Composting method and device for reducing abundance of antibiotic resistance genes in livestock and poultry manure

Abstract

The invention relates to the technical field of organic solid waste treatment, and discloses a composting method and device for reducing abundance of antibiotic resistance genes in livestock and poultry manure. The composting method comprises the following steps: mixing livestock and poultry manure with a composting auxiliary material to form a composting material, and then mixing the composting material with an extreme thermophilic microbial agent to obtain a composting raw material; sequentially carrying out ultrahigh-temperature aerobic composting treatment and low-voltage pulsed electric field composting treatment on the composting raw materials to reduce the abundance of antibiotic resistance genes; wherein the extreme thermophilic bacteria agent comprises at least one of brevibacillus brevis, pseudomonas, bacillus subtilis, saccharomonosporum and acinetobacter. According to the composting method provided by the invention, ultrahigh-temperature composting and low-voltage pulsed electric field treatment are innovatively coupled, extremely high removal efficiency is shown on various ARGs such as tetracyclines, sulfonamides and macrolides, the average removal rate can reach 97% or above, and the effect is far better than that of pure ultrahigh-temperature composting or traditional composting.

Description

Technical Field

[0001] This invention relates to the field of organic solid waste treatment technology, and in particular to a composting method and apparatus for reducing the abundance of antibiotic resistance genes in livestock and poultry manure. Background Technology

[0002] Antibiotic resistance genes (ARGs), as an emerging environmental pollutant, accumulate in large quantities in livestock and poultry manure during large-scale livestock and poultry farming due to the widespread use of veterinary antibiotics. These ARGs pose a serious threat to human health and ecological balance through the spread of environmental microorganisms. While traditional composting techniques can reduce ARG abundance to some extent during the high-temperature stage, they have significant shortcomings in practical application, leading to frequent ARG rebound during the composting period and limiting the safety and resource utilization of compost products. First, traditional composting processes struggle to maintain ultra-high temperature (>70℃) environments, where most heat-resistant ARG host bacteria can still survive, resulting in incomplete inactivation of ARGs. Second, during the composting maturation period, microbial activity gradually recovers, and some ARG host bacteria re-proliferate through horizontal gene transfer (HGT), further exacerbating the spread and residue of ARGs. Furthermore, traditional composting methods cannot effectively disrupt host cell membranes, preventing the full release and degradation of intracellular ARGs, thus posing environmental risks during subsequent utilization.

[0003] In existing technologies, although some studies have attempted to improve temperature or degradation efficiency by adding exogenous microbial agents or optimizing composting conditions, these methods are often difficult to promote on a large scale due to their complexity, high cost, susceptibility to secondary pollution, limited technical approaches, and limited adaptability. For example, CN 108033817 A proposes a method for rapidly reducing antibiotics and resistance genes (ARGs) in organic solid waste by using ultra-high temperature aerobic fermentation (temperature ≥80℃) combined with specific extreme thermophilic microbial agents. This method effectively reduces the abundance of some ARGs and alters the microbial community structure of compost by maintaining a high-temperature environment of at least 80℃ for at least 5-7 days, thus inhibiting the spread of ARGs to some extent. However, the entire ARGs reduction process in this scheme relies entirely on a single "ultra-high temperature microbial agent-high temperature fermentation" model. This model is highly dependent on the properties of the compost raw materials (such as conductivity, moisture content fluctuations, etc.), environmental conditions, and microbial agent activity. Under complex and variable actual production conditions, its stability and universality are affected.

[0004] Therefore, there is an urgent need to develop an efficient, stable and environmentally friendly composting method that can significantly reduce the abundance of ARGs and effectively suppress their rebound throughout the entire process, thereby achieving the safe treatment and resource utilization of livestock and poultry manure. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure.

[0006] The second objective of this invention is to provide a composting device.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, comprising the following steps: Animal manure is mixed with composting additives to form compost material, which is then mixed with an extreme thermophilic bacteria agent to obtain compost raw material. The compost raw material is then subjected to ultra-high temperature aerobic composting treatment and low-voltage pulsed electric field composting treatment in sequence to reduce the abundance of antibiotic resistance genes. The extreme thermophilic bacteria agent includes at least one of Bacillus breviculatus, Pseudomonas aeruginosa, Bacillus subtilis, sucralospora, and Acinetobacter bacillus.

[0008] In some embodiments of the present invention, the mass ratio of the livestock and poultry manure to the composting additive is 10:(3-5); the bacterial content of the extreme thermophilic bacteria agent is 2×10⁻⁶. 10 cfu / g-2×10 12 cfu / g; the amount of the extreme thermophilic microbial agent is 0.08%-0.12% of the mass of the compost material.

