WO2023039921A1 - Composite microbial agent for degrading petroleum hydrocarbons, and preparation method therefor and use thereof - Google Patents

Abstract

Provided are a composite microbial agent for degrading petroleum hydrocarbons, and a preparation method therefor and the use thereof. The composite microbial agent is composed of live Acinetobacter KJ-1 with the deposit number of CGMCC No. 20664, Providencia L1 with the deposit number of CGMCC No. 16526, Bacillus SWH-1 with the deposit number of GMCC No. 1950, and Sphingobacterium SWH-2 with the deposit number of CGMCC No. 1951. The prepared composite microbial agent for degrading petroleum hydrocarbons can effectively degrade petroleum hydrocarbon substances in soil and oil sludge, can be effectively applied to the biodegradation treatment of petroleum-contaminated soil, and does not induce secondary pollution on the ecological environment of the soil. By fully utilising the synergistic effect of live Acinetobacter KJ-1, Providencia L1, Bacillus SWH-1 and Sphingobacterium SWH-2, the final degradation efficiency of petroleum hydrocarbons is improved. The solid microbial agent has a simple preparation process which is easily operated, is low cost, and has wide application prospects in the field of bioremediation of petroleum-contaminated soil and petroleum product leakage accident sites.

Description

A kind of petroleum hydrocarbon degrading composite microbial bacterial agent and its preparation method and application technical field

The invention belongs to the technical field of microbes and soil remediation, and in particular relates to a petroleum hydrocarbon degrading composite microbial bacterial agent and its preparation method and application.

Background technique

Oil pollution has become a global environmental problem. According to statistics, the area of oil-contaminated soil in China's oil fields exceeds 300 million m ² , and the annual increase of oil-contaminated soil exceeds 1 million tons, and various oily sludge exceeds 10 million tons, forming very serious soil oil pollution. The quality of the ecological environment in major oilfield areas continues to decline, the accumulation of toxic and harmful petroleum pollutants continues, and the ecological risks are increasing day by day. Due to the complexity of the soil medium, remediation of oil-contaminated soil has become a bottleneck problem in the international oilfield environmental protection, and it is a symbol to measure the technical level of a country's contaminated soil treatment.

Soil oil pollution remediation is the main field of intensive research and development of environmental protection technologies in various countries in the world. In order to quickly remove oil pollutants in soil, conventional methods at home and abroad include physical and chemical remediation, such as concentrated drying, solid-liquid separation, extraction and separation, Washing method, heat treatment and thermal desorption, chemical demulsification recovery method, etc. These methods were mainly established for the recovery and treatment of crude oil in oil sludge in the early stage. Physical and chemical remediation methods are difficult to avoid secondary pollution, so that the original habitat is destroyed, and it is difficult to be effective and applied to large-scale, medium-low concentration oil pollution control, and the cost is expensive. Therefore, the original physical and chemical methods need to be further improved, enhanced, optimized or supplemented/combined with biological methods. Bioremediation usually uses endogenous or artificially added microorganisms or bacterial agents, repair plants and specific enzymes to strengthen/accelerate the degradation of petroleum hydrocarbons. Compared with physical and chemical methods, it is more environmentally friendly, and can minimize the damage to the soil ecosystem while removing pollutants, and is a sustainable restoration method.

Contents of the invention

Aiming at the deficiencies in the prior art, the present invention provides a petroleum hydrocarbon degrading composite microbial agent and its preparation method and application. The composite microbial agent can effectively degrade petroleum hydrocarbons in soil and oil sludge sand, and can be effectively applied biodegradation treatment of petroleum-contaminated soil.

The present invention is realized through the following technical solutions:

A compound microbial bacterial agent for petroleum hydrocarbon degradation, including Acinetobacter KJ-1 of preservation number CGMCC No.20664, Providencia rettii L1 of CGMCC No.16526, and Bacillus SWH-1 of GMCC No.1950 and Sphingobacterium SWH-2 of CGMCC No.1951.

