An iron carrier-mediated symbiotic degradation bacterial agent for petroleum hydrocarbon contaminated soil remediation

Abstract

This invention discloses a siderophore-mediated symbiotic degradation agent for the remediation of petroleum hydrocarbon-contaminated soil. This agent is composed of *Pseudomonas aeruginosa*, a high-side-producing bacterium. Pseudomonas aeruginosa DG-1, strain preservation number CGMCC No. 28382) and the fungus white rot fungus ( Phanerochaete chrysosporium BNCC336258 is formulated in a volume ratio of 1:(2-4) and enhances iron-limiting conditions (free iron concentration ≤1.0×10⁻⁶) through siderophore-mediated bacterial-fungal synergistic metabolism. ‑18 The remediation efficiency of this microbial agent on petroleum hydrocarbon-contaminated soil (M) was demonstrated. This agent is suitable for treating low-concentration contaminated soil with petroleum hydrocarbon concentrations of 0.5-5 wt%, achieving a total petroleum hydrocarbon removal rate of over 80% after remediation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of petroleum hydrocarbon contaminated soil remediation, and particularly relates to a siderophore-mediated symbiotic degradation bacterial agent for petroleum hydrocarbon contaminated soil remediation. BACKGROUND

[0002] In the process of oil exploitation, transportation and refining, oil hydrocarbons entering the soil environment due to accidents or improper discharge will not only seriously damage the ecological system of the soil, but also have potential harm to plant growth, animals and even human health. Compared with high-concentration oil hydrocarbons, low-concentration (≤5 wt%) oil hydrocarbon contaminated soil is more suitable for in-situ remediation by using a microbial method.

[0003] The iron element in the soil has a regulatory effect on the mechanism of microbial degradation of oil hydrocarbons. In some areas of northwest, northeast and north China, due to high pH value and carbonate content in the soil, the bioavailability of iron is poor, forming an iron-limited (free iron concentration ≤1.0×10 -18 M) environment, which is not conducive to microbial remediation of oil hydrocarbon contaminated soil. How to improve the remediation efficiency of oil hydrocarbon contaminated soil under iron-limited conditions is a key problem that needs to be solved at present.

[0004] So far, there are few reports on the development of bacterial-fungal symbiotic bacterial agents based on siderophore interaction and their application in the treatment of oil hydrocarbon contaminated soil. 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 siderophore-mediated symbiotic degradation bacterial agent for petroleum hydrocarbon contaminated soil remediation, which is applied to the treatment of low-concentration petroleum hydrocarbon contaminated soil. The present application uses high-yield siderophore bacteria (Pseudomonas aeruginosa) and fungi (white rot fungus) to construct a symbiotic degradation bacterial agent for remediation of low-concentration petroleum hydrocarbon contaminated soil under iron-limited conditions. The siderophore secreted by the bacteria can be recognized and taken up by the specific receptors on the membrane surface of the fungi as a key cross-border communication signal, realizing the stable cooperation of the bacterial flora and constructing a symbiotic system of "bacterial iron supply-fungal degradation". The bacterial agent realizes efficient remediation of petroleum hydrocarbon contaminated soil under iron-limited conditions through the siderophore-mediated and metabolic complementary effects between strains.

[0006] The present application is realized by the following technical scheme:

[0007] The siderophore-mediated symbiotic degradation bacterial agent for petroleum hydrocarbon contaminated soil remediation is composed of siderophore-producing bacteria and fungi, the bacteria used are Pseudomonas aeruginosa (DG-1), and the fungi used are white rot fungus (BNCC336258). Pseudomonas aeruginosa Phanerochaete chrysosporium DG-1), and the fungi used are white rot fungus (BNCC336258).

[0008] ​The preparation process of the siderophore-mediated symbiotic degradation bacterial agent is as follows: the bacterial seed liquid and the fungal seed liquid are respectively resuspended in 0.9% sodium chloride physiological saline to obtain corresponding resuspensions, and the resuspensions of the two bacteria are mixed in a volume ratio of 1:(2-4) to obtain the siderophore-mediated symbiotic degradation bacterial agent.

