1. The high-temperature-resistant petroleum hydrocarbon degrading bacterium is characterized by being Bacillus (Bacillus paramycoides) A4-1 or Bacillus (Bacillus paramycoides) P4-3;

wherein the bacillus A4-1 is delivered to the China center for type culture Collection with the preservation date of 2021 year, 1 month and 20 days, and the preservation number is CCTCC NO: m2021117;

the bacillus P4-3 is sent to China center for type culture Collection, the preservation date is 2021 year, 1 month and 20 days, and the preservation number is CCTCC NO: m2021118.

2. A high temperature resistant petroleum hydrocarbon degrading bacterial composition comprising Bacillus (Bacillus paramycoides) a4-1 and Bacillus (Bacillus paramycoides) P4-3 according to claim 1.

3. The high temperature resistant petroleum hydrocarbon degrading bacterial composition according to claim 2, wherein the ratio of the colony count of Bacillus (Bacillus paramycoides) a4-1 to the colony count of Bacillus (Bacillus paramycoides) P4-3 is 0.1-10: 1, preferably 1: 1.

4. Use of the high temperature resistant petroleum hydrocarbon degrading bacteria of claim 1 or the high temperature resistant petroleum hydrocarbon degrading bacteria composition of claim 2 or 3 in the preparation of high temperature resistant petroleum hydrocarbon degrading bacteria agents.

5. A high temperature resistant petroleum hydrocarbon degrading bacterial agent, wherein the high temperature resistant petroleum hydrocarbon degrading bacterial agent comprises the high temperature resistant petroleum hydrocarbon degrading bacteria of claim 1 or the high temperature resistant petroleum hydrocarbon degrading bacterial composition of claim 2 or 3.

6. Use of the high temperature resistant petroleum hydrocarbon degrading bacteria of claim 1, the high temperature resistant petroleum hydrocarbon degrading bacteria composition of claim 2 or 3, and/or the high temperature resistant petroleum hydrocarbon degrading bacteria inoculant of claim 5 for bioremediation of removal of petroleum pollutants from an environment.

7. The use of claim 6, wherein the environment comprises soil and water.

8. A method for remediating a petroleum-contaminated environment, comprising applying the high temperature resistant petroleum hydrocarbon degrading bacteria of claim 1, the high temperature resistant petroleum hydrocarbon degrading bacteria composition of claim 2 or 3, and/or the high temperature resistant petroleum hydrocarbon degrading bacteria agent of claim 5 to the petroleum-contaminated environment.

9. The remediation method of claim 8 wherein the petroleum-contaminated environment comprises petroleum-contaminated soil and petroleum-contaminated water.

10. A treatment method according to claim 8, wherein the temperature of the petroleum-contaminated environment is above 25 ℃, preferably 25-50 ℃.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Petroleum is one of the most important energy sources in modern society. The leakage accidents occurring during the exploration, exploitation, storage and transportation of petroleum and the oily wastewater discharged during the refining and processing of petroleum can cause serious environmental harm. Oil is discharged into a water body, an oil film is formed on the surface of the water body, dissolved oxygen in the water body is reduced, and an original ecological system is damaged; some volatile organic compounds in petroleum hydrocarbon can affect human health, and some components have carcinogenic, mutagenic and teratogenic effects. The method of adding chemical reagents such as oil collecting agent, settling agent and the like to solve the problem of petroleum pollution is expensive in cost and easy to cause secondary pollution, the application of the method is limited to a certain extent, and the microbial degradation technology is widely concerned due to the advantages of economy, high efficiency, simplicity in operation, no secondary pollution and the like.

The microbial degradation technology is a technology for degrading and converting pollutants through various metabolic pathways of microorganisms under a proper environment. Numerous studies have shown that microbial degradation plays an important role in the natural degradation of petroleum hydrocarbon pollution. The demulsifying bacteria with strong surface hydrophobicity coalesce oil drops dispersed in water together to realize the rapid separation of oil and water phases. Adding bacterial strains or floras with strong environmental adaptability and high degradation capability into the oil-containing wastewater is an important technical means for improving the oil degradation efficiency.

