A method for repairing norfloxacin contaminated soil by using earthworm gut contents

Abstract

The application belongs to the technical field of soil remediation, and relates to a method for remedying norfloxacin contaminated soil by using earthworm intestinal contents, which comprises the following steps: inducing earthworms to empty their intestinal contents on quartz sand by light irradiation, extracting and activating the intestinal contents by using a phosphate buffer solution, treating the intestinal contents with a nutrient solution and a modifier to obtain high-activity modified earthworm intestinal contents, granulating the high-activity modified earthworm intestinal contents, active carbon, organic acid and mineral materials according to a specific proportion, embedding and solidifying the granulation to prepare a remediation agent, and applying the remediation agent to contaminated soil. After the remediation agent is applied to the contaminated soil, the norfloxacin is efficiently decomposed and the soil ecological function is simultaneously recovered through the degradation of earthworm intestinal microorganisms, the adsorption effect of active carbon, the complexation reaction of organic acid and the physical regulation of mineral materials. The method innovatively uses the natural microbial community in the earthworm intestinal tract, has the advantages of complete degradation, environmental compatibility, low cost and simple operation, and effectively overcomes the problems of chemical residues, poor microbial adaptability and long remediation period in traditional remediation methods.

Description

Technical Field

[0001] This invention belongs to the field of soil remediation technology and relates to a method for remediating norfloxacin-contaminated soil using earthworm intestinal contents. Background Technology

[0002] Norfloxacin is a widely used fluoroquinolone antibiotic, extensively used in human medicine, livestock farming, and aquaculture. This leads to its entry into the soil environment through fecal excrement, wastewater irrigation, and drug residues. Norfloxacin exhibits high persistence and mobility in soil, is difficult to degrade naturally, and long-term residues can lead to the spread of antibiotic resistance genes, disrupt soil microbial communities, inhibit plant growth, and accumulate through the food chain, posing a potential threat to human health. With the increasing prominence of antibiotic pollution, the remediation of norfloxacin in soil has become a research hotspot in the environmental field.

[0003] Currently, remediation methods for norfloxacin-contaminated soil mainly include physical remediation, chemical remediation, and bioremediation. Physical remediation methods, such as adsorption and fixation (using adsorbents like activated carbon and clay) or soil replacement, can reduce the bioavailability of norfloxacin in the short term, but only transfer or fix the pollutant, not achieve complete degradation. Furthermore, saturated adsorbents may cause secondary pollution, resulting in high remediation costs and limited applicability. Chemical remediation methods, such as advanced oxidation technologies (e.g., Fenton reaction, ozone oxidation) or chemical leaching, can rapidly degrade norfloxacin, but require large amounts of chemical reagents, easily generating toxic intermediates, disrupting soil ecological balance, and affecting soil fertility and long-term health. Bioremediation methods, such as microbial inoculation or phytoremediation, are considered more environmentally friendly approaches. However, existing microbial agents are mostly derived from single strains screened in laboratories, resulting in low survival rates and unstable degradation efficiency in complex soil environments, and limited specific degradation capabilities for norfloxacin. Phytoremediation relies on specific plants for absorption or transformation, has a long remediation cycle, and is ineffective for high-concentration contaminated soils.

[0004] Existing remediation methods share common drawbacks: low remediation efficiency, incomplete degradation, and susceptibility to environmental factors (such as pH, temperature, and moisture); high cost and complex preparation of remediation agents, hindering large-scale application; and the potential introduction of foreign pollutants or disruption of the soil's native microecology, leading to ecological risks. Therefore, developing an efficient, economical, and eco-compatible method for remediating norfloxacin-contaminated soil is urgently needed. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for remediating norfloxacin-contaminated soil using earthworm intestinal contents, specifically comprising the following steps: Step 1: Collect healthy live earthworms and let them stand for 24-48 hours to ensure that most of the food in their intestines has been emptied. Spread the empty earthworms evenly on quartz sand with a particle size of 1-2 mm and a moisture content of 40-50%. Apply light of 300-500 lx and continue to induce for 8-14 hours. Then remove the earthworms and collect the quartz sand mixed with the earthworm intestinal contents.