[0009] In some preferred embodiments of the present invention, the mass ratio of the livestock and poultry manure to the composting additive is 10:(3-5); the bacterial content of the extreme thermophilic bacteria agent is 2×10⁻⁶. 10 cfu / g-2×10 12 cfu / g; the amount of the extreme thermophilic microbial agent is 0.09%-0.11% of the mass of the compost material.

[0010] In some embodiments of the present invention, the extreme thermophilic bacterial agent is a compound bacterial agent formed by mixing Bacillus brevis, Pseudomonas, Bacillus subtilis, Saccharomyces cerevisiae, and Acinetobacterium sp. GSS19 with accession number GDMCC No. 62979 in a mass ratio of (3-5): (1.5-2.5): (1.5-2.5): (0.8-1.2): 1.

[0011] In some preferred embodiments of the present invention, the extreme thermophilic bacterial agent is a compound bacterial agent formed by mixing Bacillus brevis, Pseudomonas, Bacillus subtilis, Saccharomyces cerevisiae, and Acinetobacterium sp. GSS19 with accession number GDMCC No. 62979 in a mass ratio of (3.5-4.5): (1.8-2.2): (1.8-2.2): (0.9-1.1): 1, wherein the Acinetobacterium sp. GSS19 is deposited at the Guangdong Provincial Microbial Culture Collection Center.

[0012] In some embodiments of the present invention, the livestock and poultry manure includes at least one of pigeon manure, pig manure, cow manure, chicken manure, and duck manure.

[0013] In some embodiments of the present invention, the composting additives include at least one of straw, rice husks, sawdust, and corn cobs.

[0014] In some embodiments of the present invention, the physicochemical parameters of the composting raw material include at least one of the following: pH=6.0-8.0; moisture content 60%-70%; carbon-nitrogen ratio of (25-30):1.

[0015] In some embodiments of the present invention, the ultra-high temperature aerobic composting treatment includes controlling the compost temperature to be no lower than 80°C for fermentation for 10-15 days; during this period, intermittent aeration is used, once every 0.8-1.2 hours, for 10-15 minutes each time, with an aeration volume of 0.01-0.1 m³. 3 / (min·kg compost material), maintain the moisture content of the compost pile at 60%-65%, and turn the pile over every 2-3 days.

[0016] In some preferred embodiments of the present invention, the ultra-high temperature aerobic composting treatment includes controlling the compost temperature to be no lower than 80°C for fermentation for 13-15 days; during this period, intermittent aeration is used, once every 0.9-1.1 hours, for 10-15 minutes each time, with an aeration volume of 0.01-0.1 m³. 3 / (min·kg compost material), maintain the moisture content of the compost pile at 62%-65%, and turn the pile over every 2-3 days.

[0017] In some embodiments of the present invention, the working voltage of the low-voltage pulse electric field composting treatment is 5-36V, the pulse frequency is 50-100Hz, and the pulse width is 10ms-20ms; an intermittent on-time mode is adopted, with the number of on-times being 1-5 times per hour, the single treatment duration being 2-10 minutes, and the total treatment time being 15-25 days.

[0018] In some preferred embodiments of the present invention, the working voltage of the low-voltage pulse electric field composting treatment is 10-36V, the pulse frequency is 50-100Hz, and the pulse width is 10ms-20ms; an intermittent on-time mode is adopted, with the number of on-times being 2-3 times per hour, the single treatment duration being 3-10 minutes, and the total treatment time being 15-20 days.

[0019] In some embodiments of the present invention, the antibiotic resistance gene includes at least one of tetB, tetL, tetM, tetO, tetQ, tetW, sulⅠ, sulⅡ, ermB, ermF, mphA, mphB, aadA1, aacC2, strA, strB, qnrA, qnrS, blaTEM, and blaOXA-1.

[0020] The basic principles of this invention are explained as follows: The composting method for reducing the abundance of antibiotic resistance genes (ARGs) in livestock and poultry manure provided by this invention is carried out in two stages. Through these two stages, which are connected and have different mechanisms of action, a comprehensive and non-rebounding deep reduction of antibiotic resistance genes (ARGs) in livestock and poultry manure is achieved. Specifically: The first stage involves adding specific extreme thermophilic bacteria to the composting raw materials. By utilizing their vigorous metabolic activity, the temperature of the compost pile is rapidly increased and maintained at an ultra-high temperature environment of 80-90℃ or even higher. The high temperature directly kills most of the heat-intolerant ARGs host microorganisms. By changing the entire microbial community structure, the risk of ARGs spreading through horizontal gene transfer is significantly reduced, thereby achieving the first significant decrease in ARGs abundance in the early stage of composting. The second stage: After ultra-high temperature treatment, the compost is transferred to a low-pressure pulsed electric field environment. The electric pulses generated by the electric field produce an "electroporation" effect on the microbial cell membrane and may induce electrochemical reactions to produce reactive oxygen species. Electropenetration can effectively destroy the cell membranes of the host bacteria that survived the ultra-high temperature stage (especially heat-resistant ones), causing their intracellular ARGs to be released. Reactive oxygen species and other substances can then directly oxidize and degrade these exposed ARGs. This stage is specifically designed for residual host bacteria and intracellular ARGs, solving the problem of ARG rebound during the maturation period in traditional composting.

