Method for strengthening removal of antibiotic resistance genes and promoting humification in aerobic composting

Abstract

The application discloses a method for strengthening removal of antibiotic resistance genes and promoting humification in aerobic composting, and belongs to the field of organic solid waste treatment. The application aims to solve the problems of incomplete reduction of ARGs in the existing aerobic composting method, easy rebound of ARGs in the cooling period, and low humification degree. The method comprises the following steps: 1, preparing a mixture; 2, preparing a composite slurry; and 3, adding the composite slurry into the mixture to perform aerobic composting. The porous structure of the attapulgite is used to provide a loading site for microorganisms, and the fulvic acid is used as an electron shuttle to regulate the metabolic network of the microorganisms. The two cooperate to continuously reduce the abundance and horizontal transfer potential of antibiotic resistance genes in the composting process, and significantly promote the humification process and increase the ratio of humic acid / fulvic acid. The application effectively reduces the spread risk of antibiotic resistance genes in the composting product, reduces the health threat after the land use of the composting, and improves the composting maturity and quality.

Description

Technical Field

[0001] This invention belongs to the field of organic solid waste treatment, specifically relating to a method for enhancing the removal of antibiotic resistance genes and promoting humification in aerobic composting. Background Technology

[0002] With the rapid development of large-scale livestock and poultry farming, antibiotics are widely used in the prevention and control of livestock and poultry diseases and in the growth promotion process, resulting in a large amount of antibiotic residues in livestock and poultry manure. These residual antibiotics create continuous selective pressure in the environment, inducing the generation of antibiotic resistance genes (ARGs) and the enrichment and proliferation of antibiotic-resistant bacteria (ARBs). ARGs spread in the environment mainly through two pathways: vertical gene transfer (VGT) and horizontal gene transfer (HGT). Horizontal gene transfer, especially mediated by mobile genetic elements (MGEs), allows ARGs to enter pathogenic bacteria, reducing the inhibitory effect of antibiotics on these bacteria. Ultimately, through the "soil-plant-animal-human" transmission chain, ARGs threaten human health. This is one of the core issues addressed by the One Health concept and has become a bottleneck restricting the green recycling of organic solid waste.

[0003] ARGs and ARBs contained in livestock and poultry manure have become major risks to its safe application as organic fertilizer. To control the spread of ARGs at its source and reduce ecological and public health risks, numerous studies have focused on ARGs reduction technologies during the resource-based treatment of livestock and poultry manure. Aerobic composting is an important way to achieve the harmless treatment and resource utilization of organic solid waste such as livestock and poultry manure and traditional Chinese medicine residues. The high-temperature stage of aerobic composting can significantly reduce the total abundance of ARGs and inactivate some resistant microorganisms, and is considered the core critical period for reducing ARGs abundance during composting. Current research often uses exogenous additives such as biochar and zero-valent iron to prolong the high-temperature duration of composting and enhance the ARGs removal effect. However, existing conventional exogenous additive regulation methods generally have obvious limitations. They cannot effectively control the occurrence and diffusion of MGEs in the composting system from the source, nor can they achieve long-term inhibition and inactivation of potential host microorganisms of ARGs. They cannot fundamentally block the horizontal transfer pathway of resistance genes, and ultimately cannot avoid the engineering pain point of ARG abundance rebound during the composting cooling stage, which seriously limits the safe return of compost products to the field and their resource utilization.

[0004] Currently, conventional aerobic composting and existing control technologies generally suffer from multiple problems, including incomplete ARGs reduction, easy rebound of ARGs abundance during the cooling period, slow composting humification process, long maturation cycle, and insufficient humus accumulation. These issues make it difficult to simultaneously address the dual technical requirements of ARGs risk management and efficient humification of organic materials. Therefore, there is an urgent need for a composite control application method that can effectively reduce ARGs abundance throughout the entire aerobic composting process, stably suppress ARGs rebound during the cooling period, and simultaneously accelerate the humification process of materials, improve the maturation quality of compost products, and enhance resource utilization safety. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of incomplete ARGs reduction, abundant ARGs during the cooling period and easy rebound, and low degree of humification in existing aerobic composting methods, and to provide a method for enhancing the removal of antibiotic resistance genes and promoting humification in aerobic composting.

