A method for in-situ bioremediation of petrochemical contaminated soil

Abstract

The present application belongs to the technical field of microbial degradation of petroleum hydrocarbon, and relates to an in-situ bioremediation method for petrochemical contaminated soil. The method comprises injecting a bioremediation material into the petrochemical contaminated soil in-situ, wherein the bioremediation material comprises freeze-dried bacteria powder of Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No. 24463. The present application utilizes the high degradation activity and strong stress resistance of the Lactobacillus paracasei to petroleum hydrocarbon, and the Lactobacillus paracasei is prepared into high-concentration and high-activity freeze-dried solid bacteria powder. The solid bacteria powder can reduce the volume of the microbial preparation by more than 99%, and is suitable for injection operation for in-situ remediation of soil, so that a better in-situ remediation effect of the contaminated soil can be achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial degradation of petroleum hydrocarbons, and particularly relates to an in-situ bioremediation method for petrochemical contaminated soil. BACKGROUND

[0002] In the process of oil exploration, exploitation, transportation and refining, part of the oil is spilled into the soil due to improper operation or accidents, causing soil pollution by petroleum hydrocarbons. About 8x10 6 t of petroleum pollutants are discharged into the environment through spilled crude oil and accidental leakage every year in the world, and petroleum hydrocarbon pollution has become a focus of the world.

[0003] Early soil remediation technologies in China mainly include cement kiln co-incineration and normal temperature desorption, which are ex-situ remediation technologies. Other technologies are mostly in the laboratory research stage, especially in-situ remediation technologies, and few engineering projects have been applied. With the transition of application scenarios from shutdown enterprises to operating enterprises, the technology method also develops from large-scale remediation to pollution control. Microbial technology can be coupled with multi-phase extraction (MPE), in-situ reaction zone (IRZ), permeable reactive barrier (PRB), and monitored natural attenuation (MNA) technology, and is very flexible in application. Microbial technology also has the characteristics of green, sustainable and low cost, and gradually plays a leading role in soil pollution control. Therefore, it is necessary to develop in-situ remediation microbial inoculants with strong applicability, in-situ remediation injection technology, equipment and products to improve the level of soil pollution control in China.

[0004] In the natural environment, the decomposition and conversion mechanism of microorganisms to organic matter is the main method of removing organic pollutants under non-human intervention conditions. This degradation and conversion mechanism is the core idea of using microorganisms to treat organic pollutants. When the pollutants on the site are chain petroleum hydrocarbons or organic pesticides, the microbial method often has good treatment effect. However, in-situ soil remediation requires higher growth and degradation ability of microorganisms due to the harsh soil environment. SUMMARY

[0005] The inventors of the present application developed a Lactobacillus paracasei (CGMCC No. 24463), and found that the Lactobacillus paracasei (CGMCC No. 24463) is a facultative anaerobe, which can maintain high growth in a low-oxygen environment; meanwhile, the Lactobacillus paracasei (CGMCC No. 24463) has good degradation activity on petroleum hydrocarbons, and the bacterial agent containing the Lactobacillus paracasei (CGMCC No. 24463) can be added to the petroleum hydrocarbon contaminated soil to repair the petroleum hydrocarbon pollution of the soil; and the Lactobacillus paracasei (CGMCC No. 24463) has strong stress resistance, and can be prepared into high-concentration and high-activity freeze-dried solid bacterial powder, which can reduce the volume of the microbial preparation by more than 99%, and is suitable for injection operation for in-situ soil repair.

[0006] Based on the above findings, the present application provides an in-situ bioremediation method for petrochemical contaminated soil, which comprises injecting a bioremediation material into the petrochemical contaminated soil in-situ, wherein the bioremediation material comprises freeze-dried bacterial powder of Lactobacillus paracasei, and the Lactobacillus paracasei has a preservation number of CGMCC No. 24463.

