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

Abstract

The invention provides an efficient-degradation psychrophilic oil removal strain, an oil removal microbial preparation, an oil removal product and application of the efficient-degradation psychrophilic oil removal strain, the oil removal microbial preparation and the oil removal product, the psychrophilic oil removal strain is classified and named as Rhodococcus sp. ZhaoKe-LT and is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20241737, 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 low-temperature environment, can maintain high degradation activity in the low-temperature environment, remarkably improves the oil stain removal effect in the low-temperature environment, and expands the application range of the microbial degradation technology.

Description

Technical Field

[0001] The invention relates to the technical field of oil removal strains, in particular to a high-efficiency degrading psychrophilic oil removal strain, an oil removal microbial preparation, an oil removal product 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. The medium temperature environment is the most suitable growth temperature range for most microorganisms. Within this temperature range, many microorganisms show good oil removal ability and can efficiently degrade various types of oil pollutants. However, in some low temperature environments (0°C to 20°C), the oil removal ability of these microorganisms is limited or even ineffective. For this reason, the present invention provides a strain that can always maintain a high oil removal ability in a low temperature environment. Summary of the invention

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

[0005] In the first aspect, a high-efficiency degradation psychrophilic oil-removing strain is provided, which is classified and named Rhodococcus sp. ZhaoKe-LT (referred to as strain ZhaoKe-LT), and is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M20241737 and a preservation date of 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 low temperature environment, can maintain high degradation activity in low temperature environment, significantly improves the oil removal effect in low temperature environment, and expands the application scope of microbial degradation technology.

[0007] In the second aspect, a method for preparing a psychrophilic 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 MSM medium conical flask in a neutral environment and at room temperature of 25 ° 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 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 Rhodococcus sp. ZhaoKe-LT 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 sent to the company for sequencing; 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 was most closely related to Rhodococcus sp., and was identified as Rhodococcus sp. ZhaoKe-LT based on physiological and biochemical mycological characteristics and phylogenetic tree analysis.

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

[0013] The strain ZhaoKe-LT was picked up and placed in 50 mL LB medium, and the strain Rhodococcus sp.ZhaoKe-LT was cultured in a constant temperature shaker at 25°C at a speed of 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 hours at 25°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 high-efficiency degradation psychrophilic 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 not only has a good oil removal effect, but also can maintain its good oil removal performance in a low temperature environment, thereby increasing the application scenarios of microbial oil removal;

[0023] 2. Prepare the strain ZhaoKe-LT 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;

[0025] 4. Through optimizing culture conditions and degradation experiments, it was proved that strain ZhaoKe-LT exhibited excellent degradation ability in the range of 5-15℃; BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] The high-efficiency degradation psychrophilic oil-removing strain provided by the present invention was deposited in the China Center for Type Culture Collection on August 5, 2024, with the deposit number CCTCC NO:M 20241737.

[0028] Figure 1 is a schematic diagram of the evolutionary tree of strain ZhaoKe-LT;

[0029] Figure 2 This is a schematic diagram of microscopic examination of strain ZhaoKe-LT;

[0030] Figure 3 is the growth and degradation curve of long-chain alkanes of strain ZhaoKe-LT at 5℃;

[0031] Figure 4 is the growth and degradation curve of long-chain alkanes of strain ZhaoKe-LT at 15°C;

[0032] Figure 5 This is the GC-MS analysis of oil pollution components after 4 days of culture of the ZhaoKe-LT resting cell system;

[0033] Figure 6 This is the degradation curve of oil pollution by the ZhaoKe-LT resting cell system. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1 provides a high-efficiency degradation psychrophilic oil removal strain, named Rhodococcus sp. ZhaoKe-LT, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241737, a deposit date of August 5, 2024, and a deposit address of Wuhan University, Wuhan, China.

[0036] The oil removal strain provided by the present invention can solve the problem that traditional biodegradation technology has poor oil degradation effect in low temperature environment, can maintain high degradation activity in low temperature environment, significantly improves the oil removal effect in low temperature environment, and expands the application scope of microbial degradation technology.

[0037] Embodiment 2 provides a method for preparing a psychrophilic oil removal strain with high efficiency degradation, specifically:

[0038] 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 in a neutral environment and at room temperature of 25° 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 Rhodococcus sp. ZhaoKe-LT described in the first embodiment.

[0039] In this example, the strain was also identified:

[0040] 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 sent to the company for sequencing; 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 Rhodococcus sp., and combined with physiological and biochemical mycological characteristics and phylogenetic tree analysis, it was named Rhodococcus sp. ZhaoKe-LT.