[0006] A method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting, the specific steps of which are as follows:

[0007] 1. Mix fresh pig manure and Chinese herbal medicine residue evenly to obtain a mixture;

[0008] 2. Weigh out 5% of the dry weight of the mixture of attapulgite and 0.2% of the dry weight of the mixture, then add the weighed attapulgite and fulvic acid to water, stir and age to form a composite slurry.

[0009] 3. Add the composite slurry to the mixture and adjust the moisture content of the compost to 60%-65%. Then carry out aerobic composting. During the aerobic composting process, the removal of antibiotic resistance genes is enhanced and humification is promoted. After the composting is completed, organic fertilizer is obtained.

[0010] The principle of this invention:

[0011] Fulvic acid (FA) is a natural small-molecule organic active substance with excellent adsorption, complexation, and chelation properties. It can act as an electron shuttle to participate in regulating microbial metabolism and material-energy conversion processes within the compost micro-ecosystem. Related studies have confirmed that adding lignite materials to aerobic composting, with fulvic acid as its core functional active component, can effectively reduce the abundance of ARGs (antibiotic-resistant genes). Attapulgite is a natural layered chain clay mineral with a large specific surface area, well-developed pore structure, and strong adsorption stability. It can retain residual antibiotics, drug-resistant bacteria, and extracellular resistance genetic material through physical adsorption and chemical complexation. The combined application of FA and attapulgite can fully leverage the synergistic regulatory effects of organic active components and inorganic mineral materials. On the one hand, it enhances the adsorption and fixation of residual antibiotics, ARGs, and MGEs (including intⅠ1 and Tn916 / 1545b), inhibits horizontal gene transfer among microorganisms, and achieves efficient reduction of ARGs and inhibition of rebound during the cooling period. On the other hand, it optimizes the microbial community structure of compost, regulates the metabolic activity of key microbial groups in humus synthesis, accelerates the humification process of materials, and improves the maturity and quality of compost products.

[0012] Compared with existing technologies, the method for enhancing the removal of antibiotic resistance genes and promoting humification in aerobic composting according to the present invention has the following beneficial effects:

[0013] I. This invention utilizes the porous structure of attapulgite soil to provide loading sites for microorganisms, while using fulvic acid as an electron shuttle to regulate the microbial metabolic network. The two work synergistically to continuously reduce the abundance and horizontal transfer potential of antibiotic resistance genes during composting, and significantly promote the humification process, increasing the humic acid / fulvic acid ratio. This invention effectively reduces the risk of antibiotic resistance gene transmission in compost products, reduces health threats after compost land use, and improves compost maturity and quality.

[0014] II. This invention significantly enhances the removal of antibiotic resistance genes and promotes humification in aerobic composting. Specifically, in Example 1, the relative abundance of ARGs during the high-temperature composting period was reduced by 62.4% compared to the control; in Example 1, the relative abundance of ARGs during the cooling composting period was reduced by 62.8% compared to the control (among which, the relative abundance of sulfonamide resistance genes decreased by 75.0%, tetracycline resistance genes by 82.1%, and macrolide resistance genes by 78.6%; in Example 1, the relative abundance of β-lactam resistance genes was below the detection limit, with a removal rate of 99.9%); in Example 1, the abundance of mobile genetic elements was significantly reduced during the cooling composting period compared to the control group (among which, the relative abundance of intⅠ1 decreased by 73.7%, and the relative abundance of Tn916 / 1545b decreased by 27.9%); at the end of composting in Example 1, the HS content of humic substances increased by 17.6% compared to the control, the HA content of humic acid increased by 27.6% compared to the control, and the HA / FA ratio of humic acid increased by 26.4% compared to the control.

[0015] Third, this invention is simple to operate, produces no secondary pollution, and significantly enhances the removal of antibiotic resistance genes in aerobic composting. It effectively inhibits the rebound of antibiotic resistance gene abundance during the composting cooling period, improves the composting quality, and has important practical significance for ensuring the agricultural safety of compost products and promoting the development of the green circular economy of organic solid waste. Attached Figure Description

[0016] Figure 1 This is a comparison chart of the relative abundance changes of antibiotic resistance genes in the FA and attapulgite soil combined addition group and the control group during the composting process of Example 1.

