A strain of Bacillus thuringiensis NH26 and its application

Abstract

This invention provides a strain of Bacillus thuringiensis NH26 and its applications. This strain was deposited on July 21, 2022, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20221149, located at Wuhan University, Wuhan, China. The Bacillus thuringiensis NH26 strain provided by this invention exhibits excellent degradation capabilities for various petroleum hydrocarbons, including alkanes and polycyclic aromatic hydrocarbons, under high salinity conditions such as seawater and other high-salinity bodies. Even under high concentrations of petroleum / petroleum hydrocarbons, it still demonstrates excellent degradation performance, making it of significant value in the remediation of petroleum pollution in high-salinity bodies such as marine environments.

Description

Technical Field

[0001] This invention belongs to the field of water microbial remediation technology, specifically involving a strain of Bacillus thuringiensis NH26 and its application. Background Technology

[0002] Petroleum is mainly composed of various alkanes and aromatics, among which polycyclic aromatic hydrocarbons (PAHs) pose hazards such as mutagenicity, teratogenicity, and carcinogenicity. Marine oil spills are a type of marine pollution with a wide scope and high degree of harm, severely damaging the marine ecosystem. Once a marine oil spill occurs, the amount of petroleum hydrocarbons in the ocean will exceed its self-cleaning capacity, directly or indirectly causing serious harm to marine life and humans.

[0003] Currently, methods for treating marine oil pollution include physical treatment, chemical treatment, and microbial remediation. Physical and chemical treatment are commonly used emergency response methods in the initial stages of marine oil spills, aiming to recover as much oil as possible from the ocean and reduce the oil pollution load. However, physical and chemical treatment methods not only struggle to completely remove spilled oil from the ocean, but also suffer from high treatment costs and the potential for secondary pollution. Compared to physical and chemical treatment, microbial remediation offers advantages such as high efficiency in hydrocarbon reduction, in-situ remediation, low treatment costs, and no secondary pollution. Therefore, microbial remediation, which utilizes microorganisms with petroleum hydrocarbon degradation capabilities to remove marine oil pollution, is widely recognized as the most promising and environmentally and economically beneficial ultimate method for treating marine oil pollution.

[0004] One of the key technologies for microbial remediation of marine oil pollution is screening and obtaining microbial strains with strong petroleum hydrocarbon degradation capabilities and high adaptability to the marine environment. Existing research indicates that one of the key factors limiting the widespread application of microbial remediation for marine oil pollution is the insufficient reserve of microbial strains with high petroleum hydrocarbon degradation efficiency and strong adaptability to the marine environment. Currently, *Acinetobacter venetianus*, *Pseudomonas mendocina*, and *Thalassospira alkalitolerans*, all capable of degrading petroleum hydrocarbons, have been isolated. *Bacillus thuringiensis*, also capable of degrading petroleum hydrocarbons, has also been isolated. Maddela NR et al. (2015) provided a *Bacillus thuringiensis* isolate-2 strain capable of degrading petroleum hydrocarbons in freshwater [Maddela NR et al., 2015. Novel diesel-oil-degrading bacteria and fungi from the Ecuadorian Amazon rainforest. Water Science & Technology, 71(10):1554-1561], but it is not suitable for the remediation of high-salinity bodies such as seawater, and the highest degradation rate is only 49.71%. Sun W et al. Sun Wet et al. (2019) provided a Bacillus thuringiensis strain CQ8-1 that can degrade petroleum hydrocarbons in freshwater [Sun Wet et al., 2019. Isolation, identification, and characterization of diesel-oil-degrading bacterial strains indigenous to Changqing oil field, China. Journal of Basic Microbiology, 59:723-734], but it is not applicable to the remediation of high-salinity bodies such as seawater, and the highest degradation rate of diesel is only 66%.

