A method for preparing and applying a biochar-based bacterial agent for reductive dechlorination.

Abstract

This invention discloses a method for preparing and applying biochar-based microbial agents for reductive dechlorination, belonging to the fields of environmental engineering and pollution treatment engineering technology. This invention solves the problems of existing anaerobic reductive dechlorination reactions where dehalogenating respiratory bacteria struggle to withstand load shocks and highly heterogeneous environmental media, and are difficult to stably and scalably prepare biochar-immobilized microbial agents. This invention utilizes surfactants to modify biomass biochar, enhancing its hydrophilicity or electron transfer efficiency, and employs mixed anaerobic cultivation to ensure a more uniform distribution of dehalogenating respiratory bacteria on the modified biochar, significantly increasing the microbial loading. The resulting biochar-immobilized microbial agent material loaded with dehalogenating respiratory bacteria integrates adsorption and bioremediation functions, increasing its continuous action time in the soil while minimizing the impact of external substances on the soil. It is an ideal in-situ soil bioremediation material.

Description

Technical Field

[0001] This invention relates to a biochar immobilized bacterial agent material for in-situ enhanced biological dechlorination, its preparation method and application, belonging to the fields of environmental engineering and pollution treatment engineering technology. Background Technology

[0002] Existing soil remediation technologies mainly include bioremediation, physicochemical remediation, and combined remediation technologies. Among them, in-situ bioremediation is less disturbing to the soil, lower in cost, and more environmentally friendly, thus it is widely studied in soil remediation engineering. In-situ soil biochlorination technology simulates the soil's self-purification process, where microorganisms undergo reductive dechlorination reactions through respiration in an anaerobic environment, achieving the dechlorination and detoxification of halogenated organic matter. However, existing anaerobic reductive dechlorination bacteria are significantly limited when facing load shocks and highly heterogeneous environmental media. Therefore, in experiments and engineering, microorganisms are usually loaded onto carriers such as biochar, wheat bran, and montmorillonite. However, the required amount of immobilized microorganisms in in-situ soil bioremediation is large, which the above-mentioned carriers cannot meet. Therefore, it is essential to provide a biochar-immobilized bacterial agent that can be stably and scalably applied to enhance in-situ soil biochlorination, providing a sanctuary for reductive dechlorination bacteria while reducing the remediation risks brought about by complex environments, thereby strengthening the in-situ soil bioremediation process. Summary of the Invention

[0003] This invention addresses the challenges of existing anaerobic reduction dechlorination reactions where dehalogenating respiratory bacteria struggle to withstand load shocks and highly heterogeneous environmental media, and where it is difficult to stably and scalably prepare biochar-immobilized bacterial agents. It provides a biochar-immobilized bacterial agent material for in-situ enhanced biological dechlorination, along with its preparation method and application. This biochar-immobilized bacterial agent material is suitable for large-scale in-situ enhanced biological dechlorination in soil, enabling enhanced and sustainable remediation of halogenated organic pollutants in soil.

[0004] The technical solution of the present invention:

[0005] One objective of this invention is to provide a method for modifying a biochar carrier with a surfactant. The method specifically involves soaking biochar in TX-100 solution or FeSO4 solution for 24 hours, filtering, washing the precipitate until neutral, and drying to obtain the modified biochar.

[0006] Further specifying, the mass concentration of the TX-100 solution is 1%.

[0007] Further specified, the FeSO4 solution concentration is 1 mol / L.

[0008] Further specifying the process, the biochar preparation process is as follows: after drying the biomass, it is placed in a tube furnace and heated to 600°C at a rate of 5°C / min, kept at that temperature for 2 hours, cooled to room temperature, and sieved to obtain 100~200 mesh biochar.

[0009] To further specify, the biomass is corn stalks.

