Method for in-situ remediation or management and control of petroleum contaminated site through micro-nano aeration reinforced microorganisms

Abstract

The invention relates to a method for in-situ remediation or management and control of a petroleum contaminated site by micro-nano aeration enhanced microorganisms, which comprises the following steps: 1) injecting micro-nano bubbles into soil or underground water, and continuously aerating for 3-5 days for 6-10 hours every day; an air source of the micro-nano bubbles is one or a combination of two of air and oxygen; 2) 12-24 hours after the step 1) is completed, a compound microbial agent is injected into the soil or the underground water, the compound microbial agent comprises compound microorganisms, and the compound microorganisms comprise pseudomonas, leucas, phenyl bacilli and brevibacillus in the viable count ratio of (1-25): (1-15): (1-5): 1. According to the method, the energy consumption is low, secondary pollution is avoided, the remediation or control effect is excellent, and the total petroleum hydrocarbon (C10-C40) pollutant concentration of the contaminated site after remediation is remarkably reduced.

Description

Technical Field

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

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

[0003] In order to increase the oxygen content in soil and groundwater, there is a prior art method of introducing oxygen into soil and groundwater by aeration. However, traditional aeration methods either rely on chemical reactions of agents to generate oxygen or aerate in the form of millimeter-sized bubbles. The former requires additional chemical agents and has poor persistence; the latter has a short bubble residence time, requires continuous aeration, and the oxygen supply effect is limited by the water solubility of oxygen, resulting in a still not high enough oxygen content in the final soil and groundwater, which is not conducive to the growth and reproduction of aerobic microorganisms, thus affecting the final remediation effect.

[0004] Micro-nano aeration technology has been applied in soilless cultivation, aquaculture, surface water environment treatment, industrial wastewater treatment, etc. at present, 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 provide an improved method for in-situ remediation or control of soil and groundwater in petroleum-contaminated sites in view of the deficiencies and drawbacks of the prior art. The method has low energy consumption, does not produce secondary pollution, and has excellent remediation or control effects, and the concentration of total petroleum hydrocarbon (C10-C40) pollutants after remediation is significantly reduced.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for in-situ remediation or control of petroleum-contaminated sites, comprising the following steps: 1) Inject micro-nano bubbles into soil or groundwater, and continuously aerate for 3-5 days, with the aeration time being 6-10 hours per day; the gas source of the micro-nano bubbles is one or a combination of two of air and oxygen; 2) After 12 - 24 hours of completing step 1), a composite microbial inoculant is injected into the soil or groundwater. The composite microbial inoculant includes composite microorganisms, and the composite microorganisms include Pseudomonas, Rheinheimera, Phenylobacterium, and Brevibacillus with a viable cell number ratio of 1 - 25:1 - 15:1 - 5:1.

[0007] In the present invention, remediation refers to the treatment after the site is contaminated, while control can monitor and prevent pollution before the site is contaminated.

[0008] In some embodiments, the particle size of the micro - nano bubbles is 90 - 120 nm.

[0009] In some embodiments, the soil or groundwater is taken as one unit for every 5 m depth. In step 1), the micro - nano bubbles are injected to a depth of 4 - 6 m in each unit. The remediation method of the present invention can be used for the remediation of contaminated sites at various depths.

[0010] In some embodiments, the viable cell number ratio of Pseudomonas, Rheinheimera, Phenylobacterium, and Brevibacillus is 2 - 10:2 - 10:1 - 3:1.

[0011] In some embodiments, the viable cell number ratio of Pseudomonas, Rheinheimera, Phenylobacterium, and Brevibacillus is 3 - 7:2 - 5:1 - 2:1.

[0012] In some embodiments, the composite microorganisms can be in liquid form or solid form. For example, it can be microbial fermentation broth, microbial freeze - dried powder, glycerol bacteria, etc. When the composite microorganisms are in liquid form, the viable cell number of the composite microorganisms is not less than 1×10 9 CFU / mL, and the composite microbial inoculant is used directly or after dilution with water; when the composite microorganisms are in solid form, before using the composite microbial inoculant, the composite microorganisms are activated and cultured until the viable cell number is not less than 1×10 9 CFU / mL.

