A method for efficient detoxification and nutrient recovery of fungus-strengthened erythromycin fermentation residue by aerobic composting

Abstract

The application discloses a kind of fungus reinforced erythromycin fermentation microbial residue aerobic compost efficient detoxification and nutrient recovery method, belong to solid waste disposal and resource recycling field.The method includes that the erythromycin fermentation microbial residue is mixed after being treated with high-temperature sterilization with corn straw powder, inoculates and carries out aerobic compost fermentation with Trichoderma asperellum.The antibiotic degrading inoculum with Trichoderma asperellum as core is inoculated to reinforce the aerobic co-composting of erythromycin fermentation microbial residue and crop straw, produces nutrient content high and non-toxic harmless organic fertilizer, to provide an efficient, economical, environmentally friendly solution to solve the problem of resource utilization of erythromycin fermentation microbial residue.

Description

Technical Field

[0001] This invention relates to the field of solid waste disposal and resource recycling, and in particular to a method for efficient detoxification and nutrient recovery from aerobic composting of erythromycin fermentation residue enhanced by fungi. Background Technology

[0002] Erythromycin is widely used in clinical medicine, livestock and poultry farming, and aquaculture for the prevention and treatment of bacterial infectious diseases. It is also an indispensable precursor for the synthesis of other novel macrolide antibiotics. The production of erythromycin relies entirely on microbial fermentation, but due to the low yield, producing one ton of erythromycin generates more than ten tons of fermentation residue. This residue is slurry-like, with a high water content (>80%), rich in nutrients, and contains high concentrations of erythromycin residue (200-1000 mg / kg, or even higher). It is highly susceptible to spoilage during storage and transportation, emitting an unpleasant odor. If not properly treated, the harmful components in the residue can leak into the soil or aquatic environment, causing a series of ecotoxicological events. More worryingly, residual erythromycin may induce the proliferation and spread of drug-resistant bacteria and antibiotic resistance genes (ARGs) in the environment, thereby increasing the risk of resistant bacteria evolution and spread, posing a serious threat to human health. Meanwhile, if the abundant nutrients contained in the fungal residue cannot be effectively converted and utilized, it will not only be a huge waste of resources but may also cause secondary environmental pollution. How to strengthen the resource utilization of antibiotic fungal residue has become a matter of great concern.

[0003] The presence of numerous drug-resistant bacteria (including erythromycin-producing bacteria and other resistant microorganisms) in erythromycin fermentation residue poses a significant risk to its safe resource utilization. Therefore, effectively eliminating these drug-resistant bacteria is a necessary pretreatment method. Although reports have indicated that high temperature and pressure, high-energy electron radiation, strong acidification, and oxidants (such as ozone, hydrogen peroxide, and persulfate) can efficiently kill drug-resistant bacteria, these methods are difficult to implement on a large scale due to high equipment investment, high operating costs, and the potential for secondary pollution from added chemicals. Therefore, the development of efficient, energy-saving, and environmentally friendly pretreatment technologies for fermentation residue sterilization is particularly urgent.

[0004] Composting technology is considered an important way to achieve deep detoxification and nutrient resource utilization of antibiotic fermentation residue. However, while traditional composting technology can partially degrade organic matter, it suffers from drawbacks such as low antibiotic detoxification efficiency, incomplete detoxification, long treatment cycles, and significant nutrient volatilization and loss. Furthermore, some microbial strains used to degrade antibiotics may pose significant risks of pathogenicity to plants and animals and the spread of drug resistance. Therefore, developing safe, efficient, and low-cost technologies for reducing drug-resistant bacteria, microbially enhanced antibiotic detoxification, and nutrient recovery and resource utilization is the core path to overcoming the dual dilemma of environmental risks and resource waste associated with erythromycin fermentation residue. Summary of the Invention

[0005] The purpose of this invention is to provide a method for efficient detoxification and nutrient recovery in aerobic composting of erythromycin fermentation residue enhanced by fungi, in order to solve the problems existing in the prior art. By inoculating the residue with an antibiotic-degrading agent with Trichoderma echinosporum as the core, the aerobic co-composting of erythromycin fermentation residue and crop straw is enhanced, producing organic fertilizer with high nutrient content and no toxicity or harm, thus providing an efficient, economical, and environmentally friendly solution to the problem of resource utilization of erythromycin fermentation residue.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for efficient detoxification and nutrient recovery in aerobic composting of erythromycin fermentation residue enhanced with fungi, comprising the steps of mixing erythromycin fermentation residue that has been sterilized at high temperature with corn straw powder, and then inoculating it with Trichoderma asperellum for aerobic composting fermentation.

