Method for biologically degrading tetracycline antibiotics in soil through cooperation of microorganisms and earthworms and application

Abstract

The invention belongs to the technical field of antibiotic degradation, soil organic pollution remediation and agriculture, and discloses a method for biodegrading tetracycline antibiotics in soil through cooperation of microorganisms and earthworms and application of the method. The method comprises the following steps that the earthworms subjected to intestine cleaning and a feed (e) inoculated with a microbial agent are added into the soil polluted by the tetracycline antibiotics together; the soil is placed in a dark fuming cupboard to be aged for 12 h and then transferred into a container with holes in the bottom, an opening of the container is covered with a nylon gauze bag, and the moisture content of the soil is kept at 30%; the soil is cultured under natural conditions, in the culture process, water is supplemented every two days to keep the water content of the soil unchanged, and culture is conducted for 30 d. The inoculation method of the microbial agent is different from the traditional culture method, the microbial agent is directly inoculated into the feed, the operation is convenient, simple and feasible, the requirement on the professional degree is not high, and the tedious steps of culture medium preparation, microbial culture, separation and purification and the like are avoided.

Description

Technical Field

[0001] This invention belongs to the fields of antibiotic degradation, soil organic pollution remediation, and agricultural technology, and in particular, it relates to a method and application of microbial synergistic earthworm biodegradation of tetracycline antibiotics in soil. Background Technology

[0002] Soil is a significant accumulation site for antibiotics. Currently, 56 types of antibiotic residues have been detected in soil, with tetracyclines being the predominant type. Tetracycline residues in soil not only pose potential ecological risks to agricultural ecosystems but can also enter the human body through the food chain and threaten human health. Furthermore, antibiotic residues in the soil may induce the generation and spread of antibiotic-resistant bacteria and antibiotic resistance genes, thereby threatening public health and safety.

[0003] Earthworms have been widely used to remediate organically contaminated soils due to their eco-friendly nature, low cost, lack of secondary pollution, and strong tolerance and resistance to organic pollutants. Currently, earthworms have been proven effective in remediating soils contaminated with various organic pollutants, including polycyclic aromatic hydrocarbons (PAHs, naphthalene, phenanthrene, anthracene, fluoranthene, etc.), polychlorinated biphenyls (Aoclor 1242, 2,2,5-trichlorobiphenyls, PCBs 91, etc.), pesticides (DDTs, pentachlorophenol, atrazine, etc.), and emerging pollutants (antibiotics, perfluorinated compounds, etc.), demonstrating broad application prospects. Microbial degradation is an effective way to reduce tetracycline antibiotics in soil ecosystems. Proteobacteria, Bacteroidetes, and Firmicutes are the main phyla to which tetracycline antibiotic-degrading bacteria belong. Microbial community enrichment culture technology was used to isolate and culture... Stenotrophomonas , Pseudomonas , Bacillus , Achromobacter Earthworms exhibit good degradation capabilities for tetracycline antibiotics. However, the degradation of antibiotics in soil by earthworms relies excessively on the synergistic effect of gut microbiota, resulting in relatively low efficiency; furthermore, microbial degradation of antibiotics in soil suffers from nutrient deficiency in the later stages of experiments. Therefore, a method for in-situ and efficient degradation of tetracycline antibiotics in soil is urgently needed.

[0004] Soil pollution is characterized by its hidden, cumulative, long-term, and irreversible nature, making traditional or single remediation technologies less effective. Combined technologies utilizing soil animals, plants, and microorganisms have emerged to address this need; earthworms can also be used in conjunction with other remediation methods (such as microorganisms). In comparison, combined technologies demonstrate superior remediation effects compared to single technologies and are expected to become a research hotspot for remediating antibiotic-contaminated soils. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method and application for the biodegradation of tetracycline antibiotics in soil by microorganisms in synergistic with earthworms.