[0009] The preparation method of the bacterial seed liquid and the fungal seed liquid is as follows: the bacteria are inoculated in LB culture medium, and the fungi are inoculated in PDA culture medium; the activated culture medium after inoculation is placed in a constant temperature incubator at 30-35 DEG C, and is oscillated and cultured at a rotation speed of 100-200 r / min for 1-5 days. 600 After inoculation, the activated culture medium of the bacteria is cultured to an OD value of 0.8-2.0 to obtain the bacterial seed liquid; and after inoculation, the activated culture medium of the fungi is cultured to a dry weight of 20-30 mg / mL to obtain the fungal seed liquid.

[0010] The application method of the siderophore-mediated symbiotic degradation bacterial agent in low-concentration petroleum hydrocarbon contaminated soil is as follows: the free iron content is ≤1.0*10 -18 M, the pH of the petroleum hydrocarbon contaminated soil is adjusted to 6.0-8.0, the petroleum hydrocarbon concentration is 0.5-5% (w / w), the siderophore-mediated symbiotic degradation bacterial agent is added to the petroleum hydrocarbon contaminated soil, wherein the addition amount of the bacterial agent is 2-10% (v / w) per ton of the petroleum hydrocarbon contaminated soil, urea and potassium dihydrogen phosphate are added as nutrient salts at the beginning of the repair, so that the mass ratio of carbon (calculated based on total petroleum hydrocarbon), nitrogen and phosphorus in the soil is adjusted to about 100:(5-10):1, the soil is turned every 1 week to ensure sufficient oxygen supply, after 45-75 days of repair under room temperature conditions, the petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil is measured by the weight method, and the total petroleum hydrocarbon removal rate is calculated.

[0011] Compared with the prior art, the beneficial effects of the present application are as follows:

[0012] The present application is a method for supplying iron nutrition through siderophore mediation and activating the degradation activity of a bacterial-fungal symbiotic system, which is used for green and efficient repair of petroleum hydrocarbon contaminated soil.

[0013] Table 1 Comparison of effects of different bacterial-fungal systems

[0014]

[0015] The present application dissolves the ubiquitous insoluble iron minerals (such as hematite and goethite) in the environment by secreting siderophores (a kind of efficient small molecule iron chelator) by Pseudomonas aeruginosa in an iron-limited environment to form a bioavailable siderophore-iron ion complex, which is recognized and taken up by specific receptors on the microbial cell membrane, thereby providing iron source for the synthesis and activation of intracellular iron-containing petroleum hydrocarbon degrading enzyme system, enhancing the degradation of petroleum hydrocarbon pollutants; at the same time, the fungus white rot fungus can rely on its non-specific extracellular enzyme system (such as lignin peroxidase and manganese peroxidase) to exhibit unique advantages in degrading high molecular weight and complex aromatic hydrocarbons, and the two can work together to ensure the iron nutrition supply of white rot fungus in an iron-limited environment to ensure the competitive advantage, thereby significantly improving the degradation efficiency. DETAILED DESCRIPTION

[0016] The present application will be described in detail below through specific examples, and the purpose of these exemplary embodiments is only to exemplify the present application, and does not constitute any form of any limitation on the actual protection scope of the present application, nor does it limit the protection scope of the present application.

[0017] Strain source:

[0018] Pseudomonas aeruginosa DG-1, preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 7, 2023, with the preservation number CGMCC No. 28382, and disclosed in the patent with the publication number CN117925473A.

[0019] White rot fungus Phanerochaete chrysosporium BNCC336258, purchased from Beina Biological Technology Co., Ltd., with the number BNCC336258.

[0020] Phanerochaete chrysosporium Phanerochaete chrysosporium CICC40719 is commercially available and obtained from Beijing Yuyi Technology Co., Ltd.

[0021] Pseudomonas sp. Pseudomonas sp. LD23, preserved in the China General Microbiological Culture Collection Center on November 17, 2015, with the preservation number CGMCC No. 11679, and disclosed in the patent with the publication number CN113717904A.

