A mixed bacterial agent for microbial oil washing and its application

Abstract

A mixed bacterial agent for microbial oil washing and its application, the present invention relates to the field of microbial technology, in particular to the technical field of a microbial oil washing agent and its application. The mixed bacterial agent for microbial oil washing of the present invention is composed of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ‑11 and Bacillus jelly-like ( Bacillus mucilaginosus The mixed bacterial agent for microbial oil washing of the present invention has excellent emulsification performance, and its fermentation liquid has low surface tension, which can greatly reduce the interfacial tension level between the oil and the crude oil, and the oil washing efficiency can reach more than 80%.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to the technical field of an oil-cleaning bacterial agent and application thereof. Background Art

[0002] Nowadays, oil pollution is mainly reflected in two aspects: one is oil pollution in the soil, and the other is oil pollution in the ocean. In particular, most of the oil spills in marine oil pollution will migrate to the coastline and eventually adhere to the beach, causing serious harm to the offshore ecological environment and resources. For example, 20 years after the Exxon Valdez oil spill, oil residues can still be detected on the beach. The use of traditional chemical oil washing is costly and prone to secondary pollution, which has an adverse effect on subsequent deep treatments such as biodegradation. The patent application "A biological cleaning agent for treating oily sludge and its use method" (Announcement No. CN108298780A) discloses a biological cleaning agent for cleaning oily solid waste and its preparation method, which is mainly composed of monorhamnolipid. Although this type of cleaning agent has the advantages of being green and environmentally friendly and easy to prepare. However, this method also has the characteristics of a long treatment cycle, a large amount of cleaning agent used (the sand-agent ratio is about 1:10), and the oil washing efficiency is only 60%.

[0003] The oil washing rate is the result of the combined effect of many factors, such as the surfactant's ability to reduce oil-water interfacial tension, its ability to emulsify crude oil, and its ability to change wettability. The oil washing rate characterizes the ability of the surfactant solution to remove the oil film from the particle surface. The larger the value, the stronger the oil washing ability of the oil washing agent. Improving the oil washing efficiency of biological oil washing agents is the future development direction of green and environmentally friendly oil washing. Summary of the invention

[0004] In order to solve the problems of large usage and low oil washing efficiency of existing biological oil cleaning agents, the present invention provides a mixed bacterial agent for microbial oil washing and application thereof.

[0005] The mixed bacterial agent for microbial oil washing of the present invention is composed of Bacillus amyloliquefaciens ( Bacillus starch liquefier ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus )ZZ-8.

[0006] Furthermore, the mixed bacterial agent contains Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus ) The number ratio of ZZ-8 is (10 8 ~10 9 ): (10 7 ~10 8 ).

[0007] Furthermore, the total colony concentration in the mixed bacterial agent is 108 ~10 9 cfu / mL.

[0008] The present invention provides a microorganism that can be used for washing oil, which is Bacillus amyloliquefaciens ( Bacillus starch liquefier ) ZZ-11, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.30788.

[0009] Another microorganism that can be used for oil washing of the present invention is Bacillus jelly-like ( Bacillus mucilaginous ) ZZ-8, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.30789.

[0010] The invention discloses an application of the mixed bacterial agent for microbial oil washing in oil washing.

[0011] Furthermore, the fermentation liquid of the mixed bacterial agent is used for oil washing.

[0012] The mixed bacterial agent used for microbial oil washing of the present invention has excellent emulsification performance, and its fermentation liquid has low surface tension, can greatly reduce the interfacial tension level between the crude oil, and the oil washing efficiency can reach more than 80%.

[0013] The mixed bacterial agent for microbial oil washing of the present invention can effectively treat marine oil pollution and can also be used for crude oil exploitation.

[0014] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), belonging to the genus Bacillus ( Bacillus ), deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC N0.30788, the deposit date is May 28, 2024, and the taxonomic name is Bacillus amyloliquefaciens Bacillus amyloliquefaciens .

[0015] Bacillus jelly-like Mucilaginous Bacillus )ZZ-8, a jelly-like bacillus, belongs to the genus Bacillus ( Bacillus ), deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC N0.30789, the deposit date is May 28, 2024, and the taxonomic name is Bacillus jelly Mucilaginous Bacillus . BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ-11 Gram staining observation diagram;

[0017] Figure 2 Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )Observation of colonies cultured on ZZ-11 plate;

[0018] Figure 3 It is a jelly-like Bacillus ( Mucilaginous Bacillus ) ZZ-8 Gram staining observation picture;

[0019] Figure 4 It is a jelly-like Bacillus ( Mucilaginous Bacillus ) Observation of colonies cultured on ZZ-8 plate;

[0020] Figure 5 This is a curve diagram of the water separation rate of ZZ-11, ZZ-8 and mixed bacterial agents in the emulsification performance test of Example 1. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0023] Specific implementation method 1: In this implementation method, a microorganism that can be used for oil washing is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ-11, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.30788.

