Method for strengthening and reducing plasmid-mediated resistance gene by using mixed magnetic resin

Abstract

The invention relates to the technical field of harmless treatment of livestock and poultry manure, in particular to a method for reinforcing and reducing plasmid-mediated resistance genes by using mixed magnetic resin. The method comprises the following steps: adding livestock and poultry manure and mixed magnetic resin into a fermentation tank, controlling the pH value of a system in the fermentation tank to be 7-8, and controlling the temperature of a reaction tank to be 37 + / -1 DEG C for anaerobic digestion; wherein the addition amount of the mixed magnetic resin is 0.1-0.5 g / kg of total suspended solids, and the mixed magnetic resin is a mixture of magnetic cation exchange resin and magnetic anion exchange resin in a mass ratio of 2: 3-3: 2. The method provided by the invention is used for treating the livestock and poultry manure, and provides a new technical support for reducing resistance genes and controlling transmission of the resistance genes.

Description

Technical Field

[0001] The invention relates to the technical field of harmless treatment of livestock and poultry excrement, and in particular to a method for strengthening and reducing plasmid-mediated resistance genes by using mixed magnetic resin. Background Art

[0002] Problems such as lack of environmental awareness, high treatment costs and lack of large-scale treatment facilities have led to the random discharge of a large amount of untreated manure, posing a severe challenge to the environment. If the complex components of livestock and poultry manure are discharged directly without proper treatment, it will seriously pollute the natural environment. Its highly leaching wastewater easily penetrates the soil and groundwater, and excessive nitrogen and phosphorus content can easily cause eutrophication of water bodies and destroy the water ecological balance. In addition, the harmful gases released by the random stacking of manure not only pollute the air and endanger human health, but also aggravate the greenhouse effect.

[0003] Anaerobic digestion refers to the technology of effectively converting biodegradable organic substances such as carbohydrates, fats, and proteins into reducing byproducts such as gases such as CH4, CO2, NH3, and H2S and organic acids through the action of anaerobic and facultative anaerobic microorganisms in an oxygen-free or low-oxygen environment. Given that livestock and poultry manure is rich in organic components and has the potential to be converted into more energy, anaerobic digestion has become one of the mainstream ways to treat such manure. This technology not only efficiently converts organic matter in manure into clean energy, but its digested product, digestate, can also be returned to farmland as high-quality organic fertilizer, realizing the recycling of resources.

[0004] At present, antibiotics are widely used in livestock and poultry farming. While preventing and treating animal diseases and promoting growth, antibiotics also give rise to drug resistance problems, prompting the production of resistant strains in livestock and poultry, and the formation of antibiotic resistance genes, becoming new pollutants. Resistance genes can be transferred and evolved between animals and humans, posing a major threat to public health. Studies have found that livestock and poultry manure is the main storage and transmission source of resistance genes. Although anaerobic digestion methods can reduce the abundance of resistant strains and resistance plasmids by generating high temperatures, they cannot completely remove all resistance genes. Therefore, the problem of resistance gene contamination in livestock and poultry manure needs to be solved urgently. Summary of the invention

[0005] In view of the technical problem that anaerobic digestion methods have limited effects on the removal of resistance genes in livestock and poultry manure, the present invention provides a method for strengthening the reduction of plasmid-mediated resistance genes using a mixed magnetic resin. In the process of anaerobic digestion of livestock and poultry manure, a mixed magnetic resin is used to strengthen the reduction of plasmid-mediated resistance genes, thereby reducing the migration, transformation and enrichment hazards of resistance genes in the environment.

[0006] The technical solution of the present invention is as follows: A method for strengthening and reducing plasmid-mediated resistance genes by using mixed magnetic resin, wherein livestock and poultry manure and mixed magnetic resin are added to a fermentation tank, the pH value of the system in the fermentation tank is controlled to be 7-8, and the temperature of the reaction tank is controlled to be 37±1°C for anaerobic digestion; Among them, the addition amount of the mixed magnetic resin is 0.1-0.5 g / kg total suspended solids, and the mixed magnetic resin is a mixture of magnetic cation exchange resin and magnetic anion exchange resin in a mass ratio of 2:3-3:2. The magnetic cation exchange resin refers to a cation exchange resin embedded with magnetic particles, and the magnetic anion exchange resin refers to an anion exchange resin embedded with magnetic particles.

