A strain with improved crude oil properties, its screening and cultivation method, and applications

Abstract

The present invention discloses a strain with improved crude oil properties, its screening and cultivation method, and application. The strain is named Tistrellamobilis JSC4535. This strain reproduces with crude oil as the sole carbon source and is obtained through the processes of enrichment, screening, and separation from produced water in oil wells. It can reduce the viscosity and surface tension of crude oil, and has a degradation effect on long-chain hydrocarbons, cholestane in naphthenes, and naphthalene components in aromatic hydrocarbons in crude oil, thereby changing the properties of crude oil and increasing its fluidity. Moreover, this strain can tolerate temperatures of 25-50°C, salinity of 1-50 g / L, and alkalinity of 5-10. The strain has a wide tolerance range and also has good adaptability to the reservoir environment in the research area.

Description

Technical Field

[0001] The invention relates to the technical field of petrochemicals, and in particular to a strain capable of improving crude oil properties, a screening and culturing method and application thereof. Background Art

[0002] In recent years, the global oil industry has been facing the problem of unsustainable exploitation. The total amount of oil obtained from so-called "easy-to-extract" oil fields has been decreasing, and it is difficult to find new replacement oil fields for mature oil fields that have been developed for many years. At the same time, the consumption of fossil energy has also increased sharply with the rapid increase in population. Oil and gas are stored in the pores of underground rocks in the form of fluids and are under negative pressure. During the initial drilling, crude oil will flow out naturally due to the pressure of the oil layer, which is called primary oil recovery. This is the cheapest way to extract oil. As the formation pressure decreases, the crude oil production decreases, and secondary oil recovery methods such as water flooding are subsequently used to make the crude oil flow from the pores to the wellhead. However, as time goes by, the reservoir enters a period of high water content, and the injected water flows along the high permeability zone, the affected area is greatly reduced, and the oil recovery efficiency is rapidly reduced. Research shows that in oil production, only 30%-40% of the oil can be obtained by primary oil production methods, and methods such as fracturing and water flooding can only increase the recovery rate by about 15%-25%, that is, there is still 35%-55% of oil remaining in the formation after the traditional oil production method. Therefore, how to efficiently extract the residual oil underground is an urgent problem that oil companies around the world need to solve.

[0003] The contradiction between my country's oil demand and production is even more serious. my country has a wide distribution of low-permeability resources that are difficult to exploit, accounting for 46% of the total oil and gas resources. Due to its complex geological conditions, the recovery rate is often unsatisfactory, but the increasing demand for crude oil forces the import volume to continue to increase. China's oil dependence on foreign countries has reached 70%, far exceeding the safety red line. Therefore, exploiting the abundant residual oil underground to make up for the gap between oil supply and demand is a breakthrough to solve the current difficulties of the oil industry, and tertiary oil recovery technology has been developed accordingly.

[0004] Microbial enhanced oil recovery (MEOR) is one of the oil recovery technologies with relatively good economic benefits in tertiary oil recovery. It is a general term for a series of technologies that use microbial reproduction and their metabolites to increase oil production. Metabolites produced by microorganisms (such as biosurfactants, polymers, etc.) can reduce the interfacial tension between oil and water, lower the viscosity of crude oil, and can change the wettability of rocks from oil-wet to water-wet. In addition, the gas produced by microorganisms can increase the reservoir pressure, reduce the viscosity, and acid substances can dissolve carbonate rocks to increase porosity. The operation method of MEOR is relatively simple, with lower costs compared to other methods, and MEOR is an environmentally friendly oil recovery technology. Petroleum microorganisms can be used to restore the surface polluted by oil and degrade the leaked crude oil. In the case of low oil prices, microbial oil extraction technology is an extremely promising oil displacement method. It has good applications for marginal oilfields, high water cut oilfields, oilfields with poor economic benefits, low permeability oilfields, etc. However, how to screen out highly efficient oil-displacing bacteria remains a topic that needs continuous research at the present stage. Summary of the Invention

[0005] In view of the above problems, the present invention aims to provide a strain with improved crude oil properties, its screening and cultivation method, and application. This strain reproduces with crude oil as the sole carbon source and may have great potential in improving oil recovery.

