Method for realizing deep degradation of petroleum hydrocarbon in contaminated soil based on metabolic division of labor

Abstract

The application discloses a method for realizing deep degradation of petroleum hydrocarbon in contaminated soil based on metabolic division, and uses a wide-substrate-spectrum bacterial agent including three kinds of fungi and one kind of bacteria, wherein the mass ratio of the bacteria in the wide-substrate-spectrum bacterial agent is Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter = 1: (0.5-2): (0.5-2): (0.5-2), the wide-substrate-spectrum bacterial agent is added into petroleum hydrocarbon contaminated soil according to a ratio of 2% to 8% of the volume of the bacterial agent to the mass of the petroleum hydrocarbon contaminated soil, the mass concentration of the petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil is 0.5% to 5%, the soil moisture content is kept at 20% to 40% wt during the remediation process, the soil is completely stirred once every 7 days to 10 days, and the culture is carried out at room temperature for 50 days to 70 days. The application can solve the problems of narrow substrate spectrum and large residual amount of heavy components such as colloid and asphaltene in the existing mixed bacterial agent of fungi and bacteria, and realizes deep remediation of multi-component petroleum hydrocarbon.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial remediation of petroleum hydrocarbon contaminated soil, and particularly relates to a method for realizing deep degradation of petroleum hydrocarbon in contaminated soil based on metabolic division. BACKGROUND

[0002] The pollution caused by the leakage of oil into the soil environment in the process of exploitation, processing and transportation has attracted widespread attention. The entry of petroleum hydrocarbon into the soil environment not only destroys the ecological function of the soil itself, but also migrates and spreads, posing a threat to plants, animals and even human health. Compared with high-concentration petroleum hydrocarbon contaminated soil which is suitable for centralized treatment by ex-situ chemical cleaning, low-concentration (usually < 5%) petroleum hydrocarbon contaminated soil is more suitable for in-situ remediation by environmentally friendly and low-cost microbial methods to achieve deep degradation of the multiple components of petroleum hydrocarbon in the contaminated soil.

[0003] The microorganisms that degrade petroleum hydrocarbon pollutants in soil are mainly bacteria and fungi. The mechanism of bacteria degrading petroleum hydrocarbon in soil is mainly to gradually decompose macromolecular hydrocarbons into small molecular compounds through specific enzymatic reactions, and finally completely mineralize them into CO2 and H2O through the tricarboxylic acid cycle. The mechanism of bacteria degrading petroleum hydrocarbon lies in the oxidation and degradation of petroleum hydrocarbon through its highly specific enzyme system and metabolic pathway, and the core is the precise attack and conversion of specific enzymes on petroleum hydrocarbon substrates. However, the degradation substrates of the bacterial-based degrading agent mainly focus on small molecular components, and the degradation ability of the degrading agent for macromolecular, structurally complex heavy petroleum hydrocarbons such as resin and asphaltene is limited, and the residual amount of resin and asphaltene in the soil after remediation is still large.

[0004] Compared with bacteria, fungi are more suitable for degrading macromolecular, structurally complex petroleum hydrocarbons, which mainly rely on two decomposition pathways of lignin and non-lignin. The lignin decomposition pathway mineralizes or partially mineralizes petroleum hydrocarbon through the extracellular oxidase secreted by fungi, and the non-lignin decomposition pathway mainly decomposes medium and low molecular weight petroleum hydrocarbons through cytochrome P450 enzymes. Studies have shown that the efficient degradation of petroleum hydrocarbon molecules by fungi depends on the cascade reaction and synergistic effect of its extracellular enzyme system. The mycelium of fungi can penetrate into the soil pores and fully contact with the insoluble macromolecular hydrocarbons, which is conducive to the remediation of petroleum hydrocarbon contaminated soil. However, a single fungal agent also cannot form a perfect metabolic division system, and the utilization of different components of petroleum hydrocarbon is still uneven, which makes it difficult to fundamentally solve the problems of narrow substrate spectrum and large residual amount of resin and asphaltene of existing degrading agents. SUMMARY

[0005] In view of the deficiencies of the prior art, the technical problem to be solved by the present application is to provide a method for realizing deep degradation of petroleum hydrocarbon in contaminated soil based on metabolic division, which can solve the problems of narrow substrate spectrum of existing mixed fungal and bacterial inoculants and large residual amount of heavy components such as colloid and asphaltene, and realize deep repair of multi-component petroleum hydrocarbon by constructing a wide-substrate-spectrum inoculant composed of specific types of fungi and bacteria.

