-order model fitting Cd(II), and 3 represents the kinetic first-order model fitting Cd(II);

[0033] Figure 15 : The experimental graph of the Pb(II) isotherm of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown. ◆ represents 5°C, ● represents 15°C, ▲ represents 30°C, 1 represents the Sips model fitting at 5°C, 2 represents the Langmuir model fitting at 5°C, 3 represents the Freundlich model fitting at 5°C, 4 represents the Sips model fitting at 15°C, 5 represents the Langmuir model fitting at 15°C, 6 represents the Freundlich model fitting at 15°C, 7 represents the Sips model fitting at 30°C, 8 represents the Langmuir model fitting at 30°C, and 9 represents the Freundlich model fitting at 30°C.

[0034] Figure 16 : The experimental graph of Cd(II) isotherm of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown. ◆ represents 5°C, ● represents 15°C, ▲ represents 30°C, 1 represents the Sips model fitting at 5°C, 2 represents the Langmuir model fitting at 5°C, 3 represents the Freundlich model fitting at 5°C, 4 represents the Sips model fitting at 15°C, 5 represents the Langmuir model fitting at 15°C, 6 represents the Freundlich model fitting at 15°C, 7 represents the Sips model fitting at 30°C, 8 represents the Langmuir model fitting at 30°C, and 9 represents the Freundlich model fitting at 30°C.

[0035] Figure 17 Graph showing the experimental results of carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent for remediation of soil Pb contamination in Example 1, A represents the control group, B represents phosphate-solubilizing bacteria, C represents the CMC-coated phosphorus-doped biochar composite material, D represents the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent prepared in Example 1, EX represents exchangeable Pb, CB represents carbonate-bound Pb, OX represents iron-manganese oxide-bound Pb, OM represents organic matter-bound Pb, and RS represents residual Pb;

[0036] Figure 18It represents the experimental diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent to repair soil Cd pollution in Example 1, A represents the control group, B represents phosphate-solubilizing bacteria, C represents the CMC-coated phosphorus-doped biochar composite material, D represents the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent prepared in Example 1, EX represents exchangeable Cd, CB represents carbonate-bound Cd, OX represents iron-manganese oxide-bound Cd, OM represents organic matter-bound Cd, and RS represents residual Cd. DETAILED DESCRIPTION

[0037] Specific embodiment 1: The preparation method of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent in this embodiment is carried out according to the following steps:

[0038] Step S1, preparing phosphorus-modified biochar:

[0039] The biomass powder and potassium dihydrogen phosphate are added into a tube furnace, heated to 470-500° C. under a nitrogen atmosphere, and kept at 470-500° C. for 110-120 minutes; after the holding period, the mixture is cooled, soaked, rinsed, and dried overnight to obtain phosphorus-modified biochar;

[0040] Step S2: preparing a BC-based biological agent loaded with PSB:

[0041] The phosphate-solubilizing bacteria culture solution is mixed with the phosphorus-modified biochar obtained in step S1, and then placed in a constant temperature oscillator and fully shaken. The mixture is then fully centrifuged and finally freeze-dried to obtain a biochar-based bioagent loaded with phosphate-solubilizing bacteria;

[0042] Step S3, preparing a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent:

[0043] The biochar-based biological agent loaded with phosphate-solubilizing bacteria obtained in step S2 is added to the sodium carboxymethyl cellulose solution, stirred evenly, and freeze-dried to obtain a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a functionalized agent of phosphate-solubilizing bacteria.

[0044] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the mass ratio of the biomass powder to potassium dihydrogen phosphate in step S1 is 1:1, and the biomass powder is corn straw powder.

[0045] The other steps are the same as those in the first embodiment.

[0046] Specific embodiment three: The difference between this embodiment and specific embodiment one or two is that: in step S2, the phosphate-solubilizing bacteria culture solution is mixed with the phosphorus-modified biochar, and then placed in a constant temperature oscillator and oscillated at a rate of 140-150 r / min for 4-5 hours, and then the mixture is centrifuged at a speed of 2900-3000 r / min for 4-5 minutes, and finally freeze-dried for 45-48 hours.