[0005] It is evident that the currently isolated Bacillus thuringiensis has a very limited ability to degrade petroleum hydrocarbons, and is only suitable for decomposing petroleum hydrocarbons in freshwater. Because the adaptability of microbial strains in non-saline terrestrial soils or freshwater differs significantly from that in marine or other high-salinity waters, they cannot be used to remove petroleum pollution from marine or high-salinity waters. Furthermore, microorganisms may lose their degradation ability due to bacterial inactivation, strain mutation, or degeneration; therefore, expanding the membership of the degradation bacterial library is essential. Summary of the Invention

[0006] This invention addresses the technical problems existing in the application of microbial remediation methods for removing oil pollution from marine and high-salinity bodies. It aims to provide a microbial strain that has good degradation capabilities for alkanes and polycyclic aromatic hydrocarbons in natural seawater and other high-salinity bodies, providing technical support for the treatment of oil pollution in marine and other high-salinity bodies.

[0007] The primary objective of this invention is to provide a strain of Bacillus thuringiensis NH26.

[0008] Another object of the present invention is to provide the application of the above-mentioned Bacillus thuringiensis NH26 strain in the degradation of petroleum.

[0009] Another object of the present invention is to provide the application of the above-mentioned Bacillus thuringiensis NH26 strain in the removal of petroleum pollution, or in the remediation of petroleum-polluted environments.

[0010] Another object of the present invention is to provide the application of the above-mentioned Bacillus thuringiensis NH26 strain in the degradation of petroleum hydrocarbons.

[0011] Another object of the present invention is to provide the application of the above-mentioned Bacillus thuringiensis NH26 strain in the degradation of petroleum hydrocarbon pollution, or in the remediation of petroleum hydrocarbon pollutants in the environment.

[0012] The present invention achieves the above-mentioned objectives through the following technical solutions:

[0013] This invention obtained a strain of Bacillus thuringiensis NH26, which can grow rapidly in natural seawater and other high-salinity waters containing petroleum hydrocarbons. It exhibits excellent degradation activity against alkanes and polycyclic aromatic hydrocarbons (PAHs). In high-salinity waters, it achieves a degradation rate of over 90% for n-dodecane to n-heptadecane in refined diesel fuel at a concentration of 20 g / L, with a total degradation rate of 93.1%. In high-salinity waters, it achieves a degradation rate of over 87% for n-dodecane to n-heptadecane in refined diesel fuel at a concentration of 50 g / L, with a total degradation rate of 88.4%. In natural seawater, it achieves a degradation rate of over 87% for n-dodecane to n-heptadecane in refined diesel fuel at a concentration of 20 g / L. The degradation rates of alkanes to n-heptane all exceeded 90%, with a total degradation rate of 93.4%. In natural seawater, the degradation rates of n-dodecane to n-heptane in refined diesel oil at a concentration of 50 g / L all exceeded 88%, with a total degradation rate of 89.4%. In high-salinity water, the removal rates of anthracene, phenanthrene, and pyrene at concentrations of 100 mg / L were 39.57%, 42.48%, and 64.79%, respectively. In natural seawater, the removal rates of anthracene, phenanthrene, and pyrene at concentrations of 100 mg / L were 40.07%, 43.88%, and 65.02%, respectively. The Bacillus thuringiensis NH26 strain of this invention exhibits excellent degradation effects on alkanes and polycyclic aromatic hydrocarbons in high-salinity water.

[0014] Therefore, the following technical solutions should all fall within the protection scope of this invention:

[0015] This invention provides a strain of Bacillus thuringiensis NH26, which was deposited at the China Center for Type Culture Collection (CCTCC) on July 21, 2022, with accession number CCTCC NO: M20221149, and the deposit address is Wuhan University, Wuhan, China, postcode: 430072.

[0016] This invention provides the application of the above-mentioned Bacillus thuringiensis NH26 strain in the degradation of petroleum.

[0017] This invention provides the application of the above-mentioned Bacillus thuringiensis NH26 strain in the removal of petroleum pollution, or in the remediation of petroleum-polluted environments.

[0018] This invention provides the application of the above-mentioned Bacillus thuringiensis NH26 strain in the degradation of petroleum hydrocarbons.