[0021] A second aspect of the present invention provides a composting apparatus for the composting method described in the first aspect of the present invention, comprising: An ultra-high temperature composting device includes an insulated tank, which is equipped with a variable speed spiral agitator and an aeration system, and the tank is equipped with a temperature monitoring and control system. A low-voltage pulsed electric field composting device includes an insulated reactor and a low-voltage pulsed electric field generator. The reactor is equipped with multiple pairs of paired electrodes, which are connected to the low-voltage pulsed electric field generator. A conveying device is used to transport the compost pile processed by the ultra-high temperature composting device to the low-voltage pulse electric field composting device.

[0022] In some embodiments of the present invention, the insulated tank has a cylindrical structure.

[0023] Specifically, compared to rectangular tanks, cylindrical tanks have a smaller surface area to volume ratio, which can effectively reduce heat loss and better maintain temperature during the rising temperature of composting. In addition, the mechanical properties of the cylindrical structure are superior, which can withstand the internal pressure changes generated during composting, prevent tank deformation, ensure the airtightness of the device, and reduce the risk of heat leakage.

[0024] In some embodiments of the present invention, the insulated tank body is a double-layer jacketed structure, including an inner shell, an outer shell, and an insulation material layer filled between the two.

[0025] In some embodiments of the present invention, the thermal insulation material includes nano-aerogel felt and polyurethane foam.

[0026] Specifically, the insulated tank with a double-layer jacket structure can greatly reduce the heat transfer to the outside.

[0027] In some embodiments of the present invention, the top of the insulated tank is provided with a feed inlet for feeding composting raw materials.

[0028] In some embodiments of the present invention, the bottom of the insulated tank is provided with a discharge port, and the discharge port is equipped with a sealing valve.

[0029] Specifically, the discharge port is equipped with a sealing valve to effectively prevent heat and gas leakage.

[0030] In some embodiments of the present invention, the ultra-high temperature composting device further includes a heating device and a cooling device; the heating device and the cooling device are respectively connected to the temperature monitoring and control system.

[0031] In some embodiments of the present invention, the heating device includes an electric heating wire.

[0032] In some embodiments of the present invention, the cooling device includes a circulating water cooling pipe.

[0033] In some embodiments of the present invention, the aeration system includes: Multiple aeration pipes are fixed to the blades of the spiral agitator, and the aeration pipes are distributed at equal intervals along the axial direction on the rotating rod of the spiral agitator. An air chamber is formed inside the rotating rod, and the aeration pipe is connected to the air chamber. The aeration pipe is provided with a plurality of aeration holes evenly distributed on it. A blower is fixedly installed on the top of the tank, and the air outlet of the blower is connected to the air chamber of the rotating rod; Additionally, a shielding assembly is provided on the outer wall of the aeration pipe to prevent composting materials from clogging the aeration holes.

[0034] In some embodiments of the invention, the rotating rod of the spiral stirrer extends to the top of the tank and is connected to a drive assembly that drives the rotating rod to rotate.

[0035] In some embodiments of the present invention, the heat-insulating reactor has a cuboid structure.

[0036] In some embodiments of the present invention, the heat-insulating reactor is externally covered with a heat-insulating layer.

[0037] In some embodiments of the present invention, the materials of the insulation layer include cotton quilts, aluminum foil self-adhesive rubber and plastic boards, high-temperature resistant aluminum silicate needle-punched blankets, ceramic fiber insulation cotton, flame-retardant rubber and plastic sponges, and glass wool.

[0038] In some embodiments of the present invention, the paired electrodes are fixed to the inner wall of the reactor by an insulating support, and the distance between the positive and negative electrodes of the paired electrodes is 15-50 cm.

[0039] In some embodiments of the present invention, the positive electrode is selected from stainless steel plate, titanium plate or graphite plate.

[0040] In some embodiments of the present invention, the negative electrode is selected from graphite plate, conductive carbon, or carbon felt.

