Composite modified biochar, immobilized microbial inoculum containing composite modified biochar, and preparation method and application of immobilized microbial inoculum

Abstract

The invention discloses composite modified charcoal, an immobilized microbial inoculum containing the composite modified charcoal as well as a preparation method and application of the immobilized microbial inoculum. The preparation method of the composite modified biochar comprises the following steps: impregnating biochar with an alkali solution, and washing and drying the impregnated biochar to obtain alkali modified biochar; and impregnating the alkali-modified biochar with a surfactant solution, washing the impregnated alkali-modified biochar, and drying to obtain the alkali-modified biochar. The specific surface area and porosity of the composite modified biochar are obviously increased, oxygen-containing groups are obviously increased, the composite modified biochar is used as a carrier to adsorb petroleum hydrocarbon degrading bacteria, and then polymer gel is used for embedding and immobilizing to prepare an immobilized microbial agent, so that the immobilization effect of the petroleum hydrocarbon degrading bacteria and the degradation effect of the immobilized petroleum hydrocarbon are greatly improved; petroleum hydrocarbon polluted water can be efficiently and stably treated in a complex underground water environment.

Description

Technical Field

[0001] This invention belongs to the field of microbial immobilization technology, and specifically relates to a composite modified biochar, an immobilization agent containing the same, its preparation method, and its application. Background Technology

[0002] Petroleum is a major energy source and chemical raw material in the world today, and it is closely related to the activities of all humankind in modern society. Oil spills during oil extraction and related operations can lead to groundwater pollution. Statistics show that over 68% of the world's freshwater is stored in ice and glaciers, with the remaining 30% underground. Oil spills seriously jeopardize the safety of groundwater resources. Therefore, the remediation of oil-contaminated groundwater is imperative.

[0003] Microbial degradation is an environmentally friendly and low-cost method for treating petroleum hydrocarbon pollution. Petroleum hydrocarbon-degrading bacteria are screened from petroleum hydrocarbon-contaminated soil. These bacteria can break down petroleum hydrocarbons into smaller molecules through enzymatic action, ultimately converting them into water and carbon dioxide. However, in actual groundwater remediation, the stress on microbial degradation is significant due to the influence of complex solution environments such as temperature and pH, resulting in inconsistent degradation efficiency.

[0004] Immobilized microbial technology, which fixes microbial cells onto a carrier, can increase the density and stability of microorganisms, ensuring their stability in complex polluted environments. Immobilized microbial technology can also reduce microbial loss, improve degradation efficiency, and reduce environmental impact. However, current microbial immobilization products suffer from poor immobilization effects and a significant decrease in the ability of immobilized microorganisms to degrade petroleum hydrocarbons, which are pressing technical problems that need to be solved. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a composite modified biochar, an immobilized bacterial agent containing the same, and the preparation method and application thereof. The immobilized bacterial agent of the present invention uses composite modified biochar co-modified with alkali and surfactant as a carrier to adsorb petroleum hydrocarbon degrading bacteria, and then immobilizes them by embedding with a polymer gel, which greatly improves the immobilization effect of petroleum hydrocarbon degrading bacteria and the petroleum hydrocarbon degradation effect after immobilization.

[0006] In a first aspect, the present invention provides a method for preparing composite modified biochar, comprising: impregnating biochar with an alkaline solution, washing and drying the impregnated biochar to obtain alkaline modified biochar; impregnating the alkaline modified biochar with a surfactant solution, washing and drying the impregnated alkaline modified biochar to obtain alkaline and surfactant co-modified biochar, which is composite modified biochar.

[0007] According to a specific embodiment of the present invention, the concentration of the alkaline solution is 0.5-2 mol / L, and the concentration of the surfactant solution is 5-50 g / L.

[0008] According to a specific embodiment of the present invention, the mass-to-volume ratio of biochar to alkaline solution is 1g:10-40mL, the mass-to-volume ratio of alkali-modified biochar to surfactant solution is 1g:5-15mL, the impregnation temperature is room temperature, and the time is 20-30h.

[0009] According to a specific embodiment of the present invention, the alkali is selected from ammonia monohydrate, sodium hydroxide, or potassium hydroxide, preferably potassium hydroxide.

[0010] According to a specific embodiment of the present invention, the surfactant is selected from rhamnolipin, sodium dodecylbenzenesulfonate or Tween 80, preferably rhamnolipin.

[0011] In a second aspect, the present invention provides a composite modified biochar prepared by the aforementioned method.

