Petroleum hydrocarbon-degrading bacterial strain 3AS20 and application thereof

Abstract

The application discloses a petroleum hydrocarbon degrading strain 3AS20, the taxonomic name of the strain 3AS20 is Ornithinimicrobium sp. , the preservation number is GDMCC NO: 64107, and the preservation date is December 4, 2023. The application also discloses application of the strain 3AS20 in preparation of diesel oil degradation products. The application verifies that the strain 3AS20 has the ability to degrade diesel oil, especially diesel oil in seawater, and the strain 3AS20 in the application can be made into products for degrading diesel oil in seawater, such as bacterial liquid, culture, microbial preparation and the like, and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of marine microbial technology, specifically relating to a petroleum hydrocarbon degrading strain 3AS20 and its applications. Background Technology

[0002] Oil pollution has significant negative effects on soil, water bodies, and marine ecosystems, and is widely recognized as one of the global environmental problems. With the continuous expansion of offshore oil exploration and development and the sustained growth of maritime oil transportation, oil spills are occurring frequently, and the problem of marine oil pollution is becoming increasingly serious, urgently requiring the development of efficient, economical, and environmentally friendly remediation technologies.

[0003] Currently, remediation technologies for petroleum hydrocarbons (such as diesel) are mainly divided into three categories: physical, chemical, and biological. Physical remediation (such as thermal desorption and gas-phase extraction) is fast and effective, but it consumes a lot of energy. Chemical remediation (such as chemical oxidation) is efficient and has a short cycle, but the injection of chemical reagents may bring the risk of secondary pollution and affect ecological functions. Bioremediation (such as microbial degradation) has significant advantages such as being environmentally friendly, low-cost, and free from secondary pollution.

[0004] Different types of microorganisms can decompose petroleum hydrocarbons into smaller intermediate products through aerobic or anaerobic pathways, utilizing a series of enzymatic reactions such as oxygenases and dehydrogenases. These intermediate products are ultimately mineralized into CO2 and H2O or transformed into the microorganisms' own cellular material. The screening and domestication of highly efficient degrading bacteria are fundamental to this research. In recent years, researchers have isolated a large number of diesel-degrading bacteria from marine sediments, oil-contaminated soils, and oily sludge, covering multiple bacterial groups including Pseudomonas, Bacillus, Acinetobacter, and Lysobacter. However, existing petroleum hydrocarbon microbial degradation technologies still have many shortcomings and bottlenecks. For example, most reported petroleum hydrocarbon degrading bacteria generally lack environmental adaptability and stress resistance. Most reported highly efficient petroleum hydrocarbon degrading bacteria are known species, and the discovery of new species is insufficient. Breakthroughs in microbial remediation technologies depend on the continuous discovery of novel microbial resources. However, there are relatively few studies on the isolation and identification of new species of microorganisms with petroleum hydrocarbon degradation activity in special ecological environments, and the reserve of microbial strains is insufficient to support the development and application of efficient remediation technologies.

[0005] Reef-building corals thrive in tropical oligotrophic waters, where their symbionts form extremely complex microenvironmental gradients (such as areas of localized anoxic conditions, high reducing potential, and organic matter enrichment). These microenvironments share highly similar physicochemical properties with the oil-water-sediment interface formed after oil pollution. Therefore, bacteria that survive long-term within coral symbionts naturally possess the tolerance and transformation potential for hydrophobic organic compounds such as hydrocarbons. A long-term co-evolutionary relationship exists between corals and symbiotic bacteria; the symbiotic bacteria not only assist the host in obtaining nutrients (such as supplementing nitrogen sources through nitrogen fixation) but also participate in the host's response to abiotic stressors. This hydrocarbon degradation metabolic network, selected through host-microbe co-evolution, represents a novel enzyme system or regulatory element distinct from ordinary marine bacteria, possessing irreplaceable scientific research and industrial value.

[0006] Microbial diversity surveys reveal a wealth of undescribed new bacterial taxonomic units (new species, genera, and even families) within coral symbiotic microorganisms. These new species often carry unique metabolic gene clusters capable of catalyzing biotransformation reactions that are impossible for known strains. However, current research on hydrocarbon-degrading bacteria in coral symbiotic organisms is extremely limited both domestically and internationally, with a severe shortage of culturable strains. Furthermore, coral-derived bacteria possessing both novel taxonomic status and hydrocarbon-degrading capabilities are currently absent from domestic and international patent databases. Summary of the Invention

[0007] The purpose of this invention is to provide a petroleum hydrocarbon degrading strain 3AS20.

