CN113231462B - A method for stimulating indigenous flora to rapidly degrade cypermethrin in soil - Google Patents

Abstract

A method for stimulating indigenous flora to rapidly degrade cypermethrin in soil. The living adult earthworms after cleaning the soil are selected, washed and placed in a dark box to clean the intestines; the earthworms after the cleaning process are put into a preparation concentration of 0.5-20 mg/kg In the cypermethrin-contaminated soil, domestication was carried out for 20 days; after taking out the culture, the earthworms were washed, the intestinal tract was dissected to remove the intestinal contents, and the intestinal wall flora was scraped; according to the mass ratio of the earthworm intestinal flora to the soil, 1:2000‑1:8000, add earthworm intestinal flora to the soil to be repaired for a period of 7‑60 days. Under the stress of cypermethrin, the targeted flora after domestication in the earthworm intestinal reactor was rich in cypermethrin pesticide-degrading microorganisms, and it was added to the cypermethrin-contaminated soil to promote the rapid degradation of cypermethrin by the indigenous flora, and the degradation rate could reach 96.1%. In order to maintain the safety of soil ecological environment.

Description

A method for stimulating indigenous flora to rapidly degrade cypermethrin in soil

technical field

The invention belongs to the technical field of pesticide-contaminated soil bioremediation, in particular to a method for cultivating targeted flora in an earthworm intestinal reactor to stimulate indigenous flora to rapidly degrade cypermethrin in soil.

Background technique

As a major country in the production and use of pesticides, my country's pesticide production reached 3.78 million tons in 2016, accounting for more than 1/3 of the world's total. In 2018, the use of pesticides reached 1.504 million tons. These pesticides have brought serious soil environment during production, transportation and use. question. As one of the most common pesticides in modern times, cypermethrin is ubiquitous in the soil environment and has a high detection rate. The main non-point source of pollution is agricultural soil. Cypermethrin is mainly used to kill pests in farmland, cultivated land, forest land and other soils. Due to a large amount and excessive use, the concentration of cypermethrin in some surface soils can reach more than 3000 mg/kg. Cypermethrin is a highly toxic organochlorine pesticide. On the one hand, the residual cypermethrin in the soil poisons soil organisms and destroys the soil ecological environment; , seriously endangering the ecological environment of the region, and there is a greater environmental risk. Therefore, it is very necessary to carry out the remediation of cypermethrin-contaminated soil.

Earthworm is one of the soil animals with the largest biomass in the soil environment, and its intestinal tract, as the main activity site, participates in various metabolic processes in the soil environment, which is of great significance to maintain the safety of the soil environment. Soil and earthworm gut are two completely different microbial living environments, colonized with microbial communities of different phenotypes and genotypes, forming two specific ecological function taxa of indigenous flora and earthworm gut flora. Indigenous flora plays an indispensable role at different trophic levels in the process of nutrient cycling, organic carbon fixation, bioaccumulation and degradation of toxic and harmful substances (pesticides) in the soil. At the same time, the intestinal flora of earthworms ingests the indigenous flora by swallowing the soil, operates and ferments through the intestinal tract, and forms ecologically specific intestinal flora. members of the group. In addition, the earthworm gut has a screening effect on the microorganisms ingested in the soil. The earthworm gut contains more heavy metal-reducing bacteria and pesticide-degrading bacteria in heavy metal and pesticide-contaminated soil, indicating that the stimulation of soil pollutants can increase the beneficial effects of the earthworm gut. Colonization of microorganisms. Therefore, as a natural bioreactor, the earthworm intestine promotes the growth and reproduction of functional bacteria, and then transports these functional bacteria into the soil to participate in the process of the indigenous flora degrading pesticides in the soil and accelerate the reduction of pollutants. It is of great significance for the ecological restoration of the soil environment to stimulate the indigenous flora to rapidly degrade soil pollutants by domesticating targeted flora in the earthworm intestinal reactor.