[0012] A third aspect of the present invention provides an immobilized bacterial agent for enhancing the degradation of petroleum hydrocarbon pollutants by microorganisms, comprising the aforementioned composite modified biochar and petroleum hydrocarbon degrading bacteria loaded on the composite modified biochar.

[0013] According to a specific embodiment of the present invention, the immobilized bacterial agent has a core-shell structure, with the aforementioned modified biochar adsorbed with petroleum hydrocarbon degrading bacteria as the core and calcium alginate gel as the outer shell.

[0014] According to a specific embodiment of the present invention, the specific surface area of ​​the immobilized bacterial agent is 300–400 m². 2 / g.

[0015] A fourth aspect of the present invention provides a method for preparing the aforementioned immobilized bacterial agent, comprising: mixing and culturing the aforementioned composite modified biochar with a bacterial broth of petroleum hydrocarbon degrading bacteria; separating the solid and liquid components of the culture product to obtain a solid; washing and drying the solid to obtain modified biochar adsorbed with petroleum hydrocarbon degrading bacteria; mixing the modified biochar adsorbed with petroleum hydrocarbon degrading bacteria with a sodium alginate solution; then adding the mixture dropwise into a calcium salt solution to allow for a complete reaction; washing and drying the mixture to obtain the immobilized bacterial agent.

[0016] According to a specific embodiment of the present invention, the concentration of the bacterial solution is 2 × 10⁻⁶. 8 ~3×10 8 CFU / mL.

[0017] According to a specific embodiment of the present invention, the mass-to-volume ratio of the composite modified biochar to the petroleum hydrocarbon degrading bacteria solution is 0.8–1.2 g: 20 mL.

[0018] According to a specific embodiment of the present invention, the culture temperature is 30°C and the culture time is 20-30 hours.

[0019] According to a specific embodiment of the present invention, the concentration of the sodium alginate solution is 3-5% w / v, the concentration of the calcium salt solution is 2-4% w / v, and the reaction time is 15-20 h. The calcium salt can be conventional calcium chloride, calcium lactate, or calcium gluconate.

[0020] A fifth aspect of the present invention provides an immobilized bacterial agent prepared by the aforementioned method.

[0021] In a sixth aspect, the present invention provides the application of the aforementioned immobilized bacterial agent in the degradation of petroleum hydrocarbon pollution in groundwater.

[0022] A seventh aspect of the present invention provides a method for degrading petroleum hydrocarbon pollution in groundwater, comprising: contacting the aforementioned immobilized bacterial agent with petroleum hydrocarbon-polluted groundwater.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. The method for preparing composite modified biochar provided by this invention involves primary modification using an alkaline solution as a modifier, followed by secondary modification using a surfactant solvent as a modifier. This significantly increases the specific surface area and porosity of the biochar, as well as the number of oxygen-containing groups, thereby greatly improving the adsorption and fixation performance of the biochar. Simultaneously, the alkaline solution used as the modifier can be recycled by appropriately adding reagents, reducing the cost of reagents and wastewater treatment.

[0025] 2. The immobilized bacterial agent provided by this invention uses composite modified biochar to adsorb and immobilize petroleum hydrocarbon-degrading bacteria, and then encapsulates them with calcium alginate gel. Specifically: 1) The composite modified biochar undergoes two-step modification treatment with alkali and surfactant. Alkali modification gives the biochar more carbonyl and hydroxyl functional groups, thereby improving the immobilization rate and enhancing the efficacy of the bacterial agent; surfactant modification improves the mass transfer efficiency at the coating interface, increases the contact area, and solves the problem of poor mass transfer caused by encapsulation. Therefore, by using composite modified biochar to adsorb and immobilize petroleum hydrocarbon-degrading bacteria, a synergistic improvement in the immobilization rate and mass transfer efficiency of petroleum hydrocarbon-degrading bacteria is achieved, thus greatly improving the final product's petroleum hydrocarbon degradation effect; 2) Encapsulation provides a good living environment for microorganisms, while mitigating the adverse effects of surfactants introduced during biochar modification on the growth of petroleum hydrocarbon-degrading bacteria, resulting in high microbial density and maintaining high microbial activity, ensuring stable degradation efficiency. Experiments show that the microbial immobilization rate of the composite modified + encapsulated immobilized bacterial agent (KT-BC-ER) of this invention is 94.5319%, and the petroleum hydrocarbon degradation rate after 7 days is 72.24%. The petroleum hydrocarbon degradation rate is significantly better than that of free bacteria (R), alkali-modified (K-BC-R), composite modified (KR-BC-R), and alkali-modified + encapsulated (K-BC-ER) petroleum hydrocarbon degradation rates of 26.65%, 63.70%, 46.08%, and 51.07%, respectively, and the degradation efficiency is improved by 45.59% compared with the free bacteria system.

