A method for micro-nano aeration-enhanced microbial in-situ remediation or control of petroleum-contaminated sites

Abstract

The present invention relates to a method for in-situ microbial remediation or management of petroleum-contaminated sites using micro-nano aeration. The method comprises the following steps: 1) injecting micro-nano bubbles into soil or groundwater for continuous aeration for 3-5 days, with a daily aeration time of 6-10 hours; the air source of the micro-nano bubbles is one or a combination of air and oxygen; and 2) injecting a composite microbial inoculum into the soil or groundwater 12-24 hours after step 1), wherein the composite microbial inoculum comprises composite microorganisms, including Pseudomonas, Reysella, Phenylobacter, and Bacillus brevis, with a viable cell count ratio of 1-25:1-15:1 to 5:1. This method has low energy consumption, does not produce secondary pollution, and has excellent remediation or management effects. After the remediation, the concentration of total petroleum hydrocarbon (C10-C40) pollutants in the contaminated site is significantly reduced.

Description

Technical Field

[0001] The present invention relates to the field of contaminated soil and groundwater treatment, and in particular to a method for micro-nano aeration-enhanced microbial in-situ remediation or control of petroleum-contaminated sites. Background Art

[0002] In the current soil and groundwater remediation industry, physical and chemical methods are mostly used in engineering projects, and a small amount of microbial technology is also used. However, physical and chemical methods have high energy consumption and may cause secondary pollution during the remediation process. When microbial technology is used for in-situ remediation of soil and groundwater in contaminated sites, including petroleum pollution, there is a problem, that is, the type of microorganisms with better remediation effects are usually aerobic, while soil and groundwater are often in anaerobic environments. The lack of oxygen inhibits the remediation effect of microorganisms, resulting in limited remediation effects.

[0003] To increase the oxygen content in soil and groundwater, existing technologies include introducing oxygen into the soil and groundwater through aeration. However, traditional aeration methods either rely on chemical reactions to produce oxygen or aeration in the form of millimeter-scale bubbles. The former requires additional chemicals and has poor sustainability. The latter has a short bubble residence time, requiring continuous aeration, and the oxygen supply is limited by the water solubility of oxygen. As a result, the oxygen content in the soil and groundwater is still insufficient, which is not conducive to the growth and reproduction of aerobic microorganisms, thus affecting the ultimate remediation effect.

[0004] Micro-nano aeration technology has been applied in soilless cultivation, aquaculture, surface water environment management, industrial wastewater treatment, etc., but it is less used in soil and groundwater environment remediation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies and shortcomings of the existing technology and provide an improved in-situ remediation or control method for soil and groundwater in petroleum-contaminated sites. The method has low energy consumption, does not produce secondary pollution, and has excellent remediation or control effects. After remediation, the concentration of total petroleum hydrocarbon (C10-C40) pollutants is significantly reduced.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for in-situ remediation or management of a petroleum-contaminated site, comprising the following steps:

[0008] 1) Injecting micro-nano bubbles into the soil or groundwater for continuous aeration for 3-5 days, with a daily aeration time of 6-10 hours; the gas source of the micro-nano bubbles is air, oxygen, or a combination of the two;

[0009] 2) 12-24 hours after step 1), injecting a composite microbial agent into the soil or groundwater, wherein the composite microbial agent includes composite microorganisms, and the composite microorganisms include Pseudomonas, Ralstonia, Phenylobacter and Brevibacillus with a viable cell count ratio of 1-25:1-15:1~5:1.

[0010] In this invention, remediation refers to the treatment of a site after it has been contaminated, while management and control can monitor and prevent pollution before the site is contaminated.

[0011] In some embodiments, the particle size of the micro-nano bubbles is 90-120 nm.

[0012] In some embodiments, the soil or groundwater is formed into a unit at a depth of 5 m, and in step 1), the micro-nano bubbles are injected into each unit at a depth of 4-6 m. The remediation method of the present invention can be used to remediate contaminated sites at various depths.

[0013] In some embodiments, the ratio of the number of viable bacteria of Pseudomonas, Ralstonia, Phenylobacterium, and Brevibacillus is 2-10:2-10:1-3:1.

[0014] In some embodiments, the ratio of the number of viable bacteria of Pseudomonas, Ralstonia, Phenylobacterium, and Brevibacillus is 3-7:2-5:1-2:1.