In the refining and processing processes of petroleum, high-temperature oily wastewater is inevitably generated, the concentration of dissolved oxygen in the high-temperature oily wastewater is reduced, the viscosity is reduced, and conventional petroleum hydrocarbon degrading bacteria are difficult to adapt to the environment, so that most of the high-temperature oily wastewater has to be provided with a cooling tower to reduce the temperature of the wastewater, the cost is increased, and the treatment difficulty is improved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a high-temperature-resistant petroleum hydrocarbon degrading bacterium, a degrading bacterium composition, a degrading bacterium agent and application thereof. The invention obtains two new strains of Bacillus (Bacillus paramycoides) A4-1 and P4-3 through screening, wherein the Bacillus A4-1 has the capability of efficiently degrading crude oil in a high-temperature environment, and the Bacillus (Bacillus paramycoides) P4-3 has an efficient demulsification effect on O/W emulsion and O/W emulsion, so that oil-water separation is rapidly realized, and the two strains are mixed for use, so that oil-water separation and petroleum degradation can be synchronously realized, and the invention has good practical application value.

The invention is realized by the following technical scheme:

in a first aspect of the invention, a high-temperature-resistant petroleum hydrocarbon degrading bacterium is provided, and is Bacillus (Bacillus paramycoides) A4-1 or Bacillus (Bacillus paramycoides) P4-3;

wherein the bacillus A4-1 is delivered to China center for type culture Collection (address: Wuhan university at Lophania city, Wuchang, Hubei), the preservation date is 2021 year, 1 month and 20 days, and the preservation number is CCTCC NO: m2021117.

The bacillus P4-3 is delivered to China center for type culture Collection (address: Wuhan university in Wuchang Lojia mountain, Wuhan, Hubei province), the preservation date is 2021 year, 1 month and 20 days, and the preservation number is CCTCC NO: m2021118.

In a second aspect of the invention, a high temperature resistant petroleum hydrocarbon degrading bacteria composition is provided, which comprises the Bacillus (Bacillus paramycoides) A4-1 and Bacillus (Bacillus paramycoides) P4-3.

The ratio of the colony number of the Bacillus (Bacillus paramycoides) A4-1 to the colony number of the Bacillus (Bacillus paramycoides) P4-3 is 0.1-10: 1, and preferably 1: 1.

In a third aspect of the invention, the application of the high temperature resistant petroleum hydrocarbon degrading bacteria or the high temperature resistant petroleum hydrocarbon degrading bacteria composition in preparing a high temperature resistant petroleum hydrocarbon degrading microbial inoculum is provided.

In a fourth aspect of the invention, a high temperature resistant petroleum hydrocarbon degrading bacterial agent is provided, which comprises the high temperature resistant petroleum hydrocarbon degrading bacteria or the high temperature resistant petroleum hydrocarbon degrading bacterial composition.

In a fifth aspect of the present invention, the application of the above-mentioned high temperature resistant petroleum hydrocarbon degrading bacteria, high temperature resistant petroleum hydrocarbon degrading bacteria composition and/or high temperature resistant petroleum hydrocarbon degrading bacteria agent in bioremediation to remove petroleum pollutants in the environment is provided.

According to the sixth aspect of the invention, a method for treating an oil-polluted environment is provided, wherein the method for treating the oil-polluted environment comprises applying the high-temperature-resistant petroleum hydrocarbon degrading bacteria, the high-temperature-resistant petroleum hydrocarbon degrading bacteria composition and/or the high-temperature-resistant petroleum hydrocarbon degrading bacteria agent to the oil-polluted environment.