[0026] 3. The immobilized bacterial agent provided by this invention can be applied to the degradation of petroleum hydrocarbon pollution in groundwater. It can efficiently and stably treat petroleum hydrocarbon polluted water in complex groundwater environments. It is a green, low-cost and simple technology that can achieve in-situ remediation. Attached Figure Description

[0027] Figure 1 SEM image of unmodified corn straw biochar (BC), EHT = 3.00 kV, WD = 4.5 mm; Mag = 20.00 KX; SignalA = InLens;

[0028] Figure 2 SEM image of unmodified corn straw biochar (BC) loaded with microbial agent, EHT = 3.00 kV, WD = 4.4 mm; Mag = 20.00 KX; SignalA = InLens;

[0029] Figure 3 SEM image of composite modified straw biochar (KR-BC), EHT = 3.00 kV, WD = 4.5 mm; Mag = 20.00 KX; SignalA = InLens;

[0030] Figure 4SEM image of composite modified straw biochar (KR-BC) loaded with microbial agent, EHT = 3.00 kV, WD = 4.6 mm; Mag = 20.00 KX; SignalA = InLens;

[0031] Figure 5 FTIR images of composite modified straw biochar (KR-BC) and unmodified biochar (BC);

[0032] Figure 6 The experimental results show the effect of different types of alkali on the degradation rate of petroleum hydrocarbons.

[0033] Figure 7 The experimental results show the effect of alkali concentration on the degradation rate of petroleum hydrocarbons.

[0034] Figure 8 The experimental results show the effect of surfactant type on the degradation rate of petroleum hydrocarbons;

[0035] Figure 9 Experimental results showing the effects of different treatment methods on the degradation rate of petroleum hydrocarbons;

[0036] Figure 10 Experimental results on the effect of biochar prepared from different raw materials on the degradation rate of petroleum hydrocarbons. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] The petroleum hydrocarbon degrading bacteria in the following examples are commercially available engineered petroleum hydrocarbon bacteria, Rhodococcus.

[0039] Example 1

[0040] This embodiment provides an immobilized bacterial agent for enhancing the microbial degradation of petroleum hydrocarbon pollutants, and the preparation method is as follows:

[0041] (1) Preparation of biochar: Corn stalks were repeatedly washed with deionized water to remove surface dust, and then dried in a drying oven at 105℃ for 12 hours. The washed and dried corn stalks were crushed, passed through a 20-mesh sieve, placed in a crucible, wrapped in aluminum foil, and then pyrolyzed in a muffle furnace at 700℃ for 2 hours to obtain biochar.

[0042] (2) Alkali modification of biochar: Prepare a 0.5 mol / L KOH aqueous solution, then add 10 g of biochar and 200 mL of KOH solution to a 500 mL beaker, place it on a magnetic stirrer at room temperature, and stir at a uniform speed of 200 rpm for 24 hours. Then, separate the solid and liquid using a vacuum filtration device, and repeatedly wash the solid with deionized water until the filtrate is neutral. Dry it in an oven at 105℃ for 24 hours to obtain alkali-modified biochar.

[0043] (3) Surfactant modification of biochar: Prepare a 5 g / L rhamnolipin aqueous solution, then add 10 g of alkali-modified biochar and 100 mL of rhamnolipin aqueous solution to a 500 mL beaker. Place the beaker on a magnetic stirrer at room temperature and stir at a uniform speed of 200 rpm for 24 hours. Subsequently, separate the solid and liquid components using a vacuum filtration device, and repeatedly wash the solid with deionized water until the filtrate is neutral. Dry the filtrate in an oven at 105 °C for 24 hours to obtain composite modified biochar.

[0044] (4) Preparation of bacterial suspension: Petroleum hydrocarbon degrading bacteria were picked and placed in 150 mL of LB liquid medium. After activation in a shaker at 30℃ and 180 rpm for 24 h, the suspension was centrifuged at 4000 rpm for 5 min. The supernatant was discarded, and the suspension was resuspended in phosphate buffer. The centrifugation and resuspension were repeated three times to prepare a bacterial concentration of 2.3 × 10⁻⁶. 8 The bacterial suspension contained CFU / mL. The LB liquid medium consisted of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl. The phosphate buffer consisted of: 1.42 g / L Na₂HPO₄, 0.27 g / L KH₂PO₄, 8 g / L NaCl, and 0.2 g / L KCl.

