Method for reducing antibiotic resistance gene abundance in pig manure anaerobic fermentation biogas residue and biogas slurry by using modified charcoal

Abstract

The invention relates to the technical field of biochar preparation and application, in particular to a method for reducing abundance of antibiotic resistance genes in pig manure anaerobic fermentation biogas residues and biogas slurry by using modified biochar, which comprises the following steps: step 1, performing high-temperature pyrolysis on a biomass raw material under an anaerobic condition, and sequentially sieving, washing and drying to obtain original biochar; 2, performing chemical modification on the original biochar by using a citric acid solution to obtain modified biochar; step 3, sequentially drying, crushing, grinding and sieving the modified biochar, then uniformly mixing pig manure and the sieved modified biochar according to a mass ratio of (9-11): 1, and adjusting the moisture content of the mixture of the pig manure and the modified biochar to 65-75% to obtain a semi-fluid mixed material; and step 4, anaerobic fermentation of the mixed material. The biochar modified by the citric acid solution and the pig manure are mixed and subjected to anaerobic fermentation, so that the abundance of antibiotic resistance genes in the pig manure is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of biochar preparation and application, and in particular to a method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid produced by anaerobic fermentation of pig manure by utilizing modified biochar. Background Art

[0002] In recent years, as the use of antibiotics has become increasingly widespread across various industries, including medicine, animal husbandry, and aquaculture, the problem of antibiotic abuse has become increasingly serious. In the livestock industry, the misuse of antibiotics not only leads to high concentrations of antibiotic residues in livestock manure but also induces the production of antibiotic resistance genes (ARGs) in livestock. ARGs are a class of genes that confer resistance to antibiotics in bacteria. These genes typically encode specific proteins or enzymes that, through various mechanisms, enable bacteria to survive and reproduce in the presence of antibiotics. ARGs can persist, spread, and proliferate in various environmental media, such as soil, water, and air, and enter the human body through the food chain, posing a threat to human health. Due to insufficient assimilation, antibiotics that enter livestock and are not fully absorbed are excreted in the form of feces, resulting in a high abundance of ARGs in livestock and poultry manure. The abundance of ARGs in pig manure is higher than in chicken and cattle manure.

[0003] Chinese patent CN116462181A discloses a potassium dihydrogen phosphate-modified biochar material, preparation method, and application. Rice husks are placed in a tubular furnace, nitrogen is introduced, and the temperature is raised to 500-600°C for carbonization. CO2 is then introduced to the furnace to produce rice husk-based biochar. The material is then placed in a potassium dihydrogen phosphate solution, stirred, allowed to stand, filtered, and centrifuged. The solution is then rinsed with water until the pH is near neutral and dried to produce potassium dihydrogen phosphate-modified biochar. This patent uses potassium dihydrogen phosphate-modified biochar as an enhancer, added to a substrate with a total solids content of 7%-9%, to reduce the abundance of resistance genes in pig manure. However, this treatment process has low efficiency and high energy consumption. Summary of the Invention

[0004] In response to the problems of low treatment efficiency and high energy consumption in the existing methods for reducing the abundance of resistance genes in pig manure, the present invention provides a method for reducing the abundance of antibiotic resistance genes in sludge and liquid from anaerobic fermentation of pig manure using modified biochar. Biochar modified with citric acid solution is mixed with pig manure and anaerobically fermented, which significantly reduces the abundance of antibiotic resistance genes in pig manure.

[0005] The technical solutions of the present invention are as follows: A method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid from anaerobic fermentation of pig manure using modified biochar comprises the following steps: Step 1: Preparation of raw biochar: pyrolysis of biomass raw materials under anaerobic conditions, followed by screening, washing and drying to obtain raw biochar; Step 2: modification of the original biochar, chemically modifying the original biochar obtained in step 1 using a citric acid solution to obtain modified biochar; Step 3: The modified biochar is dried, crushed, ground, and sieved in sequence, and then the pig manure and the sieved modified biochar are evenly mixed in a mass ratio of 9-11:1. The moisture content of the mixture of pig manure and modified biochar is adjusted to 65%-75% to obtain a semi-fluid mixture. The mass ratio of pig manure to modified biochar is preferably 10:1. Step 4: Anaerobic fermentation of the mixed material, controlling the temperature of the anaerobic fermentation at 31°C-55°C, and controlling the time of the anaerobic fermentation at 25-45 days.

