In-situ bioremediation method for petrochemical contaminated soil

Abstract

The invention 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 the steps that a bioremediation material is injected into the petrochemical contaminated soil in situ, the bioremediation material comprises freeze-dried bacterial powder of lactobacillus paracasei, and the preservation number of the lactobacillus paracasei is CGMCC (China General Microbiological Culture Collection Center) No.24463. The lactobacillus paracasei is prepared into high-concentration high-activity freeze-dried solid bacterial powder by utilizing high degradation activity and strong stress resistance of the lactobacillus paracasei on petroleum hydrocarbon, the volume of the microbial preparation can be reduced by 99% or above through the solid bacterial powder, and the solid bacterial powder is suitable for injection operation of in-situ remediation of soil, so that a better in-situ contaminated soil remediation effect is achieved.

Description

Technical Field

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

[0002] During the process of oil exploration, mining, transportation, and refining, some oil spills into the soil due to improper operation or accidental leakage, causing petroleum hydrocarbon pollution in the soil. Every year, about 8 million petroleum pollutants are discharged into the environment through crude oil landing and accidental leakage. 6 t, petroleum hydrocarbon pollution has become the focus of world attention.

[0004] Early soil remediation technologies in China were mainly cement kiln co-incineration and room temperature desorption, both of which are ex situ remediation technologies. Other technologies are mostly in the laboratory research stage, especially in situ remediation technology, and there are few engineering projects that have been applied. With the transition of application scenarios from discontinued enterprises to in-production enterprises, technical methods have also developed from large-scale remediation to pollution control. Microbial technology can be coupled with multiphase extraction (MPE), in situ reaction zone (IRZ), permeable reaction wall (PRB), monitored natural attenuation (MNA) and other technologies, and its application is very flexible; microbial technology also has the characteristics of being green, sustainable, and low-cost, and gradually plays a leading role in soil pollution control. Therefore, it is necessary to develop highly applicable in-situ remediation microbial agents and in-situ remediation injection technologies, equipment and products to improve the level of soil pollution control in my country.

[0005] In the natural environment, the decomposition and conversion mechanism of organic matter by microorganisms is the main method for removing organic pollutants without human interference. This degradation and conversion mechanism is the core idea of ​​using microbial methods to treat organic pollutants. When the pollutants on the site are chain petroleum hydrocarbons or organic pesticides, microbial methods often have better treatment effects. However, in-situ soil remediation has higher requirements on the growth and degradation ability of microorganisms because the soil environment is more demanding. Summary of the invention

[0006] The inventor of the present invention has developed a lactobacillus paracasei (preservation number CGMCC No. 24463). Research has shown that the lactobacillus paracasei (preservation number CGMCC No. 24463) is a facultative anaerobic bacterium and can maintain high growth in a low oxygen environment; at the same time, the lactobacillus paracasei (preservation number CGMCC No. 24463) has good degradation activity on petroleum hydrocarbons, and a bacterial agent containing the lactobacillus paracasei (preservation number CGMCC No. 24463) is added to petroleum hydrocarbon-contaminated soil to repair the petroleum hydrocarbon pollution of the soil; and the lactobacillus paracasei (preservation number 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 of soil in-situ repair.

[0007] Based on the above findings, the present invention 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 preservation number of the Lactobacillus paracasei is CGMCC No. 24463.

[0008] The present invention combines the environmental characteristics of soil in-situ remediation and develops a facultative anaerobic agent with two metabolic systems that can survive in aerobic and anaerobic environments in response to the technical requirements of deep soil (oxygen-poor environment) treatment and remediation; develops a microbial solid powder preparation with super stress resistance and vigorous growth in response to the technical requirements of petroleum hydrocarbon contaminated soil (high carbon environment) and low water content soil (high osmotic pressure environment); develops a high-concentration powder in response to the technical requirements of soil in-situ remediation injection of microbial agent products, which greatly reduces the volume of microbial preparations. Thus, a better in-situ contaminated soil remediation effect is achieved.