Further, the composite microbial agent is composed of a fermentation broth and a carrier peat soil, and the mass ratio of the fermentation broth to the peat soil is 1:5-15.

Further, the effective number of live bacteria of Acinetobacter KJ-1 in the fermentation broth is 10 ⁷ -10 ⁹ /ml, and the effective number of live bacteria of Providencia rettii L1 is 10 ⁷ -10 ⁹ /ml, the effective number of viable bacteria of Bacillus SWH-1 is 10 ⁸ -10 ⁹ /ml, and the effective number of viable bacteria of Sphingobacterium SWH-2 is 10 ⁷ -10 ⁹ /ml.

In the present invention, the preparation method of the petroleum hydrocarbon degrading composite microbial bacterial agent is characterized in that it comprises the following steps:

(1) The live Acinetobacter KJ-1 of CGMCC No.20664, the Providencia rettii L1 of CGMCC No.16526, the Bacillus SWH-1 of GMCC No.1950 and the sphingosine of CGMCC No.1951 Bacillus SWH-2 was activated on NB solid medium for 16-48 hours, and transferred to NB liquid medium for shake flask activation for 16-48 hours as the initial seed solution;

(2) Put the initial seed liquid of petroleum-degrading bacterium Acinetobacter KJ-1, Providencia rettgeri L1, Bacillus SWH-1 and Sphingobacterium SWH-2 according to the total inoculation amount of 3-6%. Fermentation and cultivation in the fermentation medium until the total number of oil-degrading bacteria is above 10 ⁹ CFU/mL, to obtain grade I seed liquid of oil-degrading bacteria fermentation;

(3) The oil-degrading flora fermentation grade I seed liquid in step (2) is transferred to the fermentation medium again according to the volume ratio of 3-6%, and the air is cultivated until the total number of oil-degrading flora is 10 ⁹ CFU/ ml or more, the oil-degrading flora fermentation grade II seed liquid is obtained, and the cultivation is expanded step by step, and the total number of oil-degrading flora is more than 10 ⁹ CFU/ml;

(4) Using peat soil as a carrier, mix the fermented bacteria liquid obtained in step (2) with peat soil, the mass ratio is 1:5-15, and the water content after drying is 10-15%, so as to obtain a petroleum hydrocarbon degradation compound Microbial agents.

Further, the NB culture medium described in the step (1) contains 10 g of peptone, 3 g of beef powder, 5 g of sodium chloride, 18.0 g of agar, distilled water to 1000 mL, and a pH value of 7.0 to 7.4 per liter of culture medium; _The fermentation medium described in (2) and step (3) contains KH ₂ PO ₄ 1g in every liter of fermentation medium, K ₂ HPO ₄ 1g, MgSO 0.2g, CaCl ₂ 0.03g, FeCl _0.002g , Na ₂ CO ₃ 0.2g, (NH ₄ ) ₂ SO ₄ 0.8g, nutrient broth 20g, the balance is water; the fermentation temperature is 28-37°C, the culture time is 18-24h, and the culture speed is 120～ 150rpm.

Further, the effective activity of four bacterial strains of the oil-degrading bacteria Acinetobacter aviosa KJ-1, Providencia rettgeri L1, Bacillus SWH-1 and Sphingobacterium SWH-2 described in step (2) The ratio of the number of bacteria is 1.5-2:1:1-2:2.

In the present invention, the application of the petroleum hydrocarbon degrading composite microbial bacterial agent in treating petroleum pollutants.

Further, the petroleum pollutants are petroleum-contaminated soil or treated oily sludge.

Further, when the petroleum hydrocarbon degrading compound microbial bacterial agent is used to treat oil-contaminated soil, a compound microbial bacterial agent of 2-4% by mass of petroleum hydrocarbon pollutants, 0.05-0.5% by mass of petroleum hydrocarbon pollutants, nitrogen Nitrogen and phosphorus fertilizers with a phosphorus ratio of 3-5:1, keep the moisture content between 15-25%, and treat for 30-90 days.