[0047] The other steps are the same as those in the first or second embodiment.

[0048] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the ratio of the volume of the phosphate-solubilizing bacteria culture solution to the mass of the phosphorus-modified biochar in step S2 is (20-22) mL: (1-1.2) g.

[0049] The other steps are the same as those in Specific Embodiments 1 to 3.

[0050] Specific embodiment 5: The difference between this embodiment and specific embodiments 1 to 4 is that the phosphate-solubilizing bacteria culture solution described in step S2 is prepared according to the following steps: the phosphate-solubilizing bacteria are placed in LB culture medium, and cultured at a temperature of 29 to 31°C and a speed of 150 to 160 r / min for 7 to 9 hours to obtain the phosphate-solubilizing bacteria culture solution.

[0051] The other steps are the same as those in Specific Embodiments 1 to 4.

[0052] Specific embodiment 6: This embodiment differs from specific embodiments 1 to 5 in that the LB culture medium consists of 10 g NaCl, 5 g yeast extract, 10 g peptone and 1000 mL distilled water.

[0053] The other steps are the same as those in Specific Embodiments 1 to 5.

[0054] Specific embodiment seven: This embodiment differs from specific embodiments one to six in that: in step S3, the biochar-based biological agent loaded with phosphate-solubilizing bacteria is added to the sodium carboxymethyl cellulose solution, stirred for 2 to 3 hours, and freeze-dried for 45 to 48 hours.

[0055] The other steps are the same as those in Specific Embodiments 1 to 6.

[0056] Specific embodiment eight: This embodiment differs from specific embodiments one to seven in that the ratio of the mass of the biochar-based biological agent loaded with phosphate-solubilizing bacteria to the volume of the sodium carboxymethyl cellulose solution described in step S3 is (1-1.2) g: (100-105) mL.

[0057] The other steps are the same as those in Specific Embodiments 1 to 7.

[0058] Specific embodiment nine: This embodiment differs from specific embodiments one to eight in that the sodium carboxymethyl cellulose solution consists of sodium carboxymethyl cellulose and deionized water, and the ratio of the mass of the sodium carboxymethyl cellulose to the volume of deionized water is (1.1-1.3) g: (100-105) mL.

[0059] The other steps are the same as those in Specific Embodiments 1 to 8.

[0060] Specific embodiment ten: This embodiment uses carboxymethyl cellulose coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent, and uses the carboxymethyl cellulose coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent in passivating lead and cadmium in soil.

[0061] The following examples are used to verify the beneficial effects of the present invention:

[0062] Example 1: A method for preparing a functionalized bacterial agent of phosphate-solubilizing bacteria loaded on a carboxymethyl cellulose-coated phosphorus-doped biochar composite material is carried out according to the following steps:

[0063] Step S1, preparing phosphorus-modified biochar:

[0064] The straw was washed three times, dried in an oven at 80°C for 24 hours, crushed, and passed through a 100-mesh sieve to obtain straw powder. The straw powder and potassium dihydrogen phosphate were added to a tube furnace, heated to 500°C under a nitrogen atmosphere, and kept at 500°C for 120 minutes. After the holding period, the mixture was cooled, soaked in deionized water, rinsed five times, and dried overnight to obtain phosphorus-modified biochar (PBC).

[0065] The mass ratio of the straw powder to potassium dihydrogen phosphate is 1:1;

[0066] Step S2: preparing a BC-based biological agent loaded with PSB:

[0067] The phosphate-solubilizing bacteria were placed in LB medium and cultured at 30°C with a rotation speed of 150 r / min for 8 h to obtain a phosphate-solubilizing bacteria culture solution in the logarithmic growth phase (OD 600 =1.0); the LB medium consists of 10g NaCl, 5g yeast extract, 10g peptone and 1000mL distilled water;

[0068] The phosphate-solubilizing bacteria is Enterobacter sp. P1, which was purchased from China General Microorganism Collection Center.