[0022] Pseudomonas aeruginosa X1 is purchased from Mingzhou Biological Technology Co., Ltd., with the number B279935, and is referred to as X1 in this application. Pseudomonas aeruginosa X1.

[0023] The iron carrier mediated symbiotic degradation bacterial agent for petroleum hydrocarbon contaminated soil remediation of the embodiment of the present application, the bacteria used is Pseudomonas aeruginosa (DG-1), the fungus used is white rot fungus (Trametes versicolor) Pseudomonas aeruginosa Phanerochaete chrysosporium BNCC336258).

[0024] The bacteria is inoculated in LB culture medium, and the fungus is inoculated in PDA culture medium, and the activated culture medium after inoculation is placed in a constant temperature incubator at 30-35 DEG C, and is oscillated and cultured at a rotating speed of 100-200 r / min for 1-5 days. The activated culture medium of the bacteria after inoculation is cultured to an OD 600 value of 0.8-2.0, and the seed liquid of the bacteria is obtained, and the activated culture medium of the fungus after inoculation is cultured to a dry weight of the fungus of 20-30 mg / mL, and the seed liquid of the fungus is obtained.

[0025] The process for determining the dry weight of the fungus in the seed liquid of the fungus is that a certain volume V of the activated culture medium of the fungus is sampled, and after sampling, centrifugal treatment is carried out, and the supernatant is poured out, and a dry weight m is obtained, and then the dry weight of the fungus in the current activated culture medium is m / V.

[0026] If the dry weight of the fungus is in the range of 20-30 mg / mL, the seed liquid of the fungus is obtained.

[0027] The seed liquid of the bacteria and the seed liquid of the fungus are respectively resuspended in physiological saline of 0.9% mass fraction, and the corresponding resuspension liquids are obtained.

[0028] The two resuspension liquids are uniformly mixed in a volume ratio of Pseudomonas aeruginosa: white rot fungus = 1: (2-4), and the iron carrier mediated symbiotic degradation bacterial agent is obtained.

[0029] The iron carrier mediated symbiotic degradation bacterial agent prepared above is added into petroleum hydrocarbon contaminated soil in a dosage of 2%-10% (v / w) (the ratio of the volume of the bacterial agent to the mass of the petroleum hydrocarbon contaminated soil), and the concentration of the petroleum hydrocarbon contaminated soil is 0.5%-5% (w / w) (the ratio of the mass of the petroleum hydrocarbon to the mass of the petroleum hydrocarbon contaminated soil), and the free iron content is ≤1.0 x 10 -18 M, the initial pH of the petroleum hydrocarbon contaminated soil is adjusted to 6.0-8.0, urea and potassium dihydrogen phosphate are added as nutrient salts at the beginning of the remediation, so that the mass ratio of carbon (calculated based on the total petroleum hydrocarbon), nitrogen and phosphorus in the soil is adjusted to about 100: (5-10): 1, the soil is thoroughly stirred every 5-10 days to ensure sufficient oxygen supply, and the soil is cultured at room temperature for 45-75 days. The content of the petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil is measured by the gravimetric method, and the removal rate of the petroleum hydrocarbon is calculated.

[0030] Example 1

[0031] ​This embodiment describes a siderophore-mediated symbiotic degradation agent for the remediation of petroleum hydrocarbon-contaminated soil. This agent is used to achieve deep degradation of petroleum hydrocarbon-contaminated soil. The siderophore-mediated symbiotic degradation agent comprises bacteria and fungi, wherein the bacteria are *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa DG-1), the fungus is a white-rot fungus (DG-1), Phanerochaete chrysosporium BNCC336258).

[0032] Bacteria were inoculated on LB medium, and fungi on PDA medium. The activated culture media were then placed in a constant-temperature shaker (32℃) and cultured with shaking at 150 r / min. The bacterial activation medium was cultured to OD200 after inoculation. 600 The bacterial seed culture was obtained by shaking the culture medium until the value reached 0.8. In this embodiment, the bacterial seed culture was obtained by shaking the culture medium for 1 day. After inoculation, the fungal activation medium was cultured until the fungal dry weight was 20 mg / mL. In this embodiment, the fungal seed culture was obtained by shaking the culture medium for 3 days.

