A compound hyperthermophile agent and its application in inhibiting resistance gene rebound in compost

Abstract

This invention discloses a composite thermophilic microbial agent and its application in inhibiting the rebound of resistance genes in compost. The composite thermophilic microbial agent comprises thermophilic iron-reducing bacteria, thermophilic bacteria, a basal culture medium, a carrier, and a conditioner. The thermophilic iron-reducing bacteria is *Bacillus composti* SgZ-9, and the thermophilic bacteria is *Calditerricola yamamurae* YMO722. The carrier includes iron mineral nanozymes and iron-modified biochar. Under aerobic conditions, the composite thermophilic microbial agent of this invention drives the iron-reducing bacteria to perform a redox cycle in the iron mineral nanozymes and iron-modified biochar, generating a large amount of ·OH, which destroys the DNA structure of ARGs and promotes the formation of EPFRs, thus reducing ARGs. The thermophilic bacteria continuously raise the temperature of the compost pile, directly accelerating the thermal denaturation and decomposition of extracellular free ARGs, and killing the host pathogens of ARGs. The large amount of ·OH in the compost pile, combined with high temperature conditions, promotes the continuous production and accumulation of EPFRs. By inhibiting quorum sensing among bacteria, it reduces the re-reproduction of host pathogens during the composting maturation period, thus effectively suppressing the rebound of ARGs in high-temperature composting.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a compound high-temperature bacterial agent and its application in inhibiting the rebound of resistance genes in composting. Background Technology

[0002] Antibiotic resistance genes (ARGs) are specific gene sequences within bacteria that can be horizontally transferred between different bacterial species via mobile genetic elements such as integrons and transposons. This means that a harmless bacterium can acquire ARGs through multiple pathways, thus evolving into multidrug-resistant bacteria or even "superbugs." Currently, ARG residues have been detected in soil, rivers, and even the atmosphere, with the highest variety and abundance found in organic solid waste, particularly in livestock and poultry manure, where levels can reach 10. 8 -10 9 copies / kg.

[0003] Organic solid waste refers to solid or semi-solid waste generated in daily life or production activities, including urban sludge, livestock and poultry manure, agricultural residues, and kitchen waste. Recent studies have found that organic solid waste generally contains high abundances of azo dyes (ARGs) and abundant nutrients such as nitrogen, phosphorus, and potassium, thus possessing both pollutant and resource attributes. Aerobic composting is a crucial technical means to achieve the reduction, harmlessness, and resource utilization of organic solid waste, serving as the last "barrier" against pollutants entering the environment. The high-temperature environment of traditional aerobic composting can directly destroy the DNA molecular structure of extracellular free ARGs and kill host pathogens, thereby destroying intracellular ARGs and significantly reducing the types and abundance of ARGs. However, recent studies have found that not all ARGs in organic solid waste can be completely removed through composting. Furthermore, as the composting temperature gradually decreases, the regrowth and reproduction of some host pathogens cause the abundance of ARGs to rebound, ultimately significantly reducing the harmlessness effect of composting on organic solid waste and severely restricting the practical application of compost products.

[0004] Therefore, effectively reducing various ARGs during aerobic composting and inhibiting the rebound of ARG abundance during the maturation period is of great practical significance for achieving the harmless disposal and resource utilization of organic solid waste, as well as the prevention and control of ARG pollution and ecological environmental protection. Summary of the Invention

[0005] To address the problems of low ARGs reduction efficiency and ARGs abundance rebound during the composting period in existing composting technologies, which pose a risk of secondary environmental pollution, this invention provides a composite high-temperature microbial agent and its application in promoting ARGs reduction and inhibiting rebound during composting.

