CN111748483A - Bacillus for degrading petroleum hydrocarbon and application thereof - Google Patents
Bacillus for degrading petroleum hydrocarbon and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
Abstract
The invention belongs to the technical field of environmental microbiology and bioremediation, and particularly relates to a petroleum hydrocarbon degrading bacillus PL-10 and application thereof. The strain is Bacillus subtilis (PL-10) of Bacillus, which has been preserved in China general microbiological culture Collection center (CGMCC) 3, 14 days 2019 with the preservation number of CGMCC No. 17302. The bacterial strain PL-10 with petroleum hydrocarbon degradation and biosurfactant production function is used for degrading petroleum hydrocarbon in oil-containing sludge in a microorganism-enhanced mode and treating the oil-containing sludge in an oil field; the strain PL-10 provided by the invention can be used together with nutrient solution, can effectively remove petroleum hydrocarbon pollutants in oily sludge, and has the advantages of high efficiency, low cost, environmental friendliness and the like.
Description
Technical Field
The invention belongs to the technical field of environmental microbiology and bioremediation, and particularly relates to a petroleum hydrocarbon degrading bacillus PL-10 and application thereof.
Background
Petroleum is an energy source and a chemical raw material which are important for the development of human social industry. The global demand for oil has increased since the 20 th century. In the industrial processes of oil field exploration, oil field development, crude oil refining, crude oil transportation and the like, a large amount of oily sludge (called oil sludge for short) formed by mixing crude oil, water, silt and other substances is generated. According to incomplete statistics, the total amount of various oily sludge generated in various oil fields in China exceeds 1000 million tons per year. If the oil sludge is not treated timely and effectively, the total amount of the oil sludge is accumulated year by year, which not only causes resource waste, but also causes harm to soil, water body and air quality due to compounds such as various petroleum hydrocarbons (such as polycyclic aromatic hydrocarbon) contained in the oil sludge, thereby causing serious threats to ecological environment quality and safety.
The effective treatment of the oil sludge is generally regarded by governments and oil field enterprises of all countries in the world, but the treatment and treatment difficulty is higher. As soon as 1998, China puts oil sludge into the name list of dangerous wastes (waste category HW08), and oil-containing sludge must be subjected to harmless treatment according to the requirement of the clean production promotion law of the people's republic of China. China petroleum enterprises determine strategies and principles of 'reduction', 'harmlessness' and 'resource' treatment of oily sludge. Based on the strategy and principle, most oil fields adopt various physical and chemical methods including an incineration method, a solvent extraction method, a pyrolysis method, a hot washing method and the like to treat the oily sludge, so that a better effect is achieved. However, most of the physical and chemical methods have the problems of high cost, complex process, incomplete treatment, easy secondary pollution and the like.
The ability to utilize microorganisms to degrade petroleum hydrocarbons is an important addition to, and even an alternative to, physicochemical processes for treating oil sludge. The biological method for treating the oil sludge mainly utilizes petroleum hydrocarbon degrading bacteria to grow and metabolize by taking petroleum hydrocarbon as a carbon source, so that the petroleum hydrocarbon compounds are finally and completely mineralized and converted into harmless inorganic substances (CO)2And H2O). In contrast, biological methods have been increasingly regarded as more important because of their low cost, thorough treatment and environmental friendliness. In recent years, active research is conducted by a plurality of research and development institutions and enterprise units at home and abroad around the contents of screening of efficient petroleum hydrocarbon degrading bacteria, optimization of an oil sludge treatment process and the like. For example:
CN104031870A discloses a microbial composite inoculant, a soil combined remediation agent prepared from the microbial composite inoculant, and applications of the microbial composite inoculant and the soil combined remediation agent. The microbial compound microbial inoculum comprises pseudomonas aeruginosa, pseudomonas putida, acinetobacter rufoenii, pseudomonas aeruginosa, achromobacter, pseudomonas mendocina, candida tropicalis and nocardioides albus, and the volume ratio is 1.5: 1.5: 1.5: 1.5: 1: 1: 1:1, the soil combined repairing agent is prepared from the microbial complex inoculant and rhamnolipid biosurfactant with the purity of not less than 85 wt% in a volume ratio of 4-8: 1, compounding. The soil combined repairing agent has good effects in repairing petroleum polluted soil and degrading TPH pollutants, normal alkanes, hopane and aromatic hydrocarbons in the soil.
