Method for repairing norfloxacin contaminated soil by using earthworm intestinal contents

Abstract

The invention belongs to the technical field of soil remediation, and relates to a method for remediating norfloxacin contaminated soil by using earthworm intestinal contents, which comprises the following steps: inducing earthworms to empty the intestinal contents on quartz sand by illumination, extracting by using a phosphate buffer solution, activating nutrient solution and treating by using a modifier to obtain high-activity modified earthworm intestinal contents, and granulating with activated carbon, organic acid and mineral materials according to a specific ratio, and embedding and curing to prepare the repairing agent. After the remediation agent is applied to the contaminated soil, efficient decomposition of norfloxacin and synchronous restoration of the ecological function of the soil are achieved through the degradation effect of earthworm intestinal microorganisms, the adsorption effect of activated carbon, the complex reaction of organic acid and physical regulation and control of mineral materials. The method creatively utilizes natural microbial communities in the intestinal tract of the earthworm, has the advantages of thorough degradation, environmental compatibility, low cost, simplicity and convenience in operation and the like, and effectively overcomes the problems of chemical residues, poor microbial adaptability, long repair period and the like in a traditional repair method.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of soil remediation, and relates to a method for remediation of norfloxacin contaminated soil by using earthworm intestinal contents. BACKGROUND

[0002] Norfloxacin is a widely used fluoroquinolone antibiotic, which is massively applied in human medicine, livestock breeding and aquaculture, leading to its entry into the soil environment through fecal excretion, sewage irrigation and drug residues. Norfloxacin has high persistence and mobility in soil and is difficult to degrade naturally. Long-term residues can cause the spread of antibiotic resistance genes, disrupt soil microbial community structure, inhibit plant growth, and accumulate through the food chain to pose 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] At present, the remediation methods for norfloxacin contaminated soil mainly include physical remediation, chemical remediation and biological remediation. Physical remediation methods such as adsorption fixation (using activated carbon, clay and other adsorbents) or soil replacement can reduce the biological availability of norfloxacin in the short term, but only transfer or fix the pollutants, and do not achieve complete degradation. Moreover, after the saturation of adsorbents, secondary pollution may occur, the remediation cost is high, and the applicability is limited. Chemical remediation methods such as advanced oxidation technology (such as Fenton reaction, ozone oxidation) or chemical leaching can quickly degrade norfloxacin, but a large amount of chemical reagents need to be invested, which can easily produce toxic intermediates, destroy the ecological balance of soil, and affect soil fertility and long-term health. Biological remediation methods such as microbial inoculation or phytoremediation are considered to be a more environmentally friendly approach, but existing microbial inoculants are mostly derived from single strains screened in the laboratory, which have low survival rate and unstable degradation efficiency in complex soil environment, and limited specific degradation ability for norfloxacin. Phytoremediation relies on the absorption or transformation of specific plants, and has a long remediation period and poor effect on high-concentration contaminated soil.

[0004] The common defects of existing remediation methods are: low remediation efficiency, incomplete degradation, easy to be disturbed by environmental factors (such as pH, temperature, moisture); high cost of remediation agents, complex preparation, difficult to be applied on a large scale; the remediation process may introduce foreign pollutants or destroy the original micro-ecology of soil, leading to ecological risks. Therefore, it is urgent to develop an efficient, economical and ecologically compatible remediation method for norfloxacin contaminated soil. SUMMARY

[0005] In order to solve the above problems, the application provides a method for remediation of norfloxacin contaminated soil by using earthworm intestinal contents, which specifically comprises the following steps: Step one, collect healthy live earthworms, stand for 24-48h, make sure most of the food in their intestines is emptied, evenly lay the earthworms after fasting on quartz sand with particle size of 1-2mm and water content of 40-50%, apply light of 300-500lx, remove the earthworms after 8-14h of continuous induction, and collect the quartz sand mixed with earthworm intestinal contents.

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

[0007] Preferably, the phosphate buffer 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 based on water.