[0045] (5) Adsorption: Take 1g of composite modified biochar into a 100mL conical flask, sterilize it in an autoclave at 121℃ for 20min, add 20mL of the bacterial suspension prepared in step (3), place it in a shaker at 30℃ and 180rpm for 24h, centrifuge at 1000rpm for 1min, remove the supernatant, wash it three times with phosphate buffer to remove unadsorbed free bacterial agent, freeze dry it and store it in a refrigerator at 4℃ for later use to obtain modified biochar adsorbed by petroleum degrading bacteria.

[0046] (6) Implantation: Prepare a 4% w / v sodium alginate solution and a 3% w / v CaCl2 solution. Add the modified biochar of the petroleum-degrading bacteria to a sterile syringe and mix it with the 4% w / v sodium alginate solution. Inject the mixture evenly into a certain concentration of CaCl2 solution at a rate of 1 drop / s. Crosslink under suitable conditions for 18 hours. After crosslinking, store in a 4°C refrigerator for later use to obtain the immobilized bacterial agent.

[0047] Example 2

[0048] The only difference from Example 1 is that the concentration of the rhamnolipin aqueous solution in step (3) is 10 g / L.

[0049] Example 3

[0050] The only difference from Example 1 is that the concentration of the rhamnolipin aqueous solution in step (3) is 15 g / L.

[0051] Example 4

[0052] The only difference from Example 1 is that the concentration of the rhamnolipin aqueous solution in step (3) is 25 g / L.

[0053] Example 5

[0054] The only difference from Example 1 is that the concentration of the rhamnolipin aqueous solution in step (3) is 50 g / L.

[0055] Example 6

[0056] The only difference from Example 3 is that in step (1), corn stalks are replaced with rice stalks.

[0057] Example 7

[0058] The only difference from Example 3 is that in step (1), the corn stalks are replaced with corn cobs.

[0059] Example 8

[0060] The only difference from Example 3 is that in step (1), corn stalks are replaced with rice husks.

[0061] Comparative Example 1

[0062] (1) Preparation of biochar: Same as in Example 3.

[0063] (2) Alkali modification of biochar: Prepare a 2 mol / L NH3·H2O aqueous solution, then add 10 g of biochar and 200 mL of NH3·H2O aqueous solution to a 500 mL beaker. Place the beaker on a magnetic stirrer at room temperature and stir at a uniform speed of 200 rpm for 24 hours. Subsequently, separate the solid and liquid using a vacuum filtration device, and repeatedly wash the solid with deionized water until the filtrate is neutral. Dry the filtrate in an oven at 105℃ for 24 hours to obtain alkali-modified biochar.

[0064] (3) Preparation of bacterial suspension: Same as in Example 3.

[0065] (4) Preparation of modified biochar for adsorbing petroleum-degrading bacteria: Take 1g of alkali-modified biochar into a 100mL conical flask, sterilize it in an autoclave at 121℃ for 20min, add 20mL of the bacterial suspension prepared in step (3), place it in a shaker at 30℃ and 180rpm for 24h, centrifuge at 1000rpm for 1min, remove the supernatant, wash it three times with phosphate buffer to remove unadsorbed free bacterial agent, freeze dry it and store it in a refrigerator at 4℃ for later use to obtain modified biochar for adsorbing petroleum-degrading bacteria.

[0066] Comparative Example 2

[0067] (1) Preparation of biochar: Same as in Example 3.

[0068] (2) Alkali modification of biochar: Prepare a 2 mol / L NaOH aqueous solution, then add 10 g of biochar and 200 mL of NaOH solution to a 500 mL beaker, place it on a magnetic stirrer at room temperature, and stir at a uniform speed of 200 rpm for 24 hours. Then, separate the solid and liquid using a vacuum filtration device, and repeatedly wash the solid with deionized water until the filtrate is neutral. Dry it in an oven at 105℃ for 24 hours to obtain alkali-modified biochar.

[0069] (3) Preparation of bacterial suspension: Same as in Example 3.

[0070] (4) Preparation of modified biochar for adsorbing petroleum-degrading bacteria: Take 1g of alkali-modified biochar into a 100mL conical flask, sterilize it in an autoclave at 121℃ for 20min, add 20mL of the bacterial suspension prepared in step (3), place it in a shaker at 30℃ and 180rpm for 24h, centrifuge at 1000rpm for 1min, remove the supernatant, wash it three times with phosphate buffer to remove unadsorbed free bacterial agent, freeze dry it and store it in a refrigerator at 4℃ for later use to obtain modified biochar for adsorbing petroleum-degrading bacteria.