[0007] Furthermore, the livestock and poultry manure includes at least one of pig manure, cow manure, sheep manure, chicken manure and duck manure.

[0008] Furthermore, before adding the mixed magnetic resin, livestock and poultry manure is first added to the fermentation tank, and anaerobic digestion sludge is inoculated, the mass ratio of anaerobic digestion sludge to livestock and poultry manure is 1:3-2:5, and the solid content of the system is adjusted using digestion liquid, so that the final solid content of the system is 2%-8%. The inoculated anaerobic digestion sludge and digestion liquid are both taken from the sludge anaerobic digestion tank in stable operation.

[0009] Furthermore, the anaerobic digestion time is 30-70 days.

[0010] Furthermore, the gas generated during the anaerobic digestion process is collected, and the generated gas contains methane, which can be reused after being collected.

[0011] Furthermore, the anaerobic digestion products are subjected to solid-liquid separation. The solid-liquid separation method can be centrifugation, filtration and other means. The obtained liquid (i.e., biogas slurry) is used for farmland reuse, such as for irrigation, and the obtained solid is separated by magnetic separation of mixed magnetic resin (i.e., biogas residue) and used for crop fertilization.

[0012] Furthermore, after the mixed magnetic resin separated by magnetic separation is washed with tap water, it is firstly soaked and washed with a saturated NaCl solution to achieve regeneration of the magnetic cation exchange resin, and then soaked and washed with a saturated NaOH solution to achieve regeneration of the magnetic anion exchange resin.

[0013] The beneficial effects of the present invention are: The method of the present invention prepares a mixed magnetic resin by accurately mixing magnetic cation exchange resin and magnetic anion exchange resin, which integrates the characteristics of anion and cation resins, and can effectively reduce resistance genes and plasmid-mediated horizontal transfer of resistance genes through multiple mechanisms such as passivation of heavy metals, inhibition of the activity of mobile genetic elements, and change of microbial community structure, thereby inhibiting the spread and diffusion of bacterial resistance. Combined with anaerobic digestion, it strengthens the reduction of resistance genes and the inhibition of the activity of plasmid-mediated horizontal transfer of resistance genes to a certain extent. At the same time, the magnetic resin also has magnetic separation performance and is easier to recycle.

[0014] The method of the present invention is used to treat livestock and poultry manure, which can effectively reduce the relative abundance of resistance genes by 15%-71% and reduce the relative abundance of mobile genetic elements by 30%-61.2%. It is an effective method for strengthening the reduction of resistance genes in livestock and poultry manure, and provides new technical support for reducing resistance genes and controlling the spread of resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The present invention is a flow chart of a method for enhancing the reduction of plasmid-mediated resistance genes by using a mixed magnetic resin.

[0017] Figure 2 It is a bar graph of the relative abundance of resistance genes and mobile genetic elements in the biogas residue of Example 1 of the present invention and Comparative Example 1.

[0018] Figure 3 It is a bar graph of the relative abundance of resistance genes and mobile genetic elements in the biogas residue of Example 2 of the present invention and Comparative Example 2. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0020] The magnetic cation exchange resin (hereinafter referred to as S-YANG) used in the specific embodiment of the present invention is a magnetic cation exchange resin containing carboxyl sites, and the magnetic anion exchange resin (hereinafter referred to as S-YIN) is a magnetic anion exchange resin containing two types of quaternary ammonium sites; Among them, the magnetic cation exchange resin containing carboxyl sites uses commercially available macroporous weakly acidic cation resin as the functional group monomer, Fe3O4 as the magnetic source, and acrylamide, N,N'-methylenebisacrylamide and acrylic acid as the polymerization monomer and cross-linking agent respectively, and is prepared by coprecipitation method and inverse emulsion polymerization; The magnetic anion exchange resin containing two types of quaternary ammonium sites is prepared by amination and alkylation of copolymerized magnetic microspheres containing ester monomers, Fe3O4 magnetic cores and epoxy crosslinking agents to form a type of quaternary ammonium salt, and then the epoxy groups on the copolymerized magnetic microspheres react with the first type of quaternary ammonium salt to form a second type of quaternary ammonium salt.