Further, the compound microbial bacterial agent is added to the soil twice, and the interval between the two additions is one week.

Beneficial effect

The petroleum hydrocarbon degradation composite microbial bacterial agent of the present invention can effectively degrade petroleum hydrocarbons in soil and oil sludge sand, can be effectively applied to the biodegradation treatment of petroleum-contaminated soil, and will not cause secondary pollution to the ecological environment of the soil. The synergistic effect between Acinetobacter KJ-1, Providencia rettgeri L1, Bacillus SWH-1 and Sphingobacterium SWH-2 improves the degradation efficiency of final petroleum hydrocarbons; and the preparation process of solid bacterial agent is simple and easy The operation is low in cost, and has broad application prospects in the field of bioremediation of oil-contaminated soil and oil product leakage accident sites.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

(1) the live Acinetobacter KJ-1 of deposit number CGMCC No.20664, the sheath of the Bacillus SWH-1 of CGMCC No.1950 and CGMCC No.1951 of Providencia rettii L1 of CGMCC No.16526 Ammoxacillus SWH-2 bacterial strain is carried out plate culture, shakes flask activation culture, activation, according to the total inoculum of volume ratio 5% after the activation (acinetobacter live, Providencia rettgeri L1, bacillus SWH-1 and The ratio of the effective viable count of Sphingobacterium SWH-2 is 1.5:1:1.5:2) inoculated in a 500ml Erlenmeyer conical shaker flask, each bottle contains 100ml of fermentation medium, 30°C, 150rpm shaker culture to The total number of oil-degrading bacteria is more than 10 ⁹ CFU/ml, and the oil-degrading bacteria fermentation grade I seed liquid is obtained;

(2) In a 10L fermenter equipped with a high-temperature sterilized fermentation medium, inoculate the oil-degrading flora fermentation grade I seed liquid in the step (1) according to an inoculation amount of 5% by volume, at 30° C., and ventilate at 120 rpm Cultivate, test the growth activity of the bacteria every 2 hours, cultivate until the total number of oil-degrading bacteria is above 10 ⁹ CFU/ml, and obtain the second-level seed liquid of oil-degrading bacteria fermentation; then expand the culture step by step, and finally carry out in a 500L fermenter High-density fermentation culture, using simple staining method for microscopic examination, sampling for the first time 8 hours after inoculation, and microscopic examination once every 4 hours thereafter, and finally obtaining the fermentation liquid of petroleum hydrocarbon degrading bacteria (among them, the effective live bacteria of Acinetobacter KJ-1 The count is 1.8×10 ⁸ /ml, the number of effective viable bacteria of Providencia rettii L1 is 1.3×10 ⁸ /ml, the effective number of viable bacteria of Bacillus SWH-1 is 2.0×10 ⁸ /ml, sheath The effective number of viable bacteria of Ammonia bacterium SWH-2 is 1.9× ¹⁰⁹ /ml);

(3) Using peat soil as the carrier of the oil-degrading bacterial agent, mix the fermentation liquid obtained in step (2) with the peat soil according to a mass ratio of 1:10, and dry it naturally in a cool place for 2 hours until the water content is 12 %, the final compound microbial bacterial agent for petroleum hydrocarbon degradation is obtained.

The 10L seed tank control fermentation parameters are: temperature 30°C, stirring speed 120r/min, ventilation rate 1:0.5-1, culture seed tank pressure 0.04-0.06Mpa, culture time is about 24h according to the growth condition;

The 500L fermenter control fermentation parameters are: temperature 30°C, stirring speed 120r/min, ventilation rate 1:0.6-1, culture seed tank pressure 0.04-0.06Mpa, culture time about 18-24h;

Fermentation medium composition: Each liter of fermentation medium contains KH ₂ PO ₄ 1g, K ₂ HPO ₄ 1g, MgSO ₄ 0.2g, CaCl ₂ 0.03g, FeCl ₃ 0.002g, Na ₂ CO ₃ 0.2g, (NH ₄ ) ₂ SO ₄ 0.8g, nutrient broth 20g, the balance is water;