[0033] Take 30 mL of bacterial seed culture and fungal seed culture respectively, put them into centrifuge tubes, and centrifuge at 5000 r / min for 5 min. Discard the supernatant, and resuspend the centrifuged bacterial blocks in 30 mL of 0.9% sodium chloride physiological saline to obtain resuspensions. Mix the resuspensions of the two bacteria in a volume ratio of 1:2 (Pseudomonas aeruginosa: white-rot fungi) to obtain siderophore-mediated symbiotic degradation agent.

[0034] Application in the remediation of petroleum hydrocarbon-contaminated soil: Detecting the free iron content in petroleum hydrocarbon-contaminated soil and determining whether the free iron content meets the requirement of ≤1.0×10⁻⁶. -18 If condition M is met, then the iron-carrier-mediated symbiotic degradation agent described in this embodiment will be used for soil remediation. Taking a representative contaminated soil from Changqing Oilfield as an example, due to the high pH and carbonate content in the soil, an iron-limited environment is formed, and its free iron content is measured to be 0.8 × 10⁻⁶. -18 M. The initial pH of the petroleum hydrocarbon-contaminated soil was 6.8, the moisture content was 30%, and the petroleum hydrocarbon concentration was 0.5% (w / w), where the petroleum hydrocarbon concentration is the ratio of the mass of petroleum hydrocarbons to the mass of the petroleum hydrocarbon-contaminated soil.

[0035] The prepared siderophore-mediated symbiotic degradation microbial agent was added into the petroleum hydrocarbon contaminated soil at a dosage of 2% (v / w) (ratio of microbial agent volume to petroleum hydrocarbon contaminated soil mass), and urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, so that the mass ratio of carbon (calculated based on total petroleum hydrocarbon), nitrogen and phosphorus in the soil was adjusted to about 100:5:1. The soil was thoroughly stirred every 7 days to ensure sufficient oxygen supply, and incubated at room temperature for 60 days. The content of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil was measured by the gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 85.7%.

[0036] Example 2

[0037] In this example, the resuspensions of the two bacteria were uniformly mixed in a volume ratio of 1:4 (Pseudomonas aeruginosa: white rot fungus) to prepare a siderophore-mediated symbiotic degradation microbial agent, and the other processes were the same as in Example 1.

[0038] In the application of petroleum hydrocarbon contaminated soil remediation, the representative contaminated soil of Changqing Oilfield was taken as an example: the free iron content was 0.8×10 -18 M. The initial pH was adjusted to 7.6, the water content was 30%, and the petroleum hydrocarbon concentration was 2% (w / w). The prepared siderophore-mediated symbiotic degradation microbial agent was added, and the dosage of the microbial agent in each ton of petroleum hydrocarbon contaminated soil was 5% (v / w). Urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, so that the mass ratio of carbon (calculated based on total petroleum hydrocarbon), nitrogen and phosphorus in the soil was adjusted to about 100:9:1. The soil was turned over every week to ensure sufficient oxygen supply. After 60 days of remediation at room temperature, the content of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil was measured by the gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 87.0%.

[0039] Example 3

[0040] In this example, the siderophore-mediated symbiotic degradation microbial agent was the same as in Example 1.

[0041] In the application of petroleum hydrocarbon contaminated soil remediation, the representative contaminated soil of Changqing Oilfield was taken as an example: the free iron content was 0.8×10 -18 M. The initial pH was adjusted to 7.6, the water content was 30%, and the petroleum hydrocarbon concentration was 2% (w / w). The prepared siderophore-mediated symbiotic degradation microbial agent was added, and the dosage of the microbial agent in each ton of petroleum hydrocarbon contaminated soil was 5% (v / w). Urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, so that the mass ratio of carbon (calculated based on total petroleum hydrocarbon), nitrogen and phosphorus in the soil was adjusted to about 100:9:1. The soil was turned over every week to ensure sufficient oxygen supply. After 60 days of remediation at room temperature, the content of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil was measured by the gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 87.0%. -18M. The initial pH was adjusted to 7.2, the water content was 30%, and the petroleum hydrocarbon concentration was 2% (w / w). The prepared siderophore-mediated symbiotic degradation bacterial agent was added to the petroleum hydrocarbon contaminated soil, and the bacterial agent was added at a dosage of 2% (v / w) per ton of petroleum hydrocarbon contaminated soil. Urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, and the mass ratio of carbon (calculated based on total petroleum hydrocarbons), nitrogen, and phosphorus in the soil was adjusted to about 100:7:1. The soil was turned every 1 week to ensure sufficient oxygen supply. After 60 days of remediation at room temperature, the petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil was measured by the gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 87.5%.