[0021] In other embodiments, commercially available magnetic cation exchange resins and magnetic anion exchange resins prepared by other conventional methods may be used and mixed in a mass ratio of 2:3-3:2 to prepare a mixed magnetic resin, which also has the effect of enhancing the reduction of plasmid-mediated resistance genes in livestock and poultry manure compared to using cationic resins or anionic resins alone.

[0022] Example 1 The collected fresh chicken and duck manure were mixed and sampled to determine the relative abundance of resistance genes (acc(6')-Ib-cr, sul1, sul2) and mobile genetic elements (intI1, ISCR1) in the initial manure. Figure 1 As shown, anaerobic digestion is carried out as follows: (1) Wash S-YANG and S-YIN twice with ultrapure water, and then mix them in a ratio of 2:3 to form a mixed magnetic resin MIX@C-(3 / 2); (2) Dehydrating livestock and poultry manure by centrifugation to obtain dry livestock and poultry manure; (3) Dry livestock and poultry manure was added to the fermentation tank and inoculated with anaerobic digestion sludge. The mass ratio of anaerobic digestion sludge to livestock and poultry manure was 1:3. The solid content of the system in the fermentation tank was adjusted to 2% using digestate from other stably operating anaerobic digesters. Then, MIX@C-(3 / 2) was added to the fermentation tank at a ratio of 0.1 g / kg total suspended solids. The pH value of the system was adjusted to 7 using NaOH solution. Nitrogen was introduced into each reaction tank for 5 min to exhaust the air. (4) Adjust the temperature in the reaction tank to 37±1°C and perform anaerobic fermentation for 30 days. During the process, a gas collector is used to collect the generated gas; (5) filtering the fermented product after fermentation, and using the obtained filtrate for farmland reuse; The obtained filter residue is used to recover the mixed magnetic resin by magnetic separation, and after being washed twice with ultrapure water, the mixed magnetic resin is sequentially immersed in a saturated NaCl solution and a saturated NaOH solution to respectively regenerate the magnetic cation exchange resin and the magnetic anion exchange resin. After the regeneration is completed, the two resins are reused in the anaerobic digestion process; The remaining sludge after magnetic separation can be used for fertilizing crops, and samples can be taken for DNA extraction, PCR amplification, fluorescence quantification, library construction and sequencing.

[0023] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses only one magnetic resin, S-YANG, and S-YANG is added to the fermenter at a ratio of 0.1 g / kg of total suspended solids.

[0024] The relative abundance results of the resistance genes (acc(6')-Ib-cr, sul1, sul2) and mobile genetic elements (intI1, ISCR1) of the digestate finally obtained after magnetic separation in Example 1 and Comparative Example 1 are as follows: Figure 2 It can be seen that after the action of MIX@C-(3 / 2), the relative abundance of resistance genes in livestock and poultry manure decreased by up to 37.7%, and the relative abundance of mobile genetic elements decreased by up to 46.5%; after the action of single S-YANG, the relative abundance of resistance genes in livestock and poultry manure decreased by up to 15.4%, and the relative abundance of mobile genetic elements decreased by up to 30.2%.

[0025] It can be seen that the present invention uses a mixture of magnetic cation exchange resin and magnetic anion exchange resin. Compared with using magnetic cation exchange resin alone, the removal rate of resistance genes and mobile genetic elements in livestock and poultry manure is significantly improved, indicating that the method provided by the present invention has the effect of enhancing the reduction of plasmid-mediated resistance genes.

