A strain of Acinetobacter calcoaceticus and its application

Abstract

The present invention relates to the field of microbial technology, and more particularly to a strain of acinetobacter calcoaceticus and its application. The strain was deposited in the China Type Culture Collection Center on January 17, 2022, with a deposit number of CGMCCNO.24322. Experiments show that the strain can stably degrade dibenzothiophene to generate 2-hydroxybiphenyl, without losing the fuel value of crude oil, has a high degradation rate and strong tolerance to DBT, can tolerate a high oil-water ratio and processing temperature during crude oil desulfurization treatment, and can also produce surfactants, showing an obvious emulsification and viscosity reduction effect, with oil displacement, reducing crude oil viscosity, increasing the contact area of ​​bacterial agent treatment and other advantages, the discovery of acinetobacter calcoaceticus described in the present invention, broadens the variety of crude oil desulfurization bacteria, helps to accelerate the process of large-scale industrialization of biological desulfurization (BDS), and has good application prospects in crude oil desulfurization and environmental protection technology.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Acinetobacter calcoaceticus and application thereof. Background Art

[0002] With the rapid economic development and the acceleration of industrialization, oil is widely used as the main energy source in daily life and production and construction. However, since sulfur accounts for 0.03-7.89% of the mass of crude oil, it is the largest component besides carbon and hydrogen. After combustion, a large amount of sulfur dioxide will be produced. After sulfur dioxide is discharged into the atmosphere, it will fall in the form of acid rain, causing damage to the human respiratory and digestive systems, while corroding plants and destroying the ecological environment. As people pay more and more attention to environmental pollution, countries around the world have introduced a series of increasingly stringent regulations to control the sulfur level of refined oil. Therefore, there is an urgent need for an efficient and environmentally friendly oil desulfurization technology to solve the current problem of high sulfur content in oil.

[0003] High-sulfur crude oil has the characteristics of high sulfur content, complex composition, and high viscosity. The direct refining cost is high and the desulfurization is incomplete. If the sulfur content is reduced to a lower level before crude oil smelting, the downstream processing pressure and cost can be greatly reduced. Compared with traditional physical and chemical desulfurization methods, the biological desulfurization method (BDS) requires lower operating temperature and pressure, and can effectively remove heterocyclic organic sulfur, so it has broad application prospects. Dibenzothiophene and its derivatives are important components of high-sulfur crude oil. The content of dibenzothiophene in crude oil is large and difficult to remove. It is very representative. Therefore, DBT is often used as a model compound in the study of petroleum desulfurization. With increasingly stringent environmental requirements today, biological desulfurization of petroleum is imperative. Therefore, collecting and isolating desulfurization strains from nature and screening out suitable crude oil desulfurization bacteria can not only improve the utilization rate of petroleum, but also play a very positive role in improving the ecological environment. Many microorganisms have been discovered, such as Rhodococcus, Pseudomonas, Gordonia, Corynebacterium, Mycobacterium and Arthrobacter, which can degrade DBT along the sulfur-specific pathway. They do not open the benzene ring structure of these sulfur-containing compounds, thereby retaining the calorific value of the fuel. They have great application prospects and have become a hot topic of research in recent years.

[0004] Although many strains that can be used in the petroleum BDS process have been isolated, there are still various limiting factors for the commercial application of crude oil BDS. For example, first, crude oil has a high sulfur content and complex composition, which is toxic to desulfurization bacteria and affects the activity and life of the bacterial agent; second, the temperature is often high during crude oil extraction and transportation, which inhibits the activity of biological desulfurization; third, crude oil has high viscosity, which makes it difficult for microorganisms to fully contact the crude oil, reducing the degradation efficiency. Therefore, although many crude oil desulfurization strains have been isolated, it is still necessary to continue to expand the varieties of crude oil desulfurization bacteria, develop efficient desulfurization strains and methods that have high tolerance to crude oil toxicity and temperature, can reduce crude oil viscosity and increase contact area, and accelerate the large-scale industrialization of biological desulfurization (BDS). Summary of the invention

[0005] In view of this, the present invention provides a strain of Acinetobacter calcoaceticus and application thereof, wherein the strain has obvious desulfurization and emulsification viscosity reduction effects on high-sulfur crude oil.

