Soil remediation method for jointly degrading polycyclic aromatic hydrocarbon through zoysia sinensis-degrading flora

Abstract

The invention provides a soil remediation method for jointly degrading polycyclic aromatic hydrocarbon by zoysia sinensis-degrading flora, which comprises the following steps: S1, carrying out shake culture on a soil sample in a shaking table, and then culturing in a liquid culture medium to obtain an enriched bacteria solution; s2, adding PAHs into a liquid culture medium, inoculating the enriched bacterial liquid into the liquid culture medium, and gradually increasing the concentration of the PAHs to obtain a culture solution; s3, centrifuging the culture solution, collecting thalli, performing enrichment culture again, adding the bacterial solution into glycerol, and mixing to obtain a degrading flora bacterial solution; s4, mixing the polycyclic aromatic hydrocarbon contaminated soil with nutrient soil to obtain composite soil; s5, sowing the zoysia sinensis seeds in the composite soil to obtain plants, and culturing the plants in a plant culture room; and step S6, adding activated degrading flora bacterial liquid into the plants on the 15th day and the 30th day after sowing. Through the combined degradation effect of plants and microorganisms, the content of PAHs in soil is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of environmental restoration, and in particular to a soil restoration method for jointly degrading polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are extremely harmful to human health and the environment, such as environmental pollution, bioaccumulation, air pollution, water pollution, soil pollution and fire risks; potential health effects may be cancer risks, respiratory problems, nervous system effects, reproductive problems, cardiovascular disease, immune system, birth defects, developmental delays, and skin and eye problems. The acute health effects on humans are generally of lower severity, such as vomiting, skin irritation, inflammation, diarrhea and eye irritation. Chronic health effects include kidney and liver damage, red blood cell rupture, decreased immune function, respiratory problems and cataracts.

[0003] Environmental cleanup of PAH-contaminated matrices is essential for ecological restoration and human health. The degradability, adsorption, hydrophobicity and high persistence of PAHs are the main challenges for removal, so there is a need to better understand the characteristics of PAHs at specific sites, concentration ranges, assessment and monitoring of degradability. Many methods including physical, chemical and biological treatments have been designed to restore matrices contaminated with PAHs. Existing physical and chemical methods include solvent extraction, adsorption, air sparging, thermal desorption, chemical oxidation and photooxidation, but these methods are unattractive due to their inability to completely transform PAHs, low efficiency, high cost and high labor intensity. Bioremediation is widely accepted as an economical and environmentally friendly method for remediating PAH-contaminated sites. It requires less energy than other methods, making it a favorable choice. Under aerobic conditions, single or mixed PAHs are metabolized into less toxic products of CO2 and water, while methane is produced under anaerobic conditions. The mechanism and efficiency of phytoremediation depend on factors such as the nature of the pollutants, bioavailability, soil properties, and plant species.

[0004] Different plant species work synergistically with soil microorganisms to remove pollutants from the environment through phytodegradation, phytoaccumulation and phytostabilization of pollutants without causing any disturbance to ecological biodiversity and without affecting soil fertility; root degradation mainly utilizes the interaction ability of plant rhizosphere with bacteria. The main mechanism of root degradation process occurring in soil is that plants stimulate the activity of rhizosphere bacteria to degrade organic pollutants such as PAHs; in rhizodegradation, the rhizosphere transformation and degradation of PAHs occurs both through microbial metabolism and through extracellular enzymatic reactions of plant roots; the role of rhizosphere microorganisms is to cause the oxidation of PAHs, without the need to act as a medium for attacking PAH molecules like plants, and at the same time, the enzymatic properties of root exudates mediate the further oxidation of some oxidized compounds. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, which fully utilizes the plant-microorganism combined degradation effect to reduce the content of PAHs in the soil, and the degradation rate of the polycyclic aromatic hydrocarbon pyrene can reach 69.73%.

[0006] The technical solution adopted by the present invention is:

[0007] A soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria, comprising the following steps:

[0008] Step S1. Collect a PAHs-contaminated soil sample and add the soil sample to a conical flask. Then, add water to the conical flask and place it in a shaker for 1-2 hours. Let it stand, take the supernatant after standing and place it in a beef extract peptone liquid culture medium for 2-3 days to obtain an enriched bacterial liquid.