[0006] Furthermore, in step 1, the biomass raw material is corn stalks and / or rice husks. Corn stalks are widely available, and rice husks are a byproduct of rice processing. The present invention uses corn stalks and / or rice husks as biomass raw materials, which helps reduce raw material costs.

[0007] Furthermore, in step 1, the high-temperature pyrolysis temperature is 400-650°C, preferably 420-500°C, the high-temperature pyrolysis time is 1-2.5 hours, the sieve mesh size is 80 mesh, and the drying temperature is 50-110°C. High-temperature pyrolysis not only enables the raw biochar to form a high specific surface area, a multi-level pore structure, and a highly stable aromatized structure, but also destroys residual antibiotics, pathogens, and weed seeds in the raw biomass, reducing the risk of secondary contamination.

[0008] Furthermore, in step 2, the concentration of the citric acid solution is 1-3 mol / L, preferably 1 mol / L. The use of citric acid solution to modify the original biochar can add organic groups such as carbonyl, carboxyl, and hydroxyl groups to the original biochar, while allowing the biochar to form more micropores and mesopores, significantly increasing the specific surface area, and providing a large number of adsorption sites for resistance genes and their host microorganisms, making them easier to be captured by biochar. The carbonyl groups added to the original biochar play an important role in the degradation of antibiotics in the fermentation material, and the decrease in the level of antibiotics is very beneficial to the reduction of resistance gene levels. Since citric acid solution is green and environmentally friendly, the use of citric acid solution to modify the original biochar can avoid the risk of secondary pollution.

[0009] Furthermore, in step 2, the chemical modification method is: the original biochar is evenly dispersed and soaked in a citric acid solution. The original biochar is immersed in the citric acid solution in a dispersed state, allowing it to fully contact the citric acid solution, thereby improving the modification efficiency.

[0010] Furthermore, the soaking time is 2-5 hours, preferably 3 hours.

[0011] Furthermore, the mass ratio of the raw biochar to the citric acid is 1:1 to 4. When 50 g of the raw biochar is immersed in a citric acid solution with a concentration of 1 mol / L, the amount of the citric acid solution used is preferably 500-1000 ml.

[0012] Furthermore, in step three, the particle size of the modified biochar after sieving is 0.9-1.5 mm, preferably 1 mm.

[0013] Furthermore, in step 4, anaerobic fermentation is carried out in a one-way exhaust tank. The one-way exhaust tank includes a glass tank body with a single-hole one-way air release valve at the bottle mouth of the glass tank body. The one-way exhaust tank can balance the requirements of airtightness of the anaerobic fermentation environment and gas release, playing the dual role of preventing oxygen from entering and removing metabolic gases generated during the anaerobic fermentation process, providing a stable oxygen-free environment for anaerobic microorganisms, and ensuring the efficient and safe anaerobic fermentation process.

[0014] Furthermore, the antibiotic resistance gene includes a tetracycline resistance gene and / or a sulfonamide resistance gene. The tetracycline resistance gene may be one or more of tetW, tetA and tetM, and the sulfonamide resistance gene may be at least one of sul1 and sul2.

[0015] The beneficial effects of the present invention are: The present invention provides a method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid from anaerobic fermentation of pig manure using modified biochar. The method comprises the following steps: chemically modifying the original biochar using a citric acid solution, introducing functional groups such as carbonyl, carboxyl, and hydroxyl groups into the original biochar, thereby improving the specific adsorption capacity of the biochar for tetracycline antibiotics and / or sulfonamide antibiotics remaining in pig manure, reducing the selection pressure of tetracycline antibiotics and / or sulfonamide antibiotics on host bacteria of antibiotic resistance genes, inhibiting the enrichment of resistance genes from the source, and facilitating the reduction of the abundance of tetracycline resistance genes and / or sulfonamide resistance genes in pig manure; using the citric acid solution as a modifier for the original biochar can also improve the biogas yield while avoiding the risk of secondary pollution, thereby achieving coordinated resource utilization; and controlling the temperature of the anaerobic fermentation at 35-55°C is conducive to inhibiting the activity of pathogens carrying tetracycline resistance genes and / or sulfonamide resistance genes, thereby reducing the abundance of antibiotic resistance genes. DETAILED DESCRIPTION

[0016] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0017] Example 1 A method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid from anaerobic fermentation of pig manure using modified biochar comprises the following steps: Step 1: Preparation of raw biochar: Corn stalks were selected as the biomass feedstock. Nitrogen was first introduced into the pyrolysis furnace for 10 minutes to expel oxygen from the furnace. The pyrolysis was then carried out at 500°C for 2 hours in the absence of oxygen. After pyrolysis, the stalks were sieved through an 80-mesh sieve and rinsed with deionized water. The stalks were then oven-dried at 50-110°C to produce the raw biochar. To ensure an oxygen-free environment, the nitrogen flow rate was controlled at 1.5-2.0 L / min during the initial heating phase (within 10 minutes). After stabilization, the nitrogen flow rate was adjusted to 0.5-1.0 L / min.

