Method for removing antibiotic resistance genes in straw sludge compost and application thereof

Abstract

The application discloses a method for removing antibiotic resistance genes in straw sludge compost and application thereof, and belongs to the technical field of composting. In order to remove antibiotic resistance genes in municipal sludge, the application provides a method for removing antibiotic resistance genes in straw substrate sludge compost, specifically, antibiotic resistance genes are removed through combined action of strong oxidation and high temperature in aerobic composting. The method significantly reduces the abundance of antibiotic resistance genes in the compost sample on the basis of traditional aerobic composting technology, and reduces the potential risk of human health caused by recycling of the compost product in combination with an ozone oxidation technology and corn straw as a carbon source.

Description

Technical Field

[0001] This invention belongs to the field of composting technology, specifically relating to a method for removing antibiotic resistance genes from straw sludge compost and its application. Background Technology

[0002] Municipal sludge is solid waste generated during wastewater treatment at urban wastewater treatment plants. It may contain various harmful substances and poses numerous problems, including microbial contamination, chemical pollution, and antibiotic resistance genes (ARGs), posing potential risks to the environment and human health. ARGs are genes that encode antibiotic resistance in bacteria. These genes can be transferred between different bacteria, including horizontally via genetic elements such as plasmids and transposons, leading to the spread and diffusion of resistance and making existing antibiotic treatments more difficult. Municipal sludge, a solid waste generated during urban wastewater treatment, may contain a variety of microorganisms from hospitals, households, agriculture, and other sources. Due to the widespread use of antibiotics in medicine and agriculture, these microorganisms may carry antibiotic resistance genes. Therefore, municipal sludge is considered an important pool of antibiotic resistance genes, which can be released into the environment through various pathways, such as direct application of inadequately treated sludge to farmland, or through rainwater runoff and groundwater pollution. To reduce the potential threats of antibiotic resistance genes to the environment and human health, appropriate sludge treatment and disposal measures are necessary.

[0003] Aerobic composting is an organic waste treatment technology that transforms organic matter into stable humus through the aerobic metabolism of microorganisms. This process not only reduces the volume and weight of waste but also kills pathogens and improves soil structure and fertility. Aerobic composting is generally suitable for treating various organic wastes such as kitchen waste, garden waste, manure, and municipal sludge. Studies have shown that aerobic composting can also be a major way to control the risk of antibiotics and ARGs in livestock and poultry manure. However, because composting involves multiple microorganisms and is greatly affected by composting conditions and materials, there are significant differences in the changes of different types of antibiotics and ARGs in different studies. Currently, the reduction of ARGs by conventional aerobic composting technology remains controversial. Many researchers have suggested that conventional aerobic composting technology cannot effectively control the proliferation and spread of ARGs and mobile genetic elements (MGEs). Therefore, developing more effective technologies to promote the removal of ARGs during composting is crucial.

[0004] Straw refers to the remaining stalks of crops after harvest, such as wheat straw, corn straw, and rice straw. It is a byproduct of agricultural production and an important biomass resource. The main components of straw are cellulose, hemicellulose, and lignin, which can be used for various purposes, including energy, feed, fertilizer, and building materials. Straw can be directly returned to the field as organic fertilizer or converted into high-quality organic fertilizer through composting, improving soil fertility. Therefore, using straw for composting to remove ARGs from municipal sludge will not only reduce environmental pollution but also create additional income for farmers and promote rural economic development. Summary of the Invention

[0005] To remove antibiotic resistance genes (ARGs) from municipal sewage sludge, this invention provides a method for removing ARGs from straw-based sewage sludge compost. Specifically, it removes ARGs through the combined effects of strong oxidation and high temperature during aerobic composting. Based on traditional aerobic composting technology, this method utilizes corn straw as a carbon source and combines it with ozone oxidation technology, significantly reducing ARGs in compost samples and minimizing potential health risks to humans after the compost is applied to the land.

[0006] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention specifically provides the following technical solutions:

[0007] The first objective of this invention is to provide a method for removing antibiotic resistance genes from straw sludge compost, the method comprising the following steps:

[0008] S1. Mix municipal sludge and auxiliary materials evenly to obtain a mixture;

[0009] S2. The mixture obtained in S1 is subjected to aerobic composting;

[0010] S3. After the high-temperature period of composting ends, ozone is introduced into the composting system when the temperature drops to 40-47℃. The total ozone dosage is 0.4-0.6g / kg of compost material. After ozone oxidation is completed, the compost pile will normally complete the cooling and maturation period.