[0042] Example 4

[0043] In this embodiment, the siderophore-mediated symbiotic degradation bacterial agent is the same as that in Example 1.

[0044] Application in the remediation of petroleum hydrocarbon contaminated soil: The free iron content in the petroleum hydrocarbon contaminated soil was detected to determine whether the free iron content met ≤1.0×10 -18 M. If it met, the siderophore-mediated symbiotic degradation bacterial agent described in this embodiment was used for soil remediation. A representative contaminated soil from the Dagang Oilfield was used as an example to form an iron-limited environment: the free iron content was 0.5×10 -18 M. The initial pH was adjusted to 6.8, the water content was 30%, and the petroleum hydrocarbon concentration was 5% (w / w). The prepared siderophore-mediated symbiotic degradation bacterial agent was added, and the bacterial agent was added at a dosage of 2% (v / w) per ton of petroleum hydrocarbon contaminated soil. Urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, and the mass ratio of carbon (calculated based on total petroleum hydrocarbons), nitrogen, and phosphorus in the soil was adjusted to about 100:9:1. The soil was turned every 1 week to ensure sufficient oxygen supply. After 60 days of remediation at room temperature, the petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil was measured by the gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 81.4%.

[0045] Example 5

[0046] In this embodiment, the siderophore-mediated symbiotic degradation bacterial agent is the same as that in Example 1.

[0047] Application in the remediation of petroleum hydrocarbon contaminated soil: The free iron content in the petroleum hydrocarbon contaminated soil was detected to determine whether the free iron content met ≤1.0×10 -18 M. If it met, the siderophore-mediated symbiotic degradation bacterial agent described in this embodiment was used for soil remediation. A representative contaminated soil from the Daqing Oilfield was used as an example to form an iron-limited environment: the free iron content was 0.1×10 -18M. The initial pH was adjusted to 7.2, the water content was 30%, and the petroleum hydrocarbon concentration was 0.5% (w / w), and the prepared siderophore-mediated symbiotic degradation bacterial agent was added thereto, wherein the bacterial agent was added in an amount of 5% (v / w) per ton of petroleum hydrocarbon contaminated soil, and urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, and the mass ratio of carbon (calculated based on total petroleum hydrocarbon), nitrogen, and phosphorus in the soil was adjusted to about 100:7:1, the soil was turned every 1 week to ensure sufficient oxygen supply, after 60 days of remediation under room temperature conditions, the content of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil was measured by the gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 89.0%.

[0048] Example 6

[0049] In this example, the siderophore-mediated symbiotic degradation bacterial agent was prepared by uniformly mixing the resuspensions of the two bacteria in a volume ratio of 1:3 (Pseudomonas aeruginosa: white rot fungus), and the other processes were the same as in Example 1. The remediation soil was the same as in Example 4.

[0050] Example 7

[0051] In this example, the siderophore-mediated symbiotic degradation bacterial agent was prepared by uniformly mixing the resuspensions of the two bacteria in a volume ratio of 1:3 (Pseudomonas aeruginosa: white rot fungus), and the other processes were the same as in Example 1. The remediation soil was the same as in Example 4.