[0008] Preferably, the high-temperature sterilization conditions are: heating at 80°C for 30 minutes.

[0009] Preferably, the mass ratio of the erythromycin fermentation residue to corn straw powder is (3-5):(5-7).

[0010] Preferably, the mass ratio of the erythromycin fermentation residue to corn stalk powder is 5:5.

[0011] Preferably, the erythromycin fermentation residue treated with high temperature sterilization is adjusted to a pH of 7.0-7.5 before being mixed with the corn straw powder.

[0012] Preferably, the *Trichoderma hydathodes* is *Trichoderma hydathodes* TS7-1, with accession number CGMCC No. 41372;

[0013] And / or the *Trichoderma hydathodes* is inoculated in the form of a spore suspension, and the inoculation ratio of the *Trichoderma hydathodes* spore suspension is 1 g: 10 mL based on the total mass of erythromycin fermentation residue and corn straw powder.

[0014] And / or the concentration of Trichoderma spore suspension is 10 9 per mL.

[0015] Preferably, the aerobic composting fermentation temperature is 20-25℃, the time is 30 days, and the compost is thoroughly turned over every other day during the fermentation period.

[0016] Preferably, the process also includes a step of naturally drying the solid substrate obtained after aerobic composting fermentation until the moisture content is below 30%.

[0017] The present invention also provides the application of the method in improving the degradation efficiency of erythromycin in erythromycin fermentation residue.

[0018] The present invention also provides the application of the solid matrix obtained by the method in promoting the germination of Chinese cabbage seeds and the growth of plants.

[0019] The present invention discloses the following technical effects:

[0020] Before composting, the erythromycin fermentation residue is heat-treated at 80°C for 30 minutes, which reduces the density of erythromycin-resistant bacteria (including erythromycin-producing bacteria and other resistant bacteria) to below 100 cfu / mL. This effectively kills resistant bacteria in the residue under relatively mild and low-energy conditions, and effectively controls the risk of the spread of resistant bacteria and resistance genes during subsequent fermentation of the residue.

[0021] This invention involves mixing high-temperature pretreated erythromycin fermentation residue with corn straw powder, inoculating with only a 1 / 100 suspension of *Trichoderma echinococcus* spores, and then performing aerobic composting fermentation at room temperature to efficiently degrade erythromycin, reducing its content to 50 μg / kg. Further experiments revealed that in organic fertilizer produced by enhanced fermentation of erythromycin residue with *Trichoderma echinococcus*, erythromycin is completely degraded, and its toxicity to plant seed germination and seedling growth is completely eliminated, ensuring the safety of the organic fertilizer.

[0022] Testing revealed that the organic fertilizer produced from erythromycin fermentation residue, enhanced by the fungus *Trichoderma echinosporum*, exhibited significantly higher levels of nutrient content, heavy metal residue, and seed germination index than the industry standard "Organic Fertilizer (NY 525-2021)" issued by the Ministry of Agriculture and Rural Affairs of China. Small-scale pot experiments demonstrated that the organic fertilizer produced from the fermented residue provided ample nutrients for cabbage plant growth, significantly promoting biomass, leaf area, plant height, and root length. This invention provides a novel method for the resource utilization of erythromycin fermentation residue, mitigating its environmental risks and resource waste. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The effects of inoculating Trichoderma hydathodes with different substrate mixture ratios on erythromycin residue (A) and removal rate (B);

[0025] Figure 2The changes in erythromycin residual concentration in the mixed compost substrate of fungal residue and straw at different stages of composting; A: Erythromycin concentration in organic fertilizers fermented naturally and by fungal inoculation within 0-30 days; B: Erythromycin concentration in organic fertilizers fermented by fungal inoculation within 10-30 days.