[0006] Step 2: Mix the quartz sand-earthworm intestinal contents mixture with phosphate buffer at a mass ratio of 1:(4-6), shake at 80-120 rpm for 25-35 min to fully dissolve and suspend the intestinal contents in the buffer, centrifuge at 800-1200 rpm for 4-6 min, filter to remove bottom sediment, centrifuge the supernatant at 4500-5500 rpm for 10-20 min, filter to remove the supernatant, and the precipitate is the earthworm intestinal contents.

[0007] Preferably, the phosphate buffer solution, based on water, comprises (7-9) g / L NaCl, (0.1-0.3) g / L KCl, (2-3) g / L Na2HPO4·12H2O, and (0.1-0.3) g / L KH2PO4.

[0008] Step 3: Mix the earthworm intestinal contents with the activated nutrient solution, shake at 25-35℃ and 100-120 rpm for 20-28 hours. After shaking, add the modifier, stir at 30-40℃ and 60-90 rpm for 50-70 minutes, and then centrifuge at 3500-4500 rpm for 10-20 minutes. Remove the supernatant, and the precipitate is the modified earthworm intestinal contents.

[0009] Preferably, the mass ratio of the earthworm intestinal contents, the activated nutrient solution, and the modifier solution is 1:(8-12):(8-12).

[0010] Preferably, the activated nutrient solution comprises (4-6) g / L glucose, (2-4) g / L peptone, and (1-3) g / L KH2PO4 based on water.

[0011] Preferably, the modifier comprises sodium lignosulfonate, manganese sulfate and water in a mass ratio of (0.8-1.2):(0.4-0.6):1000.

[0012] Step 4: Mix the modified earthworm intestinal contents, activated carbon (particle size 0.5-1mm), organic acid and mineral materials in a mass ratio of (7-9):(4-6):(1-2):(3-5) and granulate them to a particle size of 3-5mm. Mix the granules with the encapsulating agent, stir at 20-30 rpm for 2-3 minutes, filter, remove the filtrate, mix the filter residue with the solidifying agent, stir at 50-60 rpm for 15-20 minutes, filter, remove the filtrate, and dry the filter residue at 40-50℃ until the moisture content is ≤8% to obtain the earthworm intestinal contents repair agent.

[0013] Preferably, the organic acid is one or more of humic acid, citric acid, oxalic acid, and tartaric acid.

[0014] Preferably, the mineral material includes one or more of diatomaceous earth, bentonite, quartz sand, perlite, zeolite and vermiculite.

[0015] Preferably, the mass ratio of the particles, the encapsulating agent, and the curing agent is 1:(0.8-1.2):(2-4). Most preferably, the encapsulating agent is a sodium alginate solution with a mass fraction of 1-3%, and the curing agent is a calcium chloride solution with a mass fraction of 3-5%.

[0016] Step 5: Deeply till the norfloxacin-contaminated soil to a depth of 25-30 cm, and apply an earthworm intestinal contents repair agent during tilling at a rate of 1-1.5 kg / m³. 2 Then water until the soil moisture content is 25-30%. 15-20 days after applying the repair agent, sow repair plants in the soil at a rate of 20-30 kg / m². 2 .

[0017] Preferably, the remediation plants are ryegrass and alfalfa, with a seed mass ratio of (1-2):(1-2).

[0018] The working principle of this invention is as follows: In step one, earthworms are typical negative phototaxis animals, strongly averse to light. Continuous light will make them feel uncomfortable and stressed, stimulating their strong burrowing instinct to avoid the light source. However, the rough quartz sand matrix is ​​not an ideal burrowing environment for them. Under this stress state, in order to reduce their weight and facilitate escape, earthworms will instinctively expel heavy sand grains and contents from their intestines.