CN105753283A discloses a method for treating oil-containing silt by using a biosurfactant and a microbial agent. The preparation method of the biosurfactant and the microbial agent comprises the following steps: transferring the expanded biosurfactant producing strain and microbial agent seed solution into a sterilized fermentation tank culture medium according to the inoculation amount of 2-8% of the volume ratio, wherein the fermentation culture medium contains necessary carbon source, nitrogen source, inorganic salt and trace elements, adjusting the pH value of the fermentation culture medium, ventilating, stirring and fermenting to obtain the biosurfactant or microbial agent. The method can well emulsify the residual crude oil containing the oily silt, and has the effect of well reducing the oil content of the oily silt.
CN106734181A discloses a microbial remediation method for petroleum-contaminated soil. The method jointly repairs petroleum hydrocarbon in the petroleum-polluted soil or oil sludge by using Pseudomonas aeruginosa, achromobacter and a crude biosurfactant, the initial oil content of the tested petroleum-polluted soil is 20000mg/kg-130000mg/kg, the degradation efficiency reaches 75-91%, and the degradation effect is obvious. The method has the advantages of low treatment cost, high treatment efficiency, no secondary pollution and the like.
CN108034626A, CN108102977A, CN108048376A, CN108048374A, CN108102979A, CN108034625A, CN108102978A and CN108048375A disclose various strains with the capability of degrading petroleum hydrocarbons in oil sludge, including Acinetobacter sp.jn1, Rheinheimera sp.jn2, pseudomonas (pseudomonas. jn3, JN4, JN5, JN7, JN8) Comamonas sp.jn6. By using the strains alone, the degradation rate of total petroleum hydrocarbon in the oily sludge can reach 44-94% within 30 days.
The method for removing the petroleum hydrocarbon pollutants in the oil sludge by using a biological method has the key point that microbial strain resources with high efficiency and strong adaptability are obtained. Although there are many reports about the bacterial resources of petroleum hydrocarbon degrading bacteria, the differences of physicochemical properties, components and the like of oil sludge of different oil fields are obvious, and the differences of geographical areas and climatic conditions of different oil fields can affect the process and treatment effect of the oil sludge treated by the microbial method. And the microbial strain resources suitable for oil field oil sludge treatment are still lacked, and the microbial treatment process based on the physiological characteristics of the strains is still to be optimized and improved. These deficiencies have all limited the development of sludge biotreatment technology.
Disclosure of Invention
The invention aims to provide a petroleum hydrocarbon degrading bacillus PL-10 and application thereof, aiming at the current situation of insufficient strain resources in the current oil field oily sludge biological treatment technology.
In order to achieve the purpose, the invention adopts the technical scheme that:
a strain of Bacillus subtilis (PL-10) of Bacillus sp, which is preserved in China general microbiological culture Collection center in 3 months and 04 days in 2019 with the preservation number of CGMCC No.17302, is a petroleum hydrocarbon degrading Bacillus.
The culture conditions of the strain are 25-45 ℃ and pH 5.5-9.0.
The use of the bacillus for degrading petroleum hydrocarbon according to claim 1, in degrading petroleum hydrocarbon in oily sludge in oil field.
Further, after the oil sludge to be treated is pretreated, the activated seed liquid of PL-10 is inoculated into the pretreated oil sludge according to the inoculation amount of 1-20%, then the water content of the oil sludge is adjusted to 20-40% through an inorganic salt culture medium, the pH value is adjusted to 6.0-8.5 (preferably the pH value is 7.0-8.0), and then the oil sludge is cultured for 10-120 days at the temperature of 37-45 ℃.
