A method for bioremediation of petroleum hydrocarbon contaminated soil

Abstract

This invention discloses a microbial remediation method for petroleum hydrocarbon-contaminated soil, comprising: S1, soil pretreatment; S2, microbial screening; S3, primary remediation of petroleum hydrocarbon-contaminated soil; and S4, secondary remediation of petroleum hydrocarbon-contaminated soil. This method can effectively remediate petroleum hydrocarbon-contaminated soil, overcoming the challenges of reduced permeability, limited biodegradation, and long-term pollution effects faced by traditional remediation methods. By applying voltage and through the combined action of degrading microbial communities, the method promotes microbial growth and metabolism, overcoming the difficulties in microbial degradation of long-chain hydrocarbons and aromatic hydrocarbons, and improving the degradation rate and thoroughness.

Description

Technical Field

[0001] This invention relates to the field of soil remediation technology, specifically to a microbial remediation method for petroleum hydrocarbon-contaminated soil. Background Technology

[0002] Before changing the land use of decommissioned enterprise sites, especially those of polluting enterprises, it is particularly important to conduct environmental investigations, risk assessments, remediation, and treatment. Otherwise, there will be ecological and health risks, affecting the urban environment and the health of its residents.

[0003] In existing technologies, the remediation of petroleum hydrocarbon-contaminated soil is quite complex. This is because petroleum hydrocarbons are mixtures of various compounds, including alkanes, aromatic hydrocarbons, and polycyclic aromatic hydrocarbons (PAHs), each with varying biodegradability and toxicity. Therefore, different remediation strategies are needed for different pollutants. While biodegradation is an effective remediation method, some petroleum hydrocarbon compounds are difficult to biodegrade, especially high-molecular-weight PAHs, which are toxic to microorganisms and structurally stable, making them difficult to degrade. Furthermore, due to the presence of carbon... 10 -C 40 The range of petroleum hydrocarbons encompasses a range of compounds from light hydrocarbons (such as decane) to heavy hydrocarbons (such as high-boiling-point asphalt), with increasing molecular weight and more complex structures. The degradation of long-chain hydrocarbons and aromatic hydrocarbons is generally more difficult than that of light hydrocarbons because they are less readily recognized and broken down by microbial enzymes. Furthermore, the lower solubility of heavy petroleum hydrocarbons makes them less readily adsorbed and taken up by microorganisms, limiting their degradation capacity. High molecular weight petroleum hydrocarbons (especially C46)... 30 The long-chain alkanes and aromatic hydrocarbons mentioned above have poor biodegradability because they have more cyclic and branched structures, which are more difficult for microorganisms to degrade. Meanwhile, petroleum hydrocarbon pollution can damage soil structure, leading to soil compaction and affecting soil fertility and productivity. Furthermore, remediation through chemical methods may result in secondary pollution during the remediation process, such as the spread of pollutants during excavation and transportation, and potentially harmful byproducts generated by the remediation technology itself. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a microbial remediation method for petroleum hydrocarbon-contaminated soil.

[0005] A microbial remediation method for petroleum hydrocarbon-contaminated soil includes the following steps:

[0006] S1. Pretreated soil;

[0007] Soil samples were collected from multiple depths at various locations within the abandoned chemical plant site and mixed to obtain soil samples. These soil samples were then air-dried, crushed, and sieved to obtain pretreated soil. The abandoned chemical plant site was designated as C. 10 -C 40 Petroleum hydrocarbon contaminated sites;

[0008] S2, Microbial screening;

[0009] According to the C 10 -C 40 Petroleum hydrocarbons and the pretreated soil were used to cultivate a degrading microbial community.

[0010] S3. One-time remediation of petroleum hydrocarbon-contaminated soil;

[0011] Take a surfactant with a volume fraction of 0.5% and activated carbon with a volume fraction of 2-5% of the petroleum hydrocarbon contaminated soil. First, mix the surfactant with the petroleum hydrocarbon contaminated soil and stir for 15-20 minutes. Then, add the activated carbon and stir for 5-10 minutes. Then, let it stand for 2-5 hours.

[0012] S4. Secondary remediation of petroleum hydrocarbon-contaminated soil;

[0013] When the soil moisture content is 15-20%, a voltage is applied to the petroleum hydrocarbon contaminated soil. At the same time as the voltage is applied, the degradation bacteria obtained in S2 are added to the petroleum hydrocarbon contaminated soil and stirred evenly. After the voltage is applied, the soil moisture content is adjusted to 40-60% and maintained. Then, the soil is stirred and aerated regularly until the petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil does not exceed the standard, and the remediation is completed.

[0014] The voltage is applied for 1–5 hours, the electrode spacing is set to 30–50 cm, and the electric field strength is 1–2 V / cm; the inoculum size of the degrading bacteria is 6 × 10⁻⁶ per kilogram of soil. 7 ~1×10 8 Each cell.