[0024] In this embodiment, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ-11 was isolated and purified from the rhizosphere soil of crops in Harbin City in September 2023.

[0025] ① The separation method uses the gradient dilution method: remove the topsoil, take a few dozen grams of petroleum-contaminated soil from a depth of 5 to 20 cm, and put it into a sterilized waterproof paper bag; take 10g of petroleum-contaminated soil and shake it in a 90mL sterile water triangle bottle (shake on a shaker for 30min\standstill), then take 5mL of the supernatant and inoculate it into 95mL of sterile beef extract liquid culture medium, and culture it in a constant temperature shaker at a temperature of 30℃ and a shaker speed of 150r / min for 2 days; then take the soil sample liquid and dilute it step by step to 10 -6 Pick a single colony and inoculate it into LB medium plate, spread it evenly and culture it at 28℃.

[0026] ②Purification method: Repeated streaking culture until it is confirmed that the morphology and size of the colonies on the same plate are the same, and the purified single strain ZZ-11 is obtained.

[0027] ③ Gram staining: Pick a single colony for Gram staining and examine under a microscope to identify the type of bacteria.

[0028] ④16S sequencing identification: the nucleotide sequence of 16S rDNA of the strain is shown in SEQ ID NO: 1.

[0029] The 16S rDNA sequence of this strain was compared with that of Bacillus sp. ( Bacillus ) of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) have extremely high homology, with a homology of more than 98%, and combined with the Gram staining of the strain (such as Figure 1 The results of microscopic examination showed that it was Bacillus amyloliquefaciens and was named Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11.

[0030] In this embodiment, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ-11 can be cultured in LB medium at a temperature of 28-30°C. Bacillus amyloliquefaciens ) The colony morphology of ZZ-11 on LB solid medium is dirty white round or irregular colonies with a rough and sticky surface (such as Figure 2 Gram-positive bacteria were identified by Gram staining.

[0031] Specific embodiment 2: In this embodiment, a microorganism that can be used for oil washing is a jelly-like Bacillus ( Mucilaginous Bacillus ) ZZ-8, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.30789.

[0032] The jelly-like Bacillus ( Mucilaginous Bacillus )ZZ-8 was isolated and purified from the rhizosphere soil of crops in Harbin City in September 2023.

[0033] The separation method uses the gradient dilution method: remove the topsoil, take a few dozen grams of petroleum-contaminated soil from a depth of 5 to 20 cm, and put it into a sterilized waterproof paper bag; take 10g of petroleum-contaminated soil and shake it in a 90mL sterile water triangle bottle (shake on a shaker for 30 minutes\standstill), then take 5mL of the supernatant and inoculate it into 95mL of sterile beef extract liquid culture medium, and culture it in a constant temperature shaker at a temperature of 30℃ and a shaker speed of 150r / min for 2 days; then take the soil sample liquid and dilute it step by step to 10 -6 Pick a single colony and inoculate it into LB medium plate, spread it evenly and culture it at 28℃.

[0034] ②Purification method: Repeated streaking culture until it is confirmed that the morphology and size of the colonies on the same plate are the same, and the purified single strain ZZ-8 is obtained.

[0035] ③ Gram staining: Pick a single colony for Gram staining and examine under a microscope to identify the type of bacteria.

[0036] ④16S sequencing identification: the nucleotide sequence of 16S rDNA of the strain is shown in SEQ ID NO: 2.

[0037] The 16S rDNA sequence of this strain was compared with that of Bacillus sp. ( Bacillus ) of the jelly-like Bacillus ( Mucilaginous Bacillus ) have extremely high homology, with a homology of more than 98%, and combined with the Gram staining of the strain (such as Figure 3 The results of microscopic examination confirmed that it was a jelly-like Bacillus and named it as jelly-like Bacillus ( Mucilaginous Bacillus )ZZ-8.