At present, for the remediation of cypermethrin-contaminated soil, the microbial enhanced remediation of contaminated soil is mainly carried out by directly adding microbial groups with the function of degrading cypermethrin into the contaminated soil. After searching the patent, the strains with the function of degrading cypermethrin include Acinetobacterlwoffii strain JX-2, Acinetobacter baumannii strain ZH-14, Pseudomonas fulva strain P31, Bacillus thuringiensis Bt-1, Eurotiumcristatum strain ET1, Acinetobacter sp. strain JCX22D, Pseudomonas nitroreduction CW-7, sphingosine mono Sphingomonas trueperi strain CW3, Bacillus megalosporus strain HLJ7, Leclercia adecarboxylata strain Y4, Stenotrophomonas acidaminiphila strain ZH-01, lysine Bacillus (Lysinibacillus) strain JX-5, Bacillus licheniformis (Bacillus licheniformis), Bacillus cereus (Bacillus cereus) strain BBCP-017 and Acinetobacter (Acinetobacter sp.) strain JCX22D and so on. This type of microbial enhanced remediation technology has high requirements for microbial culture technology, and the strain culture process is complex, time-consuming and costly. In addition, different strains have inconsistent requirements for the environmental conditions of soil remediation, and a large number of experiments are needed to screen suitable bacteria for different soil conditions. , consumes more energy and labor. In addition to microbial enhanced remediation technology, the remediation of cypermethrin-contaminated soil also includes genetic engineering technology. For example, in CN200910241899.5 and CN200410042712.6, genes encoding detoxification esterase and hydrolase are introduced into plasmids to obtain engineering bacteria to repair cypermethrin-contaminated soil. The method of preparing engineered bacteria requires too much genetic engineering technology and experimental operation ability, which is difficult to realize under general laboratory conditions. In addition, formulating a composite ecological restoration agent is also a common cypermethrin restoration technology. For example, CN201710776995.4 and CN201710777042.X, mix 6 kinds of materials, and reduce cypermethrin in soil by exploring the best mixing ratio. However, the preparation process of the composite ecological restoration agent is complicated and the cost is high, and some materials will adversely affect soil organisms during the restoration process and damage the soil ecological security. CN201410136171.7, using corn stover to prepare biochar, and exploring the optimal application ratio to restore cypermethrin-contaminated soil. The preparation of biochar consumes a lot of energy, and the preparation process also causes environmental pollution, which is not only costly but also has secondary pollution. Therefore, the remediation of cypermethrin-contaminated soil lacks low-cost, low-energy-consumption, high-efficiency, green, and targeted remediation technologies.

By searching for patents on earthworms and soil pollutants degradation through earthworm intestinal reactors, it is found that most of earthworms and vermicompost are involved in the bioremediation of soil heavy metals, and very few earthworms are added to degrade pesticide pollutants in soil. Enteric reactors are involved in the degradation of cypermethrin pesticides in soil. The ones closest to the research direction of the present invention are the application numbers CN201911418253.X and CN201911409957.0. The earthworms cultivated in the laboratory are dissected to obtain the intestinal contents of fresh earthworms, which are sequentially added to different types of tetracycline and sulfonamide antibiotics in contaminated soil. , to study the ability of earthworm gut contents to reduce soil antibiotics. However, most of the microorganisms in the intestinal contents of earthworms extracted by this method are directly obtained by earthworms through eating, and the source of microorganisms is mainly soil microorganisms, rather than being completely screened and enriched by earthworm intestinal reactors to form microbes with specific functions. The microbial groups did not achieve the effect of bio-enhanced stimulation and repair, and also did not highlight the colonization and screening effect of the intestinal environment of earthworms on functional flora.

The main defects of the prior art are: the existing cypermethrin-contaminated soil remediation technologies are relatively simple, and most of them use degrading functional bacteria and engineering bacteria for remediation, but the remediation scope of this technology is limited, and the remediation soil environmental conditions are relatively high. Moreover, the discovery of degrading functional bacteria and the preparation of engineered bacteria are time-consuming and labor-intensive, which is not conducive to the rapid implementation and application of microbial remediation of contaminated soil; the preparation process of mixed remediation materials is cumbersome and costly, causing potential harm to soil organisms. Therefore, there is a lack of green, low-cost and easy-to-operate cypermethrin-contaminated soil remediation technologies.