[0009] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0011] Figure 1 It is a schematic diagram of the location of the aeration well in a specific implementation manner.

[0012] Figure 2 It is a schematic diagram of the location of the in-situ injection drilling in a specific implementation manner.

[0013] Figure 3 A schematic diagram of a sampling area in a specific implementation manner.

[0014] Biological Deposit Description

[0015] The biological material Lactobacillus paracasei F1 of the present invention is classified and named Lactobacillus paracasei, and was deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China Microbiological Culture Collection Committee on March 2, 2022, with the address being: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC NO.24463. DETAILED DESCRIPTION

[0016] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0017] The invention provides an in-situ bioremediation method for petrochemical contaminated soil. The method comprises injecting a bioremediation material into the petrochemical contaminated soil in situ. The bioremediation material comprises freeze-dried bacterial powder of Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No.24463.

[0018] The Lactobacillus paracasei (preservation number CGMCC No. 24463) used in the present invention is a facultative anaerobic bacterium, Gram-positive, and is obtained by screening and separation from polycyclic aromatic hydrocarbon-contaminated soil in Tianjin.

[0019] The specific screening and separation steps are:

[0020] (1) Domestication

[0021] Take 3-5g of soil and add it to the sterilized inorganic salt culture medium. The volume of the culture medium is 150mL / 250mL Erlenmeyer flask. Then add phenanthrene as the only carbon source, shake well, and culture in a shaking cabinet at 30℃ and 150r / min. After 5 days, take 1mL of the bacterial solution in the culture bottle for transfer, and repeat at least 3 times.

[0022] (2) Separation

[0023] After the acclimated bacterial solution is diluted to an appropriate dilution, the bacterial solution is streaked using the plate streak method. Three parallel samples are made for each concentration gradient. After streaking, the culture dish is placed in an incubator at 30°C and cultured for 3 days.

[0024] (3) Purification

[0025] Observe the various colonies growing on the surface of the agar plate, pay attention to the differences in size, shape, edge, surface structure, transparency, color, properties, etc., take photos and save them. Repeatedly purify the colonies that have not formed single bacteria until the size and shape of the colonies on the same plate are uniform.

[0026] The purified Lactobacillus paracasei (named F1) was sent to Beijing Meiji Sanger Biopharmaceutical Technology Co., Ltd. for 16S rDNA strain identification. The results of the spliced ​​gene sequence of the strain and the NCBI comparison showed that the strain had 100% homology with the gene sequence of Lactobacillus paracasei strain. The isolated strain F1 was identified as Lactobacillus paracasei.

[0027] The lactobacillus paracasei F1 colonies provided by the invention are milky white, round, mostly with a diameter of more than 1.0 mm, a smooth and raised surface, and neat edges.

[0028] The in-situ bioremediation method of the present invention adopts Lactobacillus paracasei F1 in the form of freeze-dried bacterial powder, and the freeze-dried bacterial powder can be prepared by conventional bacterial culture and freeze-drying methods, and the present invention has no particular limitation on this. In order to fully contact with soil and oxygen, the particle size of the freeze-dried bacterial powder is preferably smaller. According to a preferred embodiment of the present invention, the particle size of the freeze-dried bacterial powder is 50-200 meshes, taking into account the convenience of processing and the effect, and can be controlled at 80-100 meshes.

[0029] In the bioremediation material of the present invention, freeze-dried bacterial powder is the core component. In order to achieve a better repair effect, the active bacteria content in the freeze-dried bacterial powder is controlled within the range of 100-2000 billion cfu / g; preferably controlled within the range of 1000-1000 billion cfu / g. In order to promote the bacterial agent to better play the degradation effect, preferably, the bioremediation material also contains a microbial growth promoter. The role of the microbial growth promoter is to provide the nutrients required for the growth and reproduction of Lactobacillus paracasei. All microbial growth promoters that meet this condition can be used in the present invention, such as various substances that can be used as carbon sources or nitrogen sources, including but not limited to at least one of glucose, starch, corn slurry powder, soybean meal powder, and urea.

