Method for repairing pyrene polluted soil through combination of clover, corn and microorganisms

Abstract

The invention discloses a clover-corn-microorganism combined pyrene contaminated soil remediation method, which belongs to the technical field of contaminated soil remediation, and comprises the following steps: (1) sowing disinfected corn seeds and clover seeds in to-be-remediated soil, controlling the sowing density of the corn seeds and the clover seeds to be 50-100 plants / m < 2 > and 380-510 plants / m < 2 > respectively, and controlling the sowing density of the corn seeds and the clover seeds to be 50-100 plants / m < 2 > and the sowing density of the clover seeds to be 380-510 plants / m < 2 > respectively; regularly watering to keep the soil humidity to be 50% of the field moisture capacity, and keeping the normal growth of the plants; (2) pyrene degrading flora activating liquid is applied to rhizosphere soil on the tenth day and the 20th day after sowing, the application amount is 5-6 L / m < 2 >, corn and clover are pulled out on the 60th day after sowing, and remediation of pyrene contaminated soil is completed; according to the method, the corn and the clover are cooperatively planted, the efficient pyrene degrading flora screened in situ is combined, efficient and stable remediation of the pyrene polluted soil is achieved, and the method has high application and popularization value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of contaminated soil remediation, and particularly relates to a method for repairing pyrene-contaminated soil by trifolium pratense-corn-microorganism combination. BACKGROUND

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a kind of persistent organic pollutants widely existing in the environment, which have carcinogenic, teratogenic and mutagenic properties and are listed as priority controlled pollutants. Among them, pyrene, as a kind of four-ring polycyclic aromatic hydrocarbons, has attracted much attention due to its environmental residues and ecological risks. PAHs mainly come from incomplete combustion of fossil fuels and leakage of petrochemical products, and are often found in the soil around gas stations, oil refineries and other places, which poses a serious threat to the ecosystem and human health.

[0003] At present, the remediation technologies for soil PAHs pollution mainly include physical method, chemical method and biological method. Although physical and chemical remediation methods (such as leaching, thermal desorption, oxidation and reduction, etc.) have quick effects, they are high in cost, complex in operation, and easy to cause secondary pollution and soil structure damage. Biological remediation technology, especially plant remediation and microbial remediation, has become a research hotspot due to its low cost, environmental friendliness and simple operation. Plant remediation utilizes the absorption, enrichment and degradation of pollutants by plant roots, and promotes the decomposition of pollutants by microorganisms through rhizosphere effect. However, single plant remediation has problems such as long cycle, low efficiency, limitation by plant species and environmental conditions. For example, different plants have significant differences in pyrene degradation efficiency, and some plants can absorb pyrene but have difficulty in transferring it to the aboveground part, which limits the remediation effect. Microbial remediation utilizes the metabolic ability of microorganisms to degrade PAHs, but it often faces problems such as weak competitiveness of indigenous microorganisms, need for external addition of nutrients or inducers, and easy secondary pollution in practical application. In addition, the strains domesticated in the laboratory often have poor adaptability in the field environment, and the degradation efficiency is unstable.

[0004] In recent years, the plant-microorganism combined remediation technology shows good application prospect. The rhizosphere of plants provides growth environment and nutrients for microorganisms, and microorganisms enhance the degradation of pollutants and reduce the toxicity to plants, and the two can significantly improve the remediation efficiency. For example, the Chinese invention patent with the publication number CN111760903B discloses a method for remediation of polycyclic aromatic hydrocarbon contaminated soil by bacteria-plant combination, which applies Comamonas testosteroni agent to the soil to be remediated for 25-35 days, then plants Gramineae grass seeds in the soil, and after the grass is mature, the grass is cut, the agent and the grass are applied repeatedly until the content of polycyclic aromatic hydrocarbons in the soil is less than 1 mg / kg, so that the ecological system of the soil in a severely polluted area can be restored. However, the bacteria-plant combined remediation uses single Gramineae plant and non-in-situ domesticated agent, and the remediation efficiency and stability still need to be improved. Therefore, it is of great significance to develop a remediation method based on the combination of multiple types of plants and in-situ key microorganisms for efficient remediation of low-concentration pyrene contaminated soil. SUMMARY

[0005] The purpose of the present application is to provide a clover-corn-microorganism combined method for remediation of pyrene contaminated soil to solve the problems in the background art.

