Immobilized biological bacterial agent for long-acting activation of fenton reaction and application thereof

Abstract

This invention relates to the field of environmental remediation technology, and discloses a long-acting immobilized biological agent for activating Fenton-like reactions and its application. The biological agent is a porous carrier loaded with microorganisms capable of oxidative iron reduction. The biological agent provided by this invention, through a porous carrier loaded with microorganisms capable of oxidative iron reduction, can achieve efficient and stable iron reduction and Fenton-like reactions under aerobic conditions, overcoming the technical bottlenecks of traditional microbial iron reduction relying on anaerobic environments, difficulty in colonizing free strains, and the easy inactivation and soil structure damage of chemical remediation materials. Applied to the remediation of organically polluted water / soil, it requires no external oxidants or ferrous salts, has low remediation costs, no secondary pollution, is ecologically friendly to soil, and its free radical generation cycle can reach several weeks to several months, exhibiting significantly better long-term effectiveness than existing technologies. It also possesses broad-spectrum remediation capabilities, capable of simultaneously degrading multiple organic pollutants with different structures.

Description

Technical Field

[0001] This invention relates to the field of environmental remediation technology, and in particular to a long-acting immobilized biological agent that activates the Fenton reaction and its application. Background Technology

[0002] Dryland soils, dried paddy fields, and industrial contaminated sites commonly suffer from complex pollution problems involving arsenic and organic pollutants such as methyl orange, tetracycline, dichlorophenol, and carbamazepine. Simultaneous and efficient remediation of such complex pollution is a core technical challenge in environmental governance. Current mainstream remediation technologies have significant shortcomings: traditional Fenton oxidation technology relies on the addition of hydrogen peroxide and ferrous salts, which is costly, and ferrous ions are easily and rapidly oxidized to ferric ions, leading to inactivation, a sharp decline in free radical yield, and a short remediation cycle. Furthermore, large amounts of chemical reagents can damage soil aggregate structure, reduce soil permeability, and decrease the activity of indigenous microorganisms. Iron-based materials such as zero-valent iron also suffer from easy passivation and rapid activity decay, making it difficult to meet the needs of long-term remediation.

[0003] To avoid the drawbacks of chemical reagents, researchers attempted to use microorganisms to drive iron reduction in situ to generate ferrous iron, thereby initiating a Fenton-like reaction. However, this technology still faces two major bottlenecks: First, when aerobic iron-reducing bacteria are added in free form, they are difficult to colonize effectively in the soil. Resource competition among native microorganisms and the loss of bacteria with water migration both lead to short-lived and unstable remediation effects. Second, existing microbial iron reduction processes are highly dependent on anaerobic or flooded environments. Under aerobic conditions such as dry land, oxygen, as a preferential electron acceptor, strongly competes for the metabolic electron flow of microorganisms, inhibiting iron reductase activity and significantly reducing iron reduction efficiency, thus failing to generate sufficient ferrous iron to initiate a Fenton-like reaction.

[0004] Immobilized microbial technology provides a new approach for bacterial colonization, but existing immobilized microbial technologies only focus on the direct degradation of organic pollutants. They have not yet formed a technical solution that utilizes porous carriers to load aerobic iron-reducing bacteria and drive Fenton-like reactions under aerobic conditions to produce free radicals in a long-term manner. Furthermore, they cannot solve the industry pain point of simultaneous and long-term remediation of complex pollution under aerobic conditions. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a biological agent.

[0006] The second objective of this invention is to provide a method for preparing this biological agent.

[0007] The third objective of this invention is to provide the application of this biological agent.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a biological agent, wherein the biological agent is a porous carrier loaded with microorganisms capable of reducing iron with oxygen.

[0009] In some embodiments of the present invention, the microorganism possessing oxy-iron reduction capability is selected from Bacillus megaterium (Betagenus spp.). Bacillus megaterium Bacillus subtilis ( Bacillus subtilis ), Bacillus licheniformis ( Bacillus licheniformis Colorado pseudomycolic acid bacteria ( Amycolatopsis albidoflavus ), Streptomyces grayi ( Streptomyces griseus ), Enterococcus mongolicus ( Enterococcus mundti At least one of the following.