[0018] Step 2: Modification of the original biochar. 50 g of the original biochar obtained in step 1 was evenly dispersed in 500 ml of 1 mol / L citric acid solution and soaked for 3 hours. After taking it out, it was washed with deionized water and filtered with quantitative filter paper, and then dried in an oven at 105°C to constant weight to obtain modified biochar.

[0019] Step 3: Mixing pig manure with modified biochar. The modified biochar is dried, crushed, ground and sieved through an 80-mesh sieve in sequence. The particle size of the modified biochar obtained after sieving is about 1 mm. Then, fresh pig manure with a moisture content of 54% and the sieved modified biochar are evenly mixed in a mass ratio of 10:1. Water is added to dilute the mixture of fresh pig manure and modified biochar, and the moisture content of the mixture of fresh pig manure and modified biochar is adjusted to 65% to obtain a semi-fluid mixture.

[0020] Step 4: Anaerobic fermentation of the mixture: Add the mixture to a 2L anaerobic fermentation tank, seal it, and perform anaerobic fermentation at 35°C for 30 days. During this period, the pH value and methane production are regularly monitored to maintain the pH between 6.5 and 7.5, and the methane production is 132ml / kg. After anaerobic fermentation, biogas residue and biogas liquid are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank consists of a glass tank body with a single-hole one-way vent valve at the bottle mouth of the glass tank body.

[0021] Step 5: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to determine the absolute abundance of the tetracycline resistance gene tetW and the sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples. The absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 1.89×10 12 copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 9.34×10 12 copies / g.

[0022] Comparative Example 1 This comparative example comprises the following steps: Step 1: Add water to fresh pig manure with a moisture content of 50%-60% to dilute it, adjust the moisture content of the fresh pig manure to 65%, and obtain a semi-fluid material.

[0023] Step 2: Anaerobic fermentation of the material: 1 kg of material (after adjusting the moisture content) is added to a 2 L anaerobic fermentation tank. After sealing, anaerobic fermentation is carried out at 35°C for 30 days. During this period, the pH value and methane production are regularly monitored to maintain the pH between 6.5 and 7.5, and the methane production is 107 ml. After anaerobic fermentation is completed, biogas residue and biogas liquid are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank consists of a glass tank body with a single-hole one-way vent valve at the bottle mouth.

[0024] Step 3: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to determine the absolute abundance of the tetracycline resistance gene tetW and the sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples. The absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 3.21×10 12 copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 12.63×10 12 copies / g.

[0025] Compared with the absolute abundance of the tetracycline resistance gene tetW and the absolute abundance of the sulfonamide resistance gene sul1 in the sludge and liquid samples in Comparative Example 1, the absolute abundance of the tetracycline resistance gene tetW in the sludge and liquid samples in Example 1 decreased by 17%, and the absolute abundance of the sulfonamide resistance gene sul1 decreased by 35%.

[0026] Example 2 A method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid from anaerobic fermentation of pig manure using modified biochar comprises the following steps: Step 1: Preparation of raw biochar: Rice husks are used as the biomass feedstock. Nitrogen is first introduced into the pyrolysis furnace for 10 minutes to eliminate all oxygen. Pyrolysis is then carried out at 420°C for 2.5 hours in the absence of oxygen. After pyrolysis, the husks are sieved through an 80-mesh sieve and rinsed with deionized water. The husks are then oven-dried at 50-110°C to produce the raw biochar. To ensure an oxygen-free environment, the nitrogen flow rate is controlled at 1.5-2.0 L / min during the initial heating phase (within 10 minutes). Once the temperature stabilizes, the flow rate can be adjusted to 0.5-1.0 L / min.

[0027] Step 2: Modification of the original biochar. 50 g of the original biochar obtained in step 1 was evenly dispersed in 1000 ml of 1 mol / L citric acid solution and soaked for 3 hours. After taking it out, it was washed with deionized water and filtered with quantitative filter paper. Then, it was dried in an oven at 105°C to constant weight to obtain modified biochar.