[0041] The working principle of the composting device provided by this invention is explained as follows: The composting apparatus provided by this invention includes an ultra-high temperature composting apparatus, a low-voltage pulsed electric field composting apparatus, and a conveying device, wherein: 1) The ultra-high temperature composting device adopts a double-layer jacketed tank structure filled with heat-insulating material, which can minimize heat loss and provide a basic guarantee for the ultra-high temperature reaction. The variable speed spiral agitator inside the tank can adjust the stirring speed to ensure that the materials are fully mixed and promote uniform heat distribution and accelerate temperature rise in the early stage of composting. In the later stage of composting, it avoids excessive stirring that may damage the microbial structure. The aeration system integrated into the agitator provides the necessary oxygen for thermophilic bacteria. The temperature monitoring and control system monitors the temperature in real time and adjusts the aeration and auxiliary heating or cooling to precisely control the temperature within the ultra-high temperature range. 2) The compost pile treated by the ultra-high temperature composting device flows out from the discharge port located at the bottom of the tank and is sent to the low-voltage pulse electric field composting device for low-voltage pulse electric field composting treatment through the conveying device. 3) In the low-voltage pulse electric field composting device, the low-voltage pulse electric field generator produces a safe low-voltage (5-36V) pulse electric signal with a specific frequency (50-100Hz). The electric signal passes through multiple pairs of electrodes arranged at a specific interval (15-50cm) in the reactor to form a uniform pulse electric field in the compost. The electric field operates in an intermittent mode, which can effectively generate electroporation and electrochemical effects to achieve deep physical field treatment of materials, while avoiding energy waste, equipment overheating and microbial tolerance.

[0042] Compared with the prior art, the beneficial effects of the present invention are: 1) The composting method provided by this invention innovatively couples ultra-high temperature composting with low-voltage pulsed electric field treatment to achieve deep removal of ARGs. This method shows extremely high removal efficiency for various ARGs such as tetracyclines, sulfonamides, and macrolides, with an average removal rate of over 97%. The effect is far superior to simple ultra-high temperature composting or traditional composting, reducing the safety risks of compost products in agricultural use. Moreover, the entire process does not rely on any exogenous chemical additives, but only utilizes microorganisms and physical fields, avoiding the risk of secondary pollution and demonstrating outstanding environmental friendliness. 2) The composting device provided by this invention features an ultra-high temperature composting insulated tank that greatly reduces heat loss and lowers operating costs. The low-voltage pulse electric field uses a safe voltage (5-36V) and an intermittent working mode, which significantly reduces energy consumption and extends equipment life while ensuring treatment effect. The operating conditions are mild, parameters are easy to control precisely through an automated system, and the process flow is smooth. The two core devices are connected by a conveyor, making it suitable for large-scale, continuous industrial production applications. It provides reliable and efficient technical support for the harmless and resource-based treatment of livestock and poultry breeding waste and has great promotional value. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the composting device used in the embodiment; Figure 2 This is a schematic diagram of the ultra-high temperature composting device used in the embodiment. Figure 3 This is a schematic diagram of the aeration system of the ultra-high temperature composting device used in the embodiment. Detailed Implementation

[0044] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0045] Figure 1 This is a schematic diagram of the composting device used in the embodiment. Figure 2 This is a schematic diagram of the ultra-high temperature composting device used in the embodiment. Figure 2 (a) in the diagram is a schematic diagram of the external structure of the ultra-high temperature composting device. Figure 2 (b) is a schematic diagram of the internal structure of the ultra-high temperature composting device. Figure 3 This is a schematic diagram of the aeration system structure of the ultra-high temperature composting device used in the embodiment, wherein, Figure 3 (a) in the diagram is a schematic diagram of the aeration pipe structure. Figure 3 (b) is a schematic diagram of the shielding component structure. During operation, the composting raw materials are fed into the inlet at the top of the ultra-high temperature composting device tank, and are mixed by a variable speed spiral agitator inside the tank. A blower at the top of the tank aerates the pipe through the rotating rod air chamber, providing the necessary oxygen for the thermophilic bacteria. The temperature is monitored in real time and the aeration and auxiliary heating or cooling are adjusted to control the temperature through a temperature monitoring and control system. After the ultra-high temperature aerobic composting treatment is completed, the pile is discharged from the outlet at the bottom of the tank and transported to the low-pressure pulse electric field composting device by a conveying device. The low-pressure pulse electric field generator in the low-pressure pulse electric field composting device generates pulse electric signals. The electric signals form a uniform pulse electric field in the pile through multiple pairs of electrodes arranged in the reactor, generating electroporation and electrochemical effects, and performing physical field deep treatment on the material.