The main reasons for the defects are: (1) In the early days, my country produced and used cypermethrin in large quantities. On the one hand, a large amount of cypermethrin remains in agricultural soil during the large-scale and unreasonable use of agriculture, which endangers farmland organisms, farmland ecological environment and human health; Groundwater and rock crevices make restoration work more difficult. (2) Cypermethrin is an organochlorine pesticide, which is highly toxic, poisoning and killing non-target organisms in the soil. (3) At present, the remediation of such pesticide-contaminated soil is mainly carried out by adding degrading bacteria and engineering bacteria, but the remediation process requires high biotechnology and operational capabilities, and the cost is high, which is difficult to achieve in general laboratories. . In addition, the added degrading functional bacteria and engineering bacteria are relatively simple, and the applicable conditions for different soil environmental conditions are not clear, and further verification is required. (4) The restoration of pesticide-contaminated soil lacks simple and efficient restoration techniques and standardized restoration techniques. Therefore, exploring green, efficient, targeted and standardized microbial remediation technologies is of great significance for improving the ecological environment of pesticide-contaminated soils.

SUMMARY OF THE INVENTION

Technical problem to be solved: The present invention provides a method for stimulating indigenous flora to rapidly degrade cypermethrin in soil, the method can effectively accelerate the degradation of cypermethrin by indigenous flora, reduce the use and repair cost of soil pollution remediation materials, and reduce the impact of remediation reagents and catalysts on cypermethrin. The disturbance of the soil environment can improve the soil quality and the self-recovery ability of the ecological environment.

Technical scheme: a method for stimulating indigenous flora to rapidly degrade cypermethrin in soil, the steps include: selecting live adult earthworms after cleaning the soil and domesticating them, washing them, and placing them in a dark box to clean the intestines; 0.5-20 mg/kg of cypermethrin-contaminated soil was carried out for 20 days of domestication; after taking out the culture, the earthworms were washed, the intestines were dissected, the intestinal contents were removed, and the intestinal wall flora was scraped; The mass ratio to the soil is 1:2000-1:8000, and earthworm intestinal flora is added to the soil to be repaired for a period of 7-60 days.

Preferably, the above-mentioned earthworm species is William Coeliac.

Preferably, the above-mentioned method for stimulating indigenous flora to rapidly degrade cypermethrin in soil is to take domesticated earthworms, wash them with sterile water, and immediately put them into an ice-filled incubator to cool for 5 minutes to reduce the activity of earthworms; Put it into a 30wt.% ethanol solution with a temperature of 0 °C for anesthesia treatment, until the earthworms no longer twist, take out and wash with sterile water, remove the viscous tissue on the surface of the earthworms, and absorb the water on the surface of the earthworms; The head of the worm is fixed on the operating wax plate with a sterilized tack, with the abdomen facing upward; use a sterilized surgical blade to cut the abdominal intestine of the earthworm from 0.5 cm below the ring to the anus of the earthworm, and fix the opened intestine outward. Tissue epidermis, remove intestinal contents with a small spoon, scrape the intestinal wall flora with a razor blade, and collect them in a 5 mL centrifuge tube.

Preferably, the extraction process time of each earthworm intestinal flora does not exceed 3 minutes.

The working principle of the present invention is as follows: (1) Earthworm is one of the organisms with the largest soil biomass, and its intestinal environment participates in various soil biochemical reactions, which directly affects the soil ecological environment. (2) The amount of earthworm intestinal flora was designed with reference to the biomass of earthworms in the natural soil environment and the content of intestinal flora in the intestines of normal adult earthworms. (3) During the process of domestication of adult earthworms in low-concentration pesticide soil, the intestinal environment was gradually stabilized, and a microbial community with a certain structural composition was formed. Under the stimulation of pesticides, the intestinal environment screened the microorganisms ingested in the soil, and the microorganisms with the function of resisting the poisoning of pesticides were selectively colonized in the intestinal tract, making the intestinal tract of earthworms a reactor for the production of pesticide-degrading bacteria. (4) Most of the extracted earthworm intestinal flora exists in the natural soil environment, which can adapt to the soil environment and grow and reproduce normally. (5) The addition of the domesticated flora in the gut of earthworms increased the soil microbial biomass, increased the proportion of pesticide-degrading bacteria, and accelerated the degradation of soil pesticides.

