CN113231462A - Method for stimulating indigenous flora to rapidly degrade cypermethrin in soil - Google Patents
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Abstract
A method for stimulating indigenous flora to rapidly degrade cypermethrin in soil comprises selecting clean soil domesticated adult earthworm living body, cleaning, and placing in dark box for cleaning intestine; the earthworms after the gut purging treatment are put into cypermethrin-polluted soil with the concentration of 0.5-20 mg/kg for domestication for 20 days; taking out the earthworms after the culture, cleaning the earthworms, dissecting the intestinal tracts, removing the content in the intestinal tracts, and scraping the intestinal wall flora; adding the earthworm intestinal flora into the soil to be repaired according to the mass ratio of the put earthworm intestinal flora to the soil of 1:2000-1:8000, and repairing for 7-60 days. Under the stress of cypermethrin, the target flora domesticated by the earthworm intestinal reactor is rich in cypermethrin pesticide degrading microorganisms, and the cypermethrin pesticide degrading microorganisms are added into cypermethrin-polluted soil to promote the indigenous flora to rapidly degrade cypermethrin, so that the degradation rate can reach 96.1%, and the safety of the soil ecological environment is further maintained.
Description
Technical Field
The invention belongs to the technical field of bioremediation of pesticide-contaminated soil, and particularly relates to a method for quickly degrading cypermethrin in soil by domesticating a target flora and stimulating indigenous flora in an earthworm intestinal reactor.
Background
As a big country for producing and using pesticides, the yield of the pesticides in 2016 reaches 378 ten thousand tons, which accounts for more than 1/3 in the world, and the usage amount of the pesticides in 2018 reaches 150.4 ten thousand tons, so that the pesticides bring serious soil environment problems in the production, transportation and use processes. Cypermethrin, one of the most common modern pesticides, generally exists in soil environment and has high detectable rate, and the main pollution source is agricultural soil. The cypermethrin is mainly used for killing pests in soil such as farmlands, woodlands and the like, and the concentration of the cypermethrin in part of surface soil can reach more than 3000 mg/kg due to large-amount and excessive use. Cypermethrin is used as an organochlorine pesticide with high toxicity, and cypermethrin remaining in soil poisons soil organisms and destroys the ecological environment of the soil on the one hand; on the other hand, the pesticide flows through the ground surface after being washed by rainwater and then enters underground water after being infiltrated, so that the ecological environment of the area is seriously damaged, and a great environmental risk exists. Therefore, the remediation work of the cypermethrin-polluted soil is necessary.
The earthworms are one of soil animals with the largest biomass in the soil environment, and the intestinal tracts of the earthworms are the most important moving places, participate in various metabolic processes of the soil environment, and have important significance for maintaining the safety of the soil environment. Soil and earthworm intestinal tracts are two completely different microbial living environments, microbial communities with different phenotypes and genotypes are colonized, and two specific ecological function classification units of indigenous flora and earthworm intestinal flora are created. Indigenous flora plays an indispensable role in different nutrition levels in the processes of nutrient circulation, organic carbon fixation, biological accumulation and degradation of toxic and harmful substances (pesticides) in soil and the like. Meanwhile, the earthworm intestinal flora ingests the indigenous flora through swallowing the soil, forms the ecological specific intestinal flora through intestinal operation and fermentation, and the endogenous flora returns to the soil in the form of earthworm feces to become members of the indigenous flora again. In addition, the earthworm intestinal canal has a screening effect on microorganisms taken into soil, and contains more abundant heavy metal reducing bacteria and pesticide degrading bacteria in heavy metal and pesticide polluted soil, so that the stimulation of soil pollutants can increase the colonization of beneficial microorganisms in the earthworm intestinal canal. Therefore, the earthworm intestinal canal is used as a natural bioreactor to promote the growth and the propagation of functional bacteria, and the functional bacteria are conveyed into the soil to participate in the process of degrading pesticides in the soil by indigenous flora, so that the reduction of pollutants is accelerated. The earthworm intestinal reactor domesticates the target flora to stimulate the indigenous flora to quickly degrade soil pollutants, and has important significance for ecological restoration of the soil environment.
