WO2021077888A1

WO2021077888A1 - Preparation method and use method for heavy oil viscosity reduction and degradation mixed microbial inoculum

Info

Publication number: WO2021077888A1
Application number: PCT/CN2020/110934
Authority: WO
Inventors: 张秀霞; 辛瑞
Original assignee: 中国石油大学(华东)
Priority date: 2019-10-22
Filing date: 2020-08-25
Publication date: 2021-04-29
Also published as: CN110669692A; CN110669692B

Abstract

The present invention provides a preparation method and a use method for a heavy oil viscosity reduction and degradation mixed microbial inoculum. The mixed microbial inoculum comprises Acinetobacter sp. strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. strain RS206 and hrobactrum anthropi strain CON21, the four strains are obtained by manual screening, the former two strains are high-efficiency petroleum hydrocarbon degradation bacteria, and the latter two strains are high-efficiency heavy oil viscosity reduction bacteria. The heavy oil viscosity reduction and degradation mixed microbial inoculum has a high degradation efficiency for the heavy oil, has a good viscosity reduction effect, and has a good use prospect in environmental governance.

Description

Preparation method and application method of thick oil viscosity-reducing and degrading mixed bacterial agent

Technical field

The invention relates to a preparation method and an application method of a thick oil viscosity-reducing and degrading mixed bacterial agent, which belongs to the fields of environmental protection and pollution prevention and treatment.

Background technique

my country has 400,000 tons of heavy oil resources, and the Northeast and North China regions account for a large proportion of heavy oil reserves. Among them, the heavy oil from Bohai Oilfield accounts for more than 80% of CNOOC crude oil. Heavy oil is difficult to be developed and utilized due to its higher viscosity and more heavy components, and it is more difficult to repair the pollution caused during the mining process. The microbiological method is used to repair heavy oil pollution with lower cost and better repair effect; the final product is small molecule water, carbon dioxide or organic matter, and there is no secondary pollution problem; it can be treated in situ and ex situ and has strong applicability. Therefore, with its great advantages, microbial degradation has become a remediation method for oily sewage and soil in many developed countries.

In the process of microbial degradation of heavy oil, the hydrophobicity of crude oil is one of the main factors limiting its degradation rate. The high viscosity of heavy oil makes it difficult for microorganisms to degrade and utilize it. The degradation of heavy oil by microorganisms is mainly carried out at the oil-water interface. The emulsification of oil can greatly increase the degree of oil dispersion, increase the contact surface area between strains and oil droplets, and promote the absorption and degradation of petroleum hydrocarbons by microorganisms. The direct addition of surfactants can only change the spatial structure of heavy oil macromolecules, but cannot change its chemical composition. Therefore, the viscosity of heavy oil will be restored due to the chromatographic separation effect; in contrast, microorganisms can not only pass the glycolipids produced in the metabolic process Biosurfactants, lipopeptides, phospholipids, etc., reduce the interfacial tension of oil and water and emulsify crude oil, and can improve the biochemical activity of the heavy components of crude oil, thereby improving the viscosity of crude oil. The hydrocarbons are converted into low-molecular hydrocarbons, thereby reducing the macromolecular components in the heavy oil, reducing its average molecular weight, and reducing the viscosity more thoroughly. Studies have shown that certain bacteria can promote the emulsification of crude oil through the production of biosurfactants and passively diffuse into the cells, thereby being trapped and degraded by biological enzymes, thereby increasing the degradation rate of crude oil.

Since heavy oil is composed of compounds with complex structure and difficult to degrade, a single strain is poorly degraded. The combination of different heavy oil degrading strains can destroy the aromatic ring and heterocyclic structure and further degrade it, and at the same time produce surfactant strains Metabolized biosurfactants can emulsify and solubilize crude oil. Therefore, the compounding of petroleum hydrocarbon degrading bacteria and slime reducing bacteria can improve the uptake and degradation rate of heavy oil by the strains.

At present, there are few studies on the microbial degradation of heavy oil in China, and they mainly focus on the selection of high-efficiency degrading bacteria or the compounding of petroleum hydrocarbon degrading bacteria, or the use of external surfactants to enhance microbial remediation, but rarely degrade petroleum hydrocarbons. Study on the compounding of bacteria and myxobacteria.