[0006] The technical scheme adopted by the present application to solve the technical problem is:

[0007] The method for realizing deep degradation of petroleum hydrocarbon in contaminated soil based on metabolic division uses a wide-substrate-spectrum inoculant, which includes three types of fungi and one type of bacteria, and the mass ratio of the fungi in the wide-substrate-spectrum inoculant is Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter = 1: (0.5-2): (0.5-2): (0.5-2).

[0008] The wide-substrate-spectrum inoculant is added to the petroleum hydrocarbon contaminated soil at a ratio of 2% to 8% of the volume of the inoculant to the mass of the petroleum hydrocarbon contaminated soil, the mass concentration of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil is 0.5% to 5%, the soil moisture content is maintained at 20% wt to 40% wt during the repair process, and the soil is thoroughly stirred once every 7 to 10 days, and the culture is carried out at room temperature for 50 to 70 days.

[0009] By constructing a wide-substrate-spectrum inoculant composed of specific bacteria (Achromobacter) and three types of fungi (Fusarium oxysporum, Fusarium fujikuroi, and Talaromyces emersonii), the present application utilizes the metabolic division and cross-feeding mechanism of bacteria preferentially decomposing easily degradable components and supplying fungi, and fungi specializing in heavy components, to realize wide-spectrum and collaborative degradation of saturated hydrocarbons, aromatic hydrocarbons, colloid, and asphaltene in petroleum hydrocarbon, thereby significantly improving the efficiency and speed of deep repair of contaminated soil.

[0010] Further, the degradation efficiency of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil is measured by the gravimetric method, and after the repair is completed, the removal rate of total petroleum hydrocarbon is not less than 75%, and the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, colloid, and asphaltene components are not less than 85%, 80%, 75%, and 70%, respectively.

[0011] Further, when the ratio of Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter is 1:2: (0.5-2): (0.5-2), the ratio of the volume of the inoculant to the mass of the petroleum hydrocarbon contaminated soil is 5%, and the water content of the petroleum hydrocarbon contaminated soil is 20% wt to 40% wt, after the repair is completed, the removal rate of total petroleum hydrocarbon is not less than 90%, and the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, colloid, and asphaltene components are not less than 92%, 91%, 85%, and 80%, respectively.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] This invention presents a low-cost, environmentally friendly, and simple method for removing petroleum hydrocarbons from petroleum hydrocarbon-contaminated soil using a bacterial-fungal composite agent. Through the synergistic division of labor between different types of fungi and specific bacteria in metabolic pathways and substrate utilization, *Achromobacterium* bacteria are responsible for efficiently decomposing easily degradable components, initiating initial degradation, and supplying their metabolic products to the fungi to promote growth. Three types of fungi are responsible for decomposing complex and recalcitrant components and creating a favorable microenvironment for the bacteria. The two groups work together metabolically, cross-feeding each other, promoting each other without antagonism. By controlling the species and ratio of fungi and bacteria, a broad substrate spectrum can be used to achieve deep remediation of petroleum hydrocarbon-contaminated soil. This invention's broad substrate spectrum agent exhibits excellent degradation capabilities for saturated hydrocarbons, aromatic hydrocarbons, and heavy components such as colloids and asphaltenes, significantly broadening the substrate spectrum for petroleum hydrocarbon degradation. While achieving a high total petroleum hydrocarbon removal rate, it effectively reduces the residues of various components such as saturated hydrocarbons, aromatic hydrocarbons, colloids, and asphaltenes, thus achieving deep remediation of petroleum hydrocarbon-contaminated soil.

[0014] Compared with similar microbial technologies, under the same conditions, the method of the present invention requires less time and has higher removal efficiency for petroleum hydrocarbons, as shown in Table 1.

[0015] Table 1. Comparison of the effects of the broad substrate spectrum bacterial agent of the present invention and the comparative bacterial agent.

[0016] Detailed Implementation

[0017] The present invention will be described in detail below through specific embodiments. The uses and purposes of these exemplary embodiments are only for illustrating the present invention and are not intended to limit the actual protection scope of the present invention in any way, nor are they intended to limit the protection scope of the present invention to these embodiments.

[0018] The fungi used in the embodiments of this invention are Fusarium argentis (Fusarium argentis) Fusarium acuminatum ), Fusarium oxysporum ( Fusarium fujikuroi ) and cord-shaped basket bacteria ( Talaromyces funiculosus All were purchased from Ningbo Mingzhou Biotechnology Co., Ltd., specifically, Fusarium argentis (… Fusarium acuminatum The product number is BMZ027399, originating from Ningbo Mingzhou Biotechnology Co., Ltd., and is named... Fusarium acuminatum sp. F1; Fusarium oxysporum ( Fusarium fujikuroi The product number is BMZ147104, originating from Ningbo Mingzhou Biotechnology Co., Ltd., and is named... Fusarium fujikuroi sp. F2; *Basilella fragilis* ( Talaromyces funiculosus The product number is BMZ148324, originating from Ningbo Mingzhou Biotechnology Co., Ltd., and is named... Talaromyces funiculosus sp. F3;

[0019] The bacteria used are Achromobacter xylosoxidans (ATCC 27061) Achromobacter petrolophlilum ) also purchased from Ningbo Mingzhou Biotechnology Co., Ltd., with the number BMZ106750, also known as Achromobacter piechaophilum, named Achromobacter xylosoxidans sp. S5. Achromobacter petrolophlilum sp. S5.