At present, aiming at the remediation of cypermethrin-polluted soil, the microbial enhanced remediation of the polluted soil is mainly carried out by directly adding a microbial group with the function of degrading cypermethrin into the polluted soil. Through retrieval patents, the strains with the function of degrading cypermethrin are Acinetobacter ruckeri (Acinetobacter lwoffii) strain JX-2, Acinetobacter baumannii (Acinetobacter baumannii) strain ZH-14, Pseudomonas fulvus (Pseudomonas fulva) strain P31, Bacillus thuringiensis (Bacillus thuringiensis) Bt-1, Eurotium cristatum (Eurtimurium) strain ET1, Acinetobacter (Acinetobacter sp) strain JCX22D, nitroreducens CW-7, Sphingomonas bacteria (Sphingomonas truerai) strain CW3, Bacillus megaterium (Bacillus megaterium) strain HLJ7, Nocardia (Leclenbacia adynariae) strain Y2, Acinetobacter acidophilus strain 4, Acinetobacter jejunipes (Lactobacillus) strain K-6701, Bacillus subtilis strain BR-22D, Bacillus subtilis strain K-26, Bacillus subtilis strain BR-5, Bacillus subtilis strain and the like. The microorganism strengthening and repairing technology has higher requirements on the microorganism culture technology, and the strain culture process is complex, long in time and higher in cost; in addition, different strains have different requirements on the environment conditions for soil remediation, and a large number of experiments are needed to screen suitable bacteria under different soil conditions, so that more energy and labor are consumed. Besides the microorganism strengthening repair technology, the remediation of the cypermethrin polluted soil also comprises a gene engineering technology. For example, CN200910241899.5 and CN200410042712.6, coding genes of detoxication esterase and hydrolase are introduced into plasmids to obtain engineering bacteria to repair cypermethrin polluted soil. The method for preparing the engineering bacteria has high requirements on gene engineering technology and experimental operation capacity and is difficult to realize under the condition of a common laboratory. Besides, the preparation of the composite ecological restoration agent is also a common cypermethrin restoration technology. For example, CN201710776995.4 and CN201710777042.X, 6 materials are mixed, and cypermethrin in soil is reduced by researching the optimal mixing proportion. However, the preparation process of the composite ecological restoration agent is complex, the cost is high, and partial materials can cause adverse effects on soil organisms in the restoration process and destroy the ecological safety of the soil. CN201410136171.7, the biochar is prepared by using the corn straws, and the optimal application proportion is explored for repairing the cypermethrin polluted soil. The preparation of the biochar consumes a large amount of energy, the preparation process can cause environmental pollution, the cost is high, and secondary pollution exists. Therefore, the cypermethrin-polluted soil remediation lacks a low-cost, low-energy-consumption, efficient, green and targeted remediation technology.
Through searching patents of earthworms and soil pollutant degradation through an earthworm intestinal reactor, the discovery that the earthworms and earthworm feces are mostly involved in bioremediation of heavy metals in soil, and the discovery that earthworms are added to degrade pesticide pollutants in soil is rare, and no research and discussion about the participation of the earthworm intestinal reactor in degradation of cypermethrin pesticides in soil is found. The research directions of the invention are the closest to the application numbers CN201911418253.X and CN201911409957.0, earthworms cultured in a laboratory are dissected to obtain fresh earthworm intestinal contents, and the fresh earthworm intestinal contents are sequentially added into different types of tetracycline and sulfonamide antibiotic polluted soil to research the reduction capability of the earthworm intestinal contents on soil antibiotics. However, most of the microorganisms in the earthworm intestinal contents extracted by the method are obtained by directly feeding earthworms, the sources of the microorganisms are mainly soil microorganisms, and microorganism groups with specific functions are not formed by screening and enriching the microorganisms in an earthworm intestinal reactor completely, so that the biological strengthening stimulation repair effect is not achieved, and the colonization screening effect of the earthworm intestinal environment on functional groups is not highlighted.
The main defects of the prior art are as follows: the existing cypermethrin-polluted soil restoration technology is single, most of the cypermethrin-polluted soil restoration technology is realized by using degradation functional bacteria and engineering bacteria, but the restoration range of the technology is limited, the requirement on soil restoration environmental conditions is high, the discovery of the degradation functional bacteria and the preparation of the engineering bacteria are time-consuming and labor-consuming, and the quick implementation and application of the polluted soil microbial restoration are not facilitated; the mixed repairing material has complex preparation process and high cost, and causes potential harm to soil organisms. Therefore, a green, low-cost and convenient-to-operate cypermethrin-polluted soil remediation technology is lacked.
The main causes of defects are: (1) in early China, cypermethrin is produced and used in large quantities. On one hand, a large amount of cypermethrin is left in agricultural soil in the large-scale agricultural use and unreasonable use process, so that farmland organisms, farmland ecological environment and human health are endangered; on the other hand, with the rainfall and infiltration, cypermethrin in the soil enters underground water and rock gaps along with runoff and gaps, so that the repair work is more difficult. (2) Cypermethrin is an organochlorine pesticide with strong toxicity, and can poison and kill non-target organisms in soil. (3) At present, the pesticide-polluted soil is mainly repaired by adding degradation functional bacteria and engineering bacteria, but the repairing process needs higher biotechnology and operation capacity, the cost is high, and the repairing process is difficult to realize in a common laboratory. In addition, the added degradation functional bacteria and engineering bacteria are single, the applicable conditions to different soil environmental conditions are not clear, and further verification is needed. (4) The pesticide-polluted soil remediation lacks a simple and efficient remediation technology and a standard remediation technology operation. Therefore, the method has important significance for improving the ecological environment of pesticide-polluted soil by exploring a green, efficient, targeted and standardized microbial remediation technology.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides a method for stimulating indigenous flora to rapidly degrade cypermethrin in soil, which can effectively accelerate the indigenous flora to degrade cypermethrin, reduce the use and repair cost of soil pollution repair materials, reduce the interference of repair reagents and catalysts to the soil environment, and improve the soil quality and the ecological environment self-recovery capability.
