Bacterial strain capable of efficiently degrading petroleum hydrocarbon in high-oil and high-salt soil as well as preparation and application of bacterial strain

Abstract

The invention relates to the technical field of bioremediation, and discloses a strain capable of efficiently degrading petroleum hydrocarbon in high-oil and high-salt soil as well as preparation and application of the strain. The strain comprises Escherichia coli HJYY-1, Lactobacillus helveticus HJYY-2 and a heat-resistant bacillus strain HJYY-3, the escherichia coli HJYY-1, the lactobacillus helveticus HJYY-2 and the heat-resistant bacillus strain HJYY-3 have the advantages of having excellent adaptability to a high-oil and high-salt environment and capability of degrading petroleum hydrocarbon, being capable of remarkably improving the degradation efficiency of the petroleum hydrocarbon and the like; the problems that in the prior art, a strain is poor in degradation efficiency and adaptability in the actual high-oil and high-salt soil environment are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioremediation, and specifically relates to a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil, and the preparation and application thereof. Background Art

[0002] Petroleum hydrocarbon pollution is one of the major environmental problems facing the world, especially the pollution of high-oil and high-salinity soils, which poses a serious threat to the ecological environment and human health. Petroleum hydrocarbons mainly come from leakage and emissions during oil extraction, transportation, storage and use, including various compounds such as alkanes, cycloalkanes, and aromatic hydrocarbons. Petroleum hydrocarbons in high-oil and high-salinity soils are not only difficult to degrade naturally, but their high-salinity environment also poses a challenge to the degradation ability of most microorganisms, making traditional bioremediation methods inefficient in dealing with such pollution.

[0003] At present, the common methods for treating petroleum hydrocarbons in high-oil and high-salt soils include physical methods, chemical methods and biological methods. Although physical methods such as thermal desorption and soil washing can quickly remove pollutants, they are costly and may damage the soil structure; chemical methods such as the use of soil conditioners and chemical oxidants, although effective, may introduce new chemical pollutants; biological methods, especially microbial remediation technology, have attracted widespread attention due to their low cost, environmental friendliness, and ability to fundamentally degrade pollutants. However, traditional microbial remediation technology has problems such as poor microbial adaptability and low degradation efficiency when treating high-oil and high-salt soils, which limits its effectiveness in practical applications. Therefore, finding or screening microbial strains that can adapt to high-oil and high-salt environments and can efficiently degrade petroleum hydrocarbons has become the key to solving high-oil and high-salt soil pollution. At present, although some studies have reported some salt-tolerant or oil-tolerant single strains, the degradation efficiency and adaptability of these strains in actual high-oil and high-salt soil environments still need to be improved. In addition, most studies focus on the screening and application of single strains, and pay less attention to the synergy between strains and the construction of microbial communities, which to a certain extent limits the application effect and promotion potential of microbial remediation technology in the treatment of high-oil and high-salt soil pollution.

[0004] In summary, although some microbial remediation technologies have been applied to the treatment of petroleum hydrocarbon contaminated soil, there is still a lack of efficient and adaptable microbial strains and corresponding remediation technologies under the special environmental conditions of high-oil and high-salt soil. This is the current hot spot and difficulty in research in this field, and it is also a key issue that needs to be solved urgently. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the shortcomings of the prior art, the present invention provides a strain that can efficiently degrade petroleum hydrocarbons in high-oil and high-salt soils, as well as its preparation and application. It has the advantages of excellent adaptability to high-oil and high-salt environments and the ability to degrade petroleum hydrocarbons, and can significantly improve the degradation efficiency of petroleum hydrocarbons. It solves the problem of poor degradation efficiency and adaptability of strains in the prior art in actual high-oil and high-salt soil environments.

[0007] (II) Technical solution

[0008] In order to achieve the above-mentioned purpose of improving the degradation efficiency and adaptability in a high-oil and high-salt soil environment, the present invention provides the following technical solutions:

[0009] The present invention provides a first technical solution: a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil, the strain being a thermotolerant Bacillus strain HJYY-3, isolated from petroleum-contaminated soil in Zhongyuan Oilfield, and deposited in the General Microbiological Center of China Microbiological Culture Collection Committee CGMCC, with a deposit number of CGMCC No. 31483. The deposit unit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is July 29, 2024, and the classification name is Heyndrickxiasporothermodurans.