[0017] Figure 2 This is a comparison chart of the relative abundance changes of mobile genetic elements in the FA and attapulgite soil combined addition group and the control group during the composting process of Example 1;

[0018] Figure 3 This is a comparison chart of the changes in humus HS content and Hufu ratio HA / FA in the FA and attapulgite combined addition group and the control group during the composting process of Example 1. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Specific Implementation Method 1: This implementation method is a method for enhancing the removal of antibiotic resistance genes and promoting humification in aerobic composting. The specific steps are as follows:

[0021] 1. Mix fresh pig manure and Chinese herbal medicine residue evenly to obtain a mixture;

[0022] 2. Weigh out 5% of the dry weight of the mixture of attapulgite and 0.2% of the dry weight of the mixture, then add the weighed attapulgite and fulvic acid to water, stir and age to form a composite slurry.

[0023] 3. Add the composite slurry to the mixture and adjust the moisture content of the compost to 60%-65%. Then carry out aerobic composting. During the aerobic composting process, the removal of antibiotic resistance genes is enhanced and humification is promoted. After the composting is completed, organic fertilizer is obtained.

[0024] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the main component of the medicinal residue mentioned in step one is Polygonum multiflorum vine, supplemented with Acanthopanax senticosus and Pueraria lobata, wherein the content of the main component Polygonum multiflorum vine is greater than 70%. Other steps are the same as in Specific Implementation Method One.

[0025] Specific Implementation Method Three: The difference between this implementation method and Specific Implementation Method One or Two is that the mass ratio of pig manure and Chinese medicine residue mentioned in step one, based on dry weight, is 1:(8-10); the particle size of the mixture mentioned in step one is less than 2cm. Other steps are the same as in Specific Implementation Method One or Two.

[0026] Specific Implementation Method Four: The difference between this implementation method and one of Specific Implementation Methods One to Three is that the carbon-nitrogen ratio of the mixture in step one is (25-30):1. The other steps are the same as in Specific Implementation Methods One to Three.

[0027] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that: the concentration of the composite slurry mentioned in step two is 250-300 g / L; the aging time mentioned in step two is 24h~36h. Other steps are the same as in Specific Implementation Methods One to Four.

[0028] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that the antibiotic resistance genes mentioned in step three include sulfonamides, tetracyclines, macrolides, β-lactams, and quinolones. Its distinguishing feature is that the other steps are the same as in Specific Implementation Methods One to Five.

[0029] Specific Implementation Method Seven: The difference between this implementation method and Specific Implementation Methods One through Six is ​​that during the aerobic composting process described in step three, the compost pile is turned over every 2-3 days. During the high-temperature period, an appropriate amount of water is added daily to maintain the moisture content of the compost pile at 60%-65%. The other steps are the same as in Specific Implementation Methods One through Six.

[0030] Specific Implementation Method Eight: The difference between this implementation method and Specific Implementation Methods One through Seven is that the heating period of aerobic composting is when the temperature of the compost pile rises from the ambient temperature to 50°C, and the high-temperature period is when the temperature of the compost pile remains above 55°C for 3 days or above 50°C for 5-7 days; the cooling period begins when the temperature drops below 40°C from the high-temperature period. Other steps are the same as in Specific Implementation Methods One through Seven.

[0031] Specific Implementation Method Nine: The difference between this implementation method and Specific Implementation Methods One to Eight is that the aerobic composting process lasts for 15-25 days. The other steps are the same as in Specific Implementation Methods One to Eight.

[0032] Specific Implementation Method Ten: The difference between this implementation method and Specific Implementation Methods One to Nine is that the aeration rate is maintained at 2 L / min during the aerobic composting process. The other steps are the same as those in Specific Implementation Methods One to Nine.

[0033] The beneficial effects of the present invention are verified using the following embodiments:

[0034] Example 1: A method for enhancing the removal of antibiotic resistance genes and promoting humification in aerobic composting, the specific steps of which are as follows:

[0035] 1. Take 0.5 kg of pig manure (dry weight) and 4 kg of Chinese herbal medicine residue (dry weight), crush all materials to a particle size of less than 2 cm, mix them evenly to obtain a mixture.

[0036] The main component of the Chinese medicine residue mentioned in step one is Polygonum multiflorum vine, supplemented with Acanthopanax senticosus and Pueraria lobata, wherein the mass ratio of Polygonum multiflorum vine, Acanthopanax senticosus and Pueraria lobata is 8:1:1.

