A method for removing TNT by using biochar-fixed functional microorganisms
By immobilizing TNT degradation bacteria on biochar, the synergistic effect of biochar adsorption and microbial degradation effects are used to solve the problems of low efficiency and high cost of TNT removal in the prior art, and an efficient and low-cost TNT removal effect is achieved.
Patent Information
- Application Number
- CN202510008162.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-03
- Publication Date
- 2025-04-04
AI Technical Summary
The prior art is inefficient and costly when removing TNT pollutants, and there is a problem of secondary pollution in biological methods.
By immobilizing TNT degradation bacteria on biochar, the synergistic effect of biochar adsorption and microbial degradation can improve the removal rate of TNT and reduce the treatment cost.
It significantly improves the removal efficiency of TNT, reduces the treatment cost, realizes the resource utilization of waste, and avoids secondary pollution.
Smart Images
Figure CN119752875A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pollution control, and in particular relates to a method for removing TNT by fixing functional microorganisms with biochar. Background Art
[0002] The energetic compound TNT has multiple –NO2 groups, and the nitrogen atoms on the nitro group are in an oxidized state, which can undergo redox reactions and release a large amount of energy. It has the characteristics of high detonation velocity and high explosion heat, and has become one of the most commonly used explosive components in military and civilian use in various countries. However, during the production, use and military activities of TNT, a large amount of TNT explosive residues have entered the water or soil, causing serious environmental pollution. Energetic explosives are widely toxic to microorganisms, animals, plants and humans. It has been confirmed that TNT has teratogenic, mutagenic and carcinogenic effects, and has been listed as a Class C carcinogen by the US Environmental Protection Agency (EPA).
[0003] At present, the most commonly used methods for removing explosive wastewater are physical treatment, chemical treatment and bioremediation. Physical remediation includes adsorption, electrolysis and coagulation. Alkaline hydrolysis and oxidation are commonly used methods for chemical treatment. However, the use of physical and chemical methods has the problems of high cost and secondary pollution.
[0004] The biological method is to use plants or microorganisms to degrade pollutants. It is a green, environmentally friendly and low-cost method, but the biological method has problems such as low removal efficiency. Biochar produced from agricultural waste is considered to be a new type of environmental functional material due to its green, low-carbon, high efficiency, low cost and high benefit, and is widely used. Biochar has the characteristics of large specific surface area and dense pore structure. It can provide a shelter for microorganisms, as well as good oxygen and nutrients. It can also exert its own adsorption properties to synergistically improve the removal rate of pollutants.
[0005] Therefore, it is necessary to develop a method for removing TNT using biochar-immobilized microbial materials to provide a new way to control pollution from energetic compounds in ammunition destruction, military training, and military industrial factories. Summary of the invention
[0006] The purpose of the present invention is to address the problem of slow TNT degradation rate of existing microorganisms, use biochar to fix TNT-degrading bacteria, synergize the biochar adsorption and microbial degradation, while improving the TNT removal rate, reduce the treatment cost of explosive wastewater and realize resource utilization of waste.
[0007] In order to achieve the above object, the present invention adopts the following technical scheme:
[0008] The present invention provides a method for preparing a biochar-fixing functional microorganism for removing TNT, comprising the following steps:
[0009] S1: taking mature sludge containing explosive pollutants to prepare a sludge-water mixture; performing oscillation culture on the sludge-water mixture, and then inoculating the supernatant into an enrichment medium for oscillation culture, and then performing oscillation culture in a selection medium, separating and purifying the strain to obtain a TNT-degrading strain;
[0010] The sludge-water mixing ratio is 50-150 mL of sterile water per gram of sludge; the shaking culture conditions are constant temperature shaking culture at 25-35°C and 100-300 r / min;
[0011] The enrichment medium formula is: 1 mass part of sugar source, 5-15 mass parts of protein source, 8-12 mass parts of sodium salt, 0.05-0.2 mass parts of potassium salt, 0.01-0.1 mass parts of magnesium salt, and 0.01-0.2 mass parts of TNT;
[0012] The selection medium formula is: 0.05-0.2 mass parts of TNT, 0.5-2 mass parts of ammonium salt, 0.2-1 mass parts of sodium salt, 1-3 mass parts of potassium salt, 0.1-0.3 mass parts of magnesium salt, 0.02-0.5 mass parts of calcium salt, and 0.01-0.05 mass parts of iron salt;
[0013] S2: preparing coconut shell biochar by high temperature oxygen-limited slow pyrolysis method, grinding and passing through a sieve of not less than 100 mesh;
[0014] The specific operation is as follows: the coconut shell used to prepare the biochar raw material is washed with deionized water and then soaked in deionized water, dried at 100-120°C, crushed with a grinder after drying, and then passed through a 10-mesh sieve for later use.
