Efficient detoxification and nutrient recovery method for aerobic composting of fungus-enhanced erythromycin fermentation fungus residues

Abstract

The invention discloses an efficient detoxification and nutrient recovery method for aerobic composting of fungus-enhanced erythromycin fermentation fungus residues, and belongs to the field of solid waste disposal and resource reutilization. The method comprises the following steps: mixing erythromycin fermentation mushroom dregs subjected to high-temperature sterilization treatment with corn straw powder, inoculating trichoderma asperellum, and carrying out aerobic composting fermentation. According to the method, aerobic co-compost of erythromycin fermentation mushroom dregs and crop straws is strengthened by inoculating an antibiotic degrading microbial agent taking trichoderma asperellum as a core, and an organic fertilizer which is high in nutrient content and is non-toxic and harmless is produced, so that an efficient, economical and environment-friendly solution is provided for solving the problem of resource utilization of the erythromycin fermentation mushroom dregs.

Description

Technical Field

[0001] The present invention relates to the field of solid waste disposal and resource recycling, and particularly to a method for efficiently detoxifying and nutrient recycling of aerobic composting of fungal-enhanced erythromycin fermentation residues. Background Art

[0002] Erythromycin is widely used in the prevention and treatment of bacterial infectious diseases in the fields of clinical medicine, livestock and poultry breeding, and aquaculture. At the same time, it is also an indispensable synthetic precursor for other new macrolide antibiotics. The production process of erythromycin completely depends on microbial fermentation. However, due to the low drug production rate, more than 10 tons of fermentation residues will be discharged for every 1 ton of erythromycin produced. These residues are in the form of slurry, with high water content (>80%), rich in nutrients, and contain high concentrations of erythromycin residues (200-1000 mg / kg, or even higher). They are extremely prone to corruption and deterioration during storage and transportation, emitting an unpleasant stench. If not properly treated, the harmful components in the residues will cause a series of ecological toxicities once leaked into the soil or water environment. More worryingly, the residual erythromycin may induce the proliferation and spread of drug-resistant bacteria and antibiotic resistance genes (ARGs) in the environment, thus increasing the risk of evolution and spread of resistant bacteria and posing a serious threat to human health. At the same time, if the large amount of nutrients contained in the residues cannot be effectively converted and utilized, it will not only be a great waste of resources, but also may cause secondary environmental pollution. How to strengthen the resource utilization of antibiotic residues has become an extremely concerned issue.

[0003] The large number of drug-resistant bacteria (including erythromycin-producing bacteria, drug-resistant miscellaneous bacteria, etc.) in erythromycin residues pose a huge risk to the safe resource utilization. Therefore, effectively killing these drug-resistant bacteria has become a necessary pretreatment means. Although it has been reported that treatment methods such as high temperature and high pressure, high-energy electron radiation, strong acid acidification, and oxidants (such as ozone, hydrogen peroxide, persulfate) can efficiently kill drug-resistant bacteria, these methods are difficult to be widely applied on a large scale in practice due to problems such as large equipment investment, high operating costs, and secondary pollution caused by external chemicals. Therefore, it is particularly urgent to develop a pretreatment technology for residue sterilization with high treatment efficiency, energy conservation and environmental protection.

[0004] Composting technology is regarded as an important way to achieve deep detoxification and nutrient resource utilization of antibiotic residues. However, traditional composting technology can only partially degrade organic matter, and has obvious defects such as low efficiency of antibiotic detoxification, incomplete detoxification, long treatment cycle, serious nutrient volatilization and loss, and the risk of animal and plant pathogenicity and transmission of drug resistance of some microbial strains used to degrade antibiotics. Therefore, developing a safe, efficient, and low-cost technology for reducing drug-resistant bacteria, microbial-enhanced antibiotic detoxification, and nutrient recycling and resource utilization is the core path to break through the dual dilemmas of environmental risks and resource waste of erythromycin residues. Summary of the Invention