[0006] According to a first aspect of the present invention, a composite thermophilic microbial agent is provided, comprising thermophilic iron-reducing bacteria, thermophilic bacteria, a basal culture medium, a carrier, and a conditioner. The thermophilic iron-reducing bacteria is Bacillus composti SgZ-9, and the thermophilic bacteria is Calditerricola yamamurae YMO722. The basal culture medium comprises 2.9 g / L municipal sludge (on a dry solids basis), 2.0 g / L foaming agent, 0.5 g / L yeast extract, 0.5 g / L ammonium chloride, 0.5 g / L sucrose, 0.5 g / L soluble starch, and 0.3 g / L potassium dihydrogen phosphate. The carrier comprises iron mineral nanozyme and iron-modified biochar, and the conditioner is soybean meal powder.

[0007] In some embodiments, the iron mineral nanozyme accounts for 1% to 5% of the mass of the composite high-temperature bacterial agent, the iron mineral nanozyme is iron(III) oxide nanoparticles, the iron-modified biochar accounts for 1% to 5% of the mass of the composite high-temperature bacterial agent, the iron-modified biochar is any one of ferric sulfate modified biochar, ferric chloride modified biochar, ferric oxide modified biochar or zero-valent iron modified biochar, and the conditioner accounts for 5% of the mass of the composite high-temperature bacterial agent.

[0008] In some embodiments, the content of both the thermophilic iron-reducing bacterium Bacillus composti SgZ-9 and the thermophilic bacterium Calditerricola yamamurae YMO722 in the compound thermophilic bacterial agent is 10. 8 ~10 9 cfu / g.

[0009] According to a second aspect of the present invention, a method for preparing a composite high-temperature bacterial agent is provided, comprising the following steps: S1. Preparation of seed culture of Calditerricola yamamurae YMO722; S2. Preparation of Bacillus composti SgZ-9 seed culture; S3. Prepare fermentation medium; S4. First, inoculate the Calditerricola yamamurae YMO722 seed culture prepared in step S1 into the fermentation medium prepared in step S3, introduce air and stir for fermentation culture for 16-24 hours; then, inoculate the Bacillus composti SgZ-9 seed culture prepared in step S2 into the above fermentation medium and ferment for 48-96 hours. S5. After fermentation, add soybean cake powder as a conditioning agent, stir evenly, and then filter by plate and frame to obtain a compound high-temperature bacterial agent with a moisture content of 45%~55%.

[0010] In some embodiments, step S1 is: to prepare a solution with a bacterial content of 10... 9 A 2% inoculum of *Calditerricola yamamurae* YMO722 was inoculated into CYS medium and activated by constant temperature shaking at 75–80 °C and 160–180 rpm for 16–24 h to obtain an activated *Calditerricola yamamurae* YMO722 culture. This activated culture was then inoculated into a seed tank at a 2% inoculum and incubated at 75–80 °C and an aeration rate of 20–100 m³ / h. 3 Fermentation was carried out for 16-24 h at a stirring speed of 180-220 rpm to obtain the seed culture of Calditerricola yamamurae YMO722.

[0011] In some embodiments, step S2 is: to prepare a solution with a bacterial content of 10... 9 Bacillus compostiSgZ-9 bacterial culture at cfu / mL was inoculated into LB medium and fermented at 45–50 °C for 48–96 h. Air was introduced during fermentation at a rate of 80–100 mL / min. 3 The mixture is stirred continuously at a speed of 160~200 rpm for 1 hour. Once fermentation is complete, the Bacillus composti SgZ-9 seed culture is obtained.

[0012] In some embodiments, step S3 is as follows: Take municipal sludge, add municipal sludge, foaming agent, yeast powder, ammonium chloride, sucrose, soluble starch, and potassium dihydrogen phosphate to water according to the composition of the basic culture medium, adjust the pH to 7.0~7.5, add magnetite nanozyme and iron-modified biochar, stir evenly, sterilize, and obtain fermentation culture medium, cool for later use. The fermentation culture medium contains 2.9 g / L municipal sludge (based on dry solids), 2.0 g / L foaming agent, 0.5 g / L yeast powder, 0.5 g / L ammonium chloride, 0.5 g / L sucrose, 0.5 g / L soluble starch, 0.3 g / L potassium dihydrogen phosphate, 50 g / L magnetite nanozyme, and 50 g / L iron-modified biochar.

