A biological agent for petroleum hydrocarbon pollution in saline-alkali soil and a risk reduction method

Abstract

The present invention belongs to the field of petroleum hydrocarbon pollution control, and specifically relates to a saline-alkali soil petroleum hydrocarbon pollution biological bacterial agent and a risk reduction method. The saline-alkali soil petroleum hydrocarbon pollution biological bacterial agent of the present invention comprises Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20, the ratio of the number of live bacteria being 1: (1.0-2.5): (0.8-1.5): (0.5-1.3). The saline-alkali soil petroleum hydrocarbon pollution biological bacterial agent of the present invention contains a variety of degradation bacteria to exert a synergistic effect, has a more excellent degradation performance, significantly enhances the effect of repairing petroleum hydrocarbon pollution, and has a good market application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of petroleum hydrocarbon pollution control, and in particular relates to a biological microbial agent for treating petroleum hydrocarbon pollution in saline-alkali soil and a risk reduction method. Background Art

[0002] Saline soils are widely distributed in my country, and both natural weathering and human interference can lead to soil salinization. Many of my country's oil fields are located in coastal areas, where they are eroded by seawater, forming saline-alkali land. In the process of oil development and utilization, produced water from some oil fields is dispersed into the soil as a by-product, causing soil salinization. Soil salinization is severe in northern my country. For example, the Dagang and Shengli Oilfields are both important oil production areas, primarily composed of saline-alkali soils. These oil fields, while facing oil pollution, are also affected by salinization. The issue of soil remediation under the dual stress of salinity and petroleum hydrocarbon pollution has long attracted attention.

[0003] Soil pollution is often detected only after it threatens farmland, plants, and microbial growth. Soil also has a cumulative toxicity effect, and pollutants in water and the atmosphere are more easily volatilized and transformed. Therefore, soil pollution is more toxic and more likely to exceed national standards. Petroleum pollution can affect physical and chemical properties such as soil organic matter, total nitrogen, total phosphorus, and pH. Long-term accumulation of pollutants can affect soil microbial metabolism. If unaddressed, the expansion of soil pollution will further impact groundwater and the ecological environment.

[0004] Current remediation technologies for crude oil-contaminated soil primarily include microbial-phytoremediation, electrochemical remediation, and steam remediation. The core of existing microbial remediation methods is the rapid removal of pollutants by highly efficient degrading bacteria. Research focuses on three key areas: first, the systematic taxonomy and metabolic genes of dominant degrading bacteria; second, the optimization of microbial degradation conditions and the study of their degradation mechanisms; and third, the practical application of microbial remediation technologies in engineering projects.

[0005] There are over 200 known species of microorganisms across 70 genera capable of degrading petroleum, including 28 genera of bacteria, 30 genera of molds, and 12 genera of yeasts. When biostimulation methods are used to remediate petroleum contamination due to a lack of native degrading bacteria or insufficient degradation capacity, bioaugmentation, which involves adding exogenous degrading bacteria to achieve biodegradation, becomes a more suitable treatment technology. The coordinated growth of multiple microbial strains complicates the conditions, placing high demands on the specific types and quantities of the microorganisms used. Summary of the Invention

[0006] In view of this, the present invention provides a bio-microbial agent for petroleum hydrocarbon contamination in saline-alkali soil and a risk reduction method. The bio-microbial agent has a good effect of degrading petroleum hydrocarbons and can achieve the purpose of rapid remediation of contaminated soil.

[0007] The technical solutions of the present invention are as follows:

[0008] A biological agent for treating petroleum hydrocarbon contamination in saline-alkali soil, comprising Pseudomonas aeruginosa Ya1, Halomonas elongata ZQ1-3, Achromoobacter pulmonis-10, and Burkholderia sp. AM20;

[0009] The ratio of the viable bacterial counts of Pseudomonas aeruginosa Ya1, Halomonas elongata ZQ1-3, Achromoobacter pulmonis-10, and Burkholderia sp. AM20 is 1:(1.0-2.5):(0.8-1.5):(0.5-1.3), and the total viable bacterial count is (2.5-3.5)×10 9 cfu / g;

[0010] The Pseudomonas aeruginosa Ya1 was deposited in the General Microbiology Center of China Culture Collection Administration on September 18, 2015, with the deposit number CGMCC NO.11426;

[0011] The Halomonas elongata ZQ1-3 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 26, 2020, with the deposit number CGMCC No. 20953;

[0012] The petroleum hydrocarbon degrading bacteria (Achromoobacter pulmonis)-10 was deposited in the General Microbiology Center of China Culture Collection Administration on August 9, 2018, with the deposit number CGMCC No.16240;

[0013] The Burkholderia sp. AM20 was deposited in the General Microbiology Center of the China Culture Collection Administration on April 6, 2023, with the deposit number CGMCC NO.27032.