Beneficial effects: (1) The targeted flora cultivated in the earthworm intestinal reactor is a green and environmentally friendly repair material. Compared with other chemical repair technologies that require the addition of repair reagents and catalysts, there is no secondary environmental pollution and potential environmental pollution. Risk; saves more energy and equipment consumption than physical restoration techniques. (2) The method improves the soil quality and enhances the self-recovery ability of the soil environment. (3) The addition of targeted flora for domestication in the earthworm intestinal reactor stimulates the generation of pesticide-degrading bacteria in the soil, improves the ability of indigenous flora to resist pesticide poisoning and the degradation of soil pesticides, and accelerates the restoration of the soil ecological environment.

Description of drawings

Figure 1 is a schematic diagram of the process of earthworm intestinal acclimation targeted flora to stimulate indigenous flora to rapidly degrade cypermethrin in soil;

Figure 2 is a structural composition diagram of the target flora for domestication of the earthworm intestinal reactor in different concentrations of cypermethrin-contaminated soil;

Figure 3 shows the effect of remediation of cypermethrin-contaminated soil under the condition of different target flora addition ratios: target flora, indigenous flora and targeted flora-indigenous flora;

Figure 4 shows the effect of targeted microbiota-indigenous microbiota in remediation of cypermethrin-contaminated soil under different remediation time conditions.

Detailed ways

The following specific embodiments do not limit the technical solutions of the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1 Structural composition analysis of target flora in earthworm intestinal reactor domestication in different concentrations of cypermethrin-contaminated soil

Select healthy adult worms weighing 3-5 g, wash them with sterile water, and place them on moist filter paper in a dark box for 12 hours of bowel cleansing. After complete bowel cleansing, the worms are washed again. spare. The purchased cypermethrin was mixed into an appropriate amount of diatomaceous earth, and the clean soil was added to prepare cypermethrin-contaminated soil with concentrations of 0.5, 5, and 20 mg/kg. With reference to putting one earthworm per 50 g of soil, three experimental groups with 50 kg concentrations of 0.5, 5, and 20 mg/kg cypermethrin-contaminated soil were set up. The experiment was carried out in a dark greenhouse, and the indoor temperature was kept at about 25 °C, and the domestication experiment was carried out for 20 days. After domestication, earthworms in different treatment groups were collected, washed with sterile water, and immediately placed in an ice-filled incubator to cool for 5 min to reduce the activity of earthworms. Then put the earthworms into a 30% ethanol solution with a temperature of 0 °C for anesthesia treatment, until the earthworms no longer twist, take them out and wash with sterile water three times to remove the viscous tissue on the surface of the earthworms, and use a sterile cotton cloth to gently Absorb the water on the surface of the earthworm. Fix the head of the dry clean earthworm on the operating wax plate with a sterilized tack, with the abdomen facing up. Use a sterilized surgical blade to cut the abdominal intestine of the earthworm from 0.5 cm below the ring to the anus of the earthworm, fix the cut intestinal tissue skin outward, remove the intestinal contents with a small spoon, and scrape the intestinal wall with a blade Tissues were collected in a 5 mL centrifuge tube, added with 0.5 mL of PBS buffer, and immediately refrigerated at -80 °C. High-throughput sequencing was performed on the collected intestinal wall tissues of earthworms to obtain information on the structural composition of microorganisms.