[0010] The present invention provides a second technical solution: a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil, the strain being Escherichia coli HJYY-1, isolated from petroleum-contaminated soil in Zhongyuan Oilfield, and deposited in the General Microbiological Center of China Microbiological Culture Collection CGMCC, with a deposit number of CGMCC No. 31481. The deposit unit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is July 29, 2024, and the classification name is Escherichia coli.

[0011] The present invention provides a third technical solution: a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil, wherein the strain is Lactobacillus helveticus HJYY-2, which is isolated from petroleum-contaminated soil in Zhongyuan Oilfield and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee CGMCC, with a deposit number of CGMCC No. 31482. The deposit unit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is July 29, 2024, and the classification name is Lactobacillus helveticus.

[0012] The present invention provides a fourth technical solution: a method for preparing strains of Escherichia coli HJYY-1, Lactobacillus helveticus HJYY-2 and thermotolerant Bacillus strain HJYY-3 that can efficiently degrade petroleum hydrocarbons in high-oil and high-salt soils, comprising:

[0013] Step 1: Bacteria screening

[0014] Step 1.1: domestication and cultivation of petroleum hydrocarbon degradation microbial strains;

[0015] Step 1.2: Isolation of petroleum hydrocarbon-degrading microbial strains;

[0016] Step 1.3: Screening of petroleum hydrocarbon-degrading microorganisms;

[0017] Step 2: Identification of bacterial species

[0018] Bacterial colonies with the ability to degrade petroleum hydrocarbons were selected for PCR amplification.

[0019] As one of the preferred embodiments, the process of acclimatizing and culturing the petroleum hydrocarbon-degrading microbial strain includes:

[0020] Step 1.1.1: Weigh 10 g of petroleum hydrocarbon-contaminated soil, add it to 100 mL of MSM sterile basal salt liquid medium, and culture it in a constant temperature shaker at 37°C and 150 rpm for 7 days;

[0021] Wherein: MSM sterile basal salt liquid medium contains 0.5 mL of crude oil;

[0022] Step 1.1.2: Transfer 10 mL of the liquid culture medium after standing to 90 mL of MSM sterile basal salt liquid culture medium and continue culturing in a constant temperature shaker at 37°C and 150 rpm for 7 days;

[0023] Among them: the liquid culture medium contains 0.5% crude oil, and the MSM sterile basal salt liquid culture medium contains 1 mL crude oil;

[0024] Step 1.1.3: Finally, 10 mL of the liquid culture medium after standing was transferred to 90 mL of MSM sterile basal salt liquid culture medium, and cultured in a constant temperature shaker at 37°C and 150 rpm for 7 days before use in subsequent separation experiments;

[0025] Among them: the liquid culture medium contains 1% crude oil; the MSM sterile basal salt liquid culture medium contains 2 mL crude oil.

[0026] As one of the preferred embodiments, the process of separating the petroleum hydrocarbon degrading microbial strains includes:

[0027] Step 1.2.1: Dilute the domesticated MSM liquid culture medium to 10-7 times, take 100 μL of the 10-5 to 10-7 dilution, evenly spread it on the LB solid plate, and invert it in a 37°C constant temperature incubator for 24 hours;

[0028] Step 1.2.2: Select a single colony, streak it onto an LB solid plate, and culture it in a 37°C constant temperature incubator for 24 hours before using it for subsequent degradation activity identification experiments;

[0029] Step 1.2.3: For fungi, select a culture dish with 10 to 100 colonies;

[0030] Step 1.2.4: Then pick single colonies of different sizes and shapes from the selected plates, inoculate them on the corresponding solid plates in sequence by streaking, and culture as above;

[0031] Step 1.2.5: Repeatedly pick single colonies for purification until the colonies on the same plate are uniform in size and morphology;

[0032] Step 1.2.6: The isolated petroleum-degrading strains were cultured in test tube slants and stored at 4°C.

