High-efficiency degradation thermophilic oil removal strain, oil removal microbial preparation, oil removal product and application of high-efficiency degradation thermophilic oil removal strain

Abstract

The invention provides a thermophilic oil removal strain capable of efficiently degrading, an oil removal microbial preparation, an oil removal product and application of the thermophilic oil removal strain, the oil removal microbial preparation and the oil removal product, the thermophilic oil removal strain is classified and named as Geobacillus sp. ZhaoKe-HT and is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 20241735, and the preservation date is August 5, 2024. The oil removal strain provided by the invention can solve the problem that the traditional biodegradation technology is poor in oil stain degradation effect in a high-temperature environment, can maintain relatively high degradation activity in the high-temperature environment, remarkably improves the oil stain removal effect in the high-temperature environment, and expands the application range of a microbial degradation technology.

Description

Technical Field

[0001] The invention relates to the technical field of oil removal strains, in particular to thermophilic oil removal strains with high-efficiency degradation, oil removal microbial preparations, oil removal products and applications thereof. Background Art

[0002] Microbial oil removal technology is an environmentally friendly technology that uses microorganisms to degrade oil pollutants. It is widely used in the removal of oil pollution such as edible oil, petroleum and motor oil. This technology is based on the ability of microorganisms to decompose complex oil compounds into simple harmless substances through their metabolic activities. It has the advantages of low cost, high efficiency and environmental protection. The basic principle of oil removal is: microbial degradation of oil pollutants mainly depends on the enzymes they produce. These enzymes can break down oil into smaller molecules, making them easier to be absorbed and further degraded by microorganisms. For example, some bacteria and fungi can secrete lipase and hydrocarbon oxidase to decompose complex oil compounds into simple molecules such as fatty acids, glycerol and carbon dioxide.

[0003] Traditional microbial oil removal technology is usually carried out at room temperature, but in certain specific environments, such as high-temperature industrial wastewater treatment or oil production areas, the existence of high-temperature environments makes it difficult for conventional microorganisms to survive and work. For this reason, the present invention provides strains that can effectively degrade oil in high-temperature environments. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a strain capable of effectively removing oil under high temperature environment.

[0005] In the first aspect, a thermophilic oil-removing strain with high efficiency of degradation is provided, which is classified and named Geobacillus sp. ZhaoKe-HT (referred to as strain ZhaoKe-HT), and is deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO: M 20241735 and the deposit date: August 5, 2024.

[0006] The oil removal strain provided by the present invention can solve the problem that traditional biodegradation technology has poor oil degradation effect in a high temperature environment, can maintain a high degradation activity in a high temperature environment, significantly improves the oil removal effect in a high temperature environment, and expands the application scope of microbial degradation technology.

[0007] In the second aspect, a method for preparing a thermophilic oil removal strain with high efficiency degradation is provided, specifically:

[0008] 10 g of oil-contaminated soil sample (obtained from a petroleum sludge treatment plant in Jinzhou, Liaoning) was diluted with 20 mL of distilled water, and 5 mL of the soil sample solution was added to a 50 mL conical flask of MSM culture medium for enrichment culture in a neutral environment and 60 ° C. After 5 days, it was transferred to fresh MSM culture medium. After repeated transfers, the culture solution was diluted and spread on an inorganic salt solid culture medium with petroleum extract as the sole carbon source, and the colonies grown on the plate were rescreened, and the rescreened colonies were separated by streaking to obtain a strain that can grow with petroleum as the sole carbon source, which is the Geobacillus sp. ZhaoKe-HT described in the first aspect.

[0009] Preferably, the strain is identified:

[0010] The 16S rRNA universal primers (27F 5'-AGAGTTTGATCCTGGCTCA-3' and 1492R 5'

[0011] -AGAGTTTGATCCTGGCTCA-3') was amplified and sequenced, and the amplification results were sequenced; the nucleotide BLAST (BLASTn) in the NCBI database was used to search the 16S rRNA sequence of the strain in BLAST. The phylogenetic tree established using the neighbor-joining (NJ) method of MEG11 was as follows: Figure 1 As shown, the strain is most closely related to Geobacillus sp., and combined with physiological and biochemical mycological characteristics and phylogenetic tree analysis, it was identified as Geobacillus sp. ZhaoKe-HT.

