Method for biostimulation of petroleum hydrocarbon contaminated soil remediation and soil carbon sequestration

Abstract

This invention relates to a method for enhancing the remediation of petroleum hydrocarbon-contaminated soil and synergistically improving soil carbon sequestration using indigenous degrading bacteria, belonging to the field of ecological environmental protection and remediation technology. Highly efficient petroleum-degrading bacteria strains Acinetobacter junii A2-1 and Rhodococcus sp. A17 were isolated and screened from petroleum hydrocarbon-contaminated soil and deposited at the China General Microbiological Culture Collection Center (CGMCC). The accession number for strain A2-1 is CGMCC No. 23724, and for strain A17 is CGMCC No. 23725. The A2-1 and A17 strains were prepared into a bio-enhancing agent, which was then inoculated into petroleum hydrocarbon-contaminated soil in appropriate amounts to improve the degradation efficiency of petroleum hydrocarbons. Furthermore, vegetation restoration synergistically enhances the soil's carbon sequestration function. This invention not only accelerates the remediation process of petroleum hydrocarbon-contaminated soil but also significantly improves the carbon sequestration capacity of petroleum hydrocarbon-contaminated soil, which is of great significance for the remediation of contaminated soil and the achievement of the "carbon neutrality" goal.

Description

Technical Field

[0001] This invention belongs to the field of ecological and environmental protection technology, and relates to a method for enhancing the remediation of petroleum hydrocarbon-contaminated soil and synergistically sequestering soil carbon using indigenous degrading bacteria. Background Technology

[0002] Petroleum is a mixture of hydrocarbons, including alkanes, cycloalkanes, and aromatics. It is extremely hydrophobic and difficult to decompose under natural conditions. Petroleum pollution severely damages soil resources. Long-chain alkanes are inert and sticky, harmful to soil organisms, while polycyclic aromatic hydrocarbons are persistent and highly toxic. Short-chain alkanes in petroleum are biotoxic because they can dissolve cell membranes. Petroleum hydrocarbon pollutants present in soil have long-term impacts on soil quality, function, soil ecosystems, and food quality, making it a critical ecological and environmental issue.

[0003] In the field of oil-contaminated soil remediation, bioremediation is superior to physical and chemical treatment methods in terms of both cost and technical requirements. By utilizing specific microbial communities to enhance the oil degradation capacity of contaminated areas and prevent the formation of intermediate toxic metabolites, bioremediation is more environmentally friendly and sustainable. Selecting microorganisms with strong metabolic capacity and high activity in contaminated soil, culturing them, and then adding the degrading bacteria to the contaminated soil to increase the soil degradation rate is an effective bioaugmentation method.

[0004] Adding exogenous microorganisms is a common method to improve the degradation rate of microbial pollutants. However, the effectiveness of bioaugmentation depends on the inoculum survival rate and the metabolic activity of the microorganisms. Microbial growth is affected by environmental and biological factors, including nutrient competition with local microorganisms, temperature, and pH. Therefore, screening for indigenous microorganisms becomes an important option, as they are better able to adapt to polluted environments and enhance the bioaugmentation effect.

[0005] Oil-contaminated soils severely inhibit vegetation growth, significantly reduce photosynthetic efficiency, and consequently drastically decrease soil carbon sequestration. Biofortification can accelerate the remediation of oil-contaminated soils, rapidly restoring vegetation growth and thereby improving soil carbon sequestration efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a method for enhancing the remediation of petroleum hydrocarbon-contaminated soil and synergistically sequestering soil carbon using indigenous degrading bacteria. This method is simple to operate, environmentally friendly, and has both soil remediation and soil carbon sequestration functions.

