Bacillus subtilis 20P3 LZT, fermentation liquor and application of bacillus subtilis 20P3 LZT in microbial oil recovery

Abstract

The invention relates to the technical field of microbial oil recovery, in particular to bacillus subtilis 20P3 LZT, fermentation liquor and application of the bacillus subtilis 20P3 LZT to microbial oil recovery, the bacillus subtilis 20P3 LZT is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30366. The bacillus subtilis 20P3 LZT is found for the first time, can generate surfactant lipopeptide, can grow on the basis of molasses or corn steep liquor dry powder as a carbon source, has good emulsification and viscosity reduction performance on crude oil, has the emulsification and viscosity reduction rate of 70% on thick oil, can reduce the viscosity of the crude oil and enhance the fluidity of the crude oil, and can be used for preparing crude oil. On one hand, the oil-water fluidity ratio is reduced, and on the other hand, the emulsified crude oil has the effects of profile control and liquid flow diversion, so that the crude oil recovery rate is indirectly improved, and a new strain and a material are provided for a microbial oil recovery technology.

Description

Technical Field

[0001] This invention relates to the field of microbial enhanced oil recovery (MEOR) technology, specifically to Bacillus subtilis 20P3 LZT, its fermentation broth, and its application in MEOR. Background Technology

[0002] In the process of oil recovery, after primary oil recovery using natural energy and secondary oil recovery using water injection, a large amount of crude oil remains in the formation. Tertiary oil recovery, which utilizes physical, chemical, and biological methods to develop this portion of crude oil, has become an important technical means for improving oil recovery rates in my country. Among them, Microbial Enhanced Oil Recovery (MEOR) technology is one of the tertiary oil recovery technologies with great development potential.

[0003] Currently, the microorganisms used are mainly bacteria. These bacteria multiply in the formation and their surfactants interact with crude oil to improve oil recovery. The basic principles are: ① Surfactants, organic solvents, organic acids, biopolymers, or biogases produced by bacteria can alter the fluidity of residual oil in the reservoir; ② The metabolic processes of microorganisms directly change the properties of crude oil, such as degrading some components and causing changes in crude oil properties, making it easier to flow and improving the washing efficiency of residual oil, thereby increasing the recovery rate; ③ Biopolymers produced by bacterial metabolism can block large formation channels, improve formation seepage patterns, and expand the water drive sweep volume. Compared with other tertiary oil recovery technologies, MEOR (Metal-Oil-Retaining) has advantages such as high efficiency, economy, no secondary pollution, and no damage to the oil reservoir, making it the most promising technology for improving oil recovery.

[0004] Currently reported strains used in microbial enhanced oil recovery (DEER) are concentrated in *Pseudomonas aeruginosa*, and the main component of the surfactants produced is rhamnolipid. The variety of strains used in DEER is insufficient, and it is necessary to further expand the range of microbial strains and the types of surfactants they produce. Developing a variety of functional microorganisms with surfactant-producing, emulsifying, and chemotactic properties will provide new strains and materials for DEER technology. Summary of the Invention

[0005] This invention provides Bacillus subtilis 20P3 LZT, fermentation broth, and its application in microbial enhanced oil recovery, overcoming the shortcomings of the prior art. It discloses for the first time that Bacillus subtilis 20P3 LZT can produce surfactant lipopeptides, which have good emulsification and viscosity reduction properties, and can achieve an emulsification and viscosity reduction rate of 70% for heavy oil.

[0006] One of the technical solutions of the present invention is achieved through the following measures: a Bacillus subtilis strain 20P3 LZT, classified as Bacillus subtilis, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30366, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on April 17, 2024.

[0007] The second technical solution of the present invention is achieved through the following measures: a fermentation broth containing Bacillus subtilis 20P3 LZT.

[0008] The following are further optimizations and / or improvements to the second technical solution of the above invention: The above fermentation broth containing Bacillus subtilis 20P3 LZT was obtained by the following method: The first step was to inoculate Bacillus subtilis 20P3 LZT into seed culture medium and culture it to obtain seed solution; The second step involves inoculating a seed culture solution of 4% to 6% by volume into a fermentation medium containing a carbon source for culture, thereby obtaining a fermentation broth containing Bacillus subtilis 20P3 LZT.

