A contamination removal accelerator and use thereof

Abstract

The present application belongs to the technical field of excrement pollution prevention and treatment, and relates to a pollutant removal promoter and application thereof. The present application uses fresh earthworm body fluid and earthworm manure mixture as raw material of the pollutant removal agent, which is low in price, natural, non-toxic, easy to obtain, environmentally friendly, efficient and sustainable, simple in method and easy to operate, and suitable for wide application. The present application not only can utilize the breeding waste in a harmless way in a very short period, but also can simultaneously improve the overall reduction efficiency of nearly 500 new biological pollutants, such as 319 main resistance genes, 57 mobile genetic elements and 120 virulence genes in livestock and poultry manure, and synergistically and efficiently reduce the relative abundance and detection number of most resistance genes and virulence genes in livestock and poultry manure. The reduction rate of the functional genes related to the antibiotic resistance and pathogenic bacteria of human and animals is about 60.95-70.31% and 67.85-80.80%.

Description

Technical Field

[0001] This invention belongs to the field of fecal pollution prevention and control technology, and relates to a pollutant removal promoter and its application. Background Technology

[0002] In recent years, the migration and diffusion of novel biopollutants such as antibiotic resistance genes (ARGs) and pathogenic factor genes (VFGs) have become one of the most severe public health crises globally and a cutting-edge research area and hot topic both domestically and internationally. Pathogenic bacteria carrying ARGs and VFGs in the environment can directly or indirectly infect humans and animals through physical contact and food chain transmission, significantly increasing the risk of disease and death in humans and animals. Livestock and poultry manure is a hotspot for the coexistence of biopollutants such as ARGs and VFGs, and its application to land significantly increases the occurrence and spread of antibiotic resistance in environmental bacteria, especially having a significant impact on the emergence of multidrug-resistant zoonotic pathogens. Therefore, research on the prevention and control of biopollutants such as ARGs and VFGs in livestock and poultry manure has important scientific significance and engineering application value.

[0003] Due to the lack of efficient and reliable quantitative tools, research on the virulence genome of environmental pathogens is still in its early stages. Furthermore, research on fecal antibiotic resistance genomes is mostly limited to removing certain resistance genes in livestock and poultry manure using composting devices or pyrolysis carbonization, lacking a simple and efficient method to simultaneously remove hundreds of ARGs and VFGs. Vermicomposting, as a bio-oxidation technology that utilizes earthworms and various microorganisms under aerobic conditions to stabilize and utilize organic waste, may have significant potential in controlling various pollutants such as human and animal pathogens and ARGs. Therefore, there is an urgent need to develop a method for simultaneously reducing ARGs and VFGs during the earthworm-mediated transformation of livestock and poultry manure.

[0004] Chinese patent application document (publication number: CN108484239A) discloses a method for rapidly reducing resistance gene contamination in livestock and poultry manure. This method requires fermenting the manure to a semi-composted state and adding vitamin C and earthworms. After maintaining this state for 7 weeks, four types of ARGs (anti-reactive genes) are reduced. However, this method requires pre-fermenting the manure and measuring indicators such as C / N ratio to achieve a semi-composted state. It also requires adding vitamin C and earthworms and providing a suitable living environment for the earthworms. Therefore, it necessitates certain earthworm farming, compost preparation, and detection technologies, which not only increases the technical threshold and methodological complexity but also significantly increases the technical cost and the difficulty of widespread adoption.

[0005] Chinese patent application document (publication number: CN109923967A) discloses a method for controlling the diffusion and spread of complex resistance pollutants in vermicompost using biochar. This method reduces the amount of resistance pollutants in vermicompost by adding biochar. However, this method requires the addition of specially formulated biochar, increasing raw material costs and preparation difficulty, and is difficult to reproduce, making it unsuitable for widespread application. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned problems in the prior art by proposing a pollutant removal promoter that simultaneously reduces the pollution of resistance genes and virulence genes in livestock and poultry manure.

[0007] The objective of this invention can be achieved through the following technical solution: a pollutant removal promoter, wherein the promoter comprises fresh earthworm body fluid and earthworm castings.

[0008] In one of the above-mentioned pollutant removal promoters, the mass ratio of fresh earthworm body fluid to earthworm castings is (0.5-2):(0-1).

[0009] Preferably, the mass ratio of fresh earthworm body fluid to earthworm castings is (0.5-2):1.

[0010] In one of the pollutant removal promoters mentioned above, fresh earthworm body fluid is prepared by subjecting earthworms to electrical or thermal stimulation.

[0011] As a preferred method, vermicompost is prepared by starving earthworms and emptying their entrails.

[0012] As a preferred choice, fresh earthworms are adult Eisenia fetida earthworms with a biomass of 0.3-0.5g and a length of 5.0-5.5cm.

