A method for reducing the abundance of antibiotic resistance genes in biogas slurry of anaerobic fermentation of pig manure by using modified biochar

Abstract

The present application relates to the technical field of biochar preparation and application, and particularly relates to a method for reducing the abundance of antibiotic resistance genes in pig manure anaerobic fermentation biogas residue and biogas slurry by using modified biochar, which comprises the following steps: step one: biomass raw materials are pyrolyzed under anaerobic conditions, and then are screened, washed with water and dried in sequence to obtain original biochar; step two: the original biochar is chemically modified by using a citric acid solution to obtain modified biochar; step three: the modified biochar is dried, crushed, ground and screened in sequence, and then pig manure and the screened modified biochar are uniformly mixed at a mass ratio of 9-11:1, the water content of the mixture of pig manure and modified biochar is adjusted to 65%-75%, and a semi-liquid mixture is obtained; and step four: the mixture is subjected to anaerobic fermentation. The modified biochar by using a citric acid solution is mixed with pig manure and subjected to anaerobic fermentation, and the abundance of antibiotic resistance genes in the pig manure is significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of biochar preparation and application technology, specifically to a method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar. Background Technology

[0002] In recent years, with the increasing application of antibiotics in various industries such as pharmaceuticals, animal husbandry, and aquaculture, the problem of antibiotic abuse has become increasingly serious. In the animal husbandry industry, antibiotic abuse not only leads to high concentrations of antibiotic residues in livestock manure but also induces the production of antibiotic resistance genes (ARGs) in livestock. Antibiotic resistance genes are a class of genes that enable bacteria to resist antibiotics. These genes typically encode specific proteins or enzymes that allow bacteria to survive and reproduce in the presence of antibiotics through various mechanisms. Antibiotic resistance genes can persist, spread, and diffuse 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 are not fully absorbed by livestock are excreted in feces, resulting in a high abundance of antibiotic resistance genes in livestock manure, with pig manure showing a higher abundance than chicken and cow manure.

[0003] Chinese patent CN116462181A discloses a potassium dihydrogen phosphate modified biochar material, its preparation method, and its application. The method involves placing rice husks in a tube furnace, introducing N2, and heating to 500-600℃ for carbonization. The process is then changed to introducing CO2 to obtain rice husk-based biochar. This biochar is then placed in a potassium dihydrogen phosphate solution, stirred, allowed to stand, filtered, and centrifuged. Finally, it is washed with water until the solution pH is close to neutral and then dried to obtain the potassium dihydrogen phosphate modified biochar. This patent uses potassium dihydrogen phosphate modified biochar material as an additive to substrates with a total solids content of 7%-9% to reduce the abundance of resistance genes in pig manure, resulting in low treatment efficiency and high energy consumption. Summary of the Invention

[0004] To address the problems of low efficiency and high energy consumption in existing methods for reducing the abundance of antibiotic resistance genes in pig manure, this invention provides a method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar. The method involves mixing biochar modified with citric acid solution with pig manure and carrying out anaerobic fermentation, which significantly reduces the abundance of antibiotic resistance genes in pig manure.

[0005] The technical solution of this invention is as follows:

[0006] A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar includes the following steps:

[0007] Step 1: Preparation of raw biochar. Biomass raw materials are pyrolyzed at high temperature under anaerobic conditions, and then sieved, washed and dried in sequence to obtain raw biochar.

[0008] Step 2: Modification of raw biochar. The raw biochar obtained in Step 1 is chemically modified using citric acid solution to obtain modified biochar.

[0009] Step 3: The modified biochar is dried, crushed, ground and sieved in sequence. Then, the pig manure and the sieved modified biochar are mixed evenly at 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 preferred mass ratio of pig manure to modified biochar is 10:1.

[0010] Step 4: Anaerobic fermentation of the mixed materials. The temperature of anaerobic fermentation is controlled at 31℃-55℃, and the time of anaerobic fermentation is controlled at 25-45 days.

[0011] Furthermore, in step one, the biomass raw materials are corn stalks and / or rice husks. Corn stalks are widely available, while rice husks are a byproduct of rice processing. This invention uses corn stalks and / or rice husks as biomass raw materials, which helps to reduce raw material costs.