[0046] The extreme thermophilic bacterial agents used in the following examples and comparative examples are all compound bacterial agents formed by mixing Bacillus brevis, Pseudomonas, Bacillus subtilis, Saccharomyces cerevisiae, and Acinetobacterium sp. GSS19 (accession number GDMCC No. 62979) in a mass ratio of 4:2:2:1:1, with a bacterial content of 2×10⁻⁶. 10 cfu / g-2×10 12 cfu / g, of which Acinetobacterium sp. GSS19 is deposited at the Guangdong Provincial Center for Microbial Culture Collection and is disclosed in patent CN 119120257 A.

[0047] Example 1 This embodiment provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 80 kg of cow dung, 20 kg of pig dung, 20 kg of rice husks, and 10 kg of sawdust to obtain compost material; add 0.1 kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 11 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation for 15 days. During this period, turn the pile over every 2 days, using intermittent aeration, aerating once every 1 hour for 12 minutes each time, with an aeration rate of 0.05 m³ / h. 3 / (min·kg compost raw material), during the high-temperature period, add an appropriate amount of water daily to maintain the material moisture content at 62%; S3. After 15 days of ultra-high temperature aerobic composting treatment, the compost pile is transferred to a low-voltage pulse electric field composting device via a conveyor. The low-voltage pulse electric field is used with a pulse frequency of 50Hz, a working voltage of 10V, and a pulse width of 20ms. It is turned on twice per hour, each time lasting 10 minutes. The low-voltage pulse electric field composting treatment lasts for 15 days.

[0048] Example 2 This embodiment provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 80 kg of cow dung, 20 kg of pig dung, 20 kg of rice husks, and 10 kg of sawdust to obtain compost material; add 0.1 kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 11 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation for 15 days. During this period, turn the pile over every 2 days, using intermittent aeration, aerating once every 1 hour for 12 minutes each time, with an aeration rate of 0.05 m³ / h. 3 / (min·kg compost raw material), during the high-temperature period, add an appropriate amount of water daily to maintain the material moisture content at 62%; S3. After 15 days of ultra-high temperature aerobic composting treatment, the compost pile is transferred to a low-voltage pulse electric field composting device via a conveyor. The low-voltage pulse electric field is used with a pulse frequency of 100Hz, a working voltage of 25V, and a pulse width of 10ms. It is turned on 3 times per hour, each lasting 4 minutes. The low-voltage pulse electric field composting treatment lasts for 15 days.

[0049] Example 3 This embodiment provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120 kg of pig manure, 30 kg of corn cobs, and 15 kg of straw to obtain compost material; add 0.12 kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 12 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation for 15 days. During this period, turn the pile over every 3 days, using intermittent aeration, aerating once every 1 hour for 15 minutes each time, with an aeration volume of 0.1 m³ / h. 3 / (min·kg compost raw material), during the high-temperature period, add an appropriate amount of water daily to maintain the material moisture content at 65%; S3. After 15 days of ultra-high temperature aerobic composting treatment, the compost pile is transferred to a low-voltage pulse electric field composting device via a conveyor. The low-voltage pulse electric field is used with a pulse frequency of 50Hz, a working voltage of 36V, and a pulse width of 20ms. It is turned on twice per hour, each time lasting for 4 minutes. The low-voltage pulse electric field composting treatment lasts for 20 days.

[0050] Example 4 This embodiment provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120 kg of pig manure, 30 kg of corn cobs, and 15 kg of straw to obtain compost material; add 0.12 kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 12 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation for 15 days. During this period, turn the pile over every 3 days, using intermittent aeration, aerating once every 1 hour for 15 minutes each time, with an aeration volume of 0.1 m³ / h. 3 / (min·kg compost raw material), during the high-temperature period, add an appropriate amount of water daily to maintain the material moisture content at 65%; S3. After 15 days of ultra-high temperature aerobic composting treatment, the compost pile is transferred to a low-voltage pulse electric field composting device via a conveyor. The low-voltage pulse electric field is used with a pulse frequency of 50Hz, a working voltage of 16V, and a pulse width of 20ms. It is turned on 3 times per hour, each time lasting 6 minutes. The low-voltage pulse electric field composting treatment lasts for 20 days.