The treatment of the oil sludge to be treated is to add grass carbon or wood dust into the oil sludge to be treated; wherein the mass ratio of the grass carbon or the wood dust to the oil sludge is 1:5-1: 30.
In a still further aspect of the present invention,
(1) air-drying and grinding the oil field oil sludge, sieving, adding turf or sawdust, and uniformly mixing;
(2) inoculating OD into the mixture of the oil sludge and the grass carbon or the wood chips in the step (1) according to the inoculation amount of 1-20%6000.8-2.0 of Bacillus subtilis (PL-10) bacterial liquid, and adjusting the water content of the oil sludge to 20-40% by using an inorganic salt culture medium;
(3) and (3) placing the system for adjusting the water content in a constant-temperature incubator at 25-45 ℃ for static culture for 30-60 days at constant temperature, so as to realize the degradation of the total petroleum hydrocarbon in the oily sludge.
Activating the PL-10 seed solution: selecting Bacillus subtilis (PL-10) single colony, inoculating in a liquid culture medium containing sterilized seeds, and performing shaking culture at 180rpm and 37 deg.C for 10-72 h.
In a further aspect of the present invention,
and the PL-10 seed solution is activated by picking a Bacillus sp.PL-10 single bacterial colony in a conical flask of a sterilized seed solution culture medium, sealing the conical flask by using a breathable sealing film, and placing the conical flask in a shaking table at 180rpm and 37 ℃ for constant-temperature shaking culture for 24 hours to prepare activated Bacillus subtilis (PL-10) bacterial solution.
The inorganic salt culture medium comprises the following components: (KH)4)2SO41.0-20.0g、KCl 0.1-1.5g、NaCl 0.5-5.0g、KH2PO41.0-6.4g、K2HPO41.5-10.4g、MgSO40.1-2.5g, 0.1-1.5g of yeast extract, 0.1-2.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 6.5-8.0;
the seed liquid culture medium comprises the following components: 2.0-20.0g (KH) of glucose4)2SO45-15.0g、KCl 0.5-3.0g、NaCl 0.5-2.0g、KH2PO42.0-5.0g、K2HPO420.-6.0g、MgSO40.1-1.5g, 0.1-2.5g yeast extract, trace element liquid0.1-1.5mL of distilled water and 1000mL of distilled water, and adjusting the pH value to 6.5-8.0;
the trace element liquid comprises the following components: ZnSO40.1-0.5g、CaCl20.1-1.2g、CuSO40.2-2.5g、MnSO40.1-2.0g and 1000mL of deionized water, and sterilizing the microelement liquid at 115 ℃ for 30min for later use.
A method of degrading petroleum hydrocarbons comprising:
(1) air-drying and grinding the oil field oil sludge, sieving, adding turf or sawdust, and uniformly mixing;
(2) inoculating OD into the mixture of the oil sludge and the grass carbon or the wood chips in the step (1) according to the inoculation amount of 1-20%6000.8-2.0 of Bacillus subtilis (PL-10) bacterial liquid, and adjusting the liquid content to 20-40% by using an inorganic salt culture medium;
(3) and (3) placing the system for adjusting the water content in a constant-temperature incubator at 25-45 ℃ for static culture for 30-60 days at constant temperature, so as to realize the degradation of the total petroleum hydrocarbon in the oily sludge.