[0006] In order to achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] A strain with improved crude oil properties, characterized in that: the strain is named Tistrella mobilis JSC4535.

[0008] Further, the nucleotide sequence of the strain is as shown in SEQ: ID: NO: 1.

[0009] Further, a screening and cultivation method for a strain with improved crude oil properties, characterized by comprising the following steps,

[0010] S1: Collect oil samples and produced water samples from low permeability oilfields;

[0011] S2: Enrich the strains in the oil samples and water samples to obtain an enrichment culture solution of dominant enriched bacteria;

[0012] S3: Isolate and screen the strains in the enrichment culture solution;

[0013] S4: Purify and preserve the strains isolated and screened;

[0014] S5: Inoculate the purified strain onto an inorganic salt medium, and screen out the strain that can grow with crude oil as the sole carbon source, named JSC4535.

[0015] Furthermore, the specific operation of step S2 includes the following steps.

[0016] S201: Take 5 ml of oil sample and produced water sample, and add them into 100 ml of inorganic salt medium respectively. Culture at 42 °C and 160 rpm for 4 days to allow the strain to multiply in large numbers, obtaining a culture solution.

[0017] S202: Take 2 ml of the culture solution of the oil sample and water sample respectively, transfer them into 100 ml of fresh enrichment medium, and culture again at 42 °C and 160 rpm for 4 days.

[0018] S203: Repeat step S202 twice to obtain an enrichment culture solution enriched with dominant bacteria.

[0019] Furthermore, the inorganic salt medium described in step S201 includes a carbon source and other components. The other components include 1.5 g / L of NaNO3; 1.5 g / L of (NH4)2SO4; 1 g / L of K2HPO4; 0.5 g / L of MgSO4; 0.5 g / L of KCl; 0.002 g / L of CaCl2; 0.001 g / L of FeSO4. The carbon source is crude oil, and the volume of the crude oil accounts for 10% of the total volume of the inorganic salt medium.

[0020] Furthermore, the enrichment medium described in step S202 includes: 3.6 g / L of beef extract; 10 g / L of peptone; 5.6 g / L of NaCl.

[0021] Furthermore, the specific operation of step S3 includes the following steps.

[0022] S301: Dilute the enrichment culture solution into 4 different gradients and spread them on enrichment plates. The enrichment medium on the enrichment plates includes: 3.6 g / L of beef extract; 10 g / L of peptone; 5.6 g / L of NaCl, and 20 g / L of agar.

[0023] S302: Place the enrichment plates in an incubator at 42 °C for 4 days.

[0024] S303: Select colonies with different morphological colors on the enrichment plates for strain purification.

[0025] Furthermore, the specific operation of step S4 includes the following steps.

[0026] S401: Inoculate the morphologically different bacterial populations selected in step S303 onto a new enrichment plate by the streaking method and culture at 42 °C for 3 days.

[0027] S402: Pick the single colony at the end of the enrichment plate in step S401 again, and repeat the culturing process in step S401 three times;

[0028] S403: Inoculate the purified single bacterium onto the enrichment medium. After it becomes turbid, store it at -20 °C for later use in a ratio of bacterial liquid: 50% glycerol = 1:1.

[0029] Further, the specific operations of step S5 include the following steps.

[0030] S501: Take the strain stored in step S4 and inoculate it onto the enrichment medium for activation, and culture it at 160 rpm and 42 °C for 3 days;

[0031] S502: Pipette 1 ml of the bacterial liquid in step S501 into the inorganic salt medium, and culture it under the same conditions to screen out the strain that grows with crude oil as the sole carbon source, named JSC4535.

[0032] Further, the application of a strain with improved crude oil properties in crude oil exploitation.

[0033] The beneficial effects of the present invention are as follows: Compared with the prior art, the improvements of the present invention are as follows.

