Method for removing antibiotics and resistance genes in aerobic composting of chicken manure

Abstract

The invention relates to the technical field of solid waste treatment, in particular to a method for removing antibiotics and resistance genes in aerobic composting of chicken manure. Comprising the following steps: adding a composting auxiliary material into chicken manure to enable the carbon-nitrogen ratio to be (25-30): 1, and adjusting the initial moisture content to 55-65% to obtain an initial composting material; adding persulfate and zero-valent iron into the initial compost material, and uniformly mixing; loading the mixed material into a container for composting; during the composting period, stirring is performed regularly, and the aerobic composting time is 30-40 days. The method has the advantages that the persulfate advanced oxidation technology and the aerobic composting technology are combined, the potassium persulfate and the zero-valent iron are added to serve as the aerobic composting additive, the zero-valent iron can continuously activate the potassium persulfate, persulfate free radicals and hydroxyl free radicals are generated, and removal of antibiotics and antibiotic resistance genes is promoted; high-concentration oxytetracycline, sulfamethoxazole and ciprofloxacin in the chicken manure are effectively removed; and the abundance of antibiotic resistance genes with high risk levels is obviously reduced.

Description

Technical Field

[0001] The invention relates to the technical field of solid waste treatment, and in particular to a method for removing antibiotics and resistance genes through aerobic composting of chicken manure. Background Art

[0002] With the rapid development of livestock and poultry farming, the discharge of chicken manure has increased dramatically. Chicken manure is often used to return to the field to increase soil fertility because it contains plant nutrients such as nitrogen, phosphorus, and potassium. Antibiotics are widely used in livestock and poultry farming as animal growth promoters and therapeutic drugs. Antibiotics that enter the animal body cannot be completely absorbed and will enter the environment with feces. Aerobic composting technology can improve the utilization rate of nutrients in feces, reduce environmental pollution, and promote the development of circular agriculture. It has become an important means of resource utilization of livestock and poultry manure. However, the traditional composting process has limited removal of antibiotics and resistance genes. Persulfate advanced oxidation technology exhibits strong oxidizing properties because it can produce active free radicals, and has a good removal effect on pollutants such as antibiotics and resistance genes. The use of persulfate advanced oxidation technology in the aerobic composting process of chicken manure helps to control antibiotic and resistance gene pollution. The aerobic composting process will generate a lot of heat due to microbial activity. This heat will activate potassium persulfate, thereby generating active oxygen and promoting the removal of pollutants. Potassium persulfate added to the aerobic composting system of cow dung and garden waste promoted the degradation of antibiotics and the removal of antibiotic resistance genes.

[0003] At present, the existing technology only focuses on the removal of antibiotics and resistance genes in aerobic composting systems related to cow dung by potassium persulfate. This study focuses on the removal of antibiotics and resistance genes in aerobic composting systems related to chicken manure by persulfate advanced oxidation technology.

[0004] Current technology only activates potassium persulfate through the heat generated by aerobic composting, but in the later stage of composting, the temperature of aerobic composting will be close to room temperature, and potassium persulfate cannot be activated any more. However, at this stage, there will often be a rebound of antibiotic resistance genes, so the removal effect of adding only potassium persulfate needs to be improved. The existing technology for the removal of antibiotic pollution is based on very low concentrations, generally at the μg / kg level. However, in livestock and poultry farming, tetracyclines, sulfonamide antibiotics and fluoroquinolone antibiotics are widely used in livestock and poultry farming disease prevention and control, so the detection rate and detection amount in chicken manure are very high. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for removing antibiotics and resistance genes through aerobic composting of chicken manure.

[0006] The present invention aims to provide a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically comprises the following steps: S1. Add composting materials to chicken manure, mix thoroughly, make the carbon-nitrogen ratio 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial composting materials; S2. adding persulfate and zero-valent iron to the initial compost material and mixing them evenly to obtain a mixed material; S3. Put the mixed materials into containers for composting. During the composting period, add water to keep the moisture content at 55-65%. Stir regularly. The aerobic composting time is 30-40 days.

[0007] Preferably, the composting auxiliary material is sawdust; the persulfate is potassium persulfate; and the zero-valent iron is high-purity iron powder of 200-300 mesh.

[0008] Preferably, the amount of persulfate added is 0.1-1% of the dry weight of the initial compost material; and the amount of zero-valent iron added is 0.1-1 g / kg of the wet weight of the initial compost material.