[0005] The object of the present invention is to provide a method for efficient detoxification and nutrient recovery of aerobic composting of fungal-enhanced erythromycin fermentation residues, so as to solve the problems existing in the above-mentioned prior art. By inoculating an antibiotic-degrading bacterial agent with Trichoderma asperellum as the core, the aerobic co-composting of erythromycin fermentation residues and crop straws is enhanced to produce organic fertilizers with high nutrient content and non-toxic and harmless properties, so as to provide an efficient, economical and environmentally friendly solution for solving the problem of resource utilization of erythromycin fermentation residues.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for efficient detoxification and nutrient recovery of aerobic composting of fungal-enhanced erythromycin fermentation residues, which includes the step of mixing the erythromycin fermentation residues treated by high-temperature sterilization with corn straw powder and then inoculating Trichoderma asperellum for aerobic composting fermentation.

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

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

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

[0011] Preferably, after adjusting the pH value of the erythromycin fermentation residues treated by high-temperature sterilization to 7.0 - 7.5, they are then mixed with the corn straw powder.

[0012] Preferably, the Trichoderma asperellum is Trichoderma asperellum TS7-1, and the preservation number is CGMCC No. 41372;

[0013] and / or the Trichoderma asperellum is inoculated in the form of a spore suspension, and according to the total mass of the erythromycin fermentation residues and the corn straw powder, the inoculation ratio of the Trichoderma asperellum spore suspension is 1 g:10 mL;

[0014] and / or the concentration of the Trichoderma asperellum spore suspension is 10 9 CFU / mL.

[0015] Preferably, the temperature of the aerobic composting fermentation is 20 - 25 °C, the time is 30 days, and thorough turning is carried out every other day during the fermentation period.

[0016] Preferably, it further includes the step of naturally drying the solid matrix obtained after the completion of the aerobic composting fermentation until the water content is lower than 30%.

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

[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 the composting fermentation of the present invention, the erythromycin fermentation residues are heat-treated at 80 °C for 30 min, which can reduce the density of erythromycin-resistant bacteria (including erythromycin-producing bacteria and drug-resistant miscellaneous bacteria) to below 100 cfu / mL, effectively kill the drug-resistant bacteria in the residues under relatively mild and low-energy-consuming conditions, and effectively control the risk of the spread of drug-resistant bacteria and drug-resistant genes in the subsequent fermentation of the residues.

[0021] By mixing the high-temperature pretreated erythromycin fermentation residues with corn straw powder, the present invention only needs to inoculate 1 / 100 of the Trichoderma asperellum spore suspension and carry out aerobic composting fermentation at room temperature to efficiently degrade erythromycin and reduce the content of erythromycin to 50 μg / kg. Through further experiments, it is found that erythromycin in the organic fertilizer obtained by the enhanced fermentation of erythromycin residues with Trichoderma asperellum is completely degraded, and the toxicity to the germination of plant seeds and the growth of seedlings is completely removed, ensuring the safety of the use of organic fertilizers.

[0022] After detection, the nutrient content, heavy metal residues, seed germination index and other indicators of the organic fertilizer obtained by the enhanced fermentation of erythromycin residues with the fungus Trichoderma asperellum are much higher than the industry standard of "Organic Fertilizer (NY 525-2021)" issued by the Ministry of Agriculture and Rural Affairs of China. The small potted plant experiment shows that the organic fertilizer produced by the fermentation of residues can provide sufficient fertility for the growth of Chinese cabbage plants and significantly promote the growth of plant biomass, leaf area, plant height and root length. The present invention provides a new method for the resource utilization of erythromycin fermentation residues, which can solve the environmental risk and resource waste of erythromycin residues. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 For the inoculation of Trichoderma asperellum on the residues-straw mixed matrix with different ratios, the residual amount (A) and removal rate (B) of erythromycin;

[0025] Figure 2For the change in the residual concentration of erythromycin in the mushroom residue-straw mixed compost substrate at different stages of composting; A: Concentration of erythromycin in the organic fertilizer during natural fermentation and fungal inoculation fermentation within 0 - 30 days, B: Concentration of erythromycin in the organic fertilizer during fungal inoculation fermentation within 10 - 30 days;