[0015] In some embodiments, the composite microorganism may be in liquid or solid form. For example, it may be microbial fermentation broth, microbial freeze-dried powder, or glycerol bacteria. When the composite microorganism is in liquid form, the viable cell count of the composite microorganism is not less than 1×10 9 CFU / mL, the composite microbial agent is used directly or diluted with water; when the composite microorganism is in solid form, the composite microorganism is activated and cultured to a viable count of not less than 1×10 9 CFU / mL.

[0016] In some embodiments, the concentration of the composite microbial agent in the soil or groundwater is 0.5-10×10 8 CFU / m 3 .

[0017] In some embodiments, the pollutants in the petroleum-contaminated site include one or more combinations of benzene series, petroleum hydrocarbons, chlorinated hydrocarbons, and polycyclic aromatic hydrocarbons, wherein benzene series refers to benzene and its homologues.

[0018] In some embodiments, the benzene series can be toluene, ethylbenzene, etc.; the petroleum hydrocarbons can be petroleum hydrocarbons (C10-C40), etc.

[0019] In some embodiments, the micro-nano bubbles and the composite microbial agent are injected at the same point; alternatively, the composite microbial agent is injected downstream of the micro-nano bubbles and is less than or equal to 1 meter from the micro-nano bubble injection point. The term "downstream" is defined relative to the groundwater field.

[0020] In some embodiments, the method further comprises step 3) performing micro-nano bubble aeration every 3-4 days for 3-5 hours within 12 weeks after the injection of the composite microbial agent, and performing micro-nano bubble aeration every 6-8 days for 3-5 hours after 12 weeks.

[0021] In some embodiments, the soil is selected from silt, clay, or sand.

[0022] In some embodiments, the soil is clay.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. The in-situ remediation or control method of the present invention does not involve physical or chemical methods. It uses a specific microbial agent, namely a composite agent of Pseudomonas, Ralstonia, Phenylobacter and Bacillus brevis, for microbial technology. At the same time, the gas source of micro-nano aeration is mainly air or oxygen. The entire method has low energy consumption, a small carbon footprint, and is clean without secondary pollution.

[0025] 2. The present invention first performs sufficient micro-nano bubble aeration to improve the oxygen content of the soil and groundwater, and then uses a specific composite microbial agent to perform soil remediation or control. This can significantly reduce the concentration of petroleum hydrocarbon (C10-C40) pollutants in the soil after remediation or control, and the remediation speed is fast and the remediation effect lasts for a long time.

[0026] 3. As a bioremediation technology, microbial remediation does not require a large amount of engineering means or too much chemical consumption. It is green, low-carbon, clean and has no secondary pollution. This continuous degradation is still efficient in the early stage after the end of micro-nano bubble aeration, because the aerobic environment provided by micro-nano aeration can continue to exist for a period of time after the end of aeration. DETAILED DESCRIPTION

[0027] The present invention adopts a composite microbial agent of four specific microbial agents and controls the specific ratio of the four microbial agents, so as to realize a composite microbial agent with a significant degradation effect on pollutants in the soil and groundwater of petroleum-contaminated sites under experimental conditions.

[0028] The functions of each bacterial community and the principles of their synergy are as follows:

[0029] Pseudomonas bacteria can oxidize aromatic hydrocarbons (such as BTEX) and alkanes through first oxygenases (such as cytochrome P450) to produce intermediates such as catechols and organic acids. They secrete surfactants such as rhamnolipids, which promote the dispersion of LNAPLs (light non-aqueous liquids, organic liquid pollutants that are lighter than water and insoluble, commonly found at petroleum hydrocarbon-contaminated sites), thereby increasing the accessibility of other bacteria to hydrophobic pollutants. Furthermore, they provide other bacteria with readily degradable intermediates (such as fatty acids and alcohols) and can form fixed bacterial communities through biofilms, enhancing local degradation efficiency.

[0030] Phenylobacterium specifically targets aromatic hydrocarbon degradation, utilizing monooxygenases and dioxygenases to cleave benzene ring structures (e.g., benzene and toluene) to generate dihydroxy intermediates that are further ring-opened to organic acids (e.g., succinate). Furthermore, it can complement Pseudomonas to process complex aromatic hydrocarbon intermediates and prevent metabolic inhibition. Under hypoxic conditions, it may participate in microaerobic degradation (e.g., relying on nitrate as an electron acceptor).

