Method for emulsifying and degrading crude oil by using oil reservoir endogenous bacteria and application thereof

Abstract

The invention relates to a method for emulsifying and degrading crude oil by using oil reservoir endogenous bacteria and application thereof, and the method comprises the following steps: carrying out fermentation culture on a bacterial strain which is separated and purified from an oil reservoir and can generate a biosurfactant to obtain a bacterial strain fermentation liquid; and injecting the fermentation liquor into a crude oil reservoir. According to the method, the functional microorganisms are re-injected into the oil reservoir environment after being subjected to surface fermentation, and are mutually matched with the oil reservoir indigenous microbial community on the effects of emulsifying and degrading crude oil, so that the method has important significance on the development of a microbial oil recovery technology.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a method for emulsifying and degrading crude oil by utilizing endogenous bacteria in oil reservoirs and application thereof. Background Art

[0002] In the process of oil field development, binary flooding of chemical surfactants and chemical polymers has been widely used in tertiary oil recovery. The use of biological and chemical compounding is an emerging technology for oil field development in recent years. In the related technology, biological surfactants, chemical surfactants and chemical polymers are compounded (CN115232610A and CN109897621A) to achieve good results in improving oil recovery. However, the large-scale application of chemical agents injected into oil fields has increasingly polluted the environment, and it is necessary to conduct research on green, low-carbon and environmentally friendly technologies for improving oil recovery. At the same time, chemical agents have certain toxicity or inhibition on the growth of microorganisms, and biological surfactants using metabolites can only be fermented on the ground, which limits the scope of application of the technology.

[0003] Microbial Enhanced Oil Recovery (MEOR) technology has been gradually applied to the later stage of oilfield development. The biological surfactants produced by microbial metabolism have unique characteristics such as green environmental protection and low cost. Due to their special amphiphilic structure, biological surfactants have special effects in emulsifying crude oil. The key to microbial oil recovery technology is whether strains with efficient oil recovery functions can be obtained to achieve the benefits and prospects of mine application. The strains screened from nature have different properties. Endogenous bacteria have stronger reservoir adaptability and functional robustness, and obtain environmental adaptability through community functions. At the same time, reservoir microorganisms such as Pseudomonas and Bacillus have the characteristics of producing surfactants such as rhamnolipids and lipopeptides, but due to the influence of the oligotrophic environment in the reservoir, surface-active microorganisms are often not the dominant strains in the produced fluid. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs and its application.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for emulsifying and degrading crude oil using endogenous bacteria in an oil reservoir, the method comprising:

[0007] (1) fermenting and culturing a strain capable of producing a biosurfactant separated and purified from an oil reservoir to obtain a fermentation liquid;

[0008] (2) Injecting the fermentation liquid into the crude oil reservoir.

[0009] In oilfield development technology, the pollution of the environment caused by chemical flooding is becoming increasingly prominent. Research on improving oil recovery by using green, low-carbon and environmentally optimized oil recovery agents is urgently needed. Due to the oligotrophic environment in the reservoir, surface-producing active microorganisms are often not the dominant strains in the produced fluid. Therefore, it is beneficial to the development of the reservoir to re-inject functional microorganisms into the reservoir environment after surface fermentation. The strains of biosurfactants produced in the reservoir can cooperate with the communities in the endogenous environment of the reservoir to improve the effect of emulsification and degradation of crude oil, provide a new generation of biorecovery products for tertiary oil recovery technology, and further achieve cost reduction and efficiency improvement in oil extraction.

[0010] Preferably, the strain comprises any one or a combination of at least two of Thauerapropionica WL-2 strain with a deposit number of CGMCC No.24606, Pseudomonas aeruginosa WL-4 strain with a deposit number of CGMCC No.24711, or Bacillus subtilis WL-5 strain with a deposit number of CGMCC No.24712.

[0011] Preferably, the strain is a combination of Thauera propionica WL-2 strain with a deposit number of CGMCC No.24606, Pseudomonas aeruginosa WL-4 strain with a deposit number of CGMCC No.24711, and Bacillus subtilis WL-5 strain with a deposit number of CGMCC No.24712.

[0012] The present invention creatively discovered that when the WL-2 strain, the WL-4 strain and the WL-5 strain are added simultaneously, the effect of emulsification degradation of crude oil is the best, and there is a certain synergistic effect between the strains.

[0013] Preferably, the ratio of the viable counts of the WL-2 strain, the WL-4 strain and the WL-5 strain is (1-3):(1-3):(1-3). The specific point values ​​in (1-3) can be selected from 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0014] When the WL-2 strain, the WL-4 strain and the WL-5 strain were added in the above-mentioned specific live bacterial count ratio, the effect of emulsification degradation of crude oil was the best.