[0010] The second objective of this invention is to provide a method for loading dehalogenated respiratory bacteria onto a biochar carrier modified with the above-mentioned surfactant. Specifically, this method is as follows:

[0011] The sterile initial culture medium was placed in an anaerobic fermenter, and the target halogenated organic compound was added to a final concentration of 200 μmol / L. Then, dehalogenated respiratory bacteria were inoculated, and the surfactant-modified biochar carrier prepared as described above was added. After 14 days of cultivation, the biochar-immobilized bacterial agent material was obtained. During the cultivation period, the carbon source and the target halogenated organic compound were replenished periodically.

[0012] Further specified, the initial sterile culture medium pH is 7.0.

[0013] Furthermore, the pH of the sterile initial culture medium was adjusted using a PBS buffer system.

[0014] Further defined, dehalogenated respiratory bacteria are anaerobic microorganisms that use halogenated organic matter as electron acceptors, and the inoculation mass is 10% of the volume of the sterile initial culture medium.

[0015] To further specify, dehalogenation respiratory bacteria include, but are not limited to, *Desulfobacterium* genus of the phylum Firmicutes (…). Desulfitobacterium spp.), Dehidus genus ( Dehalobacter spp.), genus *Geobryophyte* of the phylum Proteobacteria ( Geobacter spp.), desulfurizing bacteria ( Desulfomonile spp.), dechlorinated anaerobic bacteria ( Anaeromyxobacter dehalogenans ), dechlorinated bird droppings desulfurizing Vibrio ( Desulfovibrio dechloracetivorans ) and genus Thiophyta ( Sulfosprillum spp.), Phylum Chloris Dehalococcoides spp.

[0016] To further define, dehalogenated respiratory bacteria are either complex microbial communities or isolated single species that have been domesticated to target specific halogenated organic pollutants.

[0017] Further specifying, the target halogenated organic compound is 2,4,6-TCP (2,4,6-trichlorophenol).

[0018] The third objective of this invention is to provide an application of the biochar immobilized bacterial agent material prepared by the above method, specifically for in-situ enhancement of the dechlorination and detoxification of halogenated organic pollutants in soil, surface water, groundwater, and anaerobic reactors by biological agents.

[0019] Further specifying the method for using biochar immobilized microbial agent materials for in-situ enhanced biological dechlorination in soil, the method is as follows: the biochar immobilized microbial agent materials are added to the soil by topdressing, while the soil is covered with a film to maintain an anaerobic state and prevent water evaporation, keeping the soil moisture content greater than 20%.

[0020] This invention utilizes surfactants to modify biochar, enhancing its hydrophilicity or electron transfer efficiency. A mixed anaerobic culture is employed to ensure a more uniform distribution of dehalogenated respiratory bacteria on the modified biochar, significantly increasing the microbial loading. This results in a biochar-immobilized microbial agent material that integrates adsorption and bioremediation functions. The biochar can pre-concentrate pollutants or promote microbial aggregation, thereby shortening the contact distance between pollutants and microorganisms. Furthermore, in the co-culture of different microorganisms, biochar can promote direct interspecies electron transfer, enhancing the bioremediation process. Simultaneously, biochar can reduce the concentration of pollutants in the environment, providing a favorable reaction environment for functional microorganisms. This allows the biochar-immobilized microbial agent to maintain its effect in the soil for a longer period, minimizing the impact of external substances on the soil. It is an ideal in-situ soil bioremediation material. Moreover, the method for creating the biochar-immobilized microbial agent material provided by this invention is more suitable for large-scale production and easier to apply in practical engineering remediation. Attached Figure Description

[0021] Figure 1 This is a photograph of the biochar-immobilized bacterial agent material prepared in Example 1;

[0022] Figure 2 The curves showing the changes in pollutant and product concentrations during the degradation of 2,4,6-TCP by the biochar immobilized bacterial agent material prepared in Example 1 are shown.

[0023] Figure 3 The XRD curve of the biochar-immobilized bacterial agent material prepared in Example 1;

[0024] Figure 4 The curves showing the changes in pollutant and product concentrations during the degradation of 2,4,6-TCP by the biochar-immobilized bacterial agent material prepared in Example 2 are shown.