[0006] The technical solution adopted by this invention to solve its technical problem is: A method for the biodegradation of tetracycline antibiotics in soil by microorganisms in synergistic interaction with earthworms includes the following steps: Select earthworms of similar size and weight. First, clean the earthworms with sterile water, then place them on moist sterile filter paper for 24 hours to cleanse their intestines. Do not feed the earthworms during this period. After cleaning, the earthworms are inoculated together with feed (e) inoculated with microbial agents into soil contaminated with tetracycline antibiotics. The earthworms are introduced at a ratio of 6 earthworms / kg soil, the feed (e) is added at a ratio of 80 g / kg soil, and the compound microbial agent is inoculated at a ratio of 1% based on the volume of compound microbial agent (mL) to the mass of soil (g). The soil containing earthworms and microbial inoculants (e) from the previous step was placed in a light-proof fume hood to age for 12 hours. Then it was transferred to a container with holes at the bottom. To prevent the earthworms from escaping, the container opening was covered with a 20-mesh nylon mesh bag. Water was added to maintain the soil moisture content at 30%. The soil was then placed under natural conditions for cultivation. During the cultivation process, water was added every 2 days to maintain the soil moisture content. The soil was cultivated for 30 days.

[0007] Furthermore, the microbial agent includes ammonia-oxidizing bacteria, nitrifying bacteria, nitrite-oxidizing bacteria, nitrogen-fixing bacteria, and organic matter-decomposing bacteria.

[0008] Furthermore, the ammonia-oxidizing bacteria are *Pseudomonas fluorescens*, the nitrifying bacteria are *Nitrosomonas*, the nitrite-oxidizing bacteria are *Nitrobacter vesiculosus*, the nitrogen-fixing bacteria are *Azotobacter chrysogenum*, and the organic matter-decomposing bacteria are *Bacillus subtilis* and *Bacillus cereus*. The preparation method of the microbial inoculant is as follows: six functional inoculants are mixed in an equal volume ratio of 1:1:1:1:1:1, and the OD values ​​of the six inoculants are measured. 600 The values ​​were all 0.800±0.100, resulting in the compound microbial inoculant (d).

[0009] Furthermore, the method for preparing the feed (e) inoculated with the microbial agent is as follows: (1) The crushed pig manure and cow manure were mixed with xylitol in a mass ratio of 8:2:0.01 to prepare feed (b). The particle size of the crushed pig manure and cow manure was 0.85 mm. (2) The compound microbial agent is directly inoculated. The specific cultivation steps are as follows: the compound microbial agent (d) is first premixed into the feed (b) to obtain feed containing the compound microbial agent (e).

[0010] Furthermore, the earthworm cultivation method is as follows: ① Take crushed wheat straw and corn straw and mix them evenly and spread them out in a mass ratio of 1:1. The particle size of the crushed straw is 1-3 mm. Add an appropriate amount of water by spraying with a sprayer to keep the moisture content of the mixed straw (a) at 60%-65% and age it for 2 days. ② The crushed pig manure and cow manure were mixed with xylitol at a mass ratio of 8:2:0.01 to prepare feed (b). The particle size of the crushed pig manure and cow manure was 0.85 mm. Feed (b) was placed on top of mixed straw (a). Feed (b) and mixed straw (a) were evenly spread at a mass ratio of 6:1 to form a feeding layer (c). During cultivation, new feed (b) was added every 2 days according to the earthworms' feeding situation, while keeping the weight of the feeding layer (c) constant. The temperature of the feeding layer (c) was 25±1℃ and the moisture content was 60%-65%. The total weight of the feeding layer (c) was 20 kg.

[0011] ③ Introduce Eisenia fetida seedlings of similar size and weight into the rearing layer (c), ensuring that the earthworm addition in the rearing layer (c) is (30±3) earthworms / kg of feed, and place them indoors under natural conditions for 1 month; to prevent earthworms from escaping, cover the container with a 20-mesh nylon mesh bag.

[0012] Furthermore, the tetracycline antibiotics include tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC).

[0013] Furthermore, the concentration of the tetracycline antibiotics is 20 mg / kg. -1 .

[0014] Furthermore, the earthworm in question is *Eisenia fetida*.

[0015] The application of the method described above in the degradation of tetracycline antibiotics in soil.

[0016] Furthermore, the tetracycline antibiotics include tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC).

[0017] Furthermore, the concentration of the tetracycline antibiotics is 20 mg / kg. -1 .