[0014] Furthermore, the ratio of the number of live bacteria of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 in the saline-alkali soil petroleum hydrocarbon contamination biological agent is 1:(1.0-2.5):(0.8-1.5):(0.5-1.3), and the total number of live bacteria is (2.5-3.5)×10 9 cfu / g.

[0015] Furthermore, the ratio of the number of live bacteria of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum Hydrocarbon Degrading Bacteria-10 and Burkholderia AM20 in the saline-alkali soil petroleum hydrocarbon contamination biological agent is 1:2:1:0.8, and the total number of live bacteria is 3.0×10 9 cfu / g.

[0016] The present invention provides a method for preparing a biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil, comprising the following steps:

[0017] (1) Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10, and Burkholderia spp. AM20 were activated on LB medium to obtain activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10, and Burkholderia spp. AM20;

[0018] (2) inoculating the activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10, and Burkholderia AM20 obtained in step (1) into a fermentation medium for seed culture, respectively, to obtain seed solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10, and Burkholderia AM20;

[0019] (3) inoculating the seed liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10, and Burkholderia AM20 obtained in step (2) into fermentation medium for fermentation culture, respectively, to obtain bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10, and Burkholderia AM20;

[0020] (4) The bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (3) are mixed, and bran biochar is added, mixed, and stirred evenly at a mass ratio of 1:(6-15), and the moisture content is adjusted to 10-15%. After standing at room temperature, a biological agent for petroleum hydrocarbon pollution in saline-alkali soil is obtained.

[0021] Preferably, the activation culture condition in step (1) is constant temperature shaking culture at 30°C for 48 hours.

[0022] Preferably, the components and contents of the LB medium in step (1) are as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH 7.0-7.2.

[0023] Preferably, the amount of strain seed solution added in step (2) is 5-10% of the total mass of the culture medium.

[0024] Further preferably, the amount of strain seed solution added in step (2) is 8% of the total mass of the culture medium.

[0025] Preferably, the seed culture conditions in step (2) are constant temperature shaking culture at 30° C. for 36 hours and a rotation speed of 160 rpm.

[0026] Preferably, the amount of strain seed solution added in step (3) is 5-10% of the total mass of the culture medium.

[0027] Further preferably, the amount of strain seed solution added in step (3) is 8% of the total mass of the culture medium.

[0028] Preferably, the fermentation conditions in step (3) are constant temperature shaking culture at 30° C. for 12-42 hours, and the dissolved oxygen is controlled at 45-55%.

[0029] Further preferably, in step (3), the culture is carried out for 36 hours, the dissolved oxygen is 50%, and the rotation speed is 160 rpm.

[0030] The components and contents of the fermentation medium in step (2) or step (3) are as follows: 8.5g Na2HPO4·2H2O, 3.0gKH2PO4, 1.0g NH4Cl, 0.49g MgSO4·7H2O, 0.011g CaCl2, 15g NaCl; trace elements: 0.4mgCuSO4, 1.0mg KI, 4.0mg MnSO4·H2O, 4.0mg ZnSO4·7H2O, 5.0mg H3BO3, 1.6mg H2MoO4·2H2O, 2.0mg FeCl3·6H2O; 1mol / L MgSO4·7H2O and 1mol / L CaCl2 are sterilized separately and then added to the culture medium, pH 7.0.

[0031] Preferably, the bran biochar in step (4) is prepared by pyrolyzing bran at 300° C. for 2 h in a muffle furnace.

[0032] The present invention also provides the use of the above-mentioned biological bacterial agent for petroleum hydrocarbon pollution in saline-alkali soil in repairing petroleum pollutants.

[0033] Preferably, the petroleum pollutant is petroleum-contaminated soil or treated oily sludge.

[0034] Preferably, when the saline-alkali soil petroleum hydrocarbon pollution biological agent is used to repair petroleum pollutants, 3-8% of the mass of the petroleum hydrocarbon pollutants is added to the biological agent, the water content of the petroleum hydrocarbon pollutants is maintained at 35%-45%, and the petroleum hydrocarbon pollutants are turned over, and the degradation is continued for 60-120 days.