[0019] This invention provides the application of the above-mentioned Bacillus thuringiensis NH26 strain in the removal of petroleum hydrocarbon pollution, or in the remediation of petroleum hydrocarbon-polluted environments.

[0020] To further clarify, the aforementioned environmental degradation, removal, or remediation of petroleum / petroleum hydrocarbon pollution were all carried out in a high-salinity environment.

[0021] Preferably, the high salinity environment includes high salinity water environments, including but not limited to oceans or other high salinity water bodies.

[0022] Preferably, the petroleum hydrocarbons are alkanes and polycyclic aromatic hydrocarbons.

[0023] More preferably, the alkane includes n-dodecane to n-heptadecane.

[0024] More preferably, the polycyclic aromatic hydrocarbons include anthracene, phenanthrene, and pyrene.

[0025] The technical solution of the present invention has the following beneficial effects:

[0026] (1) The Bacillus thuringiensis NH26 strain provided by the present invention has excellent degradation ability of petroleum / petroleum hydrocarbons (including alkanes and polycyclic aromatic hydrocarbons) under high salinity conditions, and is suitable for the removal of petroleum / petroleum hydrocarbons in marine and other high salinity bodies.

[0027] (2) The Bacillus thuringiensis NH26 strain provided by the present invention also has excellent degradation effect on high concentrations (up to 50 g / L of refined diesel) of petroleum / petroleum hydrocarbons (including alkanes and polycyclic aromatic hydrocarbons) under high salinity conditions.

[0028] (3) The Bacillus thuringiensis NH26 strain provided by the present invention can be used to remove petroleum / petroleum hydrocarbon pollution or to remediate petroleum / petroleum hydrocarbon polluted environments, such as natural seawater and other high-salinity bodies.

[0029] (4) The Bacillus thuringiensis NH26 strain provided by the present invention has the advantages of being easy to cultivate and growing quickly, and is suitable for large-scale production of bacterial agents. It has important value and application prospects in the remediation of oil-polluted sea areas and other high-salinity bodies. Attached Figure Description

[0030] Figure 1 The image shows the morphology of Bacillus thuringiensis NH26 strain; Figure A shows the morphology of colonies on LB agar plates; Figure B shows the morphology of the cells under an optical microscope after Gram staining.

[0031] Figure 2 This is a phylogenetic tree of Bacillus thuringiensis NH26 strain constructed using the adjacent-joining method.

[0032] Figure 3 The growth curve of Bacillus thuringiensis NH26 strain is shown.

[0033] Figure 4 The dynamic fermentation curve of Bacillus thuringiensis NH26 strain is shown.

[0034] Figure 5 Before the degradation experiment, Bacillus thuringiensis NH26 strain ( Figure 5 A) and after degradation ( Figure 5 B) Gas chromatography-mass spectrometry (GC-MS) chromatograms of n-dodecane to n-heptadecane of refined diesel fuel in high-salt liquid culture medium. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0036] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0037] Example 1: Isolation and purification of Bacillus thuringiensis NH26 strain

[0038] I. Sample Source

[0039] Topsoil / sediment samples were collected from a coastal wetland in Huizhou, Guangdong Province, which was contaminated by crude oil. The physicochemical properties of the wetland were as follows: ammonia nitrogen 12.38 μg / g, nitrite nitrogen 1.36 μg / g, nitrate nitrogen 0.94 μg / g, phosphorus content 94.05 μg / g, potassium content 23.32 μg / g, total organic carbon content 1.69%, total organic nitrogen content 0.19%, and pH 7.15.

[0040] II. Isolation and Purification of Strains

[0041] Take 10g of surface soil / sediment sample from coastal wetlands contaminated with crude oil, add it to 100mL of enrichment liquid medium, and incubate for 24 hours on a shaker at 30℃ and 180 rpm. Spread the culture onto selective agar plates and incubate for 24 hours on a shaker at 30℃ and 180 rpm. Pick single colonies. Repeat picking and culturing single colonies until effective isolation of colonies is achieved. Then, based on different colony colors and morphological characteristics, select colonies for streak plating purification using an inoculation needle. Finally, inoculate the strains into slant agar plates and store them at 4℃ for later use.