[0008] Step three, mix the earthworm intestinal contents with the activated nutrient solution, oscillate at 25-35℃ and 100-120rpm for 20-28h, after oscillation, add the modifier, stir at 30-40℃ and 60-90rpm for 50-70min, then centrifuge at 3500-4500rpm for 10-20min to remove the supernatant, and the precipitate is the modified earthworm intestinal contents.

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

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

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

[0012] Step four, the modified earthworm gut contents, activated carbon (particle size 0.5-1mm), organic acid and mineral material are mixed according to the mass ratio (7-9):(4-6):(1-2):(3-5) to granulate, the particle size of the granules is 3-5mm, the granules are mixed with embedding agent, stirred at 20-30rpm for 2-3min, filtered, the filtrate is removed, the residue is mixed with solidifying agent, stirred at 50-60rpm for 15-20min, filtered, the filtrate is removed, the residue is dried at 40-50℃ until the water content is less than or equal to 8%, and the earthworm gut contents repair agent is obtained.

[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 diatomite, bentonite, quartz sand, perlite, zeolite and vermiculite.

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

[0016] Step five, the norfloxacin contaminated soil is ploughed to a depth of 25-30cm, and the earthworm gut contents repair agent is applied to the soil at the same time, the application amount is 1-1.5kg / m 2 , then water is poured to make the water content of the soil 25-30%, and the repair plant is sown in the soil 15-20 days after the repair agent is applied, the sowing amount is 20-30kg / m 2 .

[0017] Preferably, the repair plant is blackgrass and alfalfa, and the mass ratio of the seeds is (1-2):(1-2).

[0018] The working principle of the present application is that the earthworm in step one is a typical negative phototaxis animal, which strongly dislikes light, and continuous light will make it feel uncomfortable and stressed, and it will be triggered to have a strong instinct to dig into the ground to avoid the light source. However, the rough quartz sand substrate is not its ideal digging environment, and under this stress, the earthworm will instinctively discharge the heavy sand particles and contents in the gut to facilitate escape.

[0019] In step two, the density difference between quartz sand and organic matter is utilized, and the quartz sand is removed by low-speed centrifugation, and the gut contents rich in microorganisms and active substances are obtained by high-speed centrifugation, which realizes efficient separation and purification of quartz sand and effective components.

[0020] The carbon source and nitrogen source are provided by the nutrient solution in step three, so as to stimulate the growth and enzyme synthesis of the microorganism, and enhance the expression of the norfloxacin degradation gene, for example, activate the fluoroquinolone antibiotic degradation enzyme system. The lignin sulfonate sodium in the modifier serves as a biological surfactant, improves the fluidity of the cell membrane of the microorganism, and promotes the transport of norfloxacin across the membrane; and the manganese sulfate serves as a cofactor of manganese-dependent enzyme, enhances the efficiency of the enzymatic reaction, and improves the tolerance and degradation rate of the microorganism to norfloxacin.

[0021] In step four, the earthworm gut content contains rich microorganisms which can secrete norfloxacin degradation enzymes (such as norfloxacin hydrolase) to decompose norfloxacin into low-toxic or non-toxic products through reactions such as ring opening and defluorination. The activated carbon serves as a main adsorption carrier, has a porous structure and a large specific surface area, can physically adsorb norfloxacin molecules, reduces the eluviation and bioavailability of norfloxacin, and at the same time provides a habitat for the microorganism and prolongs the survival time of the microorganism. The organic acids (such as humic acid and citric acid) can promote the decomposition of norfloxacin through complexation and redox reactions, and at the same time provide nutrition, stimulate the growth of the microorganism, and adjust the soil pH to optimize the degradation environment. The mineral material can serve as an auxiliary adsorbent and a physical skeleton, improve the pore structure of the remediation agent, prevent caking, enhance the soil permeability, help maintain the humidity of the microenvironment inside the remediation agent particles, and guarantee the activity of the microorganism.