[0009] Preferably, the persulfate is potassium persulfate; the added amount of potassium persulfate is 0.5% of the dry weight of the initial compost material; and the added amount of zero-valent iron is 0.1 g / kg of the wet weight of the initial compost material.

[0010] Preferably, the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in the chicken manure is 0-100 mg / kg.

[0011] Preferably, in step S3, the bottom of the container is ventilated during composting, and the gas flow rate is 0.2-0.6 mL / min; and the periodic stirring is stirring once every 2-3 days.

[0012] Preferably, in step S3, the compost temperature is measured by a digital thermometer, and a compost reactor heating device is added outside the compost container, and the temperature difference of the compost reactor heating device is set to follow the compost auxiliary heating, and the temperature difference is 1-2°C to achieve the insulation effect.

[0013] Preferably, the aerobic composting time is 36 days.

[0014] Compared with the prior art, the present invention can achieve the following beneficial effects: (1) Potassium persulfate and zero-valent iron are added simultaneously as aerobic composting additives. Zero-valent iron can continuously activate potassium persulfate to produce persulfate radicals and hydroxyl radicals, thereby promoting the removal of antibiotic resistance genes. (2) Adding potassium persulfate and zero-valent iron to the chicken manure aerobic composting system can effectively remove high concentrations (100 mg / kg) of oxytetracycline, sulfamethoxazole and ciprofloxacin (typical antibiotics among the three types of antibiotics) in chicken manure, with significant removal effects; (3) Significantly reduced the abundance of high-risk antibiotic resistance genes in chicken manure aerobic composting products; (4) The operation method of the present invention is simple and does not require excessive chemical reagents and costs. At the same time, iron is an essential trace nutrient element in the growth process of plants. Selecting zero-valent iron as an activator will not cause secondary pollution. It is a technology that can achieve efficient harmless treatment and resource utilization of organic solid waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The present invention is a flowchart of a method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to an embodiment of the present invention.

[0016] Figure 2 These are the changes in the total abundance of all antibiotic resistance genes and the abundance of three types of antibiotic resistance genes after the composting is completed according to Comparative Examples 1 and 2 and Example 1 of the present invention.

[0017] Figure 3 This is the result of the change in the abundance of high-risk antibiotic resistance genes after the chicken manure composting provided in Comparative Examples 1 and 2 and Example 1 of the present invention is completed.

[0018] Figure 4 This is the change result of hydroxyl radicals and sulfate radicals in the chicken manure composting process provided by Example 1 of the present invention. DETAILED DESCRIPTION

[0019] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are represented by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, the detailed description thereof will not be repeated.

[0020] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0021] The present invention provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, see Figure 1 , specifically including the following steps: S1. Add composting auxiliary materials to chicken manure, mix thoroughly, make the carbon-nitrogen ratio (C / N) to be 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial composting material; S2. adding persulfate and zero-valent iron to the initial compost material and mixing them evenly to obtain a mixed material; S3. Put the mixed materials into containers for composting. During the composting period, add water to keep the moisture content at 55-65%. Stir regularly. The aerobic composting time is 30-40 days.

[0022] Specifically, the concentrations of oxytetracycline (OTC), ciprofloxacin (CIP), and sulfamethoxazole (SMX) in chicken manure ranged from 0 to 100 mg / kg; Specifically, the composting auxiliary material is sawdust; Specifically, the persulfate is potassium persulfate; the potassium persulfate accounts for 0.1-1% of the dry weight of the initial compost material; the zero-valent iron is a high-purity iron powder of 200-300 mesh, and the addition amount is 0.1-1 g / kg of the wet weight of the initial compost material; Specifically, in step S3, ensure that the temperature inside the container is higher than the temperature outside the container to achieve the effect of heat preservation; specifically, the compost temperature is measured by a digital thermometer, and a compost reactor heating device is added outside the compost container, and the temperature difference of the compost reactor heating device is set to follow the compost auxiliary heating, and the temperature difference is 1-2°C to achieve the effect of heat preservation; during composting, the bottom of the container is ventilated, and the gas flow rate is 0.2-0.6mL / min; in a specific embodiment, the gas flow rate is 0.5mL / min; Specifically, the regular stirring in step S3 is stirring every 2 to 3 days to ensure that the composting system has sufficient oxygen and meets the conditions for aerobic composting; the aerobic composting time is 36 days.