[0015] Preferably, the fermentation culture temperature is 28-45°C, for example, 28°C, 30°C, 32°C, 35°C, 38°C, 40°C, 42°C, 45°C, etc. Other specific points within the above numerical range can be selected and will not be described here one by one.

[0016] Preferably, the fermentation culture time is 12-36h, for example, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0017] Preferably, the inoculation amount of the fermentation broth is 0.5%-1.5%, for example 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, etc. Other specific points within the above numerical range can be selected and will not be described here one by one.

[0018] Preferably, the number of viable bacteria in the fermentation broth is 1×10 7 -1×10 9 CFU / mL, for example 1×10 7 CFU / mL, 2×10 7 CFU / mL, 3×10 7 CFU / mL, 4×10 7 CFU / mL, 5×10 7 CFU / mL, 6×10 7 CFU / mL, 7×10 7 CFU / mL, 8×10 7 CFU / mL, 9×10 7 CFU / mL, 1×10 8 CFU / mL, 2×10 8 CFU / mL, 3×10 8 CFU / mL, 4×10 8 CFU / mL, 5×10 8 CFU / mL, 6×10 8 CFU / mL, 7×10 8 CFU / mL, 8×10 8 CFU / mL, 9×10 8 CFU / mL, 1×10 9 CFU / mL, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0019] In a second aspect, the present invention provides an application of the method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to the first aspect in oil production.

[0020] In a third aspect, the present invention provides an application of the method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to the first aspect in emulsifying and degrading crude oil.

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

[0022] In oilfield development technology, the pollution of the environment caused by chemical flooding is becoming increasingly prominent. Research on improving oil recovery by using green, low-carbon and environmentally optimized oil recovery agents is urgently needed. Due to the oligotrophic environment in the reservoir, surface-producing active microorganisms are often not the dominant strains in the produced fluid. Therefore, it is beneficial to the development of the reservoir to re-inject functional microorganisms into the reservoir environment after surface fermentation. The strains of biosurfactants produced in the reservoir can cooperate with the communities in the endogenous environment of the reservoir to improve the effect of emulsification and degradation of crude oil, provide a new generation of biorecovery products for tertiary oil recovery technology, and further achieve cost reduction and efficiency improvement in oil extraction.

[0023] Under the same culture time, culture system and culture method, the degradation effect of crude oil is significantly improved after the surface active microorganisms are re-injected into the oil reservoir environment through surface fermentation, and the interfacial tension is reduced. The present invention also creatively finds that the strains isolated from the oil field can cooperate with the original microbial community in the emulsification and degradation of crude oil after enrichment culture and reinjection into the oil field produced fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the emulsification degradation result of Taueella WL-2.

[0025] Figure 2 This is the emulsification degradation result of Pseudomonas aeruginosa WL-4.

[0026] Figure 3 This is the emulsification degradation result of Bacillus subtilis WL-5.

[0027] Figure 4 This is the emulsification degradation result diagram of the control group. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0029] The Thauera propionica WL-2 strain involved in the following content is deposited at the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.24606, the deposit date is March 30, 2022, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0030] The Pseudomonas aeruginosa WL-4 strain involved in the following content is deposited at the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC No. 24711, the deposit date is April 18, 2022, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] The Bacillus subtilis WL-5 strain involved in the following content is deposited at the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.24712, the deposit date is April 18, 2022, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] The Xanthomonas campestris ATCC 11672 strain involved in the following content was purchased from Guangdong Microbial Culture Collection Center.

[0033] Preparation of produced fluid culture medium (C medium): Using crude oil from Daqing Oilfield as the sole carbon source, add 0.5 g of crude oil to 50 mL of unsterilized produced fluid.

[0034] Preparation of culture medium in sterilized produced fluid (CM medium): Using crude oil from Daqing Oilfield as the sole carbon source, add 0.5 g of crude oil to 50 mL of sterilized produced fluid.

[0035] Example 1

[0036] (1) Take out the Thauera propionica WL-2 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 h. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 h. Collect the bacterial solution and adjust the viable bacterial count to 10. 7 CFU / mL, and WL-2 culture medium was obtained;

[0037] (2) Inoculate 100 μL of WL-2 culture medium into C medium and culture at 30°C and 150 rpm for 14 days.

[0038] The emulsification degradation results are as follows Figure 1 shown.

[0039] Example 2

[0040] (1) Take out the Pseudomonas aeruginosa WL-4 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 h. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 h. Collect the bacterial solution and adjust the viable count to 10. 7 CFU / mL, WL-4 culture fluid was obtained;

[0041] (2) Inoculate 100 μL of WL-4 culture medium into C medium and culture at 30°C and 150 rpm for 14 days.

[0042] The emulsification degradation results are as follows Figure 2 shown.