[0007] To achieve the above objectives, the technical solution adopted by this invention is as follows: Highly efficient petroleum-degrading bacteria are isolated and screened from petroleum hydrocarbon-contaminated soil, and prepared into a bio-enhancing bacterial agent. This agent is then appropriately inoculated into petroleum hydrocarbon-contaminated soil to improve the degradation efficiency of petroleum hydrocarbons. Furthermore, vegetation restoration synergistically enhances the soil's carbon sequestration function. The invention is characterized by the following steps:

[0008] Isolation and screening of indigenous petroleum hydrocarbon degrading bacteria: Indigenous petroleum hydrocarbon degrading bacteria were obtained by domestication and enrichment from soil contaminated with petroleum hydrocarbons. After isolation and purification, two pure culture strains capable of efficiently degrading petroleum hydrocarbons were obtained: Acinetobacter junii strain A2-1 and Rhodococcus sp. strain A17. These two strains have the function of efficiently degrading petroleum hydrocarbons, i.e., indigenous petroleum hydrocarbon degrading bacteria.

[0009] Preparation of petroleum hydrocarbon degrading bacteria inoculants: Strains A2-1 and A17 were inoculated separately into a culture medium prepared from sweet potato starch wastewater. The cultures were then aerobically incubated for 18-36 hours at 20-35℃ and pH 6.5-8.5 until the effective viable bacterial count reached 2×10⁻⁶. 9 When the concentration of CFU / mL exceeds a certain level, stop the culture and obtain A2-1 inoculum and A17 inoculum, respectively.

[0010] Preparation of enhanced remediation microbial agents: Using biochar as a carrier, petroleum hydrocarbon degradation bacteria inoculants are inoculated into biochar at a weight ratio of 1:5 to 1:1 to obtain enhanced remediation microbial agents; A2-1 inoculant and A17 inoculant can be inoculated into biochar alone to obtain A2-1 enhanced remediation microbial agents and A17 enhanced remediation microbial agents, or they can be mixed in any proportion and inoculated into biochar to obtain composite enhanced remediation microbial agents.

[0011] Bioremediation of contaminated soil: Add a bioremediation agent at a weight ratio of 0.5%-5% to petroleum hydrocarbon-contaminated soil, mix thoroughly, maintain soil moisture content at 15%-25%, and carry out bioremediation under natural environmental conditions. The bioremediation agent can be A2-1, A17, or a compound bioremediation agent. Monitor the soil inoculated with the bioremediation agent, replenish moisture as needed, and till the soil to ensure full contact between the agent and the contaminated soil until the pollutant content in the soil falls below the limit of the soil environmental quality standard.

[0012] The petroleum hydrocarbon-degrading bacteria described in the above technical solution were isolated and screened by the inventors from petroleum-contaminated soil in oil fields and preserved at the China General Microbiological Culture Collection Center (CGMCC). The preservation number of Acinetobacter junii A2-1 strain is CGMCC No. 23724, and the preservation number of Rhodococcus sp. A17 strain is CGMCC No. 23725.

[0013] Vegetation restoration synergistically enhances soil carbon sequestration: Soils that have been remediated through the above-mentioned bio-enhanced methods are then cultivated by planting native plants or crops. The plant roots grow in the soil or straw is returned to the field, further increasing the soil organic matter content, increasing soil carbon sequestration, and further improving the soil. Detailed Implementation

[0014] To better understand the technical content of the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0015] Example 1: Using A2-1 enhanced remediation microbial agent to accelerate the degradation of petroleum hydrocarbons in contaminated soil

[0016] Preparation of petroleum hydrocarbon degrading bacteria A2-1 inoculum: A2-1 strain was inoculated into a culture medium prepared from sweet potato starch wastewater and cultured aerobically for 24 hours at 30℃ and pH 7.5. The inoculum was collected when the effective viable count reached 3 × 10⁻⁶. 9 When the concentration of CFU / mL reaches a certain level, stop the culture and obtain A2-1 inoculum.

[0017] Preparation of petroleum hydrocarbon degrading bacteria A2-1 enhanced repair agent: Using biochar as a carrier, A2-1 inoculant was inoculated into the biochar at a weight ratio of 2:5 and mixed thoroughly to obtain A2-1 enhanced repair agent.