[0015] Note: The above method can effectively remediate petroleum hydrocarbon-contaminated soil, overcoming the challenges of reduced permeability, limited biodegradation, and long-term pollution effects faced by traditional remediation methods. Specifically, the addition of surfactants and activated carbon enhances the solubility and adsorption of petroleum hydrocarbons, reducing their low bioavailability and toxicity. The combined action of applying voltage and degrading microorganisms promotes microbial growth and metabolism, overcoming the challenges of microbial degradation of long-chain hydrocarbons and aromatic hydrocarbons, and improving the degradation rate and thoroughness. This not only improves the removal efficiency of petroleum hydrocarbons from the soil but also maintains a suitable microbial growth environment through regular stirring and aeration.

[0016] Furthermore, the point density mentioned in S1 is 5×5m, and the multiple depths include 0.5m, 1m and 2m.

[0017] Note: The above provides a commonly used sampling density and sampling depth.

[0018] Furthermore, the soil sample described in S1 is passed through a 50-100 mesh sieve.

[0019] Note: The above sieving process can remove large particles and impurities from the soil, making the soil sample more uniform in particle size, which is beneficial for subsequent processing and the action of microorganisms.

[0020] Furthermore, in step S3, the stirring speed for mixing the surfactant with the petroleum hydrocarbon-contaminated soil is 500–600 r / min, and the stirring speed after adding activated carbon is 100–200 r / min.

[0021] Note: The stirring speed mentioned above ensures a more uniform mixing of the surfactant and petroleum hydrocarbon-contaminated soil. Setting the stirring speed for activated carbon helps prevent the destruction of the activated carbon particles. Activated carbon has a porous structure; high-speed stirring may damage its structure and reduce its adsorption capacity.

[0022] Furthermore, the statement in S2 based on the C 10 -C 40 Petroleum hydrocarbons and pretreated soil were used to cultivate a degrading microbial community, including:

[0023] S2-1. Take 1-5g of the pretreated soil, place it on petroleum hydrocarbon culture medium, and then let it stand at 34-35℃ for 70-85h to obtain preliminary colonies.

[0024] S2-2. Then, the preliminary bacterial colonies are mixed in petroleum hydrocarbon culture medium and stirred at a temperature of 30-32℃, a stirring speed of 100-120 r / min, and a stirring time of 3-5 days to obtain the preliminary bacterial population.

[0025] S2-3, C is extracted from the soil of the abandoned chemical plant site. 10 -C 40 Petroleum hydrocarbons, C added to petroleum hydrocarbon culture medium 10 -C 40 Petroleum hydrocarbons were then collected, and the initial bacterial population was divided into three equal parts and inoculated onto C14 and C24 respectively. 10 -C 40 In petroleum hydrocarbon culture media with concentrations of 50 mg / L, 200 mg / L, and 500 mg / L, the inoculated petroleum hydrocarbon culture media were shaken at a temperature of 32–34℃ for 48–72 h.

[0026] S2-4. Determine the degradation rate of petroleum hydrocarbons in the three inoculated culture media. Select one or two preliminary strains with the highest degradation rate from each inoculated culture media. Mix equal amounts of the selected preliminary strains and place them on a shaker for 3-5 seconds of ultraviolet irradiation at a wavelength of 253-265 nm. Then, culture at 30-36℃ and 150 r / min for 3-5 days to obtain the degrading bacterial community.

[0027] Note: The degrading bacterial community obtained through the above cultivation process has high efficiency in degrading C. 10 -C 40 The biodegradation of petroleum hydrocarbons is highly specific, adaptable, and stable, maintaining efficient degradation performance under different pollution concentrations. Ultraviolet irradiation mutagenesis treatment can increase the genetic diversity of the strains, allowing for the selection of more stable and adaptable strains, thereby improving the survival and degradation capabilities of the biodegrading community in complex environments. At the same time, it is easy to operate and environmentally friendly, providing an efficient, specific, and cost-effective microbial solution for the bioremediation of petroleum hydrocarbon-contaminated soils.

[0028] Furthermore, the petroleum hydrocarbon culture medium described in S2-1, by weight, comprises 20-25 parts agar, 3-5 parts sodium chloride, 0.01-0.5 parts crude oil, 1-2 parts sodium nitrate, 1.2-1.5 parts peptone, 0.6-0.8 parts beef extract, 0.5-0.8 parts dipotassium hydrogen phosphate, 2-3 parts rhamnolipid, and 1000 parts distilled water.

[0029] Note: The above methods can provide suitable nutritional and environmental conditions for culturing microorganisms that degrade petroleum hydrocarbons, thereby helping to improve the accuracy of experiments and research efficiency.

[0030] Further, the petroleum hydrocarbon culture medium, by weight, comprises 15-20 parts glucose, 3-5 parts sodium chloride, 0.3-0.5 parts gasoline, 0.5-0.8 parts kerosene, 0.5-0.8 parts diesel, 1-1.5 parts fuel oil, 1.2-1.5 parts peptone, 0.6-0.8 parts beef extract, 0.5-0.8 parts dipotassium hydrogen phosphate, and vitamin B1. 12 0.2-0.3 parts, zinc sulfate 0.05-0.08 parts, ferrous sulfate 0.05-0.08 parts, boric acid 0.005-0.006 parts, rhamnolipid 2-3 parts, and distilled water 1000 parts.