[0034] 1. The present invention discloses a strain Tistrella mobilis JSC4535 with improved crude oil properties. This strain reproduces with crude oil as the sole carbon source. After the strain acts on crude oil for 7 days, the viscosity reduction rate of the crude oil can reach 38.6%. After the strain acts on crude oil for 14 days, the degradation rate of long-chain hydrocarbons reaches 47.2%, and the degradation rate of naphthalene components in aromatic hydrocarbons is 90.6%. The surface tension of the crude oil drops from 57.42 mN / m to 43.52 mN / m. Thus, it can be seen that this strain can change the properties of crude oil well, increase the fluidity of crude oil, and reduce costs.

[0035] 2. The strain disclosed in the present invention can tolerate temperatures of 25 - 50 °C, salinity of 1 - 50 g / L, and alkalinity of 5 - 10. The strain has a wide tolerance range and also has good adaptability to the reservoir environment in the research area. Description of the Drawings

[0036] Figure 1 This is the situation of the flora in crude oil and produced water enriched in the present invention.

[0037] Figure 2 This is the dilution situation of the flora in produced water in the inorganic salt medium of the present invention.

[0038] Figure 3 This is the dilution situation of the flora in crude oil in the inorganic salt medium of the present invention

[0039] Figure 4Schematic diagram of streaking on a flat plate for the present invention.

[0040] Figure 5 SEM image of JSC4535 for the present invention.

[0041] Figure 6 Growth curve of JSC4535 for the present invention.

[0042] Figure 7 Results of hemolytic experiment of JSC4535 for the present invention.

[0043] Figure 8 Temperature tolerance curve of JSC4535 for the present invention.

[0044] Figure 9 Salinity tolerance curve of JSC4535 for the present invention.

[0045] Figure 10 pH tolerance of JSC4535 for the present invention.

[0046] Figure 11 Degradation curve of JSC4535 for long-chain n-alkanes and cholestane for the present invention.

[0047] Figure 12 Degradation curve of JSC4535 for naphthalene for the present invention. Detailed implementation manners

[0048] In order to enable those of ordinary skill in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0049] Example 1:

[0050] A screening and culture method for strains with improved crude oil properties, comprising the following steps,

[0051] S1: Collect oil samples and produced water samples from a low-permeability oilfield;

[0052] All the sampled samples of the present invention are from Block 154 of Hujianshan Oilfield in the western Ordos Basin. The collected samples include oil samples and produced water samples. The basic reservoir parameters of the block are shown in Table 1 below.

[0053] Table 1 Reservoir parameters of Block 154

[0054]

[0055] * The current temperature of the sampled block after water flooding is about 40 - 50 °C

[0056] S2: Enrich the strains in the oil samples and water samples to obtain an enrichment culture solution with enriched dominant bacteria;

[0057] Specifically, the bacterial abundance in the sample is relatively low. To obtain the dominant bacteria, the bacteria in the 4+5 layer are first enriched.

[0058] First, 5 ml of oil sample and produced water sample are taken and added to 100 ml of inorganic salt medium respectively, and cultured at 42 °C with 160 rpm for 4 days to allow the strains to multiply in large numbers, obtaining the culture solution. Then, 2 ml of the culture solution of the oil sample and water sample are taken respectively and transferred to fresh enrichment medium for further culture. The above process is repeated twice to obtain the enrichment culture solution of the enriched dominant bacteria. As shown in the appendix Figure 1 shown, in the appendix Figure 1 , the left figure is the water sample and the right figure is the oil sample. As can be seen from the appendix Figure 1 , after culturing for 4 days, the medium becomes turbid, indicating that the strains have been enriched.

[0059] Among them, the inorganic salt medium described includes a carbon source and other components. The other components include 1.5 g / L of NaNO3; 1.5 g / L of (NH4)2SO4; 1 g / L of K2HPO4; 0.5 g / L of MgSO4; 0.5 g / L of KCl; 0.002 g / L of CaCl2; 0.001 g / L of FeSO4. The carbon source is crude oil, and the volume of crude oil accounts for 10% of the total volume of the inorganic salt medium.

[0060] The enrichment medium described includes: 3.6 g / L of beef extract; 10 g / L of peptone; 5.6 g / L of NaCl; all media need to be sterilized at 121 °C for 20 or 40 min (for crude oil). Unless otherwise specified, all experiments are carried out in a laminar flow hood.