People have conducted a lot of in-depth research on the microbial treatment of heavy oil, and have achieved many results, such as:

CN 109763803 A discloses a Pseudomonas rhamnolipo-producing QFP and its application in heavy oil exploitation, including a facultative anaerobic pseudomonas (Pseudomonas sp.) strain QFP. The application method is as follows: transfer the Pseudomonas QFP stored in the glycerol tube to the LB liquid medium, shake culture at 20-30℃ for 12 hours to obtain the seed liquid, and inoculate the seed liquid at a volume ratio of 0.5%. In the surfactant fermentation medium, continuously aerated culture at 20-30°C for 96h, and the fermentation broth is obtained after the fermentation is completed. The rhamnolipid produced by the invention has strong surfactant performance and emulsifying ability, can significantly reduce the viscosity of heavy oil, and can inhibit the growth of other bacteria. The bacteria can be applied to the repair of petroleum hydrocarbon polluted environment and oil reservoir environment It has the potential to improve oil recovery, and the produced rhamnolipids have important application value in medicine, environmental protection and energy extraction.

CN 109576191 A discloses a composite microbial inoculum for the development of heavy oil and its preparation method and application. It is proposed for the problem that the existing microbial oil extraction technology is complicated in preparation and that a single strain is not resistant to environmental impact. The compound microbial inoculum of the present invention is made by mixing Bacillus pallisi, Pantoea, and thermophilic anaerobic bacillus after fermentation. The beneficial effect is to avoid the defect that single-function strains cannot tolerate environmental impacts, and the difference between the three strains The functional expression is not inhibited, the preparation method is simple, and the cost is low.

CN 104109646 A discloses a visbreaker suitable for heavy oil wells with different mineralization degrees and its application. The visbreaker is composed of Bacillus licheniformis, Bacillus subtilis and Bacillus thuringiensis, at a temperature of 30-70°C, After culturing for 24 hours under the conditions of pH 5-10 and salinity 1000-12000mg/L and 0-30MPa, the cell concentration of each strain can reach 10 ⁸ -10 ⁹ cells/mL, and the reproductive ability is strong. The bacterial agent can reduce the viscosity of crude oil by degrading heavy components of crude oil, metabolizing biological surfactants, biogas, etc., so as to ensure stable production and increased production of heavy oil wells. The method of use includes: taking the slime reducing bacteria to ferment to produce the fermentation bacteria liquid; using the fermentation bacteria liquid to prepare the injection bacteria liquid system; squeeze the injected bacteria liquid system into the target oil layer. The microbial agent has achieved a good effect in oil well field tests under the conditions of salinity 6000～83000mg/L, pH7～8.6, and 40～60℃. The viscosity of crude oil is reduced by 50%～

65%, the surface tension of the produced liquid drops by 20%-30%, and the freezing point of crude oil drops by 1 to 3°C.

CN 104450543 A discloses an original microbial repair composite preparation and an application method thereof; the composite preparation is composed of four parts: heavy oil degrading bacteria, nutrients, composite surfactant penetrant, and oxidation promoting activator, in which heavy oil is degraded The flora is composed of aerobic rod-shaped bacteria (Lactobacillus sp.) and anaerobic rod-shaped spore bacteria (bacillus sp.) screened and isolated from heavy oil soil; spray the compound formulation on the contaminated soil after 2-3 Within months, the degradation rate of heavy oil contaminated soil reached 50-80%.

As mentioned above, although people have developed many methods for degrading heavy oil by using microorganisms, they have a single function and have a good viscosity reduction effect on heavy oil but low degradation efficiency, or high degradation efficiency but not obvious viscosity reduction effect. Therefore, there is still a need and demand for continued research on new mixed bacterial agents. This is where the power and foundation of the present invention are based.

Summary of the invention

In view of the many shortcomings of mixed bacteria in the degradation and viscosity reduction of heavy oil, the present invention provides a method for preparing a thick oil viscosity reduction and viscosity reduction mixed bacteria agent. The mixed bacterial agent can not only realize high-efficiency degradation of heavy oil, but also can obviously improve the viscosity of heavy oil.