[0026] Figure 3 For the effects of different erythromycin mushroom residue-straw mixed fermented organic fertilizers on the germination of Chinese cabbage seeds and the growth of plants; A: Phenotype of Chinese cabbage seed germination, B: Phenotype of Chinese cabbage seedling growth, C: Biomass of Chinese cabbage, D: Plant height of Chinese cabbage;

[0027] Figure 4 For the morphological characteristics of strain TS7-1 on PDA medium (A) and its aerial hyphae, conidiophores and conidia (B - C);

[0028] Figure 5 For the NJ phylogenetic tree constructed based on the ITS sequence. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0033] The terms "comprising", "including", "having", "containing", etc. used in this article are all open-ended terms, meaning including but not limited to.

[0034] The sources of Trichoderma asperellum involved in the following examples:

[0035] 1. Strain isolation

[0036] (1) Preparation of isolation medium

[0037] Dispense the solid basal salt medium MSM (1 g of NaCl, 1 g of NH4Cl, 1.5 g of K2HPO4, 0.5 g of KH2PO4, 0.2 g of MgSO4·7H2O, 15 g of agar, 1000 mL of water, pH 7.0) into Erlenmeyer flasks, 100 mL per flask. After sterilization, cool to about 60 °C and add 0.01 g of erythromycin powder to the medium and quickly shake to dissolve it completely. Pour it into 9-cm Petri dishes to prepare MSM plates containing 100 mg / L of erythromycin.

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

[0039] Collect 5 g (or 5 mL) of samples from environmental samples such as forest floor humus soil samples, solid erythromycin fermentation residues, landfill leachate, activated sludge from sewage treatment plants, and solid manure from farms. Add 45 mL of sterile water and shake on a shaker at 150 rpm for 30 minutes to prepare a suspension. Gradient dilute the suspension (10 -2 ~10 -6 ) and take 100 μL of the diluted solution and spread it on MSM plates containing 1000 mg / L of erythromycin. Repeat for each dilution 2 times. Place the plates in an incubator at 30 °C in the dark. When the colonies grow to 1 - 2 mm, pick single colonies and inoculate them onto MSM agar pieces with a diameter of 6 mm containing 100 mg / L of erythromycin, and incubate them under moisturized conditions at 30 °C in the dark for 24 h. Place the cultured agar pieces on the surface of a plate coated with the erythromycin-sensitive bacterium Bacillus velezensis L0517 (an endophytic bacterium isolated from the leaves of Illicium verum, which has been published as "Liu Lianjin, Li Zhengling, Li Xueli, et al. Screening of biocontrol strains against anthracnose of Illicium verum and their fermentation conditions [J]. Plant Protection, 2022, 48(05): 204 - 211"), and culture them at 30 °C in the dark for 12 h. Measure the width (mm) of the inhibition zone around the agar pieces 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 erythromycin agar block without inoculation - 6 mm, and D i is the width of the inhibition zone of each strain - 6 mm.

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

[0041] Inoculate the initially screened strains on a PDA plate for purification culture. After the strains produce spores, elute the spores with a small amount of sterile 2% Tween-80 solution and transfer them into a potato dextrose liquid medium (PD). Adjust the addition amount of the eluent to make the spore density in the medium reach 10 7 cells / mL; culture with shaking at 30 °C and 200 rpm for 18 h to prepare a strain seed solution. Take 0.5 mL of the seed solution and inoculate it into an MSM liquid medium containing 100 mg / L erythromycin, and culture with shaking at 30 °C, 200 rpm under dark conditions. Sample every 12 h to measure the strain biomass (mg / DW), erythromycin removal rate (%), and antibacterial activity of the degradation products.

[0042] (4) Determination of degrading bacteria biomass

[0043] Take 10 mL of the well-mixed culture solution, centrifuge at 8000 rpm for 10 minutes, and then aspirate the supernatant. Resuspend the precipitated mycelium with 5 mL of sterile water. Pre-dry a circular filter paper with a diameter of 7 cm to a constant weight and weigh its mass (m p ). Subsequently, filter the mycelium suspension with the filter paper of known mass, and rinse the residual mycelium on the centrifuge tube wall with sterile water and filter it together. Dry the filter paper with attached mycelium at 60 °C to a constant weight and weigh the total mass (m t ). The dry biomass mf (mg·DW) of the degrading bacteria = m t -m p .