[0008] Another objective of this invention is to provide the application of the above-mentioned strain 3AS20 in the preparation of degraded diesel products.

[0009] The first objective of this invention can be achieved through the following technical solution: a petroleum hydrocarbon degrading strain 3AS20, wherein the taxonomic name of strain 3AS20 is... Ornithinimicrobium sp. The accession number is GDMCC NO: 64107, and the accession date is December 4, 2023.

[0010] This invention isolates a petroleum hydrocarbon-degrading bacterial strain from corals and identifies the species. This strain belongs to the phylum Actinomycetota, class Actinomycetia, order Dermatophilales, family Ornithinimicrobiaceae, genus Ornithinimicrobium, and is a new species with diesel fuel degradation capabilities. It is named... Ornithinimicrobium sp .3AS20.

[0011] The strain 3AS20 was deposited on December 4, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, postal code 510070, with accession number GDMCC NO.64107.

[0012] The strain 3AS20 described in this invention has the ability to degrade diesel fuel, especially diesel fuel in seawater.

[0013] The second objective of the present invention can be achieved by the following technical solution: the application of the above-mentioned strain 3AS20 in the preparation of degraded diesel products.

[0014] The inventors of this application have experimentally demonstrated that strain 3AS20 of this invention has the ability to degrade diesel fuel, especially diesel fuel in seawater. Therefore, it can be prepared into related products, such as bacterial suspensions, cultures, microbial preparations, etc., using conventional methods in the art, for the purpose of degrading diesel fuel, especially diesel fuel in seawater.

[0015] Preferably, the diesel fuel is diesel fuel from seawater.

[0016] Compared with the prior art, the present invention has the following advantages: (1) The present invention isolated a petroleum hydrocarbon degrading strain from coral and identified the strain. The strain belongs to a new species of the genus Ornithinimicrobium in the family Ornithinimicrobiaceae with petroleum hydrocarbon degrading function. (2) This invention verifies that strain 3AS20 has the ability to degrade diesel, especially diesel in seawater. Under the conditions of pH 7, temperature 30℃ and substrate concentration of 2500mg / L, it can achieve a degradation rate of about 80%. (3) The strain 3AS20 in this invention can be made into products that degrade diesel in seawater, such as bacterial suspension, culture, microbial preparation, etc., which have broad application prospects. Attached Figure Description

[0017] Figure 1a This is the phylogenetic tree of strain 3AS20 constructed based on the maximum likelihood (ML) method of 16S rDNA sequence in Example 1.

[0018] Figure 1b This is the phylogenetic tree of strain 3AS20 constructed in Example 1 based on the Neighbor-Joining (NJ) method using the 16S rDNA sequence.

[0019] Figure 1cThis is the phylogenetic tree of strain 3AS20 constructed based on the minimum evolution (ME) method using the 16S rDNA sequence in Example 1.

[0020] Figure 2 This is the effect of pH on the diesel degradation rate of strain 3AS20 in Example 1.

[0021] Figure 3 This is the effect of temperature on the diesel degradation rate of strain 3AS20 in Example 1.

[0022] Figure 4 This describes the effect of substrate concentration on the diesel degradation rate of strain 3AS20 in Example 1.

[0023] Figure 5 This describes the degradation effect of strain 3AS20 in environmental seawater on diesel fuel in Example 2. Detailed Implementation

[0024] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0025] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0026] Example 1

[0027] 1. Materials and Methods 1.1 Enrichment and Isolation of Strains Coral samples were collected from the reefs near Xidao Island, Sanya, Hainan, at a depth of 3 to 5 meters using scuba diving. Coral fragments (approximately 3 x 3 cm) were rinsed three times with sterile seawater to remove non-symbiotic microorganisms from the surface. The rinsed coral samples were then wrapped in sterile zinc foil, crushed with a hammer, and ground in a grinder to obtain a homogenate. This homogenate was transferred to a 50 mL sterile centrifuge tube, and 20 mL of sterile seawater was added and mixed thoroughly to obtain the coral homogenate stock solution.