The beneficial effects of one or more of the above technical solutions are as follows:

the heat-resistant petroleum degrading bacteria obtained by screening in the technical scheme can grow by taking heavy crude oil as a unique carbon source at the high temperature of 50 ℃, and can degrade and remove petroleum pollutants without artificially adding energy sources, carbon sources, heat sources and the like, so that the process requirement is low, the application cost is low, and secondary pollution is not generated; the degradation rate of the petroleum degrading bacteria to crude oil in 7 days can reach more than 25%, the degradation efficiency is high, and petroleum pollutants in the environment can be rapidly degraded; meanwhile, the demulsifying bacteria can synchronously realize oil-water separation and petroleum degradation so as to improve the petroleum degradation efficiency, and tests prove that the high-temperature-resistant petroleum hydrocarbon degrading bacteria composition has a synergistic effect and shows excellent degradation efficiency in high-temperature (50 ℃) and low-temperature (25 ℃) environments.

The petroleum degrading bacteria provided by the technical scheme can be used for bioremediation of petroleum-polluted soil or water body, and can also be used for preparing a composite microbial inoculum together with other bacterial strains so as to efficiently and thoroughly remove petroleum pollutants in the environment, thereby having good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1A is a photograph showing the colony morphology of Petroleum-degrading bacterium A4-1.

FIG. 1B is a photograph showing the colony morphology of demulsifying bacteria P4-3.

FIG. 2 is a graph of the petroleum degradation effect of the petroleum degradation microbial inoculum A4-1 experimental group and a blank control group. Only adding petroleum into the blank control group, and not adding a petroleum degrading microbial inoculum; the experimental group is added with petroleum and petroleum degrading bacteria A4-1 at the same time.

FIG. 3 is a graph of the degradation of saturated hydrocarbons of the petroleum-degrading bacterial strains at 50 ℃, wherein A is a blank comparison graph, and B is a graph of the content of the degraded petroleum hydrocarbons.

FIG. 4 is a diagram of the demulsifying effectiveness of the demulsifying bacteria on each emulsion.

FIG. 5 is a diagram showing the treatment effect of petroleum degrading bacteria A4-1, demulsifying bacteria P4-3 and mixed bacteria on high-temperature oily wastewater.

FIG. 6 is a graph showing the effect of the mixed bacterial agent on removing petroleum hydrocarbons from high-temperature oily wastewater in an SBR reactor at 25 ℃ and 50 ℃ respectively

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

As mentioned above, in the process of refining and processing petroleum, high-temperature oily wastewater is inevitably generated, the concentration of dissolved oxygen in the high-temperature oily wastewater is reduced, the viscosity is reduced, and the conventional petroleum hydrocarbon degrading bacteria are difficult to adapt to the environment, so that most of the high-temperature oily wastewater has to be provided with a cooling tower to reduce the temperature of the wastewater, which increases the cost and increases the treatment difficulty.

The heat-resistant degrading bacteria have a high-temperature-resistant degrading enzyme system, can grow and passage by using petroleum hydrocarbon organic matters as carbon sources in a high-temperature environment, and provides possibility for efficiently and stably treating high-temperature oily wastewater. The demulsifying bacteria has high separation and removal effects on emulsified oil particles in the wastewater, so that the key point for treating the high-temperature oily wastewater is to screen heat-resistant petroleum degrading bacteria with wide substrate utilization range, particularly petroleum degrading bacteria capable of degrading heavy components, and develop a compound microbial inoculum by matching with the demulsifying bacteria.

In view of the above, in one embodiment of the present invention, a high temperature-resistant petroleum hydrocarbon degrading bacterium is provided, wherein the high temperature-resistant petroleum hydrocarbon degrading bacterium is Bacillus (Bacillus paramycoides) a4-1 or Bacillus (Bacillus paramycoides) P4-3;

wherein the bacillus A4-1 is delivered to China center for type culture Collection (address: Wuhan university at Lophania city, Wuchang, Hubei), the preservation date is 2021 year, 1 month and 20 days, and the preservation number is CCTCC NO: m2021117. The bacillus A4-1 can grow in low-temperature and high-temperature environments by taking crude oil (including heavy crude oil) as a unique carbon source, and can degrade and remove petroleum pollutants without artificially adding energy sources, carbon sources, heat sources and the like.