[0025] Figure 5 Fourier transform infrared spectrum of the biochar immobilized bacterial agent material prepared in Example 2. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0027] The corn stalks used in the following examples were produced by Beijing Hemutongxing Agricultural and Animal Husbandry Technology Co., Ltd., with a particle size of less than 60 mesh; FeSO4·7H2O was produced by Shanghai Aladdin Biochemical Technology Co., Ltd.; and Triton TX-100 was produced by Shanghai Aladdin Biochemical Technology Co., Ltd., and is a colorless, transparent, viscous liquid.

[0028] Example 1:

[0029] Step 1: Preparation of modified biochar

[0030] First, take corn stalks, wash them, and then dry them in an oven at 105℃ for 24 hours. Store the resulting powder in a brown bottle.

[0031] Then, weigh 10g of straw powder into a tube furnace to produce biochar. The specific heating program is as follows: 5℃ / min, rise to the target temperature of 600℃, and hold for 2 hours. After the pyrolysis is completed and the temperature drops to room temperature, weigh the biochar and sieve it, retaining 100~200 mesh biochar.

[0032] Finally, 10g of the above biochar was soaked in 100 mL of 1mol / L FeSO4·7H2O for 24 hours, then filtered and washed with distilled water until neutral. After drying at 70℃, the modified biochar was obtained. Figure 1 As shown.

[0033] Step 2: Preparation of biochar immobilized bacterial agent material

[0034] First, prepare LB liquid culture medium with deionized water according to the following formula: 25 g / L LB broth. Pour 0.8 L of the prepared LB liquid culture medium into a 1 L Erlenmeyer flask, seal with a sealing film, and autoclave at 121 °C for 30 min to obtain sterile LB liquid culture medium. Inoculate the bacterial strain into the sterile LB liquid culture medium and place it in an air bath shaker at 150 rpm and 30 °C for 48 h. Centrifuge the prepared bacterial suspension at 6000 rpm for 10 min, wash twice with 0.85% physiological saline solution, resuspend in physiological saline, and adjust the OD. 600 =0.1, and the dehalogenated respiratory bacteria mother liquor was obtained.

[0035] Then, prepare the anaerobic liquid culture medium with deionized water according to the following formula: 1 g / L NaCl, 0.2 g / L MgCl₂·6H₂O, 2.31 g / L Na₂HPO₄·12H₂O, 0.554 g / L NaH₂PO₄·2H₂O, 0.13 g / L NH₄Cl, 0.3 g / L KCl, 0.012 g / L CaCl₂, and 0.1 mL / L mineral elements. The mineral element base solution formula is: 0.5 mg / L MnCl₂·4H₂O, 0.5 mg / L ZnCl₂, 0.5 mg / L NiCl₂·6H₂O, 0.5 mg / L H₃BO₃, 0.5 mg / L Na₂MoO₄·2H₂O, 2.5 mg / L CoCl₂·6H₂O, 0.5 mg / L NH₄VO₃, 2.5 mg / L KI, and 10 mg / L (NaPO₃). 16 Pour 1L of the prepared anaerobic culture medium into a 1.2L serum bottle.

[0036] Next, 10g of the modified biochar carrier was added to the serum bottle, and the mixture was deoxygenated by blowing with high-purity nitrogen for 20min. The container was then sealed and autoclaved at 121℃ for 30min to obtain a sterile initial culture medium. Then, a final concentration of 2mmol / L Na2S solution, 1ml of 2mol / L sodium acetate solution, 1ml of vitamin, a final concentration of 200μmol / L 2,4,6-trichlorophenol (2,4,6-TCP) solution, and a dehalogenated respiratory bacteria stock solution (10% of the sterile initial culture medium volume) were sequentially injected into the serum bottle.

[0037] Finally, the serum bottles were placed in a 30°C incubator for culture. During this period, samples were taken regularly to detect the concentrations of 2,4,6-trichlorophenol and its dechlorination products, 2,4-dichlorophenol and 4-chlorophenol, and sodium acetate solution and 2,4,6-trichlorophenol were added periodically. Biochar-immobilized bacterial material was obtained after 14 days of culture.