[0033] As one of the preferred embodiments, the process of screening petroleum hydrocarbon degradation microorganisms includes:

[0034] The strain expanded on the LB solid plate was streaked onto a solid medium containing 1% crude oil MSM and cultured in a 37°C constant temperature incubator. The ability of the strain to degrade petroleum hydrocarbons was preliminarily determined based on the growth of the strain and the effect of degrading petroleum hydrocarbons.

[0035] As one of the preferred embodiments, the colony PCR amplification system includes: 21 μL sterile ddH2O; 25 μL 2×Taq Master Mix; 2 μL 10 μM Primer1; 2 μL 10 μM Primer2; the colony PCR amplification 16S rRNA universal primer is:

[0036] 8F (5′-GAGAGTTTGATCCTGGCTCAG-3′) and

[0037] 1492R(5′-TACGGCTACCTTGTTCTCAG-3′).

[0038] As one of the preferred embodiments, the reaction procedure of the PCR amplification system for the colonies is 95°C for 3 min, 95°C for 15 s, 1 min, 60°C for 15 s, 72°C for 60 s, and 72°C for 5 min.

[0039] The present invention provides a fifth technical solution: application of strains of Escherichia coli HJYY-1, Lactobacillus helveticus HJYY-2 and thermotolerant Bacillus strain HJYY-3 capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil in the degradation of petroleum hydrocarbons.

[0040] (III) Beneficial effects

[0041] Compared with the prior art, the present invention provides a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soils, and its preparation and application, which has the following beneficial effects:

[0042] 1. The present invention extracts and screens out thermotolerant Bacillus strain HJYY-3, Escherichia coli HJYY-1 and Lactobacillus helveticus HJYY-2 from petroleum-contaminated soil in Zhongyuan Oilfield. The thermotolerant Bacillus strain HJYY-3, Escherichia coli HJYY-1 and Lactobacillus helveticus HJYY-2 have strong adaptability to high-salt environment and can maintain activity under high salt concentration, thereby improving the application potential of microbial remediation technology in high-oil and high-salt soil;

[0043] 2. The use of microbial remediation technology by the highly efficient petroleum hydrocarbon degrading bacteria thermotolerant Bacillus subtilis strain HJYY-3, Escherichia coli HJYY-1 and Lactobacillus helveticus HJYY-2 is more in line with the concept of environmental friendliness, avoids secondary pollution, and has less impact on the ecological environment; at the same time, microbial remediation technology has low cost, is easy to operate, is suitable for large-scale soil remediation projects, and has significant economic benefits, solving the problem of poor degradation efficiency and adaptability of strains in existing technologies in actual high-oil and high-salt soil environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a diagram of the experimental process of domestication and separation of petroleum-degrading bacteria of the present invention;

[0045] Figure 2 This is a diagram of the experimental process of separating the oil-degrading microbial strains of the present invention;

[0046] Figure 3 This is a diagram of the screening experiment process of the petroleum-degrading microorganisms of the present invention;

[0047] Figure 4 This is a comparison diagram of the petroleum hydrocarbon degradation strain before and after the degradation experiment of the present invention;

[0048] Figure 5 is a graph showing the degradation efficiency of the petroleum hydrocarbon-degrading microorganisms of the present invention;

[0049] Figure 6 This is the sequence map of strain 0601 of the present invention;

[0050] Figure 7 This is the sequence map of strain 0602 of the present invention;

[0051] Figure 8 This is the sequence map of strain 0603 of the present invention;

[0052] Fig. 9 This is the sequence map of strain 0604 of the present invention;

[0053] Fig.10 This is the sequence map of strain 0607 of the present invention;

[0054] Fig.11This is the sequence map of strain 0609 of the present invention;

[0055] Fig.12 This is the sequence map of strain 0610 of the present invention.