The present invention is realized through the following technical solutions:

A method for preparing a viscous-reducing and degrading mixed bacterial agent for heavy oil, the preparation method includes the following steps:

The first step is to prepare seed culture medium;

The second step is to inoculate the following single bacteria into the seed medium respectively, and obtain the seed liquid of each single bacteria after culturing. The single bacteria include Acinetobacter sp.strain ZX-15, and Achromobacter pulmonis strain PI3-03 , Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21;

In the third step, the seed liquid of each single bacteria is mixed in proportion to obtain the viscous-reducing and degrading mixed bacteria agent of heavy oil.

The above-mentioned single bacteria are all existing strains, which can be searched in the NCBI nucleic acid database by number, such as: Acinetobacter sp.strain ZX-15 in the NCBI database with the accession number MF148465.1, Achromobacter pulmonis The accession number of strain PI3-03 in the NCBI database is MK396599.1, the accession number of Acinetobacter sp.RS206 in the NCBI database is EU912468.1, and the accession number of chrobactrum anthropi strain CON21 in the NCBI database is MK167392 .1.

Further, the preparation of the seed culture medium in the first step includes the following steps:

R1: Dissolve 5.0g beef extract, 10.0g peptone, and 5.0g sodium chloride in 1000mL deionized water, stir well to obtain mixed liquid I;

R2: To the mixed solution I, add 0.1g sodium molybdate, 0.05g aluminum nitrate, 0.04g zinc chloride, 0.06g copper sulfate, 0.03g manganese chloride, 0.09g ferrous sulfate, 0.10g magnesium nitrate and 0.06 g of potassium chloride, and then fully mixed to obtain the seed culture medium.

Further, in the third step, the mixing volume ratio of the seed liquid of Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 It is (1-2):(1-2):(1-2):(1-2), especially 2:2:1:1.

Furthermore, the Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were contaminated by heavy oil from the heavy oil mining area. The specific steps are as follows:

S1: Take the soil contaminated by heavy oil in the heavy oil mining area and add it to the enrichment/degradation medium containing heavy oil in the first phase, and then incubate it on a constant temperature shaker, then transfer the supernatant to the second phase containing heavy oil. Re-cultivation in the enrichment/degradation medium; gradually increase the concentration of heavy oil in the enrichment/degradation medium in each phase, a total of multiple acclimation cycles;

S2: Take the culture solution of the last acclimatization cycle and spread it on a plate, pick out colonies with different appearances and repeat streaking to obtain multiple different purified single strains;

S3: Degradation experiments of heavy oil were carried out with each single strain of bacteria respectively, and the two bacteria with the highest degradation rate of petroleum hydrocarbons were obtained, and the two bacteria with the best viscosity reduction effect on heavy oil were obtained by measuring the surface tension of the bacteria;

S4: Perform 16sRNA sequencing on the four selected strains, and determine that the two strains with the highest petroleum hydrocarbon degradation rate are Acinetobacter sp.strain ZX-15 and Achromobacter pulmonis strain PI3-03, which reduce the viscosity of heavy oil The two most effective strains are Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21.

Further, the preparation method of the enrichment/degradation medium is: adding 5.0g sodium chloride, 1.0g ammonium sulfate, 0.25g magnesium sulfate heptahydrate, 2.0g sodium nitrate, 4.0g potassium dihydrogen phosphate, 7.6g Dipotassium hydrogen phosphate is dissolved in 1000 mL of deionized water, and then a suitable amount of heavy oil is added.

Further, the degradation experiment in step S3 includes the following steps:

T1: Inoculate a single strain in an inorganic salt medium containing heavy oil, and culture it on a constant temperature shaker at 160r/min and 37°C for 8 days;

T2: Extract the residual oil in the culture medium to obtain the petroleum hydrocarbon degradation rate, and select the two strains with the highest petroleum hydrocarbon degradation rate;

The measurement of the surface tension of the bacterial liquid includes the following steps:

M1: Inoculate a single strain into the fermentation medium, and culture it for 3 days at 160r/min and 37°C;

M2: Centrifuge the fermentation broth at 8000r/min for 10 minutes to remove the cell bodies in the fermentation broth;

M3: The surface tension of the fermentation broth is measured, and the two strains with the smallest surface tension of the fermentation broth are selected as high-efficiency viscous oil-lowering bacteria.