[0013] According to a third aspect of the present invention, the application of the above-mentioned composite high-temperature microbial agent in suppressing ARGs rebound during high-temperature composting is provided.

[0014] According to a fourth aspect of the present invention, a method for suppressing ARGs rebound during high-temperature composting is provided, wherein the method comprises mixing the above-mentioned composite high-temperature microbial agent with the initial composting material for composting fermentation, wherein the preferred mixing mass ratio of the composite high-temperature microbial agent to the initial composting material is (1-5):(95-99).

[0015] In some implementations, the initial composting material is any one of urban sludge, livestock and poultry manure, agricultural residues, or kitchen waste.

[0016] The beneficial effects of the present invention are: (1) The composite high-temperature bacterial agent of the present invention can be used in conjunction with aeration, turning and other composting processes to change the dynamic changes of the aerobic-anoxic zone in the composting process, promote the redox cycle of iron mineral nanoenzymes and iron-modified biochar in the compost, generate ·OH (hydroxyl radicals), and promote the efficient reduction of AGRs. (2) The thermophilic iron-reducing bacteria contained in the composite high-temperature bacterial agent prepared in this invention can grow and reproduce using nutrients in compost under aerobic conditions, drive the redox cycle of iron mineral nanoenzymes and iron-modified biochar to continuously generate ·OH and promote the formation of EPFRs (environmentally persistent free radicals), destroy the DNA double-stranded structure of ARGs, and achieve further oxidative removal of ARGs. (3) The thermophilic bacteria contained in the composite high-temperature bacterial agent of the present invention can create a continuous high-temperature environment in the compost, which on the one hand directly accelerates the thermal denaturation and decomposition of extracellular free ARGs, and on the other hand can directly kill host pathogens and destroy intracellular ARGs. (4) The large amount of ·OH produced by iron mineral nanozymes and iron-modified biochar in the compost pile, combined with the favorable environmental conditions of high temperature and biochar, promotes the continuous production and accumulation of EPFRs in the compost pile, and inhibits the re-reproduction of host pathogens during the composting period by reducing quorum sensing between bacteria, thus achieving effective inhibition of ARGs rebound in high temperature composting. Attached Figure Description

[0017] Figure 1 This is a graph showing the composting temperature changes of different treatment groups and the blank control group in Example 2 of the present invention.

[0018] Figure 2 This is a graph showing the changes in hydroxyl radical concentrations in different treatment groups and the blank control group in Example 2 of the present invention.

[0019] Figure 3 This is a graph showing the changes in the concentration of persistent environmental free radicals in compost in different treatment groups and the blank control group in Example 2 of the present invention.

[0020] Figure 4 This is a graph showing the changes in the abundance of resistance genes in compost from different treatment groups and the blank control group in Example 2 of the present invention.

[0021] Figure 5 This is a graph showing the changes in the removal rate of resistance genes in compost from different treatment groups and the blank control group in Example 2 of the present invention.

[0022] Figure 6This is a graph showing the abundance changes of host pathogenic bacteria in compost from different treatment groups and a blank control group in Example 2 of the present invention. Detailed Implementation

[0023] The present invention is further described in detail through specific implementation examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available.

[0024] The thermophilic iron-reducing bacterium of this invention, Bacillus composti SgZ-9, was purchased from the China Center for Type Culture Collection (CCTCC), with accession number CCTCC AB 2012109, and the thermophilic bacterium, Calditerricola yamamurae YMO722, was purchased from the Japan Microbial Collection (JCM), with accession number JCM 14720.