[0013] In some embodiments, the concentration of the composite microbial inoculant in the soil or groundwater is 0.5 - 10×10 8 CFU / m 3 .

[0014] In some embodiments, the pollutants in the petroleum - contaminated site contain one or more combinations of benzene series compounds, petroleum hydrocarbons, chlorinated hydrocarbons, and polycyclic aromatic hydrocarbons. Among them, benzene series compounds refer to benzene and its homologues.

[0015] In some embodiments, the benzene series compounds can be toluene, ethylbenzene, etc.; the petroleum hydrocarbons can be petroleum hydrocarbons (C10 - C40), etc.

[0016] In some embodiments, 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 the distance from the injection point of the micro-nano bubbles is less than or equal to 1 m. Herein, the downstream is defined with respect to the direction of the groundwater field.

[0017] In some embodiments, the method further includes step 3) within 12 weeks after injecting the composite microbial agent, micro-nano bubble aeration is carried out every 3-4 days, and each aeration lasts for 3-5 h. After more than 12 weeks, micro-nano bubble aeration is carried out every 6-8 days, and each aeration lasts for 3-5 h.

[0018] In some embodiments, the soil is selected from silt, clay or sand.

[0019] In some embodiments, the soil is clay.

[0020] Compared with the prior art, the present invention has the following advantages: 1. The in-situ remediation or control method of the present invention does not involve physical or chemical methods. A specific microbial agent, i.e., a composite agent of Pseudomonas, Thiobacillus, Phenylobacterium and Brevibacillus, is used for microbial technology. At the same time, the gas source for micro-nano aeration is mainly air or oxygen. The whole method has low energy consumption, a small carbon footprint, and is clean without secondary pollution.

[0021] 2. By first carrying out sufficient micro-nano bubble aeration to improve the oxygen content in the soil and groundwater, and then using a specific composite microbial agent for soil remediation or control, the present invention can significantly reduce the concentration of petroleum hydrocarbon (C10-C40) pollutants in the soil after remediation or control, and has a fast remediation speed and a long-lasting remediation effect.

[0022] 3. As a bioremediation technology, microbial remediation does not require a large number of engineering means or too much consumption of chemical agents. It is green and low-carbon, clean without secondary pollution. This continuous degradation is still efficient in the initial stage after the micro-nano bubble aeration ends because the aerobic environment provided by micro-nano aeration can persist for a period of time after the aeration ends. Specific Embodiments

[0023] The present invention adopts a composite microbial agent of four specific agents and controls the specific ratio of the four agents, and can achieve a composite microbial agent with a significant degradation effect on pollutants in the soil and groundwater of petroleum-contaminated sites under experimental conditions.

[0024] The functions of each microbial community and the principle of the synergistic effect among them are as follows: Pseudomonas can oxidize aromatic hydrocarbons (such as BTEX) and alkanes through pioneer oxygenases (such as cytochrome P450) to generate intermediate products such as catechol and organic acids. It secretes surfactants such as rhamnolipids to promote the dispersion of LNAPL (light non-aqueous phase liquid, which refers to organic liquid pollutants that are lighter than water and insoluble in water, commonly found in petroleum hydrocarbon contaminated sites), and improve the contact efficiency of other bacteria with hydrophobic pollutants. In addition, it can provide other bacteria with easily degradable intermediate products (such as fatty acids and alcohols), and can form fixed bacterial communities through biofilm formation to enhance the local degradation efficiency.