[0023] Example 1 This embodiment provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically includes the following steps: S1. Add sawdust to chicken manure to make the carbon-nitrogen ratio (C / N) 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial compost material; the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in chicken manure is 100 mg / kg; S2. adding potassium persulfate and zero-valent iron to the initial compost material, mixing them evenly, to obtain a mixed material; the potassium persulfate accounts for 0.5% of the dry weight of the initial compost material; and the amount of zero-valent iron added is 0.1 g / kg of the wet weight of the initial compost material; S3. The mixed material is placed in a container for composting; during the composting period, water is added to keep the moisture content at 55-65%, and the mixture is stirred every 2-3 days. The aerobic composting time is 36 days; Specifically, in step S3, the container is a cylindrical PVC barrel of about 9 L, ventilation is performed at a flow rate of 0.5 mL / min at the bottom of the device, and a blast drying oven is used to adjust the temperature outside the PVC barrel to be slightly lower than that inside the barrel, so as to achieve a heat preservation effect.

[0024] After composting, the degradation rate of antibiotics was measured (three parallel experiments were set up in the composting, and the results were averaged). The results are shown in Table 1. As can be seen from the table, the degradation rate of OTC is 97.57%, the degradation rate of CIP is 91.64%, and the degradation rate of SMX is 98.01%, and the degradation effect is good.

[0025] The changes in the abundance of antibiotic resistance genes before and after chicken manure composting are shown in Table 2 Figure 2 and Figure 3 As shown in the table and the figure, it can be seen that the total abundance of antibiotic resistance genes and the abundance of high-risk antibiotic resistance genes are significantly reduced by composting according to the method of the present invention.

[0026] Changes of hydroxyl radicals and sulfate radicals during chicken manure composting Figure 4 As shown in the figure, it can be seen that by composting according to the method of the present invention, the persulfate is continuously activated during the aerobic composting process.

[0027] Example 2 This embodiment provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically includes the following steps: S1. Add sawdust to chicken manure to make the carbon-nitrogen ratio (C / N) 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial compost material; the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in chicken manure is 100 mg / kg; S2. adding potassium persulfate and zero-valent iron to the initial compost material, mixing them evenly, to obtain a mixed material; the potassium persulfate accounts for 0.1% of the dry weight of the initial compost material; and the amount of zero-valent iron added is 0.1 g / kg of the wet weight of the initial compost material; S3. The mixed material is placed in a container for composting; during the composting period, water is added to keep the moisture content at 55-65%, and the mixture is stirred every 2-3 days. The aerobic composting time is 36 days; Specifically, in step S3, the bottom of the container is ventilated during composting to ensure that the temperature inside the container is higher than the temperature outside the container to achieve a heat preservation effect; the gas flow rate is 0.5 mL / min.

[0028] Example 3 This embodiment provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically includes the following steps: S1. Add sawdust to chicken manure to make the carbon-nitrogen ratio (C / N) 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial compost material; the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in chicken manure is 100 mg / kg; S2. adding potassium persulfate and zero-valent iron to the initial compost material, mixing them evenly, to obtain a mixed material; the potassium persulfate accounts for 0.1% of the dry weight of the initial compost material; and the amount of zero-valent iron added is 1.0 g / kg of the wet weight of the initial compost material; S3. The mixed material is placed in a container for composting; during the composting period, water is added to keep the moisture content at 55-65%, and the mixture is stirred every 2-3 days. The aerobic composting time is 36 days; Specifically, in step S3, the bottom of the container is ventilated during composting to ensure that the temperature inside the container is higher than the temperature outside the container to achieve a heat preservation effect; the gas flow rate is 0.5 mL / min.

[0029] Example 4 This embodiment provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically includes the following steps: S1. Add sawdust to chicken manure to make the carbon-nitrogen ratio (C / N) 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial compost material; the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in chicken manure is 100 mg / kg; S2. adding potassium persulfate and zero-valent iron to the initial compost material, mixing them evenly, to obtain a mixed material; the potassium persulfate accounts for 0.5% of the dry weight of the initial compost material; and the amount of zero-valent iron added is 1.0 g / kg of the wet weight of the initial compost material; S3. The mixed material is placed in a container for composting; during the composting period, water is added to keep the moisture content at 55-65%, and the mixture is stirred every 2-3 days. The aerobic composting time is 36 days; Specifically, in step S3, the bottom of the container is ventilated during composting to ensure that the temperature inside the container is higher than the temperature outside the container to achieve a heat preservation effect; the gas flow rate is 0.5 mL / min.