[0035] Further preferably, when the saline-alkali soil petroleum hydrocarbon pollution biological agent is used to repair petroleum-contaminated soil, a biological agent of 6% by mass of the petroleum hydrocarbon pollutants is added and the degradation is continued for 90 days.

[0036] The beneficial effects achieved by the present invention are:

[0037] The saline-alkali soil petroleum hydrocarbon contamination biological bacterial agent of the present invention can efficiently degrade petroleum hydrocarbons and repair petroleum hydrocarbon-contaminated soil.

[0038] Due to the complex composition of petroleum, it is difficult to achieve ideal results by relying on a single degradation bacteria. Compared with a single bacteria, the petroleum hydrocarbon pollution biological agent of the present invention, which is composed of a combination of different degradation bacteria, has a synergistic effect of multiple degradation bacteria, has better degradation performance, and significantly enhances the effect of repairing petroleum hydrocarbon pollution, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation methods.

[0040] Figure 1 It is the content of petroleum hydrocarbons (C7-C49) remaining after the biological agent of the present invention treats the soil contaminated by petroleum hydrocarbons.

[0041] Figure 2 It is the degradation rate of petroleum hydrocarbon substances after the biological agent of the present invention treats petroleum hydrocarbon contaminated soil. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0043] In the embodiments of the present invention, Pseudomonas aeruginosa Ya1 was purchased from the General Microorganism Center of China National Culture Collection Administration Committee with a deposit number of CGMCC No. 11426; Halomonas elongata ZQ1-3 was purchased from the General Microorganism Center of China National Culture Collection Administration Committee with a deposit number of CGMCC No. 20953; Achromoobacter pulmonis-10 was purchased from the General Microorganism Center of China National Culture Collection Administration Committee with a deposit number of CGMCC No. 16240; and Burkholderia sp. AM20 was purchased from the General Microorganism Center of China National Culture Collection Administration Committee with a deposit number of CGMCC No. 27032.

[0044] Example 1

[0045] A biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil and a preparation method thereof are as follows:

[0046] (1) Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum hydrocarbon degrading bacteria-10, and Burkholderia spp. AM20 were cultured in the presence of 1:2:1:0.8 viable bacteria, respectively (the total viable bacteria count was 3.0×10 9 cfu / g) were activated in LB medium and cultured at 30°C with shaking for 48 h to obtain activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum Hydrocarbon Degrading Bacteria-10, and Burkholderia sp. AM20. The components and contents of the LB medium were as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH 7.0.

[0047] (2) The activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (1) were inoculated into fermentation medium respectively, with the strain seed solution added in an amount of 8% of the total mass of the medium, the rotation speed was 160 rpm, and the seed culture was carried out at a constant temperature of 30°C for 36 hours to obtain Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 seed solutions; the components and contents of the fermentation medium were as follows: 8.5 g Na2HPO4·2H2O, 3.0 g KH2PO4, 1.0 g NH4Cl, 0.49 g MgSO4·7H2O, 0.011 g CaCl2, 15 g NaCl; trace elements: 0.4 mg CuSO4, 1.0 mg KI, 4.0 mg MnSO4·H2O, 4.0 mg ZnSO4·7H2O, 5.0mg H3BO3, 1.6mg H2MoO4·2H2O, 2.0mg FeCl3·6H2O; 1mol / L MgSO4·7H2O and 1mol / L CaCl2 were sterilized separately and then added to the culture medium, pH 7.0.

[0048] (3) The seed solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (2) are inoculated into fermentation medium respectively, with the addition amount of the strain seed solution being 8% of the total mass of the culture medium, the rotation speed being 160 rpm, the dissolved oxygen being 50%, and the fermentation culture is carried out for 36 hours to obtain bacterial solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20; the fermentation medium is the same as the fermentation medium in step (2).

[0049] (4) The bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (3) were mixed, and bran biochar (prepared by high-temperature pyrolysis of bran at 300°C in a muffle furnace for 2h) was added, mixed, and the mass ratio was 1:15. The mixture was stirred evenly, and the moisture content was adjusted to 10%. After standing at room temperature, a bio-bacterial agent for petroleum hydrocarbon pollution in saline-alkali soil was obtained.

