Separation and screening method of crude oil degrading functional bacteria
By using a gradient carbon source inorganic salt enrichment medium and crude oil screening method, highly efficient crude oil degrading strains were screened, solving the problems of long cycle of contaminant proliferation and purification in existing technologies, and achieving efficient and stable crude oil degradation effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BAOLIHENG (TIANJIN) BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-12-26
- Publication Date
- 2026-05-08
AI Technical Summary
In existing technologies, conventional enrichment media contain readily available carbon sources, leading to excessive proliferation of fast-food contaminants, inhibiting the lytic ability of high-efficiency strains, resulting in low abundance of target bacteria, long purification cycles, and low success rates.
Using gradient carbon source inorganic salt enrichment medium and crude oil as the sole carbon source, combined with crude oil degradation inducers, crude oil degrading functional bacteria were screened through gradient dilution and selective solid medium. Species information was identified by 16S rRNA gene sequencing, and degradation performance was verified and composite bacterial strains were constructed.
It increased the abundance of target strains by 2-3 orders of magnitude, shortened the purification cycle by more than 50%, had a degradation rate variation coefficient of less than 5%, and was adaptable to high-salt, low-nutrient environments, maintaining a stable degradation rate.
Smart Images
Figure CN121991804A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of crude oil processing technology, specifically a method for isolating and screening crude oil-degrading functional bacteria. Background Technology
[0002] With the increasing severity of oil pollution, bioremediation technology has attracted much attention due to its environmental friendliness and economic viability. However, existing technologies often use conventional enrichment media containing readily available carbon sources such as yeast extract and peptone, leading to the overgrowth of readily available bacteria. This inhibits or submerges highly efficient strains capable of cleaving long-chain alkanes and aromatics, resulting in low abundance of target bacteria and long purification cycles with low success rates. To address this technical problem, a method for isolating and screening functional bacteria for oil degradation is proposed. Summary of the Invention
[0003] The purpose of this invention is to provide a method for isolating and screening crude oil degrading bacteria to solve the problems mentioned in the background art.
[0004] To achieve the above objectives, the present invention provides the following technical solution: A method for isolating and screening crude oil-degrading bacteria, comprising: S1. Collect environmental samples that have been contaminated by crude oil for a long time; S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution. S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing. S4. Pick a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate; S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
[0005] As a further aspect of the present invention: the environmental sample is crude oil contaminated soil or oily wastewater, and the surface sample is collected at a depth of 5-20cm.
[0006] As a further embodiment of the present invention: the inorganic salt enrichment culture medium uses crude oil as the sole carbon source and the crude oil concentration increases in a gradient of 5 g / L → 10 g / L → 15 g / L, while adding 0.05-0.1 g / L of crude oil degradation inducer, and continuously enriches 3-4 times under corresponding habitat simulation conditions, with each culture lasting 4-6 days, to obtain the enriched bacterial solution.
[0007] As a further embodiment of the present invention, the inorganic salt enrichment culture medium is composed of: Na2HPO4 0.6-0.8 g / L, KH2PO4 0.2-0.3 g / L, NaNO3 3.0-4.0 g / L, CaCl2 0.01-0.02 g / L, FeSO4 0.01-0.015 g / L, MgSO4 0.3-0.5 g / L, and the pH is adjusted to 7.0-8.0.
[0008] As a further embodiment of the present invention, the enrichment culture conditions are: temperature 25-35℃, rotation speed 150-200rpm, and culture time 3-7 days.
[0009] As a further embodiment of the present invention, the selective solid culture medium is prepared by adding 1.5-2.0% agar to the inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting the crude oil with n-hexane to a preset concentration, uniformly coating it on the surface of the culture medium, and obtaining the culture medium after the n-hexane has completely evaporated.
[0010] As a further aspect of the present invention: in step S5, strains with crude oil degradation rates higher than a preset threshold are selected as crude oil degradation functional bacteria, including: Strains with a crude oil degradation rate higher than 80% are identified as crude oil degrading functional bacteria.