[0006] The present invention separates and purifies the strain YJY22-13 from petroleum coke and oil storage tank sludge of Shandong Jingbo Petrochemical Co., Ltd.

[0007] The strain screening method of the present invention is as follows: the above samples are respectively subjected to gradient acclimation culture at 30-50° C. in a liquid inorganic salt basal culture medium containing 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, and 0.5 mmol / L of dibenzothiophene (DBT) for 2-3 days, and then single colonies are isolated on a solid inorganic salt culture medium containing corresponding concentrations of DBT, and single colonies that are resistant to high temperatures and tolerate high DBT concentrations are obtained as much as possible. The DBT effect and metabolic pathway verification are performed on the single colonies finally obtained. The metabolic pathway identification method is as follows: the bacterial liquid cultured in a liquid basal culture medium containing DBT for 2-3 days is centrifuged to remove the bacterial bodies, the supernatant is taken and its pH is adjusted to 8.0, a small amount of Gibb's reagent is added, and the blue color indicates that 2-hydroxybiphenyl degraded from DBT exists in the culture medium.

[0008] In the present invention, the inorganic salt basic culture medium for culture includes water and the following components: 2-3 g / L KH2PO4, 10-15 g / L Na2HPO4.12H2O, 0.3-0.5 g / L MgCl2.6H2O, 0.001-0.01 g / L CaCl2.2H2O, 0.001-0.01 g / L FeCl3.6H2O, 0.001-0.01 g / L MnCl2.4H2O, and 1-3 g / L NH4Cl.

[0009] The 16S rDNA nucleotide sequence of the YJY22-13 strain is shown in SEQ ID NO: 1. The strain was identified as Acinetobacter calcoaceticus by 16S rDNA, and its deposit number is CGMCC NO.24322.

[0010] The Acinetobacter CGMCC No. 24322 of the present invention can produce a surfactant in a two-phase system, emulsify the system and reduce the viscosity of crude oil to disperse the crude oil into small oil droplets.

[0011] The invention also provides a microbial agent, the raw material of which includes acinetobacter calcoaceticus with a preservation number of CGMCC NO.24322.

[0012] In the present invention, the microbial agent comprises: live bacteria and / or inactivated bacteria of Acinetobacter calcoaceticus described in the present invention,

[0013] And / or, the culture and extract of Acinetobacter calcoaceticus described in the present invention.

[0014] The present invention also provides a method for preparing the microbial agent, comprising: step 1: inoculating the Acinetobacter calcoaceticus with a deposit number of CGMCCNO.24322 into an inorganic salt culture medium containing DBT to obtain a cell culture fluid;

[0015] Step 2: centrifuging the cell culture fluid, collecting the cell precipitate, and resuspending the cells to obtain a resting cell suspension;

[0016] Step 3: centrifuge the cell culture solution, collect the cell precipitate, mix it with a freeze-drying protective agent, and obtain the microbial agent by freeze-drying.

[0017] In some embodiments, the concentration of DBT in the DBT-containing inorganic salt culture medium is 0.1-0.5 mmol / L, specifically 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L or 0.5 mmol / L.

[0018] In some embodiments, the culture is carried out at 30-50°C and 180 rpm for 2-3 days.

[0019] In some embodiments, in step 2, the cells are resuspended in 100 mM potassium phosphate buffer at pH 7.0 to a cell concentration of 5-20 g dry cells / L.