[0042] The enrichment liquid culture medium consisted of: 20 g / L refined diesel oil (obtained by distillation at 100℃ for 240 hours from commercially available diesel oil), 2.5 g / L urea, 10 g / L NaCl, 3 g / L Na2HPO4, 2 g / L KH2PO4, 1 g / L NH4NO3, 0.7 g / L MgSO4·7H2O, and 1 mL / L trace element solution. The pH was adjusted to 7.2, and the medium was sterilized at 121℃ for 20 minutes (a urea solution of a certain concentration was prepared beforehand and sterilized by filtration through a 0.22 μm membrane; the diesel oil was also sterilized by filtration through a 0.22 μm membrane; both were added after sterilization and cooling). The trace element solution contained: 20 mg / L CaCl2, 30 mg / L FeCl3, 0.5 mg / L CuSO4, 0.5 mg / L MnSO4·H2O, and 1 mL / L ZnSO4·7H2O. 10 mg / L.

[0043] Both slant and plate selective media used LB agar medium, with the following components: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 20 g / L agar; the pH was adjusted to 7.2, and the medium was sterilized at 121°C for 20 minutes.

[0044] III. Screening of Petroleum Hydrocarbon Degrading Bacteria Using the Oil Dissolution Circle Method

[0045] The pure bacterial strain cultured on the slant medium was inoculated onto agar enrichment medium (the specific composition is the same as the enrichment liquid medium above, but agar coagulant is added), and placed in an incubator at 30℃ for 7-10 days. The presence of oil drainage rings was observed, and the hydrocarbon degradation ability of the strain was preliminarily determined based on the ratio of the diameter of the oil drainage rings to the diameter of the colonies.

[0046] The hydrocarbon-degrading bacteria with the largest oil drainage rings selected were inoculated onto slant agar plates and stored at 4°C for later use. This strain was designated NH26.

[0047] Example 2: Identification of Bacillus thuringiensis NH26 strain

[0048] I. Morphological Identification of Strains

[0049] After culturing the NH26 strain on LB agar plates at 30°C for 24 hours, the colony morphology was observed. The morphology of the NH26 strain colonies on the plates is as follows: Figure 1 As shown in A. From Figure 1 A shows that the colonies of strain NH26 are milky white, round, slightly glossy, moist, soft, and thick. After Gram staining, the morphology of strain NH26 under a 20× optical microscope is as follows: Figure 1 As shown in B. From Figure 1B shows that the cells of strain NH26 are rod-shaped, connected in short or long chains, with a size of (2.9–5.1) μm × (1.1–1.6) μm.

[0050] II. Identification of the biochemical characteristics of NH26 strain

[0051] The cells of strain NH26 turn purple after Gram staining, indicating that it is a Gram-positive bacterium (G). + The physiological and biochemical identification of Bacillus thuringiensis strain NH26 was carried out in accordance with the methods reported in the "Manual of Systematic Identification of Common Bacteria", "Bergey's Manual of Bacterial Identification" (8th edition) and other literature. The results are listed in Table 1.

[0052] Table 1 Morphological and physiological-biochemical characteristics of Bacillus thuringiensis NH26 strain

[0053] Experimental indicators strain NH26 Colony color off white Bacterial shape rod-shaped athleticism + Gram staining + aerobic + catalase + Glucose fermentation + Sugar fermentation + Oxidase - Nitrate reduction + Starch hydrolysis + Gelatin hydrolysis + Indole formation - Methyl red test + Acetylmethylmethanol (VP) test + Methyl red (MR) test + Citrate utilization - Hydrogen sulfide generation + Catalase + Casein hydrolysis +

[0054] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0055] III. Identification of the strain's 16S rDNA

[0056] Total genomic DNA was extracted from the NH26 strain obtained after isolation and purification using a DNA extraction kit; the obtained total genomic DNA was amplified; and the amplified products were sequenced on an ABI 3730 sequencing platform.