[0044] (5) Determination of erythromycin removal rate

[0045] The concentration of erythromycin was determined and analyzed using an AB TripleTOF 5600 liquid chromatography - mass spectrometry system (AB SCIEX, USA). The chromatographic column was an XBridge HBE C18 column (2.1×100 mm, 2.5 μm, Ireland). The mobile phase consisted of 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. Mass spectrometry analysis was performed in the electrospray positive ion mode (ESI+). The specific parameter settings were as follows: the capillary temperature was 220 ± 1 °C, the positive ion spray voltage was 4000 V, and the flow rate of the nebulizing gas (N2) was 8.0 L / min. The mass spectrometry scan range was set to 50 - 1000 m / z. The detection limit of erythromycin (ERY) was 50 μg / L (signal - to - noise ratio S / N ≥ 3). A standard curve was established using an erythromycin standard, and the concentration of erythromycin (C i ) in the culture broth of each strain was calculated according to the standard curve. Taking the concentration of erythromycin (C0) in the culture broth without inoculated strains as a control, the removal rate of erythromycin was calculated: Removal rate (%) = 1 - (C i / C0) × 100.

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

[0047] The disk diffusion method was used to evaluate the biological activity of the degradation products of the strains. The erythromycin - sensitive bacterium Bacillus velezensis L0517 was evenly spread on an LB plate, and sterile dry circular filter paper disks with a diameter of 6 mm were placed on the surface of the plate at intervals of 25 mm. After the culture broth to be tested was filtered and sterilized through a 0.22 - μm sterile filter membrane, 50 μL was pipetted and added to the filter paper disks. The plate was incubated in an incubator at 30 °C in the dark for 12 h, and the size (mm) of the inhibition zone around the filter paper disks was observed and measured.

[0048] The results showed that among the fungi isolated from 5 environmental samples, 6 strains had a relative removal rate of more than 50% for 100 mg / L erythromycin after 24 h of culture. Among them, strain TS7 - 1 showed the best performance, with a relative removal rate as high as 81.0% (Table 1).

[0049] Table 1 Relative removal rates of erythromycin by the initially screened strains

[0050]

[0051]

[0052] 2. Identification of strain TS7 - 1

[0053] (1) Morphological identification

[0054] Inoculate strain TS7-1 on a PDA plate and culture it in an incubator at 28 °C for 48 h. Take a small amount of mycelium, stain it with lactophenol cotton blue, place it under an optical microscope to observe and photograph the morphological characteristics of the strain, and measure its size at the same time.

[0055] The results show that strain TS7-1 grows rapidly on the PDA medium, and the colony expansion rate reaches 3.45 cm / day. The mycelium is initially white, with well-developed aerial mycelium, spreading rapidly in a radial pattern on the surface of the medium. After 3 days of cultivation, a large number of dense granular sporulation structures are produced on the surface of the colony, and the color turns light green. The sporulation area shows a concentric ring pattern ( Figure 4 in A). The conidiophores are branched in a whorled pattern, and the ampoule-shaped sterigmata on them are arranged in a whorled pattern, with an enlarged base and a pointed tip, with a length of 6.45 - 15.23 μm and a width of 2.50 - 5.25 μm ( Figure 4 in B - D). The conidia are solitary, spherical or oval-shaped, with a size of 1.95 - 4.52 μm × 1.86 - 3.55 μm. Strain TS7-1 has the typical characteristics of fungi of the genus Trichoderma.

[0056] (2) Molecular biological identification

[0057] Use the modified CTAB method to extract the genomic DNA of strain TS7-1. Using the extracted DNA as a template, use the ITS3F / ITS4R primers to amplify the internal transcribed spacer (ITS) sequence of the fungus. 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] Input the ITS sequence of strain TS7-1 into the BLATS of GenBank for homology comparison, download the ITS sequences of 19 type strains or recognized strains, and use Protocreafarinosa CBS121551 as an outgroup strain. The NJ phylogenetic tree constructed by MEAG 11 software is as Figure 5 shown. The results show that strain TS7-1 clusters with Trichoderma asperellum with a 100% support rate. Combining morphological characteristics and molecular biological analysis, strain TS7-1 is Trichoderma asperellum.

