Bacillus licheniformis with emulsifying effect and application thereof

Abstract

The invention relates to bacillus licheniformis with an emulsifying effect and application of the bacillus licheniformis. The bacillus licheniformis is named as a ZY-1 strain, the preservation time is January 8, 2024, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.29582, and the bacillus licheniformis is classified and named as bacillus licheniformis. The bacillus licheniformis ZY-1 provided by the invention has a good emulsification effect on crude oil, a preparation of the bacillus licheniformis ZY-1 can generate a macromolecular protein emulsifier, tackifying is realized while emulsification is performed, and a good application foundation is laid for microorganism on-site oil displacement.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to a bacillus licheniformis with emulsification and application thereof. BACKGROUND

[0002] With the development of industry and economy, the demand for petroleum products has greatly increased. How to improve the oil recovery economically and environmentally is the focus of the current oil development industry. After the first oil recovery using natural energy and the second oil recovery by water injection, a large amount of crude oil remains in the formation. The third oil recovery using physical, chemical and biological methods to develop this part of the oil has become an important technical means for improving the recovery in China. At present, the development situation of oilfields has entered a difficult development stage, and the demand for development of heavy oil and heavy oil reservoirs has increased. The primary recovery rate of this type of reservoir is generally low, and the serious viscous fingering and early water breakthrough caused by the unfavorable mobility ratio make it difficult for water flooding to have an effect. The injection of oil displacement agents with emulsification effect will provide great potential for the development of such reservoirs.

[0003] Microbial enhanced oil recovery is a rapidly developing technology for improving oil recovery at home and abroad, and is also a high-tech biological technology in the 21st century. Compared with other tertiary recovery technologies, microbial oil recovery technology has the characteristics of wide application range, simple process, good economic benefit and no pollution, and thus is more and more valued by people, and is a tertiary oil recovery technology with good development prospect. As a technology for solving the development problems of difficult-to-develop reservoirs and secondary oil recovery residual reservoirs, the main oil recovery mechanisms of microbial oil recovery technology include the emulsification and degradation of microbial cells and metabolic products, the production of biogas to increase formation pressure and reduce oil viscosity, the production of acid to increase the permeability of carbonate formations, the production of biosurfactants to reduce oil-water interfacial tension and emulsify oil, and the action of other oil displacement substances.

[0004] As CN118086097A discloses a kind of high-efficiency petroleum viscosity-reducing strain and its application, the bacteria liquid prepared by using the strain can be applied in low-permeability heavy oil reservoir oil production, its preparation method is as follows: in culture medium, according to the inoculation amount of 10-40% of culture medium volume, add Bacillus licheniformis CY-121 with OD600 value of 1-2, oscillation reaction is carried out at 26-45 DEG C for 12-24h, and seed liquid is prepared;In fermentation medium, inoculate seed liquid according to 1-3% of fermentation medium volume, and the oscillation reaction time is 2d-4d, limit high-efficiency petroleum viscosity-reducing bacteria liquid;The high-efficiency petroleum viscosity-reducing strain is Bacillus licheniformis with the ability of degrading and viscosity-reducing heavy oil, can grow in high-concentration heavy oil environment, can degrade total petroleum hydrocarbon, and viscosity-reducing rate can reach more than 80%, and degradation rate can reach 50-80%;Using its degradation and viscosity-reducing heavy oil total petroleum hydrocarbon, compared with physical and chemical method, the cost is low, and there is no secondary pollution.Therefore, the screening and cultivation of high-efficiency functional bacteria is the most critical and fundamental problem for the efficient implementation of microbial oil production.

[0005] The microorganisms reported for microbial oil displacement are mainly biosurfactant-producing bacteria such as Pseudomonas aeruginosa and Bacillus subtilis. The functional bacteria for microbial emulsification oil displacement are insufficient, and it is still necessary to continue to expand the category of microbial oil production bacteria to provide new strains and genetic materials for microbial oil production technology. SUMMARY

[0006] To solve the above technical problems, the present application provides a Bacillus licheniformis with emulsifying effect and its application. The Bacillus licheniformis ZY-1 has good emulsifying effect on crude oil. The bacterial agent prepared from the strain can produce macromolecular protein emulsifiers, which can increase the viscosity of the system while emulsifying, thereby achieving the effect of viscosity increase and laying a good application foundation for microbial on-site oil displacement.