[0031] Note: The above component ratio can simulate a petroleum-polluted environment, providing microorganisms with the necessary nutrients and growth conditions, thereby effectively promoting the biodegradation process of petroleum hydrocarbons and helping to screen and cultivate highly efficient petroleum-degrading microbial strains.

[0032] Furthermore, the preparation method of the petroleum hydrocarbon culture medium is as follows: weigh each component of the liquid culture medium according to the weight parts, mix and stir until each component is evenly dispersed, then sterilize at 100-110℃ for 10-15 min, and obtain the petroleum hydrocarbon culture medium after cooling.

[0033] Note: The above method ensures the homogeneity and sterility of the culture medium.

[0034] Furthermore, the surfactant is obtained by mixing cocamidopropyl betaine, sodium dodecylbenzenesulfonate and rhamnolipid in a mass ratio of 5-7:1:1-2.

[0035] Note: The above-mentioned surfactant mixture has optimized cleaning and emulsifying properties, which helps to improve the dispersibility and bioavailability of petroleum hydrocarbons in petroleum hydrocarbon culture medium, thereby providing a more suitable environment for microbial growth and enhancing the accuracy and efficiency of the experiment.

[0036] The beneficial effects of this invention are:

[0037] The method of this invention can effectively remediate petroleum hydrocarbon-contaminated soil, overcoming the difficulties faced by traditional remediation methods such as reduced permeability, limited biodegradation, and long-term pollution effects. By adding surfactants and activated carbon, the solubility and adsorption of petroleum hydrocarbons can be enhanced, reducing their low bioavailability and toxicity. Through the combined action of applying voltage and degrading bacteria, the growth and metabolism of microorganisms are promoted, overcoming the difficulties of microbial degradation of long-chain hydrocarbons and aromatic hydrocarbons, and improving the degradation rate and thoroughness. Thus, not only is the removal effect of petroleum hydrocarbons in the soil improved, but a suitable microbial growth environment is also maintained through regular stirring and aeration. Detailed Implementation

[0038] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0039] Example 1: A microbial remediation method for petroleum hydrocarbon-contaminated soil, comprising the following steps:

[0040] S1. Pretreated soil;

[0041] Soil samples were collected from multiple depths at various locations within the abandoned chemical plant site and mixed to obtain soil samples. These soil samples were then air-dried, crushed, and sieved to obtain pretreated soil. The abandoned chemical plant site was designated as C. 10 -C 40 Petroleum hydrocarbon contaminated sites;

[0042] The sampling density is 5×5m, the multiple depths include 0.5m, 1m and 2m, and the soil sample passes through an 80-mesh sieve;

[0043] S2, Microbial screening;

[0044] According to the C 10 -C 40 Petroleum hydrocarbons and the pretreated soil were used to cultivate a degrading microbial community; specifically including:

[0045] S2-1. Take 3g of the pretreated soil and place it on a petroleum hydrocarbon culture medium. Then, let it stand at 34℃ for 80h to obtain preliminary colonies.

[0046] The petroleum hydrocarbon culture medium described in S2-1, by weight, includes 22 parts agar, 4 parts sodium chloride, 0.05 parts crude oil, 1.5 parts sodium nitrate, 1.4 parts peptone, 0.7 parts beef extract, 0.7 parts dipotassium hydrogen phosphate, 2.5 parts rhamnolipid, and 1000 parts distilled water.

[0047] S2-2. Then, the preliminary bacterial colony was mixed in the petroleum hydrocarbon culture medium and stirred at 31°C for 110 r / min for 4 days to obtain the preliminary bacterial population.

[0048] The petroleum hydrocarbon culture medium, by weight, comprises 18 parts glucose, 4 parts sodium chloride, 0.4 parts gasoline, 0.6 parts kerosene, 0.7 parts diesel, 1.2 parts fuel oil, 1.3 parts peptone, 0.7 parts beef extract, 0.7 parts dipotassium hydrogen phosphate, and vitamin B1. 12 0.25 parts, zinc sulfate 0.07 parts, ferrous sulfate 0.07 parts, boric acid 0.005 parts, rhamnolipid 2.5 parts, and distilled water 1000 parts.

[0049] The preparation method of the petroleum hydrocarbon culture medium is as follows: weigh each component of the liquid culture medium according to the weight parts, mix and stir until each component is evenly dispersed, then sterilize at 105℃ for 12 minutes, and obtain the petroleum hydrocarbon culture medium after cooling.

[0050] S2-3, C is extracted from the soil of the abandoned chemical plant site. 10 -C 40 Petroleum hydrocarbons, C added to petroleum hydrocarbon culture medium 10 -C 40 Petroleum hydrocarbons were then collected, and the initial bacterial population was divided into three equal parts and inoculated onto C14 and C24 respectively. 10 -C 40 In petroleum hydrocarbon culture media with concentrations of 50 mg / L, 200 mg / L, and 500 mg / L, the inoculated petroleum hydrocarbon culture media were shaken at a temperature of 33℃ for 60 h at a shaking speed of 130 r / min.