The bacillus P4-3 is delivered to China center for type culture Collection (address: Wuhan university in Wuchang Lojia mountain, Wuhan, Hubei province), the preservation date is 2021 year, 1 month and 20 days, and the preservation number is CCTCC NO: m2021118. The bacillus P4-3 has a demulsification function.

In yet another embodiment of the present invention, there is provided a high temperature resistant petroleum hydrocarbon degrading bacterial composition comprising the above-described Bacillus paramycetes A4-1 and Bacillus paramycetes P4-3.

The ratio of the colony number of the Bacillus (Bacillus paramycoides) A4-1 to the colony number of the Bacillus (Bacillus paramycoides) P4-3 is 0.1-10: 1, and preferably 1: 1.

In another embodiment of the present invention, the application of the above-mentioned high temperature-resistant petroleum hydrocarbon degrading bacteria or the high temperature-resistant petroleum hydrocarbon degrading bacteria composition in preparing a high temperature-resistant petroleum hydrocarbon degrading bacterial agent is provided.

In another embodiment of the invention, a refractory petroleum hydrocarbon degrading bacterial agent is provided, which comprises the above refractory petroleum hydrocarbon degrading bacteria or refractory petroleum hydrocarbon degrading bacterial composition.

In another embodiment of the present invention, the microbial agent further contains a carrier in addition to the active ingredient. The carrier may be one that is commonly used in the field of microbial agents and is biologically inert.

The carrier can be a solid carrier or a liquid carrier;

the solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, bean flour and starch; the high molecular compound can be polyvinyl alcohol or/and polyglycol;

the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane or/and dodecane.

The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

In another embodiment of the present invention, there is provided a use of the above-mentioned high temperature-resistant petroleum hydrocarbon degrading bacteria, high temperature-resistant petroleum hydrocarbon degrading bacteria composition and/or high temperature-resistant petroleum hydrocarbon degrading bacteria agent in bioremediation to remove petroleum pollutants in the environment.

In yet another embodiment of the present invention, the environment comprises soil and water.

In another embodiment of the invention, a method for treating an environment polluted by petroleum is provided, and the method comprises applying the high temperature resistant petroleum hydrocarbon degrading bacteria, the high temperature resistant petroleum hydrocarbon degrading bacteria composition and/or the high temperature resistant petroleum hydrocarbon degrading bacteria agent to the environment polluted by petroleum.

In another embodiment of the present invention, the petroleum-polluted environment includes petroleum-polluted soil and petroleum-polluted water.

In another embodiment of the invention, the temperature of the petroleum-polluted environment can be above 25 ℃, including 25-50 ℃ in the treatment process.

The present invention will be further described with reference to specific examples for better illustrating the objects, technical solutions and advantages of the present invention.

In the following examples, the composition of the culture medium used:

1) inorganic salt culture medium Na₂HPO₄·3H₂O 1.97g·L^-1,KH₂PO₄ 0.22g·L^-1,MgS0₄·7H₂O 0.2g·L^-1,NH₄Cl 1.908g·L^-1,CaCl₂ 0.02g·L^-1,FeSO₄·7H₂O 0.0lg·L^-1,NaCl 33g·L^-1。

2) The petroleum liquid culture medium is prepared by adding heavy crude oil 5 g.L to inorganic salt culture medium^-1。

3) Solid separation culture medium, adding agar 20 g.L on the basis of inorganic salt culture medium^-1Heavy crude oil 5 g.L^-1。

4) Demulsifying liquid culture medium prepared by adding liquid paraffin 4% and yeast extract 5 g.L into inorganic salt culture medium^-1，

5) LB solid culture Medium Yeast extract 3 g.L^-1Trypsin L0 g. L^-1Agar 20 g.L^-1。