The invention has the beneficial effects that:
the Bacillus subtilis (PL-10) capable of degrading petroleum hydrocarbon and producing the surfactant is screened out from oily sludge of an oil field in Xinjiang by the strain, and process parameters for removing total petroleum hydrocarbon in oil sludge by using the strain are obtained, so that strain resources and process technical support are laid for further treating the oil sludge of the oil field by using a microbiological method; the method specifically comprises the following steps:
1. the petroleum hydrocarbon degrading bacterial strain PL-10 is screened from an oil sludge piling pool of an oil field, and when the bacterial strain is used for degrading petroleum hydrocarbon pollutants in oil sludge, the bacterial strain can be well adapted to and colonized in the oil sludge environment, and is beneficial to microorganisms to exert the petroleum hydrocarbon degrading activity under the real oil sludge environment condition;
2. the petroleum hydrocarbon degrading bacteria PL-10 belongs to the genus Bacillus, has strong environmental adaptability, and can produce biosurfactant in the growth process, which plays a role in promoting the degradation of petroleum hydrocarbon.
3. The optimal degradation conditions of the strain of the invention on total petroleum hydrocarbon pollutants in oil sludge are that the inoculum size is 1-20%, the temperature is 25-45 ℃, the pH value is 6-8, and the water content of the oil sludge is 20-40%. The strain PL-10 is used and simultaneously supplemented with nutrient solution, so that petroleum hydrocarbon pollutants in the oily sludge can be effectively removed, and the method has the advantages of high efficiency, low cost, environmental friendliness and the like.
Drawings
FIG. 1 is a colony morphology map of Bacillus subtilis (PL-10) provided in example 1 of the present invention.
FIG. 2 shows the bacterial morphology under the environmental scanning electron microscope of Bacillus subtilis (PL-10) provided in example 1 of the present invention.
FIG. 3 is a graph showing the results of the inoculation of Bacillus subtilis (PL-10) according to example 2 of the present invention on the removal rate of petroleum hydrocarbons from oil sludge.
FIG. 4 is a graph showing the results of the removal rate of petroleum hydrocarbons from oil sludge by Bacillus subtilis (PL-10) according to example 2 of the present invention at different addition amounts of peat.
FIG. 5 is a graph showing the results of Bacillus subtilis (PL-10) on the removal rate of petroleum hydrocarbons from oil sludge under different water contents of the oil sludge, which is provided by embodiment 2 of the invention.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.
The strain is separated and screened from an oil-containing sludge pool of an oil field in Xinjiang, is preliminarily identified as bacillus subtilis, and has obvious degradation and removal effects on total petroleum hydrocarbon compounds in oil sludge. By means of the matched application of a nutrient and the use of an oil sludge conditioner, the strain PL-10 can realize the rapid degradation of petroleum hydrocarbon in oil sludge, and the oil content is removed by more than 40% in 40 days. Provides a new strain resource and a microorganism treatment way for oil sludge treatment.
The strain PL-10 with petroleum hydrocarbon degradation and biosurfactant production function is used for degrading petroleum hydrocarbon in oil-containing sludge in a microorganism-enhanced mode and is used for treating the oil-containing sludge in oil fields. Inoculating PL-10 bacterial liquid in logarithmic growth phase into oily sludge added with proper amount of loosening agent, and applying nutrient solution to obtain oil sludge with Total Petroleum Hydrocarbon (TPH) degrading rate higher than 40% in 30-60 days. The optimal degradation conditions of the strain on total petroleum hydrocarbon pollutants in oil sludge are that the inoculation amount is 1-20%, the temperature is 25-45 ℃, the pH value is 6-8, and the water content of the oil sludge is 20-40%. The strain PL-10 is used and simultaneously supplemented with nutrient solution, so that petroleum hydrocarbon pollutants in the oily sludge can be effectively removed, and the method has the advantages of high efficiency, low cost, environmental friendliness and the like.
Material preparation
1. Oil sludge:
a proper amount of oil sludge samples are taken from an oil sludge pool in an oil field in Xinjiang, and the samples are used for screening strains after being air-dried, ground and sieved by a 2mm sieve.
2. Peat or wood flour:
purchased in the market and used for removing impurities such as gravel, wood blocks and the like.
3. Culture medium:
the seed liquid culture medium comprises the following components: 10g glucose, (KH)4)2SO410.0g、KCl 1.1g、NaCl 1.1g、KH2PO43.4g、K2HPO44.4g、MgSO40.5g, 0.5g of yeast extract, 0.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 7.