[0037] 2. Weigh out 5% of the dry weight of the mixture of attapulgite and 0.2% of the fulvic acid, then add the weighed attapulgite and fulvic acid to water, stir and age for 24 hours to form a composite slurry with a concentration of 300 g / L. Add the composite slurry to the mixture of the mixture in step 1, and adjust the initial moisture content of the aerobic compost to 65% and the initial carbon-nitrogen ratio to 27:1.

[0038] 3. Aerobic composting of materials is carried out in a 70L foam box. An air pump is used to aerate the pile, maintaining an aeration rate of 2L / min. The heating period of aerobic composting is from ambient temperature to 50℃. The high-temperature period is when the pile temperature is above 55℃ for 3 days or above 50℃ for 5-7 days. The temperature drops below 40℃ from the high-temperature period to enter the cooling period. The aerobic composting process lasts for 18 days. Depending on the condition of the pile, it is turned over every 2 days. During the high-temperature period, an appropriate amount of water is added daily to keep the moisture content of the pile at 60%-65%. The aerobic composting process strengthens the removal of antibiotic resistance genes and promotes humification. After composting, organic fertilizer is obtained.

[0039] Samples were taken at different stages of the composting process in Example 1 using a five-point sampling method. The collected samples were used to determine the abundance of antibiotic resistance genes, the abundance of mobile genetic elements, and the content of humic substances and humic acid. The antibiotic resistance genes included sulfonamides (sul1, sul2, and sul3), tetracyclines (tetG, tetW, and tetX), macrolides (ermB and ermF), β-lactams (blaTEM and blaCTX-M), and quinolones (gyrA, parC, aac(6')-Ⅰb-cr, and qnrS); the mobile genetic elements included intⅠ1 and Tn916 / 1545b.

[0040] Control group:

[0041] The aerobic composting method used in this comparative example is as follows:

[0042] The composting materials, composting equipment, and sampling methods in the comparative example are the same as those in the example, except that no additional composite slurry of attapulgite and fulvic acid is added. In other words, step two in example 1 is changed to: adjusting the initial moisture content of the mixture to 65% and the initial carbon-nitrogen ratio to 27:1.

[0043] Methods for determining resistance genes and mobile genetic elements: DNA was extracted using a soil genomic DNA extraction kit (TIANGEN-A0813A), followed by quantitative analysis of the abundance of target resistance genes and mobile genetic elements using a BIO-RAD CFX96 real-time quantitative PCR system. The relative abundance of target genes was normalized to the 16S rRNA gene copy number to eliminate the interference of environmental background differences between different samples on the gene abundance quantification results.

[0044] like Figure 1 As shown, the relative abundance of ARGs during the high-temperature period of composting in Example 1 was reduced by 62.4% compared to the control group, and the relative abundance of ARGs during the cooling period of composting in Example 1 was reduced by 62.8% compared to the control group (among which the relative abundance of sulfonamide resistance genes decreased by 75.0%, tetracycline resistance genes decreased by 82.1%, macrolide resistance genes decreased by 78.6%, and the relative abundance of β-lactam resistance genes in Example 1 was below the detection line, with a removal rate of 99.9%). The above results indicate that the enhanced ARGs reduction method provided by the present invention can continuously and efficiently reduce multiple ARGs throughout the entire composting process, effectively suppress the rebound phenomenon of ARGs abundance during the cooling period, and significantly reduce the risk of resistance gene transmission in compost products.

[0045] like Figure 2 As shown, the relative abundance of the integron gene intⅠ1 decreased by 73.7%, and the relative abundance of the transposon gene Tn916 / 1545b decreased by 27.9%. MGEs are key vectors mediating the horizontal transfer of antibiotic resistance genes; their reduced abundance directly weakens the potential of ARGs to undergo horizontal gene transfer events such as conjugation, transformation, and transduction within compost microbial communities. These results demonstrate that the method of this invention can not only effectively reduce the absolute abundance of ARGs but also significantly inhibit their horizontal transmission risk, blocking the diffusion pathway of resistance genes at the source during the composting cooling period and subsequent land use.