[0026] Figure 3 The effects of different erythromycin-based bacterial residue-straw mixed fermentation organic fertilizers on cabbage seed germination and plant growth; A: cabbage seed germination phenotype, B: cabbage seedling growth phenotype, C: cabbage biomass, D: cabbage plant height;

[0027] Figure 4 Morphological characteristics (A) and aerial hyphae, conidiophores and conidia (BC) of strain TS7-1 on PDA medium;

[0028] Figure 5 This is a phylogenetic tree of NJ constructed based on ITS sequences. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] The following examples relate to the sources of Trichoderma echinosporum:

[0035] 1. Strains Isolation

[0036] (1) Preparation of isolation culture medium

[0037] Solid basic salt medium MSM (NaCl 1g, NH4Cl 1g, K2HPO4 1.5g, KH2PO4 0.5g, MgSO4·7H2O 0.2g, agar 15g, water 1000mL, pH 7.0) was dispensed into Erlenmeyer flasks, 100mL per flask. After sterilization, the medium was cooled to approximately 60°C. 0.01g of erythromycin powder was added to the medium and rapidly shaken to dissolve it completely. The solution was then poured into 9cm Petri dishes to prepare MSM plates containing 100mg / L erythromycin.

[0038] (2) Isolation and screening of erythromycin-degrading bacteria

[0039] Collect 5g (or 5mL) of samples from environmental samples such as forest humus layer soil, erythromycin fermentation solid bacterial residue, landfill leachate, activated sludge from sewage treatment plants, and solid manure from livestock farms. Add 45mL of sterile water and shake on a shaker at 150rpm for 30 minutes to prepare a suspension. Then, perform serial dilutions (10... -2 ~10 -6 ) Take 100 μL of the diluted solution and spread it on an MSM plate containing 1000 mg / L erythromycin. Repeat each dilution twice. Place the plate in a dark incubator at 30℃ and incubate until the colony grows to 1-2 mm. Pick a single colony and inoculate it onto a 6 mm diameter MSM agar block containing 100 mg / L erythromycin. Incubate at 30℃ in the dark for 24 h. Place the incubated agar block on the surface of a plate coated with erythromycin-sensitive Bacillus velezensis L0517 (an endophytic bacterium isolated from the leaves of Illicium verum, published in "Liu Lianjin, Li Zhengling, Li Xueli, et al. Screening and fermentation conditions of biocontrol strains for anthracnose in Illicium verum [J]. Plant Protection, 2022, 48(05): 204-211") and incubate at 30℃ in the dark for 12 h. Measure the width (mm) of the inhibition zone around the agar block and calculate the relative removal rate of erythromycin. Relative removal rate = (D0 - D...) i ) / D0×100, where D0 is the width of the inhibition zone of the uninoculated erythromycin agar block - 6mm, D i The width of the inhibition zone for each strain is -6mm.

[0040] (3) Secondary screening of erythromycin-degrading bacteria

[0041] The initially screened strains were inoculated onto PDA plates for purification culture. After sporulation, the strains were eluted with a small amount of sterile 2% Tween-80 solution and transferred to potato dextrose liquid (PD) medium. The spore density in the medium was adjusted to reach 102 by adjusting the amount of elution buffer. 7 The bacterial culture was prepared by shaking at 30℃ and 200 rpm for 18 h to obtain a seed culture. 0.5 mL of the seed culture was inoculated into MSM liquid medium containing 100 mg / L erythromycin and cultured at 30℃ and 200 rpm in the dark with shaking. Samples were taken every 12 h to determine the bacterial biomass (mg / DW), erythromycin removal rate (%), and the antibacterial activity of degradation products.

[0042] (4) Determination of biomass of degrading bacteria

[0043] Take 10 mL of thoroughly mixed culture medium, centrifuge at 8000 rpm for 10 minutes, and then remove the supernatant. Resuspend the precipitated mycelium in 5 mL of sterile water. Dry a 7 cm diameter circular filter paper to constant weight and weigh it (m). p Subsequently, the mycelial suspension was filtered through filter paper of known mass, and residual mycelia were rinsed off the centrifuge tube walls with sterile water and filtered together. The filter paper with mycelia attached was dried at 60°C to constant weight, and the total mass (m) was weighed. t The dry biomass of degrading bacteria, mf (mg·DW), is equal to m t -m p .