[0011] In one embodiment of the present invention, the auxiliary material in S1 is agricultural solid waste.

[0012] In one embodiment of the present invention, the agricultural solid waste is any one or a combination of two or more of sawdust, straw, and rice husks.

[0013] In one embodiment of the present invention, the water content of the mixture in S1 is 50% to 60%, and the carbon-nitrogen ratio is 25:1 to 30:1.

[0014] In one embodiment of the present invention, the temperature during the warming period of the aerobic composting described in S2 is between the initial temperature of the mixed materials and 50°C, and the high temperature period is above 55°C for 3 days or above 50°C for 5 to 7 days.

[0015] In one embodiment of the present invention, the aeration air volume of the aerobic composting process in step S2 is controlled at 0.05–0.2 m³ / s. 3 / min.

[0016] In one embodiment of the present invention, the ozone in S3 is introduced into the pile body under positive pressure from the bottom of the pile body, and the overall ozone injection time is 30 to 150 minutes.

[0017] In one embodiment of the present invention, the composting period described in S3 is at least 7 days.

[0018] A second objective of this invention is to provide the application of the above-described method in the treatment of municipal sludge.

[0019] A third objective of this invention is to provide compost obtained using the method described above.

[0020] The beneficial effects of this invention are:

[0021] This invention not only utilizes the high temperature during the high-temperature stage of composting to effectively reduce antibiotic resistance genes (ARGs), but also addresses the issue of increased ARG abundance during the composting maturation period by combining ozone oxidation technology to further reduce ARGs in compost materials, inhibiting the rebound of ARGs during the composting maturation period. This reduces the abundance of ARGs in compost products and decreases the frequency of microbial community integration and transformation at the end of composting, thereby reducing the potential health risks posed by compost after its application to the land.

[0022] This invention not only effectively reduces antibiotic resistance genes in compost materials but also uses straw as a carbon source to address the straw waste problem in Northeast China. By blending it with municipal sewage sludge, the moisture content and carbon-to-nitrogen ratio are balanced, achieving the goal of turning waste into treasure. Attached Figure Description

[0023] Figure 1 The graph shows the abundance of ARGs in compost obtained at different composting times in the experimental group.

[0024] Figure 2 The graph shows the abundance of ARGs in compost obtained at different composting times in the control group.

[0025] Figure 3 The graph shows the results of ARGs abundance changes in compost obtained at different composting times in the experimental and control groups.

[0026] Figure 4The graph shows the detection results of tetM gene content in compost obtained at different composting times in the experimental and control groups.

[0027] Figure 5 The graph shows the detection results of sul1 gene content in compost obtained at different composting times in the experimental and control groups.

[0028] Figure 6 The graph shows the detection results of aphA1 gene content in compost obtained at different composting times in the experimental and control groups.

[0029] Figure 7 The graph shows the detection results of mefA gene content in compost obtained at different composting times in the experimental and control groups.

[0030] Figure 8 The graph shows the detection results of the content of mobile genetic element Tn916 in compost obtained at different composting times in the experimental and control groups. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The embodiments described in this invention are not all embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, which can be obtained commercially by those skilled in the art unless otherwise specified.

[0032] This invention provides a method for removing antibiotic resistance genes from straw sludge compost, the method comprising the following steps:

[0033] S1. Mix municipal sludge with auxiliary materials evenly to obtain a mixture, wherein: the auxiliary materials are any one or more of common agricultural solid wastes (such as sawdust, straw, rice husks, etc.; if straw powder is used, it needs to be crushed to a diameter of 1-2 mm).

[0034] Aerobic composting requires suitable moisture content, C / N ratio, and oxygen supply to ensure the composting process. Adding agricultural solid waste such as sawdust, straw, and rice husks can effectively regulate the carbon source of the composting materials, reduce moisture content, increase the porosity of the compost pile, and optimize the composting process. Generally, the mixing ratio of municipal sludge and auxiliary materials is determined based on the moisture content and carbon-nitrogen ratio of the mixture. By calculating a reasonable ratio, the moisture content and carbon-nitrogen ratio of aerobic composting can reach the optimal range, that is, the moisture content is 50% to 60%, and the carbon-nitrogen ratio (C / N) is 25:1 to 30:1.