Further, the 16sRNA sequencing in step S4 is to extract the genomic DNA of the strain and perform electrophoresis detection, use universal primers for gene amplification, then perform electrophoresis PCR product identification, perform sequencing, homology comparison, and evolutionary tree analysis to determine The two strains with the highest degradation rate of petroleum hydrocarbons are Acinetobacter sp.strain ZX-15 and Achromobacter pulmonis strain PI3-03. The two strains with the best effect on reducing viscosity of heavy oil are Acinetobacter sp. RS206 and chrobactrum anthropi strain CON21.

Next, the application method of the thick oil viscosity reducing and degrading mixed bacterial agent obtained by the above preparation method is given. It is applied to the viscosity reduction and degradation of heavy oil pollution. The application temperature is controlled at 25-40℃, and the best is 35℃; pH control is applied It is 7-8, the best is 7.5; the application of inoculum volume is controlled at 3-5% by volume.

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

(1) The sieving and culturing process of the mixed inoculum related to the present invention all use conventional culture medium, no special culture environment is required, and the preparation of the mixed inoculum is simpler.

(2) The mixed bacterial agent involved in the present invention is obtained by separation, enrichment and purification from heavy oil contaminated soil. The strain itself comes from heavy oil soil and belongs to the original microbial degrading bacteria. Therefore, it is more suitable for heavy components and colloidal asphalt. The high heavy oil environment has good adaptability and high efficiency.

(3) The thick oil viscosity-reducing and degrading mixed bacterial agent disclosed in the present invention is a kind of artificially screened and constructed mixed bacterial agent, which has obvious advantages in biodegradation of pollutants compared with a natural mixed bacterial system with incomplete composition and a single strain. The bacterial composition of the bacterial agent is clear, the preparation process is simple, the degradation efficiency is high, the viscosity reduction effect is good, and it has a good application prospect.

(4) The viscosity-reducing and degrading mixed microbial agent for heavy oil disclosed in the present invention can simultaneously reduce the viscosity of heavy oil and efficiently degrade petroleum hydrocarbons, and has more comprehensive functions than the existing microbial technology.

Description of the drawings

Figure 1 is a schematic diagram of the surface tension of the bacterial liquid and the degradation rate of petroleum hydrocarbons at different culture temperatures in Example 2;

2 is a schematic diagram of the surface tension of the bacterial liquid and the degradation rate of petroleum hydrocarbons under different pH in Example 2;

3 is a schematic diagram of the surface tension of the bacterial liquid and the degradation rate of petroleum hydrocarbons under different inoculation amounts in Example 2;

4 is a schematic diagram of the surface tension and petroleum hydrocarbon degradation rate of the bacterial liquid with different mixing ratios in Example 3.

Detailed ways

The present invention will be described in detail below through specific examples, but the use and purpose of these exemplary embodiments are only to illustrate the present invention, and do not constitute any limitation on the actual protection scope of the present invention in any form, let alone the present invention. The scope of protection is limited to this.

Example 1

Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 domestication, screening and preparation of mixed bacterial agents:

(1) Take 3g of the soil contaminated by heavy oil for a long time in the heavy oil production area of Shengli Oilfield, add it to 150mL enrichment/degradation medium containing 1g/L of heavy oil, and incubate at 37℃ for 5d (160r/min) in a constant temperature shaker , Take 3mL of supernatant and transfer it to a new enrichment/degradation medium with a heavy oil content of 2g/L, and incubate again for 5 days. By analogy, gradually increase the concentration of heavy oil to 5g/L for a total of five cycles of acclimatization.

(2) Take the culture solution of the last acclimatization cycle and spread it on a plate, pick out colonies with different morphological characteristics, and repeat streaking until it is confirmed that the colonies in the same plate have the same morphology and size, and a purified single strain is obtained.

(3) Inoculate a single strain in a heavy oil-containing inorganic salt medium, culture it on a constant temperature shaker at 37°C for 8 days (160r/min), and take two bottles on each of the 3rd, 5th, and 8th days; use the degradation medium for After the petroleum ether is extracted, the petroleum hydrocarbon degradation rate is obtained, and the two strains with the highest petroleum hydrocarbon degradation rate are selected as high-efficiency petroleum hydrocarbon degrading bacteria.