[0018] The advantages and positive effects of this invention are as follows: 1. The inoculation method of the microbial agent in this invention is different from the traditional culture method. The microbial agent is directly inoculated into the feed, which is convenient, simple and feasible, does not require high professional expertise, and avoids cumbersome steps such as culture medium preparation, microbial culture, separation and purification.

[0019] 2. This invention uses a feed mixture of wheat straw, corn straw, pig manure, cow manure, and xylitol as a culture medium for microbial agents. The raw materials for the culture medium are widely available, inexpensive, and readily available, thus realizing the resource utilization of straw and livestock manure. Furthermore, the culture medium not only provides various growth factors necessary for microbial growth and metabolism but also reduces costs.

[0020] 3. This invention directly inoculates the microbial agent into the feed and colonizes the microbial agent in the earthworm's intestines through feeding or on the earthworm's skin through peristalsis, thus solving the problem of the microbial agent's difficulty in colonizing in the soil.

[0021] 4. Compared with inoculating microorganisms alone, the present invention introduces earthworms to solve the problem of poor degradation effect of direct application of microorganisms, and the degradation rate of tetracycline antibiotics increases by 3.99%-7.94%.

[0022] 5. Compared with earthworms alone, the present invention combines microorganisms to solve the problem of earthworms' excessive reliance on intestinal microorganisms for degradation, and increases the degradation rate of tetracycline antibiotics by 5.29%-10.3%.

[0023] 6. This invention dynamically monitors the concentration of tetracycline antibiotics, the main transformation products of tetracycline antibiotics in soil, which indirectly confirms the difference between earthworms and microorganisms degrading tetracycline antibiotics in soil and earthworms or microorganisms alone. At the end of the experiment, the concentration of tetracycline antibiotics increased by 4.72%-12.2%.

[0024] 7. Compared with the control, the present invention effectively reduces the C / N ratio of the soil after the application of manure by 29.4%, which can effectively prevent nitrogen deficiency in crops.

[0025] 8. This invention simultaneously solves the problem that the effects of inoculating microbial agents or earthworms alone in degrading tetracycline antibiotic residues in soil are not good. It can effectively control and reduce tetracycline antibiotic residues in farmland soil, avoid the generation and spread of antibiotic resistance, and reduce the risks to soil ecosystems and human health.

[0026] 9. This invention addresses the problem of controlling and reducing tetracycline antibiotic residues in farmland soil in soil organic pollution remediation technology, and solves the problem of poor effect of using microbial agents alone to degrade antibiotic residues in soil. Attached Figure Description

[0027] Figure 1 This is a graph showing the degradation rate of tetracycline antibiotics in soils treated differently in this invention. Detailed Implementation

[0028] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0029] The various experimental operations involved in the specific embodiments are all conventional techniques in the art. For parts not specifically annotated herein, those skilled in the art can refer to various commonly used reference books, scientific and technological literature, or related instructions and manuals prior to the filing date of this invention for implementation. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] A method for the biodegradation of tetracycline antibiotics in soil by microorganisms in synergistic interaction with earthworms includes the following steps: Select earthworms of similar size and weight. First, clean the earthworms with sterile water, then place them on moist sterilized filter paper for 24 hours to cleanse their intestines. Do not feed the earthworms during this period. After cleaning, the earthworms are inoculated together with feed (e) inoculated with microbial agents into soil contaminated with tetracycline antibiotics. The earthworms are introduced at a ratio of 6 earthworms / kg soil, the feed (e) is added at a ratio of 80 g / kg soil, and the compound microbial agent is inoculated at a ratio of 1% based on the volume of compound microbial agent (mL) to the mass of soil (g). The soil containing earthworms and microbial inoculants (e) from the previous step was placed in a light-proof fume hood to age for 12 hours. Then it was transferred to a container with holes at the bottom. To prevent the earthworms from escaping, the container opening was covered with a 20-mesh nylon mesh bag. Water was added to maintain the soil moisture content at 30%. The soil was then placed under natural conditions for cultivation. During the cultivation process, water was added every 2 days to maintain the soil moisture content. The soil was cultivated for 30 days.

[0031] Preferably, the microbial agent includes ammonia-oxidizing bacteria, nitrifying bacteria, nitrite-oxidizing bacteria, nitrogen-fixing bacteria, and organic matter-decomposing bacteria.