[0022] In step five, deep ploughing can break the soil compaction, increase the soil porosity and oxygen content, and create favorable conditions for the subsequent aerobic microorganism. The application of the remediation agent particles changes the physical structure of the soil, increases the air and water permeability. At the same time, the activated carbon and the mineral material immediately begin to play a physical adsorption role, quickly capture and fix the norfloxacin molecules in the soil. The appropriate moisture is the basis for the life activities of the microorganism, also has the effect of soil conservation, reduces the water evaporation, and maintains the stability of the soil temperature and humidity; at the same time, the dark environment can prevent the algae from breeding on the surface and compete with the functional microorganism for nutrition. The root system of the remediation plant can secrete substances such as carbohydrates and organic acids, and these secretions can stimulate the microorganism in the embedded particles and the indigenous microorganism in the soil to be more active, strengthen the degradation efficiency of norfloxacin, and further improve the physical structure of the soil.

[0023] The present application has the following advantages: 1. High-efficiency and complete degradation capacity, the earthworm gut content is rich in diversified microbial community and active enzyme system (such as norfloxacin hydrolase), which can decompose norfloxacin into low-toxic or non-toxic small molecular products through biochemical reactions such as ring opening and defluorination, so as to realize the complete mineralization of the pollutants. Compared with the limitation of single strain in the existing microbial remediation, the present application utilizes the synergistic effect of the natural microecology of the earthworm gut, has higher degradation efficiency and more complete degradation path, can significantly shorten the remediation period, and the degradation products are environmentally friendly and have no secondary pollution risk.

[0024] 2. Excellent ecological compatibility and safety, the entire source of the remediation agent is derived from biomass or natural materials (such as earthworm intestinal contents, activated carbon, organic acids and mineral materials), without introducing chemical synthetic reagents or foreign harmful substances, fully guaranteeing the authenticity and stability of the soil ecosystem. Earthworm intestinal microorganisms, as part of the soil indigenous flora, can quickly adapt to the soil environment, form mutualistic symbiosis with indigenous microorganisms, and avoid the problems of competition and exclusion or ecological disturbance of foreign strains in existing biological remediation. At the same time, after the remediation agent is granulated, the active ingredients can be slowly released through embedding and solidification treatment, reducing the negative impact on non-target organisms in the soil.

[0025] 3. Earthworms are widely available and have low cultivation costs, and the remediation agent preparation process does not require complex equipment or expensive raw materials, significantly reducing remediation costs and being suitable for the popularization and application of large-area contaminated sites such as farmland and pasture. The remediation agent only needs to be deeply plowed and watered when applied, which is simple to operate and easy for farmers or technicians to master, avoiding the dependence on professional equipment in chemical remediation or the high-frequency maintenance problems in physical remediation.

[0026] 4. Multi-mechanism synergistic enhancement of remediation effect, the activated carbon in the remediation agent provides a large specific surface area and porous structure, quickly adsorbing and fixing norfloxacin molecules, reducing their leaching and biological availability; organic acids (such as humic acid and citric acid) promote the decomposition of norfloxacin through complexation and redox reactions, while adjusting the soil pH and optimizing the microbial degradation environment; mineral materials (such as diatomite and bentonite) as a physical skeleton, improve the soil permeability and the stability of the remediation agent, ensuring the long-term activity of microorganisms. This multi-element synergistic mechanism of adsorption-degradation-regulation overcomes the shortcomings of existing single remediation methods, and shows stronger adaptability especially in complex soil conditions.

[0027] 5. The present application significantly enhances the tolerance and enzyme activity of earthworm intestinal microorganisms through modification treatment (such as the addition of sodium lignosulfonate and manganese sulfate), enabling them to maintain high degradation capacity under different soil conditions (such as different pH and humidity). The embedding structure of the remediation agent granules protects the microorganisms from external environmental impact, prolonging their action time; combined with the root exudation of remediation plants, further stimulating microbial metabolism, forming a "microbe-plant" combined remediation system, achieving simultaneous improvement of soil structure and continuous reduction of pollutants. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

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

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

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

[0032] Organic acid: humic acid and citric acid, mass ratio 2:1.

[0033] Mineral material: diatomite, bentonite, and vermiculite, mass ratio 3:2:1.