[0020] In some specific embodiments, the preparation method of the microbial agent provided by the present invention comprises:

[0021] (1) Cell culture: select Acinetobacter with a deposit number of CGMCC No. 24322 and inoculate it into an inorganic salt medium containing 0.1-0.5 mmoL / LDBT and culture it at 30-50°C and 180 rpm for 2-3 days to obtain a cell culture fluid;

[0022] (2) Preparing a resting cell suspension: centrifuge the culture solution obtained in step (1) at 4,500 rpm for 15-20 min, collect the precipitated cells, and resuspend the cells in 100 mM potassium phosphate buffer at pH 7.0. Repeat 2-3 times until the cell concentration reaches 5-20 g dry cells / L. This cell suspension is the prepared resting cell suspension;

[0023] (3) The culture solution obtained in step (1) is centrifuged at 4,500 rpm for 15-20 min, the precipitated cells are collected, mixed with a lyophilization protectant, and lyophilized to obtain a lyophilized powder, i.e., the microbial agent of the present invention.

[0024] The present invention also provides the use of the Acinetobacter calcoaceticus and the microbial agent in crude oil desulfurization.

[0025] The present invention also provides a method for desulfurizing crude oil, wherein the calcoacetic acid Acinetobacter described in the present invention, the microbial agent, or the cell culture fluid, resting cell suspension or microbial agent prepared in the preparation method of the present invention are added to an oil-water two-phase system containing crude oil to carry out a desulfurization reaction.

[0026] In some embodiments, the volume ratio of the crude oil to the water phase is 1:5-20; the conditions of the desulfurization reaction are: 30-50°C, 180 rpm oscillation for 48-72h. In some embodiments, the volume ratio of the crude oil to the water phase is preferably 1:10-20; the temperature of the desulfurization reaction is preferably 35-45°C, and the oscillation time is preferably 60-72h.

[0027] In the application of the present invention, the Acinetobacter CGMCC No. 24322 can produce a surfactant in a two-phase system, emulsify the system and reduce the viscosity of crude oil to disperse the crude oil into small oil droplets.

[0028] Compared with the prior art, the Acinetobacter CGMCC No. 24322 of the present invention has the following beneficial effects: (1) it can degrade dibenzothiophene (DBT) to generate 2-hydroxybiphenyl without losing the fuel value of crude oil, has a high degradation rate and strong tolerance, and the degradation rate of 5mmoL / LDBT reaches more than 90%, and can tolerate up to 20mmoL / LDBT; (2) it has high desulfurization activity for high-sulfur crude oil, and when the oil-water ratio is 1:20 and the temperature is 35°C, the desulfurization rate of high-sulfur crude oil (sulfur content is 34485mg / L) reaches 13.38 % (3) It can tolerate a relatively high oil-water ratio and treatment temperature, and still has desulfurization activity at an oil-water ratio of 1:10-20 and 40-50°C; the degradation rate of 5mmoL / LDBT72h at 45°C reaches 58.32%, and the desulfurization rate of high-sulfur crude oil (sulfur content of 34485mg / L) at 45°C and an oil-water ratio of 1:20 is 10.02%; (4) Finally, in a two-phase system, the calcium-acetic acid Acinetobacter described in the present invention has a significant emulsification and viscosity reduction effect on high-sulfur crude oil, forming obvious emulsified small oil droplets.

[0029] In summary, the calcoacetic acid Acinetobacter CGMCC No. 24322 obtained by the present invention can stably degrade dibenzothiophene (DBT) to generate 2-hydroxybiphenyl without losing the fuel value of crude oil, has a high degradation rate for DBT and strong tolerance, can tolerate a high oil-water ratio and treatment temperature in crude oil desulfurization treatment, and can also produce surfactants, showing an obvious emulsification and viscosity reduction effect, and has the advantages of oil displacement, reducing crude oil viscosity, increasing the contact area of ​​bacterial agent treatment, etc. The discovery of the calcoacetic acid Acinetobacter CGMCC No. 24322 described in the present invention broadens the varieties of crude oil desulfurization bacteria, helps to accelerate the process of large-scale industrialization of biological desulfurization (BDS), and has good application prospects in the fields of crude oil desulfurization and environmental protection technology.