[0007] The present application combines the environmental characteristics of in-situ soil repair, and develops a facultative anaerobe with two sets of metabolic systems, which can survive in aerobic and anaerobic environments, aiming at the technical requirements for treatment and repair of deep soil (oxygen-poor environment); develops a microbial solid bacterial powder preparation with super-strong stress resistance and vigorous growth, aiming at the technical requirements for treatment and repair of petroleum hydrocarbon contaminated soil (high-carbon environment) and low-water soil (high-osmotic pressure environment); and develops a high-concentration bacterial powder, which greatly reduces the volume of the microbial preparation, aiming at the technical requirements for injection of bacterial agent products for in-situ soil repair. Thus, a better in-situ soil repair effect is achieved.

[0008] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0009] The exemplary embodiments of the present application will be described in more detail by combining the accompanying drawings.

[0010] Figure 1 It is a schematic diagram of the position of an aeration well in an embodiment.

[0011] Figure 2 It is a schematic diagram of the position of an in-situ injection borehole in an embodiment.

[0012] Figure 3 It is a schematic diagram of a sampling area in an embodiment.

[0013] Biological preservation instructions

[0014] The biomaterial Lactobacillus paracasei F1 of the present application, which is classified as Lactobacillus paracasei, was preserved in China General Microbiological Culture Collection Center (CGMCC) on March 2, 2022, and the address of the CGMCC is No. 1, Yitian West Road, Chaoyang District, Beijing, China, and the Institute of Microbiology of Chinese Academy of Sciences, and the preservation number is CGMCC NO. 24463. DETAILED DESCRIPTION

[0015] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0016] The present application provides a method for in-situ bioremediation of petrochemical contaminated soil, which comprises injecting a bioremediation material into the petrochemical contaminated soil in-situ, wherein the bioremediation material comprises freeze-dried bacteria powder of Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No. 24463.

[0017] The Lactobacillus paracasei (preservation number CGMCC No. 24463) used in the present application is a facultative anaerobe and a gram-positive bacterium, and is obtained by screening and isolation from a certain polycyclic aromatic hydrocarbon contaminated soil in Tianjin.

[0018] The specific screening and isolation steps are as follows:

[0019] (1) Acclimation

[0020] 3-5 g of soil was taken and added to a sterilized inorganic salt culture medium, the liquid volume of the culture medium was 150 mL / 250 mL flask, and phenanthrene was added as the only carbon source, and then shaken and mixed, and placed in a 30℃, 150r / min shaking flask cabinet for culture. After 5 days, 1 mL of bacterial liquid in the culture bottle was taken for transfer, and at least 3 times were repeated.

[0021] (2) Isolation

[0022] The acclimated bacterial liquid was diluted to an appropriate dilution, and then the plate streaking method was used for streaking and isolating the bacterial liquid. Three parallel samples were made for each concentration gradient. After streaking, the culture dishes were placed in an incubator at 30℃ for 3 days.

[0023] (3) Purification

[0024] Observe the various colonies growing on the surface of the agar plate, and pay attention to the differences in size, shape, edge, surface structure, transparency, color, and properties, and take photos for preservation. Repeat the purification of the colonies that do not form single bacteria until the size and shape of the colonies in the same plate are uniform.

[0025] The purified Lactobacillus paracasei (named F1) is sent to Beijing Meijisan Ge Technology Co., Ltd. for 16S rDNA strain identification. The sequence of the gene splicing of the strain and the NCBI comparison result show that the homology of the strain and the gene sequence of Lactobacillus paracasei strain is 100%, and the isolated strain F1 is identified as Lactobacillus paracasei.

[0026] The Lactobacillus paracasei F1 provided by the application is milky white, round, and the diameter is mostly more than 1.0 mm, the surface is smooth and raised, and the edge is neat.

[0027] The in-situ biological repair method of the application uses Lactobacillus paracasei F1 in the form of freeze-dried bacteria powder, which can be prepared by conventional bacterial culture and freeze-drying method, and the application does not have special limitations. In order to fully contact with soil and oxygen, the particle size of the freeze-dried bacteria powder is preferably small. According to one preferred embodiment of the application, the particle size of the freeze-dried bacteria powder is 50-200 mesh, which takes into account the convenience and effect of treatment, and can be controlled within 80-100 mesh.