[0050] Example 2

[0051] A biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil and a preparation method thereof are as follows:

[0052] (1) Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum hydrocarbon degrading bacteria-10, and Burkholderia spp. AM20 were cultured in the presence of 1:1:1:1 viable bacteria, and the total viable bacteria count was 3.2×10 9 cfu / g) were activated in LB medium and cultured with shaking at 30°C for 48 h to obtain activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum Hydrocarbon Degrading Bacteria-10, and Burkholderia sp. AM20. The components and contents of the LB medium were as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH 7.2.

[0053] (2) The activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (1) were inoculated into fermentation medium respectively, with the strain seed solution added in an amount of 10% of the total mass of the medium, the rotation speed was 160 rpm, and the seed culture was carried out at a constant temperature of 30° C. for 36 h to obtain Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 seed solutions; the components and contents of the fermentation medium were as follows: 8.5 g Na2HPO4·2H2O, 3.0 g KH2PO4, 1.0 g NH4Cl, 0.49 g MgSO4·7H2O, 0.011 g CaCl2, 15 g NaCl; trace elements: 0.4 mg CuSO4, 1.0 mg KI, 4.0 mg MnSO4·H2O, 4.0 mg ZnSO4·7H2O, 5.0mg H3BO3, 1.6mg H2MoO4·2H2O, 2.0mg FeCl3·6H2O; 1mol / L MgSO4·7H2O and 1mol / L CaCl2 were sterilized separately and then added to the culture medium, pH 7.0.

[0054] (3) The seed solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (2) are inoculated into fermentation medium respectively, with the addition amount of the strain seed solution being 10% of the total mass of the medium, the rotation speed being 160 rpm, the dissolved oxygen being 55%, and fermentation culture being carried out for 24 hours to obtain bacterial solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20; the fermentation medium is the same as the fermentation medium in step (2).

[0055] (4) The bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (3) were mixed, and bran biochar (prepared by high-temperature pyrolysis of bran at 300°C in a muffle furnace for 2h) was added, mixed, and the mass ratio was 1:10. The mixture was stirred evenly, and the moisture content was adjusted to 15%. After standing at room temperature, a bio-bacterial agent for petroleum hydrocarbon pollution in saline-alkali soil was obtained.

[0056] Example 3

[0057] A biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil and a preparation method thereof are as follows:

[0058] (1) Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum hydrocarbon degrading bacteria-10, and Burkholderia spp. AM20 were cultured in the presence of 1:2.5:0.8:1.3 living bacteria, and the total living bacteria count was 2.5×10 9 cfu / g) were activated in LB medium and cultured with shaking at 30°C for 48 h to obtain activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum Hydrocarbon Degrading Bacteria-10, and Burkholderia species AM20. The components and contents of the LB medium were as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 1 L distilled water (pH 7.0).

[0059] (2) The activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (1) were inoculated into fermentation medium respectively, with the strain seed solution added in an amount of 8% of the total mass of the medium, the rotation speed was 160 rpm, and the seed culture was carried out at a constant temperature of 30°C for 36 hours to obtain Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 seed solutions; the components and contents of the fermentation medium were as follows: 8.5 g Na2HPO4·2H2O, 3.0 g KH2PO4, 1.0 g NH4Cl, 0.49 g MgSO4·7H2O, 0.011 g CaCl2, 15 g NaCl; trace elements: 0.4 mg CuSO4, 1.0 mg KI, 4.0 mg MnSO4·H2O, 4.0 mg ZnSO4·7H2O, 5.0mg H3BO3, 1.6mg H2MoO4·2H2O, 2.0mg FeCl3·6H2O; 1mol / L MgSO4·7H2O and 1mol / L CaCl2 were sterilized separately and then added to the culture medium, pH 7.0.

[0060] (3) The seed solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (2) were inoculated into fermentation medium respectively, with the addition amount of the strain seed solution being 8% of the total mass of the culture medium, the rotation speed being 160 rpm, the dissolved oxygen being 45%, and the fermentation culture was carried out for 42 hours to obtain bacterial solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20; the fermentation medium was the same as the fermentation medium in step (2).

[0061] (4) The bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (3) were mixed, and bran biochar (prepared by high-temperature pyrolysis of bran at 300°C in a muffle furnace for 2h) was added, mixed in a mass ratio of 1:8, stirred evenly, and the moisture content was adjusted to 10%. After standing at room temperature, a bio-bacterial agent for petroleum hydrocarbon pollution in saline-alkali soil was obtained.