[0071] Comparative Example 3

[0072] (1) Preparation of biochar: Same as in Example 3.

[0073] (2) Alkali modification of biochar: Prepare a 2 mol / L KOH aqueous solution, then add 10 g of biochar and 200 mL of KOH solution to a 500 mL beaker, place it on a magnetic stirrer at room temperature, and stir at a uniform speed of 200 rpm for 24 hours. Then, separate the solid and liquid using a vacuum filtration device, and repeatedly wash the solid with deionized water until the filtrate is neutral. Dry it in an oven at 105℃ for 24 hours to obtain alkali-modified biochar.

[0074] (3) Preparation of bacterial suspension: Same as in Example 3.

[0075] (4) Preparation of modified biochar for adsorbing petroleum-degrading bacteria: Take 1g of alkali-modified biochar into a 100mL conical flask, sterilize it in an autoclave at 121℃ for 20min, add 20mL of the bacterial suspension prepared in step (3), place it in a shaker at 30℃ and 180rpm for 24h, centrifuge at 1000rpm for 1min, remove the supernatant, wash it three times with phosphate buffer to remove unadsorbed free bacterial agent, freeze dry it and store it in a refrigerator at 4℃ for later use to obtain modified biochar for adsorbing petroleum-degrading bacteria.

[0076] Comparative Example 4

[0077] (1) Preparation of biochar: Same as in Example 3.

[0078] (2) Alkali modification of biochar: Prepare a 0.5 mol / L KOH aqueous solution, then add 10 g of biochar and 200 mL of KOH solution to a 500 mL beaker, place it on a magnetic stirrer at room temperature, and stir at a uniform speed of 200 rpm for 24 hours. Then, separate the solid and liquid using a vacuum filtration device, and repeatedly wash the solid with deionized water until the filtrate is neutral. Dry it in an oven at 105℃ for 24 hours to obtain alkali-modified biochar.

[0079] (3) Preparation of bacterial suspension: Same as in Example 3.

[0080] (4) Preparation of modified biochar for adsorbing petroleum-degrading bacteria: Take 1g of alkali-modified biochar into a 100mL conical flask, sterilize it in an autoclave at 121℃ for 20min, add 20mL of the bacterial suspension prepared in step (3), place it in a shaker at 30℃ and 180rpm for 24h, centrifuge at 1000rpm for 1min, remove the supernatant, wash it three times with phosphate buffer to remove unadsorbed free bacterial agent, freeze dry it and store it in a refrigerator at 4℃ for later use to obtain modified biochar for adsorbing petroleum-degrading bacteria.

[0081] Comparative Example 5

[0082] (1) Preparation of biochar: Same as in Example 3.

[0083] (2) Alkali modification of biochar: Prepare a 4 mol / L KOH aqueous solution, then add 10 g of biochar and 200 mL of KOH solution to a 500 mL beaker, place it on a magnetic stirrer at room temperature, and stir at a uniform speed of 200 rpm for 24 hours. Then, separate the solid and liquid using a vacuum filtration device, and repeatedly wash the solid with deionized water until the filtrate is neutral. Dry it in an oven at 105℃ for 24 hours to obtain alkali-modified biochar.

[0084] (3) Preparation of bacterial suspension: Same as in Example 3.

[0085] (4) Preparation of modified biochar for adsorbing petroleum-degrading bacteria: Take 1g of alkali-modified biochar into a 100mL conical flask, sterilize it in an autoclave at 121℃ for 20min, add 20mL of the bacterial suspension prepared in step (3), place it in a shaker at 30℃ and 180rpm for 24h, centrifuge at 1000rpm for 1min, remove the supernatant, wash it three times with phosphate buffer to remove unadsorbed free bacterial agent, freeze dry it and store it in a refrigerator at 4℃ for later use to obtain modified biochar for adsorbing petroleum-degrading bacteria.

[0086] Comparative Example 6

[0087] The only difference from Example 3 is that the concentration of sodium alginate solution in step (6) is 1% w / v.

[0088] Comparative Example 7

[0089] The only difference from Example 3 is that the concentration of sodium alginate solution in step (6) is 2% w / v.

[0090] Comparative Example 8

[0091] The only difference from Example 3 is that the concentration of sodium alginate solution in step (6) is 6% w / v.

[0092] Comparative Example 9

[0093] The only difference from Example 3 is that the surfactant in step (3) is a 5 g / L SDBS solution.