[0009] Step S2. Add a certain concentration of PAHs to the MS liquid culture medium, then take the enriched bacterial solution and inoculate it into the MS liquid culture medium, and enrich and culture it in the dark for 2-3 days, then add the enriched bacterial solution after culture into a new MS liquid culture medium, control the concentration of PAHs in the MS liquid culture medium to gradually increase, transfer it every 2 days until the last enrichment culture is completed, and obtain the culture solution;

[0010] Step S3. The culture solution in step S2 is centrifuged to collect the bacterial cells, and then the collected bacterial cells are washed with sterile water and enriched and cultured again with LB liquid medium for 2-3 days, and an appropriate amount of the enriched cultured bacterial solution is added to glycerol and mixed evenly to obtain a degrading bacterial flora solution, and the degrading bacterial flora solution is stored for later use;

[0011] Step S4. mixing the polycyclic aromatic hydrocarbons contaminated soil with nutrient soil to obtain composite soil;

[0012] Step S5. Soaking the Chinese Zoysia seeds in hydrogen peroxide, then washing with distilled water, and finally sowing the Chinese Zoysia seeds in the composite soil of a flower pot to obtain a potted plant, and culturing the potted plant in a plant cultivation room;

[0013] Step S6. 15-30 days after sowing the potted plants in step S5, add the degrading bacterial solution obtained in step S3 to the culture soil of the plants, and collect soil samples for testing 60-70 days after sowing.

[0014] Preferably, the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria is as follows: in step S1, the mass ratio of the soil sample to water is 10-30:100, and the standing time is 20-40 min; the temperature of the constant temperature shaker is 25-30°C, and the rotation speed is 150-180 r / min.

[0015] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, the PAHs in step S2 are selected from one or more of phenanthrene, pyrene, fluoranthene and benzo(a)pyrene.

[0016] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, the volume ratio of the enriched bacterial solution to the MS liquid culture medium in step S2 is 2-5:100.

[0017] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, the temperature of the enrichment culture in step S2 is 25-30° C. and the rotation speed is 150-180 r / min.

[0018] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, the centrifugal speed in step S3 is 8000-10000 rpm, and the centrifugal time is 5-10 min.

[0019] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, the volume ratio of glycerol to bacterial solution in step S3 is 0.2-0.4:1.

[0020] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, the mass ratio of nutrient soil to polycyclic aromatic hydrocarbons-contaminated soil in step S4 is 10-20:100.

[0021] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, the mass concentration of hydrogen peroxide in step S5 is 10-15%, and the soaking time is 10-15 min; the lighting time for cultivation in the plant culture room is 9-11 h / d, the temperature is 20-25°C, and the soil moisture content is 50-60%.

[0022] Preferably, in the soil remediation method for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria, an activation step is performed before the degrading bacteria solution is added to the composite soil in step S5, specifically, 20-30 μL of the degrading bacteria solution is added to 200 mL of LB liquid culture medium, and cultured in a shaking table at a temperature of 25-30°C and a rotation speed of 150-180 r / min for 36-48 hours.

[0023] Advantages of the present invention:

[0024] (1) The soil remediation method of the present invention for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia knotweed and degrading bacteria. As a grass plant, Zoysia knotweed has the advantages of short growth cycle, large biomass, wide coverage, well-developed root system, and strong stress resistance in the remediation of PAHs-contaminated soil. Through the degradation effect of Zoysia knotweed, the content of PAHs in the soil can be reduced, thereby reducing the risk of human exposure to these pollutants through the food chain or direct contact.

[0025] (2) The soil remediation method of the present invention for the combined degradation of polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria is simple to operate, easy to practice, and can make full use of the plant-microorganism combined degradation effect. The degradation rate of the polycyclic aromatic hydrocarbon pyrene can reach 69.73%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the degradation rate of contaminated soil in the experimental group and the control group in the embodiment of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments.

[0028] Example

[0029] A soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria, comprising the following steps:

[0030] Step S1. Collect PAHs-contaminated soil samples. The PAHs-contaminated soil samples are soils contaminated by polycyclic aromatic hydrocarbons around a chemical plant in Anqing. The sampling method is to collect soil 10 cm below the ground, remove visible impurities in the sample and then pass it through a 20-mesh sieve. In the same way, 5 soil samples are taken at intervals of 5-10 m and mixed evenly. The mixed soil samples are placed in a small portable liquid nitrogen tank filled with liquid nitrogen for storage. 10 g of the soil sample is added to a conical flask, and then the conical flask is added with 40 ml of water and placed in a shaker for shaking culture for 2 hours. The conical flask is allowed to stand for 30 minutes. The supernatant after standing is placed in a high-pressure sterilized beef extract peptone liquid culture medium for culture for 2 days. The culture temperature is 25° C. and the rotation speed is 150 r / min to obtain an enriched bacterial solution.