(4) Inoculate a single strain into the fermentation medium, culture it for 3 days at 160r/min and 37°C, centrifuge the fermentation broth at 8000r/min for 10min, remove the cell bodies in the fermentation broth, and use a surface tensiometer The surface tension of the fermentation broth was measured. The two strains with the smallest surface tension of the fermentation broth were selected as the high-efficiency thick oil-lowering bacteria.

(5) Perform 16sRNA sequencing on the selected strains, extract the genomic DNA of the strains and perform electrophoresis detection, use universal primers for gene amplification, then perform electrophoresis PCR product identification, perform sequencing, homology comparison and evolutionary tree analysis, It can be seen that the high-efficiency petroleum hydrocarbon degrading bacteria are Acinetobacter sp.strain ZX-15 and Achromobacter pulmonis strain PI3-03, and the high-efficiency thick oil reducing bacteria are Acinetobacter sp. RS206 and chrobactrum anthropi strain CON21;

(6) Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were inoculated into the seed culture medium at 37°C, Under the condition of 160r/min, the seed liquid was obtained by shaking at a constant temperature for 24 hours, and the seed liquid was mixed in a volume ratio of 2:2:1:1 to obtain a mixed microbial agent.

Wherein, the seed culture medium preparation step: dissolve 5.0 g beef extract, 10.0 g peptone, and 5.0 g sodium chloride in 1000 mL of deionized water, stir thoroughly to obtain mixed liquid I, and add trace elements to the mixed liquid I 0.1g sodium molybdate, 0.05g aluminum nitrate, 0.04g zinc chloride, 0.06g copper sulfate, 0.03g manganese chloride, 0.09g ferrous sulfate, 0.10g magnesium nitrate and 0.06g potassium chloride, and then mix them thoroughly.

Among them, the preparation method of the enrichment/degradation medium is as follows: 5.0g sodium chloride, 1.0g ammonium sulfate, 0.25g magnesium sulfate heptahydrate, 2.0g sodium nitrate, 4.0g potassium dihydrogen phosphate, 7.6g dipotassium hydrogen phosphate Dissolve in 1000mL deionized water, then add appropriate amount of heavy oil.

Example 2

2. Degradation and viscosity reduction performance of mixed bacterial agents under different culture conditions

Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were respectively inoculated into the seed culture medium at 37℃, 160r/ Under the condition of constant temperature and shaking for 24 hours, the seed liquid was obtained, and their seed liquids were mixed according to the volume ratio of 2:2:1:1, and inoculated into the degradation medium, and the experiments under the following degradation conditions were carried out. Among them, the degradation medium is 1.0 g of ammonium sulfate, 0.25 g of magnesium sulfate heptahydrate, 2.0 g of sodium nitrate, 4.0 g of potassium dihydrogen phosphate, and 7.6 g of dipotassium hydrogen phosphate dissolved in 1000 mL of deionized water.

1. Different temperatures

Degradation conditions: pH 7.5, heavy oil 2g/L, inoculation amount 5% (V/V), sodium chloride 4g/L; temperature: 20℃, 25℃, 30℃, 35℃, 40℃, 45℃; After 10 days of cultivation, the degradation rate of petroleum hydrocarbons was measured by ultraviolet spectrophotometry, and the surface tension of the bacterial liquid was measured by a surface tension meter.

The results show that the mixed bacteria have a high degradation rate at 25-40℃, among which 35℃ is the optimum temperature, and the degradation rate can reach the highest 44.12%. At this time, the surface tension of the bacterial liquid also reaches the lowest 43.6mN/m.

2. Different pH

Degradation conditions: temperature 37℃, heavy oil 2g/L, inoculation amount 5% (V/V), sodium chloride 4g/L; pH: 3, 5, 7, 7.5, 8, 9, 11, 12; culture for 10 days Then use ultraviolet spectrophotometry to measure the degradation rate of petroleum hydrocarbons, and use a surface tension meter to measure the surface tension of the bacterial liquid.

The results showed that the mixed bacteria had a high degradation rate at pH 7-8, and the optimum pH was 7.5. At this time, the degradation rate reached the maximum 42.14%, and the surface tension of the bacterial liquid also reached the minimum 44.1mN/m.

3. Different inoculation amount

Degradation conditions: temperature 37℃, pH 7.5, heavy oil 2g/L, sodium chloride 4g/L; inoculation amount: 1%, 2%, 5%, 10%, 15%, 20%; after 10 days of culture, use UV The degradation rate of petroleum hydrocarbons was measured by spectrophotometry, and the surface tension of the bacterial liquid was measured with a surface tension meter.