[0032] Preferably, the ammonia-oxidizing bacteria are *Pseudomonas fluorescens*, the nitrifying bacteria are *Nitrosomonas*, the nitrite-oxidizing bacteria are *Nitrobacter vesiculosus*, the nitrogen-fixing bacteria are *Azotobacter chrysogenum*, and the organic matter-decomposing bacteria are *Bacillus subtilis* and *Bacillus cereus*. The preparation method of the microbial inoculant is as follows: six functional inoculants are mixed in an equal volume ratio of 1:1:1:1:1:1, and the OD values ​​of the six inoculants are measured. 600 The values ​​were all 0.800±0.100, resulting in the compound microbial inoculant (d).

[0033] Preferably, the method for preparing the feed (e) inoculated with the microbial agent is as follows: (1) The crushed pig manure and cow manure were mixed with xylitol in a mass ratio of 8:2:0.01 to prepare feed (b). The particle size of the crushed pig manure and cow manure was 0.85 mm. (2) The compound microbial agent is directly inoculated. The specific cultivation steps are as follows: the compound microbial agent (d) is first premixed into the feed (b) to obtain feed containing the compound microbial agent (e).

[0034] Preferably, the earthworm cultivation method is as follows: ① Take crushed wheat straw and corn straw and mix them evenly and spread them out in a mass ratio of 1:1. The particle size of the crushed straw is 1-3 mm. Add an appropriate amount of water by spraying with a sprayer to keep the moisture content of the mixed straw (a) at 60%-65% and age it for 2 days. ② The crushed pig manure and cow manure were mixed with xylitol at a mass ratio of 8:2:0.01 to prepare feed (b). The particle size of the crushed pig manure and cow manure was 0.85 mm. Feed (b) was placed on top of mixed straw (a). Feed (b) and mixed straw (a) were evenly spread at a mass ratio of 6:1 to form a feeding layer (c). When using the feed, new feed (b) was added every 2 days according to the earthworms’ feeding situation, while keeping the weight of the feeding layer (c) constant. The temperature of the feeding layer (c) was 25±1℃ and the moisture content was 60%-65%. The total weight of the feeding layer (c) was 20 kg.

[0035] ③ Introduce Eisenia fetida seedlings of similar size and weight into the rearing layer (c), ensuring that the earthworm addition in the rearing layer (c) is (30±3) earthworms / kg of feed, and place them indoors under natural conditions for 30 days; to prevent earthworms from escaping, cover the container with a 20-mesh nylon mesh bag.

[0036] Preferably, the tetracycline antibiotics include tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC).

[0037] Preferably, the concentration of the tetracycline antibiotics is 20 mg / kg. -1 .

[0038] The application of the method described above in the degradation of tetracycline antibiotics in soil.

[0039] Preferably, the tetracycline antibiotics include tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC).

[0040] Preferably, the concentration of the tetracycline antibiotics is 20 mg / kg. -1 .

[0041] Specifically, the relevant preparation and testing methods are as follows: A method for the biodegradation of tetracycline antibiotics in soil by microorganisms in synergistic interaction with earthworms includes the following steps: (1) Preparation of earthworms ① Take crushed wheat straw and corn straw and mix them evenly at a mass ratio of 1:1. Spread the mixture evenly at the bottom of a gray transparent storage box. The particle size of the crushed straw should be 1-3 mm. The storage box should be 66 cm long, 44 cm wide, and 40 cm high. Add an appropriate amount of water by spraying with a spray bottle to keep the moisture content of the mixed straw (a) at 60%-65%. Let it age for 2 days. ② A feed (b) was prepared by mixing crushed pig manure, cow manure, and xylitol at a mass ratio of 8:2:0.01. The particle size of the crushed pig and cow manure was 0.85 mm. Feed (b) was placed on top of mixed straw (a), and the feed (b) and mixed straw (a) were evenly spread in a gray transparent storage box at a mass ratio of 6:1 as the feeding layer (c). New feed (b) was added every 2 days according to the earthworms' feeding behavior, while keeping the weight of the feeding layer (c) constant. The temperature of the feeding layer (c) was 25±1℃, and the moisture content was about 60%-65%. The total weight of feed (b) and straw (a) in the feeding layer (c) was 20 kg.