[0028] Take 100 μL of coral homogenate stock solution and add it to 1 L of inorganic salt enrichment medium (K2HPO4 1.6 g / L, KH2PO4 0.4 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.1 g / L, glucose 3 g / L, diesel fuel filtered through a 0.22 μm organic nylon membrane for sterilization, diesel fuel concentration 1000 mg / L, sterilized 50% aged seawater to a final volume of 1 L, pH adjusted to neutral, where aged seawater is seawater collected and left to stand for more than 7 days, the 50% aged seawater is prepared by half seawater and half ultrapure water, 50% is by mass percentage). Add glass beads and shake continuously for 15 min, then incubate at 28℃ and 160 r / min. After 7 days of enrichment, the microorganisms were inoculated into a new inorganic salt enrichment medium at a ratio of 5% (w / v). The enrichment was passaged 5 times, with the diesel concentration successively increased to 1500 mg / L, 2000 mg / L, 2500 mg / L, and 3000 mg / L to acclimatize the microorganisms in the culture system, while maintaining the same culture conditions. One mL of the enriched and acclimatized bacterial solution from the 5 passages was then added to a 10... -1 10 -2 10 -3 10 -4 Dilute the solution to the specified concentration and spread it evenly onto 2216E solid medium (recommended but not limited to Haibo Biotechnology, catalog number HB0132, hereinafter the same). Place the plates in a constant temperature incubator and incubate at 28°C for 3-7 days. After colonies have grown on the solid plates, pick out colonies and re-inoculate them onto 2216E solid medium. Repeat the streak culture several times until a purified strain is obtained.

[0029] 1.2 Screening of petroleum hydrocarbon degrading bacteria Add 3-5 drops of diesel fuel to an inorganic salt solid medium plate (K₂HPO₄ 1.6 g / L, KH₂PO₄ 0.4 g / L, MgSO₄·7H₂O 0.4 g / L, NaCl 0.1 g / L, glucose 3 g / L, agar 1.5%, and sterile 50% aged seawater to a final volume of 1 L, pH 7.2). Spread the mixture evenly with a spreader to prepare an inorganic salt solid medium plate containing diesel fuel. Inoculate purified single colonies onto this plate to screen for petroleum hydrocarbon-degrading bacteria. Place the plate in an incubator at 28°C and invert it for incubation.

[0030] Select strains that grew well on inorganic salt solid medium plates containing diesel fuel and inoculate them into inorganic salt liquid medium containing diesel fuel (K2HPO4 1.6 g / L, KH2PO4 0.4 g / L, MgSO4·7H2O 0.4 g / L, NaCl 0.1 g / L, glucose 3 g / L, diesel fuel sterilized by filtration through a 0.22 μm organic nylon membrane, diesel fuel concentration 500 mg / L, and sterilized 50% aged seawater to a final volume of 1 L, pH 7.2). Incubate in a constant temperature shaker at 28℃ and 180 r / min for 7 days, and then determine the degradation efficiency.

[0031] 1.3 Determination of Petroleum Hydrocarbon Degradation Rate The degradation rate of diesel fuel was determined using ultraviolet spectrophotometry. The specific determination method is as follows: Take 100 mg / L diesel fuel and use a quartz cuvette with n-hexane as a reference solution. Measure the absorbance at 210 nm to 320 nm using a UV spectrophotometer. Plot the absorbance curve with UV wavelength as the x-axis and absorbance as the y-axis to determine the maximum absorption wavelength. The standard oil sample has the highest absorbance at 230 nm, so 230 nm is chosen as the measurement wavelength. Using a 10 mm quartz cuvette and n-hexane as a blank, measure the absorbance of samples with different concentrations at 230 nm. Plot the diesel fuel concentration as the x-axis and absorbance as the y-axis to create a standard curve. The regression equation is: y = 0.0064x - 0.0081, R0 2 =0.9991.

[0032] The strain was inoculated into an Erlenmeyer flask containing 100 mL of inorganic salt liquid medium containing diesel fuel and cultured on a shaker at 28 °C and 180 rpm. After 7 days of culture, 50 mL of n-hexane was added to the Erlenmeyer flask, and extraction was performed on a shaker (160 rpm) for 30 min. All liquid was transferred to a separatory funnel, shaken for 5 min, and allowed to stand for 3 min to allow for phase separation. The upper organic phase was collected after phase separation. The lower aqueous phase was added to 50 mL of n-hexane and extracted once more in a separatory funnel. The organic phases from the two extractions were combined, and an appropriate amount of anhydrous Na₂SO₄ was added to remove excess water. The OD value was measured at 230 nm using a UV-Vis spectrophotometer, and the diesel fuel concentration was calculated using a standard curve. Uninoculated diesel fuel inorganic salt liquid medium served as a blank control.

[0033] Formula for calculating diesel degradation rate:

[0034] In the formula, X is the diesel degradation rate, and C X The concentration of diesel fuel in the culture medium (mg / L), C CK The diesel concentration (mg / L) is for the blank group.

[0035] Strain 3AS20 has a high ability to degrade diesel fuel. Within 7 days, the diesel fuel degradation rate of this strain is 68%±5%, which is higher than 60%, making it an ideal high-efficiency degradation strain.