[0009] In the first step above, the seed culture medium is LB liquid medium.

[0010] In the first step above, the cultivation is specifically carried out in a shaker with an oscillation culture at a temperature of 35℃ to 37℃, a culture time of 36h to 40h, and an oscillation speed of 120r / min to 150r / min.

[0011] In the second step above, the formula of the carbon-source-containing fermentation medium is as follows: carbon source 30 g / L to 50 g / L, sodium citrate 3 g / L to 5 g / L, ammonium sulfate 0.5 g / L to 1.0 g / L, dipotassium hydrogen phosphate 0.2 g / L to 0.5 g / L, potassium dihydrogen phosphate 0.2 g / L to 0.5 g / L, sodium chloride 0.5 g / L to 1.5 g / L, magnesium sulfate heptahydrate 0.2 g / L to 0.5 g / L, and ferrous sulfate heptahydrate 0.001 g / L to 0.002 g / L. The pH value of the carbon-source-containing fermentation medium is 7.2.

[0012] In the second step above, the formula of the fermentation medium containing carbon source is as follows: carbon source 40 g / L, sodium citrate 3 g / L, ammonium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium chloride 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.001 g / L, and the pH value of the fermentation medium containing carbon source is 7.2.

[0013] In the second step above, the carbon source is either molasses or corn steep liquor powder.

[0014] In the second step mentioned above, the cultivation is specifically carried out in a shaker with an oscillation speed of 100 rpm to 150 rpm, a cultivation temperature of 30℃ to 40℃, and a cultivation time of 30 h to 40 h.

[0015] In the second step above, the concentration of Bacillus subtilis 20P3 LZT in the fermentation broth containing Bacillus subtilis 20P3 LZT is 1×10⁻⁶. 8 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 10 per mL.

[0016] The third technical solution of the present invention is achieved through the following measures: the application of a fermentation broth containing Bacillus subtilis 20P3 LZT in microbial oil recovery.

[0017] This invention marks the first discovery of Bacillus subtilis 20P3 LZT, a strain capable of producing surfactant lipopeptides and growing on molasses or corn steep liquor as a carbon source. This strain exhibits good emulsification and viscosity reduction properties for crude oil, achieving a viscosity reduction rate of up to 70% for heavy oil. It reduces the viscosity of crude oil and enhances its fluidity, thereby lowering the oil-water mobility ratio. Furthermore, the emulsified crude oil plays a role in profile control and flow diversion, indirectly improving oil recovery. This invention provides a new strain and material for microbial enhanced oil recovery technology. Attached Figure Description

[0018] Figure 1 This is a growth curve of Bacillus subtilis 20P3 LZT under different carbon sources.

[0019] Figure 2 This is a bar chart showing the surface tension of the fermentation broth containing Bacillus subtilis 20P3 LZT obtained by fermentation in culture media with different carbon sources according to the present invention. Detailed Implementation

[0020] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solutions and actual conditions of this invention. Unless otherwise specified, all chemical reagents and chemicals mentioned in this invention are well-known and commonly used chemical reagents and chemicals in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; unless otherwise specified, all solutions in this invention are aqueous solutions with water as the solvent, for example, hydrochloric acid solution is an aqueous solution of hydrochloric acid; room temperature in this invention generally refers to a temperature between 15°C and 25°C, generally defined as 25°C.

[0021] The present invention will be further described below with reference to embodiments: Example 1: This Bacillus subtilis 20P3 LZT strain, classified as Bacillus subtilis, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30366. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is April 17, 2024.

[0022] Example 2: The fermentation broth containing Bacillus subtilis 20P3 LZT was obtained according to the following method: The first step was to inoculate Bacillus subtilis 20P3 LZT into seed culture medium and culture it to obtain seed solution; The second step involves inoculating a seed culture solution of 4% to 6% by volume into a fermentation medium containing a carbon source for culture, thereby obtaining a fermentation broth containing Bacillus subtilis 20P3 LZT.

