Petroleum hydrocarbon degradation complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention discloses a petroleum hydrocarbon degradation complex microbial inoculant as well as a preparation method and application thereof, and relates to the technical field of petroleum hydrocarbon degradation, the petroleum hydrocarbon degradation complex microbial inoculant is prepared by taking magnetic phosphorus-containing biochar as an immobilization carrier and loading composite petroleum hydrocarbon degradation bacteria on the surface of the magnetic phosphorus-containing biochar; wherein the magnetic phosphorus-containing biochar is prepared by firstly activating and pyrolyzing biomass shell powder and potassium feldspar powder through phytic acid to obtain phosphorus-containing biochar, and then coprecipitating the phosphorus-containing biochar and ferric salt; the composite petroleum hydrocarbon degrading bacteria are prepared by mixing bacterial suspensions of at least two of the following petroleum hydrocarbon degrading bacteria in an isovolumetric manner: pseudomonas, bacillus, micrococcus, lactobacillus, candida albicans or alcaligenes. The petroleum hydrocarbon degradation composite microbial agent provided by the invention has an excellent degradation effect on petroleum hydrocarbon, can be used in a heavy metal / petroleum hydrocarbon composite polluted soil environment, and has a good application effect.

Description

Technical Field

[0001] The present invention relates to the technical field of petroleum hydrocarbon degradation, and in particular to a petroleum hydrocarbon degradation composite bacterial agent and a preparation method and application thereof. Background Art

[0002] After entering the soil, petroleum hydrocarbons destroy the soil structure and affect the soil utilization value. In addition, petroleum hydrocarbons are environmentally persistent and difficult to degrade. The polycyclic aromatic hydrocarbons they contain have teratogenic, carcinogenic, and mutagenic effects, which are harmful to human health. Therefore, the remediation and treatment of petroleum hydrocarbon pollution in soil has become a difficult and hot technology that needs to be solved urgently in environmental protection work.

[0003] At present, the main methods for repairing petroleum hydrocarbon pollution are physical, chemical and biological methods. However, the composition and properties of petroleum hydrocarbon-contaminated soil are relatively complex, and each method has certain limitations. Compared with physical and chemical methods, microbial remediation can completely degrade petroleum hydrocarbons in the soil without causing secondary pollution and at a low cost. It is a truly green and environmentally friendly remediation method. However, due to the complex composition of petroleum hydrocarbons, different microorganisms have different degradation performance for soil petroleum hydrocarbons. Usually, a single strain can only degrade petroleum hydrocarbons within a certain range. The coordinated effect of different strains can better improve the degradation efficiency of petroleum hydrocarbons.

[0004] In actual engineering applications, free degrading bacteria are susceptible to competition from indigenous microorganisms and adverse external environmental conditions, resulting in weakened biological activity and degradation performance, which is not conducive to improving the degradation effect of petroleum hydrocarbons. Therefore, using immobilized carriers to fix degrading bacteria for the repair of petroleum hydrocarbon-contaminated soil has great application prospects. In this regard, the present invention proposes a petroleum hydrocarbon degradation composite bacterial agent and a preparation method and application thereof. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a petroleum hydrocarbon degradation composite bacterial agent and a preparation method and application thereof.

[0006] The present invention is achieved by the following technical solution, which comprises the following steps:

[0007] In a first aspect, the present invention provides a petroleum hydrocarbon degradation composite bacterial agent, wherein the petroleum hydrocarbon degradation composite bacterial agent is prepared by using magnetic phosphorus-containing biochar as an immobilization carrier and loading composite petroleum hydrocarbon degradation bacteria on its surface;

[0008] Among them, the magnetic phosphorus-containing biochar is prepared by first activating and pyrolyzing biomass shell powder and potassium feldspar powder with phytic acid to obtain phosphorus-containing biochar, and then co-precipitating the phosphorus-containing biochar with iron salt; the composite petroleum hydrocarbon degrading bacteria is prepared by mixing equal volumes of bacterial suspensions of at least two of the following petroleum hydrocarbon degrading bacteria: Pseudomonas, Bacillus, Micrococcus, Lactobacillus, Candida or Alcaligenes.