[0062] Example 4

[0063] A biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil and a preparation method thereof are as follows:

[0064] (1) Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum hydrocarbon degrading bacteria-10, and Burkholderia spp. AM20 were cultured in the presence of 1:1:1.5:1.3 living bacteria, and the total living bacteria count was 3.0×10 9 cfu / g) were activated in LB medium and cultured at 30°C with shaking for 48 h to obtain activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum Hydrocarbon Degrading Bacteria-10, and Burkholderia species AM20. The components and contents of the LB medium were as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH 7.1.

[0065] (2) The activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (1) were inoculated into fermentation medium respectively, with the strain seed solution added in an amount of 10% of the total mass of the medium, the rotation speed was 160 rpm, and the seed culture was carried out at a constant temperature of 30° C. for 36 h to obtain Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 seed solutions; the components and contents of the fermentation medium were as follows: 8.5 g Na2HPO4·2H2O, 3.0 g KH2PO4, 1.0 g NH4Cl, 0.49 g MgSO4·7H2O, 0.011 g CaCl2, 15 g NaCl; trace elements: 0.4 mg CuSO4, 1.0 mg KI, 4.0 mg MnSO4·H2O, 4.0 mg ZnSO4·7H2O, 5.0mg H3BO3, 1.6mg H2MoO4·2H2O, 2.0mg FeCl3·6H2O; 1mol / L MgSO4·7H2O and 1mol / L CaCl2 were sterilized separately and then added to the culture medium, pH 7.0.

[0066] (3) The seed solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (2) were inoculated into fermentation medium respectively, with the addition amount of the strain seed solution being 10% of the total mass of the culture medium, the rotation speed being 160 rpm, the dissolved oxygen being 45%, and the fermentation culture was carried out for 36 hours to obtain bacterial solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20; the fermentation medium was the same as the fermentation medium in step (2).

[0067] (4) The bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (3) were mixed, and bran biochar (prepared by high-temperature pyrolysis of bran at 300°C in a muffle furnace for 2h) was added, mixed in a mass ratio of 1:12, stirred evenly, and the moisture content was adjusted to 15%. After standing at room temperature, a bio-bacterial agent for petroleum hydrocarbon pollution in saline-alkali soil was obtained.

[0068] Comparative Example 1

[0069] Compared with Example 1, the only difference is that in step (1), the strains are Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3 and Burkholderia spp. AM20, the ratio of the viable counts of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3 and Burkholderia spp. AM20 is 1:2:0.8, and the total viable count is 3.0×10 9cfu / g). The preparation method is the same as that in Example 1.

[0070] Comparative Example 2

[0071] Compared with Example 1, the only difference is that in step (1), the strains are Pseudomonas aeruginosa Ya1, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 (the ratio of the number of live bacteria of Pseudomonas aeruginosa Ya1, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 is 1:1:0.8, and the total number of live bacteria is 3.0×10 9 cfu / g). The preparation method is the same as that in Example 1.

[0072] Comparative Example 3

[0073] Compared with Example 1, the only difference is that in step (1), the strains are Pseudomonas aeruginosa Ya1 and Burkholderia AM20 (the ratio of the number of viable bacteria of Pseudomonas aeruginosa Ya1 and Burkholderia AM20 is 1:0.8, and the total number of viable bacteria is 3.0×10 9 cfu / g). The preparation method is the same as that in Example 1.

[0074] Comparative Example 4

[0075] Compared with Example 1, the only difference is that in step (1), the strains are Halomonas elongatus ZQ1-3 and Burkholderia AM20 (the ratio of the viable bacteria count of Halomonas elongatus ZQ1-3 and Burkholderia AM20 is 2:0.8, and the total viable bacteria count is 3.0×10 9 cfu / g). The preparation method is the same as that in Example 1.

[0076] Comparative Example 5

[0077] Compared with Example 1, the only difference is that in step (4), the carrier is corn cob powder.

[0078] Comparative Example 6

[0079] The biological agent of Example 1 was mixed with petroleum hydrocarbon contaminated soil. The amount of the biological agent added was 6% of the petroleum hydrocarbon contaminated soil. Water was added every other day to maintain the moisture content of the petroleum hydrocarbon contaminated soil at 35%-45%. The petroleum hydrocarbon contaminated soil was turned over and degradation was continued for 90 days.