[0025] Phenylobacterium can specifically degrade aromatic hydrocarbons. It uses monooxygenase / dioxygenase to cleave the benzene ring structure (such as benzene and toluene) to generate dihydroxy intermediates and further ring-opening to organic acids (such as succinic acid). In addition, it can complement Pseudomonas to process the intermediate products of complex aromatic hydrocarbons and prevent metabolic inhibition. It may participate in microaerobic degradation under low-oxygen conditions (such as relying on nitrate as an electron acceptor).

[0026] Brevibacillus can secrete esterase / lipase to degrade long-chain alkanes (C16+) into short-chain fatty acids (such as acetic acid and propionic acid). And the spore form can tolerate stresses such as high temperature and hypoxia, stabilizing the function of the bacterial community. It can convert the recalcitrant long-chain hydrocarbons generated during the degradation process into easily utilizable small molecules for the metabolism of other bacteria. And it continues to degrade organic matter through nitrate / sulfate reduction under anaerobic conditions.

[0027] Reyranella can remove toxic intermediate products (such as phenol and aldehydes) to prevent inhibitory effects, and can provide nitrate through denitrification to support the metabolism of facultative anaerobes.

[0028] The synergistic mechanism of the four: During the process of degrading organic pollutants, in the composite bacterium agent provided by the present invention, Pseudomonas first secretes surfactants such as rhamnolipids to promote the dispersion of BTEX and alkanes, improve the contact efficiency of other bacteria with hydrophobic pollutants, and oxidize aromatic hydrocarbons (such as BTEX) and alkanes through oxygenases (such as cytochrome P450) to generate intermediate products such as catechol and organic acids. Then, Phenylobacterium uses monooxygenase / dioxygenase to cleave and process the complex intermediate products generated by the metabolism of Pseudomonas, as well as the remaining pollutants (such as BTEX), to generate dihydroxy intermediates and further ring-open them into organic acids (such as succinic acid). Then, Brevibacillus and Rheinheimera completely mineralize the intermediate products generated by the metabolism of pollutants by Pseudomonas and Phenylobacterium into CO2 / H2O. In addition, as the core bacterial community of the biofilm, Pseudomonas can form a stable biofilm framework by secreting extracellular polymeric substances (EPS) and promote the embedding of other bacterial communities to construct a microenvironment conducive to degradation. In terms of the cooperation of electron acceptors, dominated by Pseudomonas and Phenylobacterium, hydrocarbons can be degraded relying on O2, while Brevibacillus and Rheinheimera can participate in the nitrate / sulfate reduction process under low-oxygen conditions to maintain the continuity of degradation. In terms of nutrition, Brevibacillus can decompose proteins to provide a nitrogen source to support the growth of other bacteria, and Rheinheimera can remove toxic intermediate products (such as phenol and aldehydes) generated during the metabolism process to prevent these products from inhibiting the activity of functional bacterial communities. Bacterial communities such as Pseudomonas and Phenylobacterium may transfer degradation genes such as oxygenase genes through plasmids to enhance the overall function. In terms of environmental adaptability, Brevibacillus can tolerate stresses such as high temperature and hypoxia, dominate under extreme conditions, and stabilize the function of the bacterial community.

[0029] The synergistic mechanism of the four strains of the present invention is summarized as follows:

[0030] At the same time, the present invention also cooperates with the micro-nano bubble technology. Before injecting the microbial composite bacterium agent, the micro-nano bubble technology is adopted, and the initial aeration time of the micro-nano bubbles is controlled simultaneously to achieve sufficient oxygen and bubbles transported to the soil and groundwater, which is conducive to the increase of the initial oxygen content and the subsequent continuous high oxygen content. The initial aeration for 3 to 5 consecutive days is to provide a soil and groundwater environment with a relatively high oxygen content for the applied aerobic bacterium agent. First, due to the complexity and heterogeneity of the actual soil and groundwater environment, if the pre-aeration is insufficient, it is difficult to fully increase the oxygen content in the soil pores, which will hinder the proliferation and diffusion of exogenous bacterial species. Second, changing the oxygen environment in the soil and groundwater inhibits anaerobic bacteria in the soil and promotes aerobic bacteria in the soil. With the increase in depth, the soil and groundwater environment gradually changes from aerobic in the surface tillage layer to anaerobic, and the pollution scenarios faced by the present invention are usually anaerobic environments. Therefore, aeration itself also has a certain inhibitory effect on the native microbial community, which helps exogenous bacterial species establish community dominance after being added.

