Method for intensively removing antibiotic resistance genes in composting process

Abstract

The invention provides a method for intensively removing antibiotic resistance genes in a composting process, and belongs to the technical field of solid waste treatment. The method comprises the following steps: performing aerobic composting on compost materials, and when the temperature in a compost body is reduced to 38-42 DEG C, inoculating a microbial preparation; the microbial preparation is a microbial agent with a function of promoting degradation of organic matters. Based on the principle that a microbial preparation is inoculated in a compost cooling period (38-42 DEG C), a microbial community structure is changed, and ARGs host bacterium proliferation is effectively inhibited, the effect of strengthening removal of resistance genes in the compost process is remarkable, operation is easy and convenient, control and implementation are easy, secondary pollution cannot be caused in the whole process, and the method is suitable for industrial production. The method has a wide prospect of popularization and application in a large range.

Description

Technical Field

[0001] The invention relates to the technical field of solid waste treatment, in particular to a method for strengthening the removal of antibiotic resistance genes in a composting process. Background Art

[0002] Antibiotic resistance genes (ARGs) are considered a ubiquitous environmental pollutant. The global livestock industry uses a large amount of antibiotics, of which more than 70% are not completely metabolized by animals and enter the environment through excrement, making livestock and poultry manure an important storage source of ARGs. Aerobic composting, as a widely used method for treating livestock and poultry manure, has the ability to remove ARGs. However, even if only a small amount of ARGs enters the environment, it may bring significant risks of continued evolution and spread. Therefore, in the process of using composting to treat livestock and poultry waste, strict control and management must be carried out to prevent the further spread and harm of ARGs.

[0003] The core mechanism of composting is to promote the biochemical transformation of organic matter with the help of microbial decomposition. In this process, bacterial communities play a core role in regulating the dynamic changes of ARGs. In order to effectively reduce the threat posed by ARGs, an effective strategy is to strengthen the competitive relationship between host microorganisms and non-host microorganisms, thereby promoting the elimination of potential host microorganisms. According to research, compared with traditional composting methods, the removal efficiency of ARGs can be further improved by inoculating microbial preparations, with an increase of 21.5% to 35.6%. Despite this, the overall removal rate of ARGs by these microbial preparations is generally maintained in the range of 60%-85%, indicating that there is still a lot of room for improvement in further improving the removal efficiency. Therefore, there is an urgent need for a technology that can enhance the removal of ARGs during composting. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for enhancing the removal of antibiotic resistance genes in the composting process, to solve the problem of limited efficiency of resistance gene removal, and to reduce the potential risks to human health after land use.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A method for enhanced removal of antibiotic resistance genes during composting, wherein composting materials are aerobically composted, and when the temperature inside the compost body drops to 38-42°C, a microbial preparation is inoculated; the microbial preparation is a bacterial agent with the function of promoting organic matter degradation.

[0007] Preferably, the compost material includes livestock and poultry manure and auxiliary materials; the livestock and poultry manure includes one or more of pig manure, chicken manure, sheep manure or cow manure, and the auxiliary materials include one or more of wheat straw, corn straw, rice straw or wood chips.

[0008] More preferably, the moisture content of the compost material is 50%-60%, and the C / N is 20-40.

[0009] Preferably, during the aerobic composting process, the compost is turned every 2-3 days during the initial stage and the high temperature stage.

[0010] More preferably, the high temperature period refers to the temperature inside the pile rising to above 50°C and being maintained for no less than 5 days.

[0011] Preferably, the microbial preparation is selected from one or more of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus megaterium, Bacillus polymyxa, Bacillus gelatinosa, Bacillus licheniformis, Bacillus velezensis, Bacillus faecalis, lactic acid bacteria, Brucella, white rot fungi and Aspergillus niger.

[0012] Preferably, the viable count of the microbial preparation is not less than 1×10 9 / g, and the inoculation amount is 0.5%-1% of the mass of the compost material.

[0013] More preferably, the microbial preparation is in the form of powder and is evenly mixed with the compost material.

[0014] Preferably, within 7 days after the inoculation of the microbial preparation, the compost is turned every 3-5 days; thereafter, the compost is turned every 7 days until the composting is completed.