[0044] (5) Erythromycin removal rate determination

[0045] Erythromycin concentration was determined and analyzed using an AB TripleTOF 5600 LC-MS / MS system (AB SCIEX, USA). The chromatographic column was an XBridge HBE C18 column (2.1 × 100 mm, 2.5 μm, Ireland). The mobile phase was 0.1% formic acid solution (phase A) and acetonitrile (phase B). The injection volume was 1.0 μL, the flow rate was 0.3 mL / min, and the column temperature was 30 °C. Gradient elution was used: 0–1 min, 95% phase A; 1–10 min, 85% phase A; 10–11 min, 45% phase A; 11–13 min, 5% phase A; 13–16 min, 95% phase A. Electrospray ionization (ESI+) was used for mass spectrometry analysis, with the following parameters: capillary temperature 220 ± 1 °C, positive ion spray voltage 4000 V, and nebulizer gas (N2) flow rate 8.0 L / min. The mass spectrometry scanning range was set to 50-1000 m / z. The detection limit for erythromycin (ERY) was 50 μg / L (signal-to-noise ratio S / N ≥ 3). A standard curve was established using erythromycin standards, and the concentration of erythromycin in the culture medium of each bacterial strain was calculated based on the standard curve (C0). i Using the erythromycin concentration (C0) in the culture medium of uninoculated strains as a control, the erythromycin removal rate was calculated as follows: Removal rate (%) = 1 - (C0) / (C0) i / C0)×100.

[0046] (6) Determination of antibacterial activity of erythromycin degradation products

[0047] The bioactivity of the degradation products of the strain was assessed using the antimicrobial susceptibility testing disc method. Erythromycin-sensitive *Bacillus belye* L0517 was evenly spread on an LB agar plate, and sterile, dry, circular antimicrobial susceptibility testing discs (6 mm in diameter) were placed at 25 mm intervals on the plate surface. After sterilization by filtration through a 0.22 μm sterile filter, 50 μL of the test culture was added dropwise to the antimicrobial susceptibility testing discs. The plates were incubated at 30°C in the dark for 12 h, and the size (mm) of the inhibition zone around the antimicrobial susceptibility testing discs was observed and measured.

[0048] The results showed that among the fungi isolated from the five environmental samples, six strains achieved a relative removal rate of over 50% for 100 mg / L erythromycin after 24 hours of culture. Among them, strain TS7-1 performed the best, with a relative removal rate as high as 81.0% (Table 1).

[0049] Table 1. Relative removal rate of erythromycin by the initial screening strains.

[0050]

[0051]

[0052] 2. Identification of strain TS7-1

[0053] (1) Morphological identification

[0054] Strains TS7-1 were inoculated onto PDA plates and cultured in a 28°C incubator for 48 hours. A small amount of mycelium was taken, stained with lactophenol blue, and then observed and photographed under an optical microscope to capture the morphological characteristics of the strain, while also measuring its size.

[0055] The results showed that strain TS7-1 grew rapidly on PDA medium, with a colony expansion rate of 3.45 cm / day. Initially, the mycelium was white with well-developed aerial hyphae, spreading rapidly radially across the medium surface. After 3 days of culture, numerous dense, granular sporulation structures appeared on the colony surface, turning pale green, and the sporulation areas showed concentric rings. Figure 4 (A). Conidiophores are whorled, with ampoule-shaped pedicels arranged in whorls, swollen at the base and tapering to a point, measuring 6.45–15.23 μm in length and 2.50–5.25 μm in width. Figure 4 (BD). Conidia are solitary, spherical or oval, measuring 1.95–4.52 μm × 1.86–3.55 μm. Strain TS7-1 exhibits typical characteristics of Trichoderma fungi.

[0056] (2) Molecular biological identification

[0057] Genomic DNA was extracted from strain TS7-1 using a modified CTAB method. Using the extracted DNA as a template, the fungal internal transcribed spacer (ITS) sequence was amplified using ITS3F / ITS4R primers. The primer sequences are as follows.

[0058] ITS3F (SEQ ID NO.1): 5'-GCATCGATGAAGAACGCAGC-3';

[0059] ITS4R (SEQ ID NO. 2): 5'-TCCTCCGCTTATTGATATGC-3'.

[0060] The ITS sequence of strain TS7-1 was entered into GenBank's BLATS database for homology comparison. ITS sequences of 19 type strains or recognized strains were downloaded. Using Protocreafarinosa CBS121551 as the peripheral group strain, a NJ developmental tree was constructed using MEAG 11 software as follows: Figure 5 As shown in the figure. The results showed that strain TS7-1 clustered with *Trichoderma asperellum* with 100% support. Based on morphological characteristics and molecular biological analysis, strain TS7-1 was identified as *Trichoderma asperellum*.