[0023] Example 3: As an optimization of the above example, in the first step, the seed culture medium is LB liquid medium.

[0024] Example 4: As an optimization of the above example, in the first step, the culture is specifically carried out in a shaker with an oscillation culture at a temperature of 35°C to 37°C, a culture time of 36h to 40h, and an oscillation speed of 120r / min to 150r / min.

[0025] Example 5: As an optimization of the above example, in the second step, the formula of the fermentation medium containing carbon source is as follows: carbon source 30 g / L to 50 g / L, sodium citrate 3 g / L to 5 g / L, ammonium sulfate 0.5 g / L to 1.0 g / L, dipotassium hydrogen phosphate 0.2 g / L to 0.5 g / L, potassium dihydrogen phosphate 0.2 g / L to 0.5 g / L, sodium chloride 0.5 g / L to 1.5 g / L, magnesium sulfate heptahydrate 0.2 g / L to 0.5 g / L, and ferrous sulfate heptahydrate 0.001 g / L to 0.002 g / L. The pH value of the fermentation medium containing carbon source is 7.2.

[0026] Example 6: As an optimization of the above example, in the second step, the formula of the fermentation medium containing carbon source is as follows: carbon source 40 g / L, sodium citrate 3 g / L, ammonium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium chloride 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.001 g / L, and the pH value of the fermentation medium containing carbon source is 7.2.

[0027] Example 7: As an optimization of the above example, in the second step, the carbon source is either molasses or corn steep liquor powder.

[0028] Example 8: As an optimization of the above example, in the second step, the culture is specifically carried out in a shaker with an oscillation speed of 100 rpm to 150 rpm, a culture temperature of 30°C to 40°C, and a culture time of 30 h to 40 h.

[0029] Example 9: As an optimization of the above example, in the second step, the concentration of Bacillus subtilis 20P3 LZT in the fermentation broth containing Bacillus subtilis 20P3 LZT is 1×10⁻⁶. 8 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 10 per mL.

[0030] Example 10: The fermentation broth containing Bacillus subtilis 20P3 LZT was obtained according to the following method: The first step was to inoculate Bacillus subtilis 20P3 LZT into seed culture medium (LB liquid medium) and culture it in a shaker at 35℃ and 120 r / min for 36 h to obtain seed liquid; The second step involved inoculating a 4% seed culture solution into a carbon-source-containing fermentation medium and incubating it at 35°C in a shaker at 120 rpm for 36 hours to obtain a concentration of 1×10⁻⁶. 8 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 10 The fermentation broth containing Bacillus subtilis 20P3 LZT was prepared at a concentration of 1 / mL. The carbon source-containing fermentation medium was formulated with the following components: carbon source (molasses) 30 g / L to 50 g / L, sodium citrate 3 g / L to 5 g / L, ammonium sulfate 0.5 g / L to 1.0 g / L, dipotassium hydrogen phosphate 0.2 g / L to 0.5 g / L, potassium dihydrogen phosphate 0.2 g / L to 0.5 g / L, sodium chloride 0.5 g / L to 1.5 g / L, magnesium sulfate heptahydrate 0.2 g / L to 0.5 g / L, and ferrous sulfate heptahydrate 0.001 g / L to 0.002 g / L. The pH of the carbon source-containing fermentation medium was 7.2.

[0031] Example 11: The fermentation broth containing Bacillus subtilis 20P3 LZT was obtained according to the following method: The first step was to inoculate Bacillus subtilis 20P3 LZT into seed culture medium (LB liquid medium) and culture it in a shaker at 35℃ and 120 r / min for 36 h to obtain seed liquid; The second step involves inoculating a 5% seed culture solution into a carbon-source-containing fermentation medium and incubating it at 35°C in a shaker at 120 rpm for 36 hours to obtain a concentration of 1×10⁻⁶. 8 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 10The fermentation broth containing Bacillus subtilis 20P3 LZT was prepared at a concentration of 1 / mL. The carbon source-containing fermentation medium was formulated as follows: carbon source (corn steep liquor dry powder) 30 g / L to 50 g / L, sodium citrate 3 g / L to 5 g / L, ammonium sulfate 0.5 g / L to 1.0 g / L, dipotassium hydrogen phosphate 0.2 g / L to 0.5 g / L, potassium dihydrogen phosphate 0.2 g / L to 0.5 g / L, sodium chloride 0.5 g / L to 1.5 g / L, magnesium sulfate heptahydrate 0.2 g / L to 0.5 g / L, and ferrous sulfate heptahydrate 0.001 g / L to 0.002 g / L. The pH of the carbon source-containing fermentation medium was 7.2.