[0031] Brevibacillus bacteria secrete esterases and lipases, which degrade long-chain alkanes (C16+) into short-chain fatty acids (such as acetic acid and propionic acid). Their spore morphology is resistant to stresses such as high temperature and hypoxia, stabilizing bacterial colony function. They can convert the difficult-to-degrade long-chain hydrocarbons produced during the degradation process into readily usable small molecules for metabolism by other bacteria. Furthermore, under anoxic conditions, they can continue to degrade organic matter through nitrate / sulfate reduction.

[0032] Reyranella can remove toxic intermediates (such as phenols and aldehydes), prevent inhibition, and provide nitrates through denitrification to support the metabolism of facultative anaerobic bacteria.

[0033] The synergistic mechanism of the four:

[0034] During the degradation of organic pollutants, in the composite bacterial agent provided by the present invention, Pseudomonas first secretes surfactants such as rhamnolipids to promote the dispersion of BTEX and alkanes, enhancing the accessibility of other bacteria to hydrophobic pollutants. Oxygenases (such as cytochrome P450) then oxidize aromatic hydrocarbons (such as BTEX) and alkanes to produce intermediates such as catechol and organic acids. Next, Phenybacterium utilizes monooxygenases / dioxygenases to cleave the complex intermediates produced by Pseudomonas metabolism and the remaining pollutants (such as BTEX), generating dihydroxy intermediates that are further ring-opened to organic acids (such as succinic acid). Brevibacillus and Ralstonia then completely mineralize the intermediates produced by Pseudomonas and Phenybacterium metabolism into CO2 / H2O. Furthermore, as the core biofilm microbiome, Pseudomonas secretes extracellular polymeric substances (EPS) to form a stable biofilm framework, encouraging the integration of other bacterial species and creating a microenvironment conducive to degradation. In terms of electron acceptor collaboration, Pseudomonas and Phenylobacterium dominate, relying on O₂ to degrade hydrocarbons. However, Bacillus brevis and Ralstonia can participate in nitrate / sulfate reduction under hypoxic conditions, maintaining continuous degradation. Regarding nutrition, Bacillus brevis breaks down proteins to provide a nitrogen source, supporting the growth of other bacteria. Ralstonia eliminates toxic intermediates (such as phenols and aldehydes) produced during metabolism, preventing these products from inhibiting the activity of functional bacterial communities. Among bacterial communities, such as Pseudomonas and Phenylobacterium, degradation genes, such as oxygenase genes, may be transferred via plasmids, enhancing overall functionality. In terms of environmental adaptability, Bacillus brevis can tolerate stresses such as high temperature and hypoxia, gaining an advantage under extreme conditions and stabilizing bacterial community function.

[0035] The synergistic mechanism of the four strains of the present invention is summarized as follows:

[0036]

[0037] The present invention also incorporates micro-nano bubble technology. Prior to injecting the microbial composite agent, micro-nano bubble technology is employed, while the initial aeration duration of the micro-nano bubbles is controlled to ensure sufficient oxygen and bubbles are delivered to the soil and groundwater, facilitating an initial increase in oxygen content and subsequent sustained high oxygen levels. The initial aeration, which lasts for three to five days, is intended to provide a soil and groundwater environment with a high oxygen content for the applied aerobic agent. First, due to the complexity and heterogeneity of the actual soil and groundwater environment, insufficient pre-aeration will make it difficult to fully increase the oxygen content in the soil pores, hindering the proliferation and spread of exogenous bacterial species. Second, altering the oxygen environment in the soil and groundwater inhibits anaerobic soil bacteria while also promoting aerobic soil bacteria. The soil and groundwater environment gradually becomes anaerobic with increasing depth, while the contaminated environments addressed by the present invention are typically anaerobic. Therefore, aeration itself has a certain inhibitory effect on native microbial communities, helping exogenous bacterial species establish community dominance after addition.

[0038] Due to their slow rise, long residence time, and pressurized dissolution, micro-nano bubbles can create a long-term, stable, and highly oxygenated aerobic environment in soil and groundwater. This further facilitates the growth and reproduction of subsequent composite microbial agents, thereby improving the effectiveness of pollutant remediation. Micro-nano bubbles persist in water for a long time, providing long-term oxygen supply through pressurized dissolution, transforming the original anaerobic environment of soil and groundwater into an aerobic environment suitable for the proliferation and activity of aerobic bacteria.