[0043] Example 3

[0044] (1) Take out the Bacillus subtilis WL-5 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 hours. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 hours. Collect the bacterial solution and adjust the viable bacterial count to 10. 7 CFU / mL, WL-5 culture medium was obtained;

[0045] (2) Inoculate 100 μL of WL-5 culture medium into C medium and culture at 30°C and 150 rpm for 14 days.

[0046] The emulsification degradation results are as follows Figure 3 shown.

[0047] Example 4

[0048] (1) Take out the Thauera propionica WL-2 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 h. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 h. Collect the bacterial solution and adjust the viable bacterial count to 10. 7 CFU / mL, and WL-2 culture medium was obtained;

[0049] (2) Inoculate 100 μL of WL-2 culture medium into CM medium and culture at 30°C and 150 rpm for 14 days.

[0050] The emulsification degradation results are as follows Figure 1 shown.

[0051] Example 5

[0052] (1) Take out the Pseudomonas aeruginosa WL-4 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 h. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 h. Collect the bacterial solution and adjust the viable count to 10. 7 CFU / mL, WL-4 culture fluid was obtained;

[0053] (2) Inoculate 100 μL of WL-4 culture medium into CM medium and culture at 30°C and 150 rpm for 14 days.

[0054] The emulsification degradation results are as follows Figure 2 shown.

[0055] Example 6

[0056] (1) Take out the Bacillus subtilis WL-5 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 hours. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 hours. Collect the bacterial solution and adjust the viable bacterial count to 10. 7 CFU / mL, WL-5 culture medium was obtained;

[0057] (2) Inoculate 100 μL of WL-5 culture medium into CM medium and culture at 30°C and 150 rpm for 14 days.

[0058] The emulsification degradation results are as follows Figure 3 shown.

[0059] Example 7

[0060] (1) Thauera propionica WL-2 strain, Pseudomonas aeruginosa WL-4 strain and Bacillus subtilis WL-5 strain stored at -80°C were taken out and spread on LB agar medium for culture, and then cultured at 30°C for 48 hours; single colonies were picked and inoculated into 50 mL LB liquid culture medium, respectively, and cultured at 30°C and 150 rpm for 20 hours, and the bacterial solution was collected and the number of viable bacteria was adjusted to 10 7 CFU / mL, and WL-2 culture fluid, WL-4 culture fluid, and WL-5 culture fluid were obtained;

[0061] (2) 60 μL of WL-2 culture medium, 20 μL of WL-4 culture medium, and 20 μL of WL-5 culture medium were inoculated into C medium and cultured at 30°C and 150 rpm for 14 days.

[0062] Example 8

[0063] (1) Take out Thauera propionica WL-2 strain and Pseudomonas aeruginosa WL-4 strain stored at -80°C, spread them on LB agar medium, and culture them at 30°C for 48 hours; pick single colonies and inoculate them into 50 mL LB liquid culture medium, culture them at 30°C and 150 rpm for 20 hours, collect the bacterial solution and adjust the viable bacterial count to 10 7 CFU / mL, WL-2 culture medium and WL-4 culture medium were obtained;

[0064] (2) 75 μL of WL-2 culture medium and 25 μL of WL-4 culture medium were inoculated into C medium and cultured at 30°C and 150 rpm for 14 days.

[0065] Example 9

[0066] (1) Thauera propionica WL-2 strain and Bacillus subtilis WL-5 strain stored at -80°C were taken out and spread on LB agar medium for culture, and then cultured at 30°C for 48 h; a single colony was picked and inoculated into 50 mL LB liquid medium, respectively, and cultured at 30°C and 150 rpm for 20 h, and the bacterial solution was collected and the number of viable bacteria was adjusted to 10 7 CFU / mL, WL-2 culture medium and WL-5 culture medium were obtained;

[0067] (2) 75 μL of WL-2 culture medium and 25 μL of WL-5 culture medium were inoculated into C medium and cultured at 30°C and 150 rpm for 14 days.

[0068] Example 10

[0069] (1) Pseudomonas aeruginosa WL-4 strain and Bacillus subtilis WL-5 strain stored at -80°C were taken out and spread on LB agar medium for culture, and then cultured at 30°C for 48 h; single colonies were picked and inoculated into 50 mL LB liquid culture medium, respectively, and cultured at 30°C and 150 rpm for 20 h. The bacterial liquid was collected and the number of viable bacteria was adjusted to 10 7 CFU / mL, WL-4 culture medium and WL-5 culture medium were obtained;

[0070] (2) 50 μL of WL-4 culture medium and 50 μL of WL-5 culture medium were inoculated into medium C and cultured at 30°C and 150 rpm for 14 days.

[0071] Comparative Example 1

[0072] 50 μL of LB liquid medium was added to the C medium and placed at 30°C and 150 rpm for 14 days.

[0073] The emulsification degradation results are as follows Figure 4 shown.