The inorganic salt culture medium comprises the following components: (KH)4)2SO410.0g、KCl 1.1g、NaCl 1.1g、KH2PO43.4g、K2HPO44.4g、MgSO40.5g, 0.5g of yeast extract, 0.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 7.
The trace element liquid comprises the following components: ZnSO40.29g、CaCl20.24g、CuSO40.25、MnSO40.17, 1000mL of deionized water.
Screening a culture medium: glucose in the seed liquid medium component was replaced with 1g/L crude oil.
4. Laboratory apparatus
Constant temperature incubator
Constant temperature vibration incubator
Electric heating constant temperature blast air drying box
Super clean bench
Vertical pressure steam sterilizer
Gel imaging system
Miniature ultraviolet spectrophotometer
PCR instrument
Electrophoresis apparatus
Vortex oscillator
(II) activation and inoculation of strain PL-10
The PL-10 deposited strain was inoculated into a 250mL conical flask containing 100mL seed liquid medium and cultured on a constant temperature shaker at 37 ℃ and 180rpm for 24 hours.
And inoculating the PL-10 bacterial liquid into the oil sludge sample to be treated according to the inoculation ratio of 1-20% after 48 h.
The following is a detailed description of the embodiments.
Example 1
The screening method of the petroleum hydrocarbon degrading bacteria PL-10 comprises the following steps:
taking 10g of oily sludge, putting the oily sludge into a 250mL conical flask which is pre-filled with 100mL of seed liquid culture medium, and then carrying out shake culture on a constant-temperature shaking table at 37 ℃ and 180rpm for 48 h;
and after 48 hours, taking the upper layer bacterial liquid after standing, diluting the upper layer bacterial liquid by 10 times of gradient, and coating the upper layer bacterial liquid on a screening culture medium plate. The content of crude oil in the screening culture medium is 1.0 g/L;
the seed liquid culture medium comprises the following components: 10g glucose, (KH)4)2SO410.0g、KCl1.1g、NaCl 1.1g、KH2PO43.4g、K2HPO44.4g、MgSO40.5g, 0.5g of yeast extract, 0.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 7.0.
Screening a culture medium: replacing glucose in the components of the seed liquid culture medium with crude oil, wherein the dosage is 1.0 g/L; agar powder is additionally added, and the using amount is 1.5 percent.
The trace element liquid comprises the following components: ZnSO40.29g、CaCl20.24g、CuSO40.25g、MnSO40.17g and 1000mL of deionized water.
After the bacterial colony grows on the screening culture medium, the bacterial colony with the oil stain diameter larger than 1cm is picked by an aseptic inoculating loop, the bacterial colony is inoculated on a seed liquid solid culture medium by adopting a plate streaking separation method and is placed in a constant temperature incubator at 37 ℃ for culturing for 24h, a single bacterial colony is picked after the bacterial colony grows out, the streaking separation process is repeated until the bacterial colony is single in shape, the obtained bacterial colony is inoculated on a slant culture medium and is temporarily stored at 4 ℃, and then the bacterial colony is stored in the China general microbiological culture Collection center (CGMCC for short), and the preservation number of the bacterial strain is CGMCC No. 17302.
The number of the strain with the oil stain diameter larger than 1cm is PL-10, the bacterial colony is milk white, the bacterial colony is not transparent, the edge of the bacterial colony is irregular, the surface of the bacterial colony is wrinkled, and the bacterial colony morphology of the PL-10 strain is shown in figure 1. PL-10 cells were in the form of short rods, and the cell morphology is shown in FIG. 2. Homology analysis is carried out on the 16S rRNA gene sequence of the strain PL-10 and the sequence information in a GenBank database, and the strain is judged to belong to Bacillus (Bacillus). The 16S rRNA gene sequence of the PL-10 strain was:
the PL-10 strain 16S rRNA gene sequence is shown as follows by DNA sequencing:
GTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTGCATGTA
example 2
Removal test of total petroleum hydrocarbons in oil field area of Xinjiang
Taking a proper amount of oil sludge of certain oil field in Xinjiang, wherein the initial oil content of the oil sludge is 6.28%, and mixing the oil sludge with a certain amount of grass carbon after air drying, grinding and sieving by a 2mm sieve. The mass ratio of the oil sludge to the grass carbon is set to be 3 gradients which are 1:5, 1:10 and 1:20 respectively.