[0031] Due to their slow rising speed, long retention time, pressure-increasing dissolution and other characteristics, micro-nano bubbles can create a long-term stable aerobic environment with a relatively high oxygen content in soil and groundwater, which is further conducive to the growth and reproduction of subsequent composite microbial agents, thereby improving the pollutant remediation effect. Micro-nano bubbles can exist in water for a long time, providing long-term oxygen supply through bubble pressure-increasing dissolution, and transforming the anaerobic environment of the original soil and groundwater into an aerobic environment suitable for the proliferation and activity of aerobic bacteria.

[0032] Regarding the aeration time of micro-nano bubbles before injecting the microbial agent, the present invention not only considers the oxygen content in soil and groundwater, but also takes into account the storage quantity of micro-nano bubbles in the underground environment. Micro-nano bubbles can store oxygen. When the aeration of micro-nano bubbles pauses, oxygen can continue to be supplied to soil and groundwater through the delayed rupture of the bubbles. By performing sufficient aeration of micro-nano bubbles before injecting the microbial agent, the present invention enables sufficient micro-nano bubbles to be stored in soil and groundwater, so that after injecting the subsequent microbial agent, it is possible to achieve that micro-nano aeration does not need to be carried out again for a relatively long time, instead of continuously performing micro-nano bubble aeration after injecting the microbial agent.

[0033] When micro-nano bubbles burst, they will generate ROS (including hydroxyl radicals, superoxide radicals, singlet oxygen, etc., all of which have oxidizing properties), and this ROS has a certain bactericidal effect. At the same time, there will be relatively large airflow disturbances during micro-nano aeration. Therefore, in order to reduce the impact during the stage of injecting the microbial agent and reduce the weak bactericidal effect generated by the bursting of bubbles during the injection of the microbial agent, the present invention injects the microbial agent at least about 12 h after the micro-nano bubble aeration is completed.

[0034] In the present invention, within the first 12 weeks after injecting the microbial agent, the microbial agent grows and reproduces in large quantities and requires a sufficient oxygen content. After 12 weeks, the number of the microbial agent is already large after reproduction, and the pollutants in the soil and groundwater to be repaired have also been degraded to a relatively low content. At this time, the requirements for the growth and reproduction of the microorganism are not high, and the requirements for the oxygen content are also not high. The frequency of micro-nano bubble aeration can be lower than that in the first 12 weeks.

[0035] The present invention will be further described below in conjunction with embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. The technical features involved in each implementation manner 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 obtained commercially or prepared by known methods in the art.

[0036] In the present invention, the Pseudomonas can be, for example, Pseudomonas of the number BMZ339652 from Mingzhou Biology, Pseudomonas of the numbers TS278212, TS278215, TS278216, TS278217, TS278218, TS278220, TS278221, TS278222, TS278223, TS278224, etc. from Taisituo Biology, and Pseudomonas of the product number HZB112253 from Huizao Biology, etc.

[0037] The Leptothrix can be, for example, Leptothrix of the numbers DSM23428, DSM 23429 or DSM 23430 from the German Collection of Microorganisms and Cell Cultures DSMZ purchased on behalf of Huizao Biology.

[0038] The Phenylobacterium can be, for example, Phenylobacterium of the numbers B64141, B64140, B64111, B64110 from Mingzhou Biology.

[0039] The Brevibacillus brevis can be, for example, Brevibacillus brevis of the number BMZ134892 from Mingzhou Biology.