[0011] As a further aspect of the present invention, the method for isolating and screening crude oil degrading functional bacteria further includes identifying the screened functional bacteria by 16S rRNA gene sequencing to determine their species information.
[0012] As a further aspect of the present invention: the method for isolating and screening functional bacteria for crude oil degradation further includes verifying the degradation performance of the screened functional bacteria, including: The degradation rate of crude oil under different pH 5-9, temperature 20-40℃, and salinity 1-10% conditions was determined. Its degradation ability on different types of crude oil was determined.
[0013] As a further aspect of the present invention, the method for isolating and screening functional bacteria for crude oil degradation further includes constructing a composite bacterial system for the screened functional bacteria, mixing the 2-3 strains with the highest degradation rate at a volume ratio of 1:1 to 1:3, and measuring their synergistic degradation effect.
[0014] Compared with existing technologies, the beneficial effects of this invention are: by using crude oil as the sole carbon source, miscellaneous bacteria that can only utilize easily degradable carbon sources are eliminated at the source, and rare strains that truly possess crude oil-enzyme systems are enriched. The abundance of target bacteria can be increased by 2-3 orders of magnitude in the first passaging, shortening the subsequent purification cycle by more than 50%. Inorganic salt enrichment medium and crude oil are used as enrichment and screening media, with clear nutrient composition, strong reproducibility, and a degradation rate variation coefficient of <5% between different batches. Attached Figure Description
[0015] Figure 1 This is a schematic flowchart of a method for isolating and screening crude oil degrading bacteria according to an embodiment of the present invention. Detailed Implementation
[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0017] Please see Figure 1 The present invention provides a flowchart of a method for isolating and screening crude oil degrading bacteria, which includes the following steps S1-S5: S1. Collect environmental samples that have been contaminated by crude oil for a long time; In some embodiments, the environmental sample is crude oil contaminated soil or oily wastewater, and the surface sample is collected at a depth of 5-20 cm.
[0018] S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution.
[0019] In some embodiments, the inorganic salt enrichment medium uses crude oil as the sole carbon source and the crude oil concentration increases in a gradient of 5 g / L → 10 g / L → 15 g / L, while 0.05-0.1 g / L of crude oil degradation inducer is added. The medium is continuously enriched 3-4 times under corresponding habitat simulation conditions, with each culture lasting 4-6 days, to obtain the enriched bacterial solution.
[0020] In some embodiments, the inorganic salt enrichment medium comprises: Na₂HPO₄ 0.6-0.8 g / L, KH₂PO₄ 0.2-0.3 g / L, NaNO₃ 3.0-4.0 g / L, CaCl₂ 0.01-0.02 g / L, FeSO₄ 0.01-0.015 g / L, MgSO₄ 0.3-0.5 g / L, with the pH adjusted to 7.0-8.0.
[0021] The enrichment culture conditions are: temperature 25-35℃, rotation speed 150-200rpm, and culture time 3-7 days.
[0022] S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing.
[0023] The selective solid culture medium is prepared by adding 1.5-2.0% agar to the inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting the crude oil with n-hexane to a preset concentration, uniformly coating it on the surface of the culture medium, and waiting for the n-hexane to completely evaporate to obtain the final product.
[0024] S4. Select a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate.
[0025] The method for determining the crude oil degradation rate is as follows: after culturing for 7 days, the remaining crude oil is extracted with n-hexane, and the absorbance is measured at a wavelength of 225 nm using ultraviolet spectrophotometry to calculate the degradation rate.
[0026] S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
[0027] In some embodiments, step S5, screening out strains with crude oil degradation rates higher than a preset threshold, which are crude oil degradation functional bacteria, includes: Strains with a crude oil degradation rate higher than 80% are identified as crude oil degrading functional bacteria.
[0028] In some embodiments, the method for isolating and screening crude oil degrading functional bacteria further includes identifying the screened functional bacteria by 16S rRNA gene sequencing to determine their species information.
[0029] In some embodiments, the method for isolating and screening crude oil degrading functional bacteria further includes verifying the degradation performance of the screened functional bacteria, including: (1) Determine the crude oil degradation rate under different pH (5-9), temperature (20-40℃), and salinity (1-10%) conditions; (2) Determine its degradation ability for different types of crude oil (light, medium and heavy).