[0056] Among them: Figure 1 In the figure, Figure (a) is a diagram of strain shaking culture, Figure (b) is a diagram of strain enrichment, Figure (c) is a diagram of strain domestication, Figure (d) is a diagram of plate streaking, Figure (e) is a diagram of strain culture, and Figure (f) is a diagram of strain separation;

[0057] exist Figure 2 Figure (a) shows the separation of bacterial strains Figure 1 Figure (b) shows bacterial strain separation Figure 2 Figure (c) shows bacterial strain separation Figure 3 Figure (d) shows bacterial strain separation Figure 4 ;

[0058] exist Figure 3 Figure (a) shows strain screening Figure 1 Figure (b) shows strain screening Figure 2 Figure (c) shows strain screening Figure 3 Figure (d) shows strain screening Figure 4 Figure (e) shows strain screening Figure 5 Figure (f) shows strain screening Figure 6 ;

[0059] exist Figure 4 In the figure, Figure (a) is a blank control figure, Figure (b) is a figure showing the degradation of petroleum hydrocarbons by Escherichia coli, Figure (c) is a figure showing the degradation of petroleum hydrocarbons by Lactobacillus helveticus, Figure (d) is a figure showing the degradation of petroleum hydrocarbons by Lactobacillus amyloliquefaciens, Figure (e) is a figure showing the degradation of petroleum hydrocarbons by thermotolerant Bacillus, and Figure (f) is a figure showing the degradation of petroleum hydrocarbons by mixed strains. DETAILED DESCRIPTION

[0060] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Embodiment 1:

[0062] like Figure 1-Figure 12 As shown, the present invention provides a technical solution: screening of strains capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soils and degradation efficiency and preparation experiments of petroleum hydrocarbon-degrading strains, including:

[0063] Step 1: Bacteria screening

[0064] Step 1.1: Domestication and isolation of petroleum hydrocarbon degradation microbial strains

[0065] After the petroleum-contaminated soil samples were enriched and cultured three times with crude oil culture medium, the dominant strains with good growth were selected based on colony morphology, and more than 350 single colonies were isolated and purified. They were then expanded and cultured for the screening test of petroleum hydrocarbon degradation microorganisms. The specific process of domestication and isolation of petroleum-degrading bacteria is as follows: Figure 1 As shown; the separation process of oil-degrading microbial strains is as follows Figure 2 shown.

[0066] Step 1.2: Screening of petroleum hydrocarbon-degrading microorganisms

[0067] The strains expanded on the LB solid plate were streaked onto a solid medium containing 1% crude oil (v / v) MSM and cultured in a 37°C constant temperature incubator. The strains were preliminarily judged to have the ability to degrade petroleum hydrocarbons based on their growth and the effect of degrading petroleum hydrocarbons. Figure 3 shown.

[0068] 45 strains that can grow on MSM solid medium containing 1% crude oil (v / v) (part of which can degrade crude oil) were screened from more than 350 single colonies. The target strains were expanded and cultured, the liquid was frozen in a -80°C refrigerator, and the test tubes containing LB solid medium were streaked and inoculated in a refrigerator for subsequent strain identification and petroleum hydrocarbon degradation efficiency experiments.

[0069] Step 2: Identification of bacterial species

[0070] PCR primers were used for 16S rRNA gene sequence analysis and bacterial genus identification.

[0071] The PCR products were detected by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were submitted to the GenBank database, and the BLAST program was used for similarity analysis. The sequences with the highest similarity were selected, and the phylogenetic tree was drawn using the neighbor-joining method using software such as ClustalX and MEGA.

[0072] The screened petroleum hydrocarbon degrading microbial strains 06-1, 06-2, 06-3, 06-4, 06-7, 06-9, and 06-10 were subjected to 16S rRNA sequence determination, and the results are shown in Table 1.