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

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

[0063] The bacterial residue contains a large amount of erythromycin-producing bacteria and drug-resistant miscellaneous bacteria. To effectively prevent and control the environmental risks of subsequent resource conversion of bacterial residue compost, necessary pre-sterilization treatment of the bacterial residue is required.

[0064] Collection of bacterial residue samples. The tested erythromycin fermentation bacterial residue was collected from a pharmaceutical factory in Hubei. The water content of the bacterial residue was 91.2%, the total nitrogen and total organic carbon contents were 32.6 g / kg and 398.5 g / kg respectively, the carbon-nitrogen ratio was 12.3, the pH value was 5.9, and the residual concentration of erythromycin was 325.6 mg / kg, showing the characteristics of significantly high water content, high nutrient residue, and high concentration of antibiotic residue.

[0065] Optimization of high-temperature sterilization conditions for bacterial residue. Add 1 kg of erythromycin bacterial residue to an electric water heater with temperature control. Heat it at 60, 70, 80, 90, and 100 °C for 30 min respectively, then quickly cool a small amount of the bacterial residue, and perform gradient dilution with sterile water to a dilution of 10 -3 ~10 -8 . Take 100 μL and spread it on an LB medium plate containing 100 mg / L. After 48 h, perform plate counting to statistically analyze the density of culturable drug-resistant bacteria in the bacterial residue under different temperature treatments, and repeat 3 times. At the same time, calculate the energy consumption under different temperature treatments (assuming an initial temperature of 20 °C, a water heater thermal efficiency of 85%, a heat loss of 5 °C / h during heat preservation, and normal pressure).

[0066] As shown in Table 2, the results show that after the erythromycin bacterial residue is treated at a temperature above 80 °C for 30 min, the density of erythromycin drug-resistant bacteria is reduced to below 100 cfu / mL. Further increasing the heating temperature results in a small decrease in the bacterial density, and some drug-resistant bacteria have extremely strong heat resistance. At the same time, increasing the temperature leads to a rapid increase in energy consumption (13 - 24% / 10 °C), indicating that the sterilization efficiency decreases when the temperature is further increased. Therefore, considering the comprehensive operation efficiency and energy consumption of large-scale bacterial residue sterilization, the appropriate treatment condition is to heat at 80 °C for 30 min under normal pressure.

[0067] Table 2 Energy Consumption and Density of Erythromycin Drug-Resistant Bacteria in Erythromycin Bacterial Residue Treated at Different Temperatures for 30 min

[0068]

[0069] Example 2 Optimization of the Optimal Ratio of Antibiotic Bacterial Residue to Straw

[0070] (1) Preparation of the fungal agent for erythromycin degradation. Trichoderma asperellum TS7-1 was inoculated on potato dextrose agar medium (PDA) 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 culture dish was gently shaken to wash off the spores. The spore suspension was collected into a triangular flask, an appropriate amount of sterile water was added, and it was placed in a shaker and shaken at 200 rpm for 5 minutes to fully disperse the spores in water. The spore density in the suspension was counted using a hemocytometer, and the spore density was diluted with water to 10 9 spores / mL, which was used as the inoculated fungal agent.

[0071] (2) Composting fermentation of bacterial residue-straw inoculated with the fungal agent. The bacterial residue was first heated at 80 °C for 30 minutes. After cooling, the pH of the bacterial residue was adjusted to 7.0 - 7.5 with 1 mol / L NaOH solution, and then it was mixed with corn straw powder (fiber length ≤ 1 cm) in different ratios (6:4, 5:5, 4:6, 3:7, w / w) to form a solid matrix. In 1000 g of the solid matrix, Trichoderma asperellum fungal agent was inoculated at a ratio of 1 / 100 (V / W). Using the inoculation of the same volume of sterile water as the control (CK), after fully stirring evenly, it was spread out flat in a plastic tray, the thickness of the piled material was 10 cm, covered with a breathable plastic film, and placed at room temperature of 20 - 25 °C for composting fermentation. During the fermentation, a thorough turning of the pile was carried out every 1 day, and about 10 g of samples were randomly taken and stored in a -20 °C refrigerator. After 7 days, the residual amount of erythromycin was measured uniformly.