[0028] In the biological repair material of the application, the freeze-dried bacteria powder is the core component. In order to achieve better repair effect, the content of active bacteria in the freeze-dried bacteria powder is controlled within the range of 100-20000 billion cfu / g; preferably, it is controlled within the range of 1000-10000 billion cfu / g. In order to promote the better degradation of the bacterial agent, preferably, the biological repair material further comprises a microbial growth promoter. The role of the microbial growth promoter is to provide the nutrient components required for the growth and reproduction of Lactobacillus paracasei, and all microbial growth promoters that meet this condition can be used in the application, such as various substances that can be used as carbon source or nitrogen source, including but not limited to at least one of glucose, starch, corn syrup powder, soybean meal powder, and urea.

[0029] The addition amount of the microbial growth promoter is sufficient to ensure the growth of microorganisms. Generally, the weight ratio of the microbial growth promoter to the freeze-dried bacteria powder is 5-25:1.

[0030] According to one preferred embodiment of the application, the repair method is to inject the mixture of the biological repair material and water into deep soil, and optionally supply air to the deep soil through an aeration well.

[0031] Although the freeze-dried bacterial powder of the present invention can achieve treatment and remediation in soil with low oxygen environment, aeration is still the preferred operating condition. The introduction of oxygen can accelerate the degradation of pollutants by the bacteria and quickly achieve the remediation goal.

[0032] Petroleum hydrocarbon contamination in soil often extends to depths of several meters or even tens of meters. Remediating only the surface soil is insufficient to address the problem. Therefore, in practice, a mixture of bioremediation materials and water is injected into the deeper soil layers to remediate the soil within a three-dimensional space centered on the injection point. The injection depth can be determined as needed, for example, from 2 to 15 meters.

[0033] Experiments have shown that adding freeze-dried bacterial powder, water, and soil in a mass ratio of 1:(10-100):(1000-100000) achieves good remediation results. Here, "soil" refers to soil that can be remediated by 1 part by weight of freeze-dried bacterial powder. In practical applications, this weight can be converted to volume to determine different injection point locations.

[0034] The in-situ bioremediation method of this invention is applicable to petrochemical contaminated soil, which refers to petroleum hydrocarbon contaminated soil, and is especially suitable for volatile and semi-volatile petroleum hydrocarbon contaminated soil.

[0035] The present invention will be further described below with reference to the embodiments, but the scope of the present invention is not limited to these embodiments.

[0036] The following examples will help to illustrate the invention, but are not limited thereto.

[0037] Example 1

[0038] The degradation effect of Lactobacillus paracasei (accession number CGMCC No. 24463) on petroleum hydrocarbons described in this invention was rapidly evaluated.

[0039] The rapid evaluation method was as follows: ① The bacterial agent was enriched in a culture medium; ② The enriched bacterial agent was obtained by centrifugation; ③ The bacterial agent was placed in an inorganic salt culture medium, and n-hexadecane was added as the sole carbon source; ④ The bacterial agent decomposed the contaminants in a shaker at 25℃ and 150 r / min; ⑤ After 5 days, the residual contaminants in the bacterial solution were extracted with carbon tetrachloride, and the oil content was determined by an infrared oil analyzer. A blank sample was prepared at the same time, and the degradation rate was calculated. The results are shown in Table 1.

[0040] Table 1

[0041]

[0042]

[0043] Stenotrophomonas maltophilia was purchased from China General Microbiological Culture Collection Center, and the preservation number was CGMCC 1.6393.

[0044] Acinetobacter was purchased from China General Microbiological Culture Collection Center, and the preservation number was CGMCC 1.10395 T ;

[0045] Dittsia was purchased from China General Microbiological Culture Collection Center, and the preservation number was CGMCC 1.6332 T .