[0012] Furthermore, in step one, the high-temperature pyrolysis temperature is 400-650℃, preferably 420-500℃, the pyrolysis time is 1-2.5 hours, the sieve mesh size is 80 mesh, and the drying temperature is 50-110℃. High-temperature pyrolysis not only enables the original biochar to form a high specific surface area, a multi-level porous structure, and a highly stable aromatic structure, but also destroys residual antibiotics, pathogens, and weed seeds in the original biomass, reducing the risk of secondary pollution.

[0013] Furthermore, in step two, the concentration of the citric acid solution is 1-3 mol / L, preferably 1 mol / L. Modifying the original biochar with citric acid solution adds organic groups such as carbonyl, carboxyl, and hydroxyl groups, while simultaneously causing the biochar to form more micropores and mesopores, significantly increasing the specific surface area and providing numerous adsorption sites for resistance genes and their host microorganisms, making them easier for the biochar to capture. 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 antibiotic content is very beneficial to the reduction of resistance gene levels. Because citric acid solution is environmentally friendly, using it to modify the original biochar avoids the risk of secondary pollution.

[0014] Furthermore, in step two, the chemical modification method involves soaking the raw biochar in a citric acid solution while it is uniformly dispersed. The dispersed state of the raw biochar in the citric acid solution allows for sufficient contact with the solution, improving the modification efficiency.

[0015] Furthermore, the soaking time is 2-5 hours, preferably 3 hours.

[0016] Furthermore, the mass ratio of raw biochar to citric acid is 1:1-4. When 50g of raw biochar is soaked in a 1mol / L citric acid solution, the amount of citric acid solution used is preferably 500-1000ml.

[0017] Furthermore, in step three, the particle size of the modified biochar after sieving is 0.9-1.5 mm, preferably 1 mm.

[0018] Furthermore, in step four, anaerobic fermentation is carried out in a one-way exhaust tank. The one-way exhaust tank consists of a glass jar with a single-hole one-way vent valve at the mouth. The one-way exhaust tank balances the need for airtightness in the anaerobic fermentation environment with the need for gas release, playing a dual role in preventing oxygen from entering and removing metabolic gases produced during anaerobic fermentation. This provides a stable anaerobic environment for anaerobic microorganisms, ensuring that the anaerobic fermentation process is efficient and safe.

[0019] Furthermore, antibiotic resistance genes include tetracycline resistance genes and / or sulfonamide resistance genes. The tetracycline resistance gene can be one or more of tetW, tetA, and tetM, and the sulfonamide resistance gene can be at least one of sul1 and sul2.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention provides a method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar. The method involves chemically modifying the original biochar with citric acid solution, introducing functional groups such as carbonyl, carboxyl, and hydroxyl groups. This enhances the biochar's specific adsorption capacity for residual tetracycline and / or sulfonamide antibiotics in pig manure, reducing the selective pressure of tetracycline and / or sulfonamide antibiotics on host bacteria carrying antibiotic resistance genes. This inhibits the accumulation of resistance genes at the source, thus reducing the abundance of tetracycline and / or sulfonamide resistance genes in pig manure. Using citric acid solution as a modifier for the original biochar also increases biogas yield while avoiding the risk of secondary pollution, achieving synergistic resource utilization. Controlling the anaerobic fermentation temperature at 35-55℃ helps inhibit the activity of pathogens carrying tetracycline and / or sulfonamide resistance genes, further reducing the abundance of antibiotic resistance genes. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0023] Example 1

[0024] A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar includes the following steps:

[0025] Step 1: Preparation of raw biochar. Corn stalks were selected as the biomass raw material. Nitrogen gas was first introduced into the pyrolysis furnace for 10 minutes to expel the oxygen. High-temperature pyrolysis was then carried out at 500℃ under anaerobic conditions for 2 hours. After pyrolysis, the biochar was passed through an 80-mesh sieve and rinsed with deionized water. It was then dried in an oven at 50-110℃ to obtain raw biochar. To ensure an anaerobic environment, the nitrogen flow rate was controlled at 1.5-2.0 L / min during the initial heating stage (within 10 minutes). After stabilization, the nitrogen flow rate could be controlled at 0.5-1.0 L / min.