[0061] In all the operation steps of the present invention, all the instruments used are shown in Table 2 below.

[0062] Table 2 List of all instruments

[0063]

[0064] S3: Isolate and screen the strains in the enrichment culture solution;

[0065] Specifically, S301: Dilute the enrichment culture solution into 4 different gradients (10 -1 -10 -4 ), and coat them on the enrichment plates; the enrichment medium on the enrichment plates includes: 3.6 g / L of beef extract; 10 g / L of peptone; 5.6 g / L of NaCl, 20 g / L of agar;

[0066] S302: Place the enrichment plates in an incubator at 42 °C for 4 days, and the results are shown in the appendix Figure 2 and the appendix Figure 3 shown. Among them, in the appendixFigure 2 For the dilution of the bacterial flora in the produced water cultured in the inorganic salt medium, see the appendix Figure 3 For the dilution of the bacterial flora in the crude oil in the inorganic salt medium. From the appendix Figure 2 and the appendix Figure 3 It can be seen that when diluting and spreading the crude oil sample cultured in the inorganic salt medium, colonies can be directly selected without dilution. And the growth communities of the bacterial flora in the crude oil sample are generally small, mostly round milky white colonies, with smooth surfaces and regular shapes. While in the produced water sample, in addition to the milky white colonies, semi-transparent light yellow colonies can also be observed and the communities are larger and irregular in shape.

[0067] S303: Select colonies with different morphological colors on the enrichment plate for strain purification

[0068] Furthermore, S4: Purify and preserve the isolated and screened strains;

[0069] The colonies obtained after dilution coating need to be purified to obtain a single strain. In the present invention, the method of streaking dilution is used to purify the community. As shown in the appendix Figure 4 shown, specifically:

[0070] Among the colonies obtained after enrichment and coating in step S3, select colonies with different morphologies and larger colonies and streak them on a new enrichment plate, culture at 42 °C for 3 days; then select a single colony at the end of the enrichment plate again and repeat the culture process 3 times; inoculate the purified single bacteria on the enrichment medium, and store them at -20 °C for standby according to the ratio of bacterial liquid: 50% glycerol = 1:1 after turbidity.

[0071] S5: Inoculate the purified strain on the inorganic salt medium and screen out the strain that can grow with crude oil as the sole carbon source, named JSC4535.

[0072] Inoculate the purified strain on the inorganic salt medium to screen out the strain that can grow with crude oil as the sole carbon source. When the strain can grow with crude oil as the sole carbon source, it has less demand for external nutrients and better tolerance to the reservoir environment. This characteristic is of great significance for reducing the on-site oil displacement cost and simplifying the process. Specifically,

[0073] Take the preserved strain and inoculate it on the enrichment medium for activation, culture at 160 rpm and 42 °C for 3 days;

[0074] Subsequently, pipette 1 ml of the bacterial liquid in step S501 into the inorganic salt medium and culture it under the same conditions, and finally screen out the strain that can grow with crude oil as the sole carbon source.

[0075] The finally obtained strains were sent to Kehao Bioengineering Company for species identification. The 16S rRNA of prokaryotes is commonly used in biological system classification due to its high sequence conservation, abundant information, and moderate size (1.5 Kb). Sequencing the DNA sequence encoding this ribosome, the sequence of the conserved region can reflect the genetic relationship between species. Usually, when the homology of the 16S rRNA sequence is higher than 97%, it can be determined to be the same species within the genus. After extracting the DNA of the strain, PCR amplification was carried out, followed by sequencing using a 3730xl sequencer and sequence comparison on NCBI (NCBI; www.ncbi.nlm.nih.gov) to obtain the strain information. The specific identification process includes the following operations.