[0051] Example 5 This embodiment provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120kg of chicken manure, 40kg of cow manure, 30kg of duck manure, 25kg of sawdust, and 15kg of rice husks to obtain compost material; add 0.15kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 10 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation for 15 days. During this period, turn the pile over every 2 days, using intermittent aeration, aerating once every 1 hour for 10 minutes each time, with an aeration rate of 0.08 m³ / h. 3 / (min·kg compost raw material), during the high-temperature period, add an appropriate amount of water daily to maintain the material moisture content at 63%; S3. After 15 days of ultra-high temperature aerobic composting treatment, the compost pile is transferred to a low-voltage pulse electric field composting device via a conveyor. The low-voltage pulse electric field is used with a pulse frequency of 100Hz, a working voltage of 30V, and a pulse width of 10ms. It is turned on 3 times per hour, each time lasting 3 minutes. The low-voltage pulse electric field composting treatment lasts for 18 days.

[0052] Example 6 This embodiment provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120kg of chicken manure, 40kg of cow manure, 30kg of duck manure, 25kg of sawdust, and 15kg of rice husks to obtain compost material; add 0.15kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 10 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation for 15 days. During this period, turn the pile over every 2 days, using intermittent aeration, aerating once every 1 hour for 10 minutes each time, with an aeration rate of 0.08 m³ / h. 3 / (min·kg compost raw material), during the high-temperature period, add an appropriate amount of water daily to maintain the material moisture content at 63%; S3. After 15 days of ultra-high temperature aerobic composting treatment, the compost pile is transferred to a low-voltage pulse electric field composting device via a conveyor. The low-voltage pulse electric field is used with a pulse frequency of 50Hz, a working voltage of 20V, and a pulse width of 20ms. It is turned on 3 times per hour, each lasting 4 minutes. The low-voltage pulse electric field composting treatment lasts for 18 days.

[0053] Comparative Example 1 This comparative example provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 80 kg of cow dung, 20 kg of pig dung, 20 kg of rice husks, and 10 kg of sawdust to obtain compost material; add 0.1 kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 11 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation. During this process, turn the pile over every two days, using intermittent aeration, aerating once every hour for 12 minutes each time, with an aeration rate of 0.05 m³ / h. 3 / (min·kg compost raw material), during the high temperature period, add an appropriate amount of water every day to maintain the material moisture content at 62%, and perform ultra-high temperature aerobic composting treatment for 30 days.

[0054] Comparative Example 2 This comparative example provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 80kg of cow dung, 20kg of pig dung, 20kg of rice husks and 10kg of sawdust to obtain compost material; S2. Place the compost material into an ultra-high temperature composting treatment device, control the pile temperature to be no lower than 80℃ for fermentation, turn the pile over every 2 days, and use intermittent aeration, aerating once every 1 hour for 12 minutes each time, with an aeration rate of 0.05m³. 3 / (min·kg compost raw material), during the high temperature period, add an appropriate amount of water every day to maintain the material moisture content at 62%, and perform ultra-high temperature aerobic composting treatment for 30 days.

[0055] Comparative Example 3 This comparative example provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120 kg of pig manure, 30 kg of corn cobs, and 15 kg of straw to obtain compost material; add 0.12 kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 12 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation. Turn the pile over every 3 days, using intermittent aeration, aerating once every 1 hour for 15 minutes each time, with an aeration volume of 0.1 m³ / h. 3 / (min·kg compost raw material), during the high temperature period, add an appropriate amount of water every day to maintain the material moisture content at 65%, and perform ultra-high temperature aerobic composting treatment for 35 days.

[0056] Comparative Example 4 This comparative example provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120kg of pig manure, 30kg of corn cobs and 15kg of straw to obtain compost material; S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation. Turn the pile over every 3 days, using intermittent aeration, aerating once every 1 hour for 15 minutes each time, with an aeration volume of 0.1 m³ / h. 3 / (min·kg compost raw material), during the high temperature period, add an appropriate amount of water every day to maintain the material moisture content at 65%, and perform ultra-high temperature aerobic composting treatment for 35 days.

[0057] Comparative Example 5 This comparative example provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120kg of chicken manure, 40kg of cow manure, 30kg of duck manure, 25kg of sawdust, and 15kg of rice husks to obtain compost material; add 0.15kg of extreme thermophilic bacteria agent (bacterial content of 2×10⁻⁶) to the compost material. 10 The mixture of cfu / g and water content was homogeneous to obtain composting raw materials (pH=6.0-8.0, moisture content 60%-70%, carbon-nitrogen ratio (25-30):1). S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation. During this period, turn the pile over every two days, using intermittent aeration, aerating once every hour for 10 minutes each time, with an aeration rate of 0.08 m³ / h. 3 / (min·kg compost raw material), during the high temperature period, add an appropriate amount of water every day to maintain the material moisture content at 63%, and perform ultra-high temperature aerobic composting treatment for 33 days.