[0069] 20 mL of phosphate-solubilizing bacteria culture solution was mixed with 1 g of phosphorus-modified biochar, and the mixture was shaken at a rate of 150 rpm for 5 h in a constant temperature shaker. The mixture was then centrifuged at a speed of 3000 rpm for 5 min and finally freeze-dried for 48 h to obtain a biochar-based bioagent loaded with phosphate-solubilizing bacteria (PBC-S).

[0070] Step S3, preparing a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent:

[0071] 1 g of biochar-based biological agent loaded with phosphate-solubilizing bacteria was added to 100 mL of sodium carboxymethyl cellulose solution, stirred for 2 h, and freeze-dried for 48 h to obtain carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized agent (CPB-S).

[0072] The sodium carboxymethyl cellulose solution consists of 1.1 g of sodium carboxymethyl cellulose and 100 mL of deionized water.

[0073] Experimental part:

[0074] The carboxymethyl cellulose-coated phosphorus-doped biochar composite material prepared in this example was used to load a functionalized phosphate-solubilizing bacteria agent. Characterization of the functionalized agent, dissolution of soluble phosphate and pH effects, kinetics, isotherms, and soil Pb / Cd contamination remediation experiments were conducted. The specific conclusions are as follows:

[0075] 1. Characterization of functionalized microbial agents (scanning electron microscopy and elemental mapping, Fourier transform infrared spectroscopy, X-ray diffraction spectrum):

[0076] Field emission scanning electron microscopy (model: SU8020) was used to observe the CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized agents and to determine the surface elements after the functionalized agents passivated Pb / Cd.

[0077] Figure 1 The scanning electron microscope image shows the surface morphology of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized bacterial agent in Example 1. Figure 4 The surface carbon element distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent after passivation of Pb in Example 1 is shown. Figure 5 The surface phosphorus distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent after passivation of Pb in Example 1 is shown. Figure 6 The distribution diagram of lead elements on the surface of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material after the phosphate-solubilizing bacteria functionalized agent passivates Pb in Example 1 is shown. Figure 7The surface carbon element distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent after passivation of Pb in Example 1 is shown. Figure 8 The surface phosphorus distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent after passivation of Pb in Example 1 is shown. Figure 9 The surface Cd element distribution diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent after passivation of Pb in Example 1 is shown.

[0078] like Figure 1 As shown in the figure, it can be clearly observed that the smooth-surfaced short rod PSBs are dispersed or aggregated on the surface, which indicates that the PSBs are successfully immobilized on the phosphorus-doped biochar composite material loaded with the functionalized phosphate-solubilizing bacteria. Figure 4-9 The elemental mapping also further observed that the functionalized bacterial agent contained C and P components, and the uniform distribution of P and Pb / Cd indicated that Pb / Cd was successfully immobilized on the surface of the functionalized bacterial agent.

[0079] The surface functional groups of the phosphate-solubilizing bacteria functionalized agent loaded on CMC-coated phosphorus-doped biochar composites were determined by Fourier transform infrared spectroscopy (model: Nicolet 6700).

[0080] Figure 2 The Fourier transform infrared spectrum of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown; Figure 2 As shown, the wavelength 3432 cm can be found on the spectrum of the CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized agent. -1 、1623cm -1 and 1122cm -1 Three peaks are associated with -CH, -COOH and -OH groups, respectively. Compared with BC, a new peak, P-Oν4 (575 cm -1 ), which proves the successful doping of phosphorus. When PSB is further fixed on PBC, the -CH and -OH of PBC-S show different degrees of displacement changes, which fully proves the successful loading of PSB.