[0030] Example 5 This embodiment provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically includes the following steps: S1. Add sawdust to chicken manure to make the carbon-nitrogen ratio (C / N) 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial compost material; the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in chicken manure is 100 mg / kg; S2. adding potassium persulfate and zero-valent iron to the initial compost material, mixing them evenly, to obtain a mixed material; the potassium persulfate accounts for 1.0% of the dry weight of the initial compost material; and the amount of zero-valent iron added is 0.1 g / kg of the wet weight of the initial compost material; S3. The mixed material is placed in a container for composting; during the composting period, water is added to keep the moisture content at 55-65%, and the mixture is stirred every 2-3 days. The aerobic composting time is 36 days; Specifically, in step S3, the bottom of the container is ventilated during composting to ensure that the temperature inside the container is higher than the temperature outside the container to achieve a heat preservation effect; the gas flow rate is 0.5 mL / min.

[0031] Example 6 This embodiment provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically includes the following steps: S1. Add sawdust to chicken manure to make the carbon-nitrogen ratio (C / N) 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial compost material; the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in chicken manure is 100 mg / kg; S2. adding potassium persulfate and zero-valent iron to the initial compost material, mixing them evenly, to obtain a mixed material; the potassium persulfate accounts for 1.0% of the dry weight of the initial compost material; and the amount of zero-valent iron added is 1.0 g / kg of the wet weight of the initial compost material; S3. The mixed material is placed in a container for composting; during the composting period, water is added to keep the moisture content at 55-65%, and the mixture is stirred every 2-3 days. The aerobic composting time is 36 days; Specifically, in step S3, the bottom of the container is ventilated during composting to ensure that the temperature inside the container is higher than the temperature outside the container to achieve a heat preservation effect; the gas flow rate is 0.5 mL / min.

[0032] Comparative Example 1 This comparative example provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, which specifically comprises the following steps: S1. Add sawdust to chicken manure to make the carbon-nitrogen ratio (C / N) 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial compost material; the concentration of oxytetracycline, ciprofloxacin and sulfamethoxazole in chicken manure is 100 mg / kg; S2. The initial composting materials are placed in a container for composting. During the composting period, water is added to keep the moisture content at 55-65%. The composting is stirred every 2-3 days. The aerobic composting time is 36 days. The container is a cylindrical PVC barrel of about 9L. The bottom of the device is ventilated at a flow rate of 0.5mL / min. The temperature outside the PVC barrel is adjusted to be slightly lower than that inside the barrel by using a forced air drying oven to achieve a heat preservation effect.

[0033] Comparative Example 2 This comparative example provides a method for removing antibiotics and resistance genes by aerobic composting of chicken manure, wherein: in step S2, only potassium persulfate is added, and zero-valent iron is not added; the remaining steps and addition amounts refer to Example 1.

[0034] The antibiotic content and resistance gene analysis were performed on the aerobic composts prepared in Examples 1 to 6 and Comparative Examples 1 to 2; the specific process is as follows: 1. Antibiotic content determination method: Take 0.5g sample (compost is freeze-dried, ground, and sieved through a 100-mesh sieve) and put it into a 50ml plastic centrifuge tube. Add 15mL of a mixed solution of Na2EDTA-Mcllvaine buffer and acetonitrile (1:1, v / v) and 5mL of MgNO3 solution to each centrifuge tube, vortex for 30s, then ultrasonicate for 15 minutes, centrifuge at 8000rpm for 15 minutes, then filter the supernatant through a 0.45um glass fiber filter and transfer it to a 250mL round-bottom flask. Repeat the extraction twice, evaporate the mixed supernatant in a rotary evaporator at 40°C for 10min, remove the organic solvent, and dilute to about 200mL with deionized water. Then enrich with an HLB solid phase extraction column, elute with methanol, and blow nitrogen at 30°C. Finally, the volume was fixed with 1 mL of a mixed solution of acetonitrile and 0.1% formic acid (1:1, v / v), and then transferred to a 2 mL brown bottle through a 0.22 μm filter. The bottle was stored at 4 °C, and the antibiotic concentration was determined by HPLC-MSMS.