[0042] ③ Introduce Eisenia fetida seedlings of similar size and weight into the rearing layer (c), ensuring that the earthworm addition amount in the rearing layer (c) is (30±3) earthworms / kg of feed, and place them indoors under natural conditions for 30 days. To prevent earthworms from escaping from the gray transparent storage box, cover the storage box with a 20-mesh nylon mesh bag.

[0043] (2) Microbial inoculants Inoculum composition: ammonia-oxidizing bacteria (fluorescent Pseudomonas, Pseudomonas fluorescens ); Nitrifying bacteria (nitrosomonas, Nitrosomonas nitrosa ); Nitrite-oxidizing bacteria (Nitrobacterium velutipes, Nitrobacter winogradskyi ); nitrogen-fixing bacteria (Azotobacter brownii, Azotomonas ); organic matter decomposing bacteria (Bacillus subtilis, Bacillus subtilis Bacillus cereus, Bacillus cereus ).

[0044] Preparation of microbial inoculants: Six functional microbial inoculants were mixed in an equal volume ratio (1:1:1:1:1:1). The OD values ​​of the six microbial solutions were measured. 600 The concentrations were all 0.800±0.100, resulting in a compound microbial inoculant (d), which was then inoculated into the feed (b) at an inoculation ratio of 1%, based on the ratio of compound inoculant volume (mL) to soil mass (g).

[0045] Unlike traditional cultivation, the compound microbial agent (d) is produced by direct inoculation. The specific cultivation steps are as follows: the compound microbial agent (d) is first premixed into the feed (b) to obtain feed (e) containing the compound microbial agent; then the feed (e) containing the compound microbial agent is added to the soil at a ratio of 80 g / kg soil; the inoculation ratio of the compound microbial agent (d) is 1%, and the inoculation is carried out according to the ratio of compound microbial agent volume (mL) to soil mass (g).

[0046] The functional bacteria are mixed with manure and added to the feed as a substrate for inoculating earthworms. Based on this, the functional bacteria are activated. Some of the functional bacteria may colonize the earthworm's intestines by consuming the manure or colonize the earthworm's epidermis by peristalsis.

[0047] (3) Experimental design The present application will be further described in detail below with reference to the embodiments.

[0048] Example 1 included four treatments: ①CK (soil): 2.50 kg soil + 200 g feed; ②E (soil + earthworms): 2.50 kg soil + 200 g feed + 15 Eisenia fetidae; ③M (soil + 1% microbial agent): 2.50 kg soil + 200 g feed + 1% microbial agent (inoculated according to the ratio of compound microbial agent volume in mL to soil mass in g); ④EM (soil + earthworms + 1% microbial agent): 2.50 kg soil + 200 g feed + 15 Eisenia fetidae + 1% microbial agent (inoculated according to the ratio of compound microbial agent volume in mL to soil mass in g).

[0049] The feed addition ratio was 80 g / kg soil; the earthworm release ratio was 6 earthworms / kg soil; and the microbial inoculant inoculation ratio was 1%, based on the ratio of compound inoculant volume (mL) to soil mass (g).

[0050] The feed mentioned above is feed (b).

[0051] Before the experiment, 200-300 Eisenia fetidae earthworms of similar size and weight were randomly selected from the rearing layer (c) for use. The earthworms were first cleaned with sterile water, then placed on moist, sterile filter paper for 24 hours to cleanse their intestines. During this period, the earthworms were not fed. The cleaned earthworms were then added to the soil along with feed (e) inoculated with microbial agents.

[0052] Each treatment was performed in triplicate. Soil was treated with tetracycline antibiotics (TCs) at a concentration of 20 mg / kg. -1After being contaminated, the soil was aged in a light-proof fume hood for 12 hours, then transferred to plastic flowerpots (21 mm × 18 mm × 12 mm) with perforated bottoms. To prevent earthworms from escaping, the rims of the flowerpots were covered with 20-mesh nylon mesh bags, and an appropriate amount of water was added to maintain the soil moisture content at approximately 30%. The soil incubation experiment was conducted in a smart greenhouse under natural conditions for 30 days. During the incubation period, water was added every two days to maintain a constant soil moisture content. Soil samples were destructively collected at 0, 3, 7, 14, and 30 days to monitor the concentrations of total toxic substances (TCs), TC differential metabolites, total carbon, and total nitrogen in the soil.