The technical scheme is as follows: a method for stimulating indigenous flora to rapidly degrade cypermethrin in soil comprises the following steps: selecting the grown earthworm living bodies domesticated with clean soil, cleaning, and placing in a dark box for clearing intestines; the earthworms after the gut purging treatment are put into cypermethrin-polluted soil with the concentration of 0.5-20 mg/kg for domestication for 20 days; taking out the earthworms after the culture, cleaning the earthworms, dissecting the intestinal tracts, removing the content in the intestinal tracts, and scraping the intestinal wall flora; adding the earthworm intestinal flora into the soil to be repaired according to the mass ratio of the put earthworm intestinal flora to the soil of 1:2000-1:8000, and repairing for 7-60 days.
Preferably, the earthworm species is lima lachryma.
Preferably, the method for stimulating the indigenous flora to rapidly degrade the cypermethrin in the soil comprises the steps of taking domesticated earthworms, cleaning the domesticated earthworms by using sterile water, immediately putting the domesticated earthworms into an incubator filled with ice cubes, and cooling for 5 min to reduce the mobility of the earthworms; then placing the earthworms into 30wt.% ethanol solution at the temperature of 0 ℃ for anesthesia treatment until the earthworms do not twist any more, taking out the earthworms, cleaning the earthworms by using sterile water, removing viscous tissues on the surfaces of the earthworms, and sucking water on the surfaces of the earthworms; fixing the head of the treated earthworm on an operation wax tray by using a sterilization tack, wherein the belly of the earthworm is upward; cutting the abdominal intestinal tract of the earthworm to the anus of the earthworm from a position 0.5 cm below the intestinal tract by using a sterilization surgical blade, fixing the cut intestinal tissue epidermis outwards, removing the content of the intestinal tract by using a small spoon, scraping the intestinal wall flora by using the blade, and collecting the intestinal wall flora in a 5mL centrifuge tube.
Preferably, the extraction process time of each earthworm intestinal flora is not more than 3 min.
The working principle of the invention is as follows: (1) the earthworms are one of the organisms with the largest soil biomass, and the intestinal environment of the earthworms participates in various soil biochemical reactions, so that the ecological environment of the soil is directly influenced. (2) The dosage of the earthworm intestinal flora is designed by referring to the biomass of the earthworms in the natural soil environment and the content of the intestinal flora in the normal adult earthworm intestinal canal. (3) In the process of domesticating adult earthworms by using low-concentration pesticide soil, the intestinal environment is gradually stabilized to form a microbial community with a certain structural composition. The intestinal environment screens microorganisms taken into soil under the stimulation of pesticides, and microorganisms with the effect of resisting pesticide toxicity selectively colonize in the intestinal tract, so that the earthworm intestinal tract becomes a reactor for producing pesticide degrading bacteria. (4) The extracted intestinal flora of the earthworms mostly exists in natural soil environment, can adapt to the soil environment and can grow and reproduce normally. (5) The addition of the earthworm intestinal domestication flora increases the biomass of soil microorganisms, improves the proportion of pesticide degrading bacteria and accelerates the degradation of soil pesticides.
Has the advantages that: (1) the targeted flora domesticated by the earthworm intestinal reactor is a green environment-friendly repair material, and compared with other chemical repair technologies needing to add repair reagents and catalysts, the secondary environmental pollution and potential environmental risks do not exist; compared with the physical repair technology, the method saves more energy and equipment consumption. (2) The method improves soil quality and improves soil environment self-recovery capability. (3) The earthworm intestinal reactor domesticates the addition of the target flora, stimulates the generation of pesticide degrading bacteria in the soil, improves the capability of the indigenous flora in resisting pesticide toxicity and the degradation of soil pesticides, and accelerates the recovery of the ecological environment of the soil.
Drawings
FIG. 1 is a schematic diagram of a process for rapidly degrading cypermethrin in soil by stimulating indigenous flora with earthworm intestinal domestication target flora;
FIG. 2 is a structural composition diagram of the earthworm intestinal reactor domesticating the target flora in the soil polluted by cypermethrin with different concentrations;
FIG. 3 is a diagram showing the effect of remediation of cypermethrin contaminated soil by target flora, indigenous flora and target flora-indigenous flora under different target flora addition ratios;
FIG. 4 is a graph showing the effect of targeted flora-indigenous flora on remediation of cypermethrin-contaminated soil under different remediation time conditions.