[0026] Example 2 The collected fresh cow dung, pig dung, and sheep dung were mixed and sampled to determine the relative abundance of resistance genes (acc(6')-Ib-cr, sul1, sul2) and mobile genetic elements (intI1, ISCR1) in the initial dung. Figure 1 As shown, anaerobic digestion is carried out as follows: (1) Wash S-YANG and S-YIN twice with ultrapure water, and then mix them in a ratio of 3:2 to form a mixed magnetic resin MIX@C-(2 / 3); (2) Dehydrating livestock and poultry manure by centrifugation to obtain dry livestock and poultry manure; (3) Dry livestock and poultry manure was added to the fermentation tank and inoculated with anaerobic digestion sludge. The mass ratio of anaerobic digestion sludge to livestock and poultry manure was 2:5. The solid content of the system in the fermentation tank was adjusted to 8% using digestate from other stably operating anaerobic digesters. Then, MIX@C-(2 / 3) was added to the fermentation tank at a ratio of 0.5 g / kg total suspended solids. The pH value of the system was adjusted to 8 using NaOH solution. Nitrogen was introduced into each reaction tank for 5 min to exhaust the air. (4) Adjust the temperature in the reaction tank to 37±1°C and perform anaerobic fermentation for 70 days. During the process, a gas collector is used to collect the generated gas; (5) filtering the fermented product after fermentation, and using the obtained filtrate for farmland reuse; The obtained filter residue is used to recover the mixed magnetic resin by magnetic separation, and after being washed twice with ultrapure water, the mixed magnetic resin is sequentially immersed in a saturated NaCl solution and a saturated NaOH solution to respectively regenerate the magnetic cation exchange resin and the magnetic anion exchange resin. After the regeneration is completed, the two resins are reused in the anaerobic digestion process; The remaining sludge after magnetic separation can be used for fertilizing crops, and samples can be taken for DNA extraction, PCR amplification, fluorescence quantification, library construction and sequencing.

[0027] Comparative Example 2 The difference between Comparative Example 2 and Example 2 is that Comparative Example 2 uses only one magnetic resin, S-YIN, and S-YIN is added to the fermenter at a ratio of 0.5 g / kg of total suspended solids.

[0028] The relative abundance results of the resistance genes (acc(6')-Ib-cr, sul1, sul2) and mobile genetic elements (intI1, ISCR1) of the digestate finally obtained after magnetic separation in Example 2 and Comparative Example 2 are as follows: Figure 3 It can be seen that after the action of MIX@C-(2 / 3), the relative abundance of resistance genes in livestock and poultry manure decreased by up to 71.0%, and the relative abundance of mobile genetic elements decreased by up to 61.2%; after the action of single S-YIN, the relative abundance of resistance genes in livestock and poultry manure decreased by up to 39.8%, and the relative abundance of mobile genetic elements decreased by up to 38.7%.

[0029] It can be seen that the mixed use of magnetic cation exchange resin and magnetic anion exchange resin in the present invention can significantly improve the removal rate of resistance genes and mobile genetic elements in livestock and poultry manure compared with the use of magnetic anion exchange resin alone, indicating that the method provided by the present invention has the effect of enhancing the reduction of plasmid-mediated resistance genes.

[0030] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A method for enhancing the reduction of plasmid-mediated resistance genes using a mixed magnetic resin, characterized in that: Add livestock and poultry manure and mixed magnetic resin into a fermentation tank, control the pH value of the system in the fermentation tank to 7-8, and control the temperature of the reaction tank at 37±1℃ for anaerobic digestion; The added amount of the mixed magnetic resin is 0.1-0.5 g / kg of total suspended solids, and the mixed magnetic resin is a mixture of a magnetic cation exchange resin and a magnetic anion exchange resin in a mass ratio of 2:3-3:

2.

2. The method according to claim 1, characterized in that The livestock and poultry manure includes at least one of pig manure, cow manure, sheep manure, chicken manure and duck manure.

3. The method according to claim 1, characterized in that Before adding the mixed magnetic resin, add livestock and poultry manure to the fermentation tank and inoculate it with anaerobic digestion sludge. The mass ratio of anaerobic digestion sludge to livestock and poultry manure is 1:3-2:

5. Use digestion liquid to adjust the solid content of the system so that the final solid content of the system is 2%-8%.

4. The method according to claim 1, characterized in that The anaerobic digestion time is 30-70 days.

5. The method according to claim 1, characterized in that The gases produced during anaerobic digestion are collected.

6. The method according to claim 1, characterized in that The anaerobic digestion products are separated into solid and liquid, the obtained liquid is reused in farmland, and the obtained solid is separated by magnetic separation and mixed magnetic resin and then used for crop fertilization.

7. The method according to claim 6, characterized in that After the mixed magnetic resin separated by magnetic separation is washed with tap water, it is first soaked and washed with a saturated NaCl solution to achieve regeneration of the magnetic cation exchange resin, and then soaked and washed with a saturated NaOH solution to achieve regeneration of the magnetic anion exchange resin.

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