Sequencing results showed that the composition of the target microbiota domesticated by the earthworm intestinal reactor in 0.5, 5, and 20 mg/kg of cypermethrin-contaminated soil was similar, with Cupriavidus (7.9%), Microvirga (3.9%), and Gaiella (2.3%). , Gemmata (2.1%) and Tumebacillus (1.4%) are dominant genera, Proteobacteria (34.2%), Planctomycetes (17.9%), Thaumarchaeota (11.3%), Actinobacteria (11.3%), Firmicutes (8.3%), Chloroflexi (4.3%) , Bacteroidetes (4.2%), Rokubacteria (2.4%), Acidobacteria (2.2%) and Nitrospirae (0.9%) were the dominant phyla (Figure 2). However, the abundance distribution of microorganisms at the genus level was slightly different. The dominant genera of earthworm intestinal reactor domestication target flora in 0.5 mg/kg cypermethrin-contaminated soil were mainly Cupriavidus (9.9%), Microvirga (3.6%), Gaiella ( 2.6%); 5 mg/kg of cypermethrin-contaminated soil, the dominant genera of earthworm intestinal reactor for domestication of targeted flora are Cupriavidus (10.8%), Microvirga (2.6%), Gemata (2.4%); 20 mg/kg The dominant genera of the earthworm intestinal reactor domesticated target flora in the cypermethrin-contaminated soil are Cupriavidus (4.1%), Microvirga (4.0%), Gaiella (2.9%); most of these microorganisms domesticated by the earthworm intestinal reactor have Part of the dehalogenation function can use organic pesticides as a carbon source for life metabolism. In addition, the diversity and coverage of target flora showed a downward trend with the increase of pesticide concentration, such as the Simpson index and Goods_coverage of target flora domesticated in earthworm gut reactors in 0.5, 5, and 20 mg/kg cypermethrin-contaminated soil. The order was 0.9854, 0.9812, 0.9777, and 0.9954, 0.9946, 0.9922; the evenness of the target flora (Pielou_e) cultivated in the earthworm intestinal reactor in the 0.5 mg/kg cypermethrin-contaminated soil was the highest (Pielou_e) was 0.7882 (Fig. 2). The experimental results show that in soils with different concentrations of cypermethrin, the microorganisms domesticated by the earthworm intestinal reactor have the potential to degrade cypermethrin, and the degree of pesticide stress has no significant effect on the domestication of the target flora.

Example 2 Targeted flora, indigenous flora and targeted flora-indigenous flora remediation of cypermethrin-contaminated soil with different concentrations

Select healthy adult worms weighing 3-5 g, wash them with sterile water, and place them on moist filter paper in a dark box for 12 hours to clean the intestines. After complete intestinal cleaning, the earthworms are washed again. spare. The purchased analytically pure standard cypermethrin was mixed into an appropriate amount of diatomaceous earth, and the clean soil was added to prepare the cypermethrin-contaminated soil with concentrations of 0.5, 5, and 20 mg/kg. With reference to putting one earthworm per 50 g of soil, three experimental groups with 50 kg concentrations of 0.5, 5, and 20 mg/kg cypermethrin-contaminated soil were set up. The experiment was carried out in a dark greenhouse, and the indoor temperature was kept at about 25 °C, and the domestication experiment was carried out for a period of 20 days. After domestication, earthworms in different treatment groups were collected, washed with sterile water, and immediately placed in an incubator filled with ice cubes to cool for 5 min to reduce the activity of earthworms. Then put the earthworms into a 30% ethanol solution at a temperature of 0 °C for anesthesia treatment, until the earthworms no longer twist, take them out and wash with sterile water three times to remove the viscous tissue on the surface of the earthworms, and use a sterile cotton cloth to gently Absorb the water on the surface of the earthworm. Fix the head of the dry clean earthworm on the operating wax plate with a sterilized tack, with the abdomen facing up. Use a sterilized surgical blade to cut the abdominal intestine of the earthworm from 0.5 cm below the ring to the anus of the earthworm, fix the cut intestinal tissue skin outward, remove the intestinal contents with a small spoon, and scrape the intestinal wall with a blade The tissue was collected in a 5 mL centrifuge tube, added with 0.5 mL of PBS buffer, and immediately placed in a -80 °C refrigerator. According to the analysis of the bacterial flora structure in Example 1, the collected intestinal wall tissue was added to LB medium (950 mL of deionized water with 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 15 g of agar), and sterilized in a high-pressure steam cooker for 12 h. Reserve) to grow colonies in the petri dish, then transfer to liquid medium for culture, wait until logarithmic phase, and use UV spectrophotometer to measure its OD value. According to the obtained OD value, the concentration of bacterial cells was estimated, and the cultured bacterial solution was mixed with 0.5 mL of PBS buffer to obtain the targeted bacterial solution.