[0007] To achieve this purpose, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a Bacillus licheniformis with emulsifying effect, which is named ZY-1 strain, and has a preservation time of January 8, 2024, a preservation number of CGMCC No.29582, and a classification name of Bacillus licheniformis.

[0009] A new Bacillus licheniformis capable of emulsification and viscosity increase is isolated from produced fluid in a formation of a Jimsar block in Xinjiang Uygur Autonomous Region Oilfield, named ZY-1 strain, classified as Bacillus licheniformis, preserved in China General Microbiological Culture Collection Center, preserved on January 8, 2024, with a preservation number of CGMCC No.29582 and an address of No.3, Beichen West Road, Chaoyang District, Beijing. The strain has excellent emulsification capacity, and the bacterial agent obtained from the strain has emulsification effect and simultaneously increases the viscosity of the system, is suitable for most types of oil reservoirs, has a wide application range, and has a good application prospect in oil development.

[0010] The screening step of the ZY-1 strain involved in the present application is as follows:

[0011] Centrifuge 20 mL of formation water at 8000 rpm for 1 min, carefully suck off 12 mL of supernatant, mix the remaining 8 mL of formation water, take 100 μL of liquid and uniformly coat it on the formation water screening plate, cultivate at 37 DEG C for 2-3 days, pick single colonies on the plate and inoculate into the formation water culture medium, carry out subculture, observe the emulsification effect on crude oil, further streak the bacterial strain with good emulsification effect on the LB plate, cultivate at 37 DEG C, determine the emulsification index and growth curve, and further screen the bacterial strain with the optimal effect for performance evaluation;

[0012] The formation water culture medium formula and preparation method are as follows: the volume ratio of Jimsar injection water to produced fluid is 3:7-7:3, 0.2% sucrose, 0.35% sodium nitrate, 0.7% disodium hydrogen phosphate, 2% crude oil is added, sterilized, and the natural pH is obtained.

[0013] The preparation method of the formation water screening plate is that 1.5% agar is added to the formation water culture medium to prepare the formation water screening plate.

[0014] The Bacillus licheniformis ZY-1 has the following characteristics:

[0015] Gram-positive bacteria, thermophilic bacteria. After cultivation for 48-60 h, the bacterial body is rod-shaped with a size of 0.8-1.0 μm x 5-20 μm, is not motile, and has a typical eight-shaped arrangement characteristic. On the LB solid culture medium, it is milky white, the colony is round, relatively viscous, opaque, convex, and glossy. It can grow at a temperature range of 15-58 DEG C and a pH range of 5-10.

[0016] Preferably, the gene sequence of the Bacillus licheniformis is shown as SEQ ID NO. 1.

[0017] In a second aspect, the present application provides a fermentation broth of Bacillus licheniformis, which is obtained by fermentation of the Bacillus licheniformis with emulsifying effect according to the first aspect.

[0018] In a third aspect, the present application provides a preparation method of the fermentation broth according to the second aspect, which comprises the following steps:

[0019] Inoculating the seed liquid of the strain of Bacillus licheniformis into a fermentation medium for culture to obtain the fermentation broth.

[0020] Preferably, the fermentation medium comprises the following components in mass content: sucrose 0.3-8% (for example, it can be 0.5%, 1%, 2%, 3%, 4%, 5%, 6% or 7%, etc.), sodium nitrate 0.5-1.0% (for example, it can be 0.6%, 0.7%, 0.8% or 0.9%, etc.), disodium hydrogen phosphate dodecahydrate 0.3-2.5% (for example, it can be 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2% or 2.2%, etc.), potassium dihydrogen phosphate 0-0.2% (for example, it can be 0.01%, 0.05%, 0.08%, 0.1%, 0.12%, 0.15% or 0.18%, etc.), magnesium sulfate 0-0.12% (for example, it can be 0.01%, 0.02%, 0.05%, 0.08% or 0.1%, etc.), yeast powder 0-0.15% (for example, it can be 0.01%, 0.02%, 0.05%, 0.08%, 0.1% or 0.12%, etc.), and the balance is water, with pH being 6-8 (for example, it can be 6.2, 6.5, 6.8, 7, 7.2, 7.5 or 7.8, etc.).

[0021] Preferably, the fermentation medium comprises the following components in mass content: sucrose 7%, sodium nitrate 0.7%, disodium hydrogen phosphate dodecahydrate 2.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1125%, yeast powder 0.15%, and the balance is water, with pH being 7.0.

[0022] Preferably, the culture temperature is 35-50°C, for example, it can be 38°C, 40°C, 42°C, 45°C or 48°C, etc.