[0009] As a further optimization scheme of the present invention, the preparation method of the magnetic phosphorus-containing biochar is specifically as follows:

[0010] (1) Biomass shell powder, phytic acid solution with a mass concentration of 25-30% and potassium feldspar powder are fully mixed, and the temperature is increased to 300°C at a heating rate of 5-10°C / min for pyrolysis for 2h. The obtained product is washed with NaHCO3 solution until neutral and then washed with deionized water, filtered, and the obtained filter residue is dried and ground to obtain phosphorus-containing biochar;

[0011] (2) Immerse the phosphorus-containing biochar obtained in step (1) into Fe 2+ / Fe 3+ The mixed solution was stirred for 1 hour, and then NaOH solution was added to adjust the pH to 8-11, and stirring was continued for 15 minutes. After standing, it was filtered, and the obtained filter residue was subjected to the same pyrolysis and washing steps as those for preparing phosphorus-containing biochar, and the product was dried to obtain magnetic phosphorus-containing biochar.

[0012] As a further optimization scheme of the present invention, in step (1), the usage ratio of the biomass shell powder, the phytic acid solution with a mass concentration of 25-30%, and the potassium feldspar powder is 1g:50-100mL:0.5-1g.

[0013] As a further optimization scheme of the present invention, in step (1), the biomass shell powder is one of peanut shell powder, walnut shell powder or coconut shell powder.

[0014] The second aspect of the present invention provides a method for preparing the composite bacterial agent for petroleum hydrocarbon degradation as described above, comprising the following steps:

[0015] (1) inoculating each petroleum hydrocarbon degrading bacterium in an LB liquid culture medium at an inoculum amount of 1-2%, culturing to a logarithmic growth phase, washing with a sterile NaCl aqueous solution with a mass concentration of 0.9%, and then adding a NaCl aqueous solution with a mass concentration of 0.9% to obtain a bacterial suspension of each petroleum hydrocarbon degrading bacterium, and mixing the bacterial suspensions of each petroleum hydrocarbon degrading bacterium in equal volumes to obtain the composite petroleum hydrocarbon degrading bacterium;

[0016] (2) Activating and pyrolyzing biomass shell powder and potassium feldspar powder with phytic acid to obtain phosphorus-containing biochar, and then co-precipitating the phosphorus-containing biochar with iron salt to prepare magnetic phosphorus-containing biochar, sterilizing the magnetic phosphorus-containing biochar and adding the composite petroleum hydrocarbon-degrading bacteria prepared in step (1), and then culturing at 25° C. and 160 r / min for 12 h, filtering and washing the free bacteria on the surface with 0.9% saline, centrifuging for 5 min, and repeating 2-3 times to obtain the petroleum hydrocarbon-degrading composite bacterial agent.

[0017] As a further optimization scheme of the present invention, the usage ratio of the magnetic phosphorus-containing biochar to the composite petroleum hydrocarbon degrading bacteria is 1 g: 20-30 mL.

[0018] The third aspect of the present invention provides a use of any of the above-mentioned petroleum hydrocarbon degradation composite bacterial agents in remediating petroleum hydrocarbon pollution, wherein the object of the petroleum hydrocarbon pollution is soil.

[0019] As a further optimization scheme of the present invention, the specific method of using the petroleum hydrocarbon degrading composite bacterial agent to repair petroleum hydrocarbon pollution is to add the petroleum hydrocarbon degrading composite bacterial agent into water, stir and dilute it 10-20 times to obtain a repair solution, and spray the repair solution on the soil contaminated by petroleum hydrocarbons.

[0020] As a further optimization scheme of the present invention, based on the total weight of the petroleum hydrocarbon contaminated soil as 100%, the spraying amount of the petroleum hydrocarbon degradation composite bacterial agent is 10-20%.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The present invention first activates and pyrolyzes biomass shell powder and potassium feldspar powder with phytic acid to obtain phosphorus-containing biochar, then co-precipitates the phosphorus-containing biochar with iron salt to prepare magnetic phosphorus-containing biochar, and uses the magnetic phosphorus-containing biochar as an immobilized carrier, and loads composite petroleum hydrocarbon-degrading bacteria on its surface. On the one hand, the magnetic phosphorus-containing biochar provides structural support for the composite bacterial agent to stabilize the performance of the bacterial agent, and on the other hand, the surface activity of the magnetic phosphorus-containing biochar prepared by co-precipitation of phytic acid activator and iron salt is improved, and the phosphorus content is increased. Under the synergistic effect of the two, the efficiency of petroleum hydrocarbon degradation is further improved.