[0041] In this embodiment, a Gram staining experiment is also performed on the psychrophilic oil-removing strain, and the specific steps are as follows:

[0042] 1. Take a small amount of strain ZhaoKe-LT culture and make a bacterial suspension with sterile saline.

[0043] 2. Make a smear on a glass slide and allow it to dry naturally.

[0044] 3. Stain with crystal violet for 1 minute and rinse with water.

[0045] 4. Fix with iodine solution for 1 minute and rinse with water.

[0046] 5. Decolorize with 95% ethanol for 30 seconds and rinse with water.

[0047] 6. Dye with safranin for 1 minute, rinse with water and dry.

[0048] Observe through a microscope, such as Figure 2 As shown, strain ZhaoKe-LT is a Gram-positive bacillus.

[0049] In this example, an experiment on degradation of long-chain alkanes by a growth degradation system was also conducted:

[0050] The strain ZhaoKe-LT was picked up into 50 mL of LB medium, and the strain ZhaoKe-LT was cultured in a constant temperature shaker at 25° C. at a rotation speed of 200 rpm until the logarithmic growth phase.

[0051] The cells were collected by centrifugation and resuspended in MSM medium to OD = 1.0. 100ul of the resuspended seed solution of ZhaoKe-LT was inoculated into 10ml of MSM medium. Long-chain alkane C20 commonly found in oil pollution was added as the only carbon source. The cells were placed in a constant temperature shaker at 5°C and 15°C and cultured at 200rpm.

[0052] The flask containing 10 ml of culture was taken out at regular intervals to extract the oil component in the culture, and the content of C20 component was determined by GC-MS and OD600 was determined by spectrophotometer.

[0053] like Figure 3 and Figure 4 It was shown that the strain could degrade long-chain alkanes commonly found in oil pollution in a low-temperature culture growth system.

[0054] Example 3 provides a dormant cell system capable of degrading oil pollution, which is prepared from the psychrophilic oil-removing strain with high efficiency in degradation described in Example 1, and the preparation process is as follows:

[0055] The strain ZhaoKe-LT was picked up and placed in 50 mL LB medium, and the strain Rhodococcus sp.ZhaoKe-LT was cultured in a constant temperature shaker at 25°C at a speed of 200 rpm until the logarithmic growth phase;

[0056] 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;

[0057] After culturing for 4 hours at 25°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.

[0058] In this embodiment, the strain ZhaoKe-LT 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.

[0059] In order to evaluate the ability of strain Rhodococcus sp.ZhaoKe-LT to degrade oil in a resting cell system, the following degradation experiments were conducted:

[0060] The optical density of the resting cell system was measured using a UV spectrophotometer, and the OD600 was adjusted to 5 with PBS buffer;

[0061] 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.

[0062] Add 1% washing oil extract and 1% edible oil extract, place the conical flask in a constant temperature shaker and culture at 25°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.

[0063] Degradation effect detection:

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Experimental conclusion: After four days of resting the cell system, the oil content decreased significantly, and the degradation rate reached 50%. Figure 5 and Figure 6 shown.

[0068] In this example, a low-temperature oily wastewater degradation experiment was also conducted:

[0069] 1. Pick strain ZhaoKe-LT and a certain Pseudomonas into 50mL LB medium respectively, and culture strain ZhaoKe-LT in a 15℃ 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.

[0070] 2. Maintain the reaction time at 30°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.

[0071] After 3 weeks of bioremediation with strain ZhaoKe-LT, the oil in the oily wastewater was well degraded, with a degradation rate of about 40%, as shown in the following table:

[0072] Table 1 Degradation table of low temperature oily wastewater

[0073] Time / Day ZhaoKe-LT Pseudomonas 0 105.20±0.37mg / L 105.05±2.93mg / L 7 82.17±1.06mg / L 105.46±1.22mg / L 14 53.54±2.74mg / L 104.44±1.72mg / L 21 40.89±1.40mg / L 103.29±0.90mg / L

[0074] Embodiment 4 provides an oil removal microbial preparation, wherein the preparation contains the high-efficiency degradation psychrophilic oil removal strain described in the first embodiment.

[0075] 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.

[0076] 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.

[0077] 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 highly efficient psychrophilic oil removal strain characterized by: Its classification name is Rhodococcus sp. ZhaoKe-LT, and it is preserved in the China Center for Type Culture Collection. The preservation date is: CCTCC NO: M20241737, and the preservation date is: August 5, 2024.

2. An oil-removing microbial preparation, characterized in that: The preparation contains the high-efficiency degradation psychrophilic 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.

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