[0030] In some embodiments, the method for isolating and screening the crude oil degrading functional bacteria further includes constructing a composite bacterial system for the screened functional bacteria, mixing the 2-3 strains with the highest degradation rate at a volume ratio of 1:1 to 1:3, and measuring their synergistic degradation effect.
[0031] In some embodiments, the method for isolating and screening the crude oil degrading functional bacteria further includes immobilizing the screened functional bacteria, preparing immobilized bacterial balls using the sodium alginate-calcium chloride encapsulation method, and determining their reusability. After being recycled 5 times, the degradation rate still remains above 70%.
[0032] This invention uses crude oil as the sole carbon source to eliminate contaminating bacteria that can only utilize easily degradable carbon sources at the source, enriching rare strains that truly possess crude oil-enzyme systems. The first passage can increase the abundance of target bacteria by 2-3 orders of magnitude, shortening the subsequent purification cycle by more than 50%. It uses an inorganic salt enrichment medium and crude oil as the enrichment and screening medium, with clearly defined nutrient components, strong reproducibility, and a degradation rate variation coefficient of <5% between different batches. The inorganic salt ion composition simulates the near-shore and soil environment, enabling the screened strains to have native adaptability to high-salt and low-nutrient field conditions, and no additional acclimatization is required for direct application.
[0033] Example 1: The isolation and screening method for this crude oil degrading bacteria includes the following S1-S5: S1. Collect environmental samples that have been contaminated by crude oil for a long time; In some embodiments, the environmental sample is crude oil contaminated soil or oily wastewater, and the surface sample is collected at a depth of 20 cm.
[0034] S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution.
[0035] In some embodiments, the inorganic salt enrichment medium uses crude oil as the sole carbon source and the crude oil concentration increases in a gradient of 5 g / L → 10 g / L → 15 g / L, while 0.1 g / L of crude oil degradation inducer is added. The enrichment is carried out four times under corresponding habitat simulation conditions, with each culture lasting 6 days, to obtain the enriched bacterial solution.
[0036] In some embodiments, the inorganic salt enrichment medium consists of: Na₂HPO₄ 0.8 g / L, KH₂PO₄ 0.3 g / L, NaNO₃ 4.0 g / L, CaCl₂ 0.02 g / L, FeSO₄ 0.015 g / L, MgSO₄ 0.5 g / L, with the pH adjusted to 8.0.
[0037] The enrichment culture conditions were: temperature 35℃, rotation speed 200 rpm, and culture time 7 days.
[0038] S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing.
[0039] The selective solid culture medium is prepared by adding 2.0% agar to an inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting crude oil with n-hexane to a concentration of 50 g / L, spreading it evenly on the surface of the culture medium, and waiting for the n-hexane to completely evaporate to obtain the medium.
[0040] S4. Select a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate.
[0041] The method for determining the crude oil degradation rate is as follows: after culturing for 7 days, the remaining crude oil is extracted with n-hexane, and the absorbance is measured at a wavelength of 225 nm using ultraviolet spectrophotometry to calculate the degradation rate.
[0042] S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
[0043] In some embodiments, step S5, screening out strains with crude oil degradation rates higher than a preset threshold, which are crude oil degradation functional bacteria, includes: Strains with a crude oil degradation rate higher than 80% are identified as crude oil degrading functional bacteria.
[0044] The method for isolating and screening crude oil degrading bacteria also includes identifying the selected functional bacteria by 16S rRNA gene sequencing to determine their species information.
[0045] The method for isolating and screening functional bacteria for crude oil degradation also includes verifying the degradation performance of the screened functional bacteria, including: (1) Determine the crude oil degradation rate under the conditions of pH 9, temperature 40℃ and salinity 10%; (2) Determine its ability to degrade heavy crude oil.
[0046] The method for isolating and screening functional bacteria for crude oil degradation also includes constructing a composite bacterial system from the screened functional bacteria, mixing the three strains with the highest degradation rate at a volume ratio of 1:3, and measuring their synergistic degradation effect.