The soil used in the experiment was taken from the abandoned site of a cypermethrin production enterprise in Jiangyin City, Jiangsu Province. The 10-20 cm topsoil of the site was collected by the chessboard method to determine the pesticide concentration. The clean soils without pesticide pollution were selected, and cypermethrin-contaminated soils with concentrations of 20, 100 and 500 mg/kg were prepared in the laboratory, and recorded as T1, T2 and T3 concentration treatment groups in turn. (1) The degradation effect of targeted bacteria on cypermethrin in soil under the conditions of different target bacteria addition ratios. The soil samples from the T1, T2 and T3 concentration treatment groups were taken, and were sterilized at a high temperature (120 °C) in a constant temperature drying oven for 90 min, and water was added until the soil moisture content reached 20%. According to the ratio of target bacteria to soil mass ratio of 1:8000, 1:6000, 1:4000, 1:2000, it was added to T1, T2 and T3 sterilized soil in turn. (2) The degradation effect of cypermethrin by indigenous flora in different concentrations of cypermethrin-contaminated soil. The soil samples in the T1, T2 and T3 concentration treatment groups were taken, and water was added until the soil moisture content reached 20%. (3) Take the soil samples in the T1, T2 and T3 concentration treatment groups, add the targeted bacteria groups with the target bacteria group to soil mass ratio of 1:8000, 1:6000, 1:4000, 1:2000 in turn, add water to The soil moisture content reaches 20%. In the T1, T2 and T3 concentration treatment groups, the target flora with the ratio of target flora to soil mass of 1:8000, 1:6000, 1:4000, 1:2000 was added, which were denoted as T1_C1, T1_C2, T1_C3, T1_C4, T2_C1, T2_C2, T2_C3, T2_C4, T3_C1, T3_C2, T3_C3, T3_C4, where C1, C2, C3, and C4 indicate that the mass ratio of the target flora to soil is 1:8000, 1:6000, 1:4000, 1, respectively :2000. This experiment was carried out in a constant temperature greenhouse with a temperature of 25 °C, and the whole experiment was protected from light. The culture period of the three experiments was 20 days. The soil weight of each treatment group was 5 kg. Three parallel treatment groups were set up, and the soil moisture content was controlled at about 20% by weighing and adding water every day. After the test period, the concentration of cypermethrin in the soil was measured, and the degradation rate of the pesticide was calculated.