[0076] (1) DNA extraction

[0077] a) Pick a single colony into a centrifuge tube, add 800 μl of Lysis Buffer, invert and mix the sample well, and let it stand at room temperature for 3 - 10 min;

[0078] b) Centrifuge at 12000g at room temperature for 5 min;

[0079] c) Insert the DNA adsorption column into the collection tube, transfer 600 μl of the supernatant to the adsorption column, and centrifuge at 12000g at room temperature for 1 min;

[0080] d) Discard the waste liquid, transfer the adsorption column back to the collection tube, add 600 μl of Buffer W1 to the adsorption column, and centrifuge at 12000g at room temperature for 1 min;

[0081] e) Discard the waste liquid, transfer the adsorption column back to the collection tube, add 600 μl of Buffer W2 (4 times anhydrous ethanol needs to be added before use) to the adsorption column;

[0082] f) Discard the waste liquid, transfer the adsorption column back to the collection tube, and centrifuge at 12000g for 3 min to remove ethanol;

[0083] g) Discard the collection tube and place the DNA adsorption column into a new centrifuge tube;

[0084] h) Add 30 - 100 μl (sterile water or 10 mM Tris - HCl, preheated to 65 °C) to the middle of the column, let it stand for 1 min, and centrifuge at 12000g for 2 min;

[0085] i) Discard the binding column and store the DNA in the refrigerator.

[0086] (2) PCR amplification

[0087] The PCR reaction system was 20 μl (1 μl each of 27F / 1492R primers, 1 μl of DNA sample, 10 μl of MIX, and 7 μl of added water). The primers used were:

[0088] 27F: AGAGTTTGATCCTGGCTCAG

[0089] 1492R: GGTTACCTTGTTACGACTT

[0090] The PCR program was pre-denaturation at 96 °C for 5 min, denaturation at 96 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, and final extension at 72 °C for 5 min. The identification results are shown in Table 3 below. The sequence of the strain was (the same as shown in SEQ: ID: NO: 1):

[0091] GCTACTTACGTGGACGGCTGCCTCCTTACGGAGTCAGCTCACCGGCTTCG

[0092] GGTAAAACCAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGA

[0093] ACGTATTCACCGCGGCATGCTGTTCCGCGATTACTAGCGATTCCGACTTC

[0094] ATGCACTCGAGTTGCAGAGTACAATCCGAACTGAGACGACTTTTTGAGAT

[0095] TAGCTTCACCTCGCGATGTCGCTGCCCACTGTAGTCGCCATTGTAGCACG

[0096] TGTGTAGCCCAGCCCATAAGGGCCATGAGGACTTGACGTCATCCCCACCT

[0097] TCCTCCGGCTTATCACCGGCAGTTTCCCTAGAGTGCCCAGCCGAACTGAT

[0098] GGCAACTAAGGATGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCT

[0099] CACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTGACGTCCGGCCG

[0100] AACCGAAAACCCCGTCTCTGAGGTCGCGACGTCCATGTCAAGGGCTGGTA

[0101] AGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCG

[0102] GGCCCCCGTCAATTCCTTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGG

[0103] CGGAGTGCTTAACGCGTTAGCTACGACACTGCGATACTAAGTATCCCAAC

[0104] GTCCAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGT

[0105] TTGCTCCCCACGCTTTCGTGCCTCAGCGTCAGTTTCGGGCCAGGCAGCCG

[0106] CCTTCGCCACCGGTGTTCTTCCCAATATCTACGAATTTCACCTCTACACT

[0107] GGGAATTCCACTACCCTCTCCCGAACTCCAGCCTACCAGTCTCAAAAGCA

[0108] GTTCCGGAGTTGAGCCCCGGGCTTTCACTTCTGACTTGATAAGCCGCCTA

[0109] CGCACGCTTTACGCCCAGTAAATCCGAACAACGCTAGCCCCCTTCGTATT

[0110] ACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCTTCTACGGGTACCGT

[0111] CATTATCTTCCCCGTCGAAAGAGCTTTACAATCCGAAGACCTTCATCACT

[0112] CACGCGGCATTGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCAC

[0113] TGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGA

[0114] TCATCCTCTCAGACCAGCTACCGATCGTCGCCTTGGTAGGCCGTTACCCC

[0115] ACCAACTAGCTAATCGGACGCGGGCTCATCCATCTACGGCCGAAGCCTTT

[0116] CCCCCGAAGGGCGTATGCGGTATTAGCTGCAGTTTCCCGCAGTTATTCCC

[0117] CATAGATGGGCAGATTCCCACGTGTTACTCACCCGTGCGCCACGTAAGCC

[0118] GGAACCGAAGTTCCGACCCCGTCGACTGCATTGTATACA

[0119] Thus, this strain can be identified as a Tistrena mobilis strain, named JSC4535. This strain was deposited at the China Center for Type Culture Collection on August 24, 2022, with the deposit number CCTCC NO: M 20221319 and the deposit address being Wuhan University, Wuhan, China.