[0051] S2-4. Determine the degradation rate of petroleum hydrocarbons in the three inoculated culture media. Select one or two preliminary strains with the highest degradation rate from each inoculated culture media. Mix the selected preliminary strains in equal amounts and place them on a shaker for 4 seconds of ultraviolet irradiation at a wavelength of 255 nm. Then, culture them at 35°C and 150 r / min for 4 days to obtain the degrading bacterial community.

[0052] S3. One-time remediation of petroleum hydrocarbon-contaminated soil;

[0053] Take a surfactant with a volume fraction of 0.5% and activated carbon with a volume fraction of 3% of the petroleum hydrocarbon contaminated soil. First, mix the surfactant with the petroleum hydrocarbon contaminated soil and stir for 18 minutes. Then add the activated carbon and stir for 7 minutes. Then let it stand for 4 hours.

[0054] In S3, the stirring speed for mixing the surfactant with the petroleum hydrocarbon contaminated soil is 550 r / min, and the stirring speed after adding activated carbon is 150 r / min.

[0055] The surfactant is a mixture of cocamidopropyl betaine, sodium dodecylbenzenesulfonate and rhamnolipid in a mass ratio of 6:1:1.5.

[0056] S4. Secondary remediation of petroleum hydrocarbon-contaminated soil;

[0057] At a soil moisture content of 18%, a voltage is applied to the petroleum hydrocarbon contaminated soil. While applying the voltage, the degradation bacteria obtained in S2 are added to the petroleum hydrocarbon contaminated soil and stirred evenly. After the voltage is applied, the soil moisture content is adjusted to 50% and maintained. Then, the soil is stirred and aerated regularly until the petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil does not exceed the standard, and the remediation is completed.

[0058] The voltage was applied for 3 hours, the electrode spacing was 40 cm, and the electric field strength was 1.5 V / cm. The inoculum size of the degrading bacteria was 6 × 10⁻⁶ per kilogram of soil. 7 ~1×10 8 Each cell.

[0059] Example 2: The difference between this example and Example 1 is that the soil sample was passed through a 50-mesh sieve.

[0060] Example 3: The difference between this example and Example 1 is that the soil sample is passed through a 100-mesh sieve.

[0061] Example 4: This example differs from Example 1 in that the microbial screening parameters are different. S2-1: Take 1g of the pretreated soil, place it on petroleum hydrocarbon culture medium, and then let it stand at 34℃ for 70h to obtain preliminary colonies.

[0062] S2-2. Then, the preliminary bacterial colony was mixed in petroleum hydrocarbon culture medium and stirred at 32°C at a stirring speed of 120 r / min for 5 days to obtain the preliminary bacterial population.

[0063] S2-3, C is extracted from the soil of the abandoned chemical plant site. 10 -C 40 Petroleum hydrocarbons, C added to petroleum hydrocarbon culture medium 10 -C 40 Petroleum hydrocarbons were then collected, and the initial bacterial population was divided into three equal parts and inoculated onto C14 and C24 respectively. 10 -C 40 In petroleum hydrocarbon culture media with concentrations of 50 mg / L, 200 mg / L, and 500 mg / L, the inoculated petroleum hydrocarbon culture media were shaken at a temperature of 32℃ for 48 h at a shaking speed of 120 r / min.

[0064] S2-4. Determine the degradation rate of petroleum hydrocarbons in the three inoculated culture media. Select one or two preliminary strains with the highest degradation rate from each inoculated culture media. Mix the selected preliminary strains in equal amounts and place them on a shaker for 5 seconds of ultraviolet irradiation at a wavelength of 265 nm. Then, culture them at 36°C and 150 r / min for 3 days to obtain the degrading bacterial community.

[0065] Example 5: This example differs from Example 1 in that the microbial screening parameters are different. S2-1: Take 5g of the pretreated soil, place it on a petroleum hydrocarbon culture medium, and then let it stand at 35℃ for 85h to obtain preliminary colonies.

[0066] S2-2. Then, the preliminary bacterial colony is mixed in petroleum hydrocarbon culture medium and stirred at 30°C at a stirring speed of 100 r / min for 3 days to obtain the preliminary bacterial population.

[0067] S2-3, C is extracted from the soil of the abandoned chemical plant site. 10 -C 40 Petroleum hydrocarbons, C added to petroleum hydrocarbon culture medium 10 -C 40 Petroleum hydrocarbons were then collected, and the initial bacterial population was divided into three equal parts and inoculated onto C14 and C24 respectively. 10 -C 40 In petroleum hydrocarbon culture media with concentrations of 50 mg / L, 200 mg / L, and 500 mg / L, the inoculated petroleum hydrocarbon culture media were shaken at a temperature of 34℃ for 72 h at a shaking speed of 140 r / min.