The physicochemical properties of the clean soil at the abandoned site of a cypermethrin production enterprise in Jiangyin City, Jiangsu Province were as follows: the organic matter content was 30.3 g/kg, the total nitrogen content was 1.8 g/kg, the NO ₃ ^- content was 5.5 mg/kg, and the NH ₄ ⁺ content was 5.5 mg/kg. It is 1.4 mg/kg, the content of alkaline hydrolyzable nitrogen is 62.1 mg/kg, the content of available phosphorus is 16.1 mg/kg, the content of CEC is 15.6 cmol/kg, the pH is 6.8, and the moisture content is 20.0%. There was no significant change in soil physical and chemical properties before and after the experiment. After the experimental period, in the T1 treatment group, the degradation rates of cypermethrin in the soil were 33.6%, 41.4%, 57.2% and 57.5%, respectively, under the conditions of adding ratios of C1, C2, C3 and C4; T2 In the treatment group, the degradation rates of cypermethrin in the soil were 40.7%, 51.2%, 53.6% and 53.5%, respectively, under the conditions of adding ratios of C1, C2, C3, and C4; in the T3 treatment group, the target bacteria The degradation rates of cypermethrin in soil were 30.7%, 37.6%, 49.3% and 49.6%, respectively, under the conditions of adding C1, C2, C3 and C4 to the bacterial flora. In the T1, T2 and T3 treatment groups, the degradation rates of cypermethrin in soil by indigenous flora were 59.7%, 76.1%, 73.6% and 51.7%, respectively. In the T1 treatment group, the degradation rates of cypermethrin in the soil were 48.2%, 72.3%, 90.7% and 92.36%, respectively, under the conditions of adding ratios of C1, C2, C3, and C4 to the targeted flora-indigenous flora; T2 In the treatment group, the degradation rates of cypermethrin in the soil were 61.0%, 79.9%, 84.2% and 85.1%, respectively, under the conditions of adding ratios of C1, C2, C3, and C4 to the target flora-indigenous flora; T3 treatment In the group, the degradation rates of cypermethrin in soil were 43.0%, 55.4%, 76.5% and 77.23% respectively under the conditions of adding ratios of C1, C2, C3 and C4 to the target flora-indigenous flora (Fig. 3). . On the whole, the degradation effect of targeted flora-indigenous flora on cypermethrin>indigenous flora>targeted flora, indicating that the addition of targeted flora improved the degradation rate of cypermethrin in soil by indigenous flora; the higher the concentration of cypermethrin , the worse the repair effect, the higher the concentration of cypermethrin may reduce the activity of soil microorganisms; the higher the addition ratio of targeted flora, the better the repair effect. Under the conditions of the addition ratio of C3 and C4, the repair effect is not much different (Figure 3). For the purpose of saving experimental energy consumption, C3 (1:4000) was selected as the best addition ratio of target bacteria. The results of this experiment show that the targeted flora cultivated in the gut of earthworms can effectively promote the rapid degradation of cypermethrin in the soil by the indigenous flora. Effective pesticide-contaminated soil microbial remediation program.

Example 3 The effect of soil culture cycle on target flora-indigenous flora in remediation of cypermethrin-contaminated soil with different concentrations

Select healthy adult worms weighing 3-5 g, wash them with sterile water, and place them on moist filter paper in a dark box for 12 hours of bowel cleansing. After complete bowel cleansing, the worms are washed again. spare. The purchased analytically pure standard cypermethrin was mixed into an appropriate amount of diatomaceous earth, and the clean soil was added to prepare the cypermethrin-contaminated soil with concentrations of 0.5, 5, and 20 mg/kg. With reference to putting one earthworm per 50 g of soil, three experimental groups with 50 kg concentrations of 0.5, 5, and 20 mg/kg cypermethrin-contaminated soil were set up. The experiment was carried out in a dark greenhouse, and the indoor temperature was kept at about 25 °C, and the domestication experiment was carried out for 20 days. After domestication, earthworms in different treatment groups were collected, washed with sterile water, and immediately placed in an ice-filled incubator to cool for 5 min to reduce the activity of earthworms. Then put the earthworms into a 30% ethanol solution with a temperature of 0 °C for anesthesia treatment, until the earthworms no longer twist, take them out and wash with sterile water three times to remove the viscous tissue on the surface of the earthworms, and use a sterile cotton cloth to gently Absorb the water on the surface of the earthworm. Fix the head of the dry clean earthworm on the operating wax plate with a sterilized tack, with the abdomen facing up. Use a sterilized surgical blade to cut the abdominal intestine of the earthworm from 0.5 cm below the ring to the anus of the earthworm, fix the cut intestinal tissue skin outward, remove the intestinal contents with a small spoon, and scrape the intestinal wall with a blade The tissue was collected in a 5 mL centrifuge tube, added with 0.5 mL of PBS buffer, and immediately placed in a -80 °C refrigerator. According to the analysis of bacterial flora structure in Example 1, the collected intestinal wall tissue was added to LB medium (950 mL of deionized water with 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 15 g of agar), and sterilized in a high-pressure steam cooker for 12 h. Reserve) to grow colonies in the petri dish, then transfer to liquid medium for culture, wait until logarithmic phase, and use UV spectrophotometer to measure its OD value. According to the obtained OD value, the concentration of bacterial cells was estimated, and the cultured bacterial solution was mixed with 0.5 mL of PBS buffer to obtain a targeted bacterial solution.