[0035] 2. Extraction and analysis of antibiotic resistance genes: The samples of Comparative Example 1, Comparative Example 2 and Example 1 were freeze-dried and then ground and sieved. The sample DNA was extracted using a kit according to the instructions, and the extracted genomic DNA was detected by 1% agarose gel electrophoresis. Then, metagenomic sequencing was performed at Shanghai Lingen Biotechnology Co., Ltd., and the original sequence was optimized by removing the linker, quality shearing and removing contamination. Then, the high-quality sequence was used for splicing assembly and gene prediction, and a non-redundant gene set was constructed and compared with the SARG database to obtain the abundance and risk level of antibiotic resistance genes (resistance genes were graded using the three conditions of enrichment, mobility potential and pathogenicity of resistance genes in human-related environments; resistance genes that were enriched in human-related environments, had gene mobility and whose hosts were pathogenic bacteria belonged to the first level; enriched, mobile but not pathogenic bacteria belonged to the second level; the third and fourth levels were less harmful). The gene removal rate was expressed by log2FC [log2fold change = log2 (gene expression level of condition A / gene expression level of condition B)].

[0036] The degradation or removal rates of antibiotics and antibiotic resistance genes are shown in Tables 1 and 2, respectively.

[0037] Table 1 Antibiotic degradation rate (%)

[0038] Table 2 Antibiotic resistance gene removal rate (log2FC)

[0039] As can be seen from Table 1, the antibiotic degradation rates in Examples 1 to 6 are all higher than those in Comparative Examples 1 to 2, among which the degradation rates of OTC, CIP and SMX in Example 1 are 97.57%, 91.64% and 98.01%, respectively, with the best degradation effect. The changes in the abundance of antibiotic resistance genes before and after chicken manure composting in Example 1 and Comparative Examples 1 to 2 are compared, and the results are shown in Table 2; it can be seen that the removal rate of antibiotic resistance genes in Example 1 is improved compared with that in Comparative Examples 1 to 2.

[0040] The invention is aimed at combining the persulfate advanced oxidation technology with the chicken manure aerobic composting technology. In addition to being used together with zero-valent iron, the persulfate additive can also be replaced by ferric oxide, ferric oxide and the corresponding iron ore.

[0041] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0042] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for removing antibiotics and resistance genes by aerobic composting of chicken manure, characterized in that: The specific steps include: S1. Add composting materials to chicken manure, mix thoroughly, make the carbon-nitrogen ratio 25-30:1, and adjust the initial moisture content to 55-65% to obtain initial composting materials; S2. adding persulfate and zero-valent iron to the initial compost material and mixing them evenly to obtain a mixed material; S3. Put the mixed materials into containers for composting. During the composting period, add water to keep the moisture content at 55-65%. Stir regularly. The aerobic composting time is 30-40 days.

2. The method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to claim 1, characterized in that: The composting auxiliary material is sawdust; the persulfate is potassium persulfate; and the zero-valent iron is high-purity iron powder with a mesh size of 200-300.

3. The method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to claim 1, characterized in that: The added amount of the persulfate is 0.1-1% of the dry weight of the initial compost material; the added amount of the zero-valent iron is 0.1-1 g / kg of the wet weight of the initial compost material.

4. The method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to claim 1, characterized in that: The persulfate is potassium persulfate; the added amount of potassium persulfate accounts for 0.5% of the mass fraction of the dry weight of the initial compost material; the added amount of zero-valent iron is 0.1 g / kg of the wet weight of the initial compost material.

5. The method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to claim 1, characterized in that: The chicken manure contains oxytetracycline, ciprofloxacin and sulfamethoxazole at a concentration of 0-100 mg / kg.

6. The method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to claim 1, characterized in that: In the step S3, the bottom of the container is ventilated during composting, and the gas flow rate is 0.2-0.6 mL / min; the regular stirring is stirring once every 2-3 days.

7. The method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to claim 6, characterized in that: In step S3, the compost temperature is measured by a digital thermometer, and a compost reactor heating device is added outside the compost container. The temperature difference of the compost reactor heating device is set to follow the compost auxiliary heating, and the temperature difference is 1-2°C to achieve the insulation effect.

8. The method for removing antibiotics and resistance genes by aerobic composting of chicken manure according to claim 1, characterized in that: The aerobic composting time is 36 days.

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