The preparation method of the emulsion and the oily wastewater adopted in the embodiment comprises the following steps:

1) O/W emulsion: 300ml of Tween 80(1g/L) aqueous solution is mixed with 200ml of span 80(0.8g/L) kerosene solution and treated for 10 minutes by using a tissue shearer at 15000 rpm;

2) W/O emulsion: mixing 0.8% Tween 80 water solution with kerosene at a volume ratio of 1:2, and treating for 10 minutes using a tissue cutter at 15000 rpm;

3) crude oil emulsion: mixing the crude oil with deionized water at a ratio of 1:1, and treating for 10 minutes using a tissue cutter at 15000 rpm;

4) simulation of oily wastewater KH₂PO₄ 18mg·L^-1,NaNO₃ 48mg·L^-1,MgS0₄·7H₂O 1g·L^-1,NH₄Cl 106.4mg·L^-1,CaCl₂ 1g·L^-1,NaCl 15g·L^-1Crude oil 100 mg.L^-1Tween 806 mg. L^-1。

Example l

The domestication, separation and purification method of the petroleum degrading bacteria comprises the following steps:

1) and (3) putting 5g of the seabed sediment sample into a 50mL centrifuge tube, adding 25mL of physiological saline, vibrating on a vortex oscillator for 5-l0min to fully break up the soil, and standing for precipitation. Wherein the seafloor sediment sample is derived from.

2) L0mL supernatant was added to 100mL petroleum liquid medium and shake-cultured at 50 ℃ at 180r/min for 7 days.

3) After 7 days, the cells were inoculated at 5% and transferred into fresh petroleum liquid medium again, and enrichment culture was carried out for 3 times continuously in the same manner as the above culture conditions.

4) Separating by dilution coating plate method, and diluting the culture solution to 10 deg.C^-4Or 10^-5Then, 100uL of the diluted solution was applied to a fresh solid isolation medium.

5) Culturing for 48h, selecting single colonies with different colors and forms after the plate grows out, respectively inoculating to a solid separation culture medium and a petroleum liquid culture medium, and obtaining the petroleum degrading microorganism which can grow in the two oil-containing culture media.

The screened strain is streaked and inoculated into a petroleum liquid culture medium again, purified for three times, coated in an LB solid culture medium, the thallus is collected, 30 percent of glycerol is added, and the thallus is preserved at the temperature of minus 80 ℃.

Example 2

Molecular biological characterization of strains

1) And carrying out streak culture on each preserved strain in an LB solid culture medium, extracting a whole genome by using a DNA kit after culture, and carrying out PCR amplification by using primers 27-F and 1492-R. The PCR reaction system was 25uL of each of the upstream and downstream primers (10. mu.l/. mu.L), 1. mu.L of the DNA template (L0 ng/. mu.L), 12.5. mu.L of 2 XTaq Master Mix, and made up to 25. mu.L of ultrapure water. The PCR reaction condition is 94 ℃ for 5 min; 30s at 94 ℃, 30s at 58 ℃, 1min at 72 ℃ and 30s for 35 cycles; final extension at 72 ℃ for 7 min.

2) Sequencing the PCR amplification product by Shanghai Meiji biological medicine science and technology limited company, and performing homology comparison on the obtained gene sequence through GenBank to determine the classification status.

The sequencing result of the 16S rRNA gene of the strain A4-1 is shown in SEQ ID NO:1, the result shows that the length of the gene sequence is about 764 (sequence), the sequence alignment result shows that the similarity rate of the gene sequence with the 16S rRNA gene sequences of a plurality of species of Bacillus (Bacillus sp.) reaches 99%, and the result shows that the strain belongs to the Bacillus (Bacillus sp.).

GGTGCATTCGCGCTCAGTGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTACGCATTTCACCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCACCTGCGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCAGCTTATTCAACTAGCACTTGTTCTTCCCTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGACGCGGGTCCATCCATAAGTGACAGCCGAAGCCGCCTTTCAATTTCGAACCATGCGGTTCAAAATGTTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTATGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACTTCATAAGAGCAAGCTCTTAATCCATTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCATCCTGAGCAGAA (SEQ ID NO:1) strain P4-3 has the 16S rRNA gene sequencing result shown in SEQ ID NO:2, the result shows that the gene sequence length is about 1458 (sequence), the sequence alignment result shows that the similarity rate with the 16S rRNA gene sequences of a plurality of species of Bacillus (Bacillus sp.) reaches 99%, and the result shows that the strain belongs to the Bacillus (Bacillus sp.).

CCTTAGGCGGCTGGCTCCAAAAAGGTTACCCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCAGCTCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCTCCCGAAGGAGAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAACTTCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTACGCATTTCACCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCACCTGCGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCAGCTTATTCAACTAGCACTTGTTCTTCCCTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGACGCGGGTCCATCCATAAGTGACAGCCGAAGCCGCCTTTCAATTTCGAACCATGCGGTTCAAAATGTTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTATGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACTTCATAAGAGCAAGCTCTTAATCCATTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCATCCTGA(SEQ ID NO:2)

Example 3

The crude oil degradation rate of the Bacillus (Bacillus paramycoides) A4-1 is calculated by a gravimetric method. The method comprises the following specific steps of preparing a liquid culture medium, adding 0.5g (1g/100m1) of crude oil samples from Shengli oil fields in China, and sterilizing for later use. Inoculating A4 bacterial liquid growing to logarithmic phase in 4m 1-100 m1 culture medium, culturing at 50 deg.C for 7 days, and setting blank control group, the crude oil degradation effect is shown in figure 2. As can be seen from fig. 2: after the culture, the residual oil in the culture medium of the blank control group of the non-inoculated strain A4 is still a clear and visible solid heavy crude oil, and the phenomena of emulsification and dispersion are avoided; in the experimental group culture medium inoculated with the strain A4, residual oil is obvious liquid oil drops with extremely small volume and is dispersed on the surface layer of the culture medium, which shows that the strain A4 has obvious degradation capability on crude oil. Further, in order to calculate the degradation rate of the crude oil, the residual crude oil in the culture bottles of the experimental group and the control group is fully extracted by adopting petroleum ether and using a separating funnel respectively, the obtained crude oil components are dried at 40 ℃, cooled to constant weight in a drier, weighed, and the degradation rate of the crude oil is calculated according to the following formula. MO is the weight of the oil residue in the blank control group, and M1 is the weight of the oil residue in the experimental group.

Crude oil degradation rate (M0-M1)/initial crude oil weight × 100%

The crude oil degradation rate of the strain A4-1 of the invention is calculated to be 25%. The actual degradation effect graph (figure 2) and the crude oil degradation rate of 25 percent both prove that the strain has the degradation and emulsification functions of heavy crude oil and petroleum.

The degradation rate of petroleum hydrocarbons was determined by gas chromatography-mass spectrometry (GC-MS). The degradation effect is shown in FIG. 3. It is obvious from the peak images that the peaks of the saturated hydrocarbon components are obviously changed after 7 days of culture, and the degradation effect of the strain A4-1 is very obvious. There is very significant degradation of the individual paraffinic components.

Example 4

Inoculating the strain P4-3 into 100mLLB liquid culture medium, carrying out shaking culture on a 180-fold shaking table at 25 ℃ for 24 hours until the middle period of logarithmic growth to obtain first-class seeds, inoculating the first-class seeds into 300mL of emulsion breaking liquid culture medium according to the volume ratio of 5-8%, carrying out shaking culture on the 180-fold shaking table at 25 ℃ for 18-24 hours until the later period of logarithmic growth, adding 2mL of cultured bacteria whole culture solution into 5mL of prepared emulsion (W/O emulsion, O/W emulsion and crude oil emulsion), vigorously shaking 200 times to uniformly mix the solution, and placing the mixed emulsion into a 25 ℃ water bath kettle to stand for 24 hours at constant temperature. The bacterial emulsion breaking rate was calculated according to the following formula. H₀Is the emulsion layer height, H₁The total height of the mixed emulsion.