[0015] Using the high-temperature oxygen-limited slow pyrolysis method, take the crushed raw materials, heat them from room temperature to 450-550°C at a rate of 3-6°C / min, maintain constant temperature carbonization for 3-6 hours, and take them out after cooling to room temperature;
[0016] S3: After the TNT-degrading bacteria obtained in step S1 are expanded and cultured in LB medium, the TNT-degrading bacteria are collected by centrifugation;
[0017] The TNT-degrading bacteria collected by centrifugation and the biochar were dispersed in sterile water, mixed and shaken to obtain the biochar-immobilized TNT-degrading bacteria.
[0018] Preferably, in step S1, the enrichment culture medium is prepared with no less than 5 gradient concentrations of TNT content; the supernatant of the mud-water mixture is first inoculated into the enrichment culture medium with the lowest TNT concentration, and then replaced with a higher concentration TNT enrichment culture medium for cultivation.
[0019] Furthermore, the supernatant is added to the enrichment medium at an inoculation rate of 4-6%; when the medium is turbid, 0.4-0.6% is taken and transplanted to a new enrichment medium.
[0020] Preferably, in step S2, the raw materials are placed in a quartz crucible, compacted and covered, and programmed to heat in a tube furnace, first from room temperature to 500°C at a rate of 5°C / min, maintained at a constant temperature for 4 hours, and then taken out after cooling to room temperature.
[0021] Preferably, in step S3, the culture is carried out in LB liquid medium until OD 600 After the pH value is no less than 1, the culture medium is centrifuged to collect the bacteria, the bacteria are washed with sterile water and then suspended in sterile water with a volume no larger than that of the original LB liquid culture medium, and 5 to 15 g / L of biochar is added.
[0022] Preferably, the enrichment medium formula is: 1.0 mass parts of glucose, 10 mass parts of peptone, 10.5 mass parts of NaCl, 0.1 mass parts of K2HPO4, 0.05 mass parts of MgSO4·7H2O, 0.02-0.1 mass parts of TNT; and the pH is adjusted to 6.8-7.5.
[0023] Preferably, the selection medium formula is: 0.1 mass part of TNT, 1 mass part of (NH4)2SO4, 0.5 mass part of NaCl, 0.5 mass part of KH2PO4, 1.5 mass part of K2HPO4, 0.2 mass part of MgSO4·7H2O, 0.1 mass part of CaCl2·2H2O, and 0.02 mass part of FeCl3.
[0024] Preferably, the LB liquid culture medium has a formula of: 10 parts by mass of tryptone, 5 parts by mass of yeast powder, and 10 parts by mass of NaCl; and the pH is adjusted to 6.8-7.5.
[0025] In a second aspect, the present invention provides a biochar-fixing functional microorganism prepared by the method described in the first aspect.
[0026] In a third aspect, the present invention provides an application of the biochar-fixed functional microorganisms of the second aspect for removing TNT.
[0027] Beneficial effects of the present invention:
[0028] 1. Fixing TNT-degrading bacteria on biochar can effectively fix the strains, and use the strains to degrade TNT on the adsorbent, increasing the life of the adsorbent, thereby exerting adsorption and biodegradation effects at the same time, greatly improving the removal efficiency of TNT.
[0029] 2. The prepared biochar has a large specific surface area and more surface active sites, providing microorganisms with a good oxygen and nutrient transmission environment. The raw materials come from waste in life and production, which is low-cost and easy to obtain, realizing the resource utilization of waste.
[0030] 3. The prepared biochar-immobilized TNT-degrading bacteria have the advantages of low cost, high efficiency, and no secondary pollution, and have significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 It is a schematic diagram of the degradation effect of the embodiment and the comparative example;
[0032] (A) Curve of TNT concentration changing with time during the degradation process of the embodiment, comparative example and control group; (B) Graph of the final degradation effect of the embodiment and comparative example. DETAILED DESCRIPTION
[0033] The present invention is further described in detail below through specific embodiments, but the protection scope of the present invention is not limited thereto.
[0034] Example 1
[0035] A method for preparing biochar-fixed functional microorganisms for removing TNT comprises the following steps:
[0036] Step 1: Strain isolation and purification
[0037] Step 1.1, culture medium formula:
[0038] There are five enrichment media with the following formulas: glucose 1.0 g, peptone 10 g, NaCl 10.5 g, K2HPO4 0.1 g, MgSO4·7H2O 0.05 g, initial amount of TNT 20 mg (the remaining four are gradually increased to 100 mg in a gradient of 20 mg), distilled water to 1000 mL, pH 7.2, and sterilized at 121°C for 20 min.