[0030] The amount of the microbial growth promoter added is sufficient to ensure the growth of the microorganisms. Usually, the weight ratio of the microbial growth promoter to the freeze-dried bacterial powder is 5-25:1.

[0031] According to a preferred embodiment of the present invention, the remediation method is to inject a mixture of bioremediation material and water into the deep soil layer, and optionally supply air to the deep soil layer through an aeration well.

[0032] Although the freeze-dried bacterial powder of the present invention can achieve treatment and restoration in soil in an oxygen-deficient 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 restoration goal.

[0033] Petroleum hydrocarbon contamination in soil is usually several meters or even tens of meters deep. Repairing only the surface soil cannot effectively solve the soil pollution problem. Therefore, in the actual repair process, a mixture of bioremediation materials and water will be injected into the deep soil, thereby repairing the soil in the three-dimensional space centered on the injection point. The injection depth can be determined according to needs, for example, 2-15 meters.

[0034] According to the test, a good repair effect can be achieved by adding freeze-dried bacterial powder and water in a mass ratio of 1: (10-100): (1000-100000) to soil. The soil here refers to the soil that can be repaired by 1 part by weight of freeze-dried bacterial powder. In actual application, the volume can be converted according to the weight, and then the different injection points can be determined.

[0035] The petrochemical contaminated soil to which the in-situ bioremediation method of the present invention is applicable refers to petroleum hydrocarbon contaminated soil, and is particularly applicable to volatile and semi-volatile petroleum hydrocarbon contaminated soil.

[0036] The present invention will be further described below in conjunction with embodiments, but the scope of the present invention is not limited to these embodiments.

[0037] The following examples will help to illustrate the present invention but are not intended to limit its scope.

[0038] Example 1

[0039] The degradation effect of Lactobacillus paracasei (preservation number CGMCC No. 24463) of the present invention on petroleum hydrocarbons was quickly evaluated.

[0040] The rapid evaluation method is as follows: ① enrich the bacterial agent in the culture medium; ② centrifuge to obtain the enriched bacterial agent; ③ place the bacterial agent in an inorganic salt culture medium and add n-hexadecane as the only carbon source; ④ decompose the pollutants in a shaking bottle cabinet at 25°C and 150r / min; ⑤ after 5 days, extract the residual pollutants in the bacterial solution with carbon tetrachloride, determine the oil content with an infrared oil meter, and make a blank sample at the same time to calculate the degradation rate. The results are shown in Table 1.

[0041] Table 1

[0042]

[0043]

[0044] Oligotrophomonas was purchased from China General Microbiological Culture Collection Center, with the accession number CGMCC1.6393;

[0045] Acinetobacter was purchased from China General Microbiological Culture Collection Center, with the collection number CGMCC1.10395 T ;

[0046] Dietrichia coli was purchased from China General Microbiological Culture Collection Center, with the collection number CGMCC1.6332 T .

[0047] Example 2

[0048] Preparation method of freeze-dried bacterial powder:

[0049] (I) Primary fermentation (100L)

[0050] ① Pick a single clone colony from the plate, inoculate it into 100 mL of culture medium, and culture it for 18 hours to form a primary seed solution.

[0051] ② Transfer 20mL of the first-level seed solution into 1L of culture medium, transfer 5 times in total, totaling 5L, culture for 7-10h, and control the OD range to 1-3.

[0052] ③ Combine 5 1L bacterial liquids into one container, inoculate the seed liquid into a 100L fermenter, fill the liquid volume to 60%, adjust the dissolved oxygen to 30%, rotate speed to 200-600rpm, and the ventilation volume to 1-1.2vvm. Linked with dissolved oxygen, feed culture, and add 15-20L of culture medium.

[0053] (II) Secondary fermentation (1000L)

[0054] 100L of primary fermentation broth was inoculated into a 1T fermentation tank (working volume 600L) at a ratio of 1:20, the dissolved oxygen was adjusted to 30%, the rotation speed was maximum, the ventilation volume was 1-2vvm. and the dissolved oxygen was linked, and about 150-200L of culture medium was supplemented.