[0010] Specifically, the present invention described Bacillus subtilis and Bacillus licheniformis Both were purchased from the American Type Culture Collection (ATCC) Biological Standards Resource Center, with catalog numbers ATCC 6051 and ATCC12759, respectively; Bacillus megaterium , Amycolatopsis albidoflavus , Streptomyces griseus and Enterococcus mundti All samples were purchased from the Guangdong Provincial Center for Microbial Culture Collection, with accession numbers GDMCC 1.183, GDMCC 4.102, GDMCC 4.19, and GDMCC 1.981, respectively.

[0011] In some embodiments of the present invention, the particle size of the porous carrier is less than 100 mesh.

[0012] In some embodiments of the present invention, the porous carrier is selected from at least one of activated carbon, diatomaceous earth, porous ceramics, and biochar.

[0013] In some preferred embodiments of the present invention, the porous carrier is biochar.

[0014] In some embodiments of the present invention, the biochar is prepared by a method comprising the following steps: Agricultural waste is pyrolyzed under limited oxygen conditions to obtain the biochar.

[0015] In some embodiments of the present invention, the agricultural waste includes at least one of mushroom sticks, corn stalks, rice husks, and peanut shells.

[0016] In some embodiments of the present invention, the agricultural waste is further dried and crushed before use.

[0017] In some embodiments of the present invention, the pyrolysis temperature is 300-700°C and the time is 1-4 hours.

[0018] In some preferred embodiments of the present invention, the pyrolysis temperature is 300-500°C and the time is 1-3 hours.

[0019] In some embodiments of the present invention, the heating rate of the pyrolysis is 6-10 °C / min.

[0020] In some preferred embodiments of the present invention, the heating rate of the pyrolysis is 7-9°C / min.

[0021] In some embodiments of the present invention, the moisture content of the biological agent is 8%-12%.

[0022] The second aspect of the present invention provides a method for preparing the biological agent described in the first aspect of the present invention, comprising the following steps: The aforementioned biological agent is obtained by co-culturing a porous carrier with a bacterial solution of microorganisms capable of reducing ferric oxygen.

[0023] In some embodiments of the present invention, the bacterial solution of the microorganisms possessing ferric oxygen reduction capability is obtained by a method comprising the following steps: After reconstitution of the lyophilized bacterial powder, it was streaked onto LB solid plates for culture. Single colonies were picked and inoculated into LB liquid medium. The culture was carried out until the logarithmic growth phase. The bacterial cells were collected by centrifugation, washed, and resuspended in sterile water to obtain the bacterial solution.

[0024] In some embodiments of the present invention, the temperature for culturing on LB solid plates is 25-35°C and the time is 20-30 hours.

[0025] In some preferred embodiments of the present invention, the temperature for culturing on LB solid plates is 28-32°C and the time is 20-25 hours.

[0026] In some embodiments of the present invention, the LB liquid culture medium comprises the following components: 8-12 g / L tryptone, 4-6 g / L yeast extract, and 8-12 g / L sodium chloride.

[0027] In some embodiments of the present invention, the pH of the LB liquid culture medium is 7.2 ± 0.2.

[0028] In some embodiments of the present invention, the LB liquid culture medium is autoclaved at 120-125°C for 15-25 minutes before use.

[0029] In some embodiments of the present invention, the culture temperature for culturing to the logarithmic growth phase is 25-35°C, and the time is 10-20 hours.

[0030] In some preferred embodiments of the present invention, the culture temperature for culturing to the logarithmic growth phase is 28-32°C, and the time is 15-20 hours.

[0031] In some embodiments of the present invention, the process of culturing to the logarithmic growth phase is supplemented with oscillation at a speed of 150-200 rpm.

[0032] In some embodiments of the present invention, the centrifugation speed is 4000-6000 rpm and the time is 6-10 min.

[0033] In some preferred embodiments of the present invention, the centrifugation speed is 4500-5500 rpm and the time is 7-9 min.

[0034] In some embodiments of the present invention, the OD of the bacterial solution 600 The value is 0.8-1.2; the solid-liquid ratio of the porous carrier to the bacterial solution is 1g: (10-50)mL.