[0080] Comparative Example 7

[0081] The biological agent of Example 1 was mixed with petroleum hydrocarbon contaminated soil. The amount of the biological agent added was 10% of the petroleum hydrocarbon contaminated soil. Water was added every other day to maintain the moisture content of the petroleum hydrocarbon contaminated soil at 35%-45%. Degradation continued for 90 days.

[0082] Comparative Example 8

[0083] The biological agent of Example 1 was mixed with petroleum hydrocarbon contaminated soil. The amount of the biological agent added was 6% of the petroleum hydrocarbon contaminated soil. The petroleum hydrocarbon contaminated soil was turned over and the degradation was continued for 90 days.

[0084] The method for reducing petroleum pollution in saline-alkali soil of the present invention comprises mixing a biological agent with petroleum hydrocarbon-contaminated soil, wherein the amount of the biological agent added is 6% of the petroleum hydrocarbon-contaminated soil, replenishing water every other day to maintain the moisture content of the petroleum hydrocarbon-contaminated soil at 35%-45%, and turning the petroleum hydrocarbon-contaminated soil for continuous degradation for 90 days.

[0085] Verification Example

[0086] The petroleum-contaminated soil in Chunhua Town, Boxing County, Binzhou City (1000 meters west of the intersection of Caochun Road and Provincial Highway 309) was selected. The moisture content of the petroleum hydrocarbon-contaminated soil before treatment was 37.1wt%, the oil content was 8.3wt%, the solid content was 54.6wt%, the texture was rough, and the petroleum hydrocarbon (C7-C49) content in the petroleum hydrocarbon-contaminated soil was 56.3g / kg.

[0087] The biological agent of Example 1, the biological agent of Comparative Example 1, the biological agent of Comparative Example 2, the biological agent of Comparative Example 3, the biological agent of Comparative Example 4, and the biological agent of Comparative Example 5 were respectively mixed with petroleum hydrocarbon contaminated soil. The addition amount of each group of biological agents was 6% of the petroleum hydrocarbon contaminated soil. Water was added every other day to maintain the moisture content of the petroleum hydrocarbon contaminated soil at 35%-45%. The petroleum hydrocarbon contaminated soil was turned over and degradation was continued for 90 days.

[0088] After 90 days of degradation by the bio-bacteria in each group, samples were taken to test their oil content and compare the petroleum hydrocarbon content in the contaminated soil before and after degradation. After sampling and monitoring, the remaining petroleum hydrocarbon (C7-C49) content in each group of petroleum hydrocarbon contaminated soil was calculated, and the degradation rate of petroleum hydrocarbon substances was calculated. The results are shown in Figure 1 、 Figure 2 .

[0089] After the petroleum hydrocarbon-contaminated soil was treated with the biological agent in Example 1 of the present invention, the remaining petroleum hydrocarbon (C7-C49) content was 11.8 g / kg, and the degradation rate of petroleum hydrocarbon substances was 79.04%.

[0090] The effectiveness of restoring petroleum hydrocarbon-contaminated soil in Example 1 of the present invention was significantly superior to that of the biological agent in Comparative Examples 1, 2, 3, 4, and 5. In the biological agent in Example 1, Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, Petroleum Hydrocarbon Degrading Bacteria-10, and Burkholderia AM20 acted together to promote the degradation of petroleum hydrocarbons. However, the biological agents in Comparative Examples 1-4, which used only two or three of these microorganisms, significantly reduced the degradation rate of petroleum hydrocarbons.

[0091] The biological agent from Example 1 was used to treat the petroleum hydrocarbon-contaminated soil according to the methods of Comparative Examples 6, 7, and 8. After 90 days of degradation, samples were collected and tested for oil content. The petroleum hydrocarbon content in the contaminated soil after treatment according to Comparative Examples 6, 7, and 8 was compared. Through sampling and monitoring, the remaining petroleum hydrocarbon (C7-C49) content in the contaminated soil was calculated, as was the degradation rate of petroleum hydrocarbons. The results are shown in Table 1.