[0025] Example 1

[0026] A method for preparing a composite high-temperature bacterial agent includes the following steps: S1. Preparation of Calditerricola yamamurae YMO722 seed culture: The bacterial content is 10 9 A 2% inoculum of *Calditerricola yamamurae* YMO722 (cfu / mL) was inoculated into CYS medium and activated by shaking at 75 °C and 180 rpm for 16 h to obtain an activated bacterial culture of *Calditerricola yamamurae* YMO722. This activated bacterial culture was then inoculated into a seed tank at a 2% inoculum and incubated at 75 °C and 100 m³ / h aeration. 3 Fermentation was carried out for 16 h at a stirring speed of 220 rpm to obtain the seed culture of Calditerricola yamamurae YMO722; S2. Preparation of Bacillus composti SgZ-9 seed culture: The bacterial content is 10 9 A CFU / mL Bacillus composti SgZ-9 culture was inoculated into LB medium and fermented at 50 °C for 48 h. Air was introduced during fermentation at a flow rate of 80 mL / min. 3The mixture was stirred continuously at 160 rpm for 1 hour, and the fermentation was completed to obtain Bacillus composti SgZ-9 seed liquid. S3. Preparation of fermentation medium: Concentrated sludge from a wastewater treatment plant in Fujian Province (basic properties of the sludge are shown in Table 1) was collected. The following components were added to water: 2.9 g / L municipal sludge (based on dry solids), 2.0 g / L foaming agent, 2 g / L yeast powder, 0.5 g / L ammonium chloride, 0.5 g / L sucrose, 0.5 g / L soluble starch, and 0.3 g / L potassium dihydrogen phosphate. The pH was adjusted to 7.0, and then ferric oxide nanoparticles and ferric sulfate-modified biochar were added to bring the final concentration of both to 50 g / L. The mixture was stirred thoroughly and sterilized at 100°C for 45 minutes to obtain the fermentation medium, which was then cooled to 80°C for later use.

[0027] Table 1 Basic Properties of Concentrated Sludge

[0028] ; S4. First, inoculate the Calditerricola yamamurae YMO722 seed culture prepared in step S1 into the fermentation medium prepared in step S3 at an inoculation rate of 1.5% (v / v), and incubate at 75 °C with an aeration rate of 100 m³ / h. 3 Under the conditions of 180 rpm and 1 h, air was introduced and the culture was stirred for 24 hours for fermentation. Then, the Bacillus composti SgZ-9 seed culture prepared in step S2 was inoculated into the above fermentation medium at an inoculation amount of 1.5% (volume ratio), and fermented at 50 °C for 48 hours to obtain the culture. S5. After fermentation, add soybean meal powder to the culture obtained in step S4 at a ratio of 5% (by mass). After stirring evenly, filter by plate and frame press to obtain a compound high-temperature bacterial agent with a moisture content of 45%~55%.

[0029] Plate counting and test tube analysis were performed on the prepared composite thermophilic microbial agent. The results showed that the bacterial counts of *Calditerricola yamamurae* YMO722 and *Bacillus composti* SgZ-9 in the prepared composite thermophilic microbial agent were approximately 9.1 × 10⁻⁶. 8 cfu / g and 1.1×10 9 cfu / g.

[0030] Example 2: Application of compound high-temperature microbial agent in inhibiting ARGs rebound in composting

[0031] In this embodiment, the compost material was initially mixed with livestock and poultry manure and rice straw at a mass ratio of 4:1. The livestock and poultry manure was taken from a livestock and poultry farm in Fuzhou. The basic properties of the above compost material were tested using conventional testing methods in the field, and the results are shown in Table 2.

[0032] Table 2 Basic Properties of Compost Materials

[0033]

[0034] The compound microbial agent was mixed with the compost material at a mass ratio of 5%:95%. PVC aeration pipes were installed at the bottom of the composting reactor for daily aeration, using intermittent aeration with a 24-hour interval followed by a 48-hour interval. The aeration rate was 2 L / min, and the compost was turned over every 5 days. The composting experiment included treatment group 1, treatment group 2, treatment group 3, and a blank control group (compost material only, without the compound microbial agent). Treatment group 1 used the compound high-temperature microbial agent prepared in Example 1. Treatment group 2 used a compound microbial agent prepared according to the method in Example 1, containing only Calditerricola yamamurae YMO722, Bacillus composti SgZ-9, and magnetite nanozyme (i.e., iron tetroxide nanoparticles). Treatment group 3 used a compound microbial agent prepared according to the method in Example 1, containing only Calditerricola yamamurae YMO722, Bacillus composti SgZ-9, and iron-modified biochar (i.e., ferric sulfate-modified biochar).