[0030] Biological Deposit Description

[0031] YJY22-13, classification name: Acinetobacter calcoaceticus, was deposited on January 17, 2022 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 24322. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The results show the tolerance of Acinetobacter calcoaceticus CGMCC No.24322 to dibenzothiophene (DBT);

[0033] Figure 2It shows that Acinetobacter calcoaceticus CGMCC No.24322 disperses small droplets (macroscopic) in the oil-water two phases (2-a blank group, 2-b experimental group);

[0034] Figure 3 It was shown that Acinetobacter calcoaceticus CGMCC No.24322 dispersed crude oil into small droplets (microscopic) in the oil-water two phases (3-a blank group, 3-b experimental group). DETAILED DESCRIPTION

[0035] The invention discloses a strain of acinetobacter calcoaceticus and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention, to achieve and apply the technology of the present invention.

[0036] The present invention will be further described below in conjunction with embodiments:

[0037] Example 1: Screening, isolation and identification of Acinetobacter calcoaceticus according to the present invention

[0038] A. Isolation and screening of strains

[0039] The samples of petroleum coke and oil storage tank sludge taken from Shandong Jingbo Petrochemical Co., Ltd. were cultured in a gradient manner at 30-50°C in a liquid inorganic salt basal culture medium containing 0.1mmoL / L, 0.2mmoL / L, 0.3mmoL / L, 0.4mmoL / L, and 0.5mmoL / L of dibenzothiophene (DBT), respectively. After 2-3 days of gradient acclimation, single colonies were isolated on a solid inorganic salt culture medium containing corresponding concentrations of DBT, and single colonies that were resistant to high temperatures and tolerated high DBT concentrations were obtained as much as possible. The DBT effect and metabolic pathway verification were performed on the single colonies finally obtained.

[0040] The inorganic salt basic culture medium for the culture includes water and 2.44g / L KH2PO4, 14.03g / L Na2HPO4.12H2O, 0.36g / L MgCl2.6H2O, 0.001g / L CaCl2.2H2O, 0.001g / L FeCl3.6H2O, 0.004g / L MnCl2.4H2O, and 2g / L NH4Cl.

[0041] The metabolic pathway identification method is as follows: centrifuge the bacterial liquid cultured in a basic medium containing DBT for 2 to 3 days to remove the bacteria, take the supernatant and adjust its pH to 8.0, add a small amount of Gibb's reagent, and the blue color indicates that 2-hydroxybiphenyl degraded from DBT exists in the culture medium.

[0042] B. Effect Verification

[0043] The single colonies isolated in the above process A that can degrade dibenzothiophene (DBT) to generate 2-hydroxybiphenyl were gradient-acclimated and cultured in a liquid basal culture medium containing 0.1-0.5 mmoL / L of dibenzothiophene (DBT) for 2-3 days, and then inoculated with 0.1 mmoL / L, 0.2 mmoL / L, 0.3 mmoL / L, 0.4 mmoL / L, and 0.5 mmoL / L of dibenzothiophene (DBT) in a liquid inorganic salt basal culture medium, respectively, and cultured at 180 rpm at 35°C for 48-72 h to verify the effect. The DBT degradation rate was detected by absorbance spectrophotometry using the characteristic that DBT has a significant absorption peak at 330 nm, and finally a strain YJY22-13 with high DBT degradation activity was obtained, which can completely degrade 0.5 mmoL / DBT in 48 h.

[0044] C. Strain preservation and identification

[0045] The final obtained strain YJY22-13 was preserved in glycerol tubes and slants, and entrusted to Jinan Boshan Bioengineering Co., Ltd. for PCR amplification and sequencing. The 16S rDNA sequence was obtained as shown in the appendix. It was identified as calcoacetic acid Acinetobacter and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 17, 2022, with the deposit number CGMCC No. 24322.

[0046] Example 2: DBT degradation characteristics of Acinetobacter calcoaceticus of the present invention

[0047] According to the methods A and B in Example 1, the degradation characteristics of DBT by the calcium-acetic acid Acinetobacter of the present invention were further studied. The DBT concentration was increased to 0.5-5mmoL / L, the treatment temperature was 30-50°C, and the culture was carried out at 180 rpm for 48-72h. The results showed that the optimal temperature for DBT degradation by Acinetobacter CGMCC No.24322 was 35°C, at which the degradation rate of 5mmoL / LDBT in 72h reached 90.51%, but it still had a degradation effect on DBT at 40-50°C, and the degradation rate of 5mmoL / LDBT in 72h reached 58.32% at 45°C.