[0073] The strain sequences were compared with the protein library of NCBI (National Center for Biotechnology Information) one by one. The protein library of NCBI is also called NR library. The nucleic acid sequences of these strains were compared with the NR library using the Blastx program to obtain the best candidate possible strains. The results are shown in Table 1. The results showed that strain 06-1 (its nucleotide sequence is shown in SEQ ID No. 1, and its sequence map is shown in Figure 6 ), strain 06-2 is Escherichia coli HJYY-1 (its nucleotide sequence is shown in SEQ ID No.2, and the sequence map is shown in Figure 7 ), strain 06-3 (whose nucleotide sequence is shown in SEQ ID No.3, and the sequence map is shown in Figure 8 ), strain 06-4 is Lactobacillus helveticus HJYY-2 (its nucleotide sequence is shown in SEQ ID No.4, and its sequence map is shown in Fig. 9 ), strain 06-7 (whose nucleotide sequence is shown in SEQ ID No.5, and the sequence map is shown in Fig.10 ), strain 06-9 is Lactobacillus amyloliquefaciens (its nucleotide sequence is shown in SEQ ID No.6, and its sequence map is shown in Fig.11 ), strain 06-10 is thermotolerant Bacillus HJYY-3 (its nucleotide sequence is shown in SEQ ID No.7, and its sequence map is shown in Fig.12 shown).

[0074] Table 1: Strain comparison results

[0075]

[0076] Step 3: Degradation efficiency of petroleum hydrocarbon-degrading strains

[0077] The four strains selected (1# is Escherichia coli HJYY-1, 2# is Lactobacillus helveticus HJYY-2, 3# is Lactobacillus amyloliquefaciens, 4# is thermotolerant Bacillus HJYY-3) and their mixed strains were inoculated into a liquid culture medium with petroleum as the sole carbon source, and cultured in a shaking incubator at 30°C and 180 r / min. After 14 days, the content of petroleum hydrocarbons in the culture medium was determined; Comparison of the degradation experiment of petroleum hydrocarbon-degrading strains before and after Figure 4 The degradation efficiency of petroleum hydrocarbon-degrading microorganisms is shown in Figure 5 .

[0078] from Figure 5It can be seen that the four strains and their mixed strains have good degradation effects on petroleum hydrocarbons, with degradation efficiencies ranging from 76.81% to 92.82%. The experimental results show that among the four petroleum hydrocarbon degradation strains and their mixed strains, the best degradation effect is 4# Thermotolerant Bacillus HJYY-3, with a degradation efficiency of 92.82%; followed by 1# Escherichia coli HJYY-1, with a degradation efficiency of 91.62; followed by 2# Lactobacillus helveticus HJYY-2 and 3# Lactobacillus amyloliquefaciens, with degradation efficiencies of 83.71% and 83.45% respectively; and finally 5# mixed strains, with a degradation efficiency of 76.81%. Therefore, in this experiment, Thermotolerant Bacillus HJYY-3 has the best degradation effect on petroleum hydrocarbons and the degradation efficiency of a single strain is higher than that of mixed strains.

[0079] Among them, the Escherichia coli HJYY-1, Lactobacillus helveticus HJYY-2 and thermotolerant Bacillus HJYY-3 are all deposited in the General Microbiology Center of China National Committee for the Collection of Microorganisms, with the acceptance date being July 29, 2024. The deposit number of Escherichia coli HJYY-1 is CGMCC No.31481, and its nucleotide sequence is shown in SEQ ID No.2. The deposit number of Lactobacillus helveticus HJYY-2 is CGMCC No.31482, and its nucleotide sequence is shown in SEQ ID No.4. The deposit number of thermotolerant Bacillus HJYY-3 is CGMCC No.31483, and its nucleotide sequence is shown in SEQ ID No.7.

[0080] Embodiment 2:

[0081] The preparation method of the Escherichia coli HJYY-1, Lactobacillus helveticus HJYY-2 and Thermotolerant Bacillus HJYY-3 strains obtained in Example 1 that can efficiently degrade petroleum hydrocarbons in high-oil and high-salt soil comprises:

[0082] Step 1: Bacteria screening

[0083] Step 1.1: Culture and acclimatization of petroleum hydrocarbon degradation microorganisms

[0084] Step 1.1.1: Weigh 10 g of petroleum hydrocarbon-contaminated soil, add it to 100 mL of MSM sterile basal salt liquid medium (containing 0.5 mL of crude oil), and culture it in a constant temperature shaker at 37°C and 150 rpm for 7 days;