[0081] The crystal structure analysis of the phosphate-solubilizing bacteria functionalized agent loaded on CMC-coated phosphorus-doped biochar composite material was performed using an X-ray diffractometer (model: Mini Flex 600).

[0082] Figure 3 The X-ray photoelectron spectrum of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown; Figure 3As shown in the figure, KH2PO4 and graphitized carbon diffraction peaks can be observed on the energy spectrum of the CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized agent, proving that the preparation of phosphorus-doped biochar was successful.

[0083] 2. Soluble phosphate dissolution:

[0084] Experimental conditions: Insoluble calcium phosphate medium was used for cultivation. The dosage of the CMC-coated phosphorus-doped biochar composite material prepared in this example, loaded with the phosphate-solubilizing bacteria functionalized agent, was 0.5 g / L. The culture was placed in a constant temperature air shaker at 150 r / min and cultured for 48 hours.

[0085] Figure 10 The experimental diagram shows the dissolution of soluble phosphate by the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized bacterial agent in Example 1, where PSB represents phosphate-solubilizing bacteria and CPB-S represents the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized bacterial agent prepared in Example 1; Figure 10 As shown in the time-dependent phosphorus dissolution results, the concentration of dissolved phosphate remained at its lowest level within the first 3 hours, likely due to the limited biomass production of PSB during the lag phase of growth. Subsequently, the soluble phosphate content in both the CPB-S and PSB treatments increased significantly after 5 hours, likely due to the large biomass and high metabolic activity of PSB during the logarithmic growth phase. Finally, equilibrium was reached at 48 hours, with the dissolved phosphorus reaching 152.17 mg / L.

[0086] 3. pH influence:

[0087] The adsorption process is as follows: the initial concentration is 200 mg / L, the temperature is 30°C, the pH range is 2-6, the dosage of the CMC-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized bacterial agent prepared in this embodiment is 0.5 g / L, and it is placed in a constant temperature air shaker at 150 r / min for 48 hours of adsorption.

[0088] Figure 11 The experimental graph shows the effect of pH on the adsorption of Pb(II) by the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1. ◆ represents 20 mg / L, ● represents 50 mg / L, and ▲ represents 100 mg / L. Figure 12 The experimental graph shows the effect of pH on the adsorption of Cd(II) by the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1, where ◆ represents 20 mg / L, ● represents 50 mg / L, and ▲ represents 100 mg / L.

[0089] like Figure 11-12As shown in the figure, when the pH increased from 2.0 to 5.0, the adsorption capacity of the CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized agent for Pb(II) / Cd(II) increased sharply from 0.98 / 5.25 mg / g to 166.06 / 90.77 mg / g (corresponding to pollutant concentrations of 20-100 mg / L), while a decrease to varying degrees was observed at pH>6. This indicates that the initial pH value can significantly affect its adsorption effect on Pb(II) / Cd(II). The analysis showed that: (1) acidic conditions (pH≈2) triggered the protonation of the CPB-S surface, enhanced the electrostatic repulsion and inhibited the PSB activity (reduced phosphate dissolution); (2) at pH=5, the surface deprotonation reduced the repulsion, while the PSB activity increased and promoted metal fixation; (3) at pH>6, the PbOH + / CdOH + The morphological transformation leads to a decrease in adsorption capacity.

[0090] 4. Kinetic experiment:

[0091] The adsorption process is as follows: the initial concentration of the solution is 200 mg / L, pH = 5.5, temperature: 30°C, the dosage of the CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent prepared in this example is 0.5 g / L, the material is added to 100 mL of the pollutant solution, and placed in a constant temperature air shaker at 30°C for 48 hours.

[0092] Figure 13 The kinetic experimental diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown. 1 represents the kinetic Avrami model fitting Pb(II), 2 represents the kinetic second-order model fitting Pb(II), and 3 represents the kinetic first-order model fitting Pb(II); Figure 14 The kinetic experimental diagram of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown. 1 represents the kinetic Avrami model fitting Cd(II), 2 represents the kinetic second-order model fitting Cd(II), and 3 represents the kinetic first-order model fitting Cd(II).