[0013] The present invention also provides a method for simultaneously reducing the pollution of resistance genes and virulence genes in livestock and poultry manure, characterized in that the method includes the following steps: fresh livestock and poultry manure is collected, dried, and then the above-mentioned removal promoter is added; the initial moisture content is adjusted and then the manure is stirred evenly; finally, it is placed in a constant temperature and humidity environment and protected from light.

[0014] In the aforementioned method for simultaneously reducing the contamination of resistance genes and virulence genes in livestock and poultry manure, the initial moisture content is 70%-75%. This invention requires controlling the initial moisture content to 70%-75% because excessively high or low moisture content will prolong the composting process of livestock and poultry manure, and may even inhibit the degree of humification of the final fertilizer product. This is detrimental to the fertilizer utilization of livestock and poultry manure and the removal efficiency of the aforementioned two major categories of novel biological pollutants, thus affecting the effectiveness of manure pollution prevention and its harmless resource treatment.

[0015] In the aforementioned method for simultaneously reducing the contamination of resistance genes and virulence genes in livestock and poultry manure, the amount of removal accelerator added to fresh livestock and poultry manure is 30-60 mL / kg. This invention achieves optimal results with an addition of 30-60 mL / kg of the removal accelerator. Insufficient addition leads to a decrease in the removal efficiency of the relative abundance of resistance genes and virulence genes in livestock and poultry manure, affecting the safety of the final product. Excessive addition increases raw material costs and process input ratios, and may also interfere with the migration, transformation, and reduction of pollutants to some extent, thus affecting the final removal effect of the accelerator.

[0016] In the aforementioned method for simultaneously reducing the contamination of resistance genes and virulence genes in livestock and poultry manure, the temperature in the constant temperature and humidity environment is 18-22℃ and the humidity is 68-72%. This invention, by controlling the temperature in the constant temperature and humidity environment to 18-22℃ and the humidity to 68-72%, provides suitable activity conditions for the removal promoter, ensuring its highly efficient and reliable bioavailability and pollutant reduction efficiency, while also effectively promoting the composting process and humification degree of livestock and poultry manure.

[0017] In the aforementioned method for simultaneously reducing resistance and virulence gene contamination in livestock and poultry manure, the light-shielding treatment time is 7-14 days. In this invention, the light-shielding treatment time is 7-14 days. When the time is less than 7 days, the livestock and poultry manure is not fully decomposed, and the two types of pollutants cannot be effectively reduced, resulting in a high ecological and environmental risk for the manure products. Excessive treatment time (e.g., 28 to 56 days) leads to a significant increase in both the abundance and quantity of resistance and virulence genes in the livestock and poultry manure, significantly increasing the potential health threat of pollutant migration and spread in the environment.

[0018] Compared with existing technologies, this invention has the following beneficial effects: This invention utilizes a mixture of fresh earthworm body fluid and vermicompost as a raw material for pollutant removal, which is inexpensive, natural, non-toxic, and readily available. It is environmentally friendly, efficient, and sustainable, and the method is simple and easy to operate, making it suitable for widespread application. This invention can not only harmlessly utilize livestock waste within a very short period (1-2 weeks), but also simultaneously improve the overall reduction efficiency of nearly 500 new biological pollutants in livestock and poultry manure, including 319 major resistance genes (covering 11 categories of major environmental antibiotic resistance classifications), 57 mobile genetic elements (environmentally representative MGEs), and 120 virulence genes (covering 12 categories of typical human and zoonotic pathogens and their 5 major virulence functions). It synergistically and efficiently reduces the relative abundance and detection quantity of most resistance and virulence genes in livestock and poultry manure, with reduction rates of approximately 60.95-70.31% and 67.85-80.80% for various antibiotic resistance and human and zoonotic pathogen-related functional genes, respectively. Furthermore, in guiding subsequent applications, this invention can flexibly select the best method and add fresh earthworm body fluid and vermicompost alone or in combination, targeting the reduction and deceleration rates of different treatment processes for target pollutants (ARGs, MGEs and VFGs) in livestock and poultry manure. The implementation cycle is only 7-14 days, and the final product can be directly used as a safe and efficient bio-organic fertilizer. Attached Figure Description

[0019] Figure 1 This is a comparison chart showing the changes in the abundance and detection quantity of functional genes such as resistance genes and mobile genetic elements at different treatment times in Example 1 and Comparative Examples 1-3.

[0020] Figure 2 This is a comparison chart showing the changes in gene abundance and detection quantity of pathogenic bacteria to which virulence genes belong at different treatment times in Example 1 and Comparative Examples 1-3. Detailed Implementation

[0021] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0022] Example 1:

[0023] S1. Fresh vermicompost of adult Eisenia fetida earthworms with a biomass of (0.3-0.5g, 5.0-5.5cm) was collected in batches using the starvation method (24-48h jejunal treatment). Fresh body fluid of the above-mentioned jejunal earthworms was obtained in real time using the electrical stimulation method (DC voltage 5V, 30s).