[0031] Step S2. Add 25 mg / L of pyrene to the MS liquid culture medium, then take 1 mL of the enriched bacterial solution and inoculate it into 20 mL of the MS liquid culture medium, and culture it in a dark environment for 2 days at a temperature of 25° C. and a rotation speed of 150 r / min. Then, add the cultured enriched bacterial solution into a new MS liquid culture medium, control the concentration of pyrene in the liquid culture medium to 50 mg / L, culture it in a dark environment for 2 days, and then add it into the MS liquid culture medium containing 100 mg / L of pyrene and culture it for two days to obtain a culture solution;

[0032] Step S3. Take 5 mL of the culture solution in step S2 and centrifuge to collect the bacteria. The centrifugal speed is 10000 rpm and the centrifugal time is 5 min. Then the collected bacteria are washed with sterile water and enriched with 20 mL of LB liquid culture medium for 2 days. The culture temperature is 25 ° C and the rotation speed is 150 r / min. Take 1 mL of the enriched culture solution and add it to 0.4 mL of glycerol to mix evenly to obtain a degradation bacterial flora solution. The degradation bacterial flora solution is stored at -80 ° C for use.

[0033] Step S4. The polycyclic aromatic hydrocarbons-contaminated soil and the nutrient soil are mixed in a mass ratio of 100:10 to obtain a composite soil, the manufacturer of the nutrient soil is Shenyang Xingyue Trading Co., Ltd., and the nutrient soil is a general horticultural nutrient soil;

[0034] Step S5. Soak the seeds of Zoysia japonica in 10% hydrogen peroxide for 15 minutes, then wash with distilled water, and finally sow the seeds of Zoysia japonica in the composite soil of a flower pot to obtain a potted plant. The planting area of ​​each flower pot is 0.02m 2 , 3 g of Zoysia japonica seeds were evenly sown in each flower pot, and the potted plants were cultured in a plant culture room;

[0035] Step S6. On the 15th and 30th days after sowing the potted plants in step S5, the degrading bacterial solution in step S5 is activated. Specifically, 20 μL of the degrading bacterial solution is added to 200 mL of LB liquid culture medium, and cultured in a shaking incubator at a temperature of 25°C and a rotation speed of 150 r / min for 36 hours. The activated degrading bacterial solution is added to the culture soil of the potted plants, and soil samples are collected for testing on the 60th day after sowing.

[0036] The degradation bacterial liquid was set up into an experimental group and a control group, as follows:

[0037] Control group CK: No degrading bacterial solution was added, and only Zoysia sinensis was planted;

[0038] Experimental group A1: Zoysia japonica was planted, and 50mL of degrading bacteria solution with a concentration of OD600=0.2 was added to the roots of the plants; Experimental group A2: Zoysia japonica was planted, and 50mL of degrading bacteria solution with a concentration of OD600=0.4 was added to the roots of the plants; Experimental group A3: Zoysia japonica was planted, and 50mL of degrading bacteria solution with a concentration of OD600=0.6 was added to the roots of the plants; Experimental group A4: Zoysia japonica was planted, and 50mL of degrading bacteria solution with a concentration of OD600=0.8 was added to the roots of the plants, and the required concentration of the solution was obtained after dilution and adjustment by adding distilled water. Each control group and experimental group included three potted plant samples.

[0039] On the 15th and 30th days after the sowing of Zoysia japonica, activated degrading bacterial flora was added, and the potted plants were cultured in a plant culture room. The lighting time was set to 10h / d, the temperature was 25°C, and the soil moisture content was maintained at 50%-60%.

[0040] Soil samples were collected on the 60th day after sowing. 5.00 g of contaminated soil sample was taken, 30 mL of acetone-n-hexane (V:V=1:1) mixed solvent was added as the extractant, 3 g of copper powder was added for ultrasonic extraction for 30 min, and the supernatant was collected after centrifugation at 4000 rpm for 10 min. The above operation was repeated once, the extracts were combined, and concentrated to 1 mL by rotary evaporation. The samples were purified by the method described in HJ 784-2016, and the content of PAHs in the soil was determined by high performance liquid chromatography.