[0053] The contents of total chemical substances (TCs) and their differential metabolites in soil were determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). The assay method was as follows: Kinetex F5 100 Å column (100 mm × 2.1 mm × 2.6 μm); column temperature: 40℃; injection volume: 5.0 μL; flow rate: 0.40 mL·min. -1 A binary gradient pump was used, with mobile phase A being 0.1% formic acid-water solution and mobile phase B being acetonitrile, employing a gradient elution program. Ion source: electrospray ionization (ESI+); curtain gas flow rate (CUR): 30 psi; collision gas flow rate (CAD): median; spray voltage (IS): 5500 V; nebulization temperature (TEM): 550℃; nebulizing gas (GS1): 55 psi; auxiliary gas (GS2): 55 psi. Detection method: multiple reaction monitoring (MRM).

[0054] (4) Test results Table 1. Degradation rate of tetracycline antibiotics in soil under different treatments

[0055] The results are shown in Table 1 and Figure 1 As shown in Table 1 and Figure 1It was observed that the degradation rate of tetracycline antibiotics in the soil gradually increased with the introduction of earthworms and microorganisms. The degradation rates of tetracycline, oxytetracycline, and chlortetracycline in the soil treated with earthworms and microorganisms were all higher than in other treatments (P<0.05). After 30 days of cultivation, the degradation rate of tetracycline in the soil was: 92.81% (with earthworms and microorganisms) > 86.72% (with microorganisms) > 83.82% (with earthworms) > 82.10% (control) (P<0.05). The degradation rate of oxytetracycline in the soil was: 93.57% (with earthworms and microorganisms) > 85.63% (with microorganisms) > 83.26% (with earthworms) > 80.91% (control) (P<0.05). The degradation rate of chlortetracycline in soil was: 91.07% (with earthworms and microorganisms) > 87.07% (with microorganisms) > 85.77% (with earthworms) > 82.79% (control) (P<0.05).

[0056] Table 2. Concentrations of tetracycline antibiotic differential metabolites in soils under different treatments

[0057] Under the action of earthworms and microorganisms, tetracycline antibiotics undergo degradation and transformation, with aberrant metabolites being the main transformation products in the soil. Table 2 shows that with the biodegradation of tetracycline antibiotics by earthworms and microorganisms, aberrant tetracycline, aberrant oxytetracycline, and aberrant chlortetracycline were detected in all treatment groups. With prolonged culture time, the residual concentration of aberrant metabolites of tetracycline antibiotics in the soil increased significantly (P<0.05). After 30 days of culture, the concentration of aberrant tetracycline in the soil was: 17.71 mg / kg (with earthworms and microorganisms) > 16.23 mg / kg (with microorganisms) > 16.05 mg / kg (with earthworms) > 15.31 mg / kg (control) (P<0.05). The concentrations of oxytetracycline in the soil were: 16.87 mg / kg (with earthworms and microorganisms) > 16.11 mg / kg (with microorganisms) > 15.04 mg / kg (with earthworms) > 14.11 mg / kg (control) (P<0.05). The concentrations of chlortetracycline in the soil were: 13.69 mg / kg (with earthworms and microorganisms) > 12.96 mg / kg (with microorganisms) > 12.73 mg / kg (with earthworms) > 10.53 mg / kg (control) (P<0.05).

[0058] Table 3. Concentration of total nitrogen and C / N ratio in soils under different treatments

[0059] The carbon-to-nitrogen ratio (C / N) refers to the ratio of the total carbon content to the total nitrogen content in organic matter. An appropriate C / N ratio promotes microbial fermentation and decomposition; a higher C / N ratio, due to competition for nitrogen by microbial activities, can lead to nitrogen deficiency in crops. The C / N ratio in the top 15 cm of soil typically ranges from 8:1 to 15:1. Table 3 shows that, over time, the C / N ratio in the control group increased from 22.68 initially to 24.91; while the C / N ratio in the group treated with earthworms and microorganisms decreased from 22.68 initially to 16.01, falling within a suitable range.