[0023] (2) The petroleum hydrocarbon degradation composite bacterial agent provided by the present invention can simultaneously promote the degradation rate of TPHs and repair the heavy metal pollution in the soil under the condition of heavy metal / petroleum hydrocarbon composite pollution, thereby achieving better application effect. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technicians in this field can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0025] 1. Reagents and Materials

[0026] The following strains were purchased from Testo Biotech:

[0027] Pseudomonas is Pseudomonas aeruginosa, with the catalog number TS278080; Bacillus is Bacillus subtilis, with the catalog number TS276952; Micrococcus is Micrococcus luteus, with the catalog number TS278117; Lactobacillus is Lactobacillus sp., with the catalog number TS279084; Alcaligenes is Alcaligenes xylosoxidans, with the catalog number TS339392.

[0028] The Candida parapsilosis was purchased from Shanghai Bo Ke Biotechnology Co., Ltd. with the catalog number BKJ3879.

[0029] LB liquid culture medium was purchased from Shanghai YuBo Biotechnology Co., Ltd. with the catalog number YB70010LM.

[0030] Biomass shell powder chooses peanut shell powder.

[0031] Unless otherwise specified, the reagents and materials used below are commercially available.

[0032] 2. Methods

[0033] Unless otherwise specified, the following methods are conventional methods known to those skilled in the art.

[0034] 1. Preparation of composite petroleum hydrocarbon degradation bacteria

[0035] 1.1. Preparation of bacterial suspensions of various petroleum hydrocarbon-degrading bacteria

[0036] According to the culturing characteristics of various petroleum hydrocarbon degrading bacteria and the strain activation and resuscitation culture methods provided by the merchants, the purchased strains are activated and revived respectively. Subsequently, the activated and revived strains are inoculated into LB liquid culture medium at an inoculum amount of 2%, and shaken and cultured under various adaptive culturing conditions. After each strain is cultured to the logarithmic growth phase, it is centrifuged at 5000r / min for 5 minutes, the supernatant is discarded, and it is washed three times with a sterile NaCl aqueous solution with a mass concentration of 0.9%, and then a NaCl aqueous solution with a mass concentration of 0.9% is added in the same amount as the LB liquid culture medium to obtain bacterial suspensions of various petroleum hydrocarbon degrading bacteria, and the bacterial suspensions of various petroleum hydrocarbon degrading bacteria are mixed in equal volumes to obtain the composite petroleum hydrocarbon degrading bacteria.

[0037] 2. Preparation of immobilized carrier

[0038] 2.1. For the selection of the composition of the immobilized carrier, the following treatment groups were set up:

[0039] Immobilized carrier A: (1) Preparation of phosphorus-containing biochar: Peanut shell powder, phytic acid solution with a mass concentration of 25%, and potassium feldspar powder were fully mixed in a ratio of 1 g:50 mL:0.5 g. The mixture was heated to 300 °C at a heating rate of 10 °C / min and pyrolyzed for 2 h. The obtained product was washed with NaHCO3 solution until neutral and then washed with deionized water and filtered. The obtained filter residue was dried and ground (to about 200 mesh) to obtain phosphorus-containing biochar.

[0040] (2) Preparation of magnetic phosphorus-containing biochar: The obtained phosphorus-containing biochar was immersed in Fe 2+ / Fe 3+ (The molar ratio is 1:1, Fe 3+ The mixture was stirred in a mixed solution (with an ion concentration of 0.02 mol / L) for 1 h, and then a 6 mol / L NaOH solution was added to adjust the pH to 8, and stirring was continued for 15 min. The mixture was allowed to stand and then filtered. The filter residue was subjected to the same pyrolysis and washing steps as those for preparing phosphorus-containing biochar, and the product was dried to obtain magnetic phosphorus-containing biochar.