[0006] The purpose of the present application can be achieved by the following technical solutions. A clover-corn-microorganism combined method for remediation of pyrene contaminated soil, comprising the following steps: (1) Sowing the corn seeds and clover seeds treated by disinfection in the soil to be remediated, controlling the sowing density of the corn seeds and the clover seeds to be 50-100 plants / m 2 and 380-510 plants / m 2 , respectively, regularly watering to keep the soil humidity at 50% of the field water holding capacity, and keeping the plants normal growth; the corn grows fast, has large biomass and deep root system, and has obvious advantages in treating pyrene contaminated soil, but the planting density of the corn cannot be high, so the clover is used for co-planting with the corn to synergistically enhance the remediation effect on the pyrene contaminated soil; (2) Applying the pyrene degrading bacterial group activation liquid to the rhizosphere soil at the 10th day and the 20th day after sowing, the application amount being 5-6 L / m 2 , and removing the corn and the clover at the 60th day after sowing to complete the remediation of the pyrene contaminated soil.

[0007] Further, the disinfection treatment is that the seeds are soaked in 10 wt% hydrogen peroxide for 8-12 min, and then washed with distilled water.

[0008] Further, the pyrene content in the soil to be repaired is ≤20 mg / kg, and the pH is 6-8. The object to be repaired by the present application is low and medium concentration pyrene contaminated soil, such as farmland, vegetable land and residential land, etc. The present application shows a high pyrene removal rate for the pyrene concentration range of the contaminated soil; the pyrene concentration of the contaminated soil is too high, which will lead to the difficulty of corn and clover to survive, and will reduce the pyrene removal rate.

[0009] Further, the pyrene degrading bacterial population activation liquid is prepared by the following method: Step A1, collecting contaminated soil samples from a high concentration pyrene contaminated area with a depth of 20-50 cm, adding water for culture, taking supernatant after standing and stratifying, adding beef extract peptone liquid medium for enrichment culture, and obtaining enriched bacterial liquid; Step A2, taking the enriched bacterial liquid to inoculate in pyrene-containing MS liquid medium with pyrene as the only carbon source for repeated enrichment culture twice, then oscillating culture on a constant temperature shaker, collecting and washing the bacterial cells after centrifugal separation, and diluting to obtain a bacterial suspension with a cell concentration of 100 / mL; Step A3, taking the bacterial suspension to centrifugally collect the bacterial cells, washing with sterile water, and then enriching culture in LB liquid medium, mixing the cultured bacterial liquid with glycerol uniformly, and obtaining the degrading bacterial population liquid; Step A4, taking the degrading bacterial population liquid to add to LB liquid medium, and performing activation culture on a constant temperature shaker to obtain the pyrene degrading bacterial population activation liquid.

[0010] The pyrene degrading bacterial population is screened in situ from the soil of a high concentration pyrene contaminated area. The composite bacterial population not only has specific degradation ability for pyrene, but also has strong vitality. When applied to the soil to be repaired, it has obvious competitive advantage and does not excessively affect the survival of other microorganisms in the soil to be repaired. It will gradually decrease with the decrease of the pyrene content in the soil to be repaired.

[0011] Further, the use amount ratio of the contaminated soil sample to water in step A1 is 10g:50mL; and the pyrene content in the contaminated soil sample is ≥50mg / kg.

[0012] Further, the use amount ratio of the enriched bacterial liquid to pyrene-containing MS liquid medium in step A2 is 2mL:40mL; and the pyrene content in the pyrene-containing MS liquid medium is 5mg / L.