[0018] Bio-enhanced remediation of contaminated soil: A2-1 enhanced remediation microbial agent was added at a weight ratio of 2% to contaminated soil with a petroleum hydrocarbon content of 13.3 g / kg. The mixture was thoroughly prepared, and the soil moisture content was maintained at approximately 20%. Bio-enhanced remediation was carried out under natural environmental conditions. The soil inoculated with the enhanced remediation microbial agent was monitored, and water was replenished as needed. The soil was also tilled to ensure sufficient contact between the microbial agent and the contaminated soil. Soil without the enhanced remediation microbial agent served as a control. Soil samples were collected 35 days after remediation to determine the petroleum hydrocarbon content. The final petroleum hydrocarbon concentration in the control group was 11.5 g / kg, while the concentration in the soil with the enhanced remediation microbial agent was 1.7 g / kg, indicating that bio-enhanced remediation significantly improved the degradation rate of petroleum hydrocarbons in the soil.

[0019] Example 2: Using A17 enhanced remediation microbial agent to accelerate the degradation of petroleum hydrocarbons in contaminated soil

[0020] Preparation of petroleum hydrocarbon degrading bacteria A17 inoculum: A17 strain was inoculated into a culture medium prepared from sweet potato starch wastewater and cultured aerobically for 28 hours at 28℃ and pH 8.0. The inoculum was cultured until the effective viable count reached 2 × 10⁻⁶. 9 When the concentration of CFU / mL reaches a certain level, stop the culture and obtain the A17 inoculum.

[0021] Preparation of petroleum hydrocarbon degrading bacteria A17 enhanced repair agent: Using biochar as a carrier, A17 inoculant was inoculated into the biochar at a weight ratio of 3:5 and mixed thoroughly to obtain the A17 enhanced repair agent.

[0022] Bioenhanced remediation of contaminated soil: A17 enhanced remediation microbial agent was added at a weight ratio of 3% to contaminated soil with a petroleum hydrocarbon content of 12.7 g / kg. The mixture was thoroughly prepared, and the soil moisture content was maintained at approximately 20%. Bioenhanced remediation was carried out under natural environmental conditions. The soil inoculated with the enhanced remediation microbial agent was monitored, and water was replenished as needed. The soil was also tilled to ensure sufficient contact between the microbial agent and the contaminated soil. Soil without the enhanced remediation microbial agent served as a control. Soil samples were collected 35 days after remediation to determine the petroleum hydrocarbon content. The final petroleum hydrocarbon concentration in the control group was 10.6 g / kg, while the concentration in the soil with the enhanced remediation microbial agent was 1.3 g / kg, indicating that bioenhanced remediation significantly improved the degradation rate of petroleum hydrocarbons in the soil.

[0023] Example 3: Accelerating the Degradation of Petroleum Hydrocarbons in Contaminated Soil Using Composite Enhanced Remediation Microbial Agents

[0024] Preparation of a composite enhanced repair agent for petroleum hydrocarbon degrading bacteria: Using biochar as a carrier, A2-1 inoculant and A17 inoculant were inoculated into the biochar at a weight ratio of 1:2:5 and mixed thoroughly to obtain the composite enhanced repair agent.

[0025] Bioremediation of contaminated soil: A compound bioremediation agent was added at a weight ratio of 5% to contaminated soil with a petroleum hydrocarbon content of 10.9 g / kg. The mixture was thoroughly mixed, and the soil moisture content was maintained at approximately 20%. Bioremediation was carried out under natural environmental conditions. The soil inoculated with the compound bioremediation agent was monitored, and water was replenished as needed. The soil was also tilled to ensure sufficient contact between the agent and the contaminated soil, while maintaining adequate oxygen levels. Soil without the added bioremediation agent served as a control. Soil samples were collected 35 days after remediation to determine the petroleum hydrocarbon content. The final petroleum hydrocarbon concentration in the control group was 8.1 g / kg, while the concentration in the soil with the added compound bioremediation agent was 0.77 g / kg, indicating that bioremediation significantly improved the degradation rate of petroleum hydrocarbons in the soil.