[0041] Immobilized carrier B: The difference from immobilized carrier A is that (1) Preparation of phosphorus-containing biochar: Peanut shell powder, 25% phosphoric acid solution and potassium feldspar powder were fully mixed in a ratio of 1 g:50 mL:0.5 g. The mixture was heated to 300 °C at a heating rate of 10 °C / min and pyrolyzed for 2 h. The product was washed with NaHCO3 solution until neutral and then washed with deionized water and filtered. The filter residue was dried and ground (to about 200 mesh) to obtain phosphorus-containing biochar.

[0042] Immobilized carrier C: The difference from immobilized carrier A is that (1) Preparation of phosphorus-containing biochar: Peanut shell powder and potassium feldspar powder were fully mixed in a ratio of 1g:0.5g, and the temperature was raised to 300℃ at a heating rate of 10℃ / min for pyrolysis for 2h. The obtained product was washed with NaHCO3 solution until neutral and then washed with deionized water and filtered. The obtained filter residue was dried and ground (to about 200 mesh) to obtain phosphorus-containing biochar.

[0043] Immobilized carrier D: The difference from immobilized carrier A is that (1) Preparation of phosphorus-containing biochar: Peanut shell powder and phytic acid solution with a mass concentration of 25% are fully mixed in a ratio of 1 g:50 mL, and the mixture is heated to 300 °C at a heating rate of 10 °C / min and pyrolyzed for 2 h. The obtained product is washed with NaHCO3 solution until it is neutral and then washed with deionized water and filtered. The obtained filter residue is dried and ground (to about 200 mesh) to obtain phosphorus-containing biochar.

[0044] Immobilized carrier E: The difference from treatment group A is that (2) preparation of magnetic phosphorus-containing biochar was not performed.

[0045] Immobilized carrier F: Peanut shell powder was heated to 300°C at a heating rate of 10°C / min and pyrolyzed for 2h. The obtained product was washed with NaHCO3 solution until neutral and then washed with deionized water and filtered. The obtained filter residue was dried and ground (to about 200 mesh) to obtain biochar as an immobilized carrier.

[0046] 3. Preparation of composite bacterial agent for petroleum hydrocarbon degradation

[0047] After sterilizing the immobilized carriers AF at 121° C. for 20 min, the composite petroleum hydrocarbon degrading bacteria prepared in step (1) were added, with the ratio of the immobilized carrier to the composite petroleum hydrocarbon degrading bacteria being 1 g:30 mL. The immobilized carriers were then cultured at 25° C. and 160 r / min for 12 h under constant temperature shaking, and the free bacteria on the surface were filtered and washed with 0.9% saline, and the mixture was centrifuged for 5 min. The process was repeated 2-3 times to prepare the composite petroleum hydrocarbon degrading bacterial agent.

[0048] 4. Microbial soil remediation test under indoor simulated room temperature environment

[0049] The method of the reference book "Soil Microbiology Research Method" was used to conduct a test on the efficiency of petroleum hydrocarbon degradation in soil by using petroleum hydrocarbon degradation composite bacteria. The soil sample was soil that was not contaminated by petroleum hydrocarbons. The soil sample was air-dried and crushed to remove gravel and plant residues, and passed through a 60-mesh sieve. The results of the physical and chemical properties of the air-dried soil sample were as follows: pH value was 8.32, available phosphorus content was 0.46g / kg, total nitrogen content was 11.37g / kg, and organic matter content was 8.52g / kg.

[0050] Add petroleum hydrocarbons to the air-dried soil sample and mix thoroughly in a fume hood to make the petroleum hydrocarbon content in the soil 10g / kg. Weigh 10kg of artificially prepared oil-containing soil sample into a 9L red clay pot and adjust the C:N:P ratio in the soil to 100:10:1 to promote microbial proliferation.

[0051] Then, the following treatment group design was performed:

[0052] Treatment group AF: The petroleum hydrocarbon degradation composite bacterial agent immobilized by immobilized carrier AF was inoculated into the soil sample at an inoculation rate of 20% (w / w);

[0053] Control group 1: The mixed free strains were inoculated into the soil samples at 20% inoculum (w / w);

[0054] Blank control group: soil samples without any bacterial agent added.