[0020] The three fungi are inoculated in PDA culture medium, and the bacteria are inoculated in LB culture medium. The activated culture medium after inoculation is placed in a constant temperature incubator at 30-35 DEG C, and cultured at a rotation speed of 100-200 r / min for 7-10 days. The activated culture medium of the bacteria after inoculation is cultured to an OD 600 value of 0.8-2.0, to obtain the bacterial seed liquid. The activated culture medium of the fungi after inoculation is cultured to a dry weight of the fungi of 20-30 mg / mL, to obtain the fungal seed liquid.

[0021] The process for determining the dry weight of the fungi in the fungal seed liquid is as follows: a certain volume V of the activated culture medium of the fungi is sampled, and after sampling, centrifugal treatment is performed, and the supernatant is discarded, to obtain a dry weight m, and then the dry weight of the fungi in the current activated culture medium is m / V.

[0022] If the dry weight of the fungi is in the range of 20-30 mg / mL, the fungal seed liquid is obtained.

[0023] The fungal seed liquid and the bacterial seed liquid are resuspended in 0.9% sodium chloride physiological saline respectively to obtain the corresponding resuspensions.

[0024] The mass ratio of the corresponding bacteria in the resuspensions of the fungi and the bacteria can be regarded as the volume ratio of the corresponding resuspensions, which is more convenient for calculation and operation.

[0025] The four resuspensions are uniformly mixed in a volume ratio of F. oxysporum: F. fujikuroi: T. funiculosum: A. xylosoxidans = 1: (0.5-2): (0.5-2): (0.5-2) to obtain the wide-substrate-spectrum microbial agent.

[0026] In the present application, the Achromobacter xylosoxidans Achromobacter petrolophlilum sp. S5 is responsible for decomposing the easily degradable components, starting the preliminary degradation, and supplying the metabolic products to the fungi to promote the growth. Fusarium acuminatum The three fungi F. oxysporum sp. F1, F. fujikuroi sp. F2 and T. funiculosum sp. F3 are responsible for decomposing the complex and difficult-to-degrade components and creating a favorable microenvironment for the bacteria. Fusarium fujikuroi Talaromyces funiculosus

[0027] ​​The prepared broad-substrate spectrum microbial agent is added to the petroleum hydrocarbon contaminated soil with a concentration of 0.5% to 5% (w / w) (mass of petroleum hydrocarbon / mass of petroleum hydrocarbon contaminated soil) at a dosage of 2% to 8% (v / w) (volume ratio of microbial agent to mass of petroleum hydrocarbon contaminated soil), and the petroleum hydrocarbon contaminated soil is kept at a water content of 20% to 40% (wt), and stirred thoroughly every 7 to 10 days to ensure sufficient oxygen supply, and incubated at room temperature for 50 to 70 days. The content of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil is measured by the gravimetric method, and the degradation efficiency of petroleum hydrocarbon is calculated.

[0028] Example 1

[0029] The present example is based on a method for achieving deep degradation of petroleum hydrocarbons in contaminated soil through metabolic division of labor, which uses a broad-substrate spectrum microbial agent comprising fungi and bacteria, wherein the fungi are a mixture of Fusarium oxysporum (Fusarium sp. F1), Fusarium fujikuroi (Fusarium sp. F2), Talaromyces emersonii (Talaromyces sp. F3), and the bacteria are Achromobacter sp. S5. Fusarium acuminatum Fusarium fujikuroi Talaromyces funiculosus sp.F3) and bacteria (Achromobacter sp. S5). Achromobacter petrolophlilum

[0030] The fungi are inoculated in PDA medium, and the bacteria are inoculated in LB medium. The activated culture medium after inoculation is placed in a constant temperature shaker for shaking culture. The activated culture medium of bacteria is cultured after inoculation until the OD600 value reaches 0.8, to obtain bacterial seed liquid;

[0031] The activated culture medium of fungi is cultured after inoculation until the dry weight of fungi reaches 20 mg / mL, to obtain fungal seed liquid;

[0032] 30 mL of bacterial seed liquid and fungal seed liquid are taken respectively, and centrifuged at 3500 r / min for 10 min. The supernatant is discarded, and the bacterial pellet is resuspended with 30 mL of 0.9% (w / w) sodium chloride physiological saline to prepare a resuspension. The resuspensions of the four bacteria are uniformly mixed in a volume ratio of 1:2:2:2 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter sp.), to obtain a broad-substrate spectrum microbial agent.