[0057] The sequencing results showed that the 16S rRNA of this strain was 1420 bp long. A phylogenetic tree was constructed using NCBI's nt library Blast alignment and the phylogenetic analysis software MEGA11. Figure 2 The results showed that the NH26 strain described in this invention belongs to Bacillus thuringiensis and has the highest similarity (approximately 99%) to Bacillus thuringiensis VKK-BB-1 strain.

[0058] In summary, based on the above results and combined with its physiological and biochemical characteristics, it is named Bacillus thuringiensis NH26. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on July 21, 2022, with accession number CCTCC NO: M20221149, deposited at Wuhan University, Wuhan, China, 430072, China.

[0059] Example 3: Growth experiment of Bacillus thuringiensis NH26 strain

[0060] I. Determination of Growth Curve

[0061] Bacillus thuringiensis NH26 strain from LB medium was inoculated into seed culture medium. Three parallel samples were taken and cultured at 30℃ and 180 rpm. The absorbance (OD) of the culture medium at 610 nm was measured every 2 hours using a microplate reader. 610 OD changing over time 610 The curve is the growth curve of Bacillus thuringiensis NH26 strain.

[0062] The components of the seed culture medium are as follows:

[0063] 20 g / L glucose, 10 g / L yeast extract, 5 g / L ammonium sulfate, 2 g / L KH2PO4, 0.5 g / L MgSO4 7H2O and 0.5 g / L NaCl; adjust the pH to 7.0 and sterilize at 121℃ for 20 minutes.

[0064] The results of the growth curve determination are shown in [the table below]. Figure 3 .from Figure 3 It can be seen that the logarithmic growth phase of strain NH26 lasts for 2-10 hours, followed by a stable growth phase. After 34 hours, the bacteria begin to die, reflecting that Bacillus thuringiensis strain NH26 is easy to culture and grows quickly in seed culture medium.

[0065] II. Determination of Dynamic Fermentation Curve

[0066] The seed culture of Bacillus thuringiensis NH26 (cultured in seed medium for 8 hours) was transferred to liquid fermentation medium at an inoculum of 10%. The culture was incubated at 30°C and 180 rpm for 4 days. Fermentation broth samples were taken periodically during fermentation. After centrifugation, the precipitated bacterial cells were freeze-dried under vacuum, and their dry weight was measured to obtain the dry weight of Bacillus thuringiensis NH26. The dry weight of biomass, surface tension (measured using a surface tensiometer), and pH (measured using a pH meter) of the Bacillus thuringiensis NH26 fermentation broth were plotted against time to obtain the dynamic fermentation curve of Bacillus thuringiensis NH26.

[0067] The components of the liquid fermentation medium are shown below:

[0068] Soybean oil 20g / L, yeast extract 5g / L, KH2PO4 2g / L, MgSO4·7H2O 0.5g / L, (NH4)2SO4 5g / L, NaCl 0.5g / L, FeSO4·7H2O 1.5mg / L, MnSO4·H2O 0.005g / L, and L-sodium glutamate 5g / L; adjust the pH to 7.0 and sterilize at 121℃ for 20 minutes.

[0069] The results of the dynamic fermentation curve determination are shown below. Figure 4 .from Figure 4 It can be seen that after 12 hours of fermentation, the biomass of Bacillus thuringiensis NH26 strain reached its maximum, and after 40 hours the biomass tended to stabilize, reflecting that the NH26 strain has the characteristics of being easy to cultivate and growing rapidly in the fermentation liquid medium.