[0055] Each group was set up with 3 replicates and placed in a ventilated environment at 25℃ for 50 days. Deionized water was sprayed at any time to keep the soil moisture content at about 35%, and the soil was plowed every day to ensure good soil permeability. Every 10 days, 0.5g of soil samples were weighed using the chessboard five-point sampling method, and the content of TPHs in the soil samples was detected using a gas chromatograph equipped with an HP-5 column (30m×0.25mm×0.5μm) and a flame ionization detector (GC-FID). The degradation rate was calculated by the formula.

[0056]

[0057] Wherein, w is the degradation rate, %; C0 is the content of residual petroleum hydrocarbons at the sampling time point of the blank control group, mg / L; C i is the content of residual petroleum hydrocarbons at the sampling time point of each treatment group, mg / L.

[0058] The results are shown in Table 1.

[0059] Table 1. Degradation rate of TPHs in soil samples

[0060]

[0061]

[0062] As shown in Table 1, compared with the control group 1, the petroleum hydrocarbon degradation composite bacterial agents used in treatment groups AF were all fixed by immobilized carriers. From the results of TPHs degradation rate, the immobilized petroleum hydrocarbon degradation composite bacterial agents had a positive effect on improving the degradation rate of TPHs. The reason is that, for the immobilized petroleum hydrocarbon degradation composite bacterial agents, on the one hand, the immobilized carrier has a certain adsorption effect on TPHs through a large specific surface area and a complex pore structure. On the other hand, the immobilized carrier provides structural support for the microbial agent, and the microbial density is large and the performance is stable. Unlike the free bacterial agent, it is easily affected by the competition of indigenous microorganisms and adverse external conditions, resulting in weakened biological activity and degradation performance, which can effectively improve the efficiency of petroleum hydrocarbon degradation.

[0063] In addition, from the comparison of data between treatment groups AF, it can be seen that the choice of immobilized carrier has an impact on the effect of bacterial agent on TPHs degradation. The immobilized carrier used in treatment group A has undergone phytic acid activation pyrolysis and iron salt co-precipitation steps. Compared with treatment group CD which only uses phytic acid activation pyrolysis and iron salt co-precipitation, it has a better effect in improving the degradation rate of TPHs. After phytic acid activation pyrolysis, the phosphorus content on the biochar surface increased, which promoted the degradation of TPHs.

[0064] 5. Indoor simulation of microbial soil remediation test under heavy metal pollution

[0065] The method in the reference book "Soil Microbiology Research Method" was used to conduct an experiment on the degradation efficiency of petroleum hydrocarbons in soil using a petroleum hydrocarbon degradation compound bacterial agent.

[0066] The soil sample was contaminated by heavy metals (Pb 2+ and Cd 2+ The contents of 220.1mg / kg and 67.4mg / kg were respectively). The soil samples were air-dried and ground to remove gravel and plant residues, and then passed through a 60-mesh sieve. The results of the physical and chemical properties of the air-dried soil samples were as follows: pH value was 7.93, available phosphorus content was 0.21g / kg, total nitrogen content was 7.23g / kg, and organic matter content was 6.25g / kg. Petroleum hydrocarbons were added to the air-dried soil samples and mixed thoroughly in a fume hood to make the petroleum hydrocarbon content in the soil 10g / kg.

[0067] 10 kg of artificially prepared oil-containing soil sample was weighed into a 9 L red clay pot, and the C:N:P ratio in the soil was adjusted to 100:10:1 to promote microbial proliferation.

[0068] Then, the following treatment group design was performed:

[0069] Treatment group AF: The petroleum hydrocarbon degradation composite bacterial agent immobilized by immobilized carrier AF was inoculated into the soil sample at an inoculation rate of 20% (w / w);

[0070] Control group 1: The mixed free strains were inoculated into the soil samples at 20% inoculum (w / w);

[0071] Blank control group: soil samples without any bacterial agent added.

[0072] Each group was set up with 3 replicates and placed in a ventilated environment at 25℃ for 50 days. Deionized water was sprayed at any time to keep the soil moisture content at about 35%, and the soil was plowed every day to ensure good soil permeability. Every 10 days, 0.5g of soil samples were weighed using the chessboard five-point sampling method, and the content of TPHs in the soil samples was detected using a gas chromatograph equipped with an HP-5 column (30m×0.25mm×0.5μm) and a flame ionization detector (GC-FID). The degradation rate of TPHs in the soil was calculated by the following formula.