[0058] Comparative Example 6 This comparative example provides a composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, the steps of which are as follows: S1. Mix 120kg of chicken manure, 40kg of cow manure, 30kg of duck manure, 25kg of sawdust, and 15kg of rice husks to obtain compost material; S2. Place the composting materials into an ultra-high temperature composting treatment device, controlling the pile temperature to be no lower than 80℃ for fermentation. During this period, turn the pile over every two days, using intermittent aeration, aerating once every hour for 10 minutes each time, with an aeration rate of 0.08 m³ / h. 3 / (min·kg compost raw material), during the high temperature period, add an appropriate amount of water every day to maintain the material moisture content at 63%, and perform ultra-high temperature aerobic composting treatment for 33 days.

[0059] After composting was completed in Examples 1-6 and Comparative Examples 1-6, compost samples were collected. The compost samples were freeze-dried, ground, and sieved. DNA was extracted from the samples using the Rapid DNA Spin Kit for Soil (MP Corporation, USA). The DNA extraction efficiency was checked using Nanodrop-2000c. The primers designed included 20 common ARGs, including 6 tetracycline ARGs (tetB, tetL, tetM, tet0, tetQ, and tetw), 2 sulfonamide ARGs (sul I and sul II), 4 macrolide ARGs (ermB, ermF, mphA, and mphB), 4 aminoglycoside ARGs (aadAl, aacC2, strA, and strB), 2 quinolone ARGs (qnrA and qnrS), and 2 β-lactam ARGs (blaTEM and blaOXA-1). PCR amplification was achieved on the Smart ChipReal-time PCR Systems high-throughput quantitative PCR platform.

[0060] Table 1. Reduction effect of antibiotic resistance gene abundance in Examples 1 and 2 and Comparative Examples 1 and 2

[0061] Table 1 shows the reduction effect of antibiotic resistance gene abundance in Examples 1 and 2 and Comparative Examples 1 and 2. As can be seen from Table 1, the average removal rates of 20 ARGs in Examples 1 and 2 can reach 97.83% and 97.97%, respectively, indicating that the composting method provided by the present invention can achieve extremely stable and efficient ARGs removal under different combinations of low-voltage pulsed electric field parameters, and the removal rate of all single genes is above 97%. Comparative Example 1 uses a single ultra-high temperature aerobic composting treatment, and the average ARGs removal rate is 92.11%, indicating that although ultra-high temperature itself has a certain effect, there is a bottleneck in the removal ability of certain genes such as tetQ, sulⅡ, and aadA1. Comparative Example 2 uses traditional composting, and the average ARGs removal rate is only 84.82%, indicating that the traditional composting process without adding extreme thermophilic bacteria and without reaching ultra-high temperature has limited effect in reducing ARGs.

[0062] Table 2. Reduction effect of antibiotic resistance gene abundance in Examples 3 and 4 and Comparative Examples 3 and 4

[0063] Table 2 shows the reduction effect of antibiotic resistance gene abundance in Examples 3 and 4 and Comparative Examples 3 and 4. As shown in Table 2, the average removal rates of 20 ARGs in Examples 3 and 4 can reach 96.96% and 97.01%, respectively, indicating that the composting method provided by the present invention has good adaptability to different livestock and poultry manure raw materials. In Comparative Example 3, a single ultra-high temperature aerobic composting treatment was used, and the average removal rate dropped to 87.58% compared with Comparative Example 1, indicating that the effect of the single ultra-high temperature technology may be affected by the material properties and the stability is not good. In Comparative Example 2, traditional composting was used, and the average removal rate of ARGs dropped sharply to 75.37%, and several genes with extremely low removal rates appeared. This further proves that for certain specific manure (such as pig manure) and composting conditions, traditional composting not only cannot effectively reduce ARGs, but may even lead to an increase in the relative abundance of certain genes due to microbial community succession, which poses a huge environmental risk.

[0064] Table 3. Reduction effect of antibiotic resistance gene abundance in Examples 5 and 6 and Comparative Examples 5 and 6

[0065] Table 3 shows the reduction effect of antibiotic resistance gene abundance in Examples 5 and 6 and Comparative Examples 5 and 6. As can be seen from Table 3, the average removal rates of 20 ARGs in Examples 3 and 4 can reach 99.34% and 99.14%, respectively, indicating that the composting method provided by the present invention has a more significant effect on reducing the abundance of antibiotic resistance genes in complex mixed feces. Comparative Example 5 uses a single ultra-high temperature aerobic composting treatment, and Comparative Example 6 uses conventional composting. The average removal rates of ARGs are 93.33% and 86.31%, respectively, which are significantly lower than those in Examples 5 and 6. This highlights the key role of pulsed electric field in destroying cells and degrading intracellular genes, proving that the coupling of ultra-high temperature composting and low-voltage pulsed electric field composting in the present invention produces a synergistic effect of "1+1>2", which can solve the problem of ARGs pollution in complex real-world scenarios.