[0019] In step two, the density difference between quartz sand and organic matter is utilized. First, the quartz sand is removed by low-speed centrifugation, and then the intestinal contents rich in microorganisms and active substances are obtained by high-speed centrifugation. This process achieves efficient separation and purification of quartz sand and active ingredients.

[0020] In step three, the nutrient solution provides carbon and nitrogen sources to stimulate microbial growth and enzyme synthesis, enhancing the expression of genes that degrade norfloxacin, such as activating the enzyme system that degrades fluoroquinolone antibiotics. Sodium lignosulfonate, as a biosurfactant, improves the fluidity of microbial cell membranes, promoting the transmembrane transport of norfloxacin; manganese sulfate, as a cofactor for manganese-dependent enzymes, enhances the efficiency of enzymatic reactions, improving the microorganisms' tolerance to norfloxacin and its degradation rate.

[0021] In step four, the earthworm's intestinal contents contain abundant microorganisms. These microorganisms secrete norfloxacin-degrading enzymes (such as norfloxacin hydrolase), which decompose norfloxacin into low-toxicity or non-toxic products through ring-opening and defluorination reactions. Activated carbon, as the main adsorbent carrier, has a porous structure and a large specific surface area, which can physically adsorb norfloxacin molecules, reducing their leaching and bioavailability; at the same time, it provides a habitat for microorganisms, extending their survival time. Organic acids (humic acid and citric acid, etc.) can promote the decomposition of norfloxacin through complexation and redox reactions; they also provide nutrients, stimulate microbial growth, regulate soil pH, and optimize the degradation environment. Mineral materials can serve as auxiliary adsorbents and physical frameworks, improving the pore structure of the remediation agent, preventing clumping, enhancing soil permeability, helping to maintain the microenvironmental humidity inside the remediation agent particles, and ensuring microbial activity.

[0022] Step five, deep tilling, breaks up soil compaction, increases soil porosity and oxygen content, creating favorable conditions for subsequent aerobic microorganisms. The application of the remediation agent granules alters the soil's physical structure, increasing aeration and water permeability. Simultaneously, biochar and mineral materials immediately begin to exert their physical adsorption effects, rapidly capturing and immobilizing norfloxacin molecules in the soil. Appropriate moisture is fundamental for microbial activity and also has a moisture-retaining effect, reducing water evaporation and maintaining stable soil temperature and humidity; at the same time, the dark environment prevents algae from growing on the surface and competing with functional microorganisms for nutrients. The remediation plant roots secrete sugars, organic acids, and other substances. These secretions stimulate the microorganisms embedded in the granules and native soil microorganisms to become more active, enhancing the degradation efficiency of norfloxacin and further improving the soil's physical structure.

[0023] The present invention has the following advantages: 1. Highly efficient and thorough degradation capability: Earthworm intestinal contents are rich in diverse microbial communities and active enzyme systems (such as norfloxacin hydrolase), which can decompose norfloxacin into low-toxicity or non-toxic small molecule products through biochemical reactions such as ring-opening and defluorination, achieving complete mineralization of pollutants. Compared with the limitations of single strains in existing microbial remediation, this invention utilizes the synergistic effect of the natural microecology of earthworm intestines, resulting in higher degradation efficiency, a more complete pathway, significantly shortened remediation cycle, and environmentally friendly degradation products with no risk of secondary pollution.

[0024] 2. Excellent ecological compatibility and safety: The remediation agent is entirely derived from biomass or natural materials (such as earthworm gut contents, activated carbon, organic acids, and mineral materials), without introducing chemically synthesized reagents or foreign harmful substances, fully ensuring the authenticity and stability of the soil ecosystem. Earthworm gut microbiota, as part of the native soil flora, can quickly adapt to the soil environment and form a mutually beneficial symbiotic relationship with native microorganisms, avoiding the competitive exclusion or ecological disturbance problems caused by foreign strains in existing bioremediation methods. Simultaneously, after granulation, the remediation agent undergoes encapsulation and solidification treatment, enabling the slow release of active ingredients and reducing negative impacts on non-target organisms in the soil.