[0026] Example 4: Planting sunflowers after contaminated soil remediation to synergize with soil carbon sequestration

[0027] The soil from Example 1, after bio-enhanced remediation, was evenly distributed across the remediation site. After thorough tilling, sunflowers were planted, and their roots grew normally in the soil. Soil samples were collected before remediation and after sunflower harvest to determine the soil organic matter content. Sunflowers could not grow normally in the unremediated contaminated soil; however, after bio-enhanced remediation, the sunflowers grew well. Compared to the control, the organic matter content of the remediated soil was significantly increased, and the carbon sequestration of the surface soil at the remediated site increased by an average of more than 2 tons of CO2 per acre.

[0028] Example 5: Planting sorghum after contaminated soil remediation to synergize with soil carbon sequestration

[0029] The soil from Example 2, after bioaugmentation remediation, was evenly distributed across the remediation site. After thorough tilling, sorghum was planted, and its roots grew normally in the soil. Soil samples were collected before remediation and after sorghum harvest to determine the soil organic matter content. Sorghum could not grow normally in the unremediated contaminated soil; however, it grew normally after bioaugmentation remediation. Compared to the control, the organic matter content of the remediated soil was significantly increased; the carbon sequestration of the surface soil at the remediated site increased by an average of over 1.5 tons of CO2 per acre.

[0030] The invention exemplifies the use of different amounts and types of remediation microbial agents to inoculate contaminated soil, which can effectively degrade petroleum hydrocarbon pollutants in contaminated soils with varying petroleum hydrocarbon content. These examples are not listed here.

Claims

1. A method for bio-enhanced remediation of petroleum hydrocarbon-contaminated soil with synergistic soil carbon sequestration, characterized in that, Highly efficient petroleum-degrading bacteria were isolated and screened from petroleum hydrocarbon-contaminated soil and prepared into a bio-enhancing agent. This agent was then inoculated into petroleum hydrocarbon-contaminated soil in appropriate amounts to improve the degradation efficiency of petroleum hydrocarbons. Furthermore, the soil's carbon sequestration function was synergistically enhanced through vegetation restoration.

2. The highly efficient petroleum-degrading bacteria isolated and screened from petroleum hydrocarbon-contaminated soil as described in claim 1, characterized in that, The highly efficient petroleum hydrocarbon degrading bacteria were isolated and screened by the inventors from petroleum-contaminated soil in oil fields and preserved at the China General Microbiological Culture Collection Center (CGMCC). The preservation number of Acinetobacter junii A2-1 strain is CGMCC No. 23724, and the preservation number of Rhodococcus sp. A17 strain is CGMCC No. 23725.

3. The bio-enhancing microbial agent according to claim 1, characterized in that, The steps for preparing bio-enhanced microbial agents include: using biochar as a carrier, inoculating the biochar with petroleum hydrocarbon degrading bacteria inoculants at a weight ratio of 1:5 to 1:1 to obtain enhanced repair microbial agents; A2-1 inoculant and A17 inoculant can be inoculated into the biochar separately to obtain A2-1 enhanced repair microbial agents and A17 enhanced repair microbial agents, or they can be mixed in any proportion and inoculated into the biochar to obtain composite enhanced repair microbial agents.

4. The bio-enhanced method for remediating petroleum hydrocarbon-contaminated soil as described in claim 1, characterized in that, Add a bioremediation agent at a weight ratio of 0.5%-5% to petroleum hydrocarbon-contaminated soil, mix thoroughly, and maintain a soil moisture content of 15%-25%. Perform bioremediation under natural environmental conditions. The bioremediation agent can be A2-1, A17, or a compound bioremediation agent. Monitor the soil inoculated with the bioremediation agent, replenish moisture as needed, and till the soil to ensure full contact between the agent and the contaminated soil until the pollutant content in the soil falls below the limits of the soil environmental quality standards.

5. The method for bio-enhanced remediation of petroleum hydrocarbon-contaminated soil and synergistic soil carbon sequestration according to claims 1-4, characterized in that, The steps to synergistically enhance soil carbon sequestration through vegetation restoration include: adopting tillage methods, planting native plants or crops, allowing plant roots to grow in the soil or returning straw to the field, further increasing soil organic matter content, increasing soil carbon sequestration, and further improving the soil.