[0120] Table 3 Strain identification results

[0121]

[0122] Furthermore, a scanning electron microscope (SEM) was used to visually observe the morphology of the screened strain. The strain was inoculated into an enrichment medium for enrichment culture. When the OD of the strain 600 reached about 0.8, 5 ml of the bacterial solution was taken into a centrifuge tube and centrifuged at 7000 rpm for 10 min (the bacterial pellet was about the size of a mung bean). The supernatant was slowly poured out, PBS was added for rinsing, and then it was centrifuged at 6000 rpm for 10 min. The supernatant was slowly removed again, and 5 ml of 2.5% glutaraldehyde was added for fixation (the bacterial pellet needed to be suspended in the fixing solution). It was fixed at room temperature for 2 hours and then stored at 4°C. The SEM photography part was completed by Kehao Bioengineering Company, and the results are as shown Figure 5 in the appendix. The whole strain was slender rod-shaped, with an aspect ratio of length to width reaching 6:1, and there were obvious grooves on the surface of the strain.

[0123] Example 2:

[0124] Example 2 Performance evaluation was carried out on the strain JSC4535 obtained in Example 1, which specifically included four aspects: monitoring of the strain growth rate, evaluation of the strain emulsifying ability, evaluation of the strain tolerance, and the effect of the strain on crude oil.

[0125] 1. Monitoring of the strain growth rate

[0126] The growth of JSC4535 at 42°C was monitored to observe the growth rate of the strain. Strains with rapid reproduction can shorten the culture time, thereby reducing costs, which is of great significance for the application of microbial enhanced oil recovery. Its growth can be measured by measuring the absorbance value. OD (Optical Density) is the optical density. When light of a specific wavelength passes through the bacterial solution, an energy difference will be formed, and this energy difference is the absorbed energy. There is a quantitative relationship between the concentration of the bacterial solution and the absorbed energy. Therefore, the optical density value is commonly used to characterize the concentration of the strain and thus illustrate the growth of the strain. Usually, the selected wavelength is 600 nm. Take 1 ml of the strain enriched at 42°C and inoculate it on a new enriched liquid medium, and culture it with shaking at 160 rpm at 42°C. Take 3 ml of the solution at regular intervals, and measure the OD value of the bacterial solution using a UV spectrophotometer. Each sample is measured three times and the average value is taken to obtain the relationship curve of time and OD value, as shown in the appendix. As can be seen from the appendix, the absorbance of this strain can reach 0.8 after 24 h of culture, and the growth is rapid. 600 value measurement, and the average value is taken for each sample measured three times to obtain the relationship curve of time and OD 600 value, as shown in the appendix Figure 6 shown, as shown in the appendix Figure 6 It can be seen that the absorbance of this strain can reach 0.8 after 24 h of culture, and the growth is rapid.

[0127] 2. Evaluation of the strain emulsifying ability

[0128] (1) Emulsification rate (E 24 )

[0129] Inoculate JSC4535 onto a fresh beef extract peptone medium and activate it for 3 days. Take 3 ml of the bacterial solution, centrifuge it at 8000 rpm for 10 min, mix it with 3 ml of n-hexane, shake it and then vortex it for 2 minutes. Record the height of the emulsified layer after standing for 24 h. All experiments are repeated three times and the average value is taken. E 24 is determined by the following formula:

[0130]

[0131] (2) Hemolysis

[0132] The biosurfactant produced by the strain can reduce the surface tension and has the ability to lyse red blood cells, forming a transparent zone around the strain on the blood plate. The strain was inoculated onto fresh liquid NB medium and enriched for 3 days. Then, 50 μl of the bacterial solution was pipetted and spread onto the solid enrichment medium, and cultured at 42 °C for 2 days. The center point of the colony was picked with an inoculation loop and inoculated onto the surface of the blood plate (LB medium supplemented with 5% goat blood), and cultured at 42 °C for 3 days. Subsequently, the diameter of the formed transparent zone was measured and the hemolytic ability was evaluated. The hemolytic ability of the strain was rated according to the size of the hemolytic zone diameter (d) formed: (-) no hemolytic zone; (+) d ≤ 1 cm; (++) 1 cm < d < 3 cm; (+++) d ≥ 3 cm.