[0015] The invention also provides the organic fertilizer prepared by the method.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention is based on the principle of inoculating microbial preparations during the compost cooling period (38-42°C) to change the microbial community structure and effectively inhibit the proliferation of ARGs host bacteria, and has a significant effect on strengthening the removal of resistance genes during the composting process.

[0018] (2) Inoculating microbial preparations during the cooling period not only has a positive effect on promoting the secondary fermentation of the compost mixture, but also can effectively prolong the duration of the high temperature stage, thereby continuously eliminating pathogens and other harmful substances.

[0019] (3) The treatment method of the present invention is simple to operate, easy to control and implement, and will not cause secondary pollution during the entire process, and has broad prospects for promotion and application on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of temperature change during composting process in Example 1 and Comparative Example 1;

[0021] Figure 2 Schematic diagram of changes of four common resistance genes during the composting process of Example 1 and Comparative Example 1;

[0022] Figure 3 Schematic diagram of temperature change during composting process in Example 2 and Comparative Example 2;

[0023] Figure 4 Schematic diagram of the changes of four common resistance genes during the composting process of Example 2 and Comparative Example 2. DETAILED DESCRIPTION

[0024] The present invention provides a method for enhancing the removal of antibiotic resistance genes in a composting process, comprising the following steps:

[0025] (1) Material pretreatment: sorting and removing livestock and poultry manure and auxiliary materials.

[0026] In the present invention, preferably, livestock and poultry manure includes one or more of pig manure, chicken manure, sheep manure or cow manure; preferably, the auxiliary material includes one or more of wheat straw, corn straw, rice straw or sawdust, and further preferably, the auxiliary material is crushed to a length of <3 cm.

[0027] (2) Aerobic composting: The compost material prepared in step (1) is mixed and the C / N ratio and moisture content are adjusted to carry out conventional aerobic composting. The material is turned over regularly to ensure sufficient oxygen, so that the compost temperature is raised to the high temperature stage smoothly and maintained until it enters the cooling stage naturally.

[0028] In the present invention, the moisture content of the compost material is preferably 50%-60%, more preferably 52%-58%, and more preferably 55%; the C / N of the compost material is preferably 20-40, more preferably 25-35, and more preferably 30.

[0029] In the present invention, the compost is preferably turned every 2-3 days during the initial stage and high temperature stage of aerobic composting, and more preferably every 2 days; the high temperature stage (compost temperature ≥ 50°C) is preferably maintained for no less than 5 days.

[0030] (3) Inoculation of microbial preparations: When the temperature in the compost drops from the high temperature period to 38-42°C, preferably to 40°C, inoculate the microbial preparation into the compost material, turn the compost over, and stir to mix. The microbial preparation of the present invention is a bacterial agent that has the function of promoting the degradation of organic matter. Such bacterial agents can quickly decompose organic matter in the compost, such as cellulose, hemicellulose, and protein. By secreting various enzymes, they can convert complex organic matter into simple compounds, providing sufficient energy and nutrients for their own growth and reproduction. The microbial preparations used inhibit the growth of resistance gene-carrying bacteria through competitive effects, while guiding the microbial community to develop in a direction that is conducive to the removal of resistance genes, and improving the composting environment to accelerate this process. These mechanisms work together to effectively remove antibiotic resistance genes in compost.

[0031] In the present invention, the microbial preparation is preferably selected from one or more of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus velez, Bacillus megaterium, Bacillus polymyxa, Bacillus gelatinous, Bacillus licheniformis, Bacillus faecalis, lactic acid bacteria, Brucella, white rot fungi and Aspergillus niger. It is further preferred that the number of viable bacteria of the microbial preparation is not less than 1×10 9 The inoculation amount is 0.5%-1% of the mass of the compost material, and more preferably 0.8%. The microbial preparation is preferably a powder. The present invention has no special restrictions on the specific source of the microbial preparation, and it can be obtained through market channels according to demand.

[0032] In the present invention, preferably within 7 days after inoculation of the microbial preparation, the compost is turned every 3-5 days, more preferably every 4 days; thereafter, the compost is turned every 7 days until the composting is completed.

[0033] (4) Obtaining compost products: To ensure the continued efficiency of the composting process, the compost must be turned regularly until the compost has fully entered the mature stage, and finally a fully fermented compost product is obtained that meets the requirements of the NY525-2021 standard.