Detailed Description
The following detailed description does not limit the technical solutions of the present invention in any way, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the scope of the present invention. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 structural composition analysis of earthworm intestinal reactor domestication target flora in cypermethrin-contaminated soil with different concentrations
Selecting healthy adult Megalobus wilsonii with the weight of 3-5 g, cleaning with sterile water, placing on wet filter paper in a lightproof dark box for gut cleaning for 12h, and cleaning the earthworms after the gut is completely cleaned again for standby. The purchased cypermethrin is mixed into a proper amount of diatomite, and clean soil is added to prepare the cypermethrin-polluted soil with the concentration of 0.5, 5 and 20 mg/kg. Three experimental groups of cypermethrin-polluted soil with the concentration of 50 kg are set according to the condition that one earthworm is put in every 50 g of soil, and the concentration of the three experimental groups is 0.5 mg/kg, 5mg/kg and 20 mg/kg in sequence. The experiment was carried out in a dark greenhouse, the indoor temperature was kept at about 25 ℃ and the domestication experiment was carried out for 20 days. After the domestication is finished, the earthworms in different treatment groups are collected, washed by sterile water and immediately placed into an incubator filled with ice cakes to be cooled for 5 min, so that the activity of the earthworms is reduced. And then placing the earthworms into 30% ethanol solution at the temperature of 0 ℃ for anesthesia treatment until the earthworms are not twisted any more, taking out the earthworms, cleaning the earthworms for three times by using sterile water, removing viscous tissues on the surfaces of the earthworms, and slightly sucking water on the surfaces of the earthworms by using sterile cotton cloth. The head of the dry clean earthworm is fixed on an operation wax tray by using a sterilization tack, and the belly of the dry clean earthworm is upward. Cutting the abdominal intestinal tract of the earthworm to the anus of the earthworm from a position 0.5 cm below the intestinal tract by using a sterile surgical blade, fixing the cut intestinal tissue epidermis outwards, removing the content of the intestinal tract by using a spoon, scraping intestinal wall tissues by using a blade, collecting the intestinal wall tissues in a 5mL centrifuge tube, adding 0.5mL PBS buffer solution, and immediately putting the mixture into a refrigerator at the temperature of 80 ℃ below zero for refrigeration. And (3) carrying out high-throughput sequencing on the collected earthworm intestinal wall tissues to obtain the microbial structure composition information.
Sequencing results showed that the structure of the targeted flora domesticated by the earthworm intestinal reactor in the soil polluted by 0.5, 5 and 20 mg/kg of cypermethrin is similar, and cuprivatus (7.9%), Microvirga (3.9%), Gaiella (2.3%), Gemmata (2.1%) and tubebacillus (1.4%) are dominant genera, and Proteobacteria (34.2%) Planctomycetes (17.9%), thaumachasaeota (11.3%), Actinobacteria (11.3%), Firmicutes (8.3%), Chloroflexi (4.3%), bactoidites (4.2%), rokrakaactera (2.4%), aridobia (2.2%) and Nitrospirae (0.9%) are dominant phyla (fig. 2). But the abundance distribution of microorganisms at the level of the genus is slightly different, and the dominant genera of the earthworm intestinal reactor domestication target flora in the soil polluted by the cypermethrin at 0.5 mg/kg are mainly Cupriavidus (9.9%), Microvirga (3.6%) and Gaiella (2.6%); the dominant genera of the earthworm intestinal reactor domesticating the target flora in the soil polluted by 5mg/kg of cypermethrin are mainly Cupriavidus (10.8%), Microvirga (2.6%) and Gemmata (2.4%); the dominant genera of the earthworm intestinal reactor domesticating the target flora in the cypermethrin-polluted soil of 20 mg/kg are mainly Cupriavidus (4.1%), Microvirga (4.0%) and Gaiella (2.9%); most of the microorganisms domesticated by the earthworm intestinal reactor have the dehalogenation function, and part of the microorganisms can carry out life metabolism by taking organic pesticides as carbon sources. In addition, the diversity and coverage of the target flora show a descending trend along with the increase of the pesticide concentration, for example, the Simpson index and the Goods _ coverage of the target flora domesticated by the earthworm intestinal reactor in the soil polluted by the cypermethrin of 0.5, 5 and 20 mg/kg are 0.9854, 0.9812, 0.9777 and 0.9954, 0.9946 and 0.9922 in sequence; the uniformity (Pielou _ e) of the target flora domesticated by the earthworm intestinal reactor in the soil polluted by the cypermethrin at 0.5 mg/kg is 0.7882 (figure 2) at most. Experimental results show that in the cypermethrin soil with different concentrations, microorganisms domesticated by the earthworm intestinal reactor have the potential of degrading cypermethrin, and the domestication influence of pesticide stress degree on the targeted flora is not obvious.