[0068] S2-4. Determine the degradation rate of petroleum hydrocarbons in the three inoculated culture media. Select one or two preliminary strains with the highest degradation rate from each inoculated culture media. Mix the selected preliminary strains in equal amounts and place them on a shaker for 3 seconds of ultraviolet irradiation at a wavelength of 253 nm. Then, culture them at 30°C and 150 r / min for 5 days to obtain the degrading bacterial community.

[0069] Example 6: The difference between this example and Example 1 is that the composition ratio of the petroleum hydrocarbon culture medium is different. The petroleum hydrocarbon culture medium, by weight, includes 20 parts agar, 3 parts sodium chloride, 0.01 parts crude oil, 2 parts sodium nitrate, 1.5 parts peptone, 0.8 parts beef extract, 0.8 parts dipotassium hydrogen phosphate, 3 parts rhamnolipid, and 1000 parts distilled water.

[0070] Example 7: The difference between this example and Example 1 is that the composition ratio of the petroleum hydrocarbon culture medium is different. The petroleum hydrocarbon culture medium, by weight, includes 25 parts agar, 5 parts sodium chloride, 0.5 parts crude oil, 1 part sodium nitrate, 1.2 parts peptone, 0.6 parts beef extract, 0.5 parts dipotassium hydrogen phosphate, 2 parts rhamnolipid, and 1000 parts distilled water.

[0071] Example 8: This example differs from Example 1 in that the composition ratio of the petroleum hydrocarbon culture medium is different. The petroleum hydrocarbon culture medium, by weight, includes 15 parts glucose, 3 parts sodium chloride, 0.3 parts gasoline, 0.5 parts kerosene, 0.5 parts diesel, 1.5 parts fuel oil, 1.5 parts peptone, 0.8 parts beef extract, 0.8 parts dipotassium hydrogen phosphate, and vitamin B1. 12 0.3 parts, zinc sulfate 0.08 parts, ferrous sulfate 0.08 parts, boric acid 0.006 parts, rhamnolipid 3 parts, and distilled water 1000 parts.

[0072] Example 9: This example differs from Example 1 in that the composition ratio of the petroleum hydrocarbon culture medium is different. The petroleum hydrocarbon culture medium, by weight, includes 20 parts glucose, 5 parts sodium chloride, 0.5 parts gasoline, 0.8 parts kerosene, 0.8 parts diesel, 1 part fuel oil, 1.2 parts peptone, 0.6 parts beef extract, 0.5 parts dipotassium hydrogen phosphate, and vitamin B1. 12 0.2 parts, zinc sulfate 0.05 parts, ferrous sulfate 0.05 parts, boric acid 0.005 parts, rhamnolipid 2 parts, and distilled water 1000 parts.

[0073] Example 10: This example differs from Example 1 in that the preparation parameters of the petroleum hydrocarbon culture medium are different. The petroleum hydrocarbon culture medium is obtained by sterilization at 110°C for 10 minutes and cooling.

[0074] Example 11: This example differs from Example 1 in that the preparation parameters of the petroleum hydrocarbon culture medium are different. The petroleum hydrocarbon culture medium is obtained by sterilization at 100°C for 15 minutes and cooling.

[0075] Example 12: The difference between this example and Example 1 is that the proportion of materials used for the remediation of petroleum hydrocarbon-contaminated soil is different, and activated carbon is used in the example, accounting for 2% of the volume of the petroleum hydrocarbon-contaminated soil.

[0076] Example 13: The difference between this example and Example 1 is that the proportion of materials used for the remediation of petroleum hydrocarbon contaminated soil is different, and activated carbon is used in the example, accounting for 5% of the volume of the petroleum hydrocarbon contaminated soil.

[0077] Example 14: This example differs from Example 1 in that the parameters for remediating petroleum hydrocarbon-contaminated soil are different. First, the surfactant is mixed with the petroleum hydrocarbon-contaminated soil and stirred for 15 minutes. Then, activated carbon is added and stirred for 5 minutes, followed by standing for 5 hours. The stirring speed for mixing the surfactant with the petroleum hydrocarbon-contaminated soil is 500 r / min, and the stirring speed after adding activated carbon is 100 r / min.

[0078] Example 15: This example differs from Example 1 in that the parameters for remediating petroleum hydrocarbon-contaminated soil are different. First, the surfactant is mixed with the petroleum hydrocarbon-contaminated soil and stirred for 20 minutes. Then, activated carbon is added and stirred for 10 minutes, followed by standing for 2 hours. The stirring speed for mixing the surfactant with the petroleum hydrocarbon-contaminated soil is 600 r / min, and the stirring speed after adding activated carbon is 200 r / min.

[0079] Example 16: The difference between this example and Example 1 is that the surfactant composition ratio is different. The surfactant is a mixture of cocamidopropyl betaine, sodium dodecylbenzenesulfonate and rhamnolipid in a mass ratio of 5:1:2.

[0080] Example 17: The difference between this example and Example 1 is that the surfactant composition ratio is different. The surfactant is a mixture of cocamidopropyl betaine, sodium dodecylbenzenesulfonate and rhamnolipid in a mass ratio of 7:1:1.