[0093] like Figure 13-14 As shown in the figure, the adsorption of Pb(II) / Cd(II) on the CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized agent showed a two-stage characteristic: rapid adsorption reached 95.63% / 94.51% of the total amount within the first hour, attributed to the rich active sites of the material; after 10 hours, it tended to equilibrium as the sites were saturated. Avolami model (R 2 =0.97 / 0.98, SD=6.58 / 6.55) optimal fitting showed that the adsorption was dominated by chemical action, the kinetics was regulated by the distribution of active sites, and there was a multi-mechanism synergistic effect.

[0094] 5. Isotherm experiment:

[0095] Isotherm conditions: The lead solution concentration range is: 10 mg / L to 400 mg / L. The dosage of the CMC-coated phosphorus-doped biochar composite material prepared in this example, loaded with the phosphate-solubilizing bacteria functionalized bacterial agent, is 0.5 g / L. 0.05 g of the material was added to 100 mL of pollutant solutions of different concentrations, and adsorption was carried out in a constant temperature air shaker at 5°C, 15°C, and 30°C and a pH of 5.5 for 48 hours.

[0096] Figure 15 : The experimental graph of the Pb(II) isotherm of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1 is shown. ◆ represents 5°C, ● represents 15°C, ▲ represents 30°C, 1 represents the Sips model fitting at 5°C, 2 represents the Langmuir model fitting at 5°C, 3 represents the Freundlich model fitting at 5°C, 4 represents the Sips model fitting at 15°C, 5 represents the Langmuir model fitting at 15°C, 6 represents the Freundlich model fitting at 15°C, 7 represents the Sips model fitting at 30°C, 8 represents the Langmuir model fitting at 30°C, and 9 represents the Freundlich model fitting at 30°C. Figure 16 The experimental graph shows the Cd(II) isotherm of the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with the phosphate-solubilizing bacteria functionalized agent in Example 1, ◆ represents 5°C, ● represents 15°C, ▲ represents 30°C, 1 represents the Sips model fitting at 5°C, 2 represents the Langmuir model fitting at 5°C, 3 represents the Freundlich model fitting at 5°C, 4 represents the Sips model fitting at 15°C, 5 represents the Langmuir model fitting at 15°C, 6 represents the Freundlich model fitting at 15°C, 7 represents the Sips model fitting at 30°C, 8 represents the Langmuir model fitting at 30°C, and 9 represents the Freundlich model fitting at 30°C.

[0097] like Figure 15-16 As shown in Figure 2, the isotherm adsorption experiment revealed that the adsorption of Pb(II) / Cd(II) by CMC-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized agent was concentration / temperature dependent: (1) in the range of 5-30℃, the adsorption amount increased with the metal ion concentration gradient, but was limited by the competition for active sites and eventually reached equilibrium; (2) the temperature increased to promote adsorption, confirming the endothermic characteristics. Model fitting showed that Sips(R 2 =0.99 / 0.99, SD=16.97 / 6.59) and Langmuir (R 2=0.98 / 0.98, SD=18.57 / 10.95) model was optimal, indicating a monolayer nonlinear adsorption mechanism. The maximum adsorption capacity of CPB-S reached 462.73 mg / g Pb and 232.69 mg / g Cd.

[0098] 6. Remediation of soil Pb / Cd pollution experiment:

[0099] Experimental conditions: Different materials were added to the contaminated soil for treatment: (1) phosphate-solubilizing bacteria (PSB), (2) biochar (BC), (3) phosphorus-doped biochar (PBC), (4) phosphorus-doped biochar loaded with functionalized bacteria (PBC-S), (5) CMC-coated phosphorus-doped biochar composite loaded with functionalized bacteria (CPB-S), and (6) control (CK). Each treatment was divided into three replicates, and each pot consisted of 80 g of soil, and then cultivated at 25°C for 10 days. Samples were collected at 0, 1, 3, 5, 10, 20, and 30 days, and the five Pb / Cd components in the soil, namely exchangeable Pb / Cd (EX), carbonate-bound Pb / Cd (CB), iron-manganese oxide-bound Pb / Cd (OX), organic matter-bound Pb / Cd (OM), and residual Pb / Cd (RS), were analyzed using the Tessier sequential extraction method.