[0038] In this embodiment, the jelly-like Bacillus ( Mucilaginous Bacillus ) ZZ-8 can be cultured in LB medium at a temperature of 28-30°C. Mucilaginous Bacillus ) The colony morphology of ZZ-8 on LB solid medium is colorless, transparent, shiny, smooth, rounded, sticky, and has neat edges (such as Figure 4 Gram-negative bacteria were identified by Gram staining.

[0039] Specific implementation method three: The mixed bacterial agent used for microbial oil washing in this implementation method is composed of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus )ZZ-8.

[0040] Specific embodiment 4: The difference between this embodiment and specific embodiment 3 is that: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11 and Bacillus jelly-like ( Bacillus mucilaginous ) The number ratio of ZZ-8 is (10 8 ~10 9 ): (10 7 ~10 8 ). Other aspects are the same as those of the third specific implementation method.

[0041] Specific implementation method 5: The difference between this implementation method and specific implementation method 3 or 4 is that the total concentration of colonies in the mixed bacterial agent is 10 8 ~10 9 cfu / mL. Others are the same as those in the third or fourth embodiment.

[0042] Example 1

[0043] Emulsification performance test:

[0044] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11、Bacillus jelly( Mucilaginous Bacillus ) ZZ-8, mixed bacterial agent (Bacillus amyloliquefaciens ( Bacillus starch liquefier ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus ) and ZZ-8 were mixed in a 1:1 ratio and inoculated into LB liquid medium, and cultured and fermented at 28-30°C under shaking conditions until the colony concentration in the fermentation liquid reached 10 8 ~10 9 cfu / mL.

[0045] Add 5 ml of the fermentation liquid and 5 ml of crude oil to a 25 ml stoppered measuring cylinder, and then place it in a 45 °C thermostat for 20 minutes. After sealing, oscillate the stoppered measuring cylinder vigorously for 5 minutes, and then place it in a 45 °C thermostat again to stand. Start timing at the same time, record the volume of water separated in the test tube every 10 minutes, and calculate the water separation rate. Repeat the experiment three times. The formula is Ed=Vw / V, where Ed is the water separation rate; Vw is the amount of water separated, mL; V is the total water phase volume, mL.

[0046] The water separation rate is used to evaluate the stability of the water-in-oil emulsion formed by the biosurfactant acting on crude oil. The smaller the water separation rate, the more stable the emulsion and the stronger the emulsification performance of the system. It can be seen from the figure that the water separation rate of each group increases with time. After a period of time, the water separation rates of ZZ-11, ZZ-8, and mixed bacterial agents are stable, which are 47%, 44%, and 16%, respectively (such as Figure 5 as shown).

[0047] The water separation rate can be used to evaluate the emulsification performance of biosurfactants on crude oil. According to the experimental results, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ-11 and Bacillus jelly-like ( Bacillus mucilaginous ) ZZ-8 fermentation broth has good emulsifying properties, and the emulsifying properties of the mixed bacteria are more excellent and outstanding.

[0048] Example 2

[0049] Surface tension test: The surface tension of the fermentation supernatant of the strain was measured using a platinum plate.

[0050] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11、Bacillus jelly( Mucilaginous Bacillus ) ZZ-8, mixed bacterial agent (Bacillus amyloliquefaciens ( Bacillus starch liquefier ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus ) and ZZ-8 were mixed in a 1:1 ratio and inoculated into LB liquid medium, and cultured and fermented at 28-30°C under shaking conditions until the colony concentration in the fermentation liquid reached 10 8 ~10 9 cfu / mL.

[0051] 30 mL of fermentation broth was taken from each group, centrifuged at 10000×g for 10 min at room temperature, and the fermentation supernatant of each group was taken. The surface tension of the fermentation broth of each group was measured using a surface tension meter. The measurement was repeated 3 times and the average value was taken. The test results are shown in Table 1.

[0052] Surface tension is an important indicator of the ability of oil washing agent to wash crude oil. The lower the value, the stronger the washing ability. The test results show that Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus ) The surface tension of the supernatant of ZZ-8 fermentation can be reduced, and the surface tension of the supernatant of mixed bacterial fermentation is even lower.

[0053] Table 1

[0054]

[0055] Example 3

[0056] Interfacial tension test: At 45°C, the interfacial tension between the fermentation supernatant and crude oil was measured using a TX500C interfacial tension meter.

[0057] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11、Bacillus jelly( Mucilaginous Bacillus ) ZZ-8, mixed bacterial agent (Bacillus amyloliquefaciens ( Bacillus starch liquefier ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus) and ZZ-8 were mixed in a 1:1 ratio and inoculated into LB liquid medium, and cultured and fermented at 28-30°C under shaking conditions until the colony concentration in the fermentation liquid reached 10 8 ~10 9 cfu / mL.