[0026] Step 2: Modification of raw biochar. 50g of raw biochar obtained in Step 1 was uniformly dispersed in 500ml of 1mol / L citric acid solution and soaked for 3 hours. After being taken out, it was washed with deionized water and filtered with quantitative filter paper. Then it was dried in an oven at 105℃ to constant weight to obtain modified biochar.

[0027] Step 3: Mixing pig manure and modified biochar. The modified biochar is dried, crushed, ground, and sieved through an 80-mesh sieve. The particle size of the modified biochar obtained after sieving is about 1 mm. Then, fresh pig manure with a moisture content of 54% is mixed with the sieved modified biochar at a mass ratio of 10:1. Water is added to the mixture of fresh pig manure and modified biochar to dilute it and adjust the moisture content of the mixture to 65%, resulting in a semi-fluid mixture.

[0028] Step 4: Anaerobic fermentation of the mixture. Add the mixture to a 2L anaerobic fermenter, seal it, and incubate at 35℃ for 30 days. During this period, regularly monitor the pH and methane production, maintaining the pH between 6.5 and 7.5, and ensuring a methane production of 132 ml / kg. After anaerobic fermentation, biogas residue and biogas slurry are obtained. The anaerobic fermenter is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way vent valve at the opening.

[0029] Step 5: Collect biogas residue and biogas slurry samples, and use real-time 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 results showed that the absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 1.89 × 10¹² copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 9.34 × 10¹² copies / g.

[0030] Comparative Example 1

[0031] This comparative example includes the following steps:

[0032] Step 1: Dilute fresh pig manure with a moisture content of 50%-60% by adding water to adjust the moisture content of the fresh pig manure to 65%, thus obtaining a semi-fluid material.

[0033] Step Two: Anaerobic Fermentation of the Material. Add 1 kg of material (adjusted for moisture content) to a 2 L anaerobic fermenter, seal it, and incubate at 35℃ for 30 days. During this period, regularly monitor the pH and methane production, maintaining the pH between 6.5 and 7.5 and the methane production at 107 ml. After anaerobic fermentation, biogas residue and biogas slurry are obtained. The anaerobic fermenter is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way vent valve at the opening.

[0034] Step 3: Collect biogas residue and biogas slurry samples, and use real-time 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 results showed that the absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 3.21 × 10¹² copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 12.63 × 10¹² copies / g.

[0035] Compared with the absolute abundance of tetracycline resistance gene tetW and sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples in Comparative Example 1, the absolute abundance of tetracycline resistance gene tetW and sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples in Example 1 decreased by 17% and decreased by 35%.

[0036] Example 2

[0037] A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar includes the following steps:

[0038] Step 1: Preparation of raw biochar. Rice husks are selected as the biomass raw material. Nitrogen gas is first introduced into the pyrolysis furnace for 10 minutes to expel the oxygen. High-temperature pyrolysis is carried out at 420℃ under anaerobic conditions for 2.5 hours. After high-temperature pyrolysis, the biochar is passed through an 80-mesh sieve and rinsed with deionized water. Then, it is dried in an oven at 50-110℃ to obtain raw biochar. To ensure the anaerobic effect, the nitrogen flow rate is controlled at 1.5-2.0 L / min during the initial heating stage (within 10 minutes). After stabilization, the nitrogen flow rate can be controlled at 0.5-1.0 L / min.

[0039] Step 2: Modification of raw biochar. 50g of raw biochar obtained in Step 1 was uniformly dispersed in 1000ml of 1mol / L citric acid solution and soaked for 3 hours. After being taken out, it was washed with deionized water and filtered with quantitative filter paper. Then it was dried in an oven at 105℃ to constant weight to obtain modified biochar.