[0081] Example 18: The difference between this example and Example 1 is that the water content parameter in the secondary remediation of petroleum hydrocarbon contaminated soil is different. When the soil water content is 15%, a voltage is applied to the petroleum hydrocarbon contaminated soil. After the voltage is applied, the soil water content is adjusted to 40% and maintained.

[0082] Example 19: The difference between this example and Example 1 is that the water content parameter in the secondary remediation of petroleum hydrocarbon contaminated soil is different. When the soil water content is 20%, a voltage is applied to the petroleum hydrocarbon contaminated soil. After the voltage is applied, the soil water content is adjusted to 60% and maintained.

[0083] Example 20: This example differs from Example 1 in that the voltage parameters in the secondary remediation of petroleum hydrocarbon contaminated soil are different. The voltage application time is 5 hours, the electrode spacing is 30 cm, and the electric field strength is 2 V / cm.

[0084] Example 21: The difference between this example and Example 1 is that the voltage parameters in the secondary remediation of petroleum hydrocarbon contaminated soil are different. The voltage application time is 1 hour, the electrode spacing is set to 50 cm, and the electric field strength is 1 V / cm.

[0085] Experimental Example: This experimental example is based on the scheme described in Example 1, aiming to illustrate the practical application effects of the present invention. The investigated abandoned chemical plant site is a site belonging to a certain limited company, with a total area of ​​4979.9 square meters. 2 The site was investigated and found to be primarily used for the storage and transportation of heavy diesel oil and fuel oil. Testing revealed the presence of petroleum hydrocarbons (C). 10 -C 40 The detected content of ) exceeded the screening value for Class II land use in the "Supplementary Provisions (Trial) on Soil Pollution Status Investigation, Risk Assessment, Risk Control and Remediation Plan Preparation and Risk Control and Remediation Effect Evaluation of Construction Land in ×× City", with a maximum exceeding multiple of 46.75 times; therefore, the soil remediation goal is to degrade the petroleum hydrocarbon content in the soil until it does not exceed the screening value for Class II land use.

[0086] Experimental Example: 1. Soil samples were remediated and degraded using the methods of Examples 1 to 21 respectively. The degradation results after 15 days are as follows;

[0087] Tests revealed that C was extracted from the soil of the abandoned chemical plant site. 10 -C 40 Petroleum hydrocarbons include C 10 Saturated alkanes, C 12 Saturated alkanes, C 20 Saturated alkanes, C 30 Saturated alkanes, C 10 Cycloalkanes, C 30 Cycloalkanes, C 10 Olefins, C 30 Olefins and C 10 Alkynes; C 10 -C 40 Petroleum hydrocarbon degradation rate = [(initial C)] 10 -C 40 Total mass of petroleum hydrocarbons - C after degradation10 -C 40 (Total mass of petroleum hydrocarbons) / Initial C 10 -C 40 [Total mass of petroleum hydrocarbons] × 100%;

[0088] 1. Investigate the effects of different degradation pathways on C 10 -C 40 The impact of petroleum hydrocarbon degradation;

[0089] Comparative Example 1: Unlike Example 1, S1-S4 were not performed. Equal amounts of Pseudomonas spp. and Acinetobacter spp. were directly added for stirring and repair. The inoculum amount was the same as in Example 1.

[0090] Comparative Example 2: Unlike Example 1, the S3-S4 operations were not performed; instead, the degrading bacteria from S2 were directly added to the soil for degradation.

[0091] Comparative Example 3: Unlike Example 1, the primary remediation of petroleum hydrocarbon contaminated soil in S3 was not carried out; instead, the secondary remediation of petroleum hydrocarbon contaminated soil in S4 was carried out directly.

[0092] Comparative Example 4: Unlike Example 1, no voltage was applied in S4, and the degradation was carried out directly by stirring;

[0093] Comparative Example 5: Unlike Example 1, no surfactant was added;

[0094] Comparative Example 6: The difference from Example 1 is that the surfactant is fatty alcohol polyoxyethylene ether.

[0095] Comparative Example 7: Unlike Example 1, in S3, surfactants, activated carbon, and petroleum hydrocarbon-contaminated soil were mixed and stirred and left to stand.

[0096] Example 1 and Comparative Examples 1 to 6 were compared, as shown in Table 1;

[0097] Table 1 C under different processing methods 10 -C 40 Degradation results of petroleum hydrocarbons

[0098] parameter Degradation rate % Example 1 75.0 Comparative Example 1 34.5 Comparative Example 2 49.1 Comparative Example 3 52.0 Comparative Example 4 60.1 Comparative Example 5 55.6 Comparative Example 6 67.9 Comparative Example 7 71.0

[0099] As shown in Table 1, comparing Example 1 and Comparative Example 1, the degradation rate in Example 1 is higher. This is because Example 1 combines soil pretreatment, specific microbial screening, surfactants, and activated carbon bioremediation technologies, thereby improving the targeting and efficiency of the remediation process. Compared with direct addition of microorganisms for stirring remediation, this method increases the accessibility of pollutants through pretreatment, and the screened degradation bacteria are more targeted. The primary and secondary remediation steps for petroleum hydrocarbon-contaminated soil enhance the degradation effect of petroleum hydrocarbons through the comprehensive application of physical, chemical, and biological methods, improving the thoroughness and speed of the overall remediation.