Crude oil degradation rate H₀/H₁×100％

The degradation rates of the strain P4-3 of the invention on W/O emulsion, O/W emulsion and crude oil emulsion are calculated to be 82%, 90% and 91.1% respectively. The actual degradation effect graph (figure 4) proves that the strain has strong demulsification function.

Example 5

Inoculating the strain into 100mL LB liquid culture medium, carrying out shaking culture on a 180-200 r/min shaking table at 50 ℃ for 24 hours until reaching the middle stage of logarithmic phase, centrifuging for 8 minutes under the condition of 5000 r/min, respectively collecting the prepared thalli, and washing for 1-2 times by using physiological saline; then, using normal saline to prepare bacterial suspension with OD600 reaching 3 for later use; mixing Bacillus (Bacillus paramycoides) A4-1 and Bacillus (Bacillus paramycoides) P4-3 in equal volume to obtain a degradation microbial inoculum, and adding the degradation microbial inoculum to the original oil content of 50 mg.L in an adding amount of 10%^-1In the simulated oil-containing wastewater, shaking culture is carried out on a shaking table at 180-. And detecting the total petroleum hydrocarbon content in the oily wastewater according to an ultraviolet spectrophotometry.

According to calculation, the removal rates of the bacterial strains A4-1 and P4-3 of the invention to the total petroleum hydrocarbon in the high-temperature oily wastewater respectively reach 30.26 percent and 42 percent, and the removal rate of the degrading bacterial agent which is mixed by the two bacterial strains in equal volume to the total petroleum hydrocarbon in the high-temperature oily wastewater reaches 62.43 percent (figure 5). The microbial inoculum of the invention is proved to have higher total petroleum hydrocarbon removal rate compared with a single microbial inoculum.

Example 6

Selecting Bacillus (Bacillus paramycoides) A4-1 and Bacillus (Bacillus paramycoides) P4-3 to carry out the following operations: respectively inoculating the strains into 100mLLB liquid culture medium, carrying out shake culture on a shaking table at 180-200 r/min at 50 ℃ for 24 hours to the middle stage of logarithmic phase, centrifuging for 8 minutes under the condition of 5000 r/min, respectively collecting the prepared thalli, and washing for 1-2 times by using normal saline; then, using normal saline to prepare bacterial suspension with OD600 reaching 3.5 for standby; equal volumes of Bacillus (Bacillus paramycoides) A4-1 and Bacillus (Bacillus paramycoides) P4-3 are mixed to form the degradation microbial inoculum. Transferring the degrading bacteria agent into two same sequencing batch reactors, wherein the initial adding concentration of the bacteria agent in the reactors is 3g/L, and the degrading bacteria agent is used for treating simulated oily wastewater. Adjusting the aeration intensity of the sequencing batch reactor to 1L/min; setting the operation period to be 8 hours, mainly comprising aeration time controlled to be 7 hours, sedimentation for 30 minutes, and water inlet and water discharge for 30 minutes; the electromagnetic stirrer and the stirring magnetons provide mixed flow during operation; the water discharge is controlled by an electromagnetic valve, the volume exchange ratio is 50%, the hydraulic retention time is 16 hours, and the temperatures of the two reactors are respectively set to be 25 +/-3 ℃ and 50 +/-3 ℃. And detecting the total petroleum hydrocarbon content in the oily wastewater according to an ultraviolet spectrophotometry.