[0060] (5) Experimental conclusions Compared with single degradation (earthworm degradation and microbial degradation), the combined degradation of earthworms and microorganisms significantly increased the degradation rates of tetracycline, oxytetracycline, and chlortetracycline in the soil (p<0.05); the concentration of differential metabolites of tetracycline antibiotics also significantly increased (p<0.05), increasing by 10.2% compared with microbial degradation and by 6.56% compared with earthworm degradation. Compared with the control, the combined degradation of earthworms and microorganisms increased the total nitrogen content in the soil by 46.4% and decreased the C / N ratio by 29.4%.

[0061] Meanwhile, it can also be seen that earthworms and compound microbial agents have a synergistic effect in the method of the present invention, which can synergistically improve the relevant effect of degrading tetracycline antibiotics in soil. In particular, there is a significant synergistic effect between earthworms inoculated with 15 Eisenia fetidae and 1% microbial agent (inoculated according to the ratio of compound agent volume mL to soil mass g) per 2.50 kg of soil, which can significantly synergistically improve the relevant effect of degrading tetracycline antibiotics in soil.

[0062] Compared with existing technologies: 1. Previous research (Sun Pingping. Study on the mechanism of earthworms promoting the degradation of residual tetracycline in soil [D]. Yangzhou University, 2021. DOI:10.27441 / d.cnki.gyzdu.2021.001348.) found that in soils with tetracycline concentrations of 5 mg / kg and 100 mg / kg, the degradation rates of tetracycline by Eisenia fetida were 66.98% and 67.09% after 28 days, respectively. In this invention, after 30 days of cultivation, the degradation rate of tetracycline in soil with a concentration of 20 mg / kg by earthworms and synergistic microorganisms was 92.81%, which is an increase of 25.83% and 25.72% respectively compared with existing earthworm degradation methods.

[0063] 2. Previous research (Cao Jia. Synergistic mechanism of earthworms and mycorrhizal fungi in promoting oxytetracycline degradation and remediating contaminated soil [D]. China Agricultural University, 2018.) found that in soils with oxytetracycline concentrations of 1 mg / kg and 100 mg / kg, the degradation rates of oxytetracycline by Eisenia fetida were 79.91% and 72.77% after 56 days, respectively. In this invention, after 30 days of cultivation, the degradation rate of oxytetracycline in soil with a concentration of 20 mg / kg by earthworm synergistic microorganisms was 93.57%, which is an increase of 13.66% and 20.80% respectively compared with existing earthworm degradation methods.

[0064] 3. Previous research (WU X, WEI Y, ZHENG J, et al. The behavior of tetracyclines and their degradation products during swine manure composting[J]. Bioresource Technology, 2011, 102(10): 5924-5931.) found that the degradation rates of tetracycline, oxytetracycline, and chlortetracycline by microorganisms during pig manure composting were 70%, 74%, and 92%, respectively. In this invention, after 30 days of cultivation, the degradation rates of tetracycline, oxytetracycline, and chlortetracycline in the soil by earthworms and microorganisms were 92.81%, 93.57%, and 91.06%, respectively. Compared with the degradation by microorganisms during existing composting, the degradation rates of tetracycline and oxytetracycline increased by 22.81% and 19.57%, respectively.

[0065] 4. Previous research (Henan Agricultural University. Tetracycline Antibiotic Degrading Bacteria, Microbial Agents and Their Applications: 202410718602.4 [P]. 2024-08-23.) found that *Providens spp.* TX2 achieved a 40.74% degradation rate of tetracycline in soil at a concentration of 200 mg / kg. Earthworm-associated microorganisms achieved a 92.81% degradation rate of tetracycline in soil at a concentration of 20 mg / kg, an increase of 52.04% compared to existing microbial degradation methods.