[0035] In some preferred embodiments of the present invention, the OD of the bacterial solution 600 The value is 0.9-1.1; the solid-liquid ratio of the porous carrier to the bacterial solution is 1g: (10-30)mL.

[0036] In some embodiments of the present invention, the co-culture temperature is 25-35°C and the time is 2-6 hours.

[0037] In some preferred embodiments of the present invention, the co-culture temperature is 28-32°C and the time is 3-5 hours.

[0038] In some embodiments of the present invention, the co-cultivation process is supplemented with oscillation at a speed of 150-200 rpm.

[0039] In some embodiments of the present invention, after the co-culture is completed, the solid phase is further separated, collected, and dried.

[0040] In some embodiments of the present invention, the drying temperature is 30-40°C.

[0041] The third aspect of the present invention provides the application of the biological agent described in the first aspect of the present invention in the remediation of organically polluted water bodies and / or soil.

[0042] In some embodiments of the present invention, the organic pollutants include at least one of methyl orange, tetracycline, dichlorophenol, and carbamazepine.

[0043] In some embodiments of the present invention, the content of methyl orange in the organically polluted water body is 16-24 mg / L.

[0044] In some preferred embodiments of the present invention, the content of methyl orange in the organically polluted water body is 18-22 mg / L.

[0045] In some embodiments of the present invention, the tetracycline content in the organically polluted water body is 8-12 mg / L.

[0046] In some preferred embodiments of the present invention, the tetracycline content in the organically polluted water body is 9-11 mg / L.

[0047] In some embodiments of the present invention, the content of dichlorophenol in the organically polluted water body is 12-18 mg / L.

[0048] In some preferred embodiments of the present invention, the content of dichlorophenol in the organic polluted water is 13-17 mg / L.

[0049] In some embodiments of the present invention, the content of carbamazepine in the organically polluted water body is 4-6 mg / L.

[0050] In some preferred embodiments of the present invention, the content of carbamazepine in the organically polluted water body is 4.5-5.5 mg / L.

[0051] In some embodiments of the present invention, the content of methyl orange in the organically contaminated soil is 40-60 mg / kg.

[0052] In some preferred embodiments of the present invention, the content of methyl orange in the organically contaminated soil is 45-55 mg / kg.

[0053] In some embodiments of the present invention, the tetracycline content in the organically contaminated soil is 24-36 mg / kg.

[0054] In some preferred embodiments of the present invention, the tetracycline content in the organically contaminated soil is 27-33 mg / kg.

[0055] In some embodiments of the present invention, the content of dichlorophenol in the organically contaminated soil is 32-48 mg / kg.

[0056] In some preferred embodiments of the present invention, the content of dichlorophenol in the organically contaminated soil is 36-44 mg / kg.

[0057] In some embodiments of the present invention, the content of carbamazepine in the organically contaminated soil is 16-24 mg / kg.

[0058] In some preferred embodiments of the present invention, the content of carbamazepine in the organically contaminated soil is 18-22 mg / kg.

[0059] In some embodiments of the present invention, the pollutants in the organically contaminated soil are methyl orange, tetracycline, dichlorophenol, and carbamazepine.

[0060] In some embodiments of the present invention, the organic polluted water and / or soil contains iron oxides, the iron oxides including at least one of ferrihydrite, ferrihydrite, and goethite.

[0061] In some embodiments of the present invention, the water content of the organically contaminated soil is 50%-70% of field capacity.

[0062] In some embodiments of the present invention, the pH of the organically contaminated soil is 5.2-7.8.

[0063] In some preferred embodiments of the present invention, the pH of the organically contaminated soil is 5.8-7.2.

[0064] In some embodiments of the present invention, the organic matter content of the organically contaminated soil is 9-15 g / kg.

[0065] In some preferred embodiments of the present invention, the organic matter content of the organically contaminated soil is 10-13 g / kg.

[0066] In some embodiments of the present invention, the dosage of the biological agent in organically polluted water is 1-5 g / L of water.

[0067] In some preferred embodiments of the present invention, the dosage of the biological agent in organically polluted water is 1-3 g / L of water.