[0092] Table 1 Remaining petroleum hydrocarbon content and degradation rate in petroleum hydrocarbon contaminated soil after biological agent treatment

[0093]

[0094] After using the present invention's method for reducing petroleum pollution in saline-alkali soil to remediate petroleum hydrocarbon-contaminated soil, the remaining petroleum hydrocarbon content in the soil was significantly reduced. The present method for reducing petroleum pollution in saline-alkali soil has a high degradation rate for petroleum hydrocarbons. However, the methods of Comparative Examples 6, 7, and 8 were slightly less effective in treating petroleum hydrocarbon-contaminated soil. The present method for reducing petroleum pollution in saline-alkali soil has significant technical advantages.

[0095] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0096] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil. It is characterized in that These included Pseudomonas aeruginosa Ya1, Halomonas elongata ZQ1-3, Achromoobacter pulmonis-10, and Burkholderia sp. AM20; The ratio of the viable bacterial counts of Pseudomonas aeruginosa Ya1, Halomonaselongata ZQ1-3, Achromoobacter pulmonis-10 and Burkholderia sp. AM20 is 1:(1.0-2.5):(0.8-1.5):(0.5-1.3), and the total viable bacterial count is (2.5-3.5)×10 9 cfu / g; The Pseudomonas aeruginosa Ya1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 18, 2015, with the deposit number of CGMCC NO.11426; The Halomonas elongata ZQ1-3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 26, 2020, with the deposit number CGMCC No.20953; The petroleum hydrocarbon degrading bacteria (Achromoobacter pulmonis)-10 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 9, 2018, with the deposit number of CGMCC No.16240; The Burkholderia sp. AM20 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 6, 2023, with the deposit number CGMCC NO.27032.

2. The method for preparing the biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil according to claim 1, It is characterized in that The steps include: (1) activating Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 on LB medium to obtain activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20; (2) inoculating the activated Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon-degrading bacteria-10 and Burkholderia AM20 obtained in step (1) into a fermentation medium for seed culture, respectively, to obtain seed solutions of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon-degrading bacteria-10 and Burkholderia AM20; (3) inoculating the seed liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (2) into fermentation medium for fermentation culture, respectively, to obtain bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20; The bacterial liquids of Pseudomonas aeruginosa Ya1, Halomonas elongatus ZQ1-3, petroleum hydrocarbon degrading bacteria-10 and Burkholderia AM20 obtained in step (3) are mixed, and bran biochar is added, mixed, and stirred evenly at a mass ratio of 1:(6-15), and the moisture content is adjusted to 10-15%. After standing at room temperature, a biological bacterial agent for petroleum hydrocarbon pollution in saline-alkali soil is obtained.

3. The preparation method according to claim 2, It is characterized in that The activation culture condition in step (1) is constant temperature shaking culture at 30° C. for 48 hours; the components and contents of the LB culture medium in step (1) are as follows: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH 7.0-7.

2.

4. The preparation method according to claim 2, It is characterized in that The components and contents of the fermentation medium in step (2) or step (3) are as follows: 8.5 g Na 2 HPO 4 ·2H 2 O, 3.0 g KH 2 PO 4 , 1.0 g NH 4 Cl, 0.49 g MgSO 4 7H 2 O, 0.011 g CaCl 2 , 15g NaCl; trace elements: 0.4mg CuSO 4 , 1.0 mg KI, 4.0 mg MnSO 4 ·H 2 O, 4.0mgZnSO 4 7H 2 O, 5.0 mg H 3 BO 3 , 1.6 mg H 2 MoO 4 ·2H 2 O, 2.0 mg FeCl 3 6H 2 O; 1 mol / L MgSO 4 7H 2 O and 1 mol / L CaCl 2 After sterilization, add to the culture medium, pH 7.

0.

5. Use of the biological agent for treating petroleum hydrocarbon pollution in saline-alkali soil according to claim 1 in remediating petroleum pollutants.

6. The use according to claim 5, It is characterized in that The petroleum pollutant is soil contaminated by petroleum or treated oily sludge; when the saline-alkali soil petroleum hydrocarbon pollution biological bacterial agent is used to repair the petroleum pollutant, 3-8% of the mass of the petroleum hydrocarbon pollutant is added to the biological bacterial agent, the water content of the petroleum hydrocarbon pollutant is maintained at 35%-45%, and the petroleum hydrocarbon pollutant is turned over for continuous degradation for 60-120 days.

7. The use according to claim 6, It is characterized in that When the saline-alkali soil petroleum hydrocarbon pollution biological bacterial agent is used to repair petroleum pollutants, 6% of the mass of the petroleum hydrocarbon pollutants is added to the biological bacterial agent, and the degradation is continued for 90 days.

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