[0061] The strain was identified as Trichoderma asperellum TS7-1 and taxonomically named Trichoderma asperellum. It was deposited on June 17, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41372. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0062] Example 1: Optimization of High-Temperature Sterilization Conditions for Fungal Residue

[0063] The mushroom residue contains a large number of erythromycin-producing bacteria and drug-resistant bacteria. In order to effectively control the environmental risks of subsequent mushroom residue composting and resource conversion, it is necessary to carry out necessary sterilization pretreatment on the mushroom residue.

[0064] Collection of fermentation residue samples. The erythromycin fermentation residue tested was collected from a pharmaceutical factory in Hubei Province. The residue had a water content of 91.2%, total nitrogen and total organic carbon contents of 32.6 g / kg and 398.5 g / kg, respectively, a carbon-to-nitrogen ratio of 12.3, a pH value of 5.9, and an erythromycin residue concentration of 325.6 mg / kg. It was characterized by significantly high water content, high nutrient residue, and high concentration of antibiotic residue.

[0065] Optimization of high-temperature sterilization conditions for erythromycin-containing bacterial residue: 1 kg of erythromycin-containing bacterial residue was added to a temperature-controlled electric water heater and heated for 30 minutes at 60, 70, 80, 90, and 100°C, respectively. A small amount of the residue was then rapidly cooled and serially diluted with sterile water to a concentration of 10. -3 ~10 -8 The diluted solution was spread at a rate of 100 μL onto LB agar plates containing 100 mg / L of the drug. After 48 h, plate counts were performed to determine the density of culturable drug-resistant bacteria in the bacterial residue under different temperature treatments, with three replicates. Simultaneously, energy consumption under different temperature treatments was calculated (assuming an initial temperature of 20°C, a water heater thermal efficiency of 85%, a heat loss of 5°C / h during the insulation period, and atmospheric pressure).

[0066] As shown in Table 2, the results indicate that after treatment with erythromycin-resistant bacterial residue at temperatures above 80℃ for 30 minutes, the density of erythromycin-resistant bacteria decreased to below 100 CFU / mL. Further increasing the heating temperature resulted in a smaller decrease in bacterial density, indicating that some resistant bacteria exhibited extremely high heat resistance. Simultaneously, increasing the temperature led to a rapid increase in energy consumption (13–24% / 10℃), suggesting that further increases in temperature reduced sterilization efficiency. Therefore, considering both the efficiency and energy consumption of large-scale bacterial residue sterilization operations, the suitable treatment condition is heating at 80℃ under normal pressure for 30 minutes.

[0067] Table 2 Energy consumption and density of erythromycin-resistant bacteria after 30 min of treatment with erythromycin-containing bacterial residue at different temperatures.

[0068]

[0069] Example 2: Optimal Ratio Optimization of Antibiotic Inoculum Residue and Straw

[0070] (1) Preparation of erythromycin-degrading fungal inoculant. Trichoderma esculenta TS7-1 was inoculated onto potato dextrose agar (PDA) medium and cultured for 5 days. After conidia were produced on the surface, 2 mL of sterile 0.5% Tween-80 solution was added to the surface of the medium, and the spores were gently washed off by agitating the culture dish. The fungal suspension was collected into an Erlenmeyer flask, an appropriate amount of sterile water was added, and the flask was placed in a shaker and shaken at 200 rpm for 5 min to ensure the spores were fully dispersed in the water. The spore density in the suspension was counted using a hemocytometer, and the spore density was diluted with water to a concentration of 10. 9 The bacterial count was set at 1 / mL as the inoculum.