[0068] In some embodiments of the present invention, the remediation time for the organically polluted water body is 2-5 days.

[0069] In some embodiments of the present invention, the dosage of the biological agent in organically contaminated soil is 1-5 g / kg soil.

[0070] In some preferred embodiments of the present invention, the dosage of the biological agent in organically contaminated soil is 1-3 g / kg soil.

[0071] In some embodiments of the present invention, the remediation time for the organically contaminated soil is 5-10 days.

[0072] Compared with the prior art, the beneficial effects of the present invention are: 1) The biological agent provided by the present invention loads microorganisms with aerobic iron reduction ability on a porous carrier, which can achieve efficient and stable iron reduction and Fenton-like reaction under aerobic conditions, breaking through the technical bottlenecks of traditional microbial iron reduction relying on anaerobic environment, difficulty in colonization of free strains, and easy inactivation and damage to soil structure of chemical remediation materials. 2) The preparation method of the biological agent provided by this invention is simple and suitable for industrial application; 3) The biological agent provided by this invention can be applied to the remediation of organically polluted water / soil without the need for external oxidants and ferrous salts. It has low remediation costs, no secondary pollution, and is ecologically friendly to soil. The free radical generation cycle can last for several weeks to several months, and its long-term effectiveness is significantly better than that of existing technologies. At the same time, it has broad-spectrum remediation capabilities and can simultaneously degrade a variety of organic pollutants with different structures, providing an efficient, economical, and sustainable remediation solution for sites with aerobic compound pollution. Attached Figure Description

[0073] Figure 1 Here is a SEM image of the biochar from Example 1; Figure 2 Here is a SEM image of the biological agent in Example 1; Figure 3 The concentration of Fe(II) in different mineral systems after 7 days of reaction in Experiment Example 1; Figure 4 O2 at the goethite interface of different treatment groups after 1 day of reaction in Experiment Example 2 - (a) and ·OH(b) production amounts; Figure 5 The diagram shows the kinetic changes of the long-term generation of Fe(II) (a) and free radicals (b) in different systems in Experimental Example 2; Figure 6 The degradation kinetics curves of methyl orange (a), tetracycline (b), dichlorophenol (c) and carbamazepine (d) in Experimental Example 3 are shown. Figure 7 The concentration of Fe(II) in the different systems in Experiment Example 4 after 5 days of reaction; Figure 8 The degradation rates of different pollutants in different systems after 5 days of reaction in Experiment Example 4 are shown. Detailed Implementation

[0074] The present invention will be further described in detail below with reference to specific accompanying drawings and embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0075] Note: Among the bacterial strains used in the following examples, Bacillus licheniformis (B. licheniformis) Bacillus licheniformisPurchased from the ATCC Biostandard Resource Center in the United States, catalog number ATCC12759; Bacillus megaterium ( Bacillus megaterium Purchased from Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC 1.183.

[0076] Example 1 This embodiment prepares a biological agent, and the steps are as follows: (1) Preparation of biochar: Corn stalks were used as raw material. After being air-dried, the powder was crushed and passed through a 20-mesh sieve. 200g of the sieved powder was weighed, spread evenly in a ceramic crucible, compacted, and covered. The crucible was placed in a muffle furnace and heated from room temperature to 400℃ at a rate of 8℃ / min under limited oxygen conditions. The temperature was kept constant for 2 hours and then cooled naturally to room temperature. The pyrolysis product was taken out, ground, and passed through a 100-mesh sieve to obtain biochar. The biochar was then placed in a sealed bag and stored in a desiccator for later use.

[0077] (2) Preparation of bacterial culture: Bacillus licheniformis lyophilized powder was reconstituted with sterile water and streaked onto LB agar plates. After incubation at 30°C for 24 h, single colonies were picked and inoculated into 50 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.2 ± 0.2, autoclaved at 121°C for 20 min). The medium was incubated at 30°C and 170 rpm with shaking for 18 h until the logarithmic growth phase. The culture was centrifuged at 5000 rpm for 8 min, the supernatant was discarded, and the cells were washed three times with sterile water. Finally, the cells were resuspended in sterile water and the OD was adjusted. 600 =1.0, to obtain bacterial culture, which was stored at 4℃ for later use; (3) Preparation of biological agents: Take 10g of biochar and place it in a 250mL Erlenmeyer flask. Sterilize at 121℃ for 30min. After cooling, add 200mL of bacterial solution and shake in a constant temperature shaker at 30℃ and 170rpm for 4h for adsorption. After adsorption, filter and separate with sterile filter paper, collect the solid part, and dry it at 35℃ until the moisture content is about 10% to obtain the biological agent.