[0034] The present invention also provides an organic fertilizer prepared by the above method. Compared with other fertilizers, the organic fertilizer prepared by the above method has a lower ARGs content, thereby reducing the potential risk to human health after land use.

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1

[0037] A method for enhanced removal of antibiotic resistance genes in a composting process, comprising the following steps:

[0038] Composting was carried out in the Jilin Agricultural Science and Technology Demonstration Park. The compost material used was cow dung, and the auxiliary material was crushed corn stalks, which were crushed to a length of <3 cm.

[0039] Cow dung and corn stalks were mixed evenly in proportion, the moisture content of the pile was adjusted to 55%, the C / N was 30, and the compost was placed in a 100L composting reactor for composting. During the composting period, the oxygen content was ensured by turning the pile manually. The pile was turned every 2 days during the initial stage and the high temperature period. On the 17th day of composting (the pile temperature was 40°C), 1% Bacillus subtilis inoculant (Bacillus subtilis ATCC6633, purchased from Shanghai Collection Biotechnology Center, with a viable count of ≥1×10 9 / g), mix evenly, turn the compost once on the 20th and 26th days respectively, and once every 7 days thereafter. No turning is done after entering the maturity stage.

[0040] Comparative Example 1

[0041] The same as Example 1, except that 1% Bacillus subtilis inoculant was inoculated on the first day of composting.

[0042] Composting continued for 51 days, and the temperatures of the upper, middle and lower layers of the composts of Example 1 and Comparative Example 1 were measured and recorded at 1:00 p.m. every day. Compost samples were collected from different positions of the middle layer of the compost on the 1st, 5th, 15th, 21st and 51st days after the start of composting and mixed evenly. The mixed samples were stored at -20°C for microbial analysis.

[0043] Macrolide, tetracycline, quinolone and sulfonamide resistance genes are usually present in large quantities in livestock and poultry feces. Therefore, the corresponding resistance genes, including macrolide, tetracycline, chloramphenicol and sulfonamide resistance genes, were detected by qPCR. TM Real-time fluorescence quantitative PCR detection system (Thermo) was used for quantitative analysis by qPCR. The absolute abundance of ARGs in the samples was expressed as the number of copies / g of dry weight (copy number × DNA volume / weighing data g × dilution factor).

[0044] The calculation formula for the removal rate of resistance genes is:

[0045] Removal rate = 1-(P0-P) / P0×100%

[0046] P0: absolute abundance of resistance genes in samples before treatment; P: absolute abundance of resistance genes in samples after treatment.

[0047] Depend on Figure 1It can be concluded that: although the temperature of Example 1 was always lower than that of Comparative Example 1 due to the lack of inoculation of microbial preparations in the early stage, after the inoculation of microbial preparations when the temperature of the pile dropped to 40°C on the 17th day, the compost was promoted to enter secondary fermentation, the internal temperature of the compost in Example 1 increased rapidly, and the continuous degradation of the compost was promoted.

[0048] Depend on Figure 2 It can be concluded that: although the number of resistance genes in Comparative Example 1 decreased significantly at the beginning and high temperature stages of composting, when the composting entered the cooling period, at the end of the composting, the total removal rate of the four types of resistance genes in Comparative Example 1 was 89.34%, and a stronger ARGs elimination effect was obtained in Example 1, with a removal rate of 97.07%. This may be related to the fact that inoculation in the cooling stage can further prolong the composting decomposition process, thereby strengthening the removal of resistance genes during the composting process.

[0049] Example 2

[0050] The same as Example 1, except that 1% of the composite fermentation agent (mixed with Bacillus polymyxa, Bacillus jelly, Bacillus faecalis and lactic acid bacteria, with a viable count of ≥1×10 9 pcs / g, provided by Rural Energy and Ecology Institute of Jilin Academy of Agricultural Sciences).

[0051] Comparative Example 2

[0052] The same as Example 2, except that 1% of the composite fermentation agent is inoculated on the first day of composting.

[0053] Depend on Figure 3 It can be concluded that the temperature of Example 2 and Comparative Example 2 has been in a dynamic fluctuation stage. On the 16th day, the temperature of the compost of Example 2 was 41.5°C. At this time, Example 2 was inoculated with a composite fermentation agent, and the temperature rose to 45.6°C on the 7th day, which also promoted the continuous degradation of the compost.