[0040] Step 3: Mixing pig manure and modified biochar. The modified biochar is dried, crushed, ground, and sieved through an 80-mesh sieve. The particle size of the modified biochar obtained after sieving is about 1 mm. Then, fresh pig manure with a moisture content of 54% is mixed with the sieved modified biochar at a mass ratio of 10:1. Water is added to the mixture of fresh pig manure and modified biochar to dilute it and adjust the moisture content of the mixture to 65%, resulting in a semi-fluid mixture.

[0041] Step 4: Anaerobic fermentation of the mixture. Add the mixture to a 2L anaerobic fermenter, seal it, and incubate at 35℃ for 30 days. During this period, regularly monitor the pH and methane production, maintaining the pH between 6.5 and 7.5, and ensuring a methane production of 114 ml / kg. After anaerobic fermentation, biogas residue and biogas slurry are obtained. The anaerobic fermenter is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way vent valve at the opening.

[0042] Step 5: Collect biogas residue and biogas slurry samples, and use real-time quantitative PCR to determine the total absolute abundance of tetracycline resistance genes (tetA, tetM) and the absolute abundance of sulfonamide resistance gene sul2 in the biogas residue and biogas slurry samples. The results showed that the total absolute abundance of tetracycline resistance genes (tetA, tetM) in the biogas residue and biogas slurry samples was 7.32 × 10¹¹ copies / g, and the absolute abundance of sulfonamide resistance gene sul2 was 6.89 × 10¹⁰ copies / g.

[0043] Comparative Example 2

[0044] This comparative example includes the following steps:

[0045] Step 1: Dilute fresh pig manure with a moisture content of 50%-60% by adding water to adjust the moisture content of the fresh pig manure to 65%, thus obtaining a semi-fluid material.

[0046] Step Two: Anaerobic Fermentation of the Material. The material is added to a 2L anaerobic fermenter, sealed, and anaerobic fermented at 35℃ for 30 days. During this period, the pH value and methane production are monitored regularly, maintaining the pH value between 6.5 and 7.5, and the methane production at 98ml / kg. After anaerobic fermentation, biogas residue and biogas slurry are obtained. The anaerobic fermenter is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way venting valve at the opening.

[0047] Step 3: Collect biogas residue and biogas slurry samples, and use real-time quantitative PCR to determine the total absolute abundance of tetracycline resistance genes (tetA, tetM) and the absolute abundance of sulfonamide resistance gene sul2 in the biogas residue and biogas slurry samples. The results showed that the total absolute abundance of tetracycline resistance genes (tetA, tetM) in the biogas residue and biogas slurry samples was 8.85 × 10¹¹ copies / g, and the absolute abundance of sulfonamide resistance gene sul2 was 8.89 × 10¹⁰ copies / g.

[0048] Compared with the total absolute abundance of tetracycline resistance genes (tetA, tetM) and the absolute abundance of sulfonamide resistance gene sul2 in the biogas residue and biogas slurry samples in Comparative Example 2, the total absolute abundance of tetracycline resistance genes (tetA, tetM) and the absolute abundance of sulfonamide resistance gene sul2 in the biogas residue and biogas slurry samples in Example 2 decreased by 21% and 29% respectively.

[0049] Example 3

[0050] A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar includes the following steps:

[0051] Step 1: Preparation of raw biochar. Rice husks are selected as the biomass raw material. Nitrogen gas is first introduced into the pyrolysis furnace for 10 minutes to expel the oxygen. High-temperature pyrolysis is carried out at 420℃ under anaerobic conditions for 2.5 hours. After high-temperature pyrolysis, the biochar is passed through an 80-mesh sieve and rinsed with deionized water. Then, it is dried in an oven at 50-110℃ to obtain raw biochar. To ensure the anaerobic effect, the nitrogen flow rate is controlled at 1.5-2.0 L / min during the initial heating stage (within 10 minutes). After stabilization, the nitrogen flow rate can be controlled at 0.5-1.0 L / min.

[0052] Step 2: Modification of raw biochar. 500g of raw biochar obtained in Step 1 was uniformly dispersed in 10L of 1mol / L citric acid solution and soaked for 3 hours. After being taken out, it was washed with deionized water and filtered with quantitative filter paper. Then it was dried in an oven at 105℃ to constant weight to obtain modified biochar.