[0032] Example 12: The fermentation broth containing Bacillus subtilis 20P3 LZT was obtained according to the following method: The first step was to inoculate Bacillus subtilis 20P3 LZT into seed culture medium (LB liquid medium) and culture it in a shaker at 35℃ and 120 r / min for 36 h to obtain seed liquid; The second step involved inoculating a 6% seed culture solution into a carbon-source-containing fermentation medium and incubating it at 35°C in a shaker at 120 rpm for 36 hours to obtain a concentration of 1×10⁻⁶. 8 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 10 The fermentation broth containing Bacillus subtilis 20P3 LZT was prepared at a concentration of 1 / mL. The carbon source-containing fermentation medium was formulated with the following components: carbon source (molasses) 30 g / L to 50 g / L, sodium citrate 3 g / L to 5 g / L, ammonium sulfate 0.5 g / L to 1.0 g / L, dipotassium hydrogen phosphate 0.2 g / L to 0.5 g / L, potassium dihydrogen phosphate 0.2 g / L to 0.5 g / L, sodium chloride 0.5 g / L to 1.5 g / L, magnesium sulfate heptahydrate 0.2 g / L to 0.5 g / L, and ferrous sulfate heptahydrate 0.001 g / L to 0.002 g / L. The pH of the carbon source-containing fermentation medium was 7.2.

[0033] Experimental Example 1: Screening and identification of bacterial strains.

[0034] Bacillus subtilis 20P3 LZT was isolated from the produced fluid of an oil field that had long been injecting nitrate solution into the reservoir to carry out microbial oil recovery.

[0035] The specific steps for strain screening are as follows: 10 ml of oilfield produced fluid was inoculated into a 250 ml shake flask containing 100 mL of sterile enrichment medium, and cultured in a constant temperature incubator until the crude oil showed emulsification and dispersion. The enrichment medium consisted of the following components: diammonium hydrogen phosphate 0 g / L to 1 g / L, sodium nitrate 0 g / L to 3 g / L, ferrous sulfate 0 g / L to 1 g / L, magnesium sulfate hexahydrate 0 g / L to 1 g / L, calcium chloride 0.01 g / L, yeast extract 0.01 g / L, molasses 1 g / L to 10 g / L, and crude oil 1 g / L to 10 g / L. The pH was adjusted to 6 to 8, and the mixture was autoclaved at 121℃ for 20 min.

[0036] Take 5 mL of the enriched culture medium and inoculate it again into a new sterile enriched culture medium. Incubate in a constant temperature incubator until the crude oil is emulsified and dispersed. Isolate single bacteria using the dilution and plating method. Select the strain with the largest oil extrusion zone using the oil extrusion zone method and name it 20P3LZT.

[0037] The genome of the strain was extracted using a genome extraction kit. The 16S rRNA gene of the strain was obtained by PCR amplification using primers 27f (5´-AGA GTT TGA TCC TGGCTC AG-3´) and 1492r (5´-GGT TAC CTT GTTACG ACT T-3´). The PCR product was then sequenced to obtain the 16S rRNA sequence. The 16S rRNA sequence of strain 20P3 LZT was submitted to GenBank. Based on BLAST search and homology analysis, the 16S rRNA sequence is shown in SEQ ID NO:1. The 16S rRNA sequence of strain 20P3 LZT shows 99% similarity to the 16S rRNA sequence of Bacillus subtilis, indicating that this bacterium is Bacillus subtilis, and it was named Bacillus subtilis 20P3 LZT.

[0038] This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 30366.