[0012] In a third aspect, a dormant cell system capable of degrading oil pollution is provided, which is prepared from the thermophilic oil removal strain with high efficiency in degradation described in the first aspect, and the preparation process is as follows:

[0013] The strain Geobacillus sp. ZhaoKe-HT was picked up and placed in 50 mL of LB medium, and the strain Geobacillus sp. ZhaoKe-HT was cultured in a constant temperature shaker at 60°C and 200 rpm until the logarithmic growth phase;

[0014] Centrifuge at 4000 rpm for 20 min in a low-temperature centrifuge to collect resting cells, remove the supernatant and add 30 mL of PBS buffer, mix thoroughly by pipetting, centrifuge at 6000 rpm for 10 min, repeat washing 3 times, and finally resuspend with 50 mL of PBS buffer and transfer to a 250 mL conical flask;

[0015] After culturing for 4 h at 60° C. and 200 rpm in a constant temperature shaker, the cells were taken out to obtain cells in a starved state, namely, a resting cell system.

[0016] In a fourth aspect, an oil-removing microbial preparation is provided, wherein the preparation contains the thermophilic oil-removing strain with high efficiency in degradation as described in the first aspect.

[0017] In a fifth aspect, an oil removal product is provided, wherein the product contains the oil removal microbial preparation described in the fourth aspect.

[0018] Preferably, the product is a daily chemical product, a microbial detergent or a kitchen waste degrading agent.

[0019] In a sixth aspect, an application of an oil-removing microbial preparation is provided, wherein the oil-removing microbial preparation described in the fourth aspect is applied to an environment requiring oil removal to perform a degradation reaction.

[0020] Preferably, the oil pollution is oil pollution such as edible oil, petroleum or motor oil.

[0021] From the above description of the present invention, it can be seen that the present invention has the following beneficial effects:

[0022] 1. The oil removal strain provided by the present invention can solve the problem that the traditional biodegradation technology has a poor degradation effect on oil pollution in a high temperature environment, can maintain a high degradation activity in a high temperature environment, significantly improves the removal effect of oil pollution in a high temperature environment, and expands the application scope of microbial degradation technology;

[0023] 2. Prepare the strain ZhaoKe-HT into a dormant cell system to retain its biological activity, so that it can still effectively degrade oil pollution in a non-growing state;

[0024] 3. Prepare resting cell systems that are more stable during handling and storage, easier to transport and use, and reduce operating costs and complexity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] The thermophilic oil-removing strain with high efficiency of degradation provided by the present invention was deposited in the China Center for Type Culture Collection on August 5, 2024, with the deposit number being CCTCC NO: M 20241735.

[0027] Figure 1 is a schematic diagram of the evolutionary tree of strain ZhaoKe-HT;

[0028] Figure 2 This is a schematic diagram of the microscopic examination of the strain ZhaoKe-HT;

[0029] Figure 3This is the GC-MS analysis of oil pollution components after 4 days of culture in the ZhaoKe-HT resting cell system;

[0030] Figure 4 This is the degradation curve of oil pollution by the ZhaoKe-HT resting cell system. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] Example 1 provides a thermophilic oil removal strain with high efficiency degradation, named Geobacillus sp. ZhaoKe-HT, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241735 and a deposit date of August 5, 2024.

[0033] The oil removal strain provided by the present invention can solve the problem that traditional biodegradation technology has poor oil degradation effect in a high temperature environment, can maintain a high degradation activity in a high temperature environment, significantly improves the oil removal effect in a high temperature environment, and expands the application scope of microbial degradation technology.