Example 2 remediation of different concentrations of cypermethrin contaminated soil by targeting flora, indigenous flora and targeting flora-indigenous flora
Selecting healthy adult Megalobus wilsonii with the weight of 3-5 g, cleaning with sterile water, placing on wet filter paper in a lightproof dark box for gut cleaning for 12h, and cleaning the earthworms after the gut is completely cleaned again for standby. The purchased analytical pure standard cypermethrin is mixed into a proper amount of diatomite, and clean soil is added to prepare cypermethrin-polluted soil with the concentration of 0.5, 5 and 20 mg/kg. Three experimental groups of cypermethrin-polluted soil with the concentration of 50 kg are set according to the condition that one earthworm is put in every 50 g of soil, and the concentration of the three experimental groups is 0.5 mg/kg, 5mg/kg and 20 mg/kg in sequence. The experiment was carried out in a dark greenhouse, the indoor temperature was kept at about 25 ℃ and the domestication experiment was carried out for 20 days. After the domestication is finished, the earthworms in different treatment groups are collected, washed by sterile water and immediately placed into an incubator filled with ice cakes to be cooled for 5 min, so that the activity of the earthworms is reduced. And then placing the earthworms into 30% ethanol solution at the temperature of 0 ℃ for anesthesia treatment until the earthworms are not twisted any more, taking out the earthworms, cleaning the earthworms for three times by using sterile water, removing viscous tissues on the surfaces of the earthworms, and slightly sucking water on the surfaces of the earthworms by using sterile cotton cloth. The head of the dry clean earthworm is fixed on an operation wax tray by using a sterilization tack, and the belly of the dry clean earthworm is upward. Cutting the abdominal intestinal tract of the earthworm to the anus of the earthworm from a position 0.5 cm below the intestinal tract by using a sterile surgical blade, fixing the cut intestinal tissue epidermis outwards, removing the content of the intestinal tract by using a spoon, scraping intestinal wall tissues by using a blade, collecting the intestinal wall tissues in a 5mL centrifuge tube, adding 0.5mL PBS buffer solution, and immediately putting the mixture into a refrigerator at the temperature of 80 ℃ below zero for refrigeration. According to the analysis of the bacterial flora structure composition of example 1, collected intestinal wall tissues were placed in LB medium (950 mL of deionized water supplemented with tryptone 10g, yeast extract 5g, NaCl 10g and agar 15g, sterilized in an autoclave for 12 h) to grow colonies on a petri dish, and then transferred to a liquid medium for culture until logarithmic phase, and the OD value was measured using an ultraviolet spectrophotometer. And (4) estimating the concentration of the bacteria according to the obtained OD value, and mixing the cultured bacteria liquid with 0.5mL of PBS buffer solution to obtain the target bacteria liquid.
The soil for experiments is taken from a waste site of a cypermethrin production enterprise in Jiangyun city of Jiangsu province, 10-20 cm of surface soil of the site is collected by a chessboard method, and the concentration of the pesticide is measured. Selecting clean soil without pesticide pollution, preparing cypermethrin-polluted soil with the concentration of 20, 100 and 500 mg/kg in a laboratory, and sequentially marking as T1, T2 and T3 concentration treatment groups. (1) The degradation effect of the targeted flora on cypermethrin in the soil under the condition of different addition ratios of the targeted flora. And (3) taking the soil samples in the T1, T2 and T3 concentration treatment groups, sterilizing for 90 min at high temperature (120 ℃) in a constant-temperature drying box, and adding water until the water content of the soil reaches 20%. The target flora and the soil are sequentially added into T1, T2 and T3 sterilized soil according to the mass ratio of 1:8000, 1:6000, 1:4000 and 1: 2000. (2) The degradation effect of indigenous flora in the cypermethrin-polluted soil with different concentrations on the cypermethrin. And (3) taking the soil samples in the T1, T2 and T3 concentration treatment groups, and adding water until the water content of the soil reaches 20%. (3) And taking the soil samples in the T1, T2 and T3 concentration treatment groups, sequentially adding the targeted floras with the mass ratio of the targeted floras to the soil of 1:8000, 1:6000, 1:4000 and 1:2000, and adding water until the water content of the soil reaches 20%. The target flora with the target flora to soil mass ratio of 1:8000, 1:6000, 1:4000 and 1:2000 is added to the treatment groups with the concentrations of T1, T2 and T3, and is marked as T1_ C1, T1_ C2, T1_ C3, T1_ C4, T2_ C1, T2_ C2, T2_ C3, T2_ C4, T3_ C1, T3_ C2, T3_ C3 and T3_ C4, wherein C1, C2, C3 and C4 respectively indicate that the mass ratio of the target flora to the soil mass ratio of 1:8000, 1:6000, 1:4000 and 1: 2000. The experiment is carried out in a constant temperature greenhouse, the temperature is 25 ℃, and the whole experiment process is protected from light. The culture period of the three experiments is 20 days, the soil weight of each treatment group is 5 kg, three parallel treatment groups are arranged, and the three treatment groups are weighed and added with water every day to control the soil water content to be about 20%. And (4) measuring the concentration of the cypermethrin in the soil after the test period, and calculating the degradation rate of the pesticide.