[0024] S2. Mix fresh body fluid and vermicompost at a mass ratio of 2:1. Then add 60 mL / kg of the mixture to cow manure. Add water to adjust the initial moisture content to 72%. Place the mixture in a constant temperature (20℃) and constant humidity (70%) environment away from light for 56 days.

[0025] Comparative Example 1:

[0026] S1. Fresh body fluids were obtained in real time from adult Eisenia fetida earthworms with a biomass of (0.3-0.5g, 5.0-5.5cm) using an electrical stimulation method (DC voltage 5V, 30s).

[0027] S2. Then, fresh body fluid was added to cow manure at a rate of 40 mL / kg, and water was added to adjust the initial moisture content to 72%. The mixture was then placed in a constant temperature (20℃) and constant humidity (70%) environment and kept out of the light for 56 days.

[0028] Comparative Example 2:

[0029] S1. Using starvation (24-48h jejunal treatment) to obtain vermicompost from adult Eisenia fetida earthworms with biomass of (0.3-0.5g, 5.0-5.5cm) in real time;

[0030] S2. Then add earthworm castings to cow manure at a rate of 20g / kg, add water to adjust the initial moisture content to 72%, and place it in a constant temperature (20℃) and constant humidity (70%) environment in the dark for 56 days.

[0031] Comparative Example 3:

[0032] The difference from Example 1 is that in step S2, no fresh body fluids and earthworm castings were added. Instead, water was added to adjust the initial moisture content, and then a constant temperature, humidity and light-proof treatment was performed.

[0033] Multiple pollutants, including resistance genes, mobile genetic elements (MGEs) (key factors involved in the spread of resistance and virulence genes and important indicators for environmental risk assessment), and virulence genes, were measured and analyzed in fecal samples (days 0, 7, 14, 28, and 56) from Examples 1 and Comparative Examples 1-3. The reduction rate of the abundance or quantity of various functional genes in the optimized treatment samples (days 0, 7, 14, 28, and 56 of Example 1) compared to the abundance or quantity in other treatments (days 0, 7, 14, 28, and 56 of Comparative Examples 1-3) was used as the evaluation criterion. The types and classification criteria of the 319 resistance genes (ARGs), 57 mobile genetic elements (MGEs), and 120 virulence genes (VFGs) specifically detected in the four examples (treatments) are detailed in Tables 1 and 2.

[0034] The detection methods for functional genes such as resistance genes, mobile genetic elements, and virulence genes employ high-throughput biochip real-time PCR (SmartChip Real-time PCR, WaferGen, USA), also known as gene chip technology, to simultaneously and efficiently detect multiple functional genes. The antibiotic resistance gene chip uses 384 primer pairs, containing 319 resistance genes (covering most common resistance gene types), 57 mobile genetic elements (MGEs), 7 taxonomic genes, and 1 16S rRNA gene. The pathogenic bacteria virulence gene chip uses 120 primer pairs, primarily targeting typical human and animal pathogens from the environment, such as Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Salmonella enterica. It covers virulence genes involved in encoding five main functions: toxins, adhesion factors, secretion systems, immune evasion / invasion, and iron uptake.

[0035] Table 1: List of ARGs and MGEs as determined and their resistance classifications

[0036]

[0037]

[0038]

[0039]

[0040] Table 2: List of VFGs and their pathogenic bacterial classifications

[0041]

[0042] Figure 1 This is a comparative graph showing the relative abundance and number changes of functional genes such as resistance genes and mobile genetic elements in livestock and poultry manure samples collected at different treatment times (days 0 / 7 / 14 / 28 / 56) in Example 1 and Comparative Examples 1-3. Figure 1It can be seen that, compared with Comparative Example 3, the treatment with fresh earthworm body fluid and vermicompost alone or in combination can reduce the relative abundance and detection number of overall resistance genes (focusing on 319 ARGs belonging to 11 major antibiotic resistance categories) in livestock and poultry manure to a certain extent. In particular, Example 1 (body fluid + vermicompost group) showed the best reduction effect, followed by the body fluid group and the vermicompost group. After 14 days of treatment, the relative abundance of resistance genes in the manure of Comparative Example 3 increased significantly (days 0-14), while ARGs in the other three treatment groups were reduced to varying degrees. In particular, in Example 1, the abundance of ARGs and MGEs remained at a relatively low level and did not increase after 7-28 days of treatment. Among them, the degradation rates of the three major resistance genes, MLSB, peptide, and diaminopyrimidine, were as high as 70.31%, 68.40%, and 60.95% on day 14 in Example 1. As the processing time was extended (days 28-56), the overall relative abundance and detection count of ARGs and MGEs in Examples 1 and Comparative Examples 1-3 increased significantly, which significantly increased the environmental health risk of the final product. Therefore, the optimal processing time for constant temperature and humidity environment and light protection in this invention is 7-14 days.

