Efficient-degradation oil removal strain, oil removal microbial preparation, oil removal product and application of oil removal strain, oil removal microbial preparation and oil removal product
By developing the Bacillus strain ZhaoKe-MT and its rest cell system, the problem of insufficient oil removal ability in the medium-temperature environment was solved, the effect of efficient degradation of oil pollutants was achieved, and the application convenience and stability of the technology were improved.
Patent Information
- Application Number
- CN202411222884.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-02
- Publication Date
- 2025-01-10
AI Technical Summary
In the prior art, there are few strains that have efficient oil removal capabilities in medium-temperature environments, and it is difficult to meet the demand for microbial degradation of oil pollutants.
A new Bacillus strain ZhaoKe-MT was developed to have the ability to efficiently degrade oil pollutants under medium-temperature environments through specific culture and identification methods, and to prepare its resting cell system for easy application.
The oil removal effect is significantly improved under the medium temperature environment, and the application scope of microbial degradation technology is expanded. The rest cell system can still effectively degrade oil stains in the non-growth state, with high stability, convenient transportation and application, reducing operating costs and complexity sex.
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Figure CN119286675A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of oil removal strains, in particular to highly efficient oil removal strains, 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 (i.e. mesophilic temperature). Mesophilic environment (20°C to 45°C) is the most suitable growth temperature range for most microorganisms. Within this temperature range, some microorganisms show good oil removal ability and can efficiently degrade various oil pollutants. However, there are not many strains that can have high oil removal ability in mesophilic environment. Therefore, it is necessary to further obtain new oil removal strains with high degradation ability in mesophilic environment. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a new strain capable of effectively removing oil in a medium temperature environment.
[0005] In the first aspect, a highly efficient oil-removal strain is provided, which is classified and named Bacillus sp. ZhaoKe-MT (referred to as strain ZhaoKe-MT), and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M20241736 and a deposit date of August 5, 2024.
[0006] The oil removal strain provided by the present invention can have high degradation activity in a medium temperature environment, thereby expanding the application scope of microbial degradation technology.
[0007] In the second aspect, a method for preparing an oil-removing 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 30°C to 37°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 Bacillus sp. ZhaoKe-MT 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 Bacillus. Combining physiological and biochemical mycological characteristics and phylogenetic tree analysis, it was identified as Bacillus sp. ZhaoKe-MT.
[0012] In a third aspect, a dormant cell system capable of degrading oil pollution is provided, which is prepared from the highly efficient oil-removing strain described in the first aspect, and the preparation process is as follows:
[0013] The strain Bacillus sp. ZhaoKe-MT was picked up and placed in 50 mL LB medium, and the strain Bacillus sp. ZhaoKe-MT was cultured in a constant temperature shaker at 30°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 in a constant temperature shaker at 30° C. and 200 rpm, 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 highly efficient oil-removing strain 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 have a good oil removal effect under medium temperature environment;
[0023] 2. Prepare the strain ZhaoKe-MT 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 highly efficient oil-removing strain provided by the present invention is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241736 and a deposit date of August 5, 2024.
[0027] Figure 1 is a schematic diagram of the evolutionary tree of strain ZhaoKe-MT;
[0028] Figure 2 This is a schematic diagram of the microscopic examination of strain ZhaoKe-MT;
[0029] Figure 3 This is the GC-MS analysis of oil pollution components after 4 days of culture of the ZhaoKe-MT resting cell system;
[0030] Figure 4This is the degradation curve of oil by the ZhaoKe-MT 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 an efficient oil removal strain, named Bacillus sp. ZhaoKe-MT, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241736 and a deposit date of August 5, 2024.
[0033] The oil removal strain provided by the present invention has a good oil removal effect in a medium temperature environment, thereby expanding the application scope of microbial degradation technology.
[0034] Embodiment 2 provides a method for preparing an oil-removing 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 conical flask of MSM culture medium for enrichment culture in a neutral environment and 30° C. to 37° 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 Bacillus sp. ZhaoKe-MT 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 1As shown, the strain is most closely related to Bacillus sp., and combined with physiological and biochemical mycological characteristics and phylogenetic tree analysis, it was named Bacillus sp. ZhaoKe-MT.
[0038] In this embodiment, a Gram staining experiment is also performed on the oil removal strain, and the specific steps are as follows:
[0039] 1. Take a small amount of strain ZhaoKe-MT 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 dry.
[0045] Observe through a microscope, such as Figure 2 As shown, strain ZhaoKe-MT is a Gram-positive bacillus.
[0046] Example 3 provides a dormant cell system capable of degrading oil pollution, which is prepared from the highly efficient oil-removing strain described in Example 1. The preparation process is as follows:
[0047] The strain Bacillus sp. ZhaoKe-MT (i.e., strain ZhaoKe-MT) was picked up and placed in 50 mL of LB medium, and the strain Bacillus sp. ZhaoKe-MT was cultured in a constant temperature shaker at 30° 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 in a constant temperature shaker at 30° C. and 200 rpm, the cells were taken out to obtain cells in a starved state, namely, a resting cell system.
[0050] In this embodiment, the strain ZhaoKe-MT 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 strain Bacillus sp.ZhaoKe-MT 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 30°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 30%. Figure 3 and Figure 4 shown.
[0060] In this example, a medium-temperature oily wastewater degradation experiment was also conducted:
[0061] 1. Pick the strain ZhaoKe-MT into 50mL LB medium, and culture the strain ZhaoKe-MT in a 30℃ 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 room temperature of 30°C, 37°C and 45°C for 21 days, stirring regularly to ensure sufficient contact between the microorganisms and the oil pollution. After the reaction, detect the residual amount of oil pollutants in the wastewater.
[0063] After 3 weeks of bioremediation with strain ZhaoKe-MT, the oil in the oily wastewater was well degraded. The degradation rates at room temperature of 30°C, 37°C and 45°C were 30%, 35% and 28%, respectively, as shown in the following table:
[0064] Table 1 Degradation table of oily wastewater at room temperature
[0065] Time / week 30℃ 37℃ 45℃ 0 97.22±0.67mg / L 98.07±1.54mg / L 103.03±0.63mg / L 1 88.76±0.96mg / L 89.45±0.72mg / L 95.44±1.58mg / L 2 81.00±0.04mg / L 76.29±1.08mg / L 82.20±1.01mg / L 3 68.05±0.75mg / L 63.75±1.84mg / L 74.18±0.35mg / L
[0066] Embodiment 4 provides an oil removal microbial preparation, wherein the preparation contains the highly efficient oil removal strain 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. An efficient oil-removing strain characterized by: Its classification name is Bacillus sp. ZhaoKe-MT is deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO:M 20241736 and the deposit date being August 5, 2024.
2. An oil-removing microbial preparation, characterized in that: The preparation contains the highly efficient oil-removing strain according to 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.