[0094] Comparative Example 10

[0095] The only difference from Example 3 is that the surfactant in step (3) is a 10 g / L SDBS solution.

[0096] Comparative Example 11

[0097] The only difference from Example 3 is that the surfactant in step (3) is a 15 g / L SDBS solution.

[0098] Comparative Example 12

[0099] The only difference from Example 3 is that the surfactant in step (3) is a 25 g / L SDBS solution.

[0100] Comparative Example 13

[0101] The only difference from Example 3 is that the surfactant in step (3) is a 50 g / L SDBS solution.

[0102] Comparative Example 14

[0103] The only difference from Example 3 is that the surfactant in step (3) is a 5 g / L Tween 80 solution.

[0104] Comparative Example 15

[0105] The only difference from Example 3 is that the surfactant in step (3) is a 10 g / L Tween 80 solution.

[0106] Comparative Example 16

[0107] The only difference from Example 3 is that the surfactant in step (3) is a 15 g / L Tween 80 solution.

[0108] Comparative Example 17

[0109] The only difference from Example 3 is that the surfactant in step (3) is a 25 g / L Tween 80 solution.

[0110] Comparative Example 18

[0111] The only difference from Example 3 is that the surfactant in step (3) is a 50 g / L Tween 80 solution.

[0112] Comparative Example 19

[0113] The only difference from Example 3 is that step (3) is omitted.

[0114] Comparative Example 20

[0115] The only difference from Example 3 is that step (6) is omitted.

[0116] Comparative Example 21

[0117] The only difference from Example 3 is that steps (3) and (6) are omitted.

[0118] Experimental Example 1: Characterization of Composite Modified Biochar Structure and Determination of Microbial Immobilization Rate

[0119] like Figure 1 As shown, the unmodified biochar particles are relatively large, have low surface roughness, and poorly developed pores. The specific surface area measured by BET is 263.8 m². 2 / g.

[0120] like Figure 2 As shown, after unmodified biochar is loaded with microorganisms, the surface roughness of the biochar increases, and the microorganisms are loaded on the surface of the activated carbon.

[0121] like Figure 3 As shown, after two-step modification, the biochar (composite modified biochar of Example 3) is more fragmented, has a rougher surface, and more developed pores. The specific surface area measured by BET is 328.02 m². 2 / g.

[0122] like Figure 4 As shown, after two-step modification, the biomass loaded on the biochar (composite modified biochar of Example 3) increased significantly, and the microbial fixation rate was 94.5319% obtained by plate counting method.

[0123] like Figure 5 As shown, biochar (composite modified biochar of Example 3) was heated at 3420 cm⁻¹. -1 The absorption peak at 1630 cm⁻¹ belongs to the OH vibration peak, which is a stretching vibration of aldehydes, phenols, and alcohols. -1 The absorption peak at 1104 cm⁻¹ is related to the vibration of the carboxyl or carbonyl group (C=O or C=C). -1 The absorption peaks are related to the stretching of CO or aromatic groups, indicating that the modified biochar contains more oxygen-containing groups.

[0124] Experimental Example 2: Determination of Petroleum Hydrocarbon Degradation Effect

[0125] 1. Effect of Alkali Type on Petroleum Hydrocarbon Degradation Rate: 1 g of modified biochar (denoted as N-BC-R, Na-BC-R, K-BC-R, and BC-R) of petroleum-degrading bacteria in Comparative Examples 1–3, as well as free bacteria, were respectively placed in 20 mL of groundwater containing 2000 ppm petroleum hydrocarbon pollutants. After culturing for 7 days at 30℃ and 180 rpm in a shaker, the water sample was extracted with a certain volume of n-hexane. The supernatant was collected and the extraction was repeated once. The extracts were combined and filtered through a 0.22 μm organic filter membrane. The concentration of petroleum hydrocarbons was then determined by gas chromatography.

[0126] like Figure 6 As shown, different types of alkalis significantly improved the degradation of petroleum hydrocarbons, with potassium hydroxide showing the best effect, achieving a high degradation efficiency of 61.92% in about 5 days.

[0127] 2. Effect of alkali concentration on petroleum hydrocarbon degradation rate: 1 g of modified biochar (denoted as 2K-BC-R, 0.5K-BC-R, and 4K-BC-R) of comparative examples 3-5 adsorbing petroleum-degrading bacteria, as well as free bacteria, were respectively placed in 20 mL of groundwater containing 2000 ppm petroleum hydrocarbon pollutants. After culturing for 7 days in a shaker at 30℃ and 180 rpm, the water sample was extracted with a certain volume of n-hexane. The supernatant was collected and the extraction was repeated once. The extracts were combined and filtered through a 0.22 μm organic filter membrane. The petroleum hydrocarbon concentration was then determined by gas chromatography.