[0133] E 24 The results of the hemolytic experiment are shown in Table 4 below and in the appendix Figure 7 As shown, the results indicate that JSC4535 has medium surfactant-producing ability.

[0134] Table 4 Hemolytic ability of the strain and E 24 Results

[0135]

[0136] (3) Emulsification effect on crude oil

[0137] The biosurfactant produced by the strain has the effect of reducing the surface tension. By measuring the surface tension of the crude oil after the action of the strain, the emulsifying ability of the strain on the crude oil can be further evaluated. 5 ml of the enriched bacterial solution of the strain was added to 100 ml of the inorganic salt medium, with the blank medium as the control, and 10 ml of crude oil as the sole carbon source. It was cultured at 160 rpm and 42 °C for 7 days. Subsequently, the surface tension was measured using a surface tension meter (POWEREACH, Shanghai). The results showed that after the action of JSC4535, the surface tension of the crude oil decreased from 57.42 mN / m to 43.52 mN / m.

[0138] 3. Evaluation of strain tolerance

[0139] Underground reservoirs belong to extreme environments (high temperature, high salinity, anoxia, etc.) and are not suitable for the large-scale reproduction of microorganisms. Therefore, the obtained dominant strains applicable to microbial flooding must have a certain tolerance to extreme environments. Therefore, the screened strains were tested for temperature, salinity, and pH tolerance.

[0140] (1) Temperature tolerance

[0141] The JSC4535 strain was inoculated into the liquid enrichment medium, and gradient temperatures were set: 25 °C, 35 °C, 45 °C, 50 °C, 55 °C, 65 °C. It was cultured in an incubator at 160 rpm at the above temperatures for 3 days. The bacterial solution was dispersed with a pipette, and 3 ml was taken to measure the OD 600, the obtained growth curve is as shown in the appendix Figure 8 as follows. It can be seen from the appendix Figure 8 that the strain can reproduce rapidly within the range of 25°C - 50°C, the OD value exceeds 1.0, and the growth rate is the best at 35 - 40°C. While the growth rate drops rapidly at 50°C, the growth of the strain is slow, and most strains hardly grow at 65°C. The temperature of the oil wells in the study area is about 40 - 50°C after water flooding, and the dominant strains can maintain good activity during application.

[0142] (2) Tolerance to salinity

[0143] Fix the temperature at 42°C and change the NaCl concentration in the culture medium: 1, 3, 5, 7, 9, 10, 20, 30, 50 g / L, culture for 3 days at 160 rpm, and measure the activity of the strain by measuring OD 600 The experimental results are as shown in the appendix Figure 9 as follows. It can be seen from the appendix Figure 9 that with the increase of salinity, all strains show a trend of gradually slowing growth. The optimal growth salinity of the strains is between 3 g / L and 10 g / L. When the salinity reaches 50 g / L, the strains can still survive. The salinity of the formation water in the sampling area is relatively high, reaching 45 g / L. However, MEOR relies on water flooding, and the salinity of the oil wells after multiple water floodings decreases compared with the initial value. Therefore, JSC4535 can maintain good activity during application.

[0144] (3) Tolerance to pH

[0145] Set pH = 4, 5, 6, 7, 8, 9, 10, configure NB liquid medium according to the general formula, heat it in a water bath to completely dissolve the solid solutes, adjust the pH of the medium with 1 mol / L HCl and 1 mol / L NaOH (the pH can be appropriately lower than the set value when adjusting the pH, and the high-temperature sterilization will cause the pH to increase), culture at 160 rpm and 42°C for 3 days, and then measure OD 600 to measure its growth situation. The results are as shown in the appendix Figure 10 as follows. It can be seen from the appendix Figure 10 that the strain hardly grows at pH = 4. When pH > 7, the absorbance (growth value) of the strain is good for 3 days, indicating that the selected bacteria have good alkali tolerance. However, the overall OD 600 value of the strain is low, mainly because during the culture process, the strain aggregates into clusters instead of dispersing in the culture medium, resulting in a low measured OD 600 value.