[0036] 1.4 Cultivation and Microbial Characteristics of Petroleum Hydrocarbon Degrading Strain 3AS20 (1) Cultivation characteristics Strain 3AS20 grows rapidly on 2216E solid medium, forming round colonies with a diameter of approximately 0.5–1.5 mm after 24 hours of incubation at 28°C. The colonies are milky white in color, with neat edges and a moist, smooth surface. On nutrient agar medium, colonies form after 24 hours, with a diameter of 1.0–2.0 mm, milky white in color, and a smooth, round surface (see Table 1 for specific characteristics).

[0037] Table 1. Culture characteristics of strain 3AS20

[0038] (2) Physiological and biochemical characteristics Strain 3AS20 can utilize maltose, glucose, N-acetylglucosamine, gluconate, and malic acid well among the 12 carbon sources in API 20 NE (recommended but not limited to Biomerieux, catalog number 20050). However, it cannot utilize mannose, D-mannitol, arabinose, capric acid, oxalic acid, citric acid, and phenylacetic acid. The strain produces urease and cytochrome oxidase, as well as β-glucosidase, urease, protease, and β-galactosidase. It does not produce nitrate reductase, arginine dihydrolase, or indoleic acid. See Table 2 for details.

[0039] Table 2 Physiological and biochemical characteristics of strain 3AS20

[0040] 1.5 Identification of 16S rDNA of Petroleum Hydrocarbon Degrading Bacteria Single colonies of strain 3AS20 were picked and cultured in 50 mL of 2216E liquid medium (recommended but not limited to Haibo Biotechnology, catalog number HB0132-1) at 28℃ and 180 rpm for 48 h. Then, 2 μL of the bacterial culture was transferred to a 1.5 mL centrifuge tube, 30 μL of sterile water was added, and the mixture was quickly centrifuged to prevent liquid from adhering to the tube walls. The centrifuge tube was then incubated in a 100℃ metal bath for 10 min. After centrifugation at 12000 rpm for 1 min, 2 μL of the supernatant was collected as template DNA for amplification and sequencing of the 16S rRNA of the test strain.

[0041] The 16S rDNA sequence of the purified strain (e.g., SEQ ID NO:1) was amplified by PCR using universal bacterial primers: 27f (5′-AGAGTTTGAT CMTGCCTCAG-3′, e.g., SEQ ID NO:2) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′, e.g., SEQ ID NO:3). The 16S rDNA sequence was submitted to NCBI for alignment and then to the EzBioCloud model strain database (https: / / www.ezbiocloud.net / ) for phylogenetic similarity comparison with model strains.

[0042] The results showed that strain 3AS20 shared 98.74% homology with *Ornithinimicrobium faecis* strain HY1793 and *Ornithinimicrobium cryptoxanthini* strain JY.X270T in the NCBI database. However, since *Ornithinimicrobium faecis* strain HY1793 is not a type strain, and although *Ornithinimicrobium cryptoxanthini* strain JY.X270T is a type strain, its status in the EzBioCloud type strain database is "Invalid Name," therefore it cannot be used for subsequent comparisons for new species identification. Comparison with strains in the EzBioCloud type strain database revealed that strain 3AS20 is similar to the type strain *Ornithinimicrobium cavernae* CFH30183. T The two species are most closely related, sharing 98.44% homology in 16S rDNA. Furthermore, phylogenetic analysis indicates that... Figure 1a , Figure 1b and Figure 1c As shown, strain 3AS20 is related to three closely related strains. Ornithinimicrobium faecis strainHY1793, Ornithinimicrobium cryptoxanthini strain JY.X270 T and Ornithinimicrobium cavernae CFH 30183 T None of them can cluster on the same branch (the Bootstrap values ​​based on maximum likelihood, adjacency, and minimum evolution are all less than 70).

[0043] Further physiological and biochemical experimental results indicate that this bacterium belongs to the phylum Actinomycetota, class Actinomycetia, order Dermatophilales, family Ornithinimicrobiaceae. Ornithinimicrobium A new species of the genus with diesel degradation capabilities has been named Ornithinimicrobium sp. 3AS20.

[0044] Strain 3AS20 was deposited on December 4, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, postal code 510070, with accession number GDMCC NO.64107.