[0070] Example 4: Degradation of alkanes in high-salt water by Bacillus thuringiensis NH26 strain

[0071] I. Degradation capacity of Bacillus thuringiensis NH26 strain for n-dodecane to n-heptadecane in high-salt water at a refined diesel concentration of 20 g / L

[0072] The seed culture of Bacillus thuringiensis NH26 (incubated in seed medium for 8 hours) was transferred at a 10% inoculum to the enrichment liquid medium described in Example 1, but the NaCl concentration was increased to 30 g / L (equivalent to the NaCl concentration in normal seawater), with the other components remaining the same as in Example 1. The medium was cultured for 10 days at pH 7.3, 30°C, and 180 rpm. After degradation by Bacillus thuringiensis NH26, the residual alkanes in the medium (high-salt liquid medium) were first extracted with dichloromethane, and then the alkane composition was analyzed by gas chromatography-mass spectrometry (GC-MS) and compared with the refined diesel fuel composition before the degradation experiment. Figure 5 ).

[0073] The degradation rates of the n-dodecane to n-heptadecane components were calculated by comparing the peak areas before and after the degradation experiment. The results are shown in Table 2.

[0074] Table 2. Degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis NH26 strain in high-salt water at a concentration of 20 g / L.

[0075] Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) C12 94.1 C18 93.6 C24 92.9 C13 92.6 C19 92.8 C25 93.1 C14 93.4 C20 93.1 C26 92.6 C15 93.1 C21 92.4 C27 93.5 C16 93.9 C22 92.9 C17 93.2 C23 92.6

[0076] from Figure 5As shown in Table 2, the degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis strain NH26 was over 90%, with a total degradation rate of 93.1%, indicating that Bacillus thuringiensis strain NH26 has excellent degradation ability for n-dodecane to n-heptadecane in high saline environment.

[0077] II. Degradation capacity of Bacillus thuringiensis NH26 strain for n-dodecane to n-heptadecane in high-salt water at a refined diesel concentration of 50 g / L.

[0078] The seed culture of Bacillus thuringiensis NH26 (cultivated in seed medium for 8 hours) was transferred at a 10% inoculum to the enrichment liquid medium described in Example 1, but the NaCl concentration was increased to 30 g / L (equivalent to the NaCl concentration in normal seawater) and the refined diesel concentration was increased to 50 g / L, with the other components remaining the same as in Example 1. The medium was cultured for 10 days at pH 7.3, 30°C, and 180 rpm. After degradation by Bacillus thuringiensis NH26, the residual alkanes in the medium were first extracted with dichloromethane, and then the alkane composition was analyzed by GC-MS and compared with the refined diesel composition before the degradation experiment. The degradation rate was calculated by comparing the peak areas of n-dodecane to n-heptadecane before and after the degradation experiment, and the results are shown in Table 3.

[0079] Table 3. Degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis NH26 strain in refined diesel oil at a high saline concentration of 50 g / L.

[0080] Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) C12 90.8 C18 87.0 C24 89.2 C13 90.2 C19 89.0 C25 90.0 C14 90.0 C20 87.9 C26 89.4 C15 89.2 C21 88.0 C27 90.2 C16 88.7 C22 87.4 C17 87.5 C23 87.6

[0081] As shown in Table 3, the degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis strain NH26 was over 87%, with a total degradation rate of 88.4%. This indicates that Bacillus thuringiensis strain NH26 still maintains a very good degradation ability for n-dodecane to n-heptadecane in high-salt water of high-concentration (50 g / L) refined diesel.

[0082] Example 5: Degradation of alkanes in natural seawater by Bacillus thuringiensis NH26 strain

[0083] I. Degradation capacity of Bacillus thuringiensis NH26 strain for n-dodecane to n-heptadecane in natural seawater at a refined diesel concentration of 20 g / L.