[0085] Step 1.1.2: Take 10 mL of the liquid culture medium (containing 0.5% crude oil, v / v) after standing and transfer it to 90 mL of MSM sterile basal salt liquid culture medium (containing 1 mL of crude oil) and continue to culture in a constant temperature shaker at 37°C and 150 rpm for 7 days;

[0086] Step 1.1.3: Finally, 10 mL of the liquid culture medium (containing 1% crude oil, v / v) was transferred to 90 mL of MSM sterile basal salt liquid culture medium (containing 2 mL of crude oil) and cultured in a constant temperature shaker at 37°C and 150 rpm for 7 days before use in subsequent separation experiments;

[0087] Step 1.2: Isolation of petroleum hydrocarbon-degrading microbial strains

[0088] The cultivation of petroleum hydrocarbon as the sole carbon source can effectively increase the abundance of petroleum hydrocarbon-degrading microbial flora and reduce the interference of non-petroleum hydrocarbon-degrading microbial flora. The dilution coating method was used for separation.

[0089] Step 1.2.1: Dilute the cultured MSM liquid medium to 10 -7 times, take 10-5~10 -7 100 μL of the diluent was evenly spread on the LB solid plate and placed upside down in a 37°C constant temperature incubator for 24 h;

[0090] Step 1.2.2: Select a single colony, streak it onto an LB solid plate, and culture it in a 37°C constant temperature incubator for 24 hours before using it for subsequent degradation activity identification experiments;

[0091] Step 1.2.3: Select a culture dish (PDA medium) with 10 to 100 fungal colonies;

[0092] Step 1.2.4: Then pick single colonies of different sizes and shapes from the selected plates, inoculate them on the corresponding solid plates in sequence by streaking, and culture as above;

[0093] Step 1.2.5: Repeatedly pick single colonies for purification until the colonies on the same plate are uniform in size and morphology;

[0094] Step 1.2.6: The isolated petroleum-degrading strains were cultured in test tube slants and stored at 4°C.

[0095] Step 1.3: Screening of petroleum hydrocarbon-degrading microorganisms

[0096] The strain expanded on the LB solid plate was streaked onto a solid medium containing 1% crude oil (v / v) MSM and cultured in a 37°C constant temperature incubator. The strain was preliminarily judged whether it had the ability to degrade petroleum hydrocarbons based on its growth and the effect of degrading petroleum hydrocarbons.

[0097] Step 2: Identification of bacterial species

[0098] Bacterial colonies with the ability to degrade petroleum hydrocarbons were selected for PCR amplification. The reaction system was as follows:

[0099] ddH2O (sterile) 21 μL;

[0100] 2×Taq MasterMix 25 μL;

[0101] Primer1 (10 μM) 2 μL;

[0102] Primer2 (10 μM) 2 μL;

[0103] The universal primers for colony PCR amplification of 16S rRNA are:

[0104] 8F (5′-GAGAGTTTGATCCTGGCTCAG-3′) and

[0105] 1492R(5′-TACGGCTACCTTGTTCTCAG-3′);

[0106] The reaction program was 95°C for 3 min, 95°C for 15 s, 1 min, 60°C for 15 s, 72°C for 60 s, and 72°C for 5 min.

[0107] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0108] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soils, characterized by: The strain is a thermotolerant Bacillus strain HJYY-3, which is isolated from petroleum-contaminated soil in Zhongyuan Oilfield and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (CGMCC), with a deposit number of CGMCC No.31483.

2. A strain that can efficiently degrade petroleum hydrocarbons in high-oil and high-salt soils, characterized by: The strain is Escherichia coli HJYY-1, which is separated from the petroleum-contaminated soil of Zhongyuan Oilfield and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee CGMCC with a deposit number of CGMCC No.31481.

3. A strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soils, characterized by: The strain is Lactobacillus helveticus HJYY-2, which is separated from the petroleum-contaminated soil of Zhongyuan Oilfield and has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee CGMCC, with the deposit number of CGMCC No.31482.