[0039] Select culture medium: TNT 100 mg, (NH4)2SO4 1 g, NaCl 0.5 g, KH2PO4 0.5 g, K2HPO4 1.5 g, MgSO4·7H2O 0.2 g, CaCl2·2H2O 0.1 g, FeCl3 0.02 g, make up to 1000 mL with distilled water, sterilize at 121°C for 20 min.
[0040] Step 1.2, separation and purification steps:
[0041] The activated sludge samples were taken from the Shanxi ammunition destruction site. Take 10g of sludge sample and 90mL of sterile water to make a mud-water mixture. Shake and culture at 30℃ and 150r / min for 24h. After standing, take the supernatant and add it to the enrichment medium at a volume of 5mL. Shake and culture at 37℃, 150r / min, and an initial TNT concentration of 20mg / L. When the culture medium is turbid, take 5mL and transplant it to a new enrichment medium, and gradually increase the TNT concentration to 100mg / L with a gradient of 20mg / L. Continue to shake and culture under the same conditions. After that, the enrichment medium is replaced by the selection medium, and TNT is used as the only nitrogen source to screen the domesticated strains. The culture is cultured under the same conditions for 48h, and the bacterial culture fluid is finally obtained. The bacterial culture fluid is diluted and spread, cultured at 37℃ for 48h, and different strains are selected according to the colony morphology and repeatedly streaked on the same culture medium plate for separation and purification until a single colony is obtained.
[0042] Through the analysis of morphology, physiological and biochemical characteristics, it was preliminarily determined that the strain was Pseudomonas.
[0043] Step 2: Biochar preparation
[0044] The raw materials for preparing biochar are freshly discarded coconut shells from the Beijing market.
[0045] Step 2.1. Wash the surface of the raw material coconut shell used to prepare biochar with deionized water several times and then soak it in deionized water for 24 hours. Then dry it at 110°C for 24 hours. After drying, crush it with a crusher and pass it through a 10-mesh sieve for later use.
[0046] Step 2.2: Using the high-temperature oxygen-limited slow pyrolysis method, take the raw materials and put them in a quartz crucible, compact them and cover them tightly, heat them to 500°C at a rate of 5°C / min in a tube furnace, maintain constant temperature carbonization for 4 hours, and take them out after cooling to room temperature.
[0047] Step 3: Biochar immobilization strains
[0048] Pick the previously purified strain, inoculate it into 100 mL of LB liquid medium, and culture it in a shaker at 30°C and 150 r / min until OD 600 =1, then centrifuge the culture medium to collect the cells, wash the cells three times with sterile water, and finally suspend them in 100 mL of sterile water, add 1 g of biochar, and adsorb them in an oscillating incubator at 30°C and 150 r / min. After 24 hours, filter and rinse the biochar three times with sterile water to obtain a biochar-immobilized bacterial agent. The LB liquid culture medium formula is: 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, pH 7.2, distilled water to 1000 mL, and sterilized at 121°C for 20 min.
[0049] Test results
[0050] Prepare 100 mL of TNT simulated wastewater with a concentration of 100 mg / L.
[0051] 0.02 g of the biochar and TNT-degrading bacteria prepared in Example 1 as a control example, and the biochar-immobilized TNT-degrading bacteria of Example 1 were added to the simulated TNT wastewater, water samples were collected regularly, and the residual TNT concentration was determined. The results showed that the adsorption of TNT by biochar reached equilibrium in 3 hours, and the biodegradation of TNT by the biochar-immobilized bacteria reached equilibrium in about 48 hours.
[0052] Under the same conditions as above, the TNT removal rates of the biochar, TNT-degrading bacteria (T22) and biochar-immobilized TNT-degrading bacteria in Example 1 were tested respectively.
[0053] Among them, the removal rate of biochar is the lowest at 52.18%, the removal rate of TNT degrading bacteria is 22.45%, and the removal rate of TNT by biochar-immobilized TNT degrading bacteria reaches 97.26%. It can be seen that the biochar-immobilized TNT degrading bacteria of this specific embodiment has a high removal rate for TNT in water.
[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention but not to limit the protection scope of the present invention.