[0038] Take another 120 mL serum bottle and prepare the same anaerobic liquid culture medium according to the above composition. Pour 100 mL of the prepared anaerobic culture medium into the 120 mL serum bottle. Add 0.1 g of the obtained biochar immobilized bacterial agent material with attached anaerobic dechlorinating bacteria to the serum bottle. Perform deoxygenation treatment by blowing with pure nitrogen for 20 min, and then seal the container. Sequentially inject the serum bottle with a final concentration of 2 mmol / L Na₂S solution, 0.1 mL of a 2 mol / L sodium acetate solution, 0.1 mL of vitamin, and a final concentration of 200 μmol / L 2,4,6-trichlorophenol (2,4,6-TCP) solution. Periodically monitor the concentrations of the target pollutant 2,4,6-TCP and dechlorination products. The periodic monitoring results of the target pollutant are as follows: Figure 2As shown in the figure, 2,4,6-TCP was immediately and significantly adsorbed by the functional material upon initial addition, with an adsorption capacity of approximately 15%. On day 5, the concentration of 2,4,6-TCP in the solution decreased to 0, while the concentrations of the dechlorination products 2,4-dichlorophenol and 4-chlorophenol increased significantly. On day 6, the concentration of 4-chlorophenol in the solution increased to approximately 75 μM, while the concentration of 2,4-dichlorophenol decreased to 0, indicating that the material adsorbed approximately 55% of the 4-chlorophenol. These results demonstrate that the biochar-immobilized bacterial agent provided an excellent reaction site for the dehalogenating respiratory bacteria, thus enhancing the biological dechlorination reaction. Furthermore, the material exhibited excellent adsorption properties, and the adsorption-degradation pattern significantly increased the remediation efficiency.

[0039] The biochar-immobilized bacterial agent material obtained in this embodiment was characterized by XRD, and the results are as follows: Figure 3 As shown, Fe is used 2+ Iron hydroxyl oxide (FeOOH), the main component of goethite, appears on the modified biochar. Since semiconductor minerals mediate interspecies electron transfer in the biosphere, the metabolites secreted by microorganisms can also be utilized by other microorganisms. The consumption of metabolites by trophic microorganisms will accelerate the metabolism of trophic microorganisms to produce more metabolites. At the same time, minerals can mediate electron transfer between microorganisms and terminal electron acceptors. In summary, it can be determined that the formation of FeOOH on the biochar of biochar immobilized bacterial agent material has a certain promoting effect on the degradation of 2,4,6-TCP by CP-1.

[0040] Example 2:

[0041] Step 1: Preparation of modified biochar

[0042] First, take corn stalks, wash them, and dry them in an oven at 105℃ for 24 hours. Then, store the stalk powder in a brown bottle.

[0043] Then, weigh 10g of the above straw powder into a tube furnace to produce biochar. The specific heating program is as follows: 5℃ / min, rise to the target temperature of 600℃, and hold for 2 hours. After the pyrolysis is completed and the temperature drops to room temperature, weigh the biochar and sieve it, retaining 100~200 mesh biochar.

[0044] Finally, 10g of the above biochar was soaked in 100 mL of 1wt% TX-100 for 24 hours, then filtered and washed with distilled water until neutral. After drying at 70℃, the modified biochar was obtained.

[0045] Step 2: Preparation of biochar immobilized bacterial agent material

[0046] First, prepare LB liquid culture medium with deionized water according to the following formula: 25 g / L LB broth. Pour 0.8 L of the prepared LB liquid culture medium into a 1 L Erlenmeyer flask, seal with a sealing film, and autoclave at 121 °C for 30 min to obtain sterile LB liquid culture medium. Inoculate the bacterial strain into the sterile LB liquid culture medium and place it in an air bath shaker at 150 rpm and 30 °C for 48 h. Centrifuge the prepared bacterial suspension at 6000 rpm for 10 min, wash twice with 0.85% physiological saline solution, resuspend in physiological saline, and adjust the OD. 600 =0.1, and the dehalogenated respiratory bacteria mother liquor was obtained.