[0053] Step 3: Mixing pig manure and modified biochar. The modified biochar is dried, crushed, ground, and sieved through an 80-mesh sieve. 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 at a mass ratio of 10:1. Water is added to the mixture of fresh pig manure and modified biochar to dilute it and adjust the moisture content of the mixture to 65%, resulting in a semi-fluid mixture.

[0054] Step Four: Anaerobic Fermentation of the Mixed Materials. The mixed materials are added to a 15L anaerobic fermentation tank for natural fermentation. The fermentation temperature reaches a maximum of 55℃ on the fifth day. Thereafter, the fermentation temperature is gradually reduced, maintaining a temperature above 50℃ for 13 days, until the total anaerobic fermentation time is 25 days within the temperature range of 35-55℃. During this period, pH and methane production are monitored regularly, maintaining the pH between 6.5 and 7.5 and the methane production at 128 ml / kg. After anaerobic fermentation is complete, biogas residue and biogas slurry are obtained. The anaerobic fermentation tank is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way venting valve at the opening.

[0055] Step 5: Collect biogas residue and biogas slurry samples, and use real-time 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 results showed that the absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 9.32 × 10⁹ copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 8.14 × 10⁸ copies / g.

[0056] Comparative Example 3

[0057] This comparative example includes the following steps:

[0058] Step 1: Dilute fresh pig manure with a moisture content of 50%-60% by adding water to adjust the moisture content of the fresh pig manure to 65%, thus obtaining a semi-fluid material.

[0059] Step Two: Anaerobic Fermentation of the Material. The material is added to a 2L anaerobic fermentation tank, sealed, and allowed to ferment naturally. The fermentation temperature reaches a maximum of 55℃ on the fifth day. Thereafter, the fermentation temperature is gradually reduced, maintaining a temperature above 50℃ for 13 days, until the total anaerobic fermentation time is 25 days within the temperature range of 35-55℃. During this period, pH and methane production are monitored regularly, maintaining the pH between 6.5 and 7.5 and the methane production at 112 ml / kg. After anaerobic fermentation is complete, biogas residue and biogas slurry are obtained. The anaerobic fermentation tank is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way vent valve at the opening.

[0060] Step 3: Collect biogas residue and biogas slurry samples, and use real-time 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 results showed that the absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 2.51 × 10¹⁰ copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 7.63 × 10¹⁰ copies / g.

[0061] Compared with the absolute abundance of tetracycline resistance gene tetW and sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples in Comparative Example 3, the absolute abundance of tetracycline resistance gene tetW and sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples in Example 3 decreased by 2.69 times and the absolute abundance of sulfonamide resistance gene sul1 decreased by 93.73 times.

[0062] Example 4

[0063] A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar includes the following steps:

[0064] Step 1: Preparation of raw biochar. Rice husks are selected as the biomass raw material. Nitrogen gas is first introduced into the pyrolysis furnace for 10 minutes to expel the oxygen in the furnace. High-temperature pyrolysis is carried out at 500℃ under anaerobic conditions for 2 hours. After high-temperature pyrolysis, the biochar is passed through an 80-mesh sieve and rinsed with deionized water. Then, it is dried in an oven at 50-110℃ to obtain raw biochar. To ensure the anaerobic effect, the nitrogen flow rate is controlled at 1.5-2.0 L / min during the initial heating stage (within 10 minutes). After stabilization, the nitrogen flow rate can be controlled at 0.5-1.0 L / min.

[0065] Step 2: Modification of raw biochar. 50g of raw biochar obtained in Step 1 was uniformly dispersed in 500ml of 1mol / L citric acid solution and soaked for 3 hours. After being taken out, it was washed with deionized water and filtered through rapid filter paper. Then it was dried in an oven at 105℃ to constant weight to obtain modified biochar.