[0073]

[0074] Wherein, w is the degradation rate, %; C0 is the content of residual petroleum hydrocarbons at the sampling time point of the blank control group, mg / L; C i is the content of residual petroleum hydrocarbons at the sampling time point of each treatment group, mg / L.

[0075] In addition, the Pb content in the soil after 50 days of cultivation 2+ and Cd 2+The content of Pb in the soil before cultivation was determined and compared with that in the soil before cultivation. 2+ and Cd 2+ The contents were compared and the removal rates were calculated.

[0076] Specifically, the lead and cadmium content is determined by wet digestion method: weigh about 1.00g of soil sample into a 100mL conical flask, put in a few glass beads, add 10mL of mixed acid, cover and soak overnight. Add a small funnel to digest on an electric stove. If it turns brown-black, turn off the heat, carefully add mixed acid until white smoke appears, and the digestion liquid is colorless and transparent or slightly yellowish, and let it cool. Filter the digestion liquid into a 25mL volumetric flask, wash the conical flask with a small amount of water several times, combine the washing liquid in the volumetric flask, treat lead and cadmium at the same time, add 3μL of matrix modifier ammonium phosphate solution (20g / L) after treatment, dilute to the scale and mix well, and wait for measurement; at the same time, make a reagent blank.

[0077] Lead standard curve: Accurately pipette lead standard solution (500 mg / L) to prepare standard solutions with concentrations of 0.0, 10.0, 20.0, 30.0, 40.0, and 50.0 ng / L, respectively, place them in 10 mL volumetric flasks, introduce them into atomic absorption spectrophotometer, record the absorbance corresponding to the lead content, and obtain the lead standard curve: y = 0.0043x + 0.0026, R 2 =0.9963.

[0078] Cadmium standard curve: Accurately pipette cadmium standard solution (100 mg / L) to prepare standard solutions with concentrations of 0.0, 10.0, 20.0, 30.0, 40.0, and 50.0 ng / L, respectively, place them in 10 mL volumetric flasks, introduce them into atomic absorption spectrophotometer, record the absorbance corresponding to the cadmium content, and obtain the cadmium standard curve: y = 0.0134x + 0.0062, R 2 =0.995.

[0079] Degradation rate of TPHs in soil samples and Pb in soil 2+ and Cd 2+ The removal rates are shown in Table 2.

[0080] Table 2. Degradation rate of TPHs in soil samples

[0081]

[0082]

[0083] It can be seen from Table 2 that compared with Table 1, in the case of heavy metal / petroleum hydrocarbon combined pollution, the degradation effect of treatment group A on TPHs decreased but not significantly, while the degradation effect of treatment group B decreased significantly compared with Table 1. For the combined polluted soil, the immobilized bacterial agent in treatment group AF had a positive effect on improving the degradation rate of TPHs compared with control group 1. The immobilized carrier used in treatment group A was activated by phytic acid, pyrolysis and iron salt co-precipitation, and the surface activity of the immobilized carrier was enhanced. The increase in phosphorus content also promoted the degradation rate of TPHs and repaired the heavy metal pollution in the soil.

[0084] Table 3. Pb in soil 2+ and Cd 2+ Removal rate

[0085]

[0086] As shown in Table 3, compared with other treatment groups, the composite bacterial agent fixed with magnetic phosphorus-containing biochar in treatment group A has a better degradation rate of TPHs and can further effectively reduce Pb in the soil. 2+ and Cd 2+ The content of the petroleum hydrocarbon degradation composite bacterial agent provided by the present invention can obtain better application effect under the condition of heavy metal / petroleum hydrocarbon composite pollution.