[0071] (2) Composting fermentation of mycelium residue and straw inoculated with mycelium. The mycelium residue was first heated at 80℃ for 30 min, and after cooling, the pH of the mycelium residue was adjusted to 7.0-7.5 with 1 mol / L NaOH solution. Then, it was mixed with corn straw powder (fiber length ≤1cm) in different proportions (6:4, 5:5, 4:6, 3:7, w / w) to form a solid matrix. Trichoderma echinococcosis agent was inoculated into 1000g of solid matrix at a ratio of 1 / 100 (V / W). The same volume of sterile water was used as a control (CK). After thorough mixing, the mixture was spread evenly in a plastic tray with a pile thickness of 10cm, covered with breathable plastic wrap, and placed at room temperature of 20-25℃ for composting fermentation. During the fermentation period, the pile was thoroughly turned every 1 day, and about 10g of samples were randomly taken and stored in a -20℃ refrigerator. The erythromycin residue was measured uniformly after 7 days.

[0072] (3) Erythromycin extraction from compost. Accurately weigh 2.000 g of the mixed compost matrix sample, add 30 mL of a pH 6 Tris-CaCl2 mixed extraction solvent (10 mmol / L Tris, 20 mmol / L CaCl2), and sonicate for 10 minutes. Centrifuge at 8000 rpm for 5 minutes. Collect the supernatant and adjust the pH to 10 with 1 mol / L NaOH. Add the extraction / dispersant (1 mL 1,2-dichloroethane, 1 mL methanol), vortex for 1 minute, and centrifuge at 4000 rpm for 5 minutes. Collect the 1,2-dichloroethane layer, dry it under nitrogen, add 1 mL acetonitrile, vortex for 30 seconds to accelerate redissolution, and centrifuge at 13000 rpm for 10 minutes. Take the supernatant, filter it through a 0.22 μm filter membrane, and determine the erythromycin content using an LC-MS system.

[0073] The results showed that the mixing ratio of fungal residue to straw powder significantly affected the removal efficiency of erythromycin. A high ratio of fungal residue (6:4) inhibited fungal growth in the early stages, leading to slow erythromycin removal; after 7 days of fermentation, the erythromycin concentration remained as high as 48.5 mg / L, with a degradation rate below 75%. In contrast, fungal residues with ratios of 5:5, 4:6, and 3:7 showed erythromycin concentrations decreasing to 1.0–3.3 mg / L after 7 days of fermentation, with degradation rates exceeding 97%. Figure 1 (AB). Considering both erythromycin removal rate and mushroom residue treatment efficiency, the optimal mixing ratio of mushroom residue and corn straw is equal mass.

[0074] Example 3: Evaluation of the quality and safety of long-term composting fermentation organic fertilizer using antibiotic bacterial residue.

[0075] Long-term composting fermentation of mycelium residue and straw inoculated with fungal agents. The mycelium residue was first heated at 80℃ for 30 minutes, then cooled to adjust the pH to 7.0–7.5. It was then mixed with corn straw powder in equal mass to form a solid substrate. A fungal agent of *Trichoderma hygroscopicum* was inoculated at a 1 / 100 ratio (fungal inoculation fermentation), with an equal mass of sterile water used as a control (natural fermentation). Composting conditions and management were consistent with Example 2, with a total fermentation period of 30 days. Erythromycin residue was measured every 5 days during this period.

[0076] Organic fertilizer quality testing. After 30 days of fermentation, the organic fertilizer was sun-dried until the moisture content was <30%. Then, the moisture content, total nutrients, total organic matter, heavy metal content, and seed germination index of the organic fertilizer were determined according to the method of the Ministry of Agriculture and Rural Affairs standard "Organic Fertilizer (NY 525-2021)". Seed germination was tested using Chinese cabbage (Brassica rapa) seeds.

[0077] Organic fertilizer efficacy and safety testing: Organic fertilizer was mixed with garden soil at a ratio of 1 / 100 (w / w), and then 1000g was placed in each small plastic flowerpot. After thorough watering, three cabbage seedlings were planted in each pot. After 30 days, the plant height (cm), root length (cm), and leaf area (cm²) were measured. 2 Leaf area was measured using the paper-cutting and weighing method, and the biomass (g·FW) was also measured. Three replicates were performed.

[0078] The erythromycin detoxification effect showed that in the natural composting fermentation of straw without fungal inoculation, the residual concentration of erythromycin decreased rapidly in the first 5 days, but the removal rate decreased thereafter, and the residual concentration was still as high as 40.6 mg / L after 30 days of composting fermentation; while in the fermentation with the fungus *Trichoderma echinococcus*, the residual concentration of erythromycin was removed at a higher rate in the first 5 days, and the concentration was below 1.0 mg / L by the 10th day, and below the detection limit (50 μg / kg) at 30 days. Figure 2(See Table 3, AB). This indicates that the degradation by the fungus *Trichoderma hydatids* is the key driver for the rapid removal of erythromycin residues from fungal compost.