[0040] In the present invention, the types of microbial agents are preliminarily screened first to obtain a composite microbial agent that has a significant degradation effect on the pollutants in the soil and groundwater of petroleum-contaminated sites under experimental conditions. On this basis, the screened composite microbial agent is then used in the micro-nano aeration enhanced microbial remediation method of the present invention.

[0041] During the screening, the fermentation broth of each strain can be cultured according to the conventional methods in the art to obtain the fermentation broth of each strain with a viable count of not less than 1×10 9 CFU / mL. For example, the strain is first activated and cultured, and then the seed liquid is cultured. The seed liquid in the logarithmic growth phase is collected and inoculated into a fermentation tank containing a liquid medium at an inoculation ratio of 10%, and shaken well to make it evenly mixed, and an aeration device is connected to aerate the inside of the fermentation tank, and cultured at room temperature for 32 - 48 h. Samples are taken regularly to monitor its 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 measured by the plate colony counting method to obtain the fermentation broth of each strain with a viable count of not less than 1×10 9 CFU / mL.

[0042] Experiment 1: Prepare a composite microbial fermentation broth: Mix the fermentation broths of each strain according to the ratio of the viable counts of Pseudomonas, Leptothrix, Phenylobacterium, and Brevibacillus brevis being 20:15:1:1 to obtain a composite microbial fermentation broth.

[0043] After diluting the above-mentioned composite microbial fermentation broth by 10 times, it was used in the soil remediation experiment. Among them, the mass of the diluted fermentation broth was 10% of the soil mass. The specific experiment is as follows: Toluene, ethylbenzene, and C10-40 petroleum hydrocarbons were the characteristic pollutants in the soil of a certain plot. The concentration of the characteristic pollutants exceeded the standard for the first-class construction land by about 19-23 times. The pollution depth was 0-3 m below the ground surface. The site was clay. Multiple samples of clay soil from this plot were collected, and the above-mentioned diluted fermentation broth was injected into the clay soil. The average degradation rates of toluene, ethylbenzene, and petroleum hydrocarbons (C10-C40) on the 0th day (i.e., when the microbial fermentation broth was not added) and on the 7th and 14th days after adding the microbial fermentation broth were detected. The results are shown in Tables 2-4 below.

[0044] Experiment 2: It was basically the same as Experiment 1, except that: the viable cell number ratio of Pseudomonas, Rhodopseudomonas, Phenylobacterium, and Brevibacillus was adjusted to 5:3:1:1.

[0045] Experiment 3: It was basically the same as Experiment 1, except that: the viable cell number ratio of Pseudomonas, Rhodopseudomonas, Phenylobacterium, and Brevibacillus was adjusted to 1:1:5:1.

[0046] Comparative Experiment 1: It was basically the same as Experiment 1, except that: the viable cell number ratio of Pseudomonas, Rhodopseudomonas, Phenylobacterium, and Brevibacillus was adjusted to 25:20:1:1, and the total amount of the microbial agent (total amount of the fermentation broth) input into the soil was kept unchanged.

[0047] Comparative Experiment 2: It was basically the same as Experiment 1, except that: the viable cell number ratio of Pseudomonas, Rhodopseudomonas, Phenylobacterium, and Brevibacillus was adjusted to 1:1:6:2, and the total amount of the microbial agent (total amount of the fermentation broth) input into the soil was kept unchanged.

[0048] Comparative Experiment 3: It was basically the same as Experiment 1, except that: the composite microbial fermentation broth did not contain Phenylobacterium and Brevibacillus, the viable cell number ratio of Pseudomonas and Rhodopseudomonas remained unchanged, and the total amount of the microbial agent (total amount of the fermentation broth) input into the soil was kept unchanged.

[0049] Comparative Experiment 4: It was basically the same as Experiment 1, except that: the composite microbial fermentation broth did not contain Brevibacillus, the viable cell number ratio of Pseudomonas, Rhodopseudomonas, and Phenylobacterium remained unchanged, and the total amount of the microbial agent (total amount of the fermentation broth) input into the soil was kept unchanged.