[0033] ​​​The wide-substrate spectrum bacterial agent is used for deep degradation of the petroleum hydrocarbon contaminated soil. Specifically, the prepared wide-substrate spectrum bacterial agent is added into the petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 5% (v / w) (the ratio of the volume of the bacterial agent to the mass of the petroleum hydrocarbon contaminated soil), the water content of the petroleum hydrocarbon contaminated soil is kept at 30% wt during the remediation process, and the petroleum hydrocarbon contaminated soil is thoroughly stirred every 7 days to ensure sufficient oxygen supply, and the culture is incubated at room temperature for 70 days. The weight method is used to measure the content of the petroleum hydrocarbon, and the degradation efficiency of the petroleum hydrocarbon is 95.6%. Meanwhile, the degradation rates of the saturated hydrocarbon, the aromatic hydrocarbon, the resin and the asphaltene in the petroleum hydrocarbon contaminated soil are determined by using the four-component analysis method of the petroleum hydrocarbon, and the degradation rate results are as follows: the saturated hydrocarbon is 97.1%; the aromatic hydrocarbon is 95.1%; the resin is 93.8%; and the asphaltene is 92.6%.

[0034] Example 2

[0035] In this embodiment, the resuspensions of the four bacteria are uniformly mixed according to the volume ratio of 1:0.5:1:1 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter sp.) to prepare the wide-substrate spectrum bacterial agent, and the other processes are the same as those in Example 1.

[0036] The wide-substrate spectrum bacterial agent is used for deep degradation of the petroleum hydrocarbon contaminated soil, and is added into the petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 2% (v / w), the water content of the petroleum hydrocarbon contaminated soil is kept at 20% wt during the remediation process, and the other processes are the same as those in Example 1.

[0037] The weight method is used to measure the content of the petroleum hydrocarbon, and the degradation efficiency of the petroleum hydrocarbon is 86.3%. Meanwhile, the degradation rates of the saturated hydrocarbon, the aromatic hydrocarbon, the resin and the asphaltene in the petroleum hydrocarbon contaminated soil are determined by using the four-component analysis method of the petroleum hydrocarbon, and the degradation rate results are as follows: the saturated hydrocarbon is 91.3%; the aromatic hydrocarbon is 89.1%; the resin is 82.3%; and the asphaltene is 79.2%.

[0038] Example 3

[0039] In this embodiment, the resuspensions of the four bacteria are uniformly mixed according to the volume ratio of 1:2:1:2 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter sp.) to prepare the wide-substrate spectrum bacterial agent, and the other processes are the same as those in Example 1.

[0040] The wide-substrate spectrum bacterial agent is used for deep degradation of the petroleum hydrocarbon contaminated soil, and is added into the petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 2% (v / w), the water content of the petroleum hydrocarbon contaminated soil is kept at 40% wt during the remediation process, and the other processes are the same as those in Example 1.

[0041] The petroleum hydrocarbon content was measured by weight method, and the degradation efficiency of petroleum hydrocarbon was calculated as 82.4%. At the same time, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil were determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results were as follows: saturated hydrocarbon: 85.3%; aromatic hydrocarbon: 81.7%; resin: 79.3%; asphaltene: 74.1%.

[0042] Example 4

[0043] In this embodiment, the resuspensions of the four bacteria were uniformly mixed in a volume ratio of 1:1:1:1 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter xylosoxidans) to prepare a broad-substrate-spectrum bacterial agent, and other processes were the same as those in Example 1.

[0044] The broad-substrate-spectrum bacterial agent was used for deep degradation of petroleum hydrocarbon contaminated soil, and was added to petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 8% (v / w). The water content of the petroleum hydrocarbon contaminated soil was maintained at 30% wt during the remediation process, and other processes were the same as those in Example 1.

[0045] The petroleum hydrocarbon content was measured by weight method, and the degradation efficiency of petroleum hydrocarbon was calculated as 88.1%. At the same time, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil were determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results were as follows: saturated hydrocarbon: 92.7%; aromatic hydrocarbon: 89.0%; resin: 86.1%; asphaltene: 81.2%.

[0046] Example 5

[0047] In this embodiment, the resuspensions of the four bacteria were uniformly mixed in a volume ratio of 1:2:2:0.5 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter xylosoxidans), and other processes were the same as those in Example 1.