[0052] The initial pH was adjusted to 7.2, the water content was 30%, and the petroleum hydrocarbon concentration was 2% (w / w), and the prepared siderophore-mediated symbiotic degradation bacterial agent was added thereto, wherein the bacterial agent was added in an amount of 5% (v / w) per ton of petroleum hydrocarbon contaminated soil, and urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, and the mass ratio of carbon (calculated based on total petroleum hydrocarbon), nitrogen, and phosphorus in the soil was adjusted to about 100:10:1, the soil was turned every 1 week to ensure sufficient oxygen supply, after 60 days of remediation under room temperature conditions, the content of petroleum hydrocarbon in the petroleum hydrocarbon contaminated soil was measured by the gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 91.0%.

[0053] Comparative Example 1

[0054] In this comparative example, the bacterial agent used was Phanerochaete chrysosporiumCICC40719 Replacement Phanerochaete chrysosporium BNCC336258, the resuspension of the two bacteria in a volume ratio of 1:3 ( Pseudomonas aeruginosa DG-1: Phanerochaete chrysosporium CICC40719) was mixed evenly, and the preparation process was the same as in Example 1. The soil remediation and remediation process were the same as in Example 2. After 60 days of remediation at room temperature, the petroleum hydrocarbon content in the petroleum hydrocarbon-contaminated soil was measured by gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon-contaminated soil was calculated to be 49.1%.

[0055] Comparative Example 2

[0056] This comparative example uses Pseudomonas sp. LD23 replaces Pseudomonas aeruginosa DG-1, the resuspension of the two bacteria in a volume ratio of 1:3 ( Pseudomonas sp. LD23: Phanerochaete chrysosporium BNCC336258) was mixed evenly, and the preparation process was the same as in Example 1. The soil remediation and remediation process were the same as in Example 3. After 60 days of remediation at room temperature, the petroleum hydrocarbon content in the petroleum hydrocarbon-contaminated soil was measured by gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon-contaminated soil was calculated to be 39.0%.

[0057] Comparative Example 3

[0058] This comparative example uses Pseudomonas sp. LD23 replaces Pseudomonas aeruginosa DG-1, using Phanerochaete chrysosporium CICC40719 Replacement Phanerochaete chrysosporium BNCC336258, the resuspension of the two bacteria in a volume ratio of 1:3 ( Pseudomonas sp. LD23: Phanerochaete chrysosporium CICC40719) was mixed evenly, and the preparation process was the same as in Example 1. The soil remediation was the same as in Example 3, with the initial pH adjusted to 7.2, the moisture content to 30%, and the petroleum hydrocarbon concentration to 2% (w / w). The prepared microbial agent was added, with the amount of microbial agent added per ton of petroleum hydrocarbon-contaminated soil being 5% (v / w). Urea and potassium dihydrogen phosphate were added as nutrients at the beginning of remediation to adjust the mass ratio of carbon (based on total petroleum hydrocarbons), nitrogen, and phosphorus in the soil to approximately 100:5:1. The soil was turned over every week to ensure sufficient oxygen supply. After 60 days of remediation at room temperature, the petroleum hydrocarbon content in the petroleum hydrocarbon-contaminated soil was measured by gravimetric method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon-contaminated soil was calculated to be 47.2%.

[0059] Comparative Example 4

[0060] The following examples were used in the present application Pseudomonas aeruginosa X1 instead of Pseudomonas aeruginosa DG-1, using Phanerochaete chrysosporium CICC40719 instead of Phanerochaete chrysosporium BNCC336258, the resuspension of two bacteria was mixed in a volume ratio of 1:3 (1:3) Pseudomonas aeruginosa X1: Phanerochaete chrysosporium CICC40719) were mixed uniformly, and the preparation process was the same as that of Example 1. The soil for remediation was the same as that of Example 5, the initial pH was adjusted to 7.6, the water content was 30%, and the petroleum hydrocarbon concentration was 5% (w / w). The prepared bacterial agent was added to the petroleum hydrocarbon contaminated soil, and the addition amount of the bacterial agent was 5% (v / w) per ton of petroleum hydrocarbon contaminated soil. Urea and potassium dihydrogen phosphate were added as nutrient salts at the beginning of the remediation, and the mass ratio of carbon (based on total petroleum hydrocarbon), nitrogen, and phosphorus in the soil was adjusted to about 100:7:1. The soil was turned every 1 week to ensure sufficient oxygen supply. After 60 days of remediation at room temperature, the petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil was measured by the weight method, and the total petroleum hydrocarbon removal rate in the petroleum hydrocarbon contaminated soil was calculated to be 45.1%.