[0025] 3. Earthworms are widely available and have low cultivation costs. The preparation process of the remediation agent does not require complex equipment or expensive raw materials, significantly reducing remediation costs. This makes it suitable for widespread application in large-scale contaminated sites such as farmland and pastures. Application of the remediation agent only requires deep tilling and watering, making the operation simple and easy for farmers or technicians to master. This avoids the drawbacks of reliance on specialized equipment in chemical remediation or high-frequency maintenance in physical remediation.

[0026] 4. A multi-mechanism synergistic effect enhances remediation efficacy. Activated carbon in the remediation agent provides a large specific surface area and porous structure, rapidly adsorbing and immobilizing norfloxacin molecules, reducing their leaching and bioavailability. Organic acids (such as humic acid and citric acid) promote norfloxacin decomposition through complexation and redox reactions, while simultaneously regulating soil pH and optimizing the microbial degradation environment. Mineral materials (such as diatomaceous earth and bentonite) serve as the physical framework, improving soil permeability and remediation agent stability, ensuring long-term microbial activity. This multi-mechanism synergistic mechanism of adsorption-degradation-regulation overcomes the shortcomings of existing single remediation methods, exhibiting stronger adaptability, especially under complex soil conditions.

[0027] 5. This invention significantly enhances the tolerance and enzyme activity of earthworm gut microbiota through modification treatments (such as the addition of sodium lignosulfonate and manganese sulfate), enabling them to maintain high-efficiency degradation capabilities under different soil conditions (such as different pH and humidity levels). The encapsulation structure of the remediation agent particles protects the microorganisms from external environmental impacts, prolonging their action time; combined with the root secretion of remediation plants, it further stimulates microbial metabolism, forming a "microorganism-plant" joint remediation system, achieving simultaneous improvement of soil structure and continuous reduction of pollutants. Detailed Implementation

[0028] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1 Raw material preparation: Phosphate buffer: based on water, it includes 8 g / L NaCl, 0.2 g / L KCl, 2.5 g / L Na2HPO4·12H2O, and 0.2 g / L KH2PO4.

[0030] Activated nutrient solution: Based on water, it includes 5g / L glucose, 3g / L peptone, and 2g / L KH2PO4.

[0031] Modifier: sodium lignosulfonate, manganese sulfate and water, in a mass ratio of 1:0.5:1000.

[0032] Organic acids: humic acid and citric acid, in a mass ratio of 2:1.

[0033] Mineral materials: diatomaceous earth, bentonite and vermiculite, in a mass ratio of 3:2:1.

[0034] The restoration plants were ryegrass and alfalfa, with a seed weight ratio of 1:1.

[0035] Implementation method: Step 1: Collect healthy live earthworms and let them stand for 36 hours to ensure that most of the food in their intestines has been emptied. Spread the empty earthworms evenly on quartz sand with a particle size of 1-2 mm and a moisture content of 45%. Apply 400 lx of light and continue to induce for 12 hours. Then remove the earthworms and collect the quartz sand mixed with the earthworm intestinal contents.

[0036] Step 2: Mix the quartz sand-earthworm intestinal contents mixture with phosphate buffer at a mass ratio of 1:5, shake at 100 rpm for 30 min to fully dissolve and suspend the intestinal contents in the buffer, centrifuge at 1000 rpm for 5 min, filter to remove bottom sediment, centrifuge the supernatant at 5000 rpm for 15 min, filter to remove the supernatant, and the precipitate is the earthworm intestinal contents.