[0043] Figure 2 This is a comparison chart showing the relative abundance and quantity changes of the pathogenic bacteria to which the virulence genes belong in livestock and poultry manure samples collected at different treatment times (days 0 / 7 / 14 / 28 / 56) in Example 1 and Comparative Examples 1-3. Figure 2 It was found that within 14 days of treatment (0-14 days), the relative abundance of virulence genes in the feces of Comparative Example 3 increased significantly. The addition of fresh earthworm body fluid and vermicompost alone or in combination could reduce the relative abundance and detection quantity of overall virulence genes (targeting 12 types of typical human and animal pathogens in the environment and covering 5 major virulence functions) in livestock and poultry feces to varying degrees. In particular, the reduction effect of Example 1 (body fluid + vermicompost group) was the most significant, followed by the vermicompost group and body fluid group. Among them, the degradation rate of virulence genes carried by the four major types of human and animal pathogens, namely Shigella flexneri, Klebsiella pneumoniae, Acinetobacter baumannii and NMEC, was as high as 80.80%, 71.99%, 69.02% and 67.85% on days 7-14 in Example 1. As the treatment time continued to increase (days 28-56), the overall relative abundance of toxicity genes in Example 1 and Comparative Examples 1-3 increased significantly, which significantly increased the environmental health risk of the final product.

[0044] In summary, this invention, through the treatment of adding earthworm body fluid and vermicompost, can efficiently reduce the main types of biological pollutants in livestock and poultry manure within a very short time (1-2 weeks). In particular, it can simultaneously reduce the relative abundance and detection quantity of nearly 500 emerging pollutants in livestock and poultry manure, including most resistance genes (319 ARGs covering 11 major environmental antibiotic resistance categories), mobile genetic elements (57 environmentally representative MGEs), and virulence genes (120 VFGs containing 12 typical human and zoonotic pathogens in the environment and covering 5 major virulence functions). This effectively and efficiently controls the spread and diffusion of manure-borne pollutants such as resistance genes and virulence genes in the environment, providing theoretical and technical support for the harmless resource utilization of livestock manure and the sustainable green development of agriculture. This invention is crucial for the prevention and control of biological pollution from manure, ecological environmental protection, and soil-human health protection.

[0045] The embodiments described herein cover any points not exhaustively within the scope of the technical claims of this invention, as well as new technical solutions formed by equivalent substitutions of one or more technical features in the embodiments. These are all within the scope of the claims of this invention. Furthermore, in all listed or unlisted embodiments of this invention, each parameter in the same embodiment merely represents an instance (i.e., a feasible solution) of its technical solution, and there is no strict coordination or limitation relationship between the parameters. The parameters can be substituted for each other without violating axioms and the claims of this invention, unless otherwise stated.

[0046] The technical means disclosed in this invention are not limited to those described above, but also include technical solutions composed of any combination of the above technical features. The above descriptions are specific embodiments of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

[0047] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for simultaneously reducing the contamination of resistant genes and virulence genes in livestock and poultry manure, characterized in that, The method comprises the following steps: adding a removal promoter to fresh livestock and poultry manure after the livestock and poultry manure is collected and dried, uniformly stirring the livestock and poultry manure after the initial water content is adjusted, and finally placing the livestock and poultry manure in a constant temperature and humidity environment for light-proof treatment for 7-14 days. The removal promoter comprises fresh earthworm body fluid and earthworm manure in a mass ratio of (0.5-2):

1. The fresh earthworm body fluid is obtained by using a starvation method to perform 24-48h emptying treatment on the earthworm, and the fresh earthworm manure is obtained by using an electric stimulation method to obtain fresh body fluid of the emptying earthworm under a direct current voltage of 5V for 30s, the biomass of the earthworm is 0.3-0.5g, the earthworm is 5.0-5.5cm long and is adult.

2. The method for simultaneously reducing pollution of resistant genes and virulence genes in livestock and poultry manure according to claim 1, characterized in that, The initial water content is 70%-75%.

3. The method for simultaneously reducing pollution of resistant genes and virulence genes in livestock and poultry manure according to claim 1, characterized in that, The addition amount of the removal promoter in the fresh livestock and poultry manure is 30-60mL / kg.

4. The method for simultaneously reducing pollution of resistant genes and virulence genes in livestock and poultry manure according to claim 1, characterized in that, The temperature in the constant temperature and humidity environment is 18-22℃, and the humidity is 68-72%.

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