[0039] Regarding the aeration time of micro-nano bubbles before injecting the microbial agent, the present invention not only takes into account the oxygen content in the soil and groundwater, but also the storage quantity of micro-nano bubbles in the underground environment. Micro-nano bubbles can store oxygen. When micro-nano bubble aeration is paused, oxygen can continue to be supplied to the soil and groundwater through delayed bursting of the bubbles. By performing a sufficient amount of micro-nano bubble aeration before injecting the microbial agent, the present invention stores a sufficient amount of micro-nano bubbles in the soil and groundwater. This can ensure that after the subsequent microbial agent is injected, micro-nano bubble aeration needs to be performed again after a long interval, rather than continuously performing micro-nano bubble aeration after the microbial agent is injected.

[0040] When micro-nano bubbles burst, they produce ROS (including hydroxyl groups, superoxides, and singlet oxygen, all of which are oxidizing). These ROS have a certain bactericidal effect. At the same time, micro-nano aeration will cause large airflow disturbances. Therefore, in order to reduce the impact during the microbial agent injection stage and reduce the weak bactericidal effect caused by the bursting of bubbles during the microbial agent injection, the present invention injects the microbial agent after at least about 12 hours after the micro-nano bubble aeration is completed.

[0041] In the present invention, in the first 12 weeks after the injection of the microbial agent, the microbial agent grows and reproduces in large quantities, requiring sufficient oxygen content. After 12 weeks, the number of the microbial agent has increased significantly after reproduction, and the pollutants in the soil and groundwater to be repaired have also been degraded to a low content. At this time, the requirements for microbial growth and reproduction are not high, and the oxygen content requirement is also not high. The frequency of micro-nano bubble aeration can be lower than that in the first 12 weeks.

[0042] The present invention will be further described below with reference to the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use. The implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, the reagents in this application can be purchased commercially or prepared by methods known in the art.

[0043] In the present invention, Pseudomonas can be, for example, Pseudomonas genus numbered BMZ339652 from Mingzhou Bio, Pseudomonas genus numbered TS278212, TS278215, TS278216, TS278217, TS278218, TS278220, TS278221, TS278222, TS278223, TS278224, etc. from Testo Bio, and Pseudomonas genus numbered HZB112253 from Gray Algae Bio.

[0044] For example, the Ralstonia species can be obtained from the German National Culture Collection DSMZ with the number DSM23428, DSM23429 or DSM23430.

[0045] Phenylbacterium can be, for example, Phenylbacterium genus numbered B64141, B64140, B64111, or B64110 from Mingzhou Biotechnology.

[0046] The Brevibacillus can be obtained from, for example, Brevibacillus genus with the number BMZ134892 from Mingzhou Biotechnology.

[0047] The present invention first conducts a preliminary screening of microbial agents to identify composite microbial agents that demonstrate significant degradation of pollutants in the soil and groundwater of petroleum-contaminated sites under experimental conditions. This selected composite microbial agent is then used in the present invention's micro-nano aeration-enhanced microbial remediation method.

[0048] During screening, the fermentation broth of each strain can be cultured according to conventional methods in the art to obtain a viable cell count of not less than 1×10 9CFU / mL of fermentation broth of each strain. For example, the strain is first activated and then the seed liquid is cultured. The seed liquid in the logarithmic growth period is collected and inoculated into a fermentation barrel containing liquid culture medium at a 10% inoculation ratio. The mixture is fully shaken to mix evenly, and an aeration device is connected to the fermentation barrel for aeration. The fermentation is expanded and cultured at room temperature for 32 to 48 hours. Samples are taken regularly to monitor the microbial biomass to ensure the growth of the strain. After the fermentation culture is completed, the fermentation broth is concentrated and the bacterial content is determined by the plate colony count method to obtain a viable bacterial count of not less than 1×10 9 CFU / mL of fermentation broth of each strain.

[0049] Experiment 1: Preparation of composite microbial fermentation broth: According to the ratio of the number of live bacteria of Pseudomonas, Ralstonia, Phenylobacter, and Bacillus brevis of 20:15:1:1, the fermentation broths of each strain were mixed to obtain a composite microbial fermentation broth.