[0079] The final organic fertilizer quality indicators showed that the total nitrogen and total nutrient content in the organic fertilizer fermented with the fungus *Trichoderma hydathodes* reached 5.52% and 6.14%, respectively, exceeding the 27.7% and 25.1% of naturally fermented organic fertilizer. The germination index reached 99.7%, and the heavy metal content was far below the limit standard, fully meeting the standards of the Ministry of Agriculture and Rural Affairs' "Organic Fertilizer (NY 525-2021)" (Table 3). In contrast, the naturally fermented organic fertilizer suffered from severe inhibition of cabbage seed germination due to high concentrations of erythromycin residue, resulting in a final germination index of only 35.1%, failing to meet the organic fertilizer standard (Table 3). This indicates that inoculation with *Trichoderma hydathodes* can not only quickly and thoroughly remove the toxicity of erythromycin residue from the fungal residue-straw mixed compost, but also increase the nutrient content of the fertilizer, thus transforming erythromycin-containing fungal residue from hazardous waste into high-quality organic fertilizer.

[0080] Toxicity tests on cabbage seeds and plant growth using organic fertilizer showed that organic fertilizer fermented with *Trichoderma hydathodes* completely eliminated the seed toxicity of erythromycin, while naturally fermented fungal residue organic fertilizer significantly inhibited seed radicle and cotyledon development due to high residual erythromycin. Figure 3 (A). After the two types of organic fertilizers were applied to the soil, the organic fertilizer fermented by the fungus *Trichoderma hydathodes* significantly promoted the growth of Chinese cabbage plants. The biomass, leaf area, plant height, and root length of the plants were all significantly higher than those in the unfertilized control. The naturally fermented fungal residue organic fertilizer significantly inhibited the growth of Chinese cabbage plants due to the high erythromycin residue that had not been removed. Figure 3 (BD).

[0081] Table 3 Quality Indicators of Erythromycin Residue-Straw Mixed Fermentation Organic Fermentation

[0082]

[0083] Note: "—" indicates that this item is not explicitly defined in the standard "Organic Fertilizer (NY 525-2021)".

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for efficient detoxification and nutrient recovery from aerobic composting of erythromycin fermentation residue enhanced with fungi, characterized in that, The process includes mixing erythromycin fermentation residue that has been sterilized at high temperature with corn straw powder, and then inoculating it with Trichoderma asperellum for aerobic composting fermentation. The *Trichoderma* species mentioned is *Trichoderma TS7-1*, with accession number CGMCC No. 41372.

2. The method as described in claim 1, characterized in that, The conditions for high-temperature sterilization are: heating at 80°C for 30 minutes.

3. The method as described in claim 1, characterized in that, The mass ratio of the erythromycin fermentation residue to corn straw powder is (3-5):(5-7).

4. The method as described in claim 3, characterized in that, The mass ratio of the erythromycin fermentation residue to corn straw powder is 5:

5.

5. The method as described in claim 1, characterized in that, The erythromycin fermentation residue, after being treated with high-temperature sterilization, is adjusted to a pH of 7.0-7.5 and then mixed with the corn straw powder.

6. The method as described in claim 1, characterized in that, The *Trichoderma hydathodes* was inoculated in the form of a spore suspension. The inoculation ratio of the *Trichoderma hydathodes* spore suspension was 1 g: 10 mL, based on the total mass of the erythromycin fermentation residue and corn straw powder. The concentration of the Trichoderma spore suspension was 10. 9 per mL.

7. The method as described in claim 1, characterized in that, The aerobic composting fermentation temperature is 20-25℃, the time is 30 days, and the compost is thoroughly turned over every other day during the fermentation period.

8. The method as described in claim 1, characterized in that, It also includes the step of naturally drying the solid substrate obtained after aerobic composting fermentation until the moisture content is below 30%.

9. The application of the method according to any one of claims 1-8 in improving the degradation efficiency of erythromycin in erythromycin fermentation residue.

10. The application of the solid substrate obtained by the method according to any one of claims 1-8 in promoting the germination of Chinese cabbage seeds and the growth of plants.

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