The bacterial agent obtained in this example was stored at room temperature, and the detected bacteria were stored for 3, 6, and 9 months respectively, and the survival rate was calculated. The results of the survival rate test are shown in Table 1. As can be seen from Table 1, the petroleum-degrading bacterial agent prepared in Example 1 was stored at room temperature, and the survival rate reached 75% in three months, and the survival rate was 58% after half a year. Therefore, the survival period of the bacterial agent has reached the standard of biological bacterial agents;

Table 1 The survival rate of different storage periods of petroleum degrading bacterial agents

	3 months	6 months	9 months
Survival rate of petroleum degrading bacteria	75%	58%	46%

.

Comparative example 1

According to the preparation method of embodiment 1 petroleum hydrocarbon degradation composite microbial bacterial agent, prepare respectively the composite bacterial agent (composite microbial agent 1) containing Providencia rettgeri L1, bacillus SWH-1 and Sphingobacterium SWH-2 , the composite bacterial agent (composite microbial agent 2) containing bacillus SWH-1 and Sphingobacterium SWH-2, the total number of viable bacteria is not much different from the composite bacterial agent of Example 1.

Application example 1:

Get the petroleum hydrocarbon degradation composite microbial bacterial agent in the embodiment 1 and carry out the oil-contaminated soil remediation test, and the specific method is as follows:

Take the oil-contaminated soil in the isolated island of Dongying, and use the gravimetric method to detect the oil content of the soil. The oil content of the soil sample is 2.948%. Add 6.7g of ammonium nitrate and 1.8g of potassium dihydrogen phosphate to each kilogram of soil, and treat the oil-contaminated soil according to the parameters in Table 2 Carry out sample treatment, replenish water regularly, and turn tillage. The method of replenishment is to calculate the water shortage after weighing, and spray water when turning tillage;

Table 2 Treatment of oil-contaminated soil bioremediation test samples

serial number	condition parameter
1	As is - Keep moisture content at 20-25%, till tillage
2	As is - 2% bacterial agent - no water added, tilled
3	As it is - 2% bacterial agent - keep 20-25% water content, turn till
4	As it is - 2% bacterial agent - 2% peat - keep water content at 20-25%, tillage
5	As it is - 2% bacterial agent - 5% peat - keep water content at 20-25%, tillage
6	As it is - 2% bacterial agent - 5% straw - keep water content at 20-25%, plow
7	As it is - 2% bacterial agent - keep the water content at 30-35%, turn till

.

Carry out bacterial count (effective live bacterial count) and oil content determination every 15 days, wherein bacterial cell count adopts step-by-step dilution, plate counting method, bacterial count measurement result is shown in Table 3, can see according to Table 3, soil sample Adding peat, straw and other substances in the soil is conducive to the increase of the bacterial count. In the case of the same bacterial agent addition, adding 5% straw and peat to the soil sample at the same time has a better effect on the growth of the bacterial count, followed by adding 2% peat. Test 45 It reached 2.20×10 ⁸ CFU/g, 2.15×10 ⁸ CFU/g, and 1.63×10 ⁸ CFU/g in one day, and then the number of bacteria showed a downward trend. By the 90th day of the test, the bacterial content in treatment 7 was only 3.11× 10 ⁷ CFU/g, 2.79×10 ⁷ CFU/g, 19.1×10 ⁷ CFU/g;

Table 3 Determination of the number of bacteria in different test days

The determination of oil content adopts the following method: add a certain amount of dichloromethane to the oil-contaminated soil, use a Soxhlet extractor to extract the remaining oil in the soil, add a sufficient amount of anhydrous sodium sulfate to the extract for dehydration, filter and spin Steam to make dichloromethane completely volatilize, use dichloromethane to constant volume, dilute to a certain multiple, use the gas chromatography internal standard method to measure the petroleum hydrocarbon content in the soil, calculate the petroleum content in the soil according to the standard curve, and use the following formula to calculate the petroleum degradation rate:

Oil degradation rate (%)=(Wo-Wx)/Wo×100%

Wherein, Wo represents the oil content in the control soil, and Wx represents the oil content in the treated soil;

After 90 days of the above treatment, it was found that sample numbers 4, 5, and 6 had better effects, and the oil content decreased from the original 2.411% to 1.343%, 1.326% and 1.241%, and the degradation rates of petroleum hydrocarbons were 52.65%, 53.23% and 56.24% respectively . In the control group without adding exogenous bacteria, the petroleum degradation rate was only 14.98%. In addition, in the case of adding certain bacterial agents, adding peat or straw can help ensure the permeability of the soil and promote the degradation of petroleum hydrocarbons. .

Application example 2

The petroleum hydrocarbon degrading composite microbial bacterial agent in Example 1 was taken to carry out the oil-contaminated soil remediation test, and the specific method was as follows: the petroleum-contaminated soil in the isolated island area of Dongying was taken, and the oil content of the soil was detected by gravimetric method, and the oil content of the soil was 6.729%. Add 6.7g of ammonium nitrate and 1.8g of potassium dihydrogen phosphate to each kilogram of soil, carry out sample treatment on oil-contaminated soil according to the parameters in Table 4, replenish water regularly, and plow, the water supply method is to calculate the water shortage after weighing, and spray when plowing Hydrate. As shown in Table 5 to the effective viable count determination result;

Table 4 Treatment of oil-contaminated soil bioremediation test samples

Sample serial number	condition parameter
1	As is - Keep moisture content at 20-25%, till tillage
2	As is - 2% bacterial agent - no water added, tilled
3	As it is - 2% bacterial agent - keep water content at 20-25%, turn till
4	As it is - 2% peat - 2% bacterial agent - keep water at 20-25%, turn till

;

Table 5 Determination of Bacteria Content in Different Test Days

It can be seen from Table 5 that the addition of peat to the soil sample is conducive to the increase of the number of bacteria. When the amount of bacteria agent added is 2%, the effect of the growth of the number of bacteria in the soil is better, and the test reaches 3.08× in 45 days. 10 ⁸ CFU/g, and then the number of bacteria showed a decreasing trend. By the 90th day of the test, the bacterial content in treatment 4 was only 4.16×10 ⁷ CFU/g, while in the control it was only 2.35×10 ⁷ CFU/g. Treatments 2 and 3 The amount of bacteria in them were 2.62×10 ⁷ CFU/g and 3.85×10 ⁷ CFU/g respectively. It can be seen that maintaining a certain water content is of great significance to the growth of strains;

Gas chromatography was used to detect the oil degradation rate. The oil content in the sample treated with 4 decreased from 6.411% to 3.651%, and the oil degradation rate was 43.05%. The oil degradation rate of the negative control without exogenous bacterial agent was 6.22%.

Application example 3

Get the petroleum hydrocarbon degrading compound microorganism bacterium agent in the embodiment 1 and carry out petroleum hydrocarbon degradation (the petroleum-contaminated soil in the isolated island area of Dongying, add 6.7g ammonium nitrate and 1.8g potassium dihydrogen phosphate in every kilogram of soil, keep 20-25% in moisture, turn over Tillage) simulation experiments to explore the effects of the amount of bacterial agent, temperature, and fixed carrier on the degradation of petroleum hydrocarbons.

(1) The effect of the amount of bacterial agent added on the degradation of petroleum hydrocarbons

With 0%, 2%, 4% bacterial agent additions as test independent variable, observe the degradation effect, the results are shown in Table 6, find that when the bacterial agent addition is the independent variable, the results show that adding 4% of the initial bacterial agent, The degradation performance is better, and its 50-day degradation rate has reached more than 60%;

Table 6 The effect of bacterial agent addition on the degradation of petroleum hydrocarbons