[0034] Embodiment 2 provides a method for preparing a thermophilic oil removal strain with high efficiency degradation, specifically:

[0035] 10 g of oil-contaminated soil sample (obtained from a petroleum sludge treatment plant in Jinzhou, Liaoning) was diluted with 20 mL of distilled water, and 5 mL of the soil sample solution was added to a 50 mL MSM medium conical flask for enrichment culture in a neutral environment and 60° C. After 5 days, it was transferred to fresh MSM medium. After repeated transfers, the culture solution was diluted and spread on an inorganic salt solid medium with petroleum extract as the sole carbon source. The colonies grown on the plate were rescreened, and the rescreened colonies were separated by streaking to obtain a strain that can grow with petroleum as the sole carbon source, which is the Geobacillus sp. ZhaoKe-HT described in the first embodiment.

[0036] In this example, the strain was also identified:

[0037] The 16S rRNA universal primers (27F 5′-AGAGTTTGATCCTGGCTCA-3′ and 1492R 5′-AGAGTTTGATCCTGGCTCA-3′) were used for amplification and sequencing, and the amplification results were sequenced; the 16S rRNA sequences of the strains were searched in BLAST using the nucleotide BLAST (BLASTn) in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) The phylogenetic tree established using the neighbor-joining (NJ) method of MEG11 was as follows Figure 1 As shown, the strain is most closely related to Geobacillus sp., and combined with physiological and biochemical mycological characteristics and phylogenetic tree analysis, it was named Geobacillus sp. ZhaoKe-HT.

[0038] In this embodiment, a Gram staining experiment is also performed on the thermophilic oil-removing strain, and the specific steps are as follows:

[0039] 1. Take a small amount of strain ZhaoKe-HT culture and make a bacterial suspension with sterile saline.

[0040] 2. Make a smear on a glass slide and allow it to dry naturally.

[0041] 3. Stain with crystal violet for 1 minute and rinse with water.

[0042] 4. Fix with iodine solution for 1 minute and rinse with water.

[0043] 5. Decolorize with 95% ethanol for 30 seconds and rinse with water.

[0044] 6. Dye with safranin for 1 minute, rinse with water and let dry.

[0045] Observe through a microscope, such as Figure 2 As shown, strain ZhaoKe-HT is a Gram-positive bacillus.

[0046] Example 3 provides a dormant cell system capable of degrading oil pollution, which is prepared from the thermophilic oil-removing strain with high efficiency in degradation described in Example 1. The preparation process is as follows:

[0047] The strain Geobacillus sp. ZhaoKe-HT (i.e., strain ZhaoKe-HT) was picked up into 50 mL of LB medium, and the strain Geobacillus sp. ZhaoKe-HT was cultured in a constant temperature shaker at 60° C. and 200 rpm until the logarithmic growth phase;

[0048] Centrifuge at 4000 rpm for 20 min in a low-temperature centrifuge to collect resting cells, remove the supernatant and add 30 mL of PBS buffer, mix thoroughly by pipetting, centrifuge at 6000 rpm for 10 min, repeat washing 3 times, and finally resuspend with 50 mL of PBS buffer and transfer to a 250 mL conical flask;

[0049] After culturing for 4 h at 60° C. and 200 rpm in a constant temperature shaker, the cells were taken out to obtain cells in a starved state, namely, a resting cell system.

[0050] In this embodiment, the strain ZhaoKe-HT is prepared into a dormant cell system to retain its biological activity so that it can still effectively degrade oil pollution in a non-growing state. In addition, the preparation of the dormant cell system is more stable during handling and storage, easy to transport and use, and reduces operating costs and complexity.

[0051] In order to evaluate the ability of Geobacillus sp.ZhaoKe-HT to degrade oil in a resting cell system, the following experiments were conducted:

[0052] The optical density of the resting cell system was measured using a UV spectrophotometer, and the OD600 was adjusted to 5 with PBS buffer;

[0053] The resting cell suspension was divided into several 50 mL ground-mouth conical flasks. The total reaction system in the flask was 10 mL. Three parallels were made for each sampling point to reduce the influence of systematic errors and accidental errors.

[0054] Add 1% washing oil extract and 1% edible oil extract, place the conical flask in a constant temperature shaker and culture at 60°C (200rpm), take samples from the whole bottle at regular intervals to detect the concentrations of substrate and product, and add an equal volume of ethyl acetate to the sample to terminate the system reaction and extract.