[0074] Comparative Example 2

[0075] 50 μL of LB liquid medium was added to the CM medium and placed at 30°C and 150 rpm for 14 days.

[0076] The emulsification degradation results are as follows Figure 4 shown.

[0077] Comparative Example 3

[0078] (1) Take out the wild Xanthomonas campestris ATCC 11672 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 hours. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 hours. Collect the bacterial solution and adjust the viable bacterial count to 10. 7 CFU / mL, and ATCC 11672 culture fluid was obtained;

[0079] (2) Inoculate 100 μL of ATCC 11672 culture medium into medium C and culture at 30°C and 150 rpm for 14 days.

[0080] Comparative Example 4

[0081] (1) Take out the wild Xanthomonas campestris ATCC 11672 strain stored at -80°C and spread it on LB agar medium for culture. Incubate it at 30°C for 48 hours. Pick a single colony and inoculate it into 50 mL LB liquid medium. Incubate it at 30°C and 150 rpm for 20 hours. Collect the bacterial solution and adjust the viable bacterial count to 10. 7 CFU / mL, and ATCC 11672 culture fluid was obtained;

[0082] (2) Inoculate 100 μL of ATCC 11672 culture medium into CM medium and culture at 30°C and 150 rpm for 14 days.

[0083] Test Example 1

[0084] Interfacial tension testing

[0085] The interfacial tension change of oil droplets in bacterial solution was evaluated using a spinning drop interfacial tension tester (model: TEXAS-500; origin: USA). The rotation speed was 5000rpm, the temperature was 45℃, and the density difference was 0.15g / cm3 (the density difference is the difference between the density of crude oil and the density of water, which is input into the instrument when calculating the interfacial tension. The density difference refers to the reservoir temperature and crude oil density of Daqing Oilfield)

[0086] The results are shown in Table 1.

[0087] Table 1

[0088]

[0089] As can be seen from the data in Table 1, the method of emulsifying crude oil described in the present invention can improve the effect of crude oil emulsification degradation. WL-2, WL-4 and WL-5 are strains that can produce biosurfactants isolated from Daqing Oilfield. After fermentation, they are added to the produced fluid and can cooperate with the microorganisms in the produced fluid of the reservoir. The effect is better than the effect of the two acting alone, and only the strains of the same reservoir have this effect after being connected back. The injection of the fermentation liquid of the strain that can produce biosurfactants separated and purified from other reservoirs will not improve the effect of emulsification degradation. And the strains WL-2, WL-4 and WL-5 that can produce biosurfactants can cooperate with each other to improve the effect of emulsification degradation.

[0090] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the method for emulsifying and degrading crude oil by using endogenous bacteria in oil reservoirs and its application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0091] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0092] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs, characterized in that: The method comprises: (1) fermenting and culturing a strain capable of producing a biosurfactant separated and purified from an oil reservoir to obtain a fermentation liquid; (2) Injecting the fermentation liquid into the crude oil reservoir.

2. The method for emulsifying and degrading crude oil by using endogenous bacteria in oil reservoirs according to claim 1, characterized in that: The strain includes any one or a combination of at least two of Thauera propionica WL-2 strain with a deposit number of CGMCC No. 24606, Pseudomonas aeruginosa WL-4 strain with a deposit number of CGMCC No. 24711, or Bacillus subtilis WL-5 strain with a deposit number of CGMCC No. 24712.

3. The method for emulsifying and degrading crude oil by using endogenous bacteria in oil reservoirs according to claim 1 or 2, characterized in that: The strain is a combination of Thauera propionica WL-2 strain with a deposit number of CGMCC No.24606, Pseudomonas aeruginosa WL-4 strain with a deposit number of CGMCC No.24711 and Bacillus subtilis WL-5 strain with a deposit number of CGMCC No.24712.

4. The method for emulsifying and degrading crude oil by using endogenous bacteria in oil reservoirs according to claim 3, characterized in that: The ratio of the number of viable bacteria of the WL-2 strain, the WL-4 strain and the WL-5 strain is (1-3):(1-3):(1-3).

5. The method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to any one of claims 1 to 4, characterized in that: The fermentation temperature is 28-45°C.

6. The method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to any one of claims 1 to 5, characterized in that: The fermentation culture time is 12-36h.

7. The method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to any one of claims 1 to 6, characterized in that: The inoculation amount of the fermentation liquid is 0.5%-1.5%.

8. The method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to any one of claims 1 to 7, characterized in that: The viable count of the fermentation broth was 1×10 7 -1×10 9 CFU / mL.

9. Use of the method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to any one of claims 1 to 8 in oil production.

10. Use of the method for emulsifying and degrading crude oil using endogenous bacteria in oil reservoirs according to any one of claims 1 to 8 in emulsifying and degrading crude oil.

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