[0023] Preferably, the culture time is 72-96h, for example, it can be 76h, 80h, 84h, 88h or 92h, etc.

[0024] Preferably, the inoculation amount of the seed liquid is 0.5-5% in mass content, for example, it can be 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 3% or 4%, etc.

[0025] Preferably, the seed liquid is obtained by inoculating the Bacillus licheniformis with emulsifying effect as described in the first aspect into a seed culture medium and culturing at 36-38℃ (for example, it can be 36.2℃, 36.5℃, 36.8℃, 37℃, 37.2℃, 37.5℃ or 37.8℃, etc.) for 10-15h (for example, it can be 10.5h, 11h, 12h, 13h or 14h, etc.).

[0026] Preferably, the seed culture medium comprises beef extract proteose peptone medium or LB medium.

[0027] Preferably, the seed liquid is cultured in a constant temperature shaker.

[0028] Preferably, the seed liquid is cultured at a rotation speed of 100-150rpm, for example, it can be 105rpm, 110rpm, 120rpm, 130rpm or 140rpm, etc.

[0029] Preferably, the fermentation liquid is cultured in a constant temperature shaker.

[0030] Preferably, the fermentation liquid is cultured at a rotation speed of 150-250rpm, for example, it can be 160rpm, 170rpm, 180rpm, 200rpm or 220rpm, etc.

[0031] In a fourth aspect, the present application provides a microbial inoculant with emulsifying effect, wherein the strain in the microbial inoculant comprises the Bacillus licheniformis with emulsifying effect as described in the first aspect.

[0032] Preferably, the dosage form of the microbial inoculant comprises granules, solution or powder.

[0033] Preferably, in the microbial inoculant, the viable count of the Bacillus licheniformis is not less than 1×10 5 CFU / mL, for example, it can be 1.5×10 5 CFU / mL, 2×10 5 CFU / mL, 3×10 5 CFU / mL, 4×10 5 CFU / mL, 5×10 5 CFU / mL, 6×10 5 CFU / mL, 8×10 5 CFU / mL, 1×10 6 CFU / mL or 1×10 7 CFU / mL, etc.

[0034] In a fifth aspect, the present application provides a bacillus licheniformis with emulsification as described in the first aspect, a fermentation broth of the bacillus licheniformis as described in the second aspect, or a microbial inoculant with emulsification as described in the fourth aspect, for use in microbial oil displacement and / or oil emulsification.

[0035] Compared with the prior art, the present application has at least the following beneficial effects:

[0036] The bacillus licheniformis ZY-1 provided by the present application has emulsification, and after the prepared fermentation broth is vortexed and oscillated with crude oil in the Xinjiang Uygur Autonomous Region and then left standing, a clear and stable emulsion layer can be observed, and there is no obvious stratification in the emulsion layer, and the emulsification effect is good. At the same time, the biological preparation (for example, the fermentation broth thereof) containing the strain can produce macromolecular protein emulsifiers, which can increase the viscosity of the system while emulsifying, and has excellent viscosity-increasing effect, thereby laying a foundation for the application of microbial oil displacement on site. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a microscope picture of the bacillus licheniformis ZY-1 in Example 2;

[0038] Figure 2 It is a colony picture of the bacillus licheniformis ZY-1 in Example 2;

[0039] Figure 3 It is the emulsification effect of the fermentation broth of the bacillus licheniformis ZY-1 in Example 5, which is obtained by culturing at 37℃ using an emulsification medium, after being vortexed with crude oil and then left standing for 60 minutes.

[0040] The ZY-1 strain involved in the present application is classified and named as bacillus licheniformis, and is preserved in the China General Microbiological Culture Collection Center, with a preservation time of January 8, 2024, a preservation number of CGMCC No.29582, and an address of No.3, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be further described below by combining the drawings and through specific embodiments. However, the following examples are only simple examples of the present application, and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0042] The ZY-1 strain involved in the following examples is classified and named as bacillus licheniformis, and is preserved in the China General Microbiological Culture Collection Center, with a preservation time of January 8, 2024, a preservation number of CGMCC No.29582, and an address of No.3, Beichen West Road, Chaoyang District, Beijing.