[0078] Figure 1 Here is a SEM image of the biochar from Example 1. Figure 1 It can be seen that the biochar prepared in Example 1 is porous, layered, and has a rough surface. Its specific surface area was measured to be 85.6 m². 2 With a surface area of ​​ / g, it has a large number of physical attachment sites and an excellent carrier structure, making it suitable for adsorbing and immobilizing microorganisms, providing physical protection and attachment interface for the strains.

[0079] Figure 2 This is a SEM image of the biological agent in Example 1, from... Figure 2 It can be seen that the rod-shaped Bacillus licheniformis loaded on the surface and pore walls of biochar, with intact cell morphology, dense distribution, close adherence to the carrier, and no obvious detachment, proves that the cells were successfully fixed. Biochar can effectively protect the cells, reduce loss, and facilitate colonization.

[0080] Example 2 This embodiment prepares a biological agent, and the steps are as follows: (1) Preparation of porous support: Industrial-grade diatomaceous earth is repeatedly washed with deionized water to remove surface soluble salts, dust, and organic impurities. It is then allowed to settle and the supernatant is discarded until the washing water is clear and the pH is close to neutral. After pre-drying, it is calcined at 500℃ for 2 hours under air conditions, naturally cooled to room temperature, and passed through a 100-mesh sieve to obtain diatomaceous earth. It is then placed in a sealed bag and stored in a desiccator for later use.

[0081] (2) Preparation of bacterial culture: Reconstitute Bacillus megaterium lyophilized powder with sterile water and streak it onto LB agar plates. After incubation at 30°C for 24 hours, pick single colonies and inoculate them into 50 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.2 ± 0.2, autoclaved at 121°C for 20 min). Incubate at 30°C and 170 rpm with shaking for 18 hours until the logarithmic growth phase. Centrifuge the culture at 5000 rpm for 8 min, discard the supernatant, wash the cells three times with sterile water, and finally resuspend in sterile water to adjust the OD. 600 =1.0, to obtain bacterial culture, which was stored at 4℃ for later use; (3) Preparation of biological agents: Take 10g of diatomaceous earth and place it in a 250mL Erlenmeyer flask. Sterilize at 121℃ for 30min. After cooling, add 300mL of bacterial solution and shake in a constant temperature shaker at 30℃ and 170rpm for 4h for adsorption. After adsorption, filter and separate with sterile filter paper, collect the solid part, and dry it at 35℃ until the moisture content is about 10% to obtain the biological agent.

[0082] Experimental Example 1 This experimental example verifies the ability of the biological agent prepared in Example 1 to reduce iron in different typical iron oxides (hydrothermic, ferrihydrite, and goethite) under aerobic conditions: Suspensions of 1 g / L ferrihydrite (Fh), 1 g / L lepidocrocite (Lep), and 1 g / L goethite (Goe) were prepared separately, and 100 mL of each was added to a 250 mL Erlenmeyer flask to form three mineral systems. Within each mineral system, four treatment groups were set up in parallel: Control group: No materials added; Experimental group 1: 2 g / L of biochar prepared in Example 1 was added; Experimental group 2: The Bacillus licheniformis bacterial suspension prepared in Example 1 was added to make the bacterial concentration of the system 1×10⁻⁶. 7 CFU / mL; Experimental group 3: The biological agent prepared in Example 1 was added to make the bacterial concentration of the system reach 1×10⁻⁶. 7 CFU / mL.