[0047] The method for isolating and screening functional bacteria for crude oil degradation also includes immobilizing the screened functional bacteria by using the sodium alginate-calcium chloride encapsulation method to prepare immobilized bacterial balls, and measuring their reusability. After being recycled 5 times, the degradation rate still remained above 70%.
[0048] Example 2: The isolation and screening method for this crude oil degrading bacteria includes the following S1-S5: S1. Collect environmental samples that have been contaminated by crude oil for a long time; In some embodiments, the environmental sample is crude oil contaminated soil or oily wastewater, and the surface sample is collected at a depth of 5 cm.
[0049] S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution.
[0050] In some embodiments, the inorganic salt enrichment medium uses crude oil as the sole carbon source and the crude oil concentration increases in a gradient of 5 g / L → 10 g / L → 15 g / L, while 0.05 g / L of crude oil degradation inducer is added. The enrichment is carried out three times under corresponding habitat simulation conditions, with each culture lasting 4 days, to obtain the enriched bacterial solution.
[0051] In some embodiments, the inorganic salt enrichment medium consists of: Na₂HPO₄ 0.6 g / L, KH₂PO₄ 0.2 g / L, NaNO₃ 3.0 g / L, CaCl₂ 0.01 g / L, FeSO₄ 0.01 g / L, MgSO₄ 0.3 g / L, with the pH adjusted to 7.0.
[0052] The enrichment culture conditions were: temperature 25℃, rotation speed 150 rpm, and culture time 3 days.
[0053] S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing.
[0054] The selective solid culture medium is prepared by adding 1.5% agar to an inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting crude oil with n-hexane to a concentration of 50 g / L, spreading it evenly on the surface of the culture medium, and waiting for the n-hexane to completely evaporate to obtain the medium.
[0055] S4. Select a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate.
[0056] The method for determining the crude oil degradation rate is as follows: after culturing for 7 days, the remaining crude oil is extracted with n-hexane, and the absorbance is measured at a wavelength of 225 nm using ultraviolet spectrophotometry to calculate the degradation rate.
[0057] S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
[0058] In some embodiments, step S5, screening out strains with crude oil degradation rates higher than a preset threshold, which are crude oil degradation functional bacteria, includes: Strains with a crude oil degradation rate higher than 80% are identified as crude oil degrading functional bacteria.
[0059] The method for isolating and screening crude oil degrading bacteria also includes identifying the selected functional bacteria by 16S rRNA gene sequencing to determine their species information.
[0060] The method for isolating and screening functional bacteria for crude oil degradation also includes verifying the degradation performance of the screened functional bacteria, including: (1) Determine the crude oil degradation rate under the conditions of pH 5, temperature 20℃ and salinity 1%; (2) Determine its ability to degrade medium crude oil.
[0061] The method for isolating and screening functional bacteria for crude oil degradation also includes constructing a composite bacterial system from the screened functional bacteria, mixing the two strains with the highest degradation rate at a volume ratio of 1:1, and measuring their synergistic degradation effect.
[0062] The method for isolating and screening functional bacteria for crude oil degradation also includes immobilizing the screened functional bacteria by using the sodium alginate-calcium chloride encapsulation method to prepare immobilized bacterial balls, and measuring their reusability. After being recycled 5 times, the degradation rate still remained above 70%.
[0063] Example 3: The isolation and screening method for this crude oil degrading bacteria includes the following S1-S5: S1. Collect environmental samples that have been contaminated by crude oil for a long time; In some embodiments, the environmental sample is crude oil contaminated soil or oily wastewater, and the surface sample is collected at a depth of 16 cm.
[0064] S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution.
[0065] In some embodiments, the inorganic salt enrichment medium uses crude oil as the sole carbon source and the crude oil concentration increases in a gradient of 5 g / L → 10 g / L → 15 g / L, while 0.8 g / L of crude oil degradation inducer is added. The enrichment is carried out four times under corresponding habitat simulation conditions, with each culture lasting 6 days, to obtain the enriched bacterial solution.