[0043] Example 1

[0044] A bacillus licheniformis ZY-1 is screened by the following method:

[0045] After 20 mL of formation water of Xinjiang Uygur Autonomous Region Oilfield is centrifuged at 8000 rpm / min for 1 min, 12 mL of supernatant is carefully sucked off, the remaining 8 mL of formation water is mixed, 100 μL of the mixture is uniformly coated on a formation water screening plate, and the plate is cultured at 37℃ for 2 d; a single colony on the plate is picked and inoculated into a formation water culture medium for subculture, the emulsification effect on crude oil is observed, and the strain with good emulsification effect is further streaked on an LB plate and cultured at 37℃; the emulsification index and growth curve are measured, and the bacillus licheniformis with good emulsification effect is selected as the screened bacillus licheniformis ZY-1.

[0046] The formula and preparation method of the formation water culture medium are as follows: 5:5 (by volume) of Jimsar injection water and formation produced liquid, 0.2% of sucrose, 0.35% of sodium nitrate, 0.7% of disodium hydrogen phosphate, 2% of crude oil, sterilization, and natural pH.

[0047] The preparation method of the formation water screening plate is as follows: 1.5% of agar is added to the formation water culture medium to prepare the formation water screening plate.

[0048] Example 2

[0049] The strain screened in Example 1 is identified as follows in this example:

[0050] 1) Microscopic identification

[0051] After the obtained strain is picked out and cultured in an LB medium at 120 rpm / min and 37℃ for 12 h, gram staining is performed, and the result is shown in Table 1. Figure 1 The size of the strain cell is about 0.8 μm x (1.5-3.5) μm, is rod-shaped and single, and shows that the strain is gram-positive bacteria.

[0052] 2) Plate colony identification

[0053] The obtained colony is single-streaked, and the result is shown in Table 2. Figure 2 The colony formed on the culture medium is smooth, round, convex, light yellow in color, and rough and wrinkled on the surface.

[0054] 3) Molecular biology identification

[0055] The obtained colonies were identified as *Bacillus licheniformis* by Biolog fingerprinting. The genome of the strain was extracted, and PCR amplification was performed using universal bacterial primers. The amplification products were then sequenced for identification. The 16S rDNA sequence of this strain is shown in SEQ ID No:1.

[0056] Example 3

[0057] This example evaluates the temperature resistance of the ZY-1 strain obtained in Example 1:

[0058] Bacillus licheniformis ZY-1 was inoculated into LB medium and cultured in a shaker at 120 rpm and 37°C for 12 h to obtain the seed culture of strain ZY-1.

[0059] Prepare 400 mL of LB medium solution, dispense 50 mL into eight 250 mL Erlenmeyer flasks, and inoculate each flask with 2.5 mL of ZY-1 strain seed culture. Incubate at 35℃, 45℃, 50℃, and 55℃ at 150 rpm, with two replicates for each temperature. OD values ​​(600 nm wavelength) are continuously monitored, and the OD values ​​of the bacterial cultures are summarized in Table 1. The OD values ​​of the strains at different temperature gradients reflect their growth status. Comparison of the data in Table 1 shows that the strains grow best at temperatures ≤50℃, and the highest biomass is observed at 45℃. The optimal culture temperature for the strains is 45℃.

[0060] Table 1

[0061]

[0062] Example 4

[0063] This embodiment evaluates the emulsification and thickening properties of the ZY-1 fermentation broth.

[0064] The ZY-1 fermentation broth was cultured at different temperatures using the following three fermentation media:

[0065] Culture medium 1 (fermentation medium): 7% sucrose, 0.7% sodium nitrate, 2.5% disodium hydrogen phosphate dodecahydrate, 0.2% potassium dihydrogen phosphate, 0.1125% magnesium sulfate, 0.15% yeast extract, and the remainder is distilled water;

[0066] Culture medium 2: 0.35% sucrose, 0.7% sodium nitrate, 0.35% disodium hydrogen phosphate dodecahydrate, with the remainder being distilled water;

[0067] Culture medium 3: sodium nitrate 0.7%, disodium hydrogen phosphate dodecahydrate 0.35%, soybean oil 5%, and the remainder is distilled water.

[0068] The ZY-1 fermentation broth was prepared using the following method:

[0069] After sterilizing the above culture medium at 105℃ for 30 min, ZY-1 strain seed liquid was cultured at different temperatures for 24 hours at an inoculation rate of 5%. After three days of culture, the viscosity and emulsification coefficient were measured.

[0070] The seed culture of strain ZY-1 was obtained by inoculating Bacillus licheniformis ZY-1 into LB medium and culturing it in a constant temperature shaker at 120 rpm and 37°C for 12 h.