[0046] Example 2

[0047] Method for preparing freeze-dried bacterial powder:

[0048] (I) Primary fermentation (100L)

[0049] ① Single colony was picked on a plate, inoculated into 100 mL of culture medium, and cultured for 18 h to form a primary seed solution.

[0050] ② 20 mL of the primary seed solution was transferred to 1 L of culture medium, and a total of 5 transfers were made, for a total of 5 L, and cultured for 7-10 h, with an OD control range of 1-3.

[0051] ③ The 5 1 L bacterial solutions were combined in one container, and the seed solution was inoculated into a 100 L fermenter, with a liquid volume of 60%, an oxygen adjustment of 30%, a rotation speed of 200-600 rpm, a ventilation amount of 1-1.2 vvm, and an oxygen linkage, and the medium was supplemented with 15-20 L.

[0052] (II) Secondary fermentation (1000L)

[0053] 100 L of the primary fermentation solution was inoculated into a 1T fermenter (working volume 600 L) at a ratio of 1:20, with an oxygen adjustment of 30%, a maximum rotation speed, a ventilation amount of 1-2 vvm, and an oxygen linkage, and the medium was supplemented with about 150-200 L.

[0054] (III) Extraction

[0055] The bacterial cells were collected by centrifugation in a tubular centrifuge.

[0056] (IV) Refining

[0057] The collected bacterial cells were placed in a freeze-drying machine at -20℃ to -80℃, and operated for 72 h-120 h to prepare freeze-dried bacterial powder.

[0058] (V) Grinding

[0059] The particle size of the bacterial powder was controlled to be 80-100 mesh.

[0060] The embodiment of the in-situ biological remediation method of the present application is to inject microbial inoculum and microbial growth promoter into the contaminated soil in-situ to form a microbial flora with high activity in degrading petroleum hydrocarbon, and to remove the petroleum hydrocarbon pollutants in the soil rapidly by the rapid growth of the added bacteria under the condition of aeration oxygen supply, so as to achieve the purpose of purifying and remediation of the contaminated soil.

[0061] The implementation site is a certain petrochemical contaminated site in Beihai, with an area of about 200m 3 (~14m×14m).

[0062] Before the implementation, the site needs to be cleaned up, and the shrubs and weeds in the site need to be cleaned up. Before the implementation, 4 aeration wells are constructed in the site for in-situ oxygen supply to the soil in the contaminated area by injecting microbial inoculum, and the specific positions are shown in Figure 1 the light pink dots and Figure 2 the light pink dots (Q1, Q2, Q3, Q4).

[0063] The construction of the aeration well drilling is completed by a direct push soil sampling drill, a hollow auger is used to drill to 8m underground, and a well pipe is lowered; the lower 3m of the well pipe is a screen pipe, and quartz sand is filled outside the screen pipe to 20-30cm above the screen pipe; clean soil is filled to 30cm from the ground, and is fully compacted; bentonite is filled to the ground, and 20-30cm of the well pipe above the ground is left to connect the aeration pipe.

[0064] 13 injection drillings are arranged in the implementation site for injecting microbial inoculum and growth promoter, and the specific positions are shown in Figure 2 the green dots (A1-A13).

[0065] A drill rod with an injection drill bit is punched into the ground by a direct push soil sampling drill, and the injection depth is 7.5m and 6.0m; an in-situ injection system is connected, the injection pump is started, and the mixed solution of the prepared microbial inoculum and growth promoter (the mass ratio of the microbial inoculum, the growth promoter and water is 1:15:50, and the growth promoter is soybean meal powder) is punched into the ground, and the mixed solution of 50-100L is injected at each depth (determined according to the soil permeability); the injection speed is maintained at about 30L / min, and the injection pressure is not greater than 4MPa; the mixed solution injection of the 13 drillings is completed according to the above steps.

[0066] After the in-situ injection of the microbial inoculum and the growth promoter, aeration maintenance is carried out. The aeration well and the aerator are connected by a steel wire hose, and the aeration amount of each well can be controlled individually; the aeration amount is 100m 3 / h, the contaminated area is aerated for 8h per day, and the aeration is intermittent for 16h again. The maintenance time is 60d.