[0037] Step 3: Mix the earthworm intestinal contents with the activating nutrient solution, shake at 30℃ and 110 rpm for 24 hours. After shaking, add the modifier, stir at 35℃ and 75 rpm for 60 minutes, then centrifuge at 4000 rpm for 15 minutes. Remove the supernatant; the precipitate is the modified earthworm intestinal contents. The mass ratio of earthworm intestinal contents, activating nutrient solution, and modifier solution is 1:10:10.

[0038] Step four: Modified earthworm intestinal contents, activated carbon with a particle size of 0.5-1 mm, organic acid, and mineral materials are mixed and granulated at a mass ratio of 8:5:1.5:4, resulting in particle sizes of 3-5 mm. The granules are then mixed with a 2% sodium alginate solution and stirred at 25 rpm for 3 minutes. The mixture is filtered, and the filtrate is removed. The residue is mixed with a 4% calcium chloride solution and stirred at 60 rpm for 17 minutes. The mixture is then filtered, and the filtrate is removed. The residue is dried at 45°C until the moisture content is ≤8%, yielding the earthworm intestinal contents repair agent. The mass ratio of the granules, sodium alginate solution, and calcium chloride solution is 1:1:3.

[0039] Step 5: Deeply till the norfloxacin-contaminated soil to a depth of 30cm, and apply earthworm intestinal contents repair agent during tilling at a rate of 1.25kg / m³. 2 Then water until the soil moisture content reaches 28%. On the 18th day after applying the remediation agent, sow the remediation plants in the soil at a rate of 25 kg / m². 2 .

[0040] Comparative Example 1 The difference from Example 1 is that this comparative example does not use earthworm intestinal contents, but only uses a mixture of activated charcoal, organic acids and mineral materials as a repair agent.

[0041] Experimental Example 1 Soil preparation: Uncontaminated farmland soil (pH 6.5-7.5, organic matter content 1.5-2.0%) was collected, air-dried, and sieved through a 2mm sieve. Norfloxacin standard (purity ≥98%) was artificially added to prepare contaminated soil with an initial norfloxacin concentration of 10.0 mg / kg. The soil was uniformly mixed and then divided into multiple experimental pots (30 cm in diameter, 25 cm in height), with each pot containing 5.0 kg of soil. The soil was left to stand for 7 days to allow the norfloxacin to age in the soil, simulating a natural contamination state.

[0042] Experimental Design: Experimental Group: Contaminated soil was remediated according to the method in Example 1. Control Group 1: Contaminated soil was remediated according to the method in Comparative Example 1. Control Group 2: No remediation agent was applied (blank control).

[0043] Each group had 3 replicates, for a total of 9 experimental pots, which were randomly arranged in a greenhouse at a temperature of 25±2℃ and under natural light.

[0044] Sampling and Analysis: Soil samples were collected on days 0, 7, 14, 21, and 28 after the application of the remediation agent. Three soil samples (0-20 cm deep) were taken from each experimental pot, mixed evenly, air-dried, and ground through a 100-mesh sieve.

[0045] Norfloxacin residue concentration was determined by high-performance liquid chromatography (HPLC). HPLC conditions: C18 column, mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (25:75, v / v), detection wavelength: 278 nm, flow rate: 1.0 mL / min, injection volume: 10 μL. Each sample was measured in triplicate, and the average value was used to calculate the norfloxacin concentration and removal rate.

[0046] Data calculation: Norfloxacin removal rate calculation formula: Removal rate (%) = (initial concentration - residual concentration) / initial concentration × 100%.