[0050] Comparative Experiment 5: It was basically the same as Experiment 1, except that: the composite microbial fermentation broth did not contain Phenylobacterium, the viable cell number ratio of Pseudomonas, Rhodopseudomonas, and Brevibacillus remained unchanged, and the total amount of the microbial agent (total amount of the fermentation broth) input into the soil was kept unchanged.

[0051] Comparative Experiment 6: It is basically the same as Experiment 1, with the only difference being that the microorganisms in the compound microbial fermentation broth are commercially available organic pollution-degrading microbial communities, specifically the microbial agent ZWDM-W18-N developed by Guangdong Zhongwei Environmental Protection Biotechnology Co., Ltd. for petroleum hydrocarbon pollution.

[0052] The results of each experiment and comparative experiment are shown in Table 2-4 below, where " / " indicates not tested. The degradation rate test method is carried out according to the following standards: For toluene and ethylbenzene, the standard is HJ605-2011 Determination of Volatile Organic Compounds in Soil and Sediments, using purge and trap / gas chromatography-mass spectrometry; for petroleum hydrocarbons (C10-C40), the standard is HJ1021-2019 Determination of Petroleum Hydrocarbons (C10-C40) in Soil and Sediments, using gas chromatography.

[0053]

[0054]

[0055]

[0056] As can be seen from Table 2-4, the degradation rates of the experimental examples for toluene, ethylbenzene, and petroleum hydrocarbons (C10-C40) are higher than those of the comparative experimental examples. Among them, the effect of Experimental Example 2 is the best. It shows that the compound microorganisms used in the present invention can achieve significantly better pollutant remediation effects in petroleum pollution sites due to the synergy of the specific four strains and their specific ratios.

[0057] Example 1: The compound microbial fermentation broth of the above Experiment 2 is combined with the micro-nano aeration technology and used in in-situ soil remediation. The specific steps are as follows: Micro-nano bubbles are injected into groundwater through a single point, with an injection depth of 4.5 m, continuous aeration for 3 days, and an aeration time of 8 h per day. The particle size of the micro-nano bubbles is 100 nm. 12 h after the completion of aeration on the third day, the microbial agent is injected into the single point. The microbial agent is the compound microbial fermentation broth of the above Experiment 2. When used, the fermentation broth is diluted 10 times and then injected. About 200 L of the diluted bacterial solution is injected at each point. The single point where the microorganisms are injected can be the injection point of the micro-nano bubbles or within the range near the downstream of the single point where the micro-nano bubbles are injected (for example, about 1 m), and the downstream is defined relative to the upstream and downstream of the groundwater field, so that the micro-nano bubbles can play an oxygenation role along the direction of the groundwater flow. After the injection of the microbial agent, in the first 12 weeks, aeration is carried out every 3 days, each time for 4 h. After that, aeration is carried out every 7 days, each time for 4 h, and the gas source is ambient air.

[0058] The aeration equipment is an integrated micro-nano bubble generation device. For example, it can be the RJN-MN-1100 engineering model of Shanghai Rujing Environmental Protection Technology Co., Ltd. The specific parameters of this machine are as follows: Bubble water production: 1.8 T / h; Working pressure: 0.4 - 0.5 MPa; Intake air flow: 0.1 - 2.5 L / min (standard condition). Using gas-liquid dissolution technology and combined with precise shearing by a nozzle to generate bubble water, it can produce micro-nano bubbles with a minimum median particle size of 120 - 90 nm, and the bubble concentration can reach 2 - 5*10 8 per mL, the standard oxygen mass transfer efficiency SOTE can reach 82 - 85%, the potential of nano-bubbles is -58.4 to -59.6 mV, and the properties of nano-bubbles are stable.

[0059] Geological exploration was carried out on the soil in Example 1. The soil is a clay site, and its characteristics are described as follows from top to bottom: ① Plain fill layer: mainly light gray and light grayish-yellow silt, very wet, slightly dense, occasionally seeing a small amount of gravel, with uneven composition and density, and poor structure. This layer is distributed locally in the site, with a thickness of 1.00 - 3.40 m and an average of 1.99 m.