[0066] In some embodiments, the inorganic salt enrichment medium comprises: Na₂HPO₄ 0.65-0.75 g / L, KH₂PO₄ 0.23-0.27 g / L, NaNO₃ 3.3-3.7 g / L, CaCl₂ 0.013-0.017 g / L, FeSO₄ 0.011-0.014 g / L, MgSO₄ 0.35-0.45 g / L, with the pH adjusted to 7.7.
[0067] The enrichment culture conditions were: temperature 32℃, rotation speed 180 rpm, and culture time 6 days.
[0068] S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing.
[0069] The selective solid culture medium is prepared by adding 1.8% agar to an inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting crude oil with n-hexane to a concentration of 50 g / L, spreading it evenly on the surface of the culture medium, and waiting for the n-hexane to completely evaporate to obtain the medium.
[0070] S4. Select a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate.
[0071] The method for determining the crude oil degradation rate is as follows: after culturing for 7 days, the remaining crude oil is extracted with n-hexane, and the absorbance is measured at a wavelength of 225 nm using ultraviolet spectrophotometry to calculate the degradation rate.
[0072] S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
[0073] In some embodiments, step S5, screening out strains with crude oil degradation rates higher than a preset threshold, which are crude oil degradation functional bacteria, includes: Strains with a crude oil degradation rate higher than 80% are identified as crude oil degrading functional bacteria.
[0074] The method for isolating and screening crude oil degrading bacteria also includes identifying the selected functional bacteria by 16S rRNA gene sequencing to determine their species information.
[0075] The method for isolating and screening functional bacteria for crude oil degradation also includes verifying the degradation performance of the screened functional bacteria, including: (1) Determine the crude oil degradation rate under the conditions of pH 8, temperature 35℃ and salinity 7%; (2) Determine its ability to degrade light crude oil.
[0076] The method for isolating and screening functional bacteria for crude oil degradation also includes constructing a composite bacterial system from the screened functional bacteria, mixing the three strains with the highest degradation rate at a volume ratio of 1:2.5, and measuring their synergistic degradation effect.
[0077] The method for isolating and screening functional bacteria for crude oil degradation also includes immobilizing the screened functional bacteria by using the sodium alginate-calcium chloride encapsulation method to prepare immobilized bacterial balls, and measuring their reusability. After being recycled 5 times, the degradation rate still remained above 70%.
[0078] Example 4: The isolation and screening method for this crude oil degrading bacteria includes the following S1-S5: S1. Collect environmental samples that have been contaminated by crude oil for a long time; In some embodiments, the environmental sample is crude oil contaminated soil or oily wastewater, and the surface sample is collected at a depth of 8 cm.
[0079] S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution.
[0080] In some embodiments, the inorganic salt enrichment medium uses crude oil as the sole carbon source and the crude oil concentration increases in a gradient of 5 g / L → 10 g / L → 15 g / L, while 0.06 g / L of crude oil degradation inducer is added. The enrichment is carried out three times under corresponding habitat simulation conditions, with each culture lasting 4 days, to obtain the enriched bacterial solution.
[0081] In some embodiments, the inorganic salt enrichment medium consists of: Na₂HPO₄ 0.65 g / L, KH₂PO₄ 0.23 g / L, NaNO₃ 3.3 g / L, CaCl₂ 0.013 g / L, FeSO₄ 0.011 g / L, MgSO₄ 0.35 g / L, with the pH adjusted to 7.3.
[0082] The enrichment culture conditions were: temperature 28℃, rotation speed 160 rpm, and culture time 4 days.
[0083] S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing.
[0084] The selective solid culture medium is prepared by adding 1.6% agar to an inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting crude oil with n-hexane to a concentration of 50 g / L, spreading it evenly on the surface of the culture medium, and waiting for the n-hexane to completely evaporate to obtain the final product.
[0085] S4. Select a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate.
[0086] The method for determining the crude oil degradation rate is as follows: after culturing for 7 days, the remaining crude oil is extracted with n-hexane, and the absorbance is measured at a wavelength of 225 nm using ultraviolet spectrophotometry to calculate the degradation rate.