[0039] Experimental Example 2: Detection of the lipopeptide gene of Bacillus subtilis 20P3 LZT.

[0040] Using the genome of Bacillus subtilis 20P3 LZT as a template, the lipopeptide production gene sequence fragment of the strain was obtained by PCR amplification using specific primers srfAf (5′-CAAAAKCGCAKCATACCAAKTTGAG-3′) and srfAr (5′-AGCGGCAYATATTGATGCGGYTC-3′). The sequenced data was submitted to the GenBank database. According to the results of BLAST search and homology analysis, the sequence of srfA of strain 20P3 LZT is shown in SEQ ID NO:2. srfA is a lipopeptide surfactant synthase gene, indicating that Bacillus subtilis possesses a lipopeptide synthesis gene and is capable of producing surfactant lipopeptides.

[0041] Experiment 3: Investigating the growth of Bacillus subtilis 20P3 LZT in different carbon sources.

[0042] Experimental method: Bacillus subtilis 20P3 LZT strain was inoculated into 50 ml of LB liquid medium and cultured overnight. The culture was then centrifuged at 4000 r / min for 5 min, the supernatant was discarded, and the bacterial cells that had settled to the bottom of the centrifuge tube were resuspended in sterile physiological saline. The culture was centrifuged at 4000 r / min for 5 min, the supernatant was discarded, and 50 mL of sterile physiological saline was added again to suspend the bacterial cells as seed culture.

[0043] The seed culture was inoculated at a rate of 1% into enrichment media containing different carbon sources and cultured at 35°C. During the culture period, an appropriate amount of culture solution was taken at regular intervals and the absorbance was measured using a spectrophotometer to plot a time-absorbance curve. The different carbon sources included molasses, corn steep liquor powder, sucrose, glucose, acetate, and ethanol. The formulation of the fermentation medium containing carbon sources was the same as that in Example 12 of this invention.

[0044] Experimental results: The growth curves of Bacillus subtilis 20P3 LZT under different carbon sources are shown below. Figure 1 As shown, by Figure 1 It can be seen that Bacillus subtilis 20P3 LZT can grow and reproduce in different carbon sources and has potential application value. Among them, the best carbon sources are molasses and corn steep liquor powder.

[0045] Experiment Example 4: Investigating the surface tension of Bacillus subtilis 20P3 LZT under different carbon sources.

[0046] Experimental methods: The surface tension of the fermentation broth containing Bacillus subtilis 20P3 LZT prepared in Example 12 of this invention was measured. At the same time, the carbon source (molasses) in the carbon source-containing fermentation medium in Example 12 of this invention was replaced by corn steep liquor powder, sucrose, glucose, sodium acetate and ethanol, respectively, to obtain fermentation broth containing Bacillus subtilis 20P3 LZT cultured in fermentation mediums with different carbon sources. The surface tension of each fermentation broth containing Bacillus subtilis 20P3 LZT was measured.

[0047] Experimental Results: The surface tension histograms of fermentation broths containing Bacillus subtilis 20P3 LZT obtained by fermentation in media with different carbon sources are shown below. Figure 2 As shown, by Figure 2 It is known that when molasses, sucrose, glucose, and corn steep liquor powder are used as carbon sources, the surface tension of the fermentation broth containing Bacillus subtilis 20P3 LZT can be reduced from 58 mN / m before inoculation to about 30 mN / m. This indicates that the Bacillus subtilis 20P3 LZT of this invention can reduce its own surface tension, has good stability, and does not produce viscous substances, making it easy to inject into oil reservoirs.

[0048] Experimental Example 5: To investigate the viscosity-reducing effect of the fermentation broth containing Bacillus subtilis 20P3 LZT of the present invention on crude oil.

[0049] Experimental method: Add 5g of ordinary heavy oil to a 250ml conical flask. Take 100ml of the fermentation broth containing Bacillus subtilis 20P3 LZT prepared in Examples 10 to 12 of this invention and add it to the conical flask. Shake and culture at 150 r / min for 15 days. The temperature is set according to the temperature of the heavy oil in the formation (the temperature of the heavy oil is 60℃). Use the shake flask of formation water sample as a control. Calculate the viscosity reduction rate and evaluate the emulsification status through emulsification rating.