[0055] (III) Extraction

[0056] Bacteria collection: Collect bacteria by centrifugation in a tube centrifuge.

[0057] (IV) Refining

[0058] The collected bacteria were placed in a -20℃~-80℃ freeze dryer and operated for 72h-120h for freeze drying preparation.

[0059] (V) Crushing

[0060] The particle size of the bacterial powder is controlled to be 80-100 mesh.

[0061] The implementation method of the in-situ bioremediation method of the present invention is to inject microbial agents and microbial growth promoters into the contaminated soil in situ to form a microbial flora with high activity in degrading petroleum hydrocarbons, and utilize the rapid growth of added bacteria under aeration oxygen conditions to quickly remove petroleum hydrocarbon pollutants in the soil, thereby achieving the purpose of purifying and repairing the contaminated soil.

[0062] The implementation site is a petrochemical pollution site in Beihai, covering an area of ​​about 200m 3 (~14m×14m).

[0063] Before implementation, the site needs to be tidied and the shrubs and weeds on the site need to be cleared. Before implementation, four aeration wells will be built on the site to supply oxygen to the soil in the contaminated area by injecting microbial agents. The specific locations are shown in Figure 1 Medium pink dots and Figure 2 Medium pink dots (Q1, Q2, Q3, Q4).

[0064] The drilling for aeration well construction is completed by a direct-push soil sampling drill, which uses a hollow spiral drill to drill to 8m underground and lower the well pipe; the lower 3m of the well pipe is a screen pipe, and quartz sand is filled outside the pipe to 20 to 30cm above the screen pipe; clean soil is filled to 30cm from the ground and fully compacted; bentonite is filled to the ground, and 20 to 30cm of the well pipe is left on the ground to connect the aeration pipe.

[0065] 13 injection boreholes were set up in the implementation site to inject microbial agents and growth promoters. The specific locations are shown in Figure 2 Green dots (A1~A13).

[0066] A direct-push soil sampling drill was used to drive a drill rod equipped with an injection drill bit into the ground, with injection depths of 7.5 m and 6.0 m. The in-situ injection system was connected, and the injection pump was started to drive the prepared mixed solution of microbial agent and growth promoter (wherein the mass ratio of microbial agent, growth promoter and water was 1:15:50, and the growth promoter was soybean meal powder) into the ground, with 50 to 100 L of the mixed solution injected at each depth (depending on the soil permeability); the injection speed was maintained at about 30 L / min, and the injection pressure was not greater than 4 MPa; the injection of the mixed solution into 13 boreholes was completed according to the above steps.

[0067] After the microbial agent and growth promoter are injected in situ, aeration maintenance is carried out. The aeration well and the aerator are connected by a steel hose, and the aeration volume of each well can be controlled separately; 3 / h air supply, aerate the polluted area for 8 hours a day, and aerate again after 16 hours of rest. The curing time is 60 days.

[0068] Every 5 days, soil VOC data were collected at the wellheads of 13 injection wells. The analysis and detection methods are shown in Table 1, and the detailed data are shown in Table 2. On the 10th, 20th, 30th, 40th, 50th and 60th days of aeration maintenance, the contaminated soil was sampled and tested. The sampling areas are shown in Table 1. Figure 3 During sampling, two samples were collected in each of the A and B areas, namely A1 (Example 1), A2 (Example 2), B1 (Example 3), and B2 (Example 4), and the sampling depth was 7m underground. During the aeration maintenance period, a background sampling point (Comparative Example 1) was set at 150m southeast of the implementation site to collect soil samples 7m underground. The analysis and detection methods are shown in Table 2, and the soil detection data are shown in Tables 4 to 8.