[0087] S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
[0088] In some embodiments, step S5, screening out strains with crude oil degradation rates higher than a preset threshold, which are crude oil degradation functional bacteria, includes: Strains with a crude oil degradation rate higher than 80% are identified as crude oil degrading functional bacteria.
[0089] The method for isolating and screening crude oil degrading bacteria also includes identifying the selected functional bacteria by 16S rRNA gene sequencing to determine their species information.
[0090] The method for isolating and screening functional bacteria for crude oil degradation also includes verifying the degradation performance of the screened functional bacteria, including: (1) Determine the crude oil degradation rate under the conditions of pH 6, temperature 25℃ and salinity 3%; (2) Determine its ability to degrade heavy crude oil.
[0091] The method for isolating and screening functional bacteria for crude oil degradation also includes constructing a composite bacterial system from the screened functional bacteria, mixing the two strains with the highest degradation rate at a volume ratio of 1:1.5, and measuring their synergistic degradation effect.
[0092] The method for isolating and screening functional bacteria for crude oil degradation also includes immobilizing the screened functional bacteria by using the sodium alginate-calcium chloride encapsulation method to prepare immobilized bacterial balls, and measuring their reusability. After being recycled 5 times, the degradation rate still remained above 70%.
[0093] Example 5: The isolation and screening method for this crude oil degrading bacteria includes the following S1-S5: S1. Collect environmental samples that have been contaminated by crude oil for a long time; In some embodiments, the environmental sample is crude oil contaminated soil or oily wastewater, and the surface sample is collected at a depth of 12 cm.
[0094] S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution.
[0095] In some embodiments, the inorganic salt enrichment medium uses crude oil as the sole carbon source and the crude oil concentration increases in a gradient of 5 g / L → 10 g / L → 15 g / L, while 0.075 g / L of crude oil degradation inducer is added. The enrichment is carried out 3.5 times under corresponding habitat simulation conditions, with each culture lasting 5 days, to obtain the enriched bacterial solution.
[0096] In some embodiments, the inorganic salt enrichment medium consists of: Na₂HPO₄ 0.7 g / L, KH₂PO₄ 0.25 g / L, NaNO₃ 3.5 g / L, CaCl₂ 0.015 g / L, FeSO₄ 0.013 g / L, MgSO₄ 0.4 g / L, with the pH adjusted to 7.5.
[0097] The enrichment culture conditions were: temperature 30℃, rotation speed 170 rpm, and culture time 5 days.
[0098] S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing.
[0099] The selective solid culture medium is prepared by adding 1.750% agar to the inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting the crude oil with n-hexane to a concentration of 50 g / L, spreading it evenly on the surface of the culture medium, and waiting for the n-hexane to completely evaporate to obtain the medium.
[0100] S4. Select a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate.
[0101] The method for determining the crude oil degradation rate is as follows: after culturing for 7 days, the remaining crude oil is extracted with n-hexane, and the absorbance is measured at a wavelength of 225 nm using ultraviolet spectrophotometry to calculate the degradation rate.
[0102] S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
[0103] In some embodiments, step S5, screening out strains with crude oil degradation rates higher than a preset threshold, which are crude oil degradation functional bacteria, includes: Strains with a crude oil degradation rate higher than 80% are identified as crude oil degrading functional bacteria.
[0104] The method for isolating and screening crude oil degrading bacteria also includes identifying the selected functional bacteria by 16S rRNA gene sequencing to determine their species information.
[0105] The method for isolating and screening functional bacteria for crude oil degradation also includes verifying the degradation performance of the screened functional bacteria, including: (1) Determine the crude oil degradation rate under the conditions of pH 7, temperature 30℃ and salinity 5%; (2) Determine its ability to degrade heavy crude oil.
[0106] The method for isolating and screening functional bacteria for crude oil degradation also includes constructing a composite bacterial system from the screened functional bacteria, mixing the two strains with the highest degradation rate at a volume ratio of 1:2, and measuring their synergistic degradation effect.