[0013] Further, the use amount ratio of the bacterial suspension to LB liquid medium and glycerol in step A3 is 10mL:40mL; and the use amount ratio of the cultured bacterial liquid to glycerol is 2mL:0.4mL.

[0014] Further, the use amount ratio of the degrading bacterial population liquid to LB liquid medium in step A4 is 30μL:200mL.

[0015] Further, the conditions of the oscillation culture in the above step are: temperature 22 DEG C, rotation speed 200 r / min, and culture time 2 days; the conditions of the enrichment culture are: temperature 25 DEG C, rotation speed 160 r / min, and culture time 2 days; and the conditions of the activation culture are: temperature 25 DEG C, rotation speed 160 r / min, and culture time 1 day.

[0016] Beneficial effects: The present application realizes efficient, stable and green repair of pyrene contaminated soil by the synergistic planting of corn and clover and the enhanced treatment of pyrene degrading bacteria. 1. High repair efficiency: The intercropping system of corn and clover expands the rhizosphere range, enhances the soil aeration and microbial activity, and significantly improves the pyrene degradation efficiency by using specific pyrene degrading bacteria. The data show that the pyrene removal rate can reach 96.5% after 60 days of combined repair, which is much higher than the results of single plant or single microorganism treatment. 2. Synergistic repair mechanism: Corn and clover complement each other in root structure, exudates and ecological function, and jointly create a rhizosphere environment conducive to the growth of pyrene degrading bacteria. Pyrene degrading bacteria reduce the toxicity of pyrene to plants by degrading pyrene, and enhance the tolerance and absorption capacity of plants to pyrene, forming a plant-microorganism positive promotion system. 3. Good ecological adaptability of bacteria: The pyrene degrading bacteria used in the present application are derived from indigenous microorganisms in high concentration pyrene contaminated areas. After directional enrichment and activation, they show strong competitiveness and high activity in low and medium concentration pyrene contaminated soil, without the need for external addition of nutrients or inducers, avoiding secondary pollution, and having good ecological compatibility and site applicability. 4. High repair stability: By optimizing the planting density of plants and the application strategy of bacterial liquid, the present application maximizes the degradation potential of pyrene degrading bacteria while ensuring the normal growth of plants, and has stable and reliable repair effect, which is suitable for in-situ repair of large area, low and medium concentration pyrene contaminated soil. 5. Simple operation and low cost: The method does not require complex equipment or chemical agents, and the operation process is simple, which is easy to apply to practical pollution site repair engineering. In summary, the present application realizes efficient, green and sustainable repair of pyrene contaminated soil by the synergistic planting of corn and clover and the enhanced treatment of pyrene degrading bacteria. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0018] Example 1 The present embodiment provides a pyrene-degrading bacterial population activation solution, which is prepared by the following method: Step A1, the contaminated soil sample was collected from a company in Pudong, Shanghai, which is located in an area where the soil and groundwater have been found to be contaminated by high concentrations of polycyclic aromatic hydrocarbons in Shanghai. The contaminated soil sample was taken from a depth of 20-50 cm and tested for pyrene content of 50 mg / kg. The contaminated soil sample was placed in a sealed bag and stored at 4°C for later use. 10 g of the contaminated soil sample was added to a conical flask, then 50 mL of water was added to the conical flask, and the mixture was incubated in a shaking incubator at 22°C and 200 r / min for 2 h. After standing for 30 min, the supernatant was collected and added to a sterilized beef extract peptone liquid medium. The mixture was incubated at 25°C and 160 r / min for 2 days to obtain an enriched bacterial solution. Step A2, 2 mL of the enriched bacterial solution was inoculated into MS liquid medium containing 5 mg / L of pyrene as the sole carbon source, and incubated in the dark at 25°C and 160 r / min for 2 days. After replacing the MS liquid medium containing pyrene, the mixture was further incubated in the dark at 25°C and 160 r / min for 2 days. Then the mixture was incubated in a constant-temperature shaking incubator at 22°C and 200 r / min for 2 days. The bacterial cells were collected by centrifugation at 2000 r / min, washed with sterilized physiological saline for 3 times, and then diluted with sterile water to obtain a bacterial suspension with a cell concentration of 100 cells / mL, which was ready for use. Step A3, 10 mL of the bacterial suspension was centrifuged at 10000 rpm for 10 min and the bacterial cells were collected. After washing with sterile water, 40 mL of LB liquid medium was added and incubated at 25°C and 160 r / min for 2 days. 2 mL of the incubated bacterial solution was mixed with 0.4 mL of glycerol to obtain a pyrene-degrading bacterial population solution. Step A4, 30 μL of the pyrene-degrading bacterial population solution was added to 200 mL of LB liquid medium and incubated at 25°C and 160 r / min in a constant-temperature shaking incubator for 1 day to obtain a pyrene-degrading bacterial population activation solution.