[0028] Step 3: Mixing pig manure with modified biochar. The modified biochar is dried, crushed, ground and sieved through an 80-mesh sieve in sequence. The particle size of the modified biochar obtained after sieving is about 1 mm. Then, fresh pig manure with a moisture content of 54% and the sieved modified biochar are evenly mixed in a mass ratio of 10:1. Water is added to dilute the mixture of fresh pig manure and modified biochar, and the moisture content of the mixture of fresh pig manure and modified biochar is adjusted to 65% to obtain a semi-fluid mixture.

[0029] Step 4: Anaerobic fermentation of the mixed material: Add the mixed material to a 2L anaerobic fermentation tank, seal it, and perform anaerobic fermentation at 35°C for 30 days. During this period, the pH value and methane production are regularly monitored to maintain the pH between 6.5 and 7.5, and the methane production is 114ml / kg. After anaerobic fermentation, biogas residue and biogas liquid are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank consists of a glass tank body with a single-hole one-way vent valve at the bottle mouth of the glass tank body.

[0030] Step 5: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to determine the total absolute abundance of tetracycline resistance genes (tetA, tetM) and the absolute abundance of the sulfonamide resistance gene sul2 in the biogas residue and biogas slurry samples. The total absolute abundance of tetracycline resistance genes (tetA, tetM) in the biogas residue and biogas slurry samples was 7.32×10 11 copies / g, and the absolute abundance of the sulfonamide resistance gene sul2 was 6.89×10 10 copies / g.

[0031] Comparative Example 2 This comparative example comprises the following steps: Step 1: Add water to fresh pig manure with a moisture content of 50%-60% to dilute it, adjust the moisture content of the fresh pig manure to 65%, and obtain a semi-fluid material.

[0032] Step 2: Anaerobic fermentation of the material: Add the material to a 2L anaerobic fermentation tank, seal it, and perform anaerobic fermentation at 35°C for 30 days. During this period, the pH and methane production are regularly monitored to maintain the pH between 6.5 and 7.5, and the methane production is 98ml / kg. After the anaerobic fermentation is completed, the biogas residue and liquid biogas are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank consists of a glass tank body with a single-hole one-way vent valve at the bottle mouth.

[0033] Step 3: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to determine the total absolute abundance of tetracycline resistance genes (tetA, tetM) and the absolute abundance of the sulfonamide resistance gene sul2 in the biogas residue and biogas slurry samples. The total absolute abundance of tetracycline resistance genes (tetA, tetM) in the biogas residue and biogas slurry samples was 8.85×10 11 copies / g, and the absolute abundance of the sulfonamide resistance gene sul2 was 8.89×10 10 copies / g.

[0034] Compared with the total absolute abundance of tetracycline resistance genes (tetA, tetM) and the absolute abundance of sulfonamide resistance gene sul2 in the sludge and liquid biogas samples in Comparative Example 2, the total absolute abundance of tetracycline resistance genes (tetA, tetM) in the sludge and liquid biogas samples in Example 2 decreased by 21%, and the absolute abundance of sulfonamide resistance gene sul2 decreased by 29%.

[0035] Example 3 A method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid from anaerobic fermentation of pig manure using modified biochar comprises the following steps: Step 1: Preparation of raw biochar: Rice husks are used as the biomass feedstock. Nitrogen is first introduced into the pyrolysis furnace for 10 minutes to expel oxygen from the furnace. Pyrolysis is then carried out at 420°C for 2.5 hours in the absence of oxygen. After pyrolysis, the husks are sieved through an 80-mesh sieve and rinsed with deionized water. The husks are then oven-dried at 50-110°C to produce the raw biochar. To ensure an oxygen-free environment, the nitrogen flow rate is controlled at 1.5-2.0 L / min during the initial heating phase (within 10 minutes). After stabilization, the nitrogen flow rate can be controlled to 0.5-1.0 L / min.

[0036] Step 2: Modification of the original biochar. 500 g of the original biochar obtained in step 1 was evenly dispersed in 10 L of 1 mol / L citric acid solution and soaked for 3 hours. After taking it out, it was washed with deionized water and filtered with quantitative filter paper. Then, it was dried in an oven at 105°C to constant weight to obtain modified biochar.