[0067] Every 5 days, soil gas VOC data was collected at the wellhead of the 13 injection wells, the analysis and detection method is shown in Table 1, and the detailed data is shown in Table 2. On the 10th, 20th, 30th, 40th, 50th and 60th day of aeration maintenance, the contaminated soil was sampled and detected, and the sampling area is shown in Figure 3 When sampling, 2 samples were taken in each of the A and B areas, which were A1 (Example 1), A2 (Example 2), B1 (Example 3) and B2 (Example 4), respectively, and the sampling depth was 7 m underground. During the aeration maintenance, a background sampling point (Comparative Example 1) was set 150 m southeast of the implementation site, and a soil sample was collected 7 m underground. The analysis and detection method is shown in Table 2, and the soil detection data is shown in Tables 4-8.

[0068] Table 2 Analysis and detection method of soil petroleum hydrocarbon (C10-C40) and volatile organic compounds

[0069] Contaminants Detection method Standard number Petroleum hydrocarbons (C10-C40) Gas chromatography HJ 1021-2019 Volatile organic compounds Purge and trap / gas chromatography-mass spectrometry HJ 605-2011

[0070]

[0071] As can be seen from the data in Table 3, the in-situ bioremediation method of the present application greatly reduces the content of volatile organic compounds in soil gas, and from the overall trend, the content of volatile organic compounds in soil gas gradually decreases with the increase of treatment time.

[0072] Table 4 Soil detection data of A1 sampling point

[0073] Example 1 C10-C40 Benzene Toluene Ethylbenzene M, p-xylene O-xylene 0 1377 18.9 269 78.3 403 139 10 1008 8.16 113 44.8 190 54 20 868 4.98 109 34.6 174 64.2 30 491 3.27 97 30.4 149 60.2 40 264 1.28 76.9 26.8 128 51.4 50 217 0.26 12.5 15.1 60.8 21.9 60 78 0.13 8 6.93 13.8 5.9

[0074] Note: The data unit is mg / kg

[0075] Table 5 Soil detection data of A2 sampling point

[0076] Example 2 C10-C40 Benzene Toluene Ethylbenzene M, p-xylene O-xylene 0 955 4.73 124 34.8 190 64.2 10 716 3.6 107 31.4 184 59 20 466 2.3 87.8 29.8 164 53.6 30 271 1.75 75.1 25.5 162 48.8 40 251 1.22 61.3 14.6 148 26.5 50 206 0.74 11.9 10.3 57.8 20.8 60 56 0.25 3.43 3.85 8.44 4.68

[0077] Note: The data unit is mg / kg

[0078] Table 6 Soil detection data of B1 sampling point

[0079] Example 3 C10-C40 Benzene Toluene Ethylbenzene M, p-xylene O-xylene 0 1010 4.05 52.5 60.6 169 62.7 10 703 4.01 42.1 3.36 115 18.7 20 635 0.72 15.4 8.81 90 17.7 30 427 0.62 1.39 39.6 51.8 11.1 40 221 0.21 0.76 10.3 50.9 9.8 50 137 0.13 0.41 9.5 24.8 1.3 60 89 0.13 0.408 7.42 15.1 0.75

[0080] Note: The data unit is mg / kg

[0081] Table 7 Soil detection data of B2 sampling point

[0082] Example 4 C10-C40 Benzene Toluene Ethylbenzene M, p-xylene O-xylene 0 903 8.16 109 83.3 174 60.2 10 603 7.5 38.6 34.6 137 44.5 20 516 3.8 4.77 26.5 94.1 26.8 30 308 2.6 0.999 13.2 69.3 13.4 40 182 0.95 0.53 5.2 21 4.28 50 133 0.21 0.25 4.08 19.8 0.71 60 47 0.13 0.25 2.22 10 0.62

[0083] Note: The data unit is mg / kg

[0084] Table 8 Soil detection data of background sampling point

[0085] Comparative Example 1 C10-C40 Benzene Toluene Ethylbenzene M, p-xylene O-xylene 0 998 9.25 114 65.1 203 78.1 30 721 8.69 101 62.4 171 59.2 60 589 7.01 94.1 58.9 165 54.2

[0086] Note: data unit mg / kg

[0087] The comparison of the degradation rates of pollutants in the soil at 60 days is shown in Table 9.