[0019] Example 2 The present embodiment provides a method for repairing pyrene-contaminated soil by a combination of clover, corn and microorganisms, which comprises the following steps: (1) The corn seeds and clover seeds were soaked in 10 wt% hydrogen peroxide solution for 8 min, then washed with distilled water. The sterilized corn seeds and clover seeds were then sown in the pyrene-contaminated soil with a pyrene content of 20 mg / kg and a pH of 6. The sowing density of corn seeds and clover seeds was controlled at 50 plants / m 2and 445 plants / m 2 The soil was kept moist at 50% of the field water holding capacity by regular watering to maintain normal growth of the plants. (2) The pyrene-degrading bacterial population activation solution prepared in Example 1 was applied to the rhizosphere soil at 5.5 L / m 2 The corn and clover were removed on the 60th day after sowing, and the repair of the pyrene-contaminated soil was completed.

[0020] Example 3 The present example provides a method for repairing pyrene-contaminated soil by combining clover, corn and microorganisms, comprising the following steps: (1) The corn seeds and clover seeds were soaked in 10 wt% hydrogen peroxide for 10 min, then washed with distilled water, and the disinfected corn seeds and clover seeds were sown in the soil to be repaired with a pyrene content of 20 mg / kg and a pH of 7, and the sowing density of the corn seeds and clover seeds was controlled at 75 plants / m 2 and 445 plants / m 2 The soil was kept moist at 50% of the field water holding capacity by regular watering to maintain normal growth of the plants. (2) The pyrene-degrading bacterial population activation solution prepared in Example 1 was applied to the rhizosphere soil at 5.5 L / m 2 The corn and clover were removed on the 60th day after sowing, and the repair of the pyrene-contaminated soil was completed.

[0021] Example 4 The present example provides a method for repairing pyrene-contaminated soil by combining clover, corn and microorganisms, comprising the following steps: (1) The corn seeds and clover seeds were soaked in 10 wt% hydrogen peroxide for 12 min, then washed with distilled water, and the disinfected corn seeds and clover seeds were sown in the soil to be repaired with a pyrene content of 20 mg / kg and a pH of 8, and the sowing density of the corn seeds and clover seeds was controlled at 100 plants / m 2 and 510 plants / m 2 The soil was kept moist at 50% of the field water holding capacity by regular watering to maintain normal growth of the plants. (2) The pyrene-degrading bacterial population activation solution prepared in Example 1 was applied to the rhizosphere soil at 6 L / m 2 The corn and clover were removed on the 60th day after sowing, and the repair of the pyrene-contaminated soil was completed.

[0022] Comparative Example 1 The comparative example is compared with example 3, the difference is that no plant is planted, and the pyrene-degrading bacterial population activation solution prepared in example 1 is directly applied to the soil to be repaired for repair, and the remaining steps are the same.

[0023] Comparative example 2 The comparative example is compared with example 3, the difference is that clover seeds are used to replace an equal amount of corn seeds, that is, only clover is planted to repair the soil to be repaired, and the remaining steps are the same.