[0061] Table 2 Remediation conditions and effects of petroleum hydrocarbon contaminated soil in different examples

[0062]

[0063] Table 3 Remediation conditions and effects of petroleum hydrocarbon contaminated soil in different comparative examples

[0064]

[0065] The unmentioned parts of the present application are applicable to the prior art.

Claims

1. An iron-carrier-mediated symbiotic degrading bacterial agent for petroleum hydrocarbon-contaminated soil remediation, characterized in that, The siderophore-mediated symbiotic degradation bacterial agent is produced by siderophore-producing bacteria Pseudomonas aeruginosa Pseudomonas aeruginosa DG-1 and the fungus white rot fungus Phanerochaete chrysosporium consists of the following components: Pseudomonas aeruginosa The accession number of DG-1 is CGMCC No.28382.

2. The siderophore-mediated symbiotic degrading bacterial agent for the remediation of petroleum hydrocarbon-contaminated soil according to claim 1, characterized in that, The bacteria are inoculated in LB medium and the fungi are inoculated in PDA medium, and the activated medium after inoculation is placed in a constant temperature shaker at 30-35 DEG C and shaken; the activated medium of bacteria after inoculation is cultured to OD 600 value of 0.8-2.0, to obtain bacterial seed liquid; the activated medium of fungi after inoculation is cultured to a dry weight of 20-30 mg / mL, to obtain fungal seed liquid; the fungal seed liquid and the bacterial seed liquid are treated by resuspension to obtain corresponding resuspension liquids; the resuspension liquids of the two kinds of bacteria are mixed according to a volume ratio of 1:1 to obtain the siderophore-mediated symbiotic degradation microbial agent. Pseudomonas aeruginosa DG-1: fungi white rot fungus Phanerochaete chrysosporium BNCC336258=1:(2-4), and are uniformly mixed to obtain the siderophore-mediated symbiotic degradation microbial agent.

3. The use of the siderophore-mediated symbiotic degrading bacterial agent for petroleum hydrocarbon-contaminated soil remediation according to claim 1, characterized in that, The siderophore-mediated symbiotic degradation bacteria agent is used for repairing the petroleum hydrocarbon contaminated soil, nitrogen source and phosphorus source are added, so that the mass ratio of carbon, nitrogen and phosphorus in the petroleum hydrocarbon contaminated soil is 100:(5-10):1, wherein the carbon mass is calculated based on the total petroleum hydrocarbon; the nitrogen source is at least one of urea or ammonium salt, and the phosphorus source is potassium dihydrogen phosphate.

4. The use of the siderophore-mediated symbiotic degrading bacterial agent for petroleum hydrocarbon-contaminated soil remediation according to claim 1 for low concentration petroleum hydrocarbon-contaminated soil remediation, characterized in that, The siderophore-mediated symbiotic degradation bacteria agent is used for repairing the low-concentration petroleum hydrocarbon contaminated soil, wherein the total petroleum hydrocarbon content in the petroleum hydrocarbon contaminated soil is 0.5-5 wt%.

5. Use according to claim 4, characterized in that, The siderophore-mediated symbiotic degradation bacteria agent is used for repairing the low-concentration petroleum hydrocarbon contaminated soil, and the siderophore-mediated symbiotic degradation bacteria agent is added into the petroleum hydrocarbon contaminated soil according to the ratio of 2%-10% of the volume of the bacteria agent to the mass of the petroleum hydrocarbon contaminated soil, the repair time is 45-75 days, and the total petroleum hydrocarbon removal rate is greater than or equal to 80%.

6. Use according to any one of claims 3 to 5, characterized in that, The free iron concentration in the petroleum hydrocarbon contaminated soil is ≤1.0×10 -18 M.

7. Use according to any one of claims 3 to 5, characterized in that, The pH range of the petroleum hydrocarbon contaminated soil repaired by the siderophore-mediated symbiotic degradation bacteria agent is 6.0-8.0.

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