[0048] The broad-substrate-spectrum bacterial agent was used for deep degradation of petroleum hydrocarbon contaminated soil, and was added to petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 5% (v / w). The water content of the petroleum hydrocarbon contaminated soil was maintained at 20% wt during the remediation process, and other processes were the same as those in Example 1.

[0049] The petroleum hydrocarbon content was measured by weight method, and the degradation efficiency of petroleum hydrocarbon was calculated as 90.5%. At the same time, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil were determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results were as follows: saturated hydrocarbon: 92.8%; aromatic hydrocarbon: 91.3%; resin: 86.4%; asphaltene: 83.1%.

[0050] Example 6

[0051] The resuspension of the four bacteria in this embodiment was mixed uniformly according to the volume ratio of 1:2:0.5:1 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter xylosoxidans) to prepare a broad-substrate bacteria agent, and other processes were the same as in Example 1.

[0052] The broad-substrate bacteria agent was used for deep degradation of petroleum hydrocarbon contaminated soil, and was added to petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 2% (v / w). The water content of the petroleum hydrocarbon contaminated soil was maintained at 20% wt during the remediation process, and other processes were the same as in Example 1.

[0053] The weight method was used to measure the content of petroleum hydrocarbons, and the degradation efficiency of petroleum hydrocarbons was calculated to be 86.5%. At the same time, the four-component analysis method of petroleum hydrocarbons was used to determine the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, resins and asphaltenes in the petroleum hydrocarbon contaminated soil, and the degradation rates were as follows: saturated hydrocarbons: 90.8%; aromatic hydrocarbons: 89.3%; resins: 86.7%; asphaltenes: 82.3%.

[0054] Example 7

[0055] The resuspension of the four bacteria in this embodiment was mixed uniformly according to the volume ratio of 1:0.5:2:2 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter xylosoxidans), and other processes were the same as in Example 1.

[0056] The broad-substrate bacteria agent was used for deep degradation of petroleum hydrocarbon contaminated soil, and was added to petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 8% (v / w). The water content of the petroleum hydrocarbon contaminated soil was maintained at 30% wt during the remediation process, and other processes were the same as in Example 1.

[0057] The weight method was used to measure the content of petroleum hydrocarbons, and the degradation efficiency of petroleum hydrocarbons was calculated to be 81.2%. At the same time, the four-component analysis method of petroleum hydrocarbons was used to determine the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, resins and asphaltenes in the petroleum hydrocarbon contaminated soil, and the degradation rates were as follows: saturated hydrocarbons: 85.3%; aromatic hydrocarbons: 81.1%; resins: 76.2%; asphaltenes: 73.7%.

[0058] Example 8

[0059] The resuspension of the four bacteria in this embodiment was mixed uniformly according to the volume ratio of 1:2:0.5:2 (Fusarium oxysporum: Fusarium fujikuroi: Talaromyces emersonii: Achromobacter xylosoxidans) to prepare a broad-substrate bacteria agent, and other processes were the same as in Example 1.

[0060] The broad-substrate bacteria agent was used for deep degradation of petroleum hydrocarbon contaminated soil, and was added to petroleum hydrocarbon contaminated soil with a mass concentration of 2% wt at a dosage of 5% (v / w). The water content of the petroleum hydrocarbon contaminated soil was maintained at 40% wt during the remediation process, and other processes were the same as in Example 1.

[0061] The degradation efficiency of petroleum hydrocarbon is calculated as 92.6% by measuring the content of petroleum hydrocarbon by gravimetric method. Meanwhile, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil are determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results are as follows: saturated hydrocarbon: 94.8%; aromatic hydrocarbon: 92.3%; resin: 89.5%; asphaltene: 88.5%.

[0062] Comparative Example 1

[0063] In this comparative example, the sharp-top fusarium is not introduced, and only three kinds of bacteria, fusarium fujikuroi (F2), talaromyces emersonii (F3) and achromobacter (S5) are used. The resuspension of the three kinds of bacteria is uniformly mixed in a volume ratio of 1:2:2 (fusarium fujikuroi:talaromyces emersonii:achromobacter) to obtain a bacterial agent, and other processes are the same as those in Example 1. Fusarium fujikuroi Talaromyces funiculosus The bacterial agent is used for deep degradation of petroleum hydrocarbon contaminated soil, and is added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt in a dosage of 8% (v / w). The water content of the petroleum hydrocarbon contaminated soil is maintained at 30%wt during the remediation process, and other processes are the same as those in Example 1. Achromobacter petrolophlilum The degradation efficiency of petroleum hydrocarbon is calculated as 36.2% by measuring the content of petroleum hydrocarbon by gravimetric method. Meanwhile, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil are determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results are as follows: saturated hydrocarbon: 26.1%; aromatic hydrocarbon: 26.8%; resin: 46.4%; asphaltene: 42.2%.