4. A method for preparing a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil as claimed in any one of claims 1 to 3, characterized in that: include: Step 1: Bacteria screening Step 1.1: domestication and cultivation of petroleum hydrocarbon degradation microbial strains; Step 1.2: Isolation of petroleum hydrocarbon-degrading microbial strains; Step 1.3: Screening of petroleum hydrocarbon-degrading microorganisms; Step 2: Identification of bacterial species Bacterial colonies with the ability to degrade petroleum hydrocarbons were selected for PCR amplification.

5. The method for preparing a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil as claimed in claim 4, characterized in that: The process of acclimatization and cultivation of petroleum hydrocarbon degradation microbial strains includes: Step 1.1.1: Weigh 10 g of petroleum hydrocarbon-contaminated soil, add it to 100 mL of MSM sterile basal salt liquid culture medium, and culture it in a constant temperature shaker at 37°C and 150 rpm for 7 days; Wherein: MSM sterile basal salt liquid medium contains 0.5 mL of crude oil; Step 1.1.2: Transfer 10 mL of the liquid culture medium after standing to 90 mL of MSM sterile basal salt liquid culture medium and continue culturing in a constant temperature shaker at 37°C and 150 rpm for 7 days; Among them: the liquid culture medium contains 0.5% crude oil, and the MSM sterile basal salt liquid culture medium contains 1 mL crude oil; Step 1.1.3: Finally, 10 mL of the liquid culture medium after standing was transferred to 90 mL of MSM sterile basal salt liquid culture medium, and cultured in a constant temperature shaker at 37°C and 150 rpm for 7 days before use in subsequent separation experiments; Among them: the liquid culture medium contains 1% crude oil; the MSM sterile basal salt liquid culture medium contains 2 mL crude oil.

6. The method for preparing a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil as claimed in claim 4, characterized in that: The process of isolating petroleum hydrocarbon-degrading microbial strains includes: Step 1.2.1: Dilute the domesticated MSM liquid culture medium to 10-7 times, take 100 μL of the 10-5 to 10-7 dilution, evenly spread it on the LB solid plate, and invert it in a 37°C constant temperature incubator for 24 hours; Step 1.2.2: Select a single colony, streak it onto an LB solid plate, and culture it in a 37°C constant temperature incubator for 24 hours before using it for subsequent degradation activity identification experiments; Step 1.2.3: For fungi, select a culture dish with 10 to 100 colonies; Step 1.2.4: Then pick single colonies of different sizes and shapes from the selected plates, inoculate them on the corresponding solid plates in sequence by streaking, and culture as above; Step 1.2.5: Repeatedly pick single colonies for purification until the colonies on the same plate are uniform in size and morphology; Step 1.2.6: The isolated petroleum-degrading strains were cultured in test tube slants and stored at 4°C.

7. The method for preparing a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil as claimed in claim 4, characterized in that: The process of screening petroleum hydrocarbon degradation microorganisms includes The strain expanded on the LB solid plate was streaked onto a solid medium containing 1% crude oil MSM and cultured in a 37°C constant temperature incubator. The ability of the strain to degrade petroleum hydrocarbons was preliminarily determined based on the growth of the strain and the effect of degrading petroleum hydrocarbons.

8. The method for preparing a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil as claimed in claim 4, characterized in that: The PCR amplification system for the colony includes: 21 μL sterile ddH2O; 25 μL 2×Taq MasterMix; 2 μL 10 μM Primer1; 2 μL 10 μM Primer2; the universal primers for 16S rRNA in the colony PCR amplification are: 8F (5′-GAGAGTTTGATCCTGGCTCAG-3′) and 1492R(5′-TACGGCTACCTTGTTCTCAG-3′).

9. The method for preparing a strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil as claimed in claim 8, characterized in that: The reaction program of the PCR amplification system for the colonies was 95°C for 3 min, 95°C for 15 s, 1 min, 60°C for 15 s, 72°C for 60 s, and 72°C for 5 min.

10. Use of the strain capable of efficiently degrading petroleum hydrocarbons in high-oil and high-salt soil as claimed in any one of claims 1 to 3 in the degradation of petroleum hydrocarbons.

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