After one week of the test, add different concentrations of bacterial agents to the soil containing 2% bacterial agents according to Table 7, and detect the effect of adding bacterial agents on the degradation of petroleum hydrocarbons. The results are shown in Table 7. As can be seen from Table 7, test 7D Finally, the addition of 2% bacterial agent made the degradation efficiency much higher than that of the other two groups of experiments. After adding 56 days, the degradation rate of the bacterial agent to petroleum hydrocarbons reached 89.13%;

Table 7 The effect of adding bacterial agents on the degradation of petroleum hydrocarbons

(2) The effect of temperature on the degradation of petroleum hydrocarbons

As an important factor affecting the activity of bacteria, temperature needs to be controlled to determine the optimum temperature for bacterial growth. In this experiment, at the same time as the supplementary bacterial agent was processed, a comparison between room temperature (5-10°C) and constant temperature (25°C) was carried out. The test on temperature as the dependent variable was tested with room temperature and constant temperature room (25°C) as a control ( Table 8). The test shows that when only temperature is used as an independent variable, the degradation efficiency of microorganisms at 25°C is significantly better than that of the test group at room temperature;

Table 8 Effects of different temperatures on the degradation of petroleum hydrocarbons

(3) The amount of fixed carrier added

Immobilizing microorganisms can maintain the biological activity of microorganisms, and under suitable conditions, microorganisms can be rapidly multiplied in large quantities. Different carriers have a greater impact on the effects of microorganisms. Under the premise of using the same carrier at the same time, different carrier additions also have an impact. This experiment Using peat and straw as solid carriers, the effects of different carriers on their degradation rate were studied, and the results are shown in Table 9. Experiments have shown that straw is more conducive to microbial degradation of petroleum hydrocarbons when the addition amount is the same;

Table 9 Effects of different solid carrier additions on the degradation of petroleum hydrocarbons

Application example 5

Carry out the simulation test of petroleum hydrocarbon degradation with the petroleum hydrocarbon degrading bacterial agent prepared in Example 1 and Comparative Example 1 respectively (the petroleum-contaminated soil in Dongying island area, add 6.7g ammonium nitrate and 1.8g potassium dihydrogen phosphate in every kilogram of soil, 5 % straw, plowing), wherein the composite bacterial agent 1 is a compound containing Bacillus KJ-2 of CGMCC No.20665, Bacillus SWH-1 of CGMCC No.1950 and Sphingobacterium SWH-2 of CGMCC No.1951 Bacterial agent, composite bacterial agent 2 is a composite bacterial agent containing Bacillus SWH-1 of CGMCC No.1950 and Sphingobacterium SWH-2 of CGMCC No.1951, and the amount of bacterial agent added is 4% of the quality of petroleum hydrocarbon pollutants %, keep the moisture content of the soil to be treated at 15-20%, and the temperature is 25°C, record the degradation rate, the results are shown in Table 10 below. The composite bacterial agent in embodiment 1 is better than composite bacterial agent 1 and composite bacterial agent 2 to the degradation effect of petroleum hydrocarbon, further proves that live Acinetobacter KJ-1, Providencia rettgeri L1, Bacillus SWH- 1 and the synergy between Sphingobacterium SWH-2;

The influence of table 10 different bacterial agents on the degradation of petroleum hydrocarbons

Claims (10)

1. A compound microbial bacterial agent for petroleum hydrocarbon degradation, characterized in that it includes the live Acinetobacter KJ-1 of preservation number CGMCC No.20664, the spores of Providencia rettii L1 of CGMCC No.16526, and GMCC No.1950 Bacillus SWH-1 and Sphingobacterium SWH-2 of CGMCC No.1951.
2. The composite microbial agent according to claim 1, characterized in that, the composite microbial agent is composed of a fermented bacterial liquid and a carrier peat soil, and the mass ratio of the fermented bacterial liquid to the peat soil is 1:5-15.
3. According to the described composite microbial bacterial agent of claim 2, it is characterized in that, the effective number of live bacteria of Acinetobacter KJ-1 in the described fermented bacterial liquid is 10 ⁷ -10 ⁹ /ml. The effective number of viable bacteria of bacteria L1 is 10 ⁷ -10 ⁹ /ml, the effective number of viable bacteria of Bacillus SWH-1 is 10 ⁸ -10 ⁹ /ml, and the effective number of viable bacteria of Sphingobacterium SWH-2 is 10 ⁷ -10 ⁹ /ml.
4. A preparation method of the petroleum hydrocarbon degradation compound microbial bacterial agent described in any one of claims 1-3, is characterized in that, comprises the following steps:

   (1) The live Acinetobacter KJ-1 of CGMCC No.20664, the Providencia rettii L1 of CGMCC No.16526, the Bacillus SWH-1 of GMCC No.1950 and the sphingosine of CGMCC No.1951 Bacillus SWH-2 was activated on NB solid medium for 16-48 hours, and transferred to NB liquid medium for shake flask activation for 16-48 hours as the initial seed solution;

   (2) Put the initial seed liquid of petroleum-degrading bacterium Acinetobacter KJ-1, Providencia rettgeri L1, Bacillus SWH-1 and Sphingobacterium SWH-2 according to the total inoculation amount of 3-6%. Fermentation and cultivation in the fermentation medium until the total number of oil-degrading bacteria is above 10 ⁹ CFU/mL, to obtain grade I seed liquid of oil-degrading bacteria fermentation;

   (3) The oil-degrading flora fermentation grade I seed liquid in step (2) is transferred to the fermentation medium again according to the volume ratio of 3-6%, and cultured in air until the total number of oil-degrading flora reaches 10 ⁹ CFU/ ml or more, the oil-degrading flora fermentation grade II seed liquid is obtained, and the cultivation is expanded step by step, and the total number of oil-degrading flora is more than 10 ⁹ CFU/ml;

   (4) using peat soil as a carrier, mixing the fermented bacterial liquid obtained in step (2) with peat soil, the mass ratio is 1:5-15, and the water content after drying is 10-15%, so as to obtain the oil-degrading bacterial agent .
5. The preparation method according to claim 4, wherein the NB solid medium described in step (1) contains 10 g of peptone per liter of medium, 3 g of beef powder, 5 g of sodium chloride, 18.0 g of agar, and distilled water Dilute to 1000mL, pH 7.0-7.4; the fermentation medium described in step (2) and step (3) contains KH ₂ PO ₄ 1g, K ₂ HPO ₄ 1g, MgSO ₄ 0.2 per liter of fermentation medium g, CaCl ₂ 0.03g, FeCl ₃ 0.002g, Na ₂ CO ₃ 0.2g, (NH ₄ ) ₂ SO ₄ 0.8g, nutrient broth 20g, the balance is water; the fermentation temperature is 28-37°C, The culture time is 18-24 hours, and the culture speed is 120-150 rpm.
6. The preparation method according to claim 4, characterized in that, the live Acinetobacter KJ-1, Providencia rettgeri L1, Bacillus SWH-1 and Sphingobacterium SWH described in step (2) -2 The ratio of the effective number of viable bacteria of the four strains is 1.5-2:1:1-2:2.
7. An application of the petroleum hydrocarbon degrading composite microbial bacterial agent described in any one of claims 1-3 in the treatment of petroleum pollutants.
8. The application according to claim 7, characterized in that the petroleum pollutants are petroleum-contaminated soil or treated oily sludge.
9. The application according to claim 8, characterized in that, when the petroleum hydrocarbon degrading composite microbial bacterial agent is used to treat oil-contaminated soil, a composite microbial bacterial agent of 2 to 4% of the petroleum hydrocarbon pollutant quality is added, and petroleum hydrocarbon degrading compound microbial bacterial agent is added. Nitrogen and phosphorus fertilizers with a hydrocarbon pollutant mass of 0.05-0.5% and a nitrogen-to-phosphorus ratio of 3-5:1, keep the moisture content between 15-25%, and treat for 30-90 days.
10. The application according to claim 9, characterized in that, the compound microbial bacterial agent is added to the soil twice, and the interval between the two additions is one week.