[0060] ② Silt layer: light grayish-yellow and light gray, soft plastic, medium dense, no luster on the cut surface, low dry strength and toughness, and obvious water separation by shaking. This layer covers the entire site, with a thickness of 16.90 - 18.70 m and an average of 17.97 m.

[0061] ③ Silt sand layer: bluish-gray, saturated, medium dense to dense, mainly composed of quartz and feldspar as minerals, containing mica flakes, with sub-round and angular particle shapes, poor particle gradation, no dry strength and toughness, and a rapid shaking reaction. This layer covers the entire site.

[0062] Pilot test results detection: Sampling: Sampling was carried out on the treated contaminated soil. The sampling points were 1 m, 3 m, 5 m, 7 m, and 10 m horizontally from the aeration point. A total of 7 samplings were carried out, and the sampling times were 0 d (the day when the microbial inoculant was injected), 15 d, 30 d, 45 d, 75 d, 105 d, and 135 d after the injection of the microbial inoculant.

[0063] Detection indicators: The concentration of petroleum hydrocarbon (C10 - C40) pollutants and the microbial biomass. The test method for the concentration of petroleum hydrocarbon (C10 - C40) pollutants is the same as before.

[0064] The specific detection results are shown in Table 5 - 6 as follows:

[0065]

[0066] Example 2: Basically the same as Example 1, the difference is only that: micro-nano bubble continuous aeration for 5 days, 6 hours per day.

[0067] The same detection was carried out, and the results are shown in Table 7 - 8 as follows:

[0068]

[0069] Comparative Example 1: It is basically the same as Example 1, except that: micro-nano bubble continuous aeration is carried out for 2 days, 8 hours per day.

[0070] The same detection is carried out, and the results are shown in Table 9-10 below:

[0071]

[0072] Pilot test conclusion 1. Concentration of petroleum hydrocarbons (C10-C40) From the results of the change in the concentration of petroleum hydrocarbons (C10-C40), it can be seen that at 0 d, the concentrations of petroleum hydrocarbon (C10-C40) pollutants at different positions at the same distance in Example 1 and Comparative Example 1 were not very different. However, subsequently, the pollutant concentration decline rate in Example 1 was significantly higher than that in Comparative Example 1. At the 1 m position in Example 1, the concentration dropped to 3287 mg / Kg at the 30th day, meeting the requirements of the second-class land standard for construction land (less than 4500 mg / Kg). At the 105th day, the concentration dropped to 423 mg / Kg, meeting the requirements of the first-class land standard for construction land (less than 826 mg / Kg). At the 45th day at the 10 m position, it also met the requirements of the second-class land standard for construction land, and at the 165th day, it met the requirements of the first-class land standard for construction land. In Comparative Example 1, at the 1 m position, it took until the 75th day to meet the requirements of the second-class land standard for construction land, and at the 10 m position, it took until the 135th day to meet the requirements of the second-class land standard for construction land. Therefore, the remediation effect was significantly worse than that of Example 1.

[0073] 2. Microbial proliferation From the results of the change in microbial biomass, it can be seen that at 0 d, the microbial biomass at different positions at the same distance in Example 1 and Comparative Example 1 was not very different. After that, the microbial biomass in Example 1 increased rapidly, and the growth curve conformed to the characteristics of the S-shaped curve. The maximum growth rate occurred between the 15th and 30th days and basically reached the maximum at the 45th day, and the subsequent growth tended to level off. In Comparative Example 1, the microbial biomass increased slightly slower, and the growth rate was significantly lower than that in Example 1. The reason is that after 3 days of continuous micro-nano bubble aeration in Example 1, the oxygen content in the soil and groundwater was significantly higher than that in Comparative Example 1, and the soil and groundwater environment in Example 1 was more conducive to the growth and reproduction of the injected aerobic bacteria agent. In Comparative Example 1, after 2 days of micro-nano bubble aeration, the oxygen content in the soil and groundwater was not high enough, and the growth and reproduction of the injected microbial agent (aerobic bacteria agent) were slower.