[0050] The above-mentioned composite microbial fermentation liquid was diluted 10-fold and used in a soil remediation experiment, where the mass of the diluted fermentation liquid was 10% of the soil mass. The specific experiment was as follows: Toluene, ethylbenzene, and C10-40 petroleum hydrocarbons were characteristic pollutants in the soil of a certain plot, with concentrations exceeding the standard for Class I construction land by approximately 19-23 times. The contamination depth was 0-3 meters below the ground, and the site was clay. Multiple clay soil samples were collected from the plot and injected with the above-mentioned diluted fermentation liquid. The average degradation rates of toluene, ethylbenzene, and petroleum hydrocarbons (C10-C40) were measured on day 0 (i.e., without the addition of the microbial fermentation liquid) and on days 7 and 14 after the addition of the microbial fermentation liquid. The results are shown in Tables 2-4 below.

[0051] Experiment 2: basically the same as Experiment 1, except that the ratio of the number of live bacteria of Pseudomonas, Ralstonia, Phenylobacter, and Brevibacillus was adjusted to 5:3:1:1.

[0052] Experiment 3: basically the same as Experiment 1, except that the ratio of the number of live bacteria of Pseudomonas, Ralstonia, Phenylobacter, and Brevibacillus was adjusted to 1:1:5:1.

[0053] Comparative experiment 1: basically the same as experiment 1, except that the ratio of the number of live bacteria of Pseudomonas, Ralstonia, Phenylbacterium, and Brevibacillus was adjusted to 25:20:1:1, and the total amount of bacterial agent (total amount of fermentation liquid) put into the soil remained unchanged.

[0054] Comparative experiment 2: basically the same as experiment 1, the only difference is that the ratio of the number of live bacteria of Pseudomonas, Ralstonia, Phenylbacterium, and Brevibacillus was adjusted to 1:1:6:2, and the total amount of bacterial agent (total amount of fermentation liquid) put into the soil remained unchanged.

[0055] Comparative experiment 3: basically the same as experiment 1, the only difference is that the composite microbial fermentation liquid does not contain phenylbacillus and brevis, the ratio of the number of live bacteria of Pseudomonas and Ralstonia remains unchanged, and the total amount of bacterial agent (total amount of fermentation liquid) put into the soil remains unchanged.

[0056] Comparative experiment 4: basically the same as experiment 1, with the only difference being that the composite microbial fermentation liquid does not contain Brevibacillus, while the ratio of the number of live bacteria of Pseudomonas, Ralstonia, and Phenylobacter remains unchanged, and the total amount of bacterial agent (total amount of fermentation liquid) put into the soil remains unchanged.

[0057] Comparative experiment 5: basically the same as experiment 1, the only difference is that the composite microbial fermentation liquid does not contain phenylbacterium, while the ratio of the number of live bacteria of Pseudomonas, Ralstonia, and Brevibacillus remains unchanged, and the total amount of bacterial agent (total amount of fermentation liquid) put into the soil remains unchanged.

[0058] Comparative Experiment 6: Basically the same as Experiment 1, the only difference is that the microorganisms in the composite microbial fermentation broth are commercially available organic pollution-degrading bacteria, specifically the bacterial agent ZWDM-W18-N developed by Guangdong Zhongwei Environmental Protection Biotechnology Co., Ltd. for petroleum hydrocarbon pollution.

[0059] The results of the experiments and comparative experiments are shown in Tables 2-4 below, where " / " indicates not tested. Degradation rate testing was conducted according to the following standards: for toluene and ethylbenzene, the standard was HJ605-2011, Determination of Volatile Organic Compounds in Soil and Sediments, by Purge and Trap / Gas Chromatography-Mass Spectrometry; for petroleum hydrocarbons (C10-C40), the standard was HJ1021-2019, Determination of Petroleum Hydrocarbons (C10-C40) in Soil and Sediments, by Gas Chromatography.

[0060]

[0061]

[0062]

[0063] As shown in Tables 2-4, the degradation rates of toluene, ethylbenzene, and petroleum hydrocarbons (C10-C40) in the experimental examples were higher than those in the comparative examples, with Experimental Example 2 achieving the best results. This demonstrates that the composite microorganisms used in the present invention, due to the synergy and specific ratio of the four bacterial species, can achieve significantly better pollutant remediation results in petroleum-contaminated sites.