[0035] S2. The mixture obtained from S1 is subjected to aerobic composting, wherein:

[0036] Generally speaking, the temperature during the warming period of aerobic composting is between the initial temperature of the mixed materials and 50°C. During the high-temperature period of composting, it is necessary to maintain a temperature above 55°C for 3 days or above 50°C for 5 to 7 days to ensure that harmful microorganisms in the materials are effectively killed and rendered harmless.

[0037] During aerobic composting, the moisture content and carbon-to-nitrogen ratio are not separately controlled, and the aeration air volume is controlled between 0.05 and 0.2 m³ / s. 3 / min (based on each cubic meter of material), the turning frequency is adjusted according to the temperature of the pile and the discharge situation during actual operation;

[0038] S3. After the high-temperature period of composting ends, ozone is introduced into the composting system when the temperature drops to 40-47℃. After ozone oxidation is completed, the compost pile normally completes the cooling and maturation period. After the maturation period, various resistance genes in the samples are measured, including:

[0039] Ozone is introduced into the compost pile under positive pressure from the bottom. The total ozone dosage is 0.4–0.6 g / kg of compost material, and the overall ozone dosage time is 30–150 min. The ozone air volume and concentration are adjusted according to the ozone dosage time.

[0040] When the temperature of the pile body drops to room temperature, it reaches the maturity period, which generally requires a maturity period of 7 days or more.

[0041] The aeration rates during the cooling and composting periods are consistent with those during the heating and high-temperature periods.

[0042] The aforementioned resistance genes include tetracycline tetM, sulfonamide sul1, aminoglycoside aphA1, macrolide mefA, and the mobile gene element Tn916.

[0043] Example 1:

[0044] In this embodiment, the composting raw material used in the composting experiment was municipal sewage sludge, taken from a large sewage treatment plant in Harbin City, with a moisture content of 74.3%. The auxiliary material regulator was corn stalks with a moisture content of 10.2%. This embodiment was conducted in the laboratory, and various resistance genes in compost samples that met the maturity period were monitored, including tetracycline tetM, sulfonamide sul1, aminoglycoside aphA1, macrolide mefA, and the mobile gene element Tn916. The specific operation steps are as follows:

[0045] Step 1: Mix municipal sludge (74.3% moisture content) and corn stalks (10.2% moisture content, crushed to a diameter of 1-2 mm) in a 5:1 (by weight) ratio. At this point, the moisture content of the mixture will be approximately 60%.

[0046] Step 2: Place the mixture obtained in Step 1 into a composting reactor for aerobic composting. Do not adjust the moisture content or carbon-nitrogen ratio of the material during the composting process. Control the aeration rate at 0.08 L / min / kg of material. Manually turn the pile once every 5 days during the heating and high-temperature periods, and once every 7 days during the cooling and decomposition periods.

[0047] Step 3: When the composting process exhibits distinct heating, high-temperature, cooling, and maturation phases, and the pile temperature remains above 55℃ for 3 consecutive days or above 50℃ for 5-7 consecutive days, the high-temperature phase can be considered successfully completed. After the high-temperature phase, when the pile temperature drops to 40℃, ozone is introduced into the composting system using an ozone generator. Specifically, ozone is introduced into the pile through a positive pressure system at the bottom. The total ozone dosage is 0.4g / kg of compost material, the ozone flow rate is controlled at 3L / min, the ozone content is 40mg / kg, and the reaction time is 30min. After ozone oxidation is completed, the pile completes cooling and maturation (the maturation period requires 7 days). The aeration rate is consistent with that during the heating and high-temperature phases.

[0048] Comparative example:

[0049] The composting material used in the comparative composting experiment was municipal sewage sludge, taken from a large wastewater treatment plant in Harbin, with a moisture content of 74.3%. The auxiliary material regulator was corn stalks with a moisture content of 10.2%. This comparative example was conducted in the laboratory, and various resistance genes in compost samples that met the maturity period were monitored, including tetracycline tetM, sulfonamide sul1, aminoglycoside aphA1, macrolide mefA, and the mobile gene element Tn916. The difference between this comparative example and Example 1 is that ozone was not introduced. The specific operating steps of the comparative example are as follows:

[0050] Step 1: Mix municipal sludge (74.3% moisture content) and corn stalks (10.2% moisture content, crushed to a diameter of 1-2 mm) in a 5:1 (by weight) ratio. At this point, the moisture content of the mixture will be approximately 60%.