[0047] Table 1

[0048] Table 1 shows that the norfloxacin removal rate in the experimental group reached 88.20% within 28 days, significantly higher than that in control group 1 (28.50%) and control group 2 (8.80%). This indicates that the microorganisms and active ingredients in the earthworm intestinal contents played a crucial role in the degradation of norfloxacin. The norfloxacin concentration in the experimental group decreased significantly faster than that in the control group, especially between day 7 and day 21, when the removal rate rapidly increased from 25.50% to 72.50%, demonstrating the sustained effect of microbial degradation. Control group 1 (without earthworm intestinal contents) relied solely on activated carbon adsorption and organic acid action, resulting in a lower removal rate, indicating limited effects of physical adsorption and chemical degradation. The norfloxacin concentration in control group 2 (blank) showed minimal change, mainly due to natural degradation. The experiment confirms that the method of remediating norfloxacin-contaminated soil using earthworm intestinal contents has efficient and thorough degradation capabilities, good ecological compatibility, and is suitable for the remediation of large-area contaminated sites.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for remediating norfloxacin-contaminated soil using earthworm intestinal contents, characterized in that, Includes the following steps: Step 1: Collect healthy live earthworms and let them stand for 24-48 hours. Then, spread the earthworms evenly on the quartz sand, apply 300-500 lx of light, and induce them for 8-14 hours. After that, remove the earthworms and collect the quartz sand mixed with the earthworm intestinal contents. Step 2: Mix the quartz sand-earthworm intestinal contents mixture with phosphate buffer at a mass ratio of 1:4-6, shake, centrifuge to remove bottom sediment, collect the supernatant and centrifuge, filter, remove the supernatant, and the precipitate is earthworm intestinal contents; Step 3: Mix the earthworm intestinal contents with the activated nutrient solution and shake. After shaking, add the modifier, stir, centrifuge, remove the supernatant, and the precipitate is the modified earthworm intestinal contents. The mass ratio of the earthworm intestinal contents, activated nutrient solution and modifier solution is 1:8-12:8-12. Step 4: Mix the modified earthworm intestinal contents, activated carbon, organic acid and mineral materials in a mass ratio of 7-9:4-6:1-2:3-5 and granulate. Mix the granules with the encapsulating agent, filter, remove the filtrate, mix the filter residue with the solidifying agent, filter, remove the filtrate, and dry the filter residue to a moisture content of ≤8% to obtain the earthworm intestinal contents repair agent. Step 5: Deeply till the norfloxacin-contaminated soil to a depth of 25-30 cm, and apply an earthworm intestinal contents repair agent during tilling at a rate of 1-1.5 kg / m³. 2 Then water until the soil moisture content is 25-30%. Sow the repair plants in the soil 15-20 days after the repair agent is applied. The modifier mentioned in step three includes sodium lignosulfonate, manganese sulfate and water, in a mass ratio of 0.8-1.2:0.4-0.6:1000.

2. The method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 1, characterized in that, The phosphate buffer solution described in step two is based on water and includes 7-9 g / L NaCl, 0.1-0.3 g / L KCl, 2-3 g / L Na2HPO4·12H2O, and 0.1-0.3 g / L KH2PO4.

3. The method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 1, characterized in that, The activating nutrient solution described in step three, based on water, includes 4-6 g / L glucose, 2-4 g / L peptone, and 1-3 g / L KH2PO4.

4. The method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 1, characterized in that, The organic acid mentioned in step four is one or more of humic acid, citric acid, oxalic acid, and tartaric acid.

5. The method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 1, characterized in that, The mineral materials mentioned in step four include one or more of diatomaceous earth, bentonite, quartz sand, perlite, zeolite, and vermiculite.

6. The method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 1, characterized in that, The mass ratio of the particles, encapsulating agent, and curing agent in step four is 1:0.8-1.2:2-4.

7. The method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 6, characterized in that, The embedding agent is a sodium alginate solution with a mass fraction of 1-3%.

8. A method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 6, characterized in that, The curing agent is a calcium chloride solution with a mass fraction of 3-5%.

9. A method for remediating norfloxacin-contaminated soil using earthworm intestinal contents as described in claim 1, characterized in that, The restoration plants mentioned in step five are ryegrass and alfalfa, with a seed mass ratio of 1-2:1-2.

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