The method for detecting the physicochemical properties of the clean soil in the waste site of a cypermethrin production enterprise in Jiangyun city of Jiangsu province comprises the following steps: the organic matter content is 30.3 g/kg, the total nitrogen content is 1.8 g/kg, NO3 -The content of NH is 5.5mg/kg4 +The content is 1.4 mg/kg, the content of alkaline hydrolysis nitrogen is 62.1 mg/kg, the content of available phosphorus is 16.1 mg/kg, the CEC content is 15.6 cmol/kg, the pH value is 6.8, and the water content is 20.0%. Before and after the experiment, the physical and chemical properties of the soil have no obvious change. After the experimental period, in the T1 treatment group, the degradation rate of the target flora to cypermethrin in soil is 33.6%, 41.4%, 57.2% and 57.5% in sequence under the condition that the addition proportion of the target flora is C1, C2, C3 and C4; in the T2 treatment group, the degradation rate of the target flora to cypermethrin in soil is 40.7%, 51.2%, 53.6% and 53.5% in sequence under the condition that the addition proportion of the target flora is C1, C2, C3 and C4; in the T3 treatment group, the degradation rate of the target flora to cypermethrin in soil is 30.7%, 37.6%, 49.3% and 49.6% in sequence under the condition that the addition proportion of the target flora is C1, C2, C3 and C4. In the T1, T2 and T3 treatment groups, the degradation rate of the indigenous flora to the cypermethrin in the soil is 59.7%, 76.1%, 73.6% and 51.7% in sequence. Target flora-indigenous flora in the T1 treatment groupUnder the condition that the addition proportion of the flora is C1, C2, C3 and C4, the degradation rate of the cypermethrin in the soil is 48.2%, 72.3%, 90.7% and 92.36% in sequence; in the T2 treatment group, the degradation rates of the target flora-indigenous flora to cypermethrin in soil are 61.0%, 79.9%, 84.2% and 85.1% in sequence under the condition that the addition proportions of the target flora and the indigenous flora are C1, C2, C3 and C4; in the T3 treatment group, the degradation rates of cypermethrin in soil were 43.0%, 55.4%, 76.5% and 77.23% in the order of C1, C2, C3 and C4 (fig. 3). Overall, the degrading effect of the target flora-indigenous flora on cypermethrin>Indigenous flora>The target flora shows that the addition of the target flora improves the degradation rate of the indigenous flora to the cypermethrin in the soil; the higher the concentration of the cypermethrin, the poorer the repairing effect is, and the higher the concentration of the cypermethrin is, the activity of soil microorganisms is possibly reduced; the higher the addition proportion of the targeting flora is, the better the repairing effect is. Under the condition of addition ratio of C3 and C4, the repairing effect is not greatly different (figure 3), and C3 (1: 4000) is selected as the optimal addition ratio of the targeting flora for saving experimental energy consumption. The experimental result shows that the targeted flora domesticated by the intestinal tracts of the earthworms can effectively promote the indigenous flora to quickly degrade the cypermethrin in the soil, particularly the repairing effect is optimal under the condition that the adding proportion is C3, and the technology can be used as an effective microbial repairing scheme for the pesticide-polluted soil.
Example 3 Effect of the soil cultivation period on the remediation of different concentrations of Cypermethrin contaminated soil by the target flora-indigenous flora
Selecting healthy adult Megalobus wilsonii with the weight of 3-5 g, cleaning with sterile water, placing on wet filter paper in a lightproof dark box for gut cleaning for 12h, and cleaning the earthworms after the gut is completely cleaned again for standby. The purchased analytical pure standard cypermethrin is mixed into a proper amount of diatomite, and clean soil is added to prepare cypermethrin-polluted soil with the concentration of 0.5, 5 and 20 mg/kg. Three experimental groups of cypermethrin-polluted soil with the concentration of 50 kg are set according to the condition that one earthworm is put in every 50 g of soil, and the concentration of the three experimental groups is 0.5 mg/kg, 5mg/kg and 20 mg/kg in sequence. The experiment was carried out in a dark greenhouse, the indoor temperature was kept at about 25 ℃ and the domestication experiment was carried out for 20 days. After the domestication is finished, the earthworms in different treatment groups are collected, washed by sterile water and immediately placed into an incubator filled with ice cakes to be cooled for 5 min, so that the activity of the earthworms is reduced. And then placing the earthworms into 30% ethanol solution at the temperature of 0 ℃ for anesthesia treatment until the earthworms are not twisted any more, taking out the earthworms, cleaning the earthworms for three times by using sterile water, removing viscous tissues on the surfaces of the earthworms, and slightly sucking water on the surfaces of the earthworms by using sterile cotton cloth. The head of the dry clean earthworm is fixed on an operation wax tray by using a sterilization tack, and the belly of the dry clean earthworm is upward. Cutting the abdominal intestinal tract of the earthworm to the anus of the earthworm from a position 0.5 cm below the intestinal tract by using a sterile surgical blade, fixing the cut intestinal tissue epidermis outwards, removing the content of the intestinal tract by using a spoon, scraping intestinal wall tissues by using a blade, collecting the intestinal wall tissues in a 5mL centrifuge tube, adding 0.5mL PBS buffer solution, and immediately putting the mixture into a refrigerator at the temperature of 80 ℃ below zero for refrigeration. According to the analysis of the bacterial flora structure composition of example 1, collected intestinal wall tissues were placed in LB medium (950 mL of deionized water supplemented with tryptone 10g, yeast extract 5g, NaCl 10g and agar 15g, sterilized in an autoclave for 12 h) to grow colonies on a petri dish, and then transferred to a liquid medium for culture until logarithmic phase, and the OD value was measured using an ultraviolet spectrophotometer. And (4) estimating the concentration of the bacteria according to the obtained OD value, and mixing the cultured bacteria liquid with 0.5mL of PBS buffer solution to obtain the target bacteria liquid.