[0128] like Figure 7 As shown, the degradation rates after modification with 0.5 mol / L and 2 mol / L KOH solutions reached 63.87% and 63.70% respectively after seven days, which is a significant improvement compared to the 45.33% of the unmodified solution.

[0129] 3. Effect of surfactant type on petroleum hydrocarbon degradation rate: 1g of the immobilized bacterial agent prepared in Examples 1-5 and Comparative Examples 9-18 was taken into 20mL of groundwater containing 2000ppm petroleum hydrocarbon pollutants. After culturing in a shaker at 30℃ and 180rpm for 7 days, the water sample was extracted with a certain volume of n-hexane. The supernatant was collected and the extraction was repeated once. The extracts were combined and filtered through a 0.22μm organic filter membrane. The concentration of petroleum hydrocarbons was determined by gas chromatography.

[0130] like Figure 8 As shown, compared with alkali + SDBS modified biochar immobilized bacterial agents, alkali + Tween 80 and alkali + rhamnolipid modified biochar immobilized bacterial agents exhibited significantly higher degradation rates for petroleum hydrocarbons. Furthermore, with increasing concentrations of Tween 80 and rhamnolipid, the degradation rate of petroleum hydrocarbons by these two modifiers initially increased and then decreased. This is because at low concentrations, surfactants increase the contact area between the bacterial agent and petroleum hydrocarbons, thereby improving the degradation rate. However, at high concentrations, surfactants can have a certain toxic effect on the bacterial agent, and excessive surfactants may form micelles, encapsulating organic matter and reducing the contact area with the bacterial agent, thus decreasing the degradation rate. When rhamnolipid at a concentration of 15 g / L was used as the modifier in the second step, the petroleum hydrocarbon degradation rate was the highest, reaching 72.24%.

[0131] 4. Effects of modification and encapsulation on petroleum hydrocarbon degradation rate: 1g each of the following bacteria were taken: immobilized bacterial agent from Example 3 (denoted as KR-BC-ER), immobilized bacterial agent from Comparative Example 19 (denoted as K-BC-ER), modified biochar of petroleum-degrading bacteria from Comparative Example 20 (denoted as KR-BC-R), modified biochar of petroleum-degrading bacteria from Comparative Example 21 (denoted as K-BC-R), and free bacteria (denoted as R). The samples were incubated in a shaker at 30℃ and 180rpm for 7 days. The water samples were then extracted with a certain volume of n-hexane. The supernatant was collected and the extraction was repeated once. The extracts were combined and filtered through a 0.22μm organic filter membrane. The concentration of petroleum hydrocarbons was then determined by gas chromatography.

[0132] like Figure 9As shown, the degradation rate of petroleum hydrocarbons after alkali modification (K-BC-R, Comparative Example 21) was 63.70%, while the degradation rate of petroleum hydrocarbons after alkali modification + encapsulation (K-BC-ER, Comparative Example 19) was 51.07%. This is because encapsulation hinders the mass transfer of petroleum hydrocarbon pollutants and bacterial agents. When modified by the alkali-rhamnolipin two-step method without encapsulation (KR-BC-R, Comparative Example 20), the degradation rate was 46.08%, possibly because the concentration of rhamnolipin at this level has a toxic effect on the bacterial agent, weakening its activity. After modification by the alkali-rhamnolipin two-step method and encapsulation (KR-BC-ER, Example 3), the degradation rate reached 72.24%, indicating that the sodium alginate-CaCl2 encapsulation system protects the bacterial agent, weakens the toxic effect of rhamnolipin, and rhamnolipin enhances the mass transfer to a certain extent.

[0133] 5. Effect of biochar type on petroleum hydrocarbon degradation rate: 1g of the immobilized bacterial agent prepared in Examples 3 and 6-8 was respectively placed in 20mL of groundwater containing 2000ppm petroleum hydrocarbon pollutants. After culturing in a shaker at 30℃ and 180rpm for 7 days, the water sample was extracted with a certain volume of n-hexane. The supernatant was collected and the extraction was repeated once. The extracts were combined and filtered through a 0.22μm organic filter membrane. The concentration of petroleum hydrocarbons was determined by gas chromatography.