[0146] 4. The effect of the strain on crude oil

[0147] (1) Viscosity reduction effect

[0148] Transfer the culture medium, crude oil, and bacterial solution to a 150 ml conical flask at a ratio of 20:2:1, and culture them at 160 rpm and 42 °C for 7 days and 14 days respectively. Take samples of the crude oil one day before the test, transfer the crude oil to a 5 ml centrifuge tube, take three samples for each strain, and let it stand in an incubator at 42 °C overnight to completely separate the oil and water. When measuring the samples, take the upper solidified crude oil and use a rotational rheometer (Anton Paar MCR302, Austria) to measure the viscosity at 42 °C. The measurement results are averaged, and the results are shown in Table 5 below. The viscosity reduction rate of the crude oil after 7 days of culture can reach 38.66%, and after continuous culture for 14 days, the viscosity reduction rate is 47.2%. The results show that the action period of this strain is relatively long, and the viscosity reduction effect increases with the increase of the culture time.

[0149] Table 5 Viscosity measurement results after standing

[0150]

[0151] (2) Changes in crude oil components

[0152] Use GC-MS to analyze the changes in the components (saturated hydrocarbons, aromatic hydrocarbons) of the crude oil before and after the action of the strain, and quantitatively evaluate the effect of JSC4535 on the crude oil. Add 5 ml of sterile crude oil and 2 ml of the bacterial solution to 50 ml of inorganic salt medium, and culture at 160 rpm at 42 °C for 7 days. Then centrifuge at 8000 rpm for 10 minutes to remove the bacteria and impurities, and extract the supernatant with n-hexane. According to industry standards, separate saturated hydrocarbons and aromatic hydrocarbons through a chromatographic column. The GC-MS analysis chromatographic column is HP-5MS (60 m × 0.25 mm × 0.25 μm), and the conditions are set as follows: carrier gas, 99.999% He, flow rate 1 mL / min; inlet and transfer line temperature 300 °C; chromatographic column, HP-5MS (60 m × 0.25 mm × 0.25 μm); temperature program, initial temperature 50 °C, hold for 1 min, increase the temperature to 120 °C at a rate of 20 °C / min, then increase to 250 °C at a rate of 4 °C / min, and then increase to 310 °C at a rate of 3 °C / min and hold for 30 min. MS conditions Ionization mode: EI, 70 eV; filament current, 100 A; multiplier voltage, 1200 V. Data acquisition mode: Selective ion scanning (85, 123, 191, 217). The degradation rate calculation formula for different components is as follows:

[0153]

[0154] The components of the crude oil before and after the action of the strain were analyzed by GC-MS. JSC4535 has a good degradation effect on long-chain hydrocarbons (C>22), cholestane (cycloalkanes) (as shown in the attachment Figure 11 where a - degradation of n-alkanes; b - degradation of cholestane) and naphthalene components in aromatic hydrocarbons (as shown in the attachment Figure 12 shown). ∑C21- / ∑C 21+ can measure the priority of the strain's utilization of alkanes, ∑C 21- / ∑C 21+ increased from 1.29 before the action to 1.52, indicating that strain JSC4535 preferentially utilizes short-chain alkanes. The degradation rate of JSC4535 for long-chain hydrocarbons is 28.9%, and the degradation rate for naphthalene is 90.6%.

[0155] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A strain with improved crude oil properties, characterized in that: The strain is named Tistrella mobilis JSC4535; The strain was deposited at the China Center for Type Culture Collection on September 1, 2022, with the deposit number CCTCC NO: M 20221319, and the deposit address is Wuhan University, Wuhan, China.

2. Use of a strain with improved crude oil properties as described in claim 1 in crude oil exploitation.

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