Based on example 2, the optimal repair time of the target flora-indigenous flora to the cypermethrin-polluted soil with different concentrations is discussed by selecting the mass ratio of the target flora to the soil as 1: 4000. Taking 5 kg of soil samples in the T1, T2 and T3 concentration treatment groups, adding the targeting flora with the mass ratio of 1:4000 to the soil, placing in a constant temperature greenhouse at the temperature of 25 ℃, and keeping dark with black cloth. Three parallel treatment groups are respectively arranged on the T1, T2 and T3 concentration polluted soil, and the soil is weighed and added with water every day to control the water content of the soil to be about 20 percent. And (3) detecting the concentration of the cypermethrin in the soil of different treatment groups on 7 th, 14 th, 30 th and 60 th days in the experimental period, and calculating the degradation rate of the pesticide.
The experimental result shows that: in the T1 treatment group, the degradation rates of the target flora-indigenous flora to cypermethrin in soil on 7 th, 14 th, 30 th and 60 th days are 48.9%, 60.7%, 92.9% and 93.7% in sequence; in the T2 treatment group, the degradation rates of the target flora-indigenous flora to cypermethrin in soil on 7 th, 14 th, 30 th and 60 th days are 43.7%, 54.2%, 94.3% and 96.1% in sequence; in the T3-treated group, the target flora-indigenous flora degraded cypermethrin in the soil at days 7, 14, 30 and 60 in the order of 40.4%, 56.5%, 78.6% and 79.2% (fig. 4). In addition, Simpson index, Goods _ coverage index, Pielou _ e index of the microorganisms in the T1, T2, T3 treatment groups were 0.9823 and 0.9832, 0.9931 and 0.9944, 0.7718 and 0.7725, respectively, on days 0 and 60 of the experiment; the addition of the targeting flora improves the diversity, coverage and uniformity of soil microorganisms and enhances the stability of the soil ecological environment. On the whole, the degradation rate of the target flora-indigenous flora to the cypermethrin in the soil is higher along with the increase of the soil culture time; the higher the concentration of the cypermethrin, the lower the degradation rate of the targeted flora-indigenous flora to the cypermethrin in the soil; the repairing effect of 30 days and 60 days is not very different, and the best repairing period of the experiment is 30 days by considering the time cost factor. The experimental result shows that the addition of the target flora domesticated by the earthworm intestinal reactor effectively promotes the indigenous flora to rapidly degrade the cypermethrin in the soil, and the technology can be used as an effective microbial remediation scheme for the pesticide-contaminated soil.
Claims (4)
1. A method for stimulating indigenous flora to rapidly degrade cypermethrin in soil is characterized by comprising the following steps: selecting the grown earthworm living bodies domesticated with clean soil, cleaning, and placing in a dark box for clearing intestines; the earthworms after the gut purging treatment are put into cypermethrin-polluted soil with the concentration of 0.5-20 mg/kg for domestication for 20 days; taking out the earthworms after the culture, cleaning the earthworms, dissecting the intestinal tracts, removing the content in the intestinal tracts, and scraping the intestinal wall flora; adding the earthworm intestinal flora into the soil to be repaired according to the mass ratio of the put earthworm intestinal flora to the soil of 1:2000-1:8000, and repairing for 7-60 days.
2. The method for stimulating the indigenous flora to rapidly degrade cypermethrin in the soil as claimed in claim 1, wherein the earthworm species is Megascoleus williamsii.
3. The method for stimulating indigenous flora to rapidly degrade cypermethrin in soil as claimed in claim 1, wherein the domesticated earthworms are cleaned with sterile water, and immediately placed into an incubator filled with ice cubes to be cooled for 5 min so as to reduce the mobility of the earthworms; then placing the earthworms into 30wt.% ethanol solution at the temperature of 0 ℃ for anesthesia treatment until the earthworms do not twist any more, taking out the earthworms, cleaning the earthworms by using sterile water, removing viscous tissues on the surfaces of the earthworms, and sucking water on the surfaces of the earthworms; fixing the head of the treated earthworm on an operation wax tray by using a sterilization tack, wherein the belly of the earthworm is upward; cutting the abdominal intestinal tract of the earthworm to the anus of the earthworm from a position 0.5 cm below the intestinal tract by using a sterilization surgical blade, fixing the cut intestinal tissue epidermis outwards, removing the content of the intestinal tract by using a small spoon, scraping the intestinal wall flora by using the blade, and collecting the intestinal wall flora in a 5mL centrifuge tube.