[0034] The repair plant: ryegrass and alfalfa, seed mass ratio 1:1.

[0035] Implementation method: Step one, collect healthy live earthworms, stand for 36 h, ensure that most of the food in their intestines is emptied, evenly lay the earthworms after fasting on quartz sand with a particle size of 1-2 mm and a water content of 45%, apply 400 lx of light, continue to induce for 12 h, then remove the earthworms, and collect the quartz sand mixed with the earthworm intestinal contents.

[0036] Step two, mix the quartz sand-earthworm intestinal content mixture with the phosphate buffer at a mass ratio of 1:5, oscillate at 100 rpm for 30 min, so that the intestinal contents are fully dissolved and suspended in the buffer, centrifuge at 1000 rpm for 5 min, filter to remove the 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 three, mix the earthworm intestinal contents with the activated nutrient solution, oscillate at 30°C and 110 rpm for 24 h, after oscillation, add the modifier, stir at 35°C and 75 rpm for 60 min, then centrifuge at 4000 rpm for 15 min, remove the supernatant, and the precipitate is the modified earthworm intestinal contents. The mass ratio of earthworm intestinal contents, activated nutrient solution, and modifier solution is 1:10:10.

[0038] Step four, the modified earthworm gut contents, activated carbon with particle size of 0.5-1 mm, organic acid and mineral material are mixed with mass ratio of 8:5:1.5:4 to granulate, the particle size of the granules is 3-5 mm, the granules are mixed with sodium alginate solution with mass fraction of 2%, stirred at 25 rpm for 3 min, filtered, the filtrate is removed, the residue is mixed with calcium chloride solution with mass fraction of 4%, stirred at 60 rpm for 17 min, filtered, the filtrate is removed, the residue is dried at 45℃ until the water content is ≤8%, to obtain the earthworm gut contents repair agent. The mass ratio of the granules, sodium alginate solution and calcium chloride solution is 1:1:3.

[0039] Step five, the norfloxacin contaminated soil is ploughed to a depth of 30 cm, and the earthworm gut contents repair agent is applied at the same time, the application amount is 1.25 kg / m 2 , then water is poured to a soil water content of 28%, and on the 18th day after the application of the repair agent, the repair plants are sown in the soil at a sowing amount of 25 kg / m 2 .

[0040] Comparative example 1 The difference between this comparative example and example 1 is that this comparative example does not use earthworm gut contents, but only uses activated carbon, organic acid and mineral material mixed as a repair agent.

[0041] Test example 1 Soil preparation: Collect uncontaminated farmland soil (pH 6.5-7.5, organic matter content 1.5-2.0%), air dry and pass through a 2 mm sieve. Artificially add norfloxacin standard (purity ≥98%) to prepare contaminated soil with an initial concentration of 10.0 mg / kg. After mixing the soil evenly, divide it into multiple experimental pots (diameter 30 cm, height 25 cm), with 5.0 kg of soil per pot, and let the soil stand for 7 days to allow the norfloxacin to age in the soil, simulating a natural pollution state.

[0042] Experimental design: Experimental group: repair the contaminated soil according to the method of example 1. Control group 1: repair the contaminated soil according to the method of comparative example 1. Control group 2: do not apply any repair agent (blank control).

[0043] Each group has 3 replicates, a total of 9 experimental pots, randomly arranged in a greenhouse with temperature controlled at 25±2℃ and natural light.

[0044] Sampling and analysis: Collect soil samples at 0 days, 7 days, 14 days, 21 days and 28 days after the application of the repair agent. Take 3 points (depth 0-20 cm) of soil from each experimental pot, mix evenly, air dry and grind through a 100 mesh sieve.

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

[0046] Data calculation: The removal rate of norfloxacin was calculated according to the formula: removal rate (%) = (initial concentration - residual concentration) / initial concentration x 100%.