[0100] Comparing Example 1 and Comparative Example 2, it can be seen that the degradation effect of using degrading bacteria alone in Comparative Example 2 is relatively limited. In Example 1, the addition of activated carbon and surfactant can provide a good living environment for microbial degradation, and the application of voltage can further improve the degradation activity of microorganisms.

[0101] Comparing Example 1 and Comparative Example 3, it can be found that directly performing secondary remediation of petroleum hydrocarbon-contaminated soil without primary remediation leads to a decrease in degradation efficiency. This may be because primary remediation of petroleum hydrocarbon-contaminated soil involves the initial inoculation of microorganisms and the initial degradation process of petroleum hydrocarbons, which helps to reduce the concentration of pollutants in the soil. Skipping this step may lead to problems such as reduced degradation efficiency of microorganisms at higher pollutant concentrations, poor microbial adaptability, uneven distribution of pollutants, and prolonged remediation process.

[0102] Comparing Example 1 and Comparative Example 4, it can be seen that applying voltage in Example 1 has a good effect. This may be because applying voltage can enhance the migration and degradation efficiency of pollutants. Its advantage is that it promotes the directional movement of pollutants in the soil through electrodynamic action, accelerates their migration to the electrode area, thereby increasing the contact opportunities between microorganisms and pollutants, enhancing the biodegradation effect, and improving the physical and chemical properties of the soil, providing a more suitable growth environment for microorganisms, thereby improving the efficiency and success rate of the overall remediation process.

[0103] Comparing Example 1, Comparative Example 5, and Comparative Example 6, it can be seen that the surfactant in Example 1 is more preferred. This may be because, compared with fatty alcohol polyoxyethylene ether, Example 1 has stronger emulsifying ability and higher biocompatibility when remediating petroleum hydrocarbon-contaminated soil, and can more effectively promote the dispersion of petroleum hydrocarbons and the degradation by microorganisms, while reducing the potential impact on the environment.

[0104] Comparing Example 1 and Comparative Example 7, it can be found that the degradation effect of Example 1 is better. This may be because the method of Example 1 can more effectively promote the contact and emulsification of surfactant and petroleum hydrocarbons, as well as the adsorption of pollutants by activated carbon, thereby improving the overall remediation efficiency and reducing the rapid aggregation of activated carbon. In Comparative Example 7, the surfactant, activated carbon and petroleum hydrocarbon contaminated soil were mixed and stirred and left to stand, which may lead to poor adsorption and emulsification effects and easy aggregation, thus affecting the degradation effect.

[0105] 2. Investigate the effects of different parameters on;

[0106] Examples 1, 8-14, and 17-18 were compared, as shown in Table 2.

[0107] Table 1 C under different processing methods 10 -C 40 Degradation results of petroleum hydrocarbons

[0108]

[0109]

[0110] As can be seen from Table 2, comparing Examples 1, 4, and 5, the microbial screening parameters in Example 1 are more optimized. This may be because the parameters in Example 1 are suitable for the cultivation and screening of degrading bacteria, resulting in better degradation effects of the degrading bacteria obtained under the conditions of Example 1. Comparing Examples 1, 6, and 7, the composition of the petroleum hydrocarbon culture medium in Example 1 is more optimized. Comparing Examples 1, 8, and 9, the composition of the petroleum hydrocarbon culture medium in Example 1 is more optimized. Comparing Examples 1, 12, and 13, the different proportions of materials used for the one-time remediation of petroleum hydrocarbon contaminated soil in Example 1 are more optimized. Preferably, the proportion of activated carbon added in Example 1 is more suitable. Comparing Examples 14 and 15, it can be found that the stirring parameters in Example 1 are more preferred. This may be because the stirring time and stirring speed in Example 1 are more suitable for mixing activated carbon and contaminated soil, so as to reduce the occurrence of agglomeration. Comparing Examples 1, 18 and 19, it can be found that the moisture content parameter in Example 1 is more preferred. Comparing Examples 1, 20 and 21, it can be found that the voltage parameter in Example 1 is more preferred. This may be because the voltage in Example 1 has the best promoting effect on microorganisms, thereby making the microorganisms more active and the degradation performance better.