[0107] The method for isolating and screening functional bacteria for crude oil degradation also includes immobilizing the screened functional bacteria by using the sodium alginate-calcium chloride encapsulation method to prepare immobilized bacterial balls, and measuring their reusability. After being recycled 5 times, the degradation rate still remained above 70%.
[0108] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, process, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, processes, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.
[0109] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.
Claims
1. A method for isolating and screening crude oil-degrading functional bacteria, characterized in that, include: S1. Collect environmental samples that have been contaminated by crude oil for a long time; S2. The environmental sample is inoculated into a gradient carbon source inorganic salt enrichment medium for enrichment culture to obtain an enrichment culture solution. S3. The enriched culture solution is serially diluted and spread onto a selective solid culture medium containing crude oil, and single colonies are obtained by culturing. S4. Pick a single colony and inoculate it into a liquid culture medium containing crude oil, and determine its crude oil degradation rate; S5. Select strains with crude oil degradation rates higher than a preset threshold, which are then identified as crude oil degradation functional bacteria.
2. The method for isolating and screening crude oil degrading bacteria according to claim 1, characterized in that, The environmental samples are crude oil-contaminated soil or oily wastewater, and the surface samples are collected at a depth of 5-20 cm.
3. The method for isolating and screening crude oil-degrading functional bacteria according to claim 1, characterized in that, The inorganic salt enrichment medium uses crude oil as the sole carbon source, with the crude oil concentration increasing in a gradient from 5 g / L to 10 g / L to 15 g / L. At the same time, 0.05-0.1 g / L of crude oil degradation inducer is added. The medium is continuously enriched 3-4 times under corresponding habitat simulation conditions, with each culture lasting 4-6 days, to obtain the enriched bacterial solution.
4. The method for isolating and screening crude oil-degrading functional bacteria according to claim 3, characterized in that, The inorganic salt enrichment medium consists of: Na₂HPO₄ 0.6-0.8 g / L, KH₂PO₄ 0.2-0.3 g / L, NaNO₃ 3.0-4.0 g / L, CaCl₂ 0.01-0.02 g / L, FeSO₄ 0.01-0.015 g / L, MgSO₄ 0.3-0.5 g / L, with the pH adjusted to 7.0-8.
0.
5. The method for isolating and screening crude oil degrading bacteria according to claim 4, characterized in that, The enrichment culture conditions are: temperature 25-35℃, rotation speed 150-200rpm, and culture time 3-7 days.
6. The method for isolating and screening crude oil degrading functional bacteria according to claim 1, characterized in that, The selective solid culture medium is prepared by adding 1.5-2.0% agar to the inorganic salt enrichment culture medium, sterilizing and solidifying it, diluting the crude oil with n-hexane to a preset concentration, uniformly coating it on the surface of the culture medium, and waiting for the n-hexane to completely evaporate to obtain the final product.
7. The method for isolating and screening crude oil degrading functional bacteria according to claim 1, characterized in that, S5, screening out strains with crude oil degradation rates higher than a preset threshold, which are crude oil degradation functional bacteria, including: Strains with a crude oil degradation rate higher than 80% were selected as crude oil degrading functional bacteria.
8. The method for isolating and screening crude oil degrading bacteria according to claim 1, characterized in that, The method for isolating and screening crude oil degrading bacteria also includes identifying the selected functional bacteria by 16S rRNA gene sequencing to determine their species information.
9. The method for isolating and screening crude oil degrading bacteria according to claim 8, characterized in that, The method for isolating and screening functional bacteria for crude oil degradation also includes verifying the degradation performance of the screened functional bacteria, including: The degradation rate of crude oil under different pH 5-9, temperature 20-40℃, and salinity 1-10% conditions was determined. Its degradation ability on different types of crude oil was determined.
10. The method for isolating and screening crude oil-degrading functional bacteria according to claim 9, characterized in that, The method for isolating and screening functional bacteria for crude oil degradation also includes constructing a composite strain of the screened functional bacteria, mixing the 2-3 strains with the highest degradation rate at a volume ratio of 1:1 to 1:3, and measuring their synergistic degradation effect.