[0088] Table 9 Comparison of the degradation rates of pollutants in the soil

[0089] C10-C40 Benzene Toluene Ethylbenzene M, p-xylene O-xylene Example 1 94.3 99.3 97.0 91.1 96.6 95.8 Example 2 94.1 94.7 97.2 88.9 95.6 92.7 Example 3 91.2 96.8 99.2 87.8 91.1 98.8 Example 4 94.8 98.4 99.8 97.3 94.3 99.0 Comparative Example 1 41.0 24.2 17.5 9.5 18.7 30.6

[0090] Note: data unit %

[0091] As can be seen from the data in Tables 4-9, the content of C10-C40 in the soil at the four sampling points decreased significantly with the increase of the treatment time, wherein the degradation rate of C10-C40 at 60 days was more than 90%, while the degradation rate of C10-C40 at 60 days in the background sampling point was only 41%. This fully shows that the in-situ bioremediation method of the present application can greatly reduce the content of pollutants in the soil.

[0092] The above experiments show that the in-situ bioremediation method of the present application not only can reduce the content of pollutants in the soil, but also can reduce the emission of VOCs, and the effect of the method on soil remediation is remarkable.

[0093] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments.

[0094] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges and values should be interpreted as approximately between the stated values. For ranges, the endpoints are included within the range, and the ranges are inclusive of the endpoints. For individual points, the value is inclusive of the point. The ranges, endpoints, and individual points can be combined to form new ranges, endpoints, and individual points, which are also within the scope of the present disclosure.

Claims

1. An in-situ bioremediation method for petrochemical-contaminated soil, characterized in that, The method includes in-situ injection of bioremediation materials into petrochemical-contaminated soil, the bioremediation materials comprising Lactobacillus paracasei (… Lactobacillus paracasei The freeze-dried bacterial powder of Lactobacillus paracasei, wherein the preservation number of Lactobacillus paracasei is CGMCC No. 24463; The remediation method involves injecting a mixture of bioremediation material and water into deep soil, and optionally supplying air to the deep soil through aeration wells.

2. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 1, characterized in that, The content of active bacteria in the freeze-dried bacterial powder is 100-2000 billion CFU / g.

3. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 2, characterized in that, The content of active bacteria in the freeze-dried bacterial powder is 1000-10000 billion CFU / g.

4. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 1, characterized in that, The freeze-dried bacterial powder has a particle size of 50-200 mesh.

5. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 4, characterized in that, The freeze-dried bacterial powder has a particle size of 80-100 mesh.

6. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 1, characterized in that, The bioremediation material also contains microbial growth promoters.

7. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 6, characterized in that, The microbial growth promoter is a carbon source or a nitrogen source.

8. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 7, characterized in that, The microbial growth promoter is at least one of glucose, starch, corn syrup powder, soybean meal powder, and urea.

9. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 6, characterized in that, The weight ratio of the microbial growth promoter to the freeze-dried bacterial powder is 5-25:

1.

10. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 1, characterized in that, The mass ratio of the freeze-dried bacterial powder, water and soil is 1:(10-100):(1000-100000).

11. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 1, characterized in that, The injection depth is 2-15 meters.

12. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 1, characterized in that, The petrochemical-contaminated soil is petroleum hydrocarbon-contaminated soil.

13. The in-situ bioremediation method for petrochemical-contaminated soil according to claim 12, characterized in that, The in-situ bioremediation method is for the remediation of soil contaminated with volatile and semi-volatile petroleum hydrocarbons.

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