[0064] Comparative Example 2

[0065] In this comparative example, the sharp-top fusarium is not introduced, and only three kinds of bacteria, fusarium fujikuroi (F2), talaromyces emersonii (F3) and achromobacter (S5) are used. The resuspension of the three kinds of bacteria is uniformly mixed in a volume ratio of 1:0.5:1 (fusarium fujikuroi:talaromyces emersonii:achromobacter) to obtain a bacterial agent, and other processes are the same as those in Example 1.

[0066] The bacterial agent is used for deep degradation of petroleum hydrocarbon contaminated soil, and is added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt in a dosage of 2% (v / w). The water content of the petroleum hydrocarbon contaminated soil is maintained at 30%wt during the remediation process, and other processes are the same as those in Example 1.

[0067] The degradation efficiency of petroleum hydrocarbon is calculated as 36.2% by measuring the content of petroleum hydrocarbon by gravimetric method. Meanwhile, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil are determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results are as follows: saturated hydrocarbon: 26.1%; aromatic hydrocarbon: 26.8%; resin: 46.4%; asphaltene: 42.2%. Fusarium fujikuroi Talaromyces funiculosus Comparative Example 3 Achromobacter petrolophlilum In this comparative example, the sharp-top fusarium is not introduced, and only three kinds of bacteria, fusarium fujikuroi (F2), talaromyces emersonii (F3) and achromobacter (S5) are used. The resuspension of the three kinds of bacteria is uniformly mixed in a volume ratio of 1:0.5:1 (fusarium fujikuroi:talaromyces emersonii:achromobacter) to obtain a bacterial agent, and other processes are the same as those in Example 1.

[0068] The bacterial agent is used for deep degradation of petroleum hydrocarbon contaminated soil, and is added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt in a dosage of 2% (v / w). The water content of the petroleum hydrocarbon contaminated soil is maintained at 30%wt during the remediation process, and other processes are the same as those in Example 1.

[0069] ​​The degradation efficiency of petroleum hydrocarbon is 33.0% calculated by measuring the content of petroleum hydrocarbon by gravimetric method. Meanwhile, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil are determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results are as follows: saturated hydrocarbon: 22.1%; aromatic hydrocarbon: 28.3%; resin: 38.6%; asphaltene: 42.1%.

[0070] Comparative Example 3

[0071] In this comparative example, F. fujikuroi and T. funiculosum are not introduced, and only F. fujikuroi (F2) and A. xylanus (S5) are used. Fusarium fujikuroi The resuspensions of fungi and bacteria are uniformly mixed in a volume ratio of 1:1 (F. fujikuroi: A. xylanus) to obtain a microbial agent, and other processes are the same as in Example 1. Achromobacter petrolophlilum The microbial agent is used for deep degradation of petroleum hydrocarbon contaminated soil, and is added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt at a dosage of 2% (v / w), and the water content of the petroleum hydrocarbon contaminated soil is maintained at 20%wt during the remediation process, and other processes are the same as in Example 1.

[0072] The degradation efficiency of petroleum hydrocarbon is 15.3% calculated by measuring the content of petroleum hydrocarbon by gravimetric method. Meanwhile, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil are determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results are as follows: saturated hydrocarbon: 17.1%; aromatic hydrocarbon: 15.2%; resin: 13.8%; asphaltene: 12.7%.

[0073] Comparative Example 4

[0074] In this comparative example, F. fujikuroi and A. xylanus are not introduced, and only F. fujikuroi (F2) and T. funiculosum (F3) are used.

[0075] Fusarium The resuspensions of the two fungi are uniformly mixed in a volume ratio of 1:2 (F. fujikuroi: T. funiculosum) to obtain a microbial agent, and other processes are the same as in Example 1. fujikuroi Talaromyces funiculosus The microbial agent is used for deep degradation of petroleum hydrocarbon contaminated soil, and is added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt at a dosage of 5% (v / w), and the water content of the petroleum hydrocarbon contaminated soil is maintained at 40%wt during the remediation process, and other processes are the same as in Example 1.

[0076] The degradation efficiency of petroleum hydrocarbon is 15.3% calculated by measuring the content of petroleum hydrocarbon by gravimetric method. Meanwhile, the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil are determined by petroleum hydrocarbon four-component analysis method, and the degradation rate results are as follows: saturated hydrocarbon: 17.1%; aromatic hydrocarbon: 15.2%; resin: 13.8%; asphaltene: 12.7%.