[0047] Then, prepare the anaerobic liquid culture medium with deionized water according to the following formula: 1 g / L NaCl, 0.2 g / L MgCl₂·6H₂O, 2.31 g / L Na₂HPO₄·12H₂O, 0.554 g / L NaH₂PO₄·2H₂O, 0.13 g / L NH₄Cl, 0.3 g / L KCl, 0.012 g / L CaCl₂, and 0.1 mL / L mineral elements. The mineral element base solution formula is: 0.5 mg / L MnCl₂·4H₂O, 0.5 mg / L ZnCl₂, 0.5 mg / L NiCl₂·6H₂O, 0.5 mg / L H₃BO₃, 0.5 mg / L Na₂MoO₄·2H₂O, 2.5 mg / L CoCl₂·6H₂O, 0.5 mg / L NH₄VO₃, 2.5 mg / L KI, and 10 mg / L (NaPO₃). 16 Pour 1L of the prepared anaerobic culture medium into a 1.2L serum bottle.

[0048] Next, 10g of the modified biochar carrier was added to the serum bottle, and the mixture was deoxygenated by blowing with high-purity nitrogen for 20min. The container was then sealed and autoclaved at 121℃ for 30min to obtain a sterile initial culture medium. Then, a final concentration of 2mmol / L Na2S solution, 1ml of 2mol / L sodium acetate solution, 1ml of vitamin, a final concentration of 200μmol / L 2,4,6-trichlorophenol (2,4,6-TCP) solution, and a dehalogenated respiratory bacteria stock solution (10% of the sterile initial culture medium volume) were sequentially injected into the serum bottle.

[0049] Finally, the serum bottles were placed in a 30°C incubator for culture. During this period, samples were taken regularly to detect the concentrations of 2,4,6-trichlorophenol and its dechlorination products, 2,4-dichlorophenol and 4-chlorophenol, and sodium acetate solution and 2,4,6-trichlorophenol were added periodically. Biochar-immobilized bacterial material was obtained after 14 days of culture.

[0050] Take another 120 mL serum bottle and prepare the same anaerobic liquid culture medium according to the above composition. Pour 100 mL of the prepared anaerobic culture medium into the 120 mL serum bottle. Add 0.1 g of the obtained biochar immobilized bacterial agent material with attached anaerobic dechlorinating bacteria to the serum bottle. Perform deoxygenation treatment by blowing with pure nitrogen for 20 min, and then seal the container. Sequentially inject the serum bottle with a final concentration of 2 mmol / L Na₂S solution, 0.1 mL of a 2 mol / L sodium acetate solution, 0.1 mL of vitamin, and a final concentration of 200 μmol / L 2,4,6-trichlorophenol (2,4,6-TCP) solution. Periodically monitor the concentrations of the target pollutant 2,4,6-TCP and dechlorination products. The periodic monitoring results of the target pollutant are as follows: Figure 4 As shown, 2,4,6-TCP was immediately and significantly adsorbed by the functional material upon initial addition, with an adsorption capacity of approximately 10%. By day 5, the concentration of 2,4,6-TCP in the solution decreased to 0, while the concentrations of the dechlorination products 2,4-dichlorophenol and 4-chlorophenol increased significantly. By day 6, the concentration of 4-chlorophenol in the solution increased to approximately 80 μM, while the concentration of 2,4-dichlorophenol decreased to 0, indicating that the material adsorbed approximately 60% of the 4-chlorophenol. These results demonstrate that the biochar-immobilized bacterial agent provided an excellent reaction site for the dehalogenating respiratory bacteria, thereby enhancing the biological dechlorination reaction. Furthermore, the material exhibited excellent adsorption properties, and the adsorption-degradation pattern significantly increased the remediation efficiency.