[0037] Step 3: Mix the pig manure with the modified biochar. The modified biochar is dried, crushed, ground and sieved through an 80-mesh sieve in sequence. The particle size of the modified biochar obtained after sieving is about 1 mm. Then, 10 kg of fresh pig manure with a moisture content of 54% and 1 kg of sieved modified biochar are mixed evenly in a mass ratio of 10:1. Water is added to dilute the mixture of fresh pig manure and modified biochar, and the moisture content of the mixture of fresh pig manure and modified biochar is adjusted to 65% to obtain a semi-fluid mixture.

[0038] Step 4: Anaerobic fermentation of the mixed material. The mixed material is added to a 15L anaerobic fermentation tank for natural fermentation. The fermentation material reaches a maximum of 55°C on the fifth day of fermentation. Thereafter, the fermentation temperature is gradually reduced, and the fermentation temperature is maintained above 50°C for a total of 13 days, until the total number of days of anaerobic fermentation within the temperature range of 35-55°C is 25 days. During this period, the pH value and methane production are regularly monitored, and the pH value is maintained between 6.5-7.5. The methane production is 128ml / kg. After the anaerobic fermentation is completed, biogas residue and biogas liquid are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank includes a glass tank body, and a single-hole one-way air release valve is provided at the bottle mouth of the glass tank body.

[0039] Step 5: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to determine the abundance of the tetracycline resistance gene tetW and the sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples. The absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 9.32×10 9 copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 8.14×10 8 copies / g.

[0040] Comparative Example 3 This comparative example comprises the following steps: Step 1: Add water to fresh pig manure with a moisture content of 50%-60% to dilute it, adjust the moisture content of the fresh pig manure to 65%, and obtain a semi-fluid material.

[0041] Step 2: Anaerobic fermentation of the material. Add the material to a 2L anaerobic fermentation tank, seal it and ferment naturally. The fermented material reaches a maximum of 55°C on the fifth day of fermentation. Thereafter, the fermentation temperature is gradually reduced, and the fermentation temperature is maintained above 50°C for a total of 13 days, until the total number of days of anaerobic fermentation within the temperature range of 35-55°C is 25 days. During this period, the pH value and methane production are regularly monitored, and the pH value is maintained between 6.5-7.5. The methane production is 112ml / kg. After the anaerobic fermentation is completed, biogas residue and biogas liquid are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank includes a glass tank body, and a single-hole one-way air release valve is provided at the bottle mouth of the glass tank body.

[0042] Step 3: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to determine the absolute abundance of the tetracycline resistance gene tetW and the sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples. The absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 2.51×10 10 copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 7.63×10 10 copies / g.

[0043] Compared with the absolute abundance of the tetracycline resistance gene tetW and the absolute abundance of the sulfonamide resistance gene sul1 in the sludge and liquid samples in Comparative Example 3, the absolute abundance of the tetracycline resistance gene tetW in the sludge and liquid samples in Comparative Example 3 decreased by 2.69 times, and the absolute abundance of the sulfonamide resistance gene sul1 decreased by 93.73 times.

[0044] Example 4 A method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid from anaerobic fermentation of pig manure using modified biochar comprises the following steps: Step 1: Preparation of raw biochar: Rice husks are used as the biomass feedstock. Nitrogen is first introduced into the pyrolysis furnace for 10 minutes to expel oxygen from the furnace. Pyrolysis is then carried out at 500°C for 2 hours in the absence of oxygen. After pyrolysis, the husks are sieved through an 80-mesh sieve and rinsed with deionized water. The husks are then oven-dried at 50-110°C to produce the raw biochar. To ensure an oxygen-free environment, the nitrogen flow rate is controlled at 1.5-2.0 L / min during the initial heating phase (within 10 minutes). Once the flow rate stabilizes, the nitrogen flow rate can be adjusted to 0.5-1.0 L / min.

[0045] Step 2: Modification of the original biochar. 50 g of the original biochar obtained in step 1 was evenly dispersed in 500 ml of 1 mol / L citric acid solution and soaked for 3 hours. After taking it out, it was washed with deionized water and filtered with a quick filter paper. Then, it was dried in an oven at 105°C to constant weight to obtain modified biochar.

[0046] Step 3: Mix the pig manure with the modified biochar. The modified biochar is dried, crushed, ground and sieved through an 80-mesh sieve in sequence. The particle size of the modified biochar obtained after sieving is about 1 mm. Then, 1 kg of fresh pig manure with a moisture content of 55% and 100 g of the sieved modified biochar are mixed evenly in a mass ratio of 10:1. Water is added to dilute the mixture of fresh pig manure and modified biochar, and the moisture content of the mixture of fresh pig manure and modified biochar is adjusted to 75% to obtain a semi-fluid mixture.