[0047] Table 1

[0048] As shown in Table 1, the removal rate of norfloxacin in the experimental group reached 88.20% within 28 days, which was 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 play a key role in the degradation of norfloxacin. The concentration of norfloxacin in the experimental group decreased significantly faster over time than in the control groups, especially during the period from the 7th day to the 21st day, when the removal rate rapidly increased from 25.50% to 72.50%, reflecting the sustained effect of microbial degradation. The removal rate in control group 1 (without earthworm intestinal contents) was low, which indicates that the effect of physical adsorption and chemical degradation is limited. The change in the concentration of norfloxacin in control group 2 (blank) was small, mainly due to natural degradation. The experiment proves that the method for repairing norfloxacin-contaminated soil using earthworm intestinal contents has high and complete degradation capacity, and is ecologically compatible, and is suitable for the repair of large-area contaminated sites.

[0049] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended 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 gut contents, characterized in that, The method comprises the following steps: Step one, collect healthy live earthworms, stand for 24-48h, then evenly lay the earthworms on quartz sand, apply 300-500lx light, remove the earthworms after 8-14h of induction, collect the quartz sand mixed with the earthworm intestinal contents; Step two, mix the quartz sand-earthworm intestinal contents mixture with phosphate buffer solution at a mass ratio of 1:(4-6), shake, remove the bottom sediment by centrifugation, collect the supernatant and centrifuge, filter, remove the supernatant, and the precipitate is the earthworm intestinal contents; Step three, mix the earthworm intestinal contents with activated nutrient solution, shake, add modifier after shaking is completed, 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 four, mix the modified earthworm intestinal contents, activated carbon, organic acid and mineral material at a mass ratio of (7-9):(4-6):(1-2):(3-5), mix and stir the granules with embedding agent, filter, remove the filtrate, mix and stir the filter residue with solidifying agent, filter, remove the filtrate, dry the filter residue to a water content of ≤8%, and obtain the earthworm intestinal contents repair agent; Step five, deep plough the norfloxacin contaminated soil 25-30 cm, with ploughing, apply the earthworm gut content remediation agent, the application amount is 1-1.5 kg / m 2 Then water to the soil water content 25-30%, on the 15-20 days after the application of the remediation agent, sow the remediation plant in the soil, the sowing amount is 20-30 kg / m 2 .

2. A method for remediating norfloxacin contaminated soil using earthworm gut contents as claimed in claim 1, wherein, The phosphate buffer solution in step two 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 based on water.

3. A method for remediating norfloxacin contaminated soil using earthworm gut contents as claimed in claim 1, wherein, The activated nutrient solution in step three comprises (4-6)g / L glucose, (2-4)g / L peptone and (1-3)g / L KH2PO4 based on water.

4. A method for remediating norfloxacin contaminated soil using earthworm gut contents as claimed in claim 1, wherein, The modifier in step three comprises sodium lignosulfonate, manganese sulfate and water, and the mass ratio is (0.8-1.2):(0.4-0.6):1000.

5. A method for remediation of norfloxacin contaminated soil using earthworm gut contents as claimed in claim 1, wherein, The organic acid in step four is one or more of humic acid, citric acid, oxalic acid and tartaric acid.

6. A method for remediation of norfloxacin contaminated soil using earthworm gut contents as claimed in claim 1, wherein, The mineral material in step four comprises one or more of diatomite, bentonite, quartz sand, perlite, zeolite and vermiculite.

7. A method for remediation of norfloxacin contaminated soil using earthworm gut contents as claimed in claim 1, wherein, The mass ratio of the granules, embedding agent and solidifying agent in step four is 1:(0.8-1.2):(2-4).

8. A method for remediating norfloxacin contaminated soil using earthworm gut contents according to claim 7, characterized in that, The embedding agent is a sodium alginate solution with a mass fraction of 1-3%.

9. A method for remediation of norfloxacin contaminated soil using earthworm gut contents as claimed in claim 7, wherein, The solidifying agent is a calcium chloride solution with a mass fraction of 3-5%.

10. A method for remediation of norfloxacin contaminated soil using earthworm gut contents as claimed in claim 1, wherein, The repair plants in step five are blackgrass and alfalfa, and the seed mass ratio is (1-2):(1-2).

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