[0071] The viscosity was obtained using a DV-Ⅲ viscometer, with a rotor of size 18 measuring at 5 rotation speeds.

[0072] The emulsification coefficient was tested as follows: 5 mL of crude oil and 5 ± 0.8 mL of fermentation broth from different culture media were placed in a 10 mL graduated test tube (the specific amounts added are shown in Table 2). The tube was vortexed for 30 seconds. The water phase precipitation was recorded at 5 min, 10 min, 20 min, 40 min, 60 min, 3 h, 6 h, and 24 h. The less water precipitation, the better the emulsification performance.

[0073] The results of the emulsification coefficient and viscosity tests are summarized in Table 2.

[0074] Table 2

[0075]

[0076]

[0077] Based on the test results in Table 2, it can be concluded that the Bacillus licheniformis ZY-1 provided by this invention has emulsifying properties. By using a specific emulsifying culture medium and culturing at a preferred temperature, the obtained Bacillus licheniformis ZY-1 fermentation broth has a superior emulsifying effect on crude oil. Even after standing for 40 minutes, there is no water separation or stratification phenomenon. At the same time, the viscosity-enhancing effect is better, which can better meet the application needs of oilfield development.

[0078] Example 5

[0079] This embodiment evaluates the emulsification effect of the ZY-1 fermentation broth:

[0080] 70 mL of fermentation broth obtained from the same culture system (37°C and fermentation medium) in Example 4 was mixed with 30 mL of crude oil in a shake flask, vortexed for 30 seconds, and allowed to stand for 60 minutes. The results are as follows: Figure 3 As shown, after the fermentation broth was vortexed and allowed to settle with crude oil from Xinjiang Uygur Autonomous Region, a clear and stable emulsion layer was observed, and there was no obvious stratification within the emulsion layer. This confirms that the ZY-1 strain and its fermentation broth provided by this invention have good emulsification effects and have broad application prospects in microbial enhanced oil recovery or petroleum emulsification.

[0081] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A Bacillus licheniformis having an emulsifying action, characterized by, The bacillus licheniformis is named ZY-1 strain, the preservation time is January 8, 2024, the preservation number is CGMCC No.29582, and the classification name is bacillus licheniformis.

2. The Bacillus licheniformis according to claim 1, characterized in that, The gene sequence of the bacillus licheniformis is shown as SEQ ID NO.

1.

3. A fermentation broth of Bacillus licheniformis, characterized in that, The fermentation liquor of the bacillus licheniformis is obtained by fermentation of the bacillus licheniformis with emulsification effect according to claim 1 or 2.

4. A method for producing a fermentation broth as claimed in claim 3, characterized in that, The preparation method comprises the following steps: inoculating the seed liquid of the bacillus licheniformis strain into a fermentation medium for culture to obtain the fermentation liquor.

5. The production method according to claim 4, characterized by, The fermentation medium comprises the following components in mass content: sucrose 0.3-8%, sodium nitrate 0.5-1.0%, dodecahydrate disodium hydrogen phosphate 0.3-2.5%, potassium dihydrogen phosphate 0-0.2%, magnesium sulfate 0-0.12%, yeast powder 0-0.15%, and the balance is water, and the pH is 6-8; Preferably, the culture temperature is 35-50°C; Preferably, the culture time is 72-96h.

6. The production method according to claim 4 or 5, characterized by, The inoculation amount of the seed liquid is 0.5-5% in mass content; Preferably, the seed liquid is inoculated into a seed culture medium by the bacillus licheniformis with emulsification effect according to claim 1 or 2, and cultured at 36-38°C for 10-15h to obtain; Preferably, the seed culture medium comprises beef extract proteose peptone medium or LB medium.

7. A microbial inoculant having an emulsifying effect, characterized by, The strain in the microbial agent comprises the bacillus licheniformis with emulsification effect according to claim 1 or 2.

8. The microbial inoculant of claim 7, wherein, The dosage form of the microbial agent comprises granules, solution or powder.

9. The microbial inoculant of claim 7 or 8, wherein, In the microbial inoculant, the viable cell count of the Bacillus licheniformis is not less than 1 x 10 5 CFU / mL.

10. Application of the bacillus licheniformis with emulsification effect according to claim 1 or 2, the fermentation liquor of the bacillus licheniformis according to claim 3, or the microbial agent with emulsification effect according to any one of claims 7-9 in microbial oil displacement and / or oil emulsification.

Images