[0077] ​The petroleum hydrocarbon content was measured using a gravimetric method, and the degradation efficiency of petroleum hydrocarbons was calculated to be 10.9%. Simultaneously, a four-component analysis method for petroleum hydrocarbons was used to determine the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, colloids, and asphaltenes in petroleum hydrocarbon-contaminated soil. The degradation rates were as follows: saturated hydrocarbons: 5.3%; aromatic hydrocarbons: 7.9%; colloids: 18.3%; asphaltenes: 17.8%.

[0078] Comparative Example 5

[0079] In this comparative example, Achromobacterium ( Achromobacter petrolophlilum sp.S5) was replaced with Acinetobacter (sp.S5) Acinetobacter lwoffii ), Fusarium argentis ( Fusarium acuminatum replace sp. F1) with white rot fungi ( Trametes versicolor The resuspensions of the four bacteria were uniformly mixed in a volume ratio of 1:2:1:1 (white rot fungi: Fusarium tumefaciens: Basilaria cordifolia: Acinetobacter bacillus) to obtain the bacterial agent. Other processes were the same as in Example 1.

[0080] The bacterial agent was used to deeply degrade petroleum hydrocarbon contaminated soil. It was added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt at a dosage of 5% (v / w). During the remediation process, the moisture content of the petroleum hydrocarbon contaminated soil was maintained at 30%wt. Other processes were the same as in Example 1.

[0081] The petroleum hydrocarbon content was measured using the gravimetric method, and the degradation efficiency of petroleum hydrocarbons was calculated to be 33.49%. Simultaneously, the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, colloids, and asphaltenes in petroleum hydrocarbon-contaminated soil were determined using a four-component analysis method. The degradation rates were as follows: saturated hydrocarbons: 41.2%; aromatic hydrocarbons: 35.1%; colloids: 28.3%; asphaltenes: 23.8%.

[0082] Comparative Example 6

[0083] In this comparative example, Achromobacterium ( Achromobacter petrolophlilum sp.S5) was replaced with Enterobacter ( Enterobacter sp. ), the rope-shaped basket bacteria ( Talaromyces funiculosus replace sp. F3 with Candida albicans (sp. F3) Pseudozyma The resuspensions of the four bacteria were uniformly mixed in a volume ratio of 1:0.5:0.5:0.5 (Fusarium racifolium: Fusarium fusiforme: Candida albicans: Enterobacter) to obtain the bacterial agent. Other processes were the same as in Example 1.

[0084] The bacterial agent was used to deeply degrade petroleum hydrocarbon contaminated soil. It was added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt at a dosage of 5% (v / w). During the remediation process, the moisture content of the petroleum hydrocarbon contaminated soil was maintained at 30%wt. Other processes were the same as in Example 1.

[0085] The weight method is used to measure the content of petroleum hydrocarbon, and the degradation efficiency of petroleum hydrocarbon is 21.66%. At the same time, the four-component analysis method of petroleum hydrocarbon is used to measure the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil, and the degradation rate results are as follows: saturated hydrocarbon: 25.3%; aromatic hydrocarbon: 23.1%; resin: 17.3%; asphaltene: 16.2%.

[0086] Comparative Example 7

[0087] In the present comparative example, the achromobacter sp. S5 is replaced by Serratia marcescens, and the Fusarium fujikuroi sp. F2 is replaced by Aspergillus sp., and the resuspensions of the four bacteria are uniformly mixed in a volume ratio of 1:1:1:2 (Fusarium oxysporum: Aspergillus sp.: Talaromyces emersonii: Serratia marcescens) to obtain a bacterial agent, and the other processes are the same as in Example 1. Achromobacter petrolophlilum Serratia marcescens The bacterial agent is used for deep degradation of petroleum hydrocarbon contaminated soil, and is added to petroleum hydrocarbon contaminated soil with a mass concentration of 2%wt at a dosage of 5% (v / w), and the water content of the petroleum hydrocarbon contaminated soil is maintained at 30%wt during the remediation process, and the other processes are the same as in Example 1. Fusarium fujikuroi Aspergillus The weight method is used to measure the content of petroleum hydrocarbon, and the degradation efficiency of petroleum hydrocarbon is 21.66%. At the same time, the four-component analysis method of petroleum hydrocarbon is used to measure the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil, and the degradation rate results are as follows: saturated hydrocarbon: 25.3%; aromatic hydrocarbon: 23.1%; resin: 17.3%; asphaltene: 16.2%.

[0088] The weight method is used to measure the content of petroleum hydrocarbon, and the degradation efficiency of petroleum hydrocarbon is 21.66%. At the same time, the four-component analysis method of petroleum hydrocarbon is used to measure the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil, and the degradation rate results are as follows: saturated hydrocarbon: 25.3%; aromatic hydrocarbon: 23.1%; resin: 17.3%; asphaltene: 16.2%.