[0100] Figure 17 Graph showing the experimental results of carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent for repairing soil Pb contamination in Example 1, A represents the control group, B represents phosphate-solubilizing bacteria, C represents biochar, D represents CMC-coated phosphorus-doped biochar composite material, E represents phosphorus-doped biochar loaded with phosphate-solubilizing bacteria functionalized bacterial agent, F represents carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent prepared in Example 1, EX represents exchangeable Pb, CB represents carbonate-bound Pb, OX represents iron-manganese oxide-bound Pb, OM represents organic matter-bound Pb, and RS represents residual Pb; Figure 18 It represents the experimental diagram of the repair of soil Cd pollution by carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent in Example 1, A represents the control group, B represents phosphate-solubilizing bacteria, C represents biochar, D represents CMC-coated phosphorus-doped biochar composite material, E represents phosphorus-doped biochar loaded with phosphate-solubilizing bacteria functionalized bacterial agent, F represents carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with phosphate-solubilizing bacteria functionalized bacterial agent prepared in Example 1, EX represents exchangeable Cd, CB represents carbonate-bound Cd, OX represents iron-manganese oxide-bound Cd, OM represents organic matter-bound Cd, and RS represents residual Cd.

[0101] like Figure 17-18As shown in Table 1-2, in the original state, the unstable state (EX+CB) accounted for 36.49% (Pb) and 44.76% (Cd), which were significantly higher than the stable state (RS: Pb 21.64% / Cd 18.87%). After 30 days of CPB-S remediation, the unstable state of Pb / Cd decreased sharply by 67.69% / 57.15% (EX) and 64.06% / 50.80% (CB), and the stable state RS increased by 48.66% / 58.45%, with the passivation efficiency reaching 65.91% (Pb) and 53.69% (Cd); compared with the remediation effects of each precursor, the CPB-S group had the highest proportion of stable state; the remediation effect of the PBC-S group without CMC stabilization was similar to that of the PBC group, indicating that CMC stabilization can affect the occurrence form of Pb / Cd in the soil; the remediation effect of the PBC group was slightly better than that of the BC group. Combined with the above-mentioned relevant characterization results, it can be seen that phosphorus modification can improve the adsorption performance of biochar to a certain extent. In addition, the additionally added phosphate component can be utilized by phosphate-solubilizing bacteria, which can increase the survival rate of phosphate-solubilizing bacteria in the composite bacterial agent.

[0102] Table 1: Pb species proportion in contaminated soil 30 days after remediation;

[0103] form(%) CK PSB BC PBC PBC-S CPB-S RS 21.64 22.49 22.91 23.64 25.32 32.17 OM 20.62 22.8 25.93 26.12 23.9 27.23 OX 21.25 23.96 24.9 26.23 26.79 28.16 CB 17.89 15.71 13.01 12.09 11.8 6.43 EX 18.6 15.04 13.25 11.92 12.19 6.01

[0104] Table 2: Proportion of Cd forms in contaminated soil 30 days after remediation;

[0105] form(%) CK PSB BC PBC PBC-S CPB-S RS 18.87 19.49 20.47 24.56 24.56 29.90 OM 16.20 16.80 18.40 19.39 20.39 22.21 OX 20.17 20.96 20.57 23.21 24.21 27.16 CB 24.41 23.71 22.28 18.27 17.27 12.01 EX 20.35 19.04 18.28 14.57 13.57 8.72

[0106] The mechanism is attributed to: (1) the oxygen-containing functional groups of CPB-S fix heavy metals through chelation; (2) PSB induces biomineralization to produce stable minerals (Pb5(PO4)3OH / Cd5(PO4)3OH), and the dual mechanism significantly reduces the mobility of heavy metals.