Based on Example 2, the mass ratio of target flora and soil was selected as 1:4000 to explore the optimal restoration time of target flora-indigenous flora on soil contaminated with cypermethrin with different concentrations. Take 5 kg of soil samples from the T1, T2 and T3 concentration treatment groups, add targeted bacteria with a ratio of targeted bacteria to soil mass of 1:4000, and place them in a constant temperature greenhouse at a temperature of 25 °C, with a black cloth protected from light. . Three parallel treatment groups were set for each of T1, T2, and T3 contaminated soils, and the soil moisture content was controlled at about 20% by weighing and adding water daily. The concentration of cypermethrin in the soil of different treatment groups was detected on the 7th, 14th, 30th and 60th days of the experimental period, and the pesticide degradation rate was calculated.

The experimental results showed that: in the T1 treatment group, the degradation rates of cypermethrin in the soil were 48.9%, 60.7%, 92.9% and 93.7% by the targeted flora-indigenous flora on the 7th, 14th, 30th and 60th days; T2 In the treatment group, the degradation rates of cypermethrin in the soil by the target flora-indigenous flora on the 7th, 14th, 30th and 60th days were 43.7%, 54.2%, 94.3% and 96.1%, respectively; in the T3 treatment group, the target On days 7, 14, 30, and 60, the degradation rates of cypermethrin in soil were 40.4%, 56.5%, 78.6%, and 79.2%, respectively (Fig. 4). In addition, on the 0th and 60th day of the experiment, the Simpson index, Goods_coverage index, and Pielou_e index of microorganisms in the T1, T2, and T3 treatment groups were 0.9823 and 0.9832, 0.9931 and 0.9944, 0.7718 and 0.7725, respectively; indicating the addition of targeted flora It improves the diversity, coverage and uniformity of soil microorganisms, and enhances the stability of the soil ecological environment. On the whole, with the increase of soil culture time, the degradation rate of cypermethrin in soil by targeted flora-indigenous flora was higher; the higher the concentration of cypermethrin, the higher the degradation rate of cypermethrin in soil by targeted flora-indigenous flora. Low; the repair effect of 30 days and 60 days is not much different. Considering the time cost factor, the best repair period in this experiment is 30 days. The results of this experiment show that the addition of the targeted flora domesticated by the earthworm intestinal reactor can effectively promote the rapid degradation of cypermethrin in the soil by the indigenous flora, which proves that this technology can be used as an effective solution for microbial remediation of pesticide-contaminated soil.

Claims (1)

1. a method for stimulating native flora to degrade cypermethrin in the soil rapidly, it is characterized in that, step comprises: select the adult earthworm living body after cleaning soil domestication, earthworm species is William columbaria, placed in dark box after cleaning to clear intestines ; The earthworms after the bowel cleansing treatment were put into the cypermethrin-contaminated soil with a concentration of 0.5-20 mg/kg for 20 days of domestication; the domesticated earthworms were taken, washed with sterile water, and immediately placed in a warmer filled with ice cubes. The earthworms were cooled in the box for 5 min to reduce the activity of the earthworms; then the earthworms were placed in a 30wt.% ethanol solution at a temperature of 0 °C for anesthesia treatment, until the earthworms no longer twisted, removed and washed with sterile water to remove the surface of the earthworms Stick the viscous tissue and absorb the water on the surface of the earthworm; fix the head of the treated earthworm on the operation wax plate with sterilized tacks, with the abdomen facing upward; use a sterilized surgical blade to cut the abdomen of the earthworm from 0.5cm below the ring belt From the intestine to the anus of the earthworm, fix the cut intestinal tissue epidermis outward, remove the intestinal content with a spoon, scrape the intestinal wall flora with a blade, and collect it in a 5 mL centrifuge tube. The extraction process time of the colony does not exceed 3 minutes; according to the mass ratio of the earthworm intestinal flora to the soil is 1:2000-1:8000, the earthworm intestinal flora is added to the soil to be repaired for a period of 7-60 days. repair.

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