Claims
1. A method for removing TNT by using biochar-fixed functional microorganisms, characterized in that: The preparation of a biochar-fixing functional microorganism for removing TNT comprises the following steps: S1: taking sludge containing explosive pollutants to prepare a sludge-water mixture; culturing the sludge-water mixture, then inoculating the supernatant into an enrichment medium for shaking culture, and then culturing in a selective medium, separating and purifying the strain to obtain a TNT-degrading strain; The mud-water mixture is prepared by mixing each gram of sludge with 50-150 mL of sterile water; the shaking culture conditions are constant temperature shaking culture at 25-35°C and 100-300 r / min; The enrichment medium formula is: 1 mass part of sugar source, 5-15 mass parts of protein source, 8-12 mass parts of sodium salt, 0.05-0.2 mass parts of potassium salt, 0.01-0.1 mass parts of magnesium salt, and 0.01-0.2 mass parts of TNT; The selection medium formula is: 0.05-0.2 mass parts of TNT, 0.5-2 mass parts of ammonium salt, 0.2-1 mass parts of sodium salt, 1-3 mass parts of potassium salt, 0.1-0.3 mass parts of magnesium salt, 0.02-0.5 mass parts of calcium salt, and 0.01-0.05 mass parts of iron salt; S2: Prepare coconut shell biochar by high temperature oxygen-limited slow pyrolysis method, grind and sieve; The specific operation is as follows: the raw material coconut shell used to prepare biochar is washed with deionized water and then fully soaked with deionized water, dried at 100-120°C, and crushed after drying; Using the high-temperature oxygen-limited slow pyrolysis method, take the crushed raw materials, heat them from room temperature to 450-550°C at a rate of 3-6°C / min, maintain constant temperature carbonization for 3-6 hours, and take them out after cooling to room temperature; S3: After the TNT-degrading bacteria obtained in step S1 are expanded and cultured in LB medium, the TNT-degrading bacteria are collected by centrifugation; The TNT-degrading bacteria collected by centrifugation and the biochar were dispersed in sterile water, mixed and shaken to obtain the biochar-immobilized TNT-degrading bacteria.
2. The method for preparing a biochar-fixing functional microorganism for removing TNT according to claim 1, characterized in that: In step S1, the enrichment culture medium is prepared with no less than 5 gradient concentrations of TNT content; the supernatant of the mud-water mixture is first inoculated into the enrichment culture medium with the lowest TNT concentration, and then replaced with a higher concentration TNT enrichment culture medium for cultivation.
3. The method for preparing a biochar-fixing functional microorganism for removing TNT according to claim 2, characterized in that: The supernatant is added to the enrichment medium at an inoculation rate of 4-6%; when the medium is turbid, 0.4-0.6% is taken and transplanted to a new enrichment medium.
4. The method for preparing a biochar-fixing functional microorganism for removing TNT according to claim 1, characterized in that: In step S2, the raw materials are placed in a quartz crucible, compacted and covered tightly, and programmed to heat in a tubular furnace from room temperature to 500° C. at a rate of 5° C. / min, and carbonized at a constant temperature for 4 hours, and then taken out after cooling to room temperature.
5. The method for preparing a biochar-fixing functional microorganism for removing TNT according to claim 1, characterized in that: In step S3, the cells were cultured in LB liquid medium until OD 600 After the pH value is no less than 1, the culture medium is centrifuged to collect the bacteria, the bacteria are washed with sterile water and then suspended in sterile water with a volume no larger than that of the original LB liquid culture medium, and 5 to 15 g / L of biochar is added.
6. The method for preparing a biochar-fixing functional microorganism for removing TNT according to claim 1, characterized in that: The enrichment culture medium comprises: 1.0 parts by mass of glucose, 10 parts by mass of peptone, 10.5 parts by mass of NaCl, 0.1 parts by mass of K2HPO4, 0.05 parts by mass of MgSO4·7H2O, and 0.02-0.1 parts by mass of TNT; and the pH is adjusted to 6.8-7.
5.
7. The method for preparing a biochar-fixing functional microorganism for removing TNT according to claim 1, characterized in that: The selection culture medium formula is: 0.1 mass part of TNT, 1 mass part of (NH4)2SO4, 0.5 mass part of NaCl, 0.5 mass part of KH2PO4, 1.5 mass part of K2HPO4, 0.2 mass part of MgSO4·7H2O, 0.1 mass part of CaCl2·2H2O, and 0.02 mass part of FeCl3.
8. The method for preparing a biochar-fixing functional microorganism for removing TNT according to claim 1, characterized in that: The LB liquid culture medium is formulated as follows: 10 parts by mass of tryptone, 5 parts by mass of yeast powder, and 10 parts by mass of NaCl; the pH is adjusted to 6.8-7.
5.
9. A biochar-fixing functional microorganism prepared by the method according to any one of claims 1 to 8.
10. Use of the biochar-fixing functional microorganism according to claim 9 for removing TNT.