[0047] Step 4: Anaerobic fermentation of the mixed material: Add the mixed material to a 2L anaerobic fermentation tank, seal it, and perform anaerobic fermentation at 31°C for 45 days. During this period, the pH value and methane production are regularly monitored to maintain the pH between 6.5 and 7.5 and the methane production at 132ml / kg. After anaerobic fermentation, biogas residue and biogas liquid are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank consists of a glass tank body with a single-hole one-way vent valve at the bottle mouth.

[0048] Step 5: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to measure the abundance of the tetracycline resistance gene tetW and the sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples. The absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 2.03×10 12 copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 2.65×10 13 copies / g.

[0049] Comparative Example 4 This comparative example comprises the following steps: Step 1: Add water to fresh pig manure with a moisture content of 75% to dilute it, adjust the moisture content of the fresh pig manure to 75%, and obtain a semi-fluid material.

[0050] Step 2: Anaerobic fermentation of the material: Add the material to a 2L anaerobic fermentation tank, seal it, and perform anaerobic fermentation at 31°C for 45 days. During this period, the pH value and methane production are regularly monitored to maintain the pH between 6.5-7.5 and the methane production at 109ml / kg. After anaerobic fermentation, biogas residue and biogas liquid are obtained. The anaerobic fermentation tank is a one-way exhaust tank. The one-way exhaust tank consists of a glass tank body with a single-hole one-way vent valve at the bottle mouth of the glass tank body.

[0051] Step 3: Collect biogas residue and biogas slurry samples and use real-time fluorescence quantitative PCR to determine the absolute abundance of the tetracycline resistance gene tetW and the sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples. The absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 1.86×10 12copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 9.56×10 12 copies / g.

[0052] Compared with the absolute abundance of the tetracycline resistance gene tetW and the absolute abundance of the sulfonamide resistance gene sul1 in the sludge and liquid samples in Comparative Example 4, the absolute abundance of the tetracycline resistance gene tetW in the sludge and liquid samples in Comparative Example 4 increased by 8.37%, and the absolute abundance of the sulfonamide resistance gene sul1 increased by 63.92%.

[0053] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid from anaerobic fermentation of pig manure using modified biochar, characterized in that: The steps include: Step 1: Preparation of raw biochar: pyrolysis of biomass raw materials under anaerobic conditions, followed by screening, washing and drying to obtain raw biochar; Step 2: modification of the original biochar, chemically modifying the original biochar obtained in step 1 using a citric acid solution to obtain modified biochar; Step 3: The modified biochar is dried, crushed, ground, and sieved in sequence, and then the pig manure and the sieved modified biochar are evenly mixed in a mass ratio of 9-11:

1. The moisture content of the mixture of pig manure and modified biochar is adjusted to 65%-75% to obtain a semi-fluid mixture. Step 4: Anaerobic fermentation of the mixed material, controlling the temperature of the anaerobic fermentation at 31°C-55°C, and controlling the time of the anaerobic fermentation at 25-45 days.

2. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 1, characterized in that: In step 1, the biomass raw material is corn stalks and / or rice husks.

3. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 1, characterized in that: In step 1, the high-temperature pyrolysis temperature is 400-650° C., the high-temperature pyrolysis time is 1-2.5 hours, the sieving mesh size is 80 mesh, and the drying temperature is 50-110° C.

4. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 1, characterized in that: In step 2, the concentration of the citric acid solution is 1-3 mol / L.

5. A method for reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 1 or 4, characterized in that: In step 2, the chemical modification method is: the original biochar is evenly dispersed and soaked in a citric acid solution.

6. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 5, characterized in that: The soaking time is 2-5 hours.

7. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 5, characterized in that: The mass ratio of original biochar to citric acid is 1:1-4.

8. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 5, characterized in that: In step 3, the particle size of the modified biochar after screening is 0.9-1.5 mm.

9. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 1, characterized in that: In step 4, anaerobic fermentation is carried out in a one-way exhaust tank.

10. The method of reducing the abundance of antibiotic resistance genes in biogas residue and liquid biogas from anaerobic fermentation of pig manure using modified biochar according to claim 1, characterized in that: Antibiotic resistance genes include tetracycline resistance genes and / or sulfonamide resistance genes.