[0083] All systems were treated with 5 mmol / L glucose as an electron donor and sealed with a breathable sealing film to ensure an aerobic environment. They were then placed in a constant temperature shaking incubator at 30℃ and 150 rpm for 7 consecutive days. Samples were taken on days 0, 1, 3, 5, and 7, and the Fe(II) content was determined by the o-phenanthroline colorimetric method. The volume ratio of sample to colorimetric reagent was 4:1.

[0084] Figure 3 The Fe(II) concentration in different mineral systems after 7 days of reaction in Experiment Example 1 is determined by... Figure 3 It can be seen that in different mineral systems, the Fe(II) concentration in experimental group 1 was always below 0.1 mg / L throughout the entire culture process, and no obvious iron reduction occurred; in experimental group 2, Bacillus licheniformis could reduce iron to different iron oxides by 2-15 mg / L; in experimental group 3, after treatment with biological agents, the aerobic iron reduction efficiency was further improved, and the iron reduction reached 4-25 mg / L. This proves that biochar loading significantly promotes aerobic iron reduction and efficiently generates Fe(II) under aerobic conditions, which is the key to the initiation of the Fenton-like reaction.

[0085] Experimental Example 2 This experimental example verifies whether Fe(II) generated by the reduction of ferric oxide can drive a Fenton-like reaction at the iron oxide interface and generate superoxide anions (O2) over a long period. - ) and hydroxyl radicals (·OH): Prepare a 1 g / L goethite suspension by adding 1 mmol / L benzoic acid (·OH scavenger) and 50 μmol / L nitrotetrazole blue chloride (NBT, O2). - (Capture agent), forming a mineral system; Four groups of processes are set up in parallel: Control group: No materials added; Experimental group 1: 2 g / L of biochar prepared in Example 1 was added; Experimental group 2: The Bacillus licheniformis bacterial suspension prepared in Example 1 was added to make the bacterial concentration of the system 1×10⁻⁶. 7 CFU / mL; Experimental group 3: The biological agent prepared in Example 1 was added to make the bacterial concentration of the system reach 1×10⁻⁶. 7 CFU / mL.

[0086] All systems were treated with 5 mmol / L glucose as an electron donor, sealed with breathable sealing film to ensure an aerobic environment, and then placed in a constant temperature shaking incubator at 30℃ and 150 rpm for 28 consecutive days. Samples were taken and measured on days 1, 3, 5, 7, and 28. Hydroxyl radicals: The concentration of p-hydroxybenzoic acid (p-HBA), an oxidation product of benzoic acid, was determined by high performance liquid chromatography. Superoxide anion: The absorbance of formazan, the NBT reduction product, at 560 nm was determined by ultraviolet-visible spectrophotometry.

[0087] Figure 4 O2 at the goethite interface of different treatment groups after 1 day of reaction in Experiment Example 2 - The amounts of (a) and ·OH(b) produced are determined by... Figure 4 It can be seen that experimental group 1 had almost no O2 after biochar treatment. - The amount of ·OH produced was approximately 0.5 μmol / L; after treatment with Bacillus licheniformis in experimental group 2, O2... - The amount produced was approximately 0.3 μmol / L, and the amount of ·OH produced was approximately 0.8 μmol / L, slightly higher than that of experimental group 1; after treatment with the biological agent of Example 1, the O2 in experimental group 3 was... - The amounts of ·OH and ·OH produced increased significantly, reaching 3.8 µmol / L and 2.4 µmol / L, respectively, indicating that the bio-agent in Example 1 can promote the reduction of iron to Fe(II) by oxygen and drive a Fenton-like reaction at the iron oxide interface to generate O2 over a long period. - With ·OH, this biological agent possesses strong oxidative degradation potential.

[0088] Figure 5 This is a kinetic diagram showing the long-term formation of Fe(II) (a) and free radicals (b) in different systems in Experimental Example 2. Figure 5 It can be seen that after treatment with the biological agent in Example 1, experimental group 3 was able to continuously and stably produce Fe(II) and free radicals (O2). - The system showed no degradation for 28 days and its production was consistently higher than that of the experimental group 2, indicating that the system has long-term self-sustaining capabilities, overcomes the shortcomings of short-term effectiveness and easy deactivation of chemical materials, and can achieve long-term repair.