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A petroleum hydrocarbon degradation composite bacterial agent, characterized in that: The petroleum hydrocarbon degradation composite bacterial agent is prepared by using magnetic phosphorus-containing biochar as an immobilization carrier and loading composite petroleum hydrocarbon degradation bacteria on its surface; Among them, the magnetic phosphorus-containing biochar is prepared by first activating and pyrolyzing biomass shell powder and potassium feldspar powder with phytic acid to obtain phosphorus-containing biochar, and then co-precipitating the phosphorus-containing biochar with iron salt; the composite petroleum hydrocarbon degrading bacteria is prepared by mixing equal volumes of bacterial suspensions of at least two of the following petroleum hydrocarbon degrading bacteria: Pseudomonas, Bacillus, Micrococcus, Lactobacillus, Candida or Alcaligenes.

2. A petroleum hydrocarbon degradation composite bacterial agent according to claim 1, characterized in that: The preparation method of the magnetic phosphorus-containing biochar is specifically as follows: (1) Biomass shell powder, phytic acid solution with a mass concentration of 25-30% and potassium feldspar powder are fully mixed, and the temperature is increased to 300°C at a heating rate of 5-10°C / min for pyrolysis for 2h. The obtained product is washed with NaHCO3 solution until neutral and then washed with deionized water, filtered, and the obtained filter residue is dried and ground to obtain phosphorus-containing biochar; (2) Immerse the phosphorus-containing biochar obtained in step (1) into Fe 2+ / Fe 3+ The mixed solution was stirred for 1 hour, and then NaOH solution was added to adjust the pH to 8-11, and stirring was continued for 15 minutes. After standing, it was filtered, and the obtained filter residue was subjected to the same pyrolysis and washing steps as those for preparing phosphorus-containing biochar, and the product was dried to obtain magnetic phosphorus-containing biochar.

3. A petroleum hydrocarbon degradation composite bacterial agent according to claim 2, characterized in that: In step (1), the usage ratio of the biomass shell powder, the phytic acid solution with a mass concentration of 25-30%, and the potassium feldspar powder is 1g:50-100mL:0.5-1g.

4. A method for preparing a petroleum hydrocarbon degradation composite bacterial agent as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: (1) inoculating each petroleum hydrocarbon degrading bacterium in an LB liquid culture medium at an inoculum amount of 1-2%, culturing to a logarithmic growth phase, washing with a sterile NaCl aqueous solution with a mass concentration of 0.9%, and then adding a NaCl aqueous solution with a mass concentration of 0.9% to obtain a bacterial suspension of each petroleum hydrocarbon degrading bacterium, and mixing the bacterial suspensions of each petroleum hydrocarbon degrading bacterium in equal volumes to obtain the composite petroleum hydrocarbon degrading bacterium; (2) Activating and pyrolyzing biomass shell powder and potassium feldspar powder with phytic acid to obtain phosphorus-containing biochar, and then co-precipitating the phosphorus-containing biochar with iron salt to prepare magnetic phosphorus-containing biochar, sterilizing the magnetic phosphorus-containing biochar and adding the composite petroleum hydrocarbon-degrading bacteria prepared in step (1), and then culturing at 25° C. and 160 r / min for 12 h, filtering and washing the free bacteria on the surface with 0.9% saline, centrifuging for 5 min, and repeating 2-3 times to obtain the petroleum hydrocarbon-degrading composite bacterial agent.

5. The method for preparing a petroleum hydrocarbon degradation composite bacterial agent according to claim 4, characterized in that: The usage ratio of the magnetic phosphorus-containing biochar to the composite petroleum hydrocarbon degrading bacteria is 1g:20-30mL.

6. Use of the petroleum hydrocarbon degradation composite bacterial agent as claimed in any one of claims 1 to 3 in remediating petroleum hydrocarbon pollution, characterized in that: The object of the petroleum hydrocarbon pollution is soil.

7. The use according to claim 6, characterized in that: The specific method of using the petroleum hydrocarbon degradation composite bacterial agent to repair petroleum hydrocarbon pollution is to add the petroleum hydrocarbon degradation composite bacterial agent into water, stir and dilute it 10-20 times to obtain a repair solution, and spray the repair solution on the soil contaminated by petroleum hydrocarbons.

8. The use according to claim 6, characterized in that: Based on the total weight of the petroleum hydrocarbon-contaminated soil as 100%, the spraying amount of the petroleum hydrocarbon-degrading composite bacterial agent is 10-20%.

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