[0064] Example 1: The composite microbial fermentation broth of Experiment 2 above was combined with micro-nano aeration technology to be used for in-situ soil remediation. The specific steps are as follows:

[0065] Micro-nano bubbles were injected into the groundwater at a single point depth of 4.5 m. Aeration was continued for three days, with a daily aeration period of 8 hours. The micro-nano bubbles had a particle size of 100 nm. On the third day, 12 hours after aeration, a microbial inoculant was injected into the single point. The inoculant was the composite microbial fermentation broth from Experiment 2. This broth was diluted 10-fold before injection, with approximately 200 L of the diluted broth injected into each point. The single point of microbial injection could be either the micro-nano bubble injection point or a location downstream (e.g., approximately 1 m) of the micro-nano bubble injection point. This downstream area is defined relative to the upstream and downstream of the groundwater flow, allowing the micro-nano bubbles to oxygenate the groundwater downward. After microbial inoculant injection, aeration was performed every three days for four hours for the first 12 weeks, and then every seven days for four hours. The air source was ambient air.

[0066] The aeration equipment is a micro-nano bubble generating integrated device, for example, the RJN-MN-1100 engineering model from Shanghai Rujing Environmental Protection Technology Co., Ltd. The specific parameters of the machine are as follows:

[0067] Bubble water output: 1.8 T / h; working pressure: 0.4-0.5 MPa; air flow rate: 0.1-2.5 L / min (standard condition). The gas-liquid dissolution technology is used in conjunction with the nozzle precision shearing to generate bubble water, which can produce micro-nano bubbles with a minimum median particle size of 120-90nm and a bubble concentration of 2-5*10 8 / mL, the standard oxygen mass transfer efficiency SOTE can reach 82-85%, the nanobubble potential is -58.4 to -59.6mV, and the nanobubble properties are stable.

[0068] The soil in Example 1 was surveyed and found to be clay. The characteristics of the soil are described from top to bottom as follows:

[0069] ① Plain fill layer: mainly light grey or light grey-yellow silt, very wet, slightly dense, with occasional small amounts of gravel, uneven composition and density, and poor structural properties. This layer is distributed locally, with a thickness of 1.00 to 3.40 m, averaging 1.99 m.

[0070] ②Silt layer: Light grayish yellow to light gray, soft and plastic, medium density, dull cross-section, low dry strength and toughness, and significant water release upon shaking. This layer is found throughout the site, with a thickness of 16.90 to 18.70 m, averaging 17.97 m.

[0071] ③Silt layer: bluish-gray, saturated, medium-dense to dense, primarily composed of quartz and feldspar, with mica flakes. The particles are sub-rounded and angular, with poor particle gradation, little dry strength and toughness, and responds quickly to shaking. This layer is found throughout the site.

[0072] Pilot test results:

[0073] Sampling: The treated contaminated soil was sampled at 1m, 3m, 5m, 7m and 10m horizontally from the aeration point. Sampling was conducted 7 times in total. The sampling times were 0d (the day of injection of microbial agents), 15d, 30d, 45d, 75d, 105d and 135d after injection of microbial agents.

[0074] Test indicators: Petroleum hydrocarbon (C10-C40) pollutant concentration and microbial biomass. The test method for petroleum hydrocarbon (C10-C40) pollutant concentration is the same as before.

[0075] The specific test results are shown in Table 5-6 below:

[0076]

[0077]

[0078] Example 2: basically the same as Example 1, except that the micro-nano bubbles were aerated continuously for 5 days, 6 hours per day.

[0079] The same test was carried out, and the results are shown in Table 7-8 below:

[0080]

[0081]

[0082] Comparative Example 1: basically the same as Example 1, except that the micro-nano bubbles were aerated continuously for 2 days, 8 hours per day.

[0083] The same test was carried out, and the results are shown in Table 9-10 below:

[0084]

[0085]

[0086] Pilot test conclusion

[0087] 1. Petroleum hydrocarbon (C10-C40) concentration

[0088] From the results of the change in petroleum hydrocarbon (C10-C40) concentration, it can be seen that the difference in the concentration of petroleum hydrocarbon (C10-C40) pollutants at different points at the same distance between Example 1 and Comparative Example 1 is not significant on day 0, but the subsequent pollutant concentration decrease rate of Example 1 is significantly higher than that of Comparative Example 1, and the concentration at the 1m point of Example 1 drops to 3287 mg / Kg on day 30, meeting the requirements of the Class II construction land standard (less than 4500 mg / Kg), and drops to 423 mg / Kg on day 105, meeting the requirements of the Class I construction land standard (less than 826 mg / Kg), and the 10m point also meets the requirements of the Class II construction land standard on day 45, and meets the requirements of the Class I construction land standard on day 165. In comparative example 1, the 1m point needs to wait 75 days to meet the requirements of the second-class construction land standard, and the 10m point needs to wait 135 days to meet the requirements of the second-class construction land standard. Therefore, the repair effect is significantly worse than that of embodiment 1.