[0084] The seed culture of Bacillus thuringiensis NH26 strain (cultivated in seed medium for 8 hours) was transferred to the enrichment liquid medium described in Example 1 at an inoculation rate of 10%, but the added NaCl and distilled water were adjusted to natural seawater (obtained by filtering natural seawater from the South China Sea through a 0.22 μm filter membrane, with a salinity of 35‰), while the other components remained the same as in Example 1. The medium was cultured for 10 days at pH 7.3, 30°C, and 180 rpm. After degradation by Bacillus thuringiensis NH26 strain, the residual alkanes in the medium were first extracted with dichloromethane, and then the alkane composition was analyzed by gas chromatography-mass spectrometry (GC-MS) and compared with the refined diesel fuel composition before the degradation experiment. The degradation rate was calculated by comparing the peak areas of n-dodecane to n-heptadecane before and after the degradation experiment, and the results are shown in Table 4.

[0085] Table 4. Degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis NH26 strain in natural seawater at a refined diesel concentration of 20 g / L.

[0086] Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) Alkane carbon number Degradation rate (%) C12 94.2 C18 93.8 C24 92.9 C13 92.4 C19 92.9 C25 93.3 C14 93.1 C20 92.8 C26 92.4 C15 93.3 C21 92.7 C27 93.6 C16 94.0 C22 93.1 C17 93.3 C23 92.8

[0087] As shown in Table 4, the degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis strain NH26 was over 90%, with a total degradation rate of 93.4%, indicating that Bacillus thuringiensis strain NH26 has excellent degradation ability for n-dodecane to n-heptadecane in natural seawater.

[0088] II. Degradation capacity of Bacillus thuringiensis NH26 strain for n-dodecane to n-heptadecane in natural seawater at a refined diesel concentration of 50 g / L.

[0089] The seed culture of Bacillus thuringiensis NH26 strain (cultivated in seed medium for 8 hours) was transferred to the enrichment liquid medium described in Example 1 at an inoculation rate of 10%. However, the added NaCl and distilled water were adjusted to natural seawater (obtained by filtering natural seawater from the South China Sea through a 0.22 μm filter membrane, with a salinity of 35‰), and the concentration of refined diesel oil was increased to 50 g / L. The remaining components were the same as in Example 1. The medium was cultured for 10 days at pH 7.3, 30°C, and 180 rpm. After degradation by Bacillus thuringiensis NH26 strain, the residual alkanes in the medium were first extracted with dichloromethane, and then the alkane composition was analyzed by GC-MS and compared with the refined diesel oil composition before the degradation experiment. The degradation rate was calculated by comparing the peak areas of n-dodecane to n-heptadecane before and after the degradation experiment. The results are shown in Table 5.

[0090] Table 5. Degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis NH26 strain in natural seawater at a refined diesel concentration of 50 g / L.

[0091]

[0092]

[0093] Table 5 shows that the degradation rate of n-dodecane to n-heptadecane by Bacillus thuringiensis strain NH26 was over 88%, with a total degradation rate of 89.4%. This indicates that Bacillus thuringiensis strain NH26 still maintains good degradation ability for n-dodecane to n-heptadecane in natural seawater with high concentration (50 g / L) refined diesel, but the degradation rate is slightly lower than that when the concentration of refined diesel is 20 g / L.

[0094] Example 6: Degradation of polycyclic aromatic hydrocarbons in high-salt water by Bacillus thuringiensis NH26 strain

[0095] I. Degradation rates of anthracene, phenanthrene, and pyrene by Bacillus thuringiensis strain NH26 in high-salt water at concentrations of 100 mg / L

[0096] The seed culture of NH26 strain (cultivated in seed medium for 8 hours) was transferred at a 10% inoculum to the enrichment liquid medium described in Example 1, but the NaCl concentration was increased to 30 g / L (equivalent to the NaCl concentration in normal seawater) and the refined diesel oil was replaced with polycyclic aromatic hydrocarbons (anthracene, phenanthrene, and pyrene, all at 100 mg / L), while the other components remained the same as in Example 1. The medium was cultured for 30 days at pH 7.3, 30°C, and 180 rpm. The residual amounts of anthracene, phenanthrene, and pyrene in the medium were measured every 5 days to calculate their degradation rate. The specific detection method was as follows: extraction with dichloromethane followed by GC-MS analysis of the residual amounts of anthracene, phenanthrene, and pyrene, compared with those before the degradation experiment. The degradation rate was calculated by comparing the peak areas of anthracene, phenanthrene, and pyrene before and after the degradation experiment, and the results are shown in Table 6.