Claims

1. A composting method for reducing the abundance of antibiotic resistance genes in livestock and poultry manure, characterized in that, Includes the following steps: Animal manure is mixed with composting additives to form compost material, which is then mixed with an extreme thermophilic bacteria agent to obtain compost raw material. The compost raw material is then subjected to ultra-high temperature aerobic composting treatment and low-voltage pulsed electric field composting treatment in sequence to reduce the abundance of antibiotic resistance genes. The extreme thermophilic bacteria agent includes at least one of Bacillus breviculatus, Pseudomonas aeruginosa, Bacillus subtilis, sucralospora, and Acinetobacter bacillus.

2. The composting method according to claim 1, characterized in that, The mass ratio of livestock and poultry manure to composting additives is 10:(3-5); the bacterial content of the extreme thermophilic bacteria agent is 2×10⁻⁶. 10 cfu / g-2×10 12 cfu / g; the amount of the extreme thermophilic microbial agent is 0.08%-0.12% of the mass of the compost material.

3. The composting method according to claim 2, characterized in that, The extreme thermophilic bacterial agent is a compound bacterial agent formed by mixing Bacillus brevis, Pseudomonas, Bacillus subtilis, Saccharomyces cerevisiae, and Acinetobacterium sp. GSS19 (accession number GDMCC No. 62979) in a mass ratio of (3-5): (1.5-2.5): (1.5-2.5): (0.8-1.2):

1.

4. The composting method according to claim 2, characterized in that, The physicochemical parameters of the compost raw materials include at least one of the following: pH = 6.0-8.0; moisture content 60%-70%; carbon-nitrogen ratio of (25-30):

1.

5. The composting method according to claim 1, characterized in that, The ultra-high temperature aerobic composting treatment involves controlling the compost temperature to be no lower than 80℃ for 10-15 days of fermentation; during this period, intermittent aeration is used, once every 0.8-1.2 hours, for 10-15 minutes each time, with an aeration volume of 0.01-0.1 m³. 3 / (min·kg compost material), maintain the moisture content of the compost pile at 60%-65%, and turn the pile over every 2-3 days.

6. The composting method according to claim 1, characterized in that, The low-voltage pulsed electric field composting treatment operates at a voltage of 5-36V, a pulse frequency of 50-100Hz, and a pulse width of 10ms-20ms. It adopts an intermittent start-up mode, starting 1-5 times per hour, with a single treatment duration of 2-10 minutes and a total treatment time of 15-25 days.

7. The composting method according to any one of claims 1-6, characterized in that, The antibiotic resistance gene includes at least one of tetB, tetL, tetM, tetO, tetQ, tetW, sulⅠ, sulⅡ, ermB, ermF, mphA, mphB, aadA1, aacC2, strA, strB, qnrA, qnrS, blaTEM, and blaOXA-1.

8. A composting apparatus for use in the composting method according to any one of claims 1-7, characterized in that, include: An ultra-high temperature composting device includes an insulated tank, which is equipped with a variable speed spiral agitator and an aeration system, and the tank is equipped with a temperature monitoring and control system. A low-voltage pulsed electric field composting device includes an insulated reactor and a low-voltage pulsed electric field generator. The reactor is equipped with multiple pairs of paired electrodes, which are connected to the low-voltage pulsed electric field generator. A conveying device is used to transport the compost pile processed by the ultra-high temperature composting device to the low-voltage pulse electric field composting device.

9. The composting apparatus according to claim 8, characterized in that, The aeration system includes: Multiple aeration pipes are fixed to the blades of the spiral agitator, and the aeration pipes are distributed at equal intervals along the axial direction on the rotating rod of the spiral agitator. An air chamber is formed inside the rotating rod, and the aeration pipe is connected to the air chamber. The aeration pipe is provided with a plurality of aeration holes evenly distributed on it. A blower is fixedly installed on the top of the tank, and the air outlet of the blower is connected to the air chamber of the rotating rod; Additionally, a shielding assembly is provided on the outer wall of the aeration pipe to prevent composting materials from clogging the aeration holes.

10. The composting apparatus according to claim 8, characterized in that, The paired electrodes are fixed to the inner wall of the reactor by an insulating support, and the distance between the positive and negative electrodes of the paired electrodes is 15-50cm.

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