Through calculation, the total petroleum hydrocarbon content in the effluent of the two reactors is lower than 10mg/L (figure 6), the average removal rate of the microbial inoculum of the invention on the total petroleum hydrocarbon in the oily wastewater respectively reaches 93.64 percent (25 ℃) and 93.44 percent (50 ℃), and the microbial inoculum of the invention is proved to have good petroleum hydrocarbon removal function on the oily wastewater.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Shandong university

<120> high-temperature-resistant petroleum hydrocarbon degrading bacteria, degrading bacteria composition, degrading bacteria microbial inoculum and application thereof

<130>

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 764

<212> DNA

<213> Bacillus strain A4-116S rDNA

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agtctcccag tttccaatga ccctccacgg ttgagccgtg ggctttcaca tcagacttaa 180

gaaaccacct gcgcgcgctt tacgcccaat aattccggat aacgcttgcc acctacgtat 240

taccgcggct gctggcacgt agttagccgt ggctttctgg ttaggtaccg tcaaggtgcc 300

agcttattca actagcactt gttcttccct aacaacagag ttttacgacc cgaaagcctt 360

catcactcac gcggcgttgc tccgtcagac tttcgtccat tgcggaagat tccctactgc 420

tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg tggccgatca ccctctcagg 480

tcggctacgc atcgttgcct tggtgagccg ttacctcacc aactagctaa tgcgacgcgg 540

gtccatccat aagtgacagc cgaagccgcc tttcaatttc gaaccatgcg gttcaaaatg 600

ttatccggta ttagccccgg tttcccggag ttatcccagt cttatgggca ggttacccac 660

gtgttactca cccgtccgcc gctaacttca taagagcaag ctcttaatcc attcgctcga 720

cttgcatgta ttaggcacgc cgccagcgtt catcctgagc agaa 764

<210> 2

<211> 1458

<212> DNA

<213> Bacillus strain P4-316S rDNA

<400> 2

ccttaggcgg ctggctccaa aaaggttacc ccaccgactt cgggtgttac aaactctcgt 60

ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc 120

gattactagc gattccagct tcatgtaggc gagttgcagc ctacaatccg aactgagaac 180

ggttttatga gattagctcc acctcgcggt cttgcagctc tttgtaccgt ccattgtagc 240

acgtgtgtag cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg 300

gtttgtcacc ggcagtcacc ttagagtgcc caactaaatg atggcaacta agatcaaggg 360

ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca 420

ccacctgtca ctctgctccc gaaggagaag ccctatctct agggttgtca gaggatgtca 480

agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg 540

ggcccccgtc aattcctttg agtttcagcc ttgcggccgt actccccagg cggagtgctt 600

aatgcgttaa cttcagcact aaagggcgga aaccctctaa cacttagcac tcatcgttta 660

cggcgtggac taccagggta tctaatcctg tttgctcccc acgctttcgc gcctcagtgt 720

cagttacaga ccagaaagtc gccttcgcca ctggtgttcc tccatatctc tacgcatttc 780

accgctacac atggaattcc actttcctct tctgcactca agtctcccag tttccaatga 840

ccctccacgg ttgagccgtg ggctttcaca tcagacttaa gaaaccacct gcgcgcgctt 900

tacgcccaat aattccggat aacgcttgcc acctacgtat taccgcggct gctggcacgt 960

agttagccgt ggctttctgg ttaggtaccg tcaaggtgcc agcttattca actagcactt 1020

gttcttccct aacaacagag ttttacgacc cgaaagcctt catcactcac gcggcgttgc 1080

tccgtcagac tttcgtccat tgcggaagat tccctactgc tgcctcccgt aggagtctgg 1140

gccgtgtctc agtcccagtg tggccgatca ccctctcagg tcggctacgc atcgttgcct 1200

tggtgagccg ttacctcacc aactagctaa tgcgacgcgg gtccatccat aagtgacagc 1260

cgaagccgcc tttcaatttc gaaccatgcg gttcaaaatg ttatccggta ttagccccgg 1320

tttcccggag ttatcccagt cttatgggca ggttacccac gtgttactca cccgtccgcc 1380

gctaacttca taagagcaag ctcttaatcc attcgctcga cttgcatgta ttaggcacgc 1440

cgccagcgtt catcctga 1458