[0089] 2. Microbial proliferation

[0090] The results of the change in microbial biomass show that at day 0, the difference in microbial biomass between Example 1 and Comparative Example 1 at different points at the same distance was not significant. Thereafter, the microbial biomass of Example 1 grew rapidly, and the growth curve conformed to the characteristics of an S-shaped curve. The maximum growth rate occurred between days 15 and 30, and it essentially reached its maximum value on day 45, with subsequent growth tending to be gentle. In Comparative Example 1, however, the microbial biomass grew slightly slower, with a growth rate significantly lower than that of Example 1. This is because after three consecutive days of micro-nano bubble aeration in Example 1, the oxygen content in the soil and groundwater was significantly higher than that in Comparative Example 1. The soil and groundwater environment in Example 1 was more conducive to the growth and reproduction of the injected aerobic bacterial agent. However, after two days of micro-nano bubble aeration in Comparative Example 1, the oxygen content in the soil and groundwater was still not high enough, and the growth and reproduction of the injected microbial agent (aerobic agent) was even slower.

[0091] 3. Microbial proliferation

[0092] The changes in microbial biomass at different distances between Example 1 and Comparative Example 1 indicate that the microbial diffusion rate at Example 1 is faster than that at Comparative Example 1, and the resulting impact range is also larger. In Example 1, the impact range reaches 7 to 10 m after 105 days. However, the impact range at Comparative Example 1 only reaches 5 m after 105 days, with the impact and diffusion being even slower at 7 to 10 m.

[0093] As can be seen, by performing micro-nano bubble aeration of the contaminated site for a sufficiently long period of time before injecting the microbial agent, the present invention ensures that sufficient oxygen is dissolved in the soil and groundwater of the contaminated site. This also stores a sufficient amount of micro-nano bubbles for subsequent continuous oxygen release, thereby facilitating the growth and reproduction of the injected aerobic agent. This improves the final remediation effect and reduces the cost of subsequent micro-nano bubble aeration. Furthermore, the combination of four selected microbial agents of specific species and specific ratios ensures significantly superior remediation results when applied to petroleum-contaminated sites.

[0094] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A method for in-situ remediation or management of petroleum-contaminated sites, characterized in that: The following steps are involved: 1) Injecting micro-nano bubbles into the soil or groundwater for continuous aeration for 3-5 days, with a daily aeration time of 6-10 hours; the gas source of the micro-nano bubbles is air, oxygen, or a combination of the two; 2) 12-24 hours after step 1), injecting a composite microbial agent into the soil or groundwater, wherein the composite microbial agent comprises composite microorganisms, and the composite microorganisms comprise Pseudomonas, Ralstonia, Phenylobacter, and Bacillus brevis at a live cell count ratio of 1-25:1-15:1-5:

1.

2. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The particle size of the micro-nano bubbles is 90-120 nm.

3. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The soil or groundwater is formed into a unit with a depth of 5 m. In step 1), the micro-nano bubbles are injected into each unit at a depth of 4-6 m.

4. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The ratio of the number of live bacteria of Pseudomonas, Ralstonia, Phenylobacterium and Brevibacillus is 2-10:2-10:1-3:

1.

5. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The ratio of the number of live bacteria of Pseudomonas, Ralstonia, Phenylobacterium and Brevibacillus is 3-7:2-5:1-2:

1.

6. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The concentration of the composite microbial agent in the soil or groundwater is 0.5-10×10 8 CFU / m 3 .

7. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The pollutants in the petroleum-contaminated site include one or more combinations of benzene series, petroleum hydrocarbons, chlorinated hydrocarbons, and polycyclic aromatic hydrocarbons.

8. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The micro-nano bubbles and the composite microbial agent are injected through the same point; or, the injection point of the composite microbial agent is downstream of the injection point of the micro-nano bubbles and is less than or equal to 1m away from the injection point of the micro-nano bubbles.

9. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The method further comprises step 3) performing micro-nano bubble aeration every 3-4 days for 3-5 hours within 12 weeks after the injection of the composite microbial agent, and performing micro-nano bubble aeration every 6-8 days for 3-5 hours after 12 weeks.

10. The method for in-situ remediation or management of a petroleum-contaminated site according to claim 1, characterized in that: The soil is selected from silt, clay or sand.

Images