The results showed that when the inoculation amount was higher than 5%, the degradation rate of petroleum hydrocarbons did not change significantly compared with the inoculation amount, the degradation rate of petroleum hydrocarbons in the culture medium increased insignificantly by the mixed bacteria, and the decrease in surface tension was reduced. When the inoculation amount is less than 3%, the degradation rate of the strain to petroleum hydrocarbons is lower and the surface tension is higher.

Example 3

Degradation and viscosity reduction performance of bacterial agents in different mixing ratios

Acinetobacter sp.strain ZX-15, Achromobacter pulmonis strain PI3-03, Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 were inoculated into the seed culture medium at 37℃, 160r/min Under the condition of constant temperature shaking for 24 hours, the seed liquid was obtained, and the seed liquid was mixed according to the following proportions according to the volume ratio:

The results showed that the degradation rate of petroleum hydrocarbon in combination D was the highest, 43.84%, and the surface tension was 41.2mN/m, the surface tension of combination E was the lowest, 38.7mN/m, and the degradation rate of petroleum hydrocarbon was 40.58%. The difference in surface tension and degradation rate of the two combinations is relatively small. Combination D has the best degradability for heavy oil and has a better viscosity reduction effect. It is most suitable for biological treatment of heavy oil pollution. Therefore, combination D is the best. Best mixing ratio.

It can be seen from Example 2 and Example 3 that the degradation and viscosity reduction effect of the four strains after mixing is significantly greater than that of a single strain. The highest degradation rate of heavy oil after the strains is mixed can reach 43.84%, while the highest degradation rate of a single strain for 10 days is 28.96%, the degradation effect is increased by 34%, and the lowest surface tension can reach 38.7mN/m, while the single strain is cultured in heavy oil. The lowest surface tension of 10d in the base is about 54.1mN/m. In summary, the combination of strains has a synergistic effect, which not only enhances the degradability of heavy oil, but also enhances the effect of heavy oil emulsification and dispersion, and the dual effects work together to improve the fluidity of heavy oil.

The above are only typical embodiments of the present invention, and those skilled in the art may use the above-explained technical solutions to modify the present invention or modify them into equivalent technical solutions. Therefore, any simple modification or equivalent replacement made according to the technical solution of the present invention shall fall within the scope of protection of the present invention.

Claims

A preparation method of a viscous-reducing and degrading mixed microbial agent for heavy oil, characterized in that the preparation method comprises the following steps:

The first step is to prepare seed culture medium;

The second step is to inoculate the following single bacteria into the seed medium respectively, and obtain the seed liquid of each single bacteria after culturing. The single bacteria include Acinetobacter sp.strain ZX-15, and Achromobacter pulmonis strain PI3-03 , Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21;

In the third step, the seed liquid of each single bacteria is mixed in proportion to obtain the viscous-reducing and degrading mixed bacteria agent of heavy oil.
The preparation method of the thick oil viscosity-reducing and degrading mixed microbial agent according to claim 1, wherein the preparation of the seed culture medium in the first step comprises the following steps:

R1: Dissolve 5.0g beef extract, 10.0g peptone, and 5.0g sodium chloride in 1000mL deionized water, stir well to obtain mixed liquid I;

R2: To the mixed solution I, add 0.1g sodium molybdate, 0.05g aluminum nitrate, 0.04g zinc chloride, 0.06g copper sulfate, 0.03g manganese chloride, 0.09g ferrous sulfate, 0.10g magnesium nitrate and 0.06 g of potassium chloride, and then fully mixed to obtain the seed culture medium.
The preparation method of the thick oil viscosity-reducing and degrading mixed bacterial agent according to claim 1, characterized in that in the third step, Acinetobacter sp. strain ZX-15, Achromobacter pulmonis strain PI3-03, The mixing volume ratio of the seed liquid of Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21 is (1-2): (1-2): (1-2): (1-2).
The preparation method of the thick oil viscosity-reducing and degrading mixed bacterial agent according to claim 1, wherein the Acinetobacter sp. strain ZX-15, Achromobacter pulmonis strain PI3-03, and Acinetobacter Acinetobacter sp.RS206 and chrobactrum anthropi strain CON21 were domesticated and screened from the soil contaminated by heavy oil in the heavy oil production area. The specific steps are as follows:

S1: Take the soil contaminated by heavy oil in the heavy oil mining area and add it to the enrichment/degradation medium containing heavy oil in the first phase, and then incubate it on a constant temperature shaker, then transfer the supernatant to the second phase containing heavy oil. Re-cultivation in the enrichment/degradation medium; gradually increase the concentration of heavy oil in the enrichment/degradation medium in each phase, a total of multiple acclimation cycles;

S2: Take the culture solution of the last acclimatization cycle and spread it on a plate, pick out colonies with different appearances and repeat streaking to obtain multiple different purified single strains;

S3: Degradation experiments of heavy oil were carried out with each single strain of bacteria respectively, and the two bacteria with the highest degradation rate of petroleum hydrocarbons were obtained, and the two bacteria with the best viscosity reduction effect on heavy oil were obtained by measuring the surface tension of the bacteria;

S4: Perform 16sRNA sequencing on the four selected strains, and determine that the two strains with the highest petroleum hydrocarbon degradation rate are Acinetobacter sp.strain ZX-15 and Achromobacter pulmonis strain PI3-03, which reduce the viscosity of heavy oil The two most effective strains are Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21.
The preparation method of the thick oil viscosity-reducing and degrading mixed bacterial agent according to claim 4, wherein the preparation method of the enrichment/degradation medium is: adding 5.0 g of sodium chloride, 1.0 g of ammonium sulfate, and 0.25 g magnesium sulfate heptahydrate, 2.0 g sodium nitrate, 4.0 g potassium dihydrogen phosphate, 7.6 g dipotassium hydrogen phosphate are dissolved in 1000 mL of deionized water, and then an appropriate amount of heavy oil is added.
The preparation method of the thick oil viscosity-reducing and degrading mixed bacterial agent according to claim 4, wherein the degradation experiment in step S3 includes the following steps:

T1: Inoculate a single strain in an inorganic salt medium containing heavy oil, and culture it on a constant temperature shaker at 160r/min and 37°C for 8 days;

T2: Extract the residual oil in the culture medium to obtain the petroleum hydrocarbon degradation rate, and select the two strains with the highest petroleum hydrocarbon degradation rate;

The measurement of the surface tension of the bacterial liquid includes the following steps:

M1: Inoculate a single strain into the fermentation medium, and culture it for 3 days at 160r/min and 37°C;

M2: Centrifuge the fermentation broth at 8000r/min for 10 minutes to remove the cell bodies in the fermentation broth;

M3: The surface tension of the fermentation broth is measured, and the two strains with the smallest surface tension of the fermentation broth are selected as high-efficiency viscous oil reduction bacteria.
The method for preparing a mixed bacterial agent for viscosity reduction and degradation of heavy oil according to claim 4, wherein the 16sRNA sequencing in step S4 is to extract the genomic DNA of the strain and perform electrophoresis detection, use universal primers for gene amplification, and then Perform electrophoresis PCR product identification, sequencing, homology comparison and phylogenetic tree analysis to determine that the two strains with the highest petroleum hydrocarbon degradation rate are Acinetobacter sp.strain ZX-15 and Achromobacter pulmonis strain PI3-03 The two strains with the best viscosity-reducing effect on heavy oil are Acinetobacter sp. RS206 and Chrobactrum anthropi strain CON21.
The application method of the thick oil viscosity reduction and degradation mixed bacterial agent obtained by the preparation method of any one of claims 1-7, characterized in that it is applied to the viscosity reduction and degradation of heavy oil pollution, and its application temperature is controlled at 25-40°C .
The application method of the thick oil viscosity reducing and degrading mixed bacterial agent according to claim 8, characterized in that it is applied to the viscosity reduction and degradation of heavy oil pollution, and the application pH is controlled to be 7-8.
The application method of the thick oil viscosity reduction and degradation mixed bacterial agent according to claim 8, characterized in that it is applied to the viscosity reduction and degradation of heavy oil pollution, and the applied inoculum is controlled as the volume of inoculated bacterial liquid/liquid culture after inoculation The volume fraction of the total volume of the base is 3-5%.