[0045] 1.6 Effects of different conditions on the diesel degradation rate of the strain To investigate the effects of different factors on the degradation efficiency of the strain, seven pH levels (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0), six temperature levels (15℃, 20℃, 25℃, 30℃, 35℃, and 40℃), and nine substrate concentration levels (250 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L, 2500 mg / L, 3000 mg / L, 3500 mg / L, and 4000 mg / L) were selected, and orthogonal experiments were designed to optimize the degradation conditions. The bacterial culture (same as in Example 1, 1.5) was inoculated at a 5% (volume percentage) in 100 mL of inorganic salt liquid medium containing diesel oil (containing 500 mg / L diesel oil) and cultured on a shaker at 28℃ and 180 rpm. Samples were taken after 7 days of cultivation, the remaining diesel content was measured, and the diesel degradation rate was calculated.

[0046] The results are as follows Figure 2-4 As shown: At pH 5-8, the degradation rate of diesel fuel by strain 3AS20 is relatively high, exceeding 60%, with the degradation rate exceeding 80% at pH 7 and 8; when the pH is below 4, the degradation rate decreases sharply. Figure 2 ).

[0047] The degradation rate of diesel fuel by the strain was lowest at 15℃, at 16% ± 3%, and increased with increasing temperature. At 25℃, 30℃, and 35℃, the degradation rate was above 70%, while at 40℃, it decreased to 61% ± 5%. Figure 3 ).

[0048] When the substrate concentrations were 1500 mg / L, 2000 mg / L, and 2500 mg / L, the diesel degradation rate of the strain was higher than 80%. The degradation rate decreased with increasing substrate concentration, but when the substrate concentration reached 5000 mg / L, the degradation rate decreased to 28% ± 4%. Figure 4 ).

[0049] To further optimize the degradation conditions of diesel-degrading bacteria, based on the above experimental results, orthogonal experiments were conducted with three gradients of pH (6, 7, 8), temperature (25℃, 30℃, 35℃), and substrate concentration (1500mg / L, 2000mg / L, 2500mg / L) to explore the optimal degradation conditions for diesel degradation by strain 3AS20.

[0050] Table 3. Results of the orthogonal experiment

[0051] As shown in Table 3, using the diesel degradation rate of the strain at 7 days as an indicator, the range analysis yielded: R pH >R 温度 > R 底物浓度 Therefore, within the selected gradient range of factors in the experiment, the order of influence on the degradation performance of the strain is pH > temperature > substrate. Thus, within the selected gradient range, pH and temperature are the main factors affecting the degradation of diesel fuel by strain 3AS20.

[0052] Based on the above analysis, the optimal conditions for the degradation of diesel by strain 3AS20 are inferred to be: pH 7, temperature 30℃, and substrate concentration 2500 mg / L.

[0053] Example 2

[0054] Degradation effect of strain 3AS20 on diesel fuel in environmental seawater Single colonies of strain 3AS20 were picked and incubated in 100 mL of 2216E liquid medium at 28°C and 180 rpm for 48 h to obtain the seed culture. At a 10% inoculum rate, 100 mL of the seed culture was inoculated into 1 L of 2216E liquid medium and incubated at 28°C and 180 rpm for 48 h. The bacterial cells were collected to prepare a bacterial suspension with a concentration of 1 × 10⁻⁶ cells / mL. 10 cells / mL. One liter of nearshore surface seawater was taken, and diesel oil was added to a final concentration of 500 mg / L. 10 mL of bacterial suspension was added, and seawater samples without bacterial inoculation served as negative controls. Each treatment was repeated in triplicate. The samples were incubated at 28℃ and 180 rpm on a shaker. Samples were taken after 7 days to determine the diesel oil degradation.

[0055] The degradation effect of strain 3AS20 on diesel fuel in environmental seawater is as follows: Figure 5As shown in the figure, after a 7-day simulated seawater remediation experiment, the diesel degradation rate in the inoculated seawater reached 44% ± 8% compared to the uninoculated seawater, indicating that strain 3AS20 has a good degradation ability for diesel in seawater. In the control experiment without inoculation, the natural degradation rate was only 3% ± 2%. This shows that strain 3AS20 has a good remediation effect on diesel residues in seawater.

[0056] It should be noted that the above embodiments are merely further illustrations of the present invention and not limitations. Any adjustments or changes made by those skilled in the art within the equivalent meaning and scope of the technical solutions of the present invention should be considered as included within the protection scope of the present invention.

Claims

1. A petroleum hydrocarbon degrading strain 3AS20, characterized in that, The taxonomic name of strain 3AS20 is... Ornithinimicrobium sp. The accession number is GDMCC NO: 64107, and the accession date is December 4, 2023.

2. The application of strain 3AS20 according to claim 1 in the preparation of degraded diesel products.

3. The application according to claim 2, characterized in that, The diesel fuel is diesel fuel from seawater.

Images