[0035] Composting temperature was monitored in real time, and samples were taken on days 0, 8, 16, and 32 of composting. The yields of ·OH and EPFRs during composting were determined using standard methods in this field. The abundance of ARGs in the compost was detected using metagenomic sequencing and analysis. DNA extracted from different compost samples was sequenced using Illumina sequencing technology in a paired-end (PE) configuration. After sequencing, Illumina PE libraries were constructed, and the obtained sequencing data underwent quality control and ARGs analysis was performed using biological analysis techniques. Results are as follows: Figures 1-6 As shown.

[0036] Temperature changes in different composting treatment groups and the blank control group are as follows: Figure 1 As shown, compared with the blank control group, the composting temperature of treatment group 1 (as described in Example 1) was significantly higher than that of the blank control group, and the high-temperature period lasted longer. The temperatures of treatment groups 2 and 3 were both higher than those of the blank control group, but still lower than those of treatment group 1 (which had the compound high-temperature microbial agent added).

[0037] Free radical generation in different composting treatment groups, such as Figure 2 and Figure 3As shown in the figure, treatment group 1 produced the most ·OH, with a maximum concentration of 1610.5 μmol / kg, significantly higher than the ·OH concentration in the blank control group (951.7 μmol / kg). Treatment group 2 produced a maximum ·OH concentration of 1564.6 μmol / kg, only slightly lower than treatment group 1. Treatment group 3 produced a maximum ·OH concentration of 1197.2 μmol / kg, significantly lower than treatment group 1. Therefore, treatment group 1 produced the most ·OH, treatment group 2 produced a similar amount to treatment group 1, followed by treatment group 3 and the blank control group. Furthermore, the concentration of EPFRs continued to increase in all four treatment groups. Notably, the overall EPFR concentration in treatment group 1 was higher than that in the blank control group (9.7 * 10⁻⁶). 15 (spins / g), accumulating to 12.5*10 during the composting maturation period. 15 spins / g. The concentration of EPFRs in treatment group 3 was significantly increased, ultimately reaching 11.4*10^6 spins / g during the composting maturation period. 15 The concentration of spins / g in treatment group 2 was higher than that in the blank control group and treatment group 2. The overall EPFR concentration in treatment group 2 was similar to that in the blank control group, ultimately reaching 10.2*10^6. 15 The number of spins / g indicates that EPFRs were most abundant in treatment group 1, followed by treatment group 2, treatment group 3, and the blank control group. In conclusion, the addition of iron mineral nanozymes and iron-modified biochar promoted the production of ·OH and EPFRs during composting, respectively.

[0038] During composting, the abundance trends of ARGs varied among different treatment groups, as detailed in the following figures. Figure 4 and Figure 5In treatment group 1, the abundance of ARGs in the compost decreased continuously, from 6031.4 TPM at the beginning of composting to 232.4 TPM, and there was no rebound in abundance during the composting maturation period, with an effective removal rate of 96.2%. In contrast, the abundance of ARGs in the blank control group decreased continuously in the early stage of composting, but began to rebound significantly on day 16. By day 32 at the end of composting, the abundance of ARGs was significantly higher than that on day 8 after reduction, and finally reached 714.7 TPM during the composting maturation period, with an effective removal rate of 85.2%, significantly lower than that of treatment group 1. In treatment group 2, the abundance of ARGs in the compost decreased rapidly in the early stage, but rebounded significantly on day 16 of composting. By day 32 at the end of composting, the abundance of ARGs was significantly higher than that on day 8 after reduction, and finally reached 558.1 TPM, with an effective removal rate of 87.8%. Compared to treatment groups 1 and 2, the abundance of ARGs in the compost of treatment group 3 decreased more slowly in the early stage and did not show a significant rebound in the later stage of composting. The final ARG abundance reached 540.6 TPM, with an effective removal rate of 91.0%. The effective removal rates of ARGs in treatment groups 2 and 3 were both better than the blank control group, but significantly lower than those in treatment group 1. Comparison of treatment groups 1-3 revealed that adding the composite high-temperature microbial agent prepared in Example 1 could simultaneously increase ·OH (with a maximum concentration of 1610.5 μmol / kg) and EPFRs (12.5*10). 15 The generation of spins / g enhanced the synergistic effect of ·OH in the early stage of composting on the removal of ARGs and the later stage of EPFRs on the rebound inhibition of ARGs, ultimately improving the effective removal efficiency of ARGs (96.2%).