Claims

1. A method for microbial remediation of petroleum hydrocarbon-contaminated soil, characterized in that, Includes the following steps: S1. Pretreated soil; Soil samples were collected from multiple depths at various locations within the abandoned chemical plant site and mixed to obtain soil samples. These soil samples were then air-dried, crushed, and sieved to obtain pretreated soil. The abandoned chemical plant site was designated as C. 10 -C 40 Petroleum hydrocarbon contaminated sites; S2, Microbial screening; According to the C 10 -C 40 Petroleum hydrocarbons and the pretreated soil were cultured to obtain a degrading microbial community; including: S2-1. Take 1-5g of the pretreated soil, place it on petroleum hydrocarbon culture medium, and then let it stand at 34-35℃ for 70-85h to obtain preliminary colonies. S2-2. Then, the preliminary bacterial colonies are mixed in petroleum hydrocarbon culture medium and stirred at a temperature of 30-32℃, a stirring speed of 100-120 r / min, and a stirring time of 3-5 days to obtain the preliminary bacterial population. S2-3, Extract C from the soil of the abandoned chemical plant site. 10 -C 40 Petroleum hydrocarbons, C added to petroleum hydrocarbon culture medium 10 -C 40 Petroleum hydrocarbons were then collected, and the initial bacterial population was divided into three equal parts and inoculated onto C14 and C24 respectively. 10 -C 40 In petroleum hydrocarbon culture media with concentrations of 50 mg / L, 200 mg / L, and 500 mg / L, the inoculated petroleum hydrocarbon culture media were shaken at a temperature of 32–34℃ for 48–72 h. S2-4. Determine the degradation rate of petroleum hydrocarbons in the three inoculated culture media. Select one or two preliminary strains with the highest degradation rate from each inoculated culture media. Mix the selected preliminary strains in equal amounts and place them on a shaker for 3-5 seconds of ultraviolet irradiation at a wavelength of 253-265 nm. Then, culture them at 30-36℃ and 150 r / min for 3-5 days to obtain the degrading bacterial community. S3. One-time remediation of petroleum hydrocarbon-contaminated soil; Take a surfactant at a volume fraction of 0.5% and activated carbon at a volume fraction of 2-5% of the petroleum hydrocarbon-contaminated soil. First, mix the surfactant with the petroleum hydrocarbon-contaminated soil and stir for 15-20 minutes. Then, add the activated carbon and stir for 5-10 minutes, followed by standing for 2-5 hours. The surfactant is a mixture of cocamidopropyl betaine, sodium dodecylbenzene sulfonate, and rhamnolipid in a mass ratio of 5-7:1:1-2. S4. Secondary remediation of petroleum hydrocarbon-contaminated soil; When the soil moisture content is 15-20%, a voltage is applied to the petroleum hydrocarbon contaminated soil. At the same time as the voltage is applied, the degradation bacteria obtained in S2 are added to the petroleum hydrocarbon contaminated soil and stirred evenly. After the voltage is applied, the soil moisture content is adjusted to 40-60% and maintained. Then, the soil is stirred and aerated regularly until the petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil does not exceed the standard, and the remediation is completed. The voltage is applied for 1–5 hours, the electrode spacing is set to 30–50 cm, and the electric field strength is 1–2 V / cm; the inoculum size of the degrading bacteria is 6 × 10⁻⁶ per kilogram of soil. 7 ~1×10 8 Each cell.

2. The microbial remediation method for petroleum hydrocarbon-contaminated soil as described in claim 1, characterized in that, The point density mentioned in S1 is 5×5m, and the multiple depths include 0.5m, 1m and 2m.

3. The microbial remediation method for petroleum hydrocarbon-contaminated soil as described in claim 1, characterized in that, The soil sample described in S1 was passed through a 50-100 mesh sieve.

4. The microbial remediation method for petroleum hydrocarbon-contaminated soil as described in claim 1, characterized in that, In step S3, the stirring speed for mixing the surfactant with the petroleum hydrocarbon-contaminated soil is 500–600 r / min, and the stirring speed after adding activated carbon is 100–200 r / min.

5. The microbial remediation method for petroleum hydrocarbon-contaminated soil as described in claim 1, characterized in that, The petroleum hydrocarbon culture medium described in S2-1, by weight, comprises 20-25 parts agar, 3-5 parts sodium chloride, 0.01-0.5 parts crude oil, 1-2 parts sodium nitrate, 1.2-1.5 parts peptone, 0.6-0.8 parts beef extract, 0.5-0.8 parts dipotassium hydrogen phosphate, 2-3 parts rhamnolipid, and 1000 parts distilled water.

6. The microbial remediation method for petroleum hydrocarbon-contaminated soil as described in claim 1, characterized in that, The petroleum hydrocarbon culture medium, by weight, comprises 15-20 parts glucose, 3-5 parts sodium chloride, 0.3-0.5 parts gasoline, 0.5-0.8 parts kerosene, 0.5-0.8 parts diesel, 1-1.5 parts fuel oil, 1.2-1.5 parts peptone, 0.6-0.8 parts beef extract, 0.5-0.8 parts dipotassium hydrogen phosphate, and vitamin B1. 12 0.2-0.3 parts, zinc sulfate 0.05-0.08 parts, ferrous sulfate 0.05-0.08 parts, boric acid 0.005-0.006 parts, rhamnolipid 2-3 parts, and distilled water 1000 parts.

7. The microbial remediation method for petroleum hydrocarbon-contaminated soil as described in claim 6, characterized in that, The preparation method of the petroleum hydrocarbon culture medium is as follows: weigh each component of the liquid culture medium according to the weight parts, mix and stir until each component is evenly dispersed, then sterilize at 100-110℃ for 10-15 minutes, and obtain the petroleum hydrocarbon culture medium after cooling.

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