[0055] Degradation effect detection:

[0056] Take the supernatant of the extracted sample and filter it with a 0.22μm filter membrane to use it as a GC-MS detection sample.

[0057] Agilent 7890 Gas Chromatography was used as the analytical instrument, equipped with 5977 Mass Spectrometer mass spectrometer, and measured using HP-5MS 30m x 0.25mm x 0.25μm capillary column.

[0058] Helium was used as the carrier gas, and the detection program was initially set to 70°C for 5 min, then increased to 240°C at a rate of 15°C / min, and then increased to 300°C at a rate of 20°C / min, with an injection volume of 2 μl.

[0059] Experimental conclusion: After four days of resting the cell system, the oil content decreased significantly, and the degradation rate reached 40%. Figure 3 and Figure 4 shown.

[0060] In this example, a high-temperature oily wastewater degradation experiment was also conducted:

[0061] 1. Pick the strain ZhaoKe-HT into 50mL LB medium, and culture the strain ZhaoKe-HT in a 60℃ constant temperature shaker at 200rpm until the logarithmic growth phase. Centrifuge at 4000rpm for 20min to collect the cells, add them to the oily wastewater, and adjust the OD to about 0.4.

[0062] 2. Maintain the reaction time at 55°C, 60°C and 65°C for 3 days, stirring regularly to ensure sufficient contact between the microorganisms and the oil pollution. After the reaction, detect the residual oil pollutants in the wastewater.

[0063] After 3 days of bioremediation with strain ZhaoKe-HT, the oil in the oily wastewater was well degraded. The degradation rates at 55°C, 60°C and 65°C were 37.7%, 39.4% and 39% respectively, as shown in the following table:

[0064] Table 1 Degradation of high temperature oily wastewater

[0065] Time / Day 55℃ 60℃ 65℃ 0 100.28±0.82mg / L 102.90±0.73mg / L 95.07±1.83mg / L 1 84.54±1.07mg / L 87.57±1.22mg / L 86.78±0.94mg / L 2 76.87±1.45mg / L 74.06±0.20mg / L 70.57±0.92mg / L 3 62.51±0.48mg / L 62.38±0.49mg / L 58.04±0.35mg / L

[0066] Embodiment 4 provides an oil removal microbial preparation, wherein the preparation contains the thermophilic oil removal strain with high efficiency degradation as described in the first embodiment.

[0067] Embodiment 5 provides an oil removal product, the product containing the oil removal microbial preparation described in embodiment 4. Preferably, the product is a daily chemical product, a microbial detergent or a kitchen waste degrading agent.

[0068] In the sixth embodiment, an application of an oil-removing microbial preparation is provided, wherein the oil-removing microbial preparation described in the fourth embodiment is applied to an environment requiring oil removal to perform a degradation reaction. Preferably, the oil pollution is oil pollution such as edible oil, petroleum or motor oil.

[0069] Although the above embodiments have been shown and described, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. Changes, modifications, substitutions and variations of the above embodiments by those of ordinary skill in the art are all within the scope of protection of the present invention.

Claims

1. A thermophilic oil removal strain with high efficiency degradation, characterized in that: Its classification name is Geobacillus sp. ZhaoKe-HT, and it is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 20241735 and the preservation date is August 5, 2024.

2. An oil-removing microbial preparation, characterized in that: The preparation contains the thermophilic oil-removing strain with high efficiency of degradation as claimed in claim 1.

3. A degreasing product, characterized in that: The product contains the oil-removing microbial preparation according to claim 2.

4. A degreasing product according to claim 3, characterized in that: The product is a daily chemical product, a microbial detergent or a kitchen waste degrading agent.

5. A use of the oil removal microbial preparation as claimed in claim 2, characterized in that: The oil removal microbial preparation is used for degrading oil pollution.

6. The use according to claim 5, characterized in that: The oil pollution is oil pollution such as edible oil, petroleum or motor oil.

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