[0089] The weight method is used to measure the content of petroleum hydrocarbon, and the degradation efficiency of petroleum hydrocarbon is 21.66%. At the same time, the four-component analysis method of petroleum hydrocarbon is used to measure the degradation rates of saturated hydrocarbon, aromatic hydrocarbon, resin and asphaltene in the petroleum hydrocarbon contaminated soil, and the degradation rate results are as follows: saturated hydrocarbon: 25.3%; aromatic hydrocarbon: 23.1%; resin: 17.3%; asphaltene: 16.2%.

[0090] The remediation conditions and effects of petroleum hydrocarbon contaminated soil in the above examples and comparative examples are shown in Tables 2 and 3:

[0091] Table 2

[0092]

[0093] Table 3

[0094]

[0095] The present application is directed to the problem that single bacteria or fungi have narrow substrate spectrum for degrading petroleum hydrocarbon, and is based on metabolic division of labor between strains, and utilizes fungi (Fusarium oxysporum, Fusarium fujikuroi, Talaromyces emersonii) and bacteria (achromobacter sp.) to form a bacterial agent with wide substrate spectrum. The substrate preference and metabolic division of labor of specific types of fungi and bacteria in the bacterial agent are utilized to achieve efficient removal of saturated hydrocarbon, aromatic hydrocarbon and non-hydrocarbon components in petroleum components, and thus deep degradation of petroleum hydrocarbon contaminated soil is achieved. ​​

[0096] The application is applicable to the prior art where not otherwise indicated.

Claims

1. A method for deep degradation of petroleum hydrocarbons in contaminated soil based on metabolic specialization, characterized in that, The method uses a broad substrate spectrum bacterial agent, which includes three fungi and one bacterium. The mass ratio of bacteria in the broad substrate spectrum bacterial agent is Fusarium argentis: Fusarium fusiforme: Basilaria cordifolia: Achromobacterium = 1:(0.5-2):(0.5-2):(0.5-2). The broad substrate spectrum bacterial agent was added to the petroleum hydrocarbon contaminated soil at a ratio of 2% to 8% by volume of the bacterial agent to the mass of the petroleum hydrocarbon contaminated soil. The mass concentration of petroleum hydrocarbons in the petroleum hydrocarbon contaminated soil was 0.5% to 5%. During the remediation process, the soil moisture content was maintained at 20% wt% to 40% wt. The soil was thoroughly stirred every 7 to 10 days and cultured at room temperature for 50 to 70 days.

2. The method for deep degradation of petroleum hydrocarbons in contaminated soil based on metabolic division of labor according to claim 1, characterized in that, The degradation efficiency of petroleum hydrocarbons in petroleum hydrocarbon-contaminated soil was measured by gravimetric method. After remediation, the removal rate of total petroleum hydrocarbons was not less than 75%, of which the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, colloidal and asphaltenes were not less than 85%, 80%, 75% and 70%, respectively.

3. The method for deep degradation of petroleum hydrocarbons in contaminated soil based on metabolic division of labor according to claim 1, characterized in that, The three fungi were cultured under the same conditions: inoculated on PDA medium and bacteria on LB medium. The activated culture media were placed in a constant temperature shaker at 30–35°C and cultured for 7–10 days at a speed of 100–200 r / min. After inoculation, the activated bacterial culture medium was cultured to OD0.

05. 600 The bacterial seed culture was obtained by setting the concentration to 0.8-2.0; after inoculation, the fungal activation medium was cultured until the dry weight of the fungus was 20-30 mg / mL to obtain the fungal seed culture. After resuspension treatment, fungal seed culture and bacterial seed culture were used to obtain corresponding resuspensions; The resuspensions of the four bacteria were uniformly mixed in a volume ratio of Fusarium raffinosa: Fusarium fusiforme: Basilaria fasciata: Achromobacterium = 1:(0.5-2):(0.5-2):(0.5-2) to obtain the broad substrate spectrum bacterial agent.

4. The method for deep degradation of petroleum hydrocarbons in contaminated soil based on metabolic division of labor according to claim 3, characterized in that, When the ratio of Fusarium raffinosum: Fusarium tumefaciens: Basilaria fasciata: Achromobacterium is 1:2:(0.5-2):(0.5-2), the volume ratio of the inoculant to the mass of the petroleum hydrocarbon-contaminated soil is 5%, and the moisture content of the petroleum hydrocarbon-contaminated soil is 20%wt~40%wt, after remediation, the removal rate of total petroleum hydrocarbons is not less than 90%, of which the degradation rates of saturated hydrocarbons, aromatic hydrocarbons, colloidal and asphaltenes are not less than 92%, 91%, 85% and 80%, respectively.

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