[0054] Depend on Figure 4 It can be concluded that after the temperature inside the pile decreased on the 16th day, compared with the increase in the number of resistance genes in Comparative Example 2, Example 2 effectively inhibited the proliferation of resistance genes due to the inoculation of the composite fermentation agent until the end of the composting process. On the 51st day, the total removal rate of the four types of resistance genes in Comparative Example 2 was 84.84%, and a stronger ARGs elimination effect was obtained in Example 2, with a removal rate of 93.92%, which also strengthened the removal of resistance genes during the composting process.

[0055] Example 3

[0056] The manure sources used for composting materials are cow dung, chicken dung and sheep dung, and the auxiliary materials are crushed corn stalks and wheat stalks, which are crushed to a length of ≤3cm. The manure and auxiliary materials are evenly mixed in proportion, and the moisture content of the pile is adjusted to 50%, and the C / N is 40, and then loaded into a 100L composting reactor for composting. During composting, the oxygen content is ensured by manual turning of the pile. The pile is turned every 3 days during the initial period and the high temperature period. When the temperature of the pile drops to 42°C, 0.8% of the composite fermentation agent (mixed with Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus Velez, Bacillus megaterium, white rot fungi and Aspergillus niger, with a viable count of ≥1×10 9 / g), mix evenly, turn the compost once on the 3rd and 7th days after inoculating the fermentation bacteria, and then turn the compost once every 7 days. No turning is done after entering the maturity stage.

[0057] Example 4

[0058] The manure used for composting is cow manure and pig manure, and the auxiliary materials are crushed corn stalks, rice stalks and sawdust. The manure and auxiliary materials are mixed evenly in proportion, and the moisture content of the pile is adjusted to 60%, the C / N is 20, and then loaded into a 100L composting reactor for composting. During the composting period, the oxygen content is ensured by manual turning of the pile. The pile is turned every 2 days during the initial period and the high temperature period. When the temperature of the pile drops to 38°C, 0.5% of the composite fermentation agent (mixed with Brucella and Bacillus licheniformis, with a viable count of ≥1×10 9 / g), mix evenly, turn the compost once on the 4th and 7th day after inoculating the fermentation bacteria, and then turn the compost once every 7 days. No turning is done after entering the maturity stage.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for enhancing the removal of antibiotic resistance genes in a composting process, characterized in that: The composting materials are aerobic composted, and when the temperature inside the compost body drops to 38-42° C., a microbial preparation is inoculated; the microbial preparation is a bacterial agent with the function of promoting the degradation of organic matter.

2. The method according to claim 1, characterized in that The composting material includes livestock and poultry manure and auxiliary materials; the livestock and poultry manure includes one or more of pig manure, chicken manure, sheep manure or cow manure, and the auxiliary materials include one or more of wheat straw, corn straw, rice straw or wood chips.

3. The method according to claim 1 or 2, characterized in that: The moisture content of the compost material is 50%-60%, and the C / N is 20-40.

4. The method according to claim 1, characterized in that During the aerobic composting process, the compost is turned every 2-3 days during the initial stage and the high temperature stage.

5. The method according to claim 4, characterized in that The high temperature period refers to the temperature inside the pile rising to above 50°C, and the high temperature period is maintained for no less than 5 days.

6. The method according to claim 1, characterized in that The microbial preparation is selected from one or more of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus megaterium, Bacillus polymyxa, Bacillus gelatinosa, Bacillus licheniformis, Bacillus velezensis, Bacillus faecalis, lactic acid bacteria, Brucella, white rot fungi and Aspergillus niger.

7. The method according to claim 1, characterized in that The number of viable bacteria of the microbial preparation is not less than 1×10 9 / g, and the inoculation amount is 0.5%-1% of the mass of the compost material.

8. The method according to claim 1 or 7, characterized in that: The microbial preparation is in the form of powder and is evenly mixed with the compost material.

9. The method according to claim 1, characterized in that: Within 7 days after inoculation of the microbial preparation, the compost is turned every 3-5 days; thereafter, the compost is turned every 7 days until the composting is completed.

10. An organic fertilizer prepared by the method according to any one of claims 1 to 9.

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