[0058] Take 30 mL of fermentation broth from each group, centrifuge at 10,000 × g for 10 min at room temperature, take the fermentation supernatant from each group, and measure it with a TX500C interfacial tension meter at 45°C. Take the same volume of LB liquid culture medium as the blank group. -3 When the interfacial tension is within 0.133 N / m, the measured system can be considered to have reached equilibrium and the final interfacial tension is recorded. The test results are shown in Table 2.

[0059] Table 2

[0060]

[0061] From the experimental results in Table 2, it can be seen that compared with the blank, Bacillus amyloliquefaciens ( Bacillus starch liquefier )ZZ-11、Bacillus jelly( Mucilaginous Bacillus ) ZZ-8 has good interfacial activity, with the oil-water interface as low as 1.92 mN / m and 1.90 mN / m, which is a low interfacial tension level. The interfacial activity of the mixed bacterial agent is more prominent, as low as 0.68 mN / m.

[0062] Example 4

[0063] Oil washing experiment:

[0064] Crude oil and quartz sand were mixed evenly at a mass ratio of 1:5 to prepare oil sand, which was placed in a 60°C oven for aging for 24 h, taken out and sealed for later use.

[0065] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11、Bacillus jelly( Mucilaginous Bacillus ) ZZ-8, mixed bacterial agent (2 groups, Bacillus amyloliquefaciens ( Bacillus starch liquefier ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus ) ZZ-8 were mixed in a ratio of 1:1 and 3:2) and inoculated into LB liquid medium respectively, and cultured and fermented at 28-30°C under shaking conditions until the colony concentration in the fermentation broth reached 10 8 ~10 9 cfu / mL.

[0066] Pour 30 mL of fermentation liquid from each group into different centrifuge tubes, and take another 30 mL of LB liquid culture medium of the same volume as the blank group; then put 15 g of the aged oil sand (initial oil content of the oil sand is 2.5 g) into each centrifuge tube, seal it, shake it evenly for 5 times, put it into a 60°C oven and let it stand for 24 hours. After pouring out the upper liquid, rinse it repeatedly with 20-25°C distilled water until the water phase is colorless, and collect and combine the rinse liquid.

[0067] The oil sand is then dried and weighed (centrifuge tube + oil sand), the mass is m1, and the mass of the centrifuge tube is m0.

[0068] Oil washing efficiency the Calculate using the following formula:

[0069] the =(15-(m1-m0)) / 2.5

[0070] The results of the oil washing efficiency tests of each group are shown in Table 3. Bacillus starch liquefier ) ZZ-11 and Bacillus jelly-like ( Mucilaginous Bacillus ) and ZZ-8 fermentation broths all have excellent oil washing efficiency, and the oil washing efficiency of the mixed bacterial agent of the present invention is even better, reaching 84.39% and 93.32 respectively.

[0071] Table 3

[0072]

[0073] The test results of Examples 1 to 4 show that Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )ZZ-11、Bacillus jelly( Mucilaginous Bacillus ) ZZ-8 can be used as biological oil-washing bacteria; the mixed bacterial agent of the present invention can separate oil from the surface of oil sands. After washing the oil, the oil sands are clean and loose, which can prevent crude oil from adhering again and has a very excellent oil-washing effect.

Claims

1. The application of mixed bacterial agent in oil washing is characterized by: The mixed bacterial agent is composed of Bacillus amyloliquefaciens ZZ-11, taxonomically named Bacillus amyloliquefaciens Bacillus amyloliquefaciens , the deposit number is CGMCC No. 30788 and jelly-like Bacillus ZZ-8, and the taxonomic name is jelly-like Bacillus Bacillus mucilaginosus , deposit number CGMCCN0.30789.

2. The use of the mixed bacterial agent in washing oil according to claim 1, characterized in that: The fermentation liquid of mixed bacterial agents is used for oil washing.

3. The use of the mixed bacterial agent in washing oil according to claim 1, characterized in that: The ratio of Bacillus amyloliquefaciens ZZ-11 to Bacillus gelatinous ZZ-8 in the mixed bacterial agent is 10 8 ~10 9 :10 7 ~10 8 .

4. The use of the mixed bacterial agent in washing oil according to claim 1 or 3, characterized in that: The total colony concentration in the mixed bacterial agent is 10 8 ~10 9 cfu / mL.

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