[0134] like Figure 10 As shown, immobilized microbial agents prepared from biochar made from different raw materials all exhibited high petroleum hydrocarbon degradation rates. Among them, the immobilized microbial agent prepared from corn straw biochar had the highest petroleum hydrocarbon degradation rate, followed by rice straw, corn cob, and rice husk.

[0135] Experimental Study on Sodium Alginate Concentration in Experiment Example 3

[0136] The experimental results also showed that when the sodium alginate concentration was 1% w / v (Comparative Example 6) and 2% w / v (Comparative Example 7), the viscosity was too low, causing droplets to tail and failing to form spherical shapes. At 4% w / v (Example 3), perfectly round spherical shapes could be formed, meeting the requirements. However, when the sodium alginate concentration was 6% w / v (Comparative Example 8), the liquid was too viscous, and the syringe could not dispense it. Therefore, 4% w / v sodium alginate was selected.

[0137] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing composite modified biochar, characterized in that, The preparation method includes: Biochar is impregnated with an alkaline solution, and the impregnated biochar is washed and dried to obtain alkali-modified biochar. Alkali-modified biochar is impregnated with a surfactant solution, and the impregnated alkali-modified biochar is washed and dried to obtain alkali and surfactant co-modified biochar, which is the composite modified biochar.

2. The preparation method according to claim 1, characterized in that, The concentration of the alkaline solution is 0.5–2 mol / L, and the concentration of the surfactant solution is 5–50 g / L.

3. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of biochar to alkaline solution is 1g:10-40mL, the mass-to-volume ratio of alkali-modified biochar to surfactant solution is 1g:5-15mL, and the impregnation temperature is room temperature for 20-30h.

4. The preparation method according to claim 1, characterized in that, The alkali is selected from ammonia monohydrate, sodium hydroxide, or potassium hydroxide.

5. The preparation method according to claim 1, characterized in that, The surfactant is selected from rhamnolipin, sodium dodecylbenzenesulfonate, or Tween 80.

6. The composite modified biochar prepared by the method according to any one of claims 1 to 5.

7. An immobilized bacterial agent for enhancing the microbial degradation of petroleum hydrocarbon pollutants, characterized in that, The immobilized microbial agent comprises the composite modified biochar of claim 6, and petroleum hydrocarbon degrading bacteria loaded on the composite modified biochar.

8. The immobilized bacterial agent according to claim 7, characterized in that, The immobilized bacterial agent has a core-shell structure, with modified biochar adsorbed with petroleum hydrocarbon degrading bacteria as the core and calcium alginate gel as the outer shell. The modified biochar is the composite modified biochar as described in claim 6.

9. The immobilized bacterial agent according to claim 8, characterized in that, The specific surface area of ​​the immobilized bacterial agent is 300-400 m². 2 / g.

10. A method for preparing the immobilized bacterial agent according to claim 8 or 9, characterized in that, The preparation method includes: The composite modified biochar of claim 6 is mixed with the bacterial culture of petroleum hydrocarbon degrading bacteria and cultured. The culture product is separated into solid and liquid components, and the solid is washed and dried to obtain modified biochar adsorbed with petroleum hydrocarbon degrading bacteria. Modified biochar adsorbed with petroleum hydrocarbon degrading bacteria was mixed with sodium alginate solution, and then added dropwise to calcium salt solution to allow for full reaction. After washing and drying, the immobilized bacterial agent was obtained.

11. The preparation method according to claim 10, characterized in that, The concentration of the bacterial solution is 2×10⁻⁶. 8 ~3×10 8 CFU / mL.

12. The preparation method according to claim 10, characterized in that, The mass-to-volume ratio of the composite modified biochar to the petroleum hydrocarbon degrading bacteria solution is 0.8–1.2 g: 20 mL.

13. The preparation method according to claim 10, characterized in that, The culture temperature is 30℃ and the culture time is 20-30h.

14. The preparation method according to claim 10, characterized in that, The concentration of the sodium alginate solution is 3-5% w / v, the concentration of the calcium salt solution is 2-4% w / v, and the reaction time is 15-20 h.

15. The immobilized bacterial agent prepared by the method according to any one of claims 10 to 14.

16. The application of the immobilized bacterial agent according to any one of claims 7 to 9 or 15 in the degradation of petroleum hydrocarbon pollution in groundwater.

17. A method for degrading petroleum hydrocarbon pollution in groundwater, characterized in that, The method includes contacting the immobilized bacterial agent according to any one of claims 7 to 9 or 15 with petroleum hydrocarbon-contaminated groundwater.

Images