[0066] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for the biodegradation of tetracycline antibiotics in soil by microorganisms in synergistic action with earthworms, characterized in that: Includes the following steps: Select earthworms of similar size and weight. First, clean the earthworms with sterile water, then place them on moist sterile filter paper for 24 hours to cleanse their intestines. Do not feed the earthworms during this period. After cleaning, add the earthworms and feed (e) inoculated with microbial agents to the soil contaminated with tetracycline antibiotics. The earthworms were added at a ratio of 6 earthworms / kg soil, the feed (e) was added at a ratio of 80 g / kg soil, and the compound microbial agent was inoculated at a ratio of 1% based on the volume of compound microbial agent (mL) to the mass of soil (g). Soil containing earthworms and inoculated microbial agents was placed in a light-proof fume hood and aged for 12 hours. It was then transferred to a container with holes at the bottom. To prevent earthworms from escaping, the container opening was covered with a 20-mesh nylon mesh bag. Water was added to maintain the soil moisture content at 30%. The soil was then placed under natural conditions for cultivation. During the cultivation process, water was added every 2 days to maintain the soil moisture content. The soil was cultivated for 30 days.

2. The method according to claim 1, characterized in that: The microbial agents include ammonia-oxidizing bacteria, nitrifying bacteria, nitrite-oxidizing bacteria, nitrogen-fixing bacteria, and organic matter-decomposing bacteria.

3. The method according to claim 2, characterized in that: The ammonia-oxidizing bacteria are *Pseudomonas fluorescens*, the nitrifying bacteria are *Nitrosomonas*, the nitrite-oxidizing bacteria are *Nitrobacter vesiculosus*, the nitrogen-fixing bacteria are *Azotobacter chrysogenum*, and the organic matter-decomposing bacteria are *Bacillus subtilis* and *Bacillus cereus*. The preparation method of the microbial inoculant is as follows: six functional inoculants are mixed in an equal volume ratio of 1:1:1:1:1:1, and the OD values ​​of the six inoculants are measured. 600 The values ​​were all 0.800±0.100, resulting in the compound microbial inoculant (d).

4. The method according to claim 1, characterized in that: The method for preparing the feed (e) inoculated with the microbial agent is as follows: (1) The crushed pig manure and cow manure were mixed with xylitol in a mass ratio of 8:2:0.01 to prepare feed (b). The particle size of the crushed pig manure and cow manure was 0.85 mm. (2) The compound microbial agent is directly inoculated. The specific cultivation steps are as follows: the compound microbial agent (d) is first premixed into the feed (b) to obtain feed containing the compound microbial agent (e).

5. The method according to claim 1, characterized in that: The earthworm cultivation method is as follows: ① Take crushed wheat straw and corn straw and mix them evenly and spread them out. The particle size of the crushed straw is 1-3mm. Add water by spraying with a spray bottle to keep the moisture content of the mixed straw (a) at 60%-65% and age it for 2 days. ② The crushed pig manure and cow manure were mixed with xylitol at a mass ratio of 8:2:0.01 to prepare feed (b). The particle size of the crushed pig manure and cow manure was 0.85 mm. Feed (b) was placed on top of mixed straw (a), and the feed (b) and mixed straw (a) were evenly spread at a mass ratio of 6:1 to form a feeding layer (c). When using the feed, new feed (b) was added every 2 days according to the earthworms' feeding situation, while keeping the weight of the feeding layer (c) constant. The temperature of the feeding layer (c) was 25±1℃ and the moisture content was 60%-65%. ③ Introduce Eisenia fetida seedlings of similar size and weight into the rearing layer (c), ensuring that the earthworm addition in the rearing layer (c) is (30±3) earthworms / kg of feed, and place them indoors under natural conditions for 1 month; to prevent earthworms from escaping, cover the container with a 20-mesh nylon mesh bag.

6. The method according to any one of claims 1 to 5, characterized in that: The tetracycline antibiotics include tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC).

7. The method according to claim 6, characterized in that: The concentration of all the tetracycline antibiotics was 20 mg / kg. -1 .

8. The application of the method according to any one of claims 1 to 7 in the degradation of tetracycline antibiotics in soil.

9. The application according to claim 8, characterized in that: The tetracycline antibiotics include tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC).

10. The application according to claim 8 or 9, characterized in that: The concentration of all the tetracycline antibiotics was 20 mg / kg. -1 .

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