Taking PL-10 strain preserved in a glycerol tube at the temperature of minus 80 ℃ and streaking and inoculating the strain in a pre-sterilized solid plate culture medium in an ultra-clean workbench, wherein the culture medium is a seed liquid culture medium and comprises the following components: 10g glucose, (KH)4)2SO410.0g、KCl 1.1g、NaCl1.1g、KH2PO43.4g、K2HPO44.4g、MgSO40.5g, 0.5g of yeast extract, 15g of agar powder, 0.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 7. Colonies on the plates were picked and transferred to an appropriate volume of liquid seed medium and activated at 37 ℃ for 24h at 180 rpm.
Weighing 200g of the above oil mud grass carbon mixed sample in a plastic beaker, inoculating activated OD into the plastic beaker according to the inoculation proportion of 5%6001.5 strain of Bacillus subtilis (PL-10). In the treatment group with turf usage amount of 10% and water content of 30%, a treatment without inoculating PL-10 bacteria liquid is additionally provided. The water content of the mixed sample is adjusted by an inorganic salt culture medium, and 4 gradients are set, wherein the gradients are respectively 20%, 25%, 30% and 35%. After sealing with a breathable sealing film, placing the plastic beaker in a constant-temperature incubator at 37 ℃ for static culture at constant temperature. And (4) measuring the content of the total petroleum hydrocarbon in the oil sludge by adopting a Soxhlet extraction method-gravimetric method every 10 days.
As seen from the measurement results, the treatment by using the strain PL-10 has a significantly better effect of removing the total petroleum hydrocarbons in the oil sludge than the treatment without inoculation (see figure 3); the strain PL-10 has the best degradation effect on petroleum hydrocarbons in oil sludge under the conditions of turf addition amount of 10% (shown in figure 4) and water content of 30% (shown in figure 5), and the degradation rate on Total Petroleum Hydrocarbons (TPH) of the oil sludge is 42.9% within 40 days.
Example 3
Test for removing total petroleum hydrocarbon in oil sludge in certain oil field block in northwest
Taking a proper amount of oil sludge of a certain western oil field, air-drying, grinding, sieving by a 2mm sieve, and mixing with a certain amount of grass carbon, wherein the mass ratio of the oil sludge to the grass carbon is 1:10, the oil content of the oil sludge is 5.62 percent.
Taking PL-10 strain preserved in a glycerol tube at the temperature of-70 ℃, streaking and inoculating the strain in a pre-sterilized solid plate culture medium in an ultra-clean workbench, wherein the culture medium is a seed liquid culture medium and comprises the following components: 10g glucose, (KH)4)2SO410.0g、KCl 1.1g、NaCl1.1g、KH2PO43.4g、K2HPO44.4g、MgSO40.5g, 0.5g of yeast extract, 15g of agar powder, 0.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 7. Colonies on the plates were picked and transferred to an appropriate volume of liquid seed medium and activated at 37 ℃ for 24h at 180 rpm.
Weighing 200g of the oil sludge grass carbon mixed soil in a plastic beaker, inoculating activated OD (optical density) into the oil sludge grass carbon mixed soil according to the inoculation proportion of 5 percent6001.2 strain of Bacillus subtilis (PL-10), adjusting the content of the strain to 30% with an inorganic salt culture medium, and sealing with a breathable sealing film. Placing the plastic beaker into a constant-temperature incubator at 37 ℃ for static culture. And (4) measuring the content of the total petroleum hydrocarbon in the oil sludge by adopting a Soxhlet extraction method-gravimetric method every 10 days.