[0041] Test results such as Figure 1 As shown in the figure, during the 60-day experiment, compared with the control group, the PAHs degradation efficiency of different experimental groups was improved; in comparison, the degradation effect of pyrene in the experiment of this embodiment was stronger than that of benzo(a)pyrene; in general, the degradation effect of PAHs gradually increased with the increase of the concentration of the degrading bacterial flora; the experimental group A3 had the strongest degradation effect on pyrene, with a degradation rate of 69.73%.

[0042] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria, characterized in that: The following steps are involved: Step S1. Collect a PAHs-contaminated soil sample and add the soil sample to a conical flask. Then, add water to the conical flask and place it in a shaker for 1-2 hours. Let it stand, take the supernatant after standing and place it in a beef extract peptone liquid culture medium for 2-3 days to obtain an enriched bacterial liquid. Step S2. Add a certain concentration of PAHs to the MS liquid culture medium, then take the enriched bacterial solution and inoculate it into the MS liquid culture medium, and enrich and culture it in the dark for 2-3 days, then add the enriched bacterial solution after culture into a new MS liquid culture medium, control the concentration of PAHs in the MS liquid culture medium to gradually increase, transfer it every 2 days until the last enrichment culture is completed, and obtain the culture solution; Step S3. The culture solution in step S2 is centrifuged to collect the bacterial cells, and then the collected bacterial cells are washed with sterile water and enriched and cultured again with LB liquid medium for 2-3 days, and an appropriate amount of the enriched cultured bacterial solution is added to glycerol and mixed evenly to obtain a degrading bacterial flora solution, and the degrading bacterial flora solution is stored for later use; Step S4. mixing the polycyclic aromatic hydrocarbons contaminated soil with nutrient soil to obtain composite soil; Step S5. Soaking the Chinese Zoysia seeds in hydrogen peroxide, then washing with distilled water, and finally sowing the Chinese Zoysia seeds in the composite soil of a flower pot to obtain a potted plant, and culturing the potted plant in a plant cultivation room; Step S6. 15-30 days after sowing the potted plants in step S5, add the degrading bacterial solution obtained in step S3 to the culture soil of the plants, and collect soil samples for testing 60-70 days after sowing.

2. The soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria according to claim 1, characterized in that: In step S1, the mass ratio of the soil sample to water is 10-30:100, the standing time is 20-40 min; the temperature of the constant temperature shaker is 25-30° C., and the rotation speed is 150-180 r / min.

3. The soil remediation method for degrading polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria according to claim 1, characterized in that: In step S2, the PAHs are selected from one or more of phenanthrene, pyrene, fluoranthene and benzo(a)pyrene.

4. The soil remediation method for degrading polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria according to claim 1, characterized in that: In step S2, the volume ratio of the enriched bacterial solution to the MS liquid culture medium is 2-5:

100.

5. The soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria according to claim 1, characterized in that: The temperature of the enrichment culture in step S2 is 25-30° C. and the rotation speed is 150-180 r / min.

6. The soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria according to claim 1, characterized in that: In step S3, the centrifugal speed is 8000-10000 rpm, and the centrifugal time is 5-10 min.

7. The soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria according to claim 1, characterized in that: In step S3, the volume ratio of glycerol to bacterial liquid is 0.2-0.4:

1.

8. The soil remediation method for degrading polycyclic aromatic hydrocarbons by combining Zoysia japonica and degrading bacteria according to claim 1, characterized in that: In step S4, the mass ratio of nutrient soil to polycyclic aromatic hydrocarbons contaminated soil is 10-20:

100.

9. The soil remediation method for degrading polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria according to claim 1, characterized in that: In step S5, the mass concentration of hydrogen peroxide is 10-15%, and the soaking time is 10-15 minutes; the lighting time in the plant culture room is 9-11 hours / day, the temperature is 20-25° C., and the soil moisture content is 50-60%.

10. The soil remediation method for degrading polycyclic aromatic hydrocarbons by Zoysia japonica-degrading bacteria according to claim 1, characterized in that: Before adding the degrading bacterial liquid to the composite soil in step S6, an activation step is performed, specifically: 20-30 μL of the degrading bacterial liquid is added to 200 mL of LB liquid culture medium and cultured in a shaking incubator at a temperature of 25-30° C. and a rotation speed of 150-180 r / min for 36-48 hours.

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