[0048] Table 1 Degradation of 5mmoL / LDBT by Acinetobacter calcoaceticus of the present invention

[0049]

[0050]

[0051] Example 3: Tolerance of Acinetobacter calcoaceticus of the present invention to DBT

[0052] According to the methods A and B in Example 1, the tolerance of the Acinetobacter calcoaceticus of the present invention to DBT was further studied. The DBT concentration was increased to 5-20 mmoL / L, the treatment temperature was 35°C, and the culture was carried out at 180 rpm for 72 hours. The results showed that Acinetobacter CGMCC No.24322 had a high tolerance to DBT, and the degradation rates of 5, 8, 10, 15, and 20 mmoL / L DBT in 72 hours were 90.24%, 86.89%, 61.03%, 30.80%, and 3.93%, respectively. Figure 1 and Table 2.

[0053] Table 2 Tolerance of Acinetobacter calcoaceticus to DBT

[0054] DBT concentration mmoL / L 72h degradation rate % 5 90.24 8 86.89 10 61.03 15 30.80 20 3.93

[0055] Example 4: A method for removing organic sulfur from crude oil using the resting cells of Acinetobacter calcoaceticus of the present invention, wherein the volume ratio of crude oil to water phase is 1:10, 1:15, 1:20, respectively, and the treatment temperature is 35°C

[0056] The application of the Acinetobacter calcoaceticus of the present invention in the original desulfurization, the specific method and steps are as follows:

[0057] (1) Cell culture: Acinetobacter with a deposit number of CGMCC No. 24322 was selected and inoculated into an inorganic salt medium containing 0.5 mmoL / L DBT and cultured at 35°C and 180 rpm for 2 days to obtain a cell culture medium;

[0058] (2) Preparing a resting cell suspension: centrifuge the culture medium obtained in step (1) at 4,500 rpm for 15 min, collect the precipitated cells, and resuspend the cells in 100 mM potassium phosphate buffer (pH 7.0). Repeat this process three times until the bacterial cell concentration reaches 10 g dry cells / L. This cell suspension is the prepared resting cell suspension;

[0059] (3) Sample treatment: In the reaction system, the resting cell suspension obtained in step (2) is used as the aqueous phase, which is added to the crude oil to form a two-phase system, so that the volume ratio of the crude oil to the aqueous phase is 1:5-20, and the sample is treated at 35° C. and 180 rpm for 72 h;

[0060] (4) separating the sample: centrifuging the sample treated in step (3) at 10,000 rpm for 10 minutes to obtain an upper crude oil sample;

[0061] (5) Sample testing: The residual sulfur content in the crude oil was measured using a WK-2D microcoulometer, and then compared with the sulfur content of the untreated sample to obtain the desulfurization rate of the high-sulfur crude oil (sulfur content of 34485 mg / L) treated with the Acinetobacter calcoaceticus CGMCC No. 24322.

[0062] The inorganic salt culture medium for culture in the present invention includes water, 2.44g / L KH2PO4, 14.03g / LNa2HPO4.12H2O, 0.36g / L MgCl2.6H2O, 0.001g / L CaCl2.2H2O, 0.001g / L FeCl3.6H2O, 0.004g / L MnCl2.4H2O, and 2g / L NH4Cl.

[0063] The calcoacetic acid bacterium of the present invention still has desulfurization activity for high-sulfur crude oil at an oil-water ratio of 1:10-20, and the desulfurization rates for high-sulfur crude oil (sulfur content of 34485 mg / L) at 35° C. are 3.45%, 8.87% and 13.38% at oil-water ratios of 1:10, 1:15 and 1:20, respectively.