According to the measurement result, the bacterial strain PL-10 has a good degradation effect on petroleum hydrocarbons in the oil sludge, and the degradation rate of Total Petroleum Hydrocarbons (TPH) in the oil sludge is 62.5% within 40 days.
Example 4
Test for removing total petroleum hydrocarbon in oil sludge in certain northeast oil field block
Taking a proper amount of oil sludge of Xinjiang certain oilfield, air-drying, grinding, sieving by a 2mm sieve, and mixing with a certain amount of grass carbon, wherein the mass ratio of the oil sludge to the grass carbon is 1: 12, the total petroleum hydrocarbon content of the oil sludge is 4.85 percent.
Taking PL-10 strain preserved in-70 deg.C glycerol tube, streaking in ultra-clean bench, inoculating into pre-sterilized solid plate culture medium, wherein the culture medium is seed liquid culture medium, and comprises:10 g glucose, (KH)4)2SO410.0g、KCl 1.1g、NaCl1.1g、KH2PO43.4g、K2HPO44.4g、MgSO40.5g, 0.5g of yeast extract, 15g of agar powder, 0.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 7.2. Colonies on the plates were picked and transferred to an appropriate volume of liquid seed medium and activated at 37 ℃ for 24h at 180 rpm.
Weighing 200g of the oil sludge grass carbon mixed soil in a plastic beaker, inoculating activated OD (optical density) into the oil sludge grass carbon mixed soil according to the inoculation proportion of 10%6001.3 strain of Bacillus subtilis (PL-10), adjusting the content of the strain to 32% by using an inorganic salt culture medium, and sealing by using a breathable sealing film. Placing the plastic beaker into a constant-temperature incubator at 37 ℃ for static culture. And (4) measuring the content of the total petroleum hydrocarbon in the oil sludge by adopting a Soxhlet extraction method-gravimetric method every 10 days.
According to the determination result, the bacterial strain PL-10 has a good degradation effect on petroleum hydrocarbons in the oil sludge, and the degradation rate of Total Petroleum Hydrocarbons (TPH) in the oil sludge is 60.9% within 50 days.
Sequence listing
<110> Shenyang application ecological research institute of Chinese academy of sciences
<120> Bacillus strain for degrading petroleum hydrocarbon and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1403
<212>DNA
<213> Bacillus subtilis L-10)
<400>1
gttacctcac cgacttcggg tgttacaaac tctcgtggtg tgacgggcgg tgtgtacaag 60
gcccgggaac gtattcaccg cggcatgctg atccgcgatt actagcgatt ccagcttcac 120
gcagtcgagt tgcagactgc gatccgaact gagaacagat ttgtgggatt ggcttaacct 180
cgcggtttcg ctgccctttg ttctgtccat tgtagcacgt gtgtagccca ggtcataagg 240
ggcatgatga tttgacgtca tccccacctt cctccggttt gtcaccggca gtcaccttag 300
agtgcccaac tgaatgctgg caactaagat caagggttgc gctcgttgcg ggacttaacc 360
caacatctca cgacacgagc tgacgacaac catgcaccac ctgtcactct gcccccgaag 420
gggacgtcct atctctagga ttgtcagagg atgtcaagac ctggtaaggt tcttcgcgtt 480
gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt 540
tcagtcttgc gaccgtactc cccaggcgga gtgcttaatg cgttagctgc agcactaagg 600
ggcggaaacc ccctaacact tagcactcat cgtttacggc gtggactacc agggtatcta 660
atcctgttcg ctccccacgc tttcgctcct cagcgtcagt tacagaccag agagtcgcct 720
tcgccactgg tgttcctcca catctctacg catttcaccg ctacacgtgg aattccactc 780