[0024] Comparative example 3 The comparative example is compared with example 3, the difference is that corn seeds are used to replace an equal amount of clover seeds, that is, only corn is planted to repair the soil to be repaired, and the remaining steps are the same.

[0025] Comparative example 4 The comparative example is compared with example 3, the difference is that the planting density of corn seeds and clover seeds is 25 plants / m 2 and 320 plants / m 2 respectively, and the remaining steps are the same.

[0026] Comparative example 5 The comparative example is compared with example 3, the difference is that the planting density of corn seeds and clover seeds is 125 plants / m 2 and 640 plants / m 2 respectively, and the remaining steps are the same.

[0027] Comparative example 6 The comparative example is compared with example 3, the difference is that the pyrene-degrading bacterial population activation solution prepared in example 1 is not added, and the remaining steps are the same.

[0028] Comparative example 7 The comparative example is compared with example 3, the difference is that the pyrene-degrading bacterial population activation solution prepared in example 1 is replaced with an equal amount of a commonly commercially available Pseudomonas activation solution, and the remaining steps are the same.

[0029] The set conditions for repairing pyrene-contaminated soil in the methods of example 2 to example 4 and comparative examples 1 to 7 are shown in Table 1: Table 1 The soil repaired in example 2 to example 4 and comparative examples 1 to 7 is freeze-dried, then ground and passed through a 1 mm sieve, the pyrene content in the soil sample after repair treatment is detected, and the pyrene removal rate is calculated, and the results are shown in Table 2: Table 2 As can be seen from the data in Table 2, the corn and clover are synergistically planted in combination with the specific pyrene-degrading bacterial population of the application to reinforce the treatment of the pyrene-contaminated soil, and the residual pyrene in the remediated contaminated soil is significantly reduced to 0.7-1.22 mg / kg, and the pyrene removal rate is as high as 96.48%, and the pyrene degradation is more thorough, and the pyrene-contaminated soil remediation effect is excellent; As can be seen from the data in Comparative Examples 1-3, only the pyrene-degrading bacterial population can only reduce the pyrene concentration to 9.16 mg / kg, and the pyrene removal rate is 54.18%, and the remediation effect is poor; and the single corn planting or single clover planting in combination with the pyrene-degrading bacterial population can slightly increase the pyrene removal rate to 71.45% and 75.65%, respectively, but the pyrene concentration in the remediated soil is still high, and the pyrene degradation is not thorough; As can be seen from the data in Comparative Examples 4 and 5, the planting density of corn and clover directly affects the remediation effect of the pyrene-contaminated soil, and the planting density lower or higher than the corn and clover planting density within the protection scope of the application cannot achieve the remediation effect of the application, and the reason is that the planting density is too low, and the plant root system cannot completely cover the contaminated soil range, resulting in poor remediation effect, and the planting density is too high, which causes the corn and clover to compete with each other, resulting in negative impact on the normal growth of the plants; As can be seen from the data in Comparative Examples 6 and 7, only the corn and clover synergistic planting can not achieve the level of the application for remediation of the pyrene-contaminated soil, and the pyrene removal rate in the contaminated soil cannot achieve the level of the application; and the use of commercially available Pseudomonas to replace the pyrene-degrading bacterial population of the application for reinforcement treatment also cannot achieve the level of the application, and the reason is that the microorganism artificially screened in the laboratory has weak vitality in the field and poor competitiveness with local microorganisms, and the pyrene-degrading bacterial population collected and screened from the high-concentration pyrene contaminated area has stronger adaptability and competitiveness in the low-concentration pyrene-contaminated soil, and therefore can bring better remediation effect.

[0030] It should be noted that, in this document, relational terms such as first and second and the like can be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0031] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and adaptions can be effected without departing from the spirit and scope of the present application, which is not limited to the exact construction and arrangement described. It is intended, therefore, to cover all modifications and adaptions that fall within the scope of the claims and their equivalents.