[0089] Experimental Example 3 This experimental example verifies the degradation ability of the biological agent prepared in Example 1 on four typical organic pollutants: methyl orange, tetracycline, dichlorophenol, and carbamazepine. Prepare aqueous solutions containing the target pollutants: methyl orange 20 mg / L, tetracycline 10 mg / L, dichlorophenol 15 mg / L, and carbamazepine 5 mg / L. Take 100 mL of each and add them to 250 mL Erlenmeyer flasks to form different pollution systems. Add 0.5 g / L goethite and 5 mmol / L glucose to each system. Four groups of processes are set up in parallel: Control group: No materials added; Experimental group 1: 2 g / L of biochar prepared in Example 1 was added; Experimental group 2: The Bacillus licheniformis bacterial suspension prepared in Example 1 was added to make the bacterial concentration of the system 1×10⁻⁶. 7 CFU / mL; Experimental group 3: The biological agent prepared in Example 1 was added to make the bacterial concentration of the system reach 1×10⁻⁶. 7 CFU / mL.

[0090] The container was sealed with a breathable sealing film to ensure an aerobic environment; then it was placed in a constant temperature shaking incubator at 30℃ and 150rpm for 72 hours, and samples were taken at 0, 12, 24, 48, and 72 hours to determine the residual concentration of the corresponding pollutants. Methyl orange: Absorbance at 464 nm was determined by ultraviolet-visible spectrophotometry; Tetracycline, dichlorophenol, and carbamazepine were detected using high-performance liquid chromatography.

[0091] Figure 6 The degradation kinetic curves for methyl orange (a), tetracycline (b), dichlorophenol (c), and carbamazepine (d) in Experimental Example 3 are shown below. Figure 6 It can be seen that the biochar in experimental group 1 has almost no degradation ability for the four organic pollutants; the Bacillus licheniformis in experimental group 2 has a certain degradation effect on methyl orange, tetracycline, dichlorophenol and carbamazepine, but the degradation rate at 72h is less than 60%; the biological agent in experimental group 3 has a degradation rate of nearly 100% for methyl orange at 72h, and the degradation rates of tetracycline, dichlorophenol and carbamazepine at 72h reach 84%, 80% and 91% respectively. This indicates that the biological agent has a broad-spectrum and high-efficiency degradation ability for dyes, antibiotics, phenols and drugs, and has the potential to be applied to the remediation of water bodies polluted by the above pollutants.

[0092] Test Example 4 This experimental example verifies the actual remediation effect of the biological agent prepared in Example 1 on real dryland soil with compound pollution under aerobic conditions: Soil preparation for the test: Dryland soil was selected, air-dried, and sieved through a 2mm sieve. The pH was measured to be 6.5 and the organic matter content was 12g / kg. Preparation of contaminated soil: Add 50 mg / kg methyl orange, 30 mg / kg tetracycline, 40 mg / kg dichlorophenol, and 20 mg / kg carbamazepine to the soil, mix thoroughly, and age for 7 days; Four groups of processes are set up in parallel: Control group: No materials added; Experimental group 1: 2 g / L of biochar prepared in Example 1 was added; Experimental group 2: The Bacillus licheniformis bacterial suspension prepared in Example 1 was added to make the bacterial concentration of the system 1×10⁻⁶. 7 CFU / mL; Experimental group 3: The biological agent prepared in Example 1 was added to make the bacterial concentration of the system reach 1×10⁻⁶. 7 CFU / mL; Adjust the soil moisture content to 60% of field capacity, seal with breathable sealing film to ensure an aerobic environment, and incubate at a constant temperature of 25℃. Turn the soil every 5 days to maintain the aerobic environment. After 30 days of incubation, take samples to determine the Fe(II) concentration and the residual concentrations of various pollutants. Fe(II) concentration: The sample was mixed with 6 mol / L hydrochloric acid at a volume ratio of 1:1; the sample was soaked in hydrochloric acid for 2 days until it was completely dissolved, and the filtrate was collected by filtration; the filtrate was diluted with an acetate-sodium acetate buffer solution at pH 5.0 so that the iron concentration fell within the linear range of 0-20 mg / L of the Fe(II) standard curve; the Fe(II) content was determined by the o-phenanthroline colorimetric method with a sample to colorimetric reagent volume ratio of 4:1; finally, the original concentration of hydrochloric acid-extracted Fe(II) in the system was calculated by back-calculation based on the dilution factor; Methyl orange: determined by ultrasonic extraction-ultraviolet spectrophotometry; Tetracycline, dichlorophenol, and carbamazepine were detected using accelerated solvent extraction-high performance liquid chromatography-tandem mass spectrometry.