[0072] (3) Extraction of erythromycin in the compost. Accurately weigh 2.000 g of the mixed compost matrix sample, add 30 mL of Tris-CaCl2 mixed extractant with pH 6 (10 mmol / L Tris, 20 mmol / L CaCl2), ultrasonically extract for 10 minutes, and centrifuge at 8000 rpm for 5 minutes. Collect the supernatant, and adjust the pH to 10 with 1 mol / L NaOH. Add the extractant / dispersant (1 mL of 1,2-dichloroethane, 1 mL of methanol), vortex for 1 minute, and then centrifuge at 4000 rpm for 5 minutes. Collect the 1,2-dichloroethane layer, dry it under nitrogen blowing, add 1 mL of acetonitrile, vortex for 30 s to accelerate re-dissolution, and centrifuge at 13000 rpm for 10 minutes. Take the supernatant, filter it through a 0.22 μm filter membrane, and then detect the erythromycin content using an LC-MS system.

[0073] The results showed that the mixing ratio of mushroom residue to straw powder had a significant effect on the removal of erythromycin. A high proportion of mushroom residue (6:4) inhibited the growth of fungi in the early stage, resulting in slow removal of erythromycin. After 7 days of fermentation, the erythromycin concentration was still as high as 48.5 mg / L, and the degradation rate was less than 75%. After 7 days of fermentation, the erythromycin concentration in the mushroom residue decreased to 1.0 - 3.3 mg / L at the ratios of 5:5, 4:6, and 3:7, and the degradation rate was as high as over 97% ( Figure 1 in Figure A - B). Considering the erythromycin removal rate and the treatment efficiency of mushroom residue comprehensively, the best mixing ratio of mushroom residue to corn straw was equal - mass mixing.

[0074] Example 3 Quality and Safety Evaluation of Long - cycle Compost Fermentation Organic Fertilizer from Antibiotic Mushroom Residue

[0075] Long - cycle compost fermentation of mushroom residue - straw inoculated with microbial agents. The mushroom residue was first heated at 80 °C for 30 min, cooled, and then the pH was adjusted to 7.0 - 7.5. Then it was mixed with corn straw powder in equal mass to form a solid matrix, and Trichoderma asperellum fungal agent was inoculated at a ratio of 1 / 100 (fungal inoculation fermentation), with the same mass of sterile water inoculated as the control (natural fermentation). The composting conditions and management were the same as in Example 2, and the total fermentation cycle was 30 days. The residual amount of erythromycin was detected by sampling every 5 days during this period.

[0076] Quality detection of organic fertilizer. After 30 days of fermentation, the organic fertilizer was sun - dried until the water content was < 30%, and then the water content, total nutrients, total organic matter, heavy metal content, seed germination index and other indicators in the organic fertilizer were determined with reference to the method of the Ministry of Agriculture and Rural Affairs standard "Organic Fertilizer (NY 525 - 2021)". Among them, Chinese cabbage (Brassica rapa) seeds were used as the test materials for seed germination.

[0077] Fertilizer efficiency and safety detection of organic fertilizer. The organic fertilizer was mixed with garden soil at a ratio of 1 / 100 (w / w), and then filled into small plastic flower pots, 1000 g per pot; after watering thoroughly, 3 Chinese cabbage seedlings were planted in each pot. After 30 days, the plant height (cm), root length (cm), leaf area (cm 2 ) and fresh biomass (g·FW) of the Chinese cabbage plants were measured. Among them, the leaf area was measured by the paper - cutting and weighing method. Three replicates were set.