[0039] The abundance changes of ARGs host pathogens in different treatment groups are as follows: Figure 6 As shown in the figure, the composting of treatment group 1 promoted the removal of host pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae, Vibrio cholerae, and Escherichia coli, and inhibited their re-reproduction during the composting period. On day 32 after the end of composting, the total abundance of host pathogens in the compost of treatment group 1 was significantly lower than that in the blank control group. In addition, the composting of treatment group 2 had a good removal effect on host pathogens in the early stage, but there was a significant problem of re-reproduction during the composting period. The composting of treatment group 3 had a similar effect on the removal of host pathogens as the blank control group, but no significant re-reproduction was observed in the later stage of composting. In summary, compared with the other three groups, treatment group 1 can efficiently remove host pathogens in the early stage of composting and significantly inhibit their re-reproduction in the later stage of composting, thereby inhibiting the rebound of ARG abundance in the later stage of composting.

[0040] The composite high-temperature microbial agent of this invention creates a dynamic aerobic-anaerobic cycle in the compost pile during the initial stage of composting through intermittent aeration. Under aerobic conditions, thermophilic iron-reducing bacteria utilize the nutrients in the compost pile for growth and reproduction, driving the redox cycle of iron ions in iron mineral nanozymes and iron-modified biochar, generating a large amount of ·OH, which destroys the DNA structure of ARGs and promotes the formation of EPFRs, achieving efficient reduction of ARGs. Under the continuous action of thermophilic bacteria, the compost pile continues to heat up. The high-temperature environment can not only directly accelerate the thermal denaturation and decomposition of extracellular free ARGs, but also kill the host pathogens of ARGs and destroy intracellular ARGs, further removing ARGs from the compost. At the same time, the iron-modified biochar in the compost pile and the large amount of ·OH generated, combined with the high-temperature conditions, promote the continuous production and accumulation of EPFRs. By inhibiting quorum sensing among bacteria, it reduces the re-reproduction of host pathogens during the compost maturation period, effectively inhibiting the rebound of ARGs in high-temperature composting.

[0041] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A compound high-temperature bacterial agent, characterized in that, The compound high-temperature bacterial agent includes thermophilic iron-reducing bacteria, thermophilic bacteria, basal culture medium, carrier, and conditioner. The thermophilic iron-reducing bacteria is Bacillus composti SgZ-9, and the thermophilic bacteria is Calditerricola yamamurae YMO722. The basal culture medium consists of 2.9 g / L municipal sludge (on a dry solids basis), 2.0 g / L foaming agent, 0.5 g / L yeast powder, 0.5 g / L ammonium chloride, 0.5 g / L sucrose, 0.5 g / L soluble starch, and 0.3 g / L potassium dihydrogen phosphate. The carrier includes iron mineral nanozyme and iron-modified biochar. The conditioner is soybean meal powder.

2. The composite high-temperature bacterial agent according to claim 1, characterized in that, The iron mineral nanozyme accounts for 1% to 5% of the mass of the composite high-temperature bacterial agent. The iron mineral nanozyme is iron(III) oxide nanoparticle. The iron-modified biochar accounts for 1% to 5% of the mass of the composite high-temperature bacterial agent. The iron-modified biochar is any one of ferric sulfate modified biochar, ferric chloride modified biochar, ferric oxide modified biochar, or zero-valent iron modified biochar. The conditioner accounts for 5% of the mass of the composite high-temperature bacterial agent.