[0064] Table 3 Desulfurization of crude oil at different oil-water ratios at 35°C by Acinetobacter calcoaceticus of the present invention

[0065] Oil-water ratio 72h desulfurization rate % 1:5 0 1:10 3.45 1:15 8.87 1:20 13.38

[0066] Example 5

[0067] The method for removing organic sulfur from crude oil by using the calcoacetic acid Acinetobacter cell culture solution of the present invention comprises the following steps: the volume ratio of crude oil to water phase is 1:20, and the treatment temperature is 35°C.

[0068] The application of the Acinetobacter calcoaceticus in crude oil desulfurization according to the present invention includes the following specific methods and steps:

[0069] (1) Cell culture: Acinetobacter with a deposit number of CGMCC No. 24322 was selected and inoculated into an inorganic salt medium containing 0.5 mmoL / L DBT and cultured at 35°C and 180 rpm for 2 days to obtain a cell culture medium;

[0070] (2) adding the cell culture fluid to a liquid inorganic salt medium to make the bacterial cell concentration reach 12 g dry cells / L to obtain an aqueous phase;

[0071] (3) Sample treatment: In the reaction system, the aqueous phase containing the cell culture medium obtained in step (2) is added to the crude oil to form a two-phase system, so that the volume ratio of the crude oil to the aqueous phase is 1:5-20, and the sample is treated at 35° C. and 180 rpm for 72 h;

[0072] (4) separating the sample: centrifuging the sample treated in step (3) at 10,000 rpm for 10 minutes to obtain an upper crude oil sample;

[0073] (5) Sample testing: The residual sulfur content in the crude oil was measured using a WK-2D microcoulometer, and then compared with the sulfur content of the untreated sample to obtain the desulfurization rate of the high-sulfur crude oil (sulfur content of 34485 mg / L) treated with the Acinetobacter calcoaceticus CGMCC No. 24322.

[0074] The inorganic salt basal culture medium for culture in the present invention comprises water, 2.44 g / L KH2PO4, 14.03 g / L Na2HPO4.12H2O, 0.36 g / L MgCl2.6H2O, 0.001 g / L CaCl2.2H2O, 0.001 g / L FeCl3.6H2O, 0.004 g / L MnCl2.4H2O, and 2 g / L NH4Cl.

[0075] The calcoacetic acid Acinetobacter cell culture solution of the present invention has a desulfurization rate of 13.16% for high-sulfur crude oil (sulfur content of 34485 mg / L) at 35° C. and an oil-water ratio of 1:20, which is equivalent to the desulfurization effect of a resting cell suspension.

[0076] Example 6

[0077] The method for removing organic sulfur from crude oil by using the resting cells of Acinetobacter calcoaceticus of the present invention, wherein the volume ratio of crude oil to water phase is 1:20, and the treatment temperatures are 40, 45, and 50° C.

[0078] The application of the Acinetobacter calcoaceticus in crude oil desulfurization according to the present invention includes the following specific methods and steps:

[0079] (1) Cell culture: Acinetobacter with a deposit number of CGMCC No. 24322 was selected and inoculated into an inorganic salt medium containing 0.5 mmoL / L DBT and cultured at 35°C and 180 rpm for 2 days to obtain a cell culture medium;

[0080] (2) Preparing a resting cell suspension: centrifuge the culture medium obtained in step (1) at 4,500 rpm for 15 min, collect the precipitated cells, and resuspend the cells in 100 mM potassium phosphate buffer (pH 7.0). Repeat this process three times until the bacterial cell concentration reaches 10 g dry cells / L. This cell suspension is the prepared resting cell suspension;

[0081] (3) Sample treatment: In the reaction system, the resting cell suspension obtained in step (2) is used as the aqueous phase, which is added to the crude oil to form a two-phase system, so that the volume ratio of the crude oil to the aqueous phase is 120, and the sample is treated by shaking at 180 rpm for 72 h at 40, 45, and 50° C.;

[0082] (4) separating the sample: centrifuging the sample treated in step (3) at 10,000 rpm for 10 minutes to obtain an upper crude oil sample;

[0083] (5) Sample testing: The residual sulfur content in the crude oil was measured using a WK-2D microcoulometer, and then compared with the sulfur content of the untreated sample to obtain the desulfurization rate of the high-sulfur crude oil (sulfur content of 34485 mg / L) treated with the Acinetobacter calcoaceticus CGMCC No. 24322.