[0093] Figure 7 The Fe(II) concentration in the different systems in Experiment Example 4 after 5 days of reaction is given by... Figure 7 It can be seen that after treatment with the biological agent prepared in Example 1, the Fe(II) production in experimental group 3 was significantly higher than that in experimental group 1 with only biochar added and experimental group 2 with only Bacillus licheniformis added. This proves that under actual dryland soil, aerobic, and complex substrate conditions, the biological agent provided by this invention can still efficiently drive aerobic iron reduction and has site applicability.

[0094] Figure 8 The degradation rates of different pollutants in different systems after 5 days of reaction in Experiment Example 4 are given by... Figure 8It can be seen that after treatment with the biological agent prepared in Example 1, the degradation rate of methyl orange, tetracycline, dichlorophenol and carbamazepine in the soil of experimental group 3 was significantly higher than that of experimental group 1 with only biochar added and experimental group 2 with only Bacillus licheniformis added. This proves that the biological agent provided by the present invention is suitable for real organic compound polluted soil and can simultaneously, efficiently and stably remediate multiple organic pollutants.

[0095] The above results indicate that this invention uses porous materials such as biochar, activated carbon, diatomaceous earth, and porous ceramics as carriers to load microorganisms with aerobic iron reduction capabilities to construct biological agents. The high specific surface area and strong adsorption of the porous carriers provide physical protection and a stable microenvironment for the strains, enhancing their colonization ability and stress resistance in aerobic environments. Under aerobic conditions, the strains continuously reduce Fe(III) in the system to Fe(II). Fe(II) drives a Fenton-like reaction at the iron oxide interface, sustainably generating superoxide anions and hydroxyl radicals. Without the addition of external chemical reagents, the synergistic degradation of organic pollutants such as methyl orange, tetracycline, dichlorophenol, and carbamazepine can be achieved simultaneously, making it suitable for long-term remediation of aerobic compound pollution sites such as dry land and dried paddy fields.

[0096] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A biological agent, characterized in that, The biological agent is a porous carrier loaded with microorganisms capable of reducing ferric oxygen.

2. The biological agent according to claim 1, characterized in that, The microorganisms possessing the ability to reduce ferric oxygen are selected from at least one of Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Amycosis discolor, Streptomyces griseus, and Enterococcus montelukastii.

3. The biological agent according to claim 1, characterized in that, The porous carrier is selected from at least one of activated carbon, diatomaceous earth, porous ceramics, and biochar.

4. The method for preparing the biological agent according to any one of claims 1-3, characterized in that, Includes the following steps: The aforementioned biological agent is obtained by co-culturing a porous carrier with a bacterial solution of microorganisms capable of reducing ferric oxygen.

5. The preparation method according to claim 4, characterized in that, The OD of the bacterial solution 600 The value is 0.8-1.2; the solid-liquid ratio of the porous carrier to the bacterial solution is 1g: (10-50)mL.

6. The use of the biological agent according to any one of claims 1-3 in the remediation of organically polluted water bodies and / or soil.

7. The application according to claim 6, characterized in that, The organic pollutants mentioned include at least one of methyl orange, tetracycline, dichlorophenol, and carbamazepine.

8. The application according to claim 6, characterized in that, The organic polluted water and / or soil contains iron oxides, including at least one of ferrihydrite, lepidocrocite, and goethite.

9. The application according to claim 6, characterized in that, The organically contaminated soil has a water content of 50%-70% of field capacity.

10. The application according to claim 6, characterized in that, The dosage of the biological agent in organically polluted water is 1-5 g / L of water. And / or, the dosage of the biological agent in organically contaminated soil is 1-5 g / kg soil.

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