[0051] Step 2: Place the mixture obtained in Step 1 into a composting reactor for aerobic composting. Do not adjust the moisture content or carbon-nitrogen ratio of the material during the composting process. Control the aeration rate at 0.08 L / min / kg of material. Manually turn the pile once every 5 days during the heating and high-temperature periods, and once every 7 days during the cooling and decomposition periods.

[0052] Step 3: When the composting process exhibits distinct heating, high-temperature, cooling, and maturation phases, and the pile temperature remains above 55℃ for 3 consecutive days or above 50℃ for 5-7 consecutive days, the high-temperature phase can be considered successfully completed. Then, the pile completes cooling and maturation (the maturation phase takes 7 days), and the aeration rate is consistent with that of the heating and high-temperature phases.

[0053] During the composting process, the content of various resistance genes in the compost samples obtained from the examples (experimental group) and the comparative examples (control group) at various time points was measured.

[0054] At the end of composting, there were significant differences in ARGs in the compost obtained from the experimental group and the control group. Figure 1 , Figure 2 and Figure 3 It was found that at the start of composting, the relative abundance of total ARGs in the control group was 0.297 copies / 16S rRNA, while in the experimental group it was 0.275 copies / 16S rRNA. At the end of composting, the relative abundance of total ARGs in the control group was 0.046 copies / 16S rRNA, while in the experimental group it was 0.016 copies / 16S rRNA. This shows that the experimental group reduced ARGs by 94.15%, while the control group reduced them by 84.51%. During the period from day 14 to day 30 of composting, the experimental group continued to reduce ARGs by 33.33%, while the control group showed a rebound of 70.37%.

[0055] The above experimental results demonstrate that ozone treatment can effectively reduce the abundance of total ARGs at the end of composting. In Example 1, after ozone treatment, only three target ARGs were detected at the end of composting: tetracycline tetM, aminoglycoside aphA1, and macrolide mefA. The mobile gene element Tn916 and the sulfonamide sul1 gene were below detection levels and became undetectable (see [link to example 1]). Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 This indicates that ozone post-treatment can effectively reduce the types of ARGs, control the rebound of ARGs in the later stages of composting, and reduce the risk of ARGs being horizontally transferred to other microorganisms and pathogens.

[0056] In summary, this invention can effectively reduce ARGs by utilizing the high temperature of the compost pile during the high-temperature period of composting. Furthermore, to address the issue of increased ARG abundance during the composting maturation period, it combines ozone oxidation technology to further reduce ARGs in compost materials, inhibit the rebound of ARGs during the composting maturation period, and ultimately reduce the abundance of ARGs in compost products. It also reduces the frequency of microbial community integration and transformation at the end of composting, thereby reducing the potential health risks posed by compost after its application to the land.

[0057] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for removing antibiotic resistance genes from straw sludge compost, characterized in that, Includes the following steps: S1. Mix municipal sludge and auxiliary materials evenly to obtain a mixture; S2. The mixture obtained in S1 is subjected to aerobic composting; S3. After the high-temperature period of composting ends, ozone is introduced into the composting system when the temperature drops to 40-47℃. The total ozone dosage is 0.4-0.6g / kg of compost material. After ozone oxidation is completed, the compost pile will normally complete the cooling and maturation period.

2. The method according to claim 1, characterized in that, The auxiliary material mentioned in S1 is agricultural solid waste.

3. The method according to claim 2, characterized in that, The agricultural solid waste is any one or a combination of two or more of sawdust, straw, and rice husks.

4. The method according to claim 1, characterized in that, The mixture described in S1 has a pH of 6-8, a water content of 50%-60%, and a carbon-to-nitrogen ratio of 25:1-30:

1.

5. The method according to claim 1, characterized in that, The temperature during the warming period of the aerobic composting described in S2 is between the initial temperature of the mixed materials and 50°C, with the high-temperature period lasting for 3 days above 55°C or 5-7 days above 50°C.

6. The method according to claim 1, characterized in that, The aeration air volume for the aerobic composting described in S2 is controlled at 0.05–0.2 m³ / s. 3 / min.

7. The method according to claim 1, characterized in that, The ozone described in S3 is introduced into the reactor body through positive pressure from the bottom of the reactor body, and the overall ozone injection time is 30 to 150 minutes.

8. The method according to claim 1, characterized in that, The decomposition period described in S3 is at least 7 days.

9. The application of the method according to any one of claims 1-8 in the treatment of municipal sludge.

10. Compost obtained by the method described in any one of claims 1-8.

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