4. The method for stimulating indigenous flora to rapidly degrade cypermethrin in soil according to claim 1, wherein the extraction process time of each earthworm intestinal flora is not more than 3 min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111893061A (en) * | 2020-07-20 | 2020-11-06 | 华南农业大学 | Pyrethrin insecticide degradation strain, fungicide and degradation process thereof |
| CN114522975A (en) * | 2022-02-24 | 2022-05-24 | 农业农村部环境保护科研监测所 | Application of earthworm foregut and/or midgut contents in repairing beta-lactam antibiotics and/or drug-resistant gene contaminated soil |
| CN115141764A (en) * | 2022-05-20 | 2022-10-04 | 四川师范大学 | Bacillus cereus microbial inoculum and application thereof in eliminating cypermethrin in animal bodies |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101823074A (en) * | 2010-04-09 | 2010-09-08 | 华南农业大学 | Method for remedying soil contaminated by DDT as pesticide residue through earthworm-based bioaugmentation and biodegradation |
| JP2013144271A (en) * | 2012-01-13 | 2013-07-25 | Japan Research Institute Ltd | Soil improving method |
| CN106434452A (en) * | 2016-10-08 | 2017-02-22 | 西华大学 | Preparation method of cypermethrin degrading bacterium powder and application of cypermethrin degrading bacterium powder in soil |
| CN110025919A (en) * | 2019-04-15 | 2019-07-19 | 陕西省微生物研究所 | Pesticide cypermethrin microbial inoculum for degrading and preparation method thereof |
| CN111069275A (en) * | 2019-12-31 | 2020-04-28 | 南京农业大学 | Method for reinforcing reduction of sulfonamide antibiotics in soil by earthworm intestinal contents |
| CN111069276A (en) * | 2019-12-31 | 2020-04-28 | 南京农业大学 | Method for enhancing tetracycline antibiotic reduction in soil by earthworm intestinal content |
-
2021
- 2021-04-20 CN CN202110425930.1A patent/CN113231462B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101823074A (en) * | 2010-04-09 | 2010-09-08 | 华南农业大学 | Method for remedying soil contaminated by DDT as pesticide residue through earthworm-based bioaugmentation and biodegradation |
| JP2013144271A (en) * | 2012-01-13 | 2013-07-25 | Japan Research Institute Ltd | Soil improving method |
| CN106434452A (en) * | 2016-10-08 | 2017-02-22 | 西华大学 | Preparation method of cypermethrin degrading bacterium powder and application of cypermethrin degrading bacterium powder in soil |
| CN110025919A (en) * | 2019-04-15 | 2019-07-19 | 陕西省微生物研究所 | Pesticide cypermethrin microbial inoculum for degrading and preparation method thereof |
| CN111069275A (en) * | 2019-12-31 | 2020-04-28 | 南京农业大学 | Method for reinforcing reduction of sulfonamide antibiotics in soil by earthworm intestinal contents |
| CN111069276A (en) * | 2019-12-31 | 2020-04-28 | 南京农业大学 | Method for enhancing tetracycline antibiotic reduction in soil by earthworm intestinal content |
Non-Patent Citations (4)
| Title |
|---|
| 开建荣等: "赤子爱胜蚓对氯氰菊酯暴露的生态毒性响应", 《农业环境科学学报》 * |
| 晁会珍 等: "蚯蚓肠道细菌生态功能及毒理学研究进展", 《生态毒理学报》 * |
| 熊张平 等: "不同污染方式进入土壤的氯氰菊酯在蚯蚓体内的蓄积特征及其生长毒性", 《生态毒理学报》 * |
| 赵丽倩等: "亚致死剂量高效氯氰菊酯对蚯蚓体内生化指标的影响", 《生态科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111893061A (en) * | 2020-07-20 | 2020-11-06 | 华南农业大学 | Pyrethrin insecticide degradation strain, fungicide and degradation process thereof |
| CN111893061B (en) * | 2020-07-20 | 2023-01-06 | 华南农业大学 | Pyrethrin insecticide degradation strain, bactericide thereof and degradation process |
| CN114522975A (en) * | 2022-02-24 | 2022-05-24 | 农业农村部环境保护科研监测所 | Application of earthworm foregut and/or midgut contents in repairing beta-lactam antibiotics and/or drug-resistant gene contaminated soil |
| CN115141764A (en) * | 2022-05-20 | 2022-10-04 | 四川师范大学 | Bacillus cereus microbial inoculum and application thereof in eliminating cypermethrin in animal bodies |
| CN115141764B (en) * | 2022-05-20 | 2023-06-20 | 四川师范大学 | Bacillus cereus microbial inoculum and application thereof in elimination of cypermethrin in animal body |
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