[0097] Table 6. Degradation rates of anthracene, phenanthrene, and pyrene by Bacillus thuringiensis strain NH26 in high-salt water at concentrations of 100 mg / L.

[0098]

[0099]

[0100] As shown in Table 6, the removal rates of anthracene, phenanthrene, and pyrene by strain NH26 all showed an increasing trend with the extension of time; after 30 days of degradation, the removal rates of anthracene, pyrene, and phenanthrene by strain NH26 were 39.57%, 42.48%, and 64.79%, respectively.

[0101] II. Degradation capacity of Bacillus thuringiensis NH26 strain for anthracene, phenanthrene, and pyrene in natural seawater at a concentration of 100 mg / L.

[0102] The seed culture of strain NH26 (cultivated in seed medium for 8 hours) was transferred at a 10% inoculum to the enrichment liquid medium described in Example 1. However, the added NaCl and distilled water were adjusted to natural seawater (obtained by filtering natural seawater from the South China Sea through a 0.22 μm filter membrane, with a salinity of 35‰), and the refined diesel oil was adjusted to polycyclic aromatic hydrocarbons (anthracene, phenanthrene, and pyrene, all 100 mg / L). The remaining components were the same as in Example 1. The medium was cultured for 30 days at pH 7.3, 30°C, and 180 rpm. The residual amounts of anthracene, phenanthrene, and pyrene in the medium were measured every 5 days to calculate their degradation rate. The specific detection method was as follows: extraction with dichloromethane was performed first, followed by GC-MS analysis of the residual amounts of anthracene, phenanthrene, and pyrene, and the results were compared with those before the degradation experiment. The degradation rate was calculated by comparing the peak areas of anthracene, phenanthrene, and pyrene before and after the degradation experiment, and the results are shown in Table 7.

[0103] Table 7. Degradation rates of anthracene, phenanthrene, and pyrene by Bacillus thuringiensis strain NH26 in natural seawater at concentrations of 100 mg / L.

[0104] Time (days) Anthracene degradation rate (%) Degradation rate of pyrene (%) Degradation rate of phenanthrene (%) 5 2.73 4.91 9.85 10 12.87 15.02 20.51 15 20.94 26.95 42.93 20 33.01 36.02 54.70 25 38.68 40.79 60.81 30 40.07 43.88 65.02

[0105] As shown in Table 7, the removal rates of anthracene, phenanthrene, and pyrene by strain NH26 all showed an increasing trend with the extension of time; after 30 days of degradation, the removal rates of anthracene, pyrene, and phenanthrene by strain NH26 were 40.07%, 43.88%, and 65.02%, respectively.

[0106] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus thuringiensis (Bt) Bacillus thuringiensis NH26 strain, characterized in that, It was deposited at the China Center for Type Culture Collection on July 21, 2022, with accession number CCTCC NO: M20221149.

2. The application of the Bacillus thuringiensis NH26 strain according to claim 1 in the degradation of petroleum hydrocarbons under high-salt conditions, wherein the petroleum hydrocarbons include alkanes and polycyclic aromatic hydrocarbons; wherein the alkanes are n-dodecane to n-heptadecane; and wherein the polycyclic aromatic hydrocarbons are anthracene, phenanthrene, and pyrene.

3. The application of the Bacillus thuringiensis NH26 strain according to claim 1 in the removal of petroleum hydrocarbon pollution under high salinity conditions, or in the remediation of petroleum hydrocarbon pollution in environments under high salinity conditions, wherein the petroleum hydrocarbons include alkanes and polycyclic aromatic hydrocarbons; wherein the alkanes are n-dodecane to n-heptadecane; and wherein the polycyclic aromatic hydrocarbons are anthracene, phenanthrene, and pyrene.

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