[0084] The basic culture medium for the culture in the present invention includes water, 2.44g / L KH2PO4, 14.03g / LNa2HPO4.12H2O, 0.36g / L MgCl2.6H2O, 0.001g / L CaCl2.2H2O, 0.001g / L FeCl3.6H2O, 0.004g / L MnCl2.4H2O, and 2g / L NH4Cl.

[0085] The calcoacetic acid bacterium of the present invention still has desulfurization activity for high-sulfur crude oil (sulfur content of 34485 mg / L) at 40-50°C when the oil-water ratio is 1:20, wherein the desulfurization rates at 40°C, 45°C and 50°C are 11.28%, 10.02% and 6.33% respectively.

[0086] Table 4 Crude oil desulfurization rate of Acinetobacter calcoaceticus at different temperatures when the oil-water ratio is 1:20

[0087] Temperature ℃ 72h desulfurization rate % 40 11.28 45 10.02 50 6.33

[0088] Example 7: Application of the Acinetobacter calcoaceticus of the present invention to emulsification and viscosity reduction of high-sulfur crude oil

[0089] The growing cell culture fluid of calcoacetic acid acinetobacter CGMCC No.24322 was added to an oil-water two-phase system containing crude oil, and the blank was compared with the same basic culture fluid, so that the volume ratio of crude oil to water phase was 1:5-20, and the mixture was shaken at 180 rpm for 72 hours at 30-50°C, and the macroscopic and microscopic states of emulsification and viscosity reduction were observed by naked eyes and microscope, respectively, and the emulsification viscosity was measured. The results showed that the calcoacetic acid acinetobacter of the present invention had an obvious emulsification and viscosity reduction effect on high-sulfur crude oil, forming obvious emulsified small oil droplets (see Figures 2-3 ), at 45°C and oil-water ratio of 1:5, the emulsification viscosity reduction rate was 33.91%.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Acinetobacter calcoaceticus with a deposit number of CGMCC NO. 24322 ( Acinetobacter calcoaceticus ).

2. A microbial agent, characterized in that: The raw materials include calcium-acetic acid acinetobacter whose preservation number is CGMCC NO. 24322.

3. The method for preparing the microbial agent according to claim 2, characterized in that: include: Step 1: inoculating the Acinetobacter calcoaceticus with a deposit number of CGMCC NO. 24322 into an inorganic salt medium containing dibenzothiophene DBT to obtain a cell culture fluid; Step 2: centrifuging the cell culture fluid, collecting the cell precipitate, and resuspending the cells to obtain a resting cell suspension; Step 3: centrifuge the resting cell suspension, collect the cell precipitate, mix it with a freeze-drying protective agent, and obtain the microbial agent by freeze-drying.

4. The preparation method according to claim 3, characterized in that: In step 1, the culture is carried out at 30-50° C. and 180 rpm for 2-3 days.

5. The preparation method according to claim 3, characterized in that: In step 2, resuspend the cells in 100 mM potassium phosphate buffer, pH 7.0, to a concentration of 5-20 g dry cells / L.

6. Use of the calcoacetic acid acinetobacter according to claim 1, the microbial agent according to claim 2, or the microbial agent prepared by the preparation method according to any one of claims 3 to 5 in crude oil desulfurization.

7. A method for desulfurizing crude oil, characterized in that: The calcoacetic acid Acinetobacter described in claim 1, the microbial agent described in claim 2, or the cell culture fluid, resting cell suspension or microbial agent prepared in the preparation method according to any one of claims 3 to 5 is added to an oil-water two-phase system containing crude oil to carry out a desulfurization reaction.

8. The method according to claim 7, characterized in that The volume ratio of the crude oil to the water phase is 1:5-20; the conditions of the desulfurization reaction are: 30-50°C, 180 rpm oscillation for 48-72 hours.

Images