[0069] Table 2 Sampling and testing of soil petroleum hydrocarbons (C10-C40) and volatile organic compounds analysis and testing methods

[0070] Pollutants Detection Methods Standard No. Petroleum hydrocarbons (C10-C40) Gas chromatography HJ 1021-2019 Volatile organic compounds Purge and Trap / Gas Chromatography-Mass Spectrometry HJ 605-2011

[0071]

[0072] It can be seen from the data in Table 3 that the in-situ bioremediation method of the present invention significantly reduces the volatile organic matter content of soil gas, and from the overall trend, the volatile organic matter content of soil gas gradually decreases with the increase of treatment time.

[0073] Table 4A1 Soil testing data at sampling points

[0074] 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

[0075] Note: Data unit: mg / kg

[0076] Table 5A2 Soil test data at sampling points

[0077] 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

[0078] Note: Data unit: mg / kg

[0079] Table 6B1 Soil testing data at sampling points

[0080] 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

[0081] Note: Data unit: mg / kg

[0082] Table 7B2 Soil testing data at sampling points

[0083] 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

[0084] Note: Data unit: mg / kg

[0085] Table 8 Soil test data of background sampling points

[0086] 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

[0087] Note: Data unit: mg / kg

[0088] The comparison of the degradation rates of pollutants in the soil of Examples 1 to 4 and Comparative Example 1 at the soil sampling point at 60 days is shown in Table 9.

[0089] Table 9 Comparison of pollutant degradation rates in soil

[0090] 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

[0091] Note: Data unit: %

[0092] It can be seen from the data in Tables 4 to 9 that the C10-C40 content in the soil of the four sampling points decreased significantly with the increase of treatment time, among which the degradation rate of the C10-C40 content on the 60th day was all above 90%, while the degradation rate of the C10-C40 content in the background sampling point on the 60th day was only 41%. This fully demonstrates that the in-situ bioremediation method of the present invention greatly reduces the content of pollutants in the soil.

[0093] The above experiments show that the in-situ bioremediation method of the present invention can not only reduce the content of pollutants in the soil, but also reduce the emission of VOCs. This method has a significant effect on soil remediation.

[0094] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

[0095] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

Claims

1. An in-situ bioremediation method for petrochemical contaminated soil, characterized in that: The method comprises injecting a bioremediation material into petrochemical contaminated soil in situ, wherein the bioremediation material comprises freeze-dried bacterial powder of Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No.24463.

2. The in-situ bioremediation method for petrochemical contaminated soil according to claim 1, characterized in that: The active bacteria content in the freeze-dried bacterial powder is 100-2000 billion cfu / g, preferably 100-1000 billion cfu / g.

3. The in-situ bioremediation method for petrochemical contaminated soil according to claim 1, characterized in that: The particle size of the freeze-dried bacterial powder is 50-200 mesh, preferably 80-100 mesh.

4. The in-situ bioremediation method for petrochemical contaminated soil according to claim 1, characterized in that: The bioremediation material further comprises a microbial growth promoter.

5. The in-situ bioremediation method for petrochemical contaminated soil according to claim 4, characterized in that: The microbial growth promoter is a carbon source or a nitrogen source, preferably at least one of glucose, starch, corn syrup powder, soybean meal powder and urea.

6. The in-situ bioremediation method for petrochemical contaminated soil according to claim 4, characterized in that: The weight ratio of the microbial growth promoter to the freeze-dried bacterial powder is 5-25:

1.

7. The in-situ bioremediation method for petrochemical contaminated soil according to claim 1, characterized in that: The remediation method is to inject a mixture of bioremediation materials and water into deep soil, and optionally supply air to the deep soil through an aeration well.

8. The in-situ bioremediation method for petrochemical contaminated soil according to claim 7, characterized in that: The mass ratio of the freeze-dried bacterial powder, water and soil is 1:(10-100):(1000-100000).

9. The in-situ bioremediation method for petrochemical contaminated soil according to claim 7, characterized in that: The injection depth is 2-15 meters.

10. 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, preferably the restoration of volatile or semi-volatile petroleum hydrocarbon contaminated soil.

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