[0050] Experimental results: As shown in Table 1, the fermentation broth containing Bacillus subtilis 20P3 LZT of the present invention has good emulsification and viscosity reduction properties for ordinary heavy oil, with an emulsification and viscosity reduction rate of over 70%.

[0051] In summary, this invention is the first to discover Bacillus subtilis 20P3 LZT, a strain capable of producing surfactant lipopeptides and growing using molasses or corn steep liquor as a carbon source. This strain exhibits good emulsification and viscosity-reducing properties for crude oil, achieving an emulsification and viscosity-reducing rate of up to 70% for heavy oil. It reduces the viscosity of crude oil and enhances its fluidity, thereby lowering the oil-water mobility ratio and, on the other hand, allowing the emulsified crude oil to play a role in profile control and flow diversion, indirectly improving oil recovery. This provides a new strain and material for microbial enhanced oil recovery technology.

[0052] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A Bacillus subtilis 20P3 LZT, characterized in that... This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30366.

2. A fermentation broth containing Bacillus subtilis 20P3 LZT as described in claim 1.

3. The fermentation broth containing Bacillus subtilis 20P3 LZT according to claim 2, characterized in that... Obtained using the following method: The first step was to inoculate Bacillus subtilis 20P3 LZT into seed culture medium and culture it to obtain seed solution; The second step involves inoculating a seed culture solution of 4% to 6% by volume into a fermentation medium containing a carbon source for culture, thereby obtaining a fermentation broth containing Bacillus subtilis 20P3 LZT.

4. The fermentation broth containing Bacillus subtilis 20P3 LZT according to claim 3, characterized in that... In the first step, the seed culture medium is LB liquid medium.

5. The fermentation broth containing Bacillus subtilis 20P3 LZT according to claim 3 or 4, characterized in that... In the first step, the cultivation is carried out in a shaker at a temperature of 35℃ to 37℃ for 36h to 40h, with a shaking speed of 120r / min to 150r / min.

6. The fermentation broth containing Bacillus subtilis 20P3 LZT according to claim 3, 4, or 5, characterized in that... In the second step, the fermentation medium containing carbon source is formulated as follows: carbon source 30 g / L to 50 g / L, sodium citrate 3 g / L to 5 g / L, ammonium sulfate 0.5 g / L to 1.0 g / L, dipotassium hydrogen phosphate 0.2 g / L to 0.5 g / L, potassium dihydrogen phosphate 0.2 g / L to 0.5 g / L, sodium chloride 0.5 g / L to 1.5 g / L, magnesium sulfate heptahydrate 0.2 g / L to 0.5 g / L, and ferrous sulfate heptahydrate 0.001 g / L to 0.002 g / L. The pH value of the fermentation medium containing carbon source is 7.

2.

7. The fermentation broth containing Bacillus subtilis 20P3 LZT according to claim 6, characterized in that... In the second step, the formula of the fermentation medium containing carbon source is as follows: carbon source 40 g / L, sodium citrate 3 g / L, ammonium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium chloride 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.001 g / L, and the pH value of the fermentation medium containing carbon source is 7.

2.

8. The fermentation broth containing Bacillus subtilis 20P3 LZT according to any one of claims 3 to 7, characterized in that... In the second step, the carbon source is either molasses or corn steep liquor powder.

9. The fermentation broth containing Bacillus subtilis 20P3 LZT according to any one of claims 3 to 8, characterized in that... In the second step, the cultivation is specifically carried out in a shaker at a speed of 100 to 150 rpm, a temperature of 30 to 40°C, and a cultivation time of 30 to 40 hours; or / and, in the second step, the concentration of Bacillus subtilis 20P3 LZT in the fermentation broth containing Bacillus subtilis 20P3 LZT is 1 × 10⁻⁶. 8 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 10 per mL.

10. The application of a fermentation broth containing Bacillus subtilis 20P3 LZT according to any one of claims 3 to 8 in microbial oil recovery.

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