tcctcttctg cactcaagtt ccccagtttc caatgaccct ccccggttga gccgggggct 840
ttcacatcag acttaagaaa ccgcctgcga gccctttacg cccaataatt ccggacaacg 900
cttgccacct acgtattacc gcggctgctg gcacgtagtt agccgtggct ttctggttag 960
gtaccgtcaa ggtaccgccc tattcgaacg gtacttgttc ttccctaaca acagagcttt 1020
acgatccgaa aaccttcatc actcacgcgg cgttgctccg tcagactttc gtccattgcg 1080
gaagattccc tactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc 1140
cgatcaccct ctcaggtcgg ctacgcatcg ttgccttggt gagccgttac ctcaccaact 1200
agctaatgcg ccgcgggtcc atctgtaagtggtagccgaa gccacctttt atgtttgaac 1260
catgcggttc aaacaaccat ccggtattag ccccggtttc ccggagttat cccagtctta 1320
caggcaggtt acccacgtgt tactcacccg tccgccgcta acatcaggga gcaagctccc 1380
atctgtccgc tcgactgcat gta 1403
Claims (7)
1. A petroleum hydrocarbon degrading Bacillus is characterized in that a strain is Bacillus subtilis (PL-10) of Bacillus, which is preserved in China general microbiological culture Collection center in 3 months and 04 days in 2019 with the preservation number of CGMCC No. 17302.
2. A petroleum hydrocarbon degrading Bacillus according to claim 1, wherein said strain is cultured at a temperature of 25-45 ℃ and a pH of 5.5-9.0.
3. The use of the bacillus for degrading petroleum hydrocarbon according to claim 1, wherein the strain is used for degrading petroleum hydrocarbon in oil-containing sludge of oil fields.
4. The use of a Bacillus for degrading petroleum hydrocarbons as claimed in claim 3, wherein the oil sludge to be treated is pretreated, then the activated PL-10 seed solution is inoculated thereto in an inoculum size of 1-20%, then the water content of the oil sludge is adjusted to 20-40% by an inorganic salt medium, the pH is adjusted to 6.0-8.5, and then the oil sludge is cultured at 37-45 ℃ for 10-120 days.
5. Use of a petroleum hydrocarbon degrading bacillus according to claim 4, wherein said PL-10 seed solution activates: selecting Bacillus subtilis (PL-10) single colony, inoculating in a liquid culture medium containing sterilized seeds, and performing shaking culture at 180rpm and 37 deg.C for 10-72 h.
6. The use of a Bacillus for degrading petroleum hydrocarbons as claimed in claim 4, wherein the treatment of the oil sludge to be treated is by adding peat or wood chips to the oil sludge to be treated; wherein the mass ratio of the grass carbon or the wood dust to the oil sludge is 1:5-1: 30.
7. The use of a Bacillus petroleum hydrocarbon degrading bacterium according to claim 4 wherein the inorganic salt medium comprises: (KH)4)2SO41.0-20.0g、KCl 0.1-1.5g、NaCl 0.5-5.0g、KH2PO41.0-6.4g、K2HPO41.5-10.4g、MgSO40.1-2.5g, 0.1-1.5g of yeast extract, 0.1-2.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 6.5-8.0;
the seed liquid culture medium comprises the following components: 2.0-20.0g (KH) of glucose4)2SO45-15.0g、KCl 0.5-3.0g、NaCl 0.5-2.0g、KH2PO42.0-5.0g、K2HPO420.-6.0g、MgSO40.1-1.5g, 0.1-2.5g of yeast extract, 0.1-1.5mL of trace element liquid and 1000mL of distilled water, and the pH is adjusted to 6.5-8.0;
the trace element liquid comprises the following components: ZnSO40.1-0.5g、CaCl20.1-1.2g、CuSO40.2-2.5g、MnSO40.1-2.0g and 1000mL of deionized water, and sterilizing the microelement liquid at 115 ℃ for 30min for later use.
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