[0066] Step 3: Mixing pig manure and modified biochar. The modified biochar is dried, crushed, ground, and sieved through an 80-mesh sieve. 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 sieved modified biochar are mixed evenly at a mass ratio of 10:1. Water is added to the mixture of fresh pig manure and modified biochar to dilute it and adjust the moisture content of the mixture to 75%, resulting in a semi-fluid mixture.

[0067] Step 4: Anaerobic fermentation of the mixture. Add the mixture to a 2L anaerobic fermenter, seal it, and incubate at 31℃ for 45 days. During this period, regularly monitor the pH and methane production, maintaining the pH between 6.5 and 7.5 and the methane production at 132 ml / kg. After anaerobic fermentation, biogas residue and biogas slurry are obtained. The anaerobic fermenter is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way vent valve at the opening.

[0068] Step 5: Collect biogas residue and biogas slurry samples, and use real-time 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 results showed that the absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 2.03 × 10¹² copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 2.65 × 10¹³ copies / g.

[0069] Comparative Example 4

[0070] This comparative example includes the following steps:

[0071] Step 1: Dilute fresh pig manure with water to a moisture content of 75% to obtain a semi-fluid material.

[0072] Step Two: Anaerobic Fermentation of the Material. The material is added to a 2L anaerobic fermenter, sealed, and anaerobic fermented at 31℃ for 45 days. During this period, pH and methane production are monitored regularly, maintaining the pH between 6.5 and 7.5 and the methane production at 109 ml / kg. After anaerobic fermentation, biogas residue and biogas slurry are obtained. The anaerobic fermenter is a one-way venting tank. The one-way venting tank consists of a glass container with a single-hole one-way vent valve at the opening.

[0073] Step 3: Collect biogas residue and biogas slurry samples, and use real-time 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 results showed that the absolute abundance of the tetracycline resistance gene tetW in the biogas residue and biogas slurry samples was 1.86 × 10¹² copies / g, and the absolute abundance of the sulfonamide resistance gene sul1 was 9.56 × 10¹² copies / g.

[0074] Compared with the absolute abundance of tetracycline resistance gene tetW and sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples in Comparative Example 4, the absolute abundance of tetracycline resistance gene tetW and sulfonamide resistance gene sul1 in the biogas residue and biogas slurry samples in Example 4 increased by 8.37% and increased by 63.92%.

[0075] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar, characterized in that, Includes the following steps: Step 1: Preparation of raw biochar. Biomass raw materials are pyrolyzed at high temperature under anaerobic conditions, and then sieved, washed and dried in sequence to obtain raw biochar. Step 2: Modification of raw biochar. The raw biochar obtained in Step 1 is chemically modified using citric acid solution to obtain modified biochar. Step 3: The modified biochar is dried, crushed, ground and sieved in sequence. Then, the pig manure and the sieved modified biochar are mixed evenly at 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 materials. The temperature of anaerobic fermentation is controlled at 35℃-55℃, and the time of anaerobic fermentation is controlled at 25-45 days.

2. The method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 1, characterized in that, In step one, the biomass raw materials are corn stalks and / or rice husks.

3. The method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 1, characterized in that, In step one, the high-temperature pyrolysis temperature is 400-650℃, the high-temperature pyrolysis time is 1-2.5 hours, the sieve mesh size is 80 mesh, and the drying temperature is 50-110℃.

4. The method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 1, characterized in that, In step two, the concentration of the citric acid solution is 1-3 mol / L.

5. A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 1 or 4, characterized in that, In step two, the chemical modification method is to soak the raw biochar in a citric acid solution while it is uniformly dispersed.

6. The method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 5, characterized in that, Soaking time is 2-5 hours.

7. The method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 5, characterized in that, The mass ratio of raw biochar to citric acid is 1:1-4.

8. A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 5, characterized in that, In step three, the particle size of the modified biochar after sieving is 0.9-1.5 mm.

9. A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 1, characterized in that, In step four, anaerobic fermentation is carried out in a one-way exhaust tank.

10. A method for reducing the abundance of antibiotic resistance genes in anaerobic fermentation residue and slurry of pig manure using modified biochar as described in claim 1, characterized in that, Antibiotic resistance genes include tetracycline resistance genes and / or sulfonamide resistance genes.