[0078] The erythromycin detoxification effect showed that in the natural compost fermentation of mushroom residue - straw without fungal inoculation, the residual concentration of erythromycin decreased rapidly in the first 5 days, but the removal rate decreased afterwards. After 30 days of compost fermentation, the residual concentration was still as high as 40.6 mg / L; while in the fermentation with inoculation of Trichoderma asperellum, the removal rate of the residual concentration of erythromycin was higher in the first 5 days. By the 10th day, the concentration was already lower than 1.0 mg / L, and by the 30th day, it was lower than the detection limit (50 μg / kg) ( Figure 2Among A - B, Table 3). It shows that the degradation by Trichoderma asperellum is the key driving force for the rapid removal of erythromycin residues in mushroom residue compost.

[0079] The final quality indicators of the organic fertilizer show that the total nitrogen and total nutrient contents in the organic fertilizer fermented with Trichoderma asperellum reach 5.52% and 6.14% respectively, exceeding those of natural fermentation by 27.7% and 25.1%. The germination index reaches 99.7%, and the heavy metal content is far lower than the limit standard, fully meeting the standards of "Organic Fertilizer (NY 525 - 2021)" of the Ministry of Agriculture and Rural Affairs (Table 3). Due to the high - concentration erythromycin residues in the naturally fermented organic fertilizer, the germination of Chinese cabbage seeds is severely inhibited, and the final germination index is only 35.1%, not meeting the organic fertilizer standard (Table 3). It shows that inoculating Trichoderma can not only quickly and thoroughly remove the residual toxicity of erythromycin in the mushroom residue - straw mixed compost, but also increase the nutrient content in the fertilizer, realizing the transformation of erythromycin - containing mushroom residue from hazardous waste to high - quality organic fertilizer.

[0080] The toxicity tests of organic fertilizer on Chinese cabbage seed germination and plant growth show that the organic fertilizer fermented with inoculated Trichoderma asperellum completely removes the seed toxicity of erythromycin, while the high residual erythromycin in the naturally fermented mushroom residue organic fertilizer significantly inhibits the development of seed radicles and cotyledons ( Figure 3 in A). After the two kinds of organic fertilizers are applied to the soil, the organic fertilizer fermented with Trichoderma asperellum significantly promotes the growth of Chinese cabbage plants. The biomass, leaf area, plant height and root length of the plants are all significantly higher than those of the non - fertilized control treatment. The naturally fermented mushroom residue organic fertilizer significantly inhibits the growth of Chinese cabbage plants due to the high residual erythromycin not being removed ( Figure 3 in B - D).

[0081] Table 3 Quality indicators of the mushroom residue - straw mixed fermented organic fertilizer containing erythromycin

[0082]

[0083] Note: "-" indicates that there is no clear standard for this item in the standard "Organic Fertilizer (NY 525 - 2021)".

[0084] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for efficient detoxification and nutrient recovery of aerobic composting of fungal-enhanced erythromycin fermentation residues, characterized in that, It includes the step of mixing the heat-sterilized erythromycin fermentation residue with corn straw powder, and then inoculating Trichoderma asperellum for aerobic composting fermentation.

2. The method according to claim 1, wherein The conditions for the heat sterilization are: heating at 80 °C for 30 min.

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

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

5.

5. The method according to claim 1, characterized in that After adjusting the pH value of the heat-sterilized erythromycin fermentation residue to 7.0 - 7.5, it is then mixed with the corn straw powder.

6. The method according to claim 1, wherein The Trichoderma asperellum is Trichoderma asperellum TS7-1, and the preservation number is CGMCC No. 41372; and / or the Trichoderma asperellum is inoculated in the form of a spore suspension, and according to the total mass of the erythromycin fermentation residue and the corn straw powder, the inoculation ratio of the Trichoderma asperellum spore suspension is 1 g:10 mL; and / or the concentration of the Trichoderma asperellum spore suspension is 10 9 CFU / mL.

7. The method according to claim 1, characterized in that, The temperature of the aerobic composting fermentation is 20 - 25 °C, the time is 30 days, and thorough turning is carried out every other day during the fermentation period.

8. The method according to claim 1, wherein It also includes the step of naturally drying the solid substrate obtained after the completion of the aerobic composting fermentation until the water content is lower than 30%.

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