[0046] Methods for determining humic substances (HS) and humic acid (HA): Weigh 2g of compost samples after grinding during the heating, high-temperature, and cooling periods, respectively. Add 20mL of alkaline extraction solution (0.1mol / L NaOH and 0.1mol / L sodium pyrophosphate mixed in a 1:1 volume ratio), shake at room temperature for 2h, centrifuge at 4000rpm for 10min, and collect the supernatant. Repeat the extraction three times with the residue, and combine the supernatants to obtain the total humic substances (HS) extract. Take a certain volume of the HS solution, acidify it with concentrated hydrochloric acid to pH 1.0-2.0, let it stand overnight, centrifuge, and the precipitate is humic acid (HA), which is dissolved and diluted to volume with 0.1mol / L KOH. Use a TOC analyzer to determine the carbon content of HS and HA, and calculate their concentrations.

[0047] like Figure 3 As shown, compared with the control group, the HS content of Example 1 increased by 17.6%, the HA content increased by 27.6%, and the humic acid-to-fatty acid (HA / FA) ​​ratio increased by 26.4%. In the early stages of composting, Example 1 had a lower initial HA / FA ratio than the control group due to the exogenous addition of 0.2% fulvic acid (FA). However, as the composting process progressed, the HA / FA ratio of Example 1 gradually surpassed and remained higher than the control group after the high-temperature period. This "low at first, then high" trend fully demonstrates that the exogenously added FA did not remain in the composting system in a free form for a long time, but was effectively transformed by microorganisms into humic acid with a more complex structure and larger molecular weight under the synergistic effect of attapulgite soil. As a natural small-molecule organic active substance, FA has excellent adsorption, complexation and chelation properties. It can act as an electron shuttle to participate in the regulation of microbial metabolism and material and energy conversion processes in the compost micro-ecosystem, and promote the biocondensation of FA to HA. Attapulgite, as a natural layered chain clay mineral, has the characteristics of large specific surface area, well-developed pore structure and strong adsorption stability. It can provide stable loading sites for functional microorganisms. The combined application of the two fully leverages the synergistic regulatory effect of organic active components and inorganic mineral materials, thereby accelerating the conversion of fulvic acid to humic acid, increasing the HA / FA ratio, promoting composting maturity and significantly improving the quality of humus.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting, characterized in that, The specific steps of the method are as follows:

1. Mix fresh pig manure and Chinese herbal medicine residue evenly to obtain a mixture; 2. Weigh out 5% of the dry weight of the mixture of attapulgite and 0.2% of the dry weight of the mixture, then add the weighed attapulgite and fulvic acid to water, stir and age to form a composite slurry.

3. Add the composite slurry to the mixture and adjust the moisture content of the compost to 60%-65%. Then carry out aerobic composting. During the aerobic composting process, the removal of antibiotic resistance genes is enhanced and humification is promoted. After the composting is completed, organic fertilizer is obtained.

2. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 1, characterized in that... The main component of the Chinese medicine residue mentioned in step one is Polygonum multiflorum vine, supplemented with Acanthopanax senticosus and Pueraria lobata, with the content of the main component Polygonum multiflorum vine being greater than 70%.

3. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 1, characterized in that... The mass ratio of pig manure and Chinese medicine residue mentioned in step one, based on dry weight, is 1:(8-10); the particle size of the mixture mentioned in step one is less than 2cm.

4. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 1, characterized in that... The carbon-to-nitrogen ratio of the mixture in step one is (25-30):

1.

5. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 1, characterized in that... The concentration of the composite slurry mentioned in step two is 250-300 g / L; the aging time mentioned in step two is 24h~36h.

6. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 1, characterized in that... The antibiotic resistance genes mentioned in step three include sulfonamides, tetracyclines, macrolides, β-lactams, and quinolones.

7. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 1, characterized in that... During the aerobic composting process described in step three, the compost pile should be turned over every 2-3 days. During periods of high temperature, an appropriate amount of water should be added daily to maintain the moisture content of the compost pile at 60%-65%.

8. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 7, characterized in that... The warming period of aerobic composting is when the temperature of the compost pile rises from the ambient temperature to 50°C. The high-temperature period is when the temperature of the compost pile remains above 55°C for 3 days or above 50°C for 5-7 days. The cooling period begins when the temperature drops below 40°C from the high-temperature period.

9. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 7, characterized in that... The aerobic composting process lasts 15-25 days.

10. The method for enhancing antibiotic resistance gene removal and promoting humification in aerobic composting according to claim 7, characterized in that... Maintain an aeration rate of 2 L / min during the aerobic composting process.

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