Claims

1. A method for the co-remediation of pyrene-contaminated soil using clover-corn-microorganisms, characterized in that, Includes the following steps: (1) Sow the disinfected corn seeds and clover seeds in the soil to be restored, and control the sowing density of corn seeds and clover seeds to be 50-100 plants / m². 2 and 380-510 plants / m 2 Water regularly to maintain soil moisture at 50% of field capacity to ensure normal plant growth; (2) Apply pyrene-degrading bacterial activation solution to the rhizosphere soil on the 10th and 20th day after sowing, at a rate of 5-6 L / m². 2 The corn and clover were removed 60 days after sowing to complete the remediation of the pyrene-contaminated soil.

2. The method for co-remediation of pyrene-contaminated soil using clover-maize-microorganisms according to claim 1, characterized in that, The disinfection process involves soaking the seeds in 10wt% hydrogen peroxide for 8-12 minutes, and then rinsing them with distilled water.

3. The method for co-remediation of pyrene-contaminated soil using clover-maize and microorganisms according to claim 1, characterized in that, The pyrene content in the soil to be remediated is ≤20mg / kg, and the pH is 6-8.

4. The method for co-remediation of pyrene-contaminated soil using clover-maize-microorganisms according to claim 1, characterized in that, The pyrene-degrading bacterial community activation solution was prepared by the following method: Step A1: Collect contaminated soil samples from a high-concentration pyrene contaminated area at a depth of 20-50cm, add them to water for culture, allow them to stand and separate into layers, take the supernatant, add it to beef extract peptone liquid medium for enrichment culture, and obtain enriched bacterial solution. Step A2: Take the enriched bacterial solution and inoculate it into MS liquid medium containing pyrene with pyrene as the sole carbon source. Repeat the enrichment culture twice in the dark, and then culture it on a constant temperature shaker. After centrifugation to collect the bacterial cells and washing, dilute it to obtain a bacterial suspension with a cell concentration of 100 cells / mL. Step A3: Collect bacterial cells by centrifugation of bacterial suspension, wash with sterile water and enrich in LB liquid medium. Add glycerol to the cultured bacterial solution and mix well to obtain the degrading bacterial culture solution. Step A4: Add the degrading bacterial culture solution to LB liquid medium and activate it on a constant temperature shaker to obtain the pyrene degrading bacterial culture activation solution.

5. The method for co-remediation of pyrene-contaminated soil using clover-corn-microorganisms according to claim 4, characterized in that, The ratio of the contaminated soil sample to water was 10g:50mL; the pyrene content in the contaminated soil sample was ≥50mg / kg.

6. The method for co-remediation of pyrene-contaminated soil using clover-maize and microorganisms according to claim 4, characterized in that, The ratio of the enriched bacterial solution to the pyrene-containing MS liquid culture medium is 2 mL: 40 mL; the pyrene content in the pyrene-containing MS liquid culture medium is 5 mg / L.

7. The method for co-remediation of pyrene-contaminated soil using clover-corn-microorganisms according to claim 4, characterized in that, The ratio of the bacterial suspension to LB liquid medium and glycerol is 10 mL: 40 mL; the ratio of the cultured bacterial solution to glycerol is 2 mL: 0.4 mL.

8. The method for co-remediation of pyrene-contaminated soil using clover-maize and microorganisms according to claim 4, characterized in that, The ratio of the degrading bacterial culture solution to LB liquid culture medium is 30 μL: 200 mL.

9. The method for co-remediation of pyrene-contaminated soil using clover-maize and microorganisms according to claim 4, characterized in that, The conditions for the shaking culture are: temperature 22℃, rotation speed 200r / min, and culture time 2 days; the conditions for the enrichment culture are: temperature 25℃, rotation speed 160r / min, and culture time 2 days; the conditions for the activation culture are: temperature 25℃, rotation speed 160r / min, and culture time 1 day.

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