Claims

1. A method for preparing a functionalized bacterial agent of phosphate-solubilizing bacteria loaded with a carboxymethyl cellulose-coated phosphorus-doped biochar composite material, characterized in that The preparation method is carried out according to the following steps: Step S1, preparing phosphorus-modified biochar: The biomass powder and potassium dihydrogen phosphate are added into a tube furnace, heated to 470-500° C. under a nitrogen atmosphere, and kept at 470-500° C. for 110-120 minutes; after the holding period, the mixture is cooled, soaked, rinsed, and dried overnight to obtain phosphorus-modified biochar; Step S2: preparing a BC-based biological agent loaded with PSB: The phosphate-solubilizing bacteria culture solution is mixed with the phosphorus-modified biochar obtained in step S1, and then placed in a constant temperature oscillator and fully shaken. The mixture is then fully centrifuged and finally freeze-dried to obtain a biochar-based bioagent loaded with phosphate-solubilizing bacteria; Step S3, preparing a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent: The biochar-based biological agent loaded with phosphate-solubilizing bacteria obtained in step S2 is added to the sodium carboxymethyl cellulose solution, stirred evenly, and freeze-dried to obtain a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a functionalized agent of phosphate-solubilizing bacteria.

2. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 1, characterized in that The mass ratio of the biomass powder to potassium dihydrogen phosphate in step S1 is 1:1, and the biomass powder is corn straw powder.

3. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 1, characterized in that In step S2, the phosphate-solubilizing bacteria culture solution is mixed with the phosphorus-modified biochar, and then placed in a constant temperature oscillator and oscillated at a rate of 140-150 r / min for 4-5 hours. The mixture is then centrifuged at a speed of 2900-3000 r / min for 4-5 minutes, and finally freeze-dried for 45-48 hours.

4. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 3, characterized in that The ratio of the volume of the phosphate-solubilizing bacteria culture solution to the mass of the phosphorus-modified biochar in step S2 is (20-22) mL: (1-1.2) g.

5. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 3 or 4, characterized in that The phosphate-solubilizing bacteria culture solution in step S2 is prepared according to the following steps: placing the phosphate-solubilizing bacteria in LB culture medium, shaking and culturing at a temperature of 29-31° C. and a rotation speed of 150-160 r / min for 7-9 hours to obtain the phosphate-solubilizing bacteria culture solution.

6. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 5, characterized in that The LB medium consists of 10 g NaCl, 5 g yeast extract, 10 g peptone and 1000 mL distilled water.

7. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 1, characterized in that In step S3, the biochar-based biological agent loaded with phosphate-solubilizing bacteria is added to the sodium carboxymethyl cellulose solution, stirred for 2 to 3 hours, and freeze-dried for 45 to 48 hours.

8. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 1 or 7, characterized in that The ratio of the mass of the biochar-based biological agent loaded with phosphate-solubilizing bacteria to the volume of the sodium carboxymethyl cellulose solution described in step S3 is (1-1.2) g: (100-105) mL.

9. The method for preparing the carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized bacterial agent according to claim 8, characterized in that The sodium carboxymethyl cellulose solution consists of sodium carboxymethyl cellulose and deionized water, and the ratio of the mass of the sodium carboxymethyl cellulose to the volume of the deionized water is (1.1-1.3) g: (100-105) mL.

10. Use of a carboxymethyl cellulose-coated phosphorus-doped biochar composite material loaded with a phosphate-solubilizing bacteria functionalized agent prepared by the method according to any one of claims 1 to 9, characterized in that The carboxymethyl cellulose-coated phosphorus-doped biochar composite material is used to load a phosphate-solubilizing bacteria functionalized bacterial agent in passivating lead and cadmium in soil.

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