[0074] 3. Microbial diffusion From the results of the changes in the microbial biomass at different distances from the points in Example 1 and Comparative Example 1, it can be seen that the microbial diffusion rate at the point in Example 1 is faster than that at the point in Comparative Example 1, and the final influence range is also larger. In Example 1, it can reach 7 - 10 m in 105 days. However, for the point in Comparative Example 1, the influence range only reaches 5 m at 105 days, and the influence and diffusion at 7 - 10 m are slower.

[0075] It can be seen that before injecting the microbial agent, the contaminated site to be treated is aerated with micro - nano bubbles for a sufficient long time in the present invention, so that enough oxygen is dissolved in the soil and groundwater in the contaminated site, and a sufficient amount of micro - nano bubbles are stored for subsequent continuous oxygen release. This is conducive to the growth and reproduction of the injected aerobic microbial agent, thereby improving the effect of the final pollution remediation and saving the cost of subsequent micro - nano bubble aeration. At the same time, combined with the four microbial agents with specific species and specific ratios screened out, it can ensure significantly excellent remediation effects when used in petroleum - contaminated sites.

[0076] The above - mentioned embodiments are only used to illustrate the technical concept and characteristics of the present invention, and their purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for in-situ remediation or control of petroleum-contaminated sites, characterized in that, It includes the following steps: 1) Inject micro-nano bubbles into the soil or groundwater, and conduct continuous aeration for 3 - 5 days, with the aeration time being 6 - 10 hours per day; the gas source of the micro-nano bubbles is one or a combination of air and oxygen; 2) 12 - 24 hours after step 1) is completed, inject a composite microbial inoculant into the soil or groundwater. The composite microbial inoculant includes composite microorganisms, and the composite microorganisms include Pseudomonas, Rheinheimera, Phenylobacterium, and Brevibacillus with a viable cell number ratio of 1 - 25:1 - 15:1 - 5:

1.

2. The method for in-situ remediation or control of petroleum-contaminated sites 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 control of petroleum-contaminated sites according to claim 1, wherein: The soil or groundwater is taken as one unit for every 5 m depth. In step 1), the micro-nano bubbles are injected to a depth of 4 - 6 m in each unit.

4. The method for in-situ remediation or control of petroleum-contaminated sites according to claim 1, characterized in that: The viable cell number ratio of Pseudomonas, Rheinheimera, Phenylobacterium, and Brevibacillus is 2 - 10:2 - 10:1 - 3:

1.

5. The method for in-situ remediation or control of petroleum-contaminated sites according to claim 1, wherein: The viable cell number ratio of Pseudomonas, Rheinheimera, Phenylobacterium, and Brevibacillus is 3 - 7:2 - 5:1 - 2:

1.

6. The method for in-situ remediation or control of petroleum-contaminated sites according to claim 1, wherein: The concentration of the composite microbial inoculum in the soil or groundwater is 0.5 - 10×10 8 CFU / m 3 .

7. The method for in-situ remediation or control of petroleum-contaminated sites according to claim 1, characterized in that: The pollutants in the petroleum-contaminated site contain one or a combination of benzene series compounds, petroleum hydrocarbons, chlorinated hydrocarbons, and polycyclic aromatic hydrocarbons.

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

9. The method for in-situ remediation or control of petroleum-contaminated sites according to claim 1, wherein: The method further includes step 3) within 12 weeks after injecting the composite microbial inoculant, conduct micro-nano bubble aeration every 3 - 4 days, with each aeration lasting for 3 - 5 hours. After more than 12 weeks, conduct micro-nano bubble aeration every 6 - 8 days, with each aeration lasting for 3 - 5 hours.

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

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