[0051] The biochar-immobilized bacterial agent material obtained in this embodiment was characterized by Fourier transform infrared spectroscopy, and the results are as follows: Figure 5 As shown, the main absorption peak of the modified biochar material is located at 3400 cm⁻¹. -1 1600cm -1 1100cm -1 The corresponding functional groups in the vicinity are OH, C=O, and CO, respectively. Carboxyl (-COOH), hydroxyl (-OH), and carbonyl (-C=O) groups are all strongly polar groups, and substances with polar groups exhibit better hydrophilicity. Fourier transform infrared spectroscopy shows that the functional group intensity of the modified biochar is enhanced, which is beneficial for improving hydrophilicity. Since many microorganisms are hydrophilic, porous carriers with strong hydrophilicity exhibit good stability of attached microorganisms and will show a higher biofilm formation rate. Simultaneously, modification improves the hydrophilicity of biochar, increasing the adsorption capacity for hydrophilic organic matter while decreasing the adsorption capacity for hydrophobic organic matter. The pollutant 2,4,6-TCP involved in the experiment is strongly hydrophobic, while the hydrophilicity of the products 2,4-DCP and 4-CP gradually increases with the removal of chloride ions, which is consistent with the adsorption effect of biochar on 2,4,6-TCP and 4-CP in the experiment.

[0052] In summary, as demonstrated in Examples 1 and 2, both FeSO4·7H2O and Triton TX-100 modified biochar immobilized bacterial agents can enhance the dechlorination reaction of 2,4,6-trichlorophenol by dehalogenating respiratory bacteria. The biochar immobilized bacterial agent modified with Triton TX-100 exhibits a faster degradation rate due to the increased hydrophilicity of the biochar and the increased microbial loading. Furthermore, a 1.2L system was used to simulate the step of loading dehalogenating respiratory bacteria onto modified biochar during production, and the results showed that this method is also applicable to large-scale production. The preparation method of the biochar immobilized bacterial agent, the loading method of dehalogenating respiratory bacteria, and the in-situ bioremediation method for halogenated organic pollution in soil provided by this invention all have significant advantages in large-scale applications and are worthy of further promotion.

[0053] The above description is only a preferred embodiment of the present invention. Given that those skilled in the art can make appropriate changes and modifications to the above embodiments, the present invention is not limited to the specific embodiments described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A method for producing a biochar-based bacterial agent material, characterized in that, Includes the following steps: After drying, the corn stalks were placed in a tube furnace and heated to 600°C at a rate of 5°C / min. The temperature was maintained for 2 hours, then cooled to room temperature and sieved to obtain 100-200 mesh biochar. Then, the biochar was soaked in TX-100 solution or FeSO4 solution for 24 hours, filtered, the precipitate was washed until neutral, and dried to obtain the modified biochar. The sterile initial culture medium was placed in an anaerobic fermenter, and the target halogenated organic compound with a final concentration of 200 μmol / L was added. Then, dehalogenated respiratory bacteria were inoculated, and modified biochar was added. After being cultured at a constant temperature of 30℃ for 14 days, biochar-based bacterial agent material was obtained. The mass concentration of the TX-100 solution is 1%; The FeSO4 solution concentration is 1 mol / L.

2. The method for using biochar-based bacterial agent materials according to claim 1, characterized in that, The initial sterile culture medium had a pH of 7.0, and the pH of the initial sterile culture medium was adjusted using a PBS buffer system.

3. The method for using biochar-based bacterial agent materials according to claim 1, characterized in that, Dehalogenated respiratory bacteria are anaerobic microorganisms that use halogenated organic matter as electron acceptors. The inoculation volume is 10% of the initial sterile culture medium volume.

4. The application of a biochar-based bacterial agent material prepared by the method of claim 1, characterized in that, It is used to enhance the reductive dechlorination and detoxification of halogenated organic pollutants in soil, surface water, groundwater and anaerobic reactors by organisms.

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