3. The composite high-temperature bacterial agent according to claim 1, characterized in that, The compound high-temperature bacterial agent contains 10% of both the thermophilic iron-reducing bacterium Bacillus composti SgZ-9 and the thermophilic bacterium Calditerricola yamamurae YMO722. 8 ~10 9 cfu / g.

4. A method for preparing a composite high-temperature bacterial agent according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Preparation of seed culture of Calditerricola yamamurae YMO722; S2. Preparation of Bacillus composti SgZ-9 seed culture; S3. Prepare fermentation medium; S4. First, inoculate the Calditerricola yamamurae YMO722 seed culture prepared in step S1 into the fermentation medium prepared in step S3, introduce air and stir for fermentation culture for 16-24 hours; then, inoculate the Bacillus composti SgZ-9 seed culture prepared in step S2 into the above fermentation medium and ferment for 48-96 hours. S5. After fermentation, add soybean cake powder as a conditioning agent, stir evenly, and then filter by plate and frame to obtain a compound high-temperature bacterial agent with a moisture content of 45%~55%.

5. The method for preparing a composite high-temperature bacterial agent according to claim 4, characterized in that, Step S1 is: The bacterial content is 10... 9 A 2% inoculum of *Calditerricola yamamurae* YMO722 (cfu / mL) was inoculated into CYS medium and activated by constant temperature shaking at 75–80 °C and 160–180 rpm for 16–24 h to obtain an activated bacterial culture of *Calditerricola yamamurae* YMO722. This activated bacterial culture was then inoculated into a seed tank at a 2% inoculum and incubated at 75–80 °C with an aeration rate of 20–100 m³ / h. 3 Fermentation was carried out for 16-24 h at a stirring speed of 180-220 rpm to obtain Calditerricola yamamurae YMO722 seed culture.

6. The method for preparing a composite high-temperature bacterial agent according to claim 4, characterized in that, Step S2 is: The bacterial content is 10... 9 Bacillus composti SgZ-9 bacterial culture at cfu / mL was inoculated into LB medium and fermented at 45–50 °C for 48–96 h. Air was introduced during fermentation at a rate of 80–100 m³ / mL. 3 The mixture is stirred continuously at a speed of 160~200 rpm for 1 hour. Once fermentation is complete, the Bacillus composti SgZ-9 seed culture is obtained.

7. The method for preparing a composite high-temperature bacterial agent according to claim 4, characterized in that, Step S3 is as follows: Take urban sludge, add urban sludge, foaming agent, yeast powder, ammonium chloride, sucrose, soluble starch, and potassium dihydrogen phosphate to water according to the composition of the basic culture medium described in claim 1, adjust the pH to 7.0~7.5, add magnetite nanozyme and iron-modified biochar, stir evenly, sterilize, and obtain fermentation culture medium, cool for later use. The fermentation culture medium contains 2.9 g / L urban sludge (based on dry solids), 2.0 g / L foaming agent, 0.5 g / L yeast powder, 0.5 g / L ammonium chloride, 0.5 g / L sucrose, 0.5 g / L soluble starch, 0.3 g / L potassium dihydrogen phosphate, 50 g / L magnetite nanozyme, and 50 g / L iron-modified biochar.

8. The application of the composite high-temperature microbial agent according to any one of claims 1 to 3, and the composite high-temperature microbial agent prepared by the method according to any one of claims 4 to 7, in inhibiting ARGs rebound during high-temperature composting.

9. A method for suppressing ARGs rebound during high-temperature composting, characterized in that, The method involves mixing a composite high-temperature microbial agent according to any one of claims 1 to 3 with initial composting materials for composting fermentation, wherein the preferred mixing mass ratio of the composite high-temperature microbial agent to the initial composting materials is (1-5):(95-99).

10. The method according to claim 9, characterized in that, The initial composting material is any one of urban sludge, livestock and poultry manure, agricultural residues, or kitchen waste.

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