Stenotrophomonas sp. BRS7-2 for degrading petroleum hydrocarbon in saline-alkali environment and application of stenotrophomonas sp. BRS7-2

Abstract

The invention relates to the field of environmental governance and bioremediation, in particular to stenotrophomonas sp. BRS7-2 for degrading petroleum hydrocarbon in a saline-alkali environment and application, the stenotrophomonas sp. BRS7-2 is preserved in China Center for Type Culture Collection on March 20, 2025, and the preservation number is CCTCC NO: M 2025545; the strain can produce glycolipid surfactants, and can degrade crude oil, toluene, isooctane, n-hexadecane, naphthalene, phenanthrene, pyrene and benzopyrene. The strain has the characteristic of saline-alkaline tolerance, and can degrade 71.01% of petroleum hydrocarbon under the conditions that the pH is 7.4 and the crude oil concentration is 3%; under the high saline-alkaline conditions that the pH is 8.0, the NaCl concentration is 7.5% and the crude oil concentration is 3%, 54.16% of petroleum hydrocarbon is degraded, by means of the excellent saline-alkaline tolerance and efficient petroleum hydrocarbon degradation performance, a powerful tool is provided for treatment and efficient remediation of saline-alkaline and petroleum polluted environments, and the huge potential of large-scale application in the field of ecological remediation is shown.

Description

Technical Field

[0001] The present invention relates to the fields of environmental management and bioremediation, and in particular to a strain of Stenotrophomonas BRS7-2 capable of degrading petroleum hydrocarbons in saline-alkali environments and applications thereof. Background Art

[0002] Oil pollution refers to the accidental release or discharge of petroleum and its derivatives (such as gasoline, diesel, and lubricants) into the environment, resulting in damage to soil, water, air, and ecosystems. Currently, physical, chemical, and biological remediation technologies can mitigate these impacts. However, traditional physical and chemical remediation technologies have drawbacks such as high costs, high environmental burdens, limited effectiveness, long cycles, and poor adaptability (Ambaye et al., 2022).

[0003] Microbial remediation, as a low-cost, environmentally friendly method for oil pollution control, has garnered widespread attention worldwide in recent years (Lu et al., 2014). Microbial remediation utilizes the metabolic activity of microorganisms to degrade petroleum contaminants into harmless or low-toxic substances, ultimately achieving soil purification (Partovinia et al., 2018). Identifying and cultivating microbial strains that are both saline-tolerant and highly efficient in degrading petroleum hydrocarbons is key to improving the remediation efficiency of oil-contaminated saline-alkali soils.

[0004] In response to the above technical problems, the present invention provides a strain of Stenotrophomonas BRS7-2 for degrading petroleum hydrocarbons in a saline-alkali environment and its application. The Stenotrophomonas BRS7-2 was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M 2025545. The colony is off-white with smooth edges, produces glycolipid surfactants, and can degrade crude oil, toluene, isooctane, n-hexadecane, naphthalene, phenanthrene, pyrene, and benzopyrene. It has the characteristics of salt and alkali resistance, and can degrade 71.01% of petroleum hydrocarbons under the conditions of pH 7.4 and crude oil concentration of 3%, and can degrade 54.16% of petroleum hydrocarbons under high salinity and alkali conditions of pH 8.0, NaCl concentration of 7.5%, and crude oil concentration of 3%. With its excellent salt and alkali resistance and efficient petroleum hydrocarbon degradation performance, this strain provides a way for efficient remediation of high-salinity and alkaline petroleum-contaminated environments, and demonstrates its great potential for large-scale application in the field of ecological restoration.

[0005] References:

[0006] Ambaye TG, Chebbi A, Formicola F, et al. Remediation of soil polluted with petroleum hydrocarbons and its reuse for agriculture: Recent progress, challenges, and perspectives [J]. Chemosphere, 2022, 293: 133572.

[0007] Lu L,Huggins T,Jin S,et al.Microbial metabolism and communitystructure in response to bioelectrochemically enhanced remediation ofpetroleum hydrocarbon-contaminated soil[J].Environmental Science&Technology,2014,48(7):4021-4029.

[0008] Partovinia A,Rasekh B.Review of the immobilized microbial cellsystems for bioremediation of petroleum hydrocarbons polluted environments[J].Critical Reviews in Environmental Science and Technology, 2018,48(1):1-38.

[0009] Li Yongxia, Huang Ying, Xu Minmin, et al. Research progress in remediation technology of oil-contaminated saline-alkali soil[J]. Earth and Environment, 2013, 41(05): 583-588.

[0010] Zhao Huihui, Xiao Xian, Pei Meng, et al. Effects of long-term petroleum pollution on the molecular ecological network of microbial communities in saline-alkali soils[J]. Environmental Science, 2016, 37(09): 3582-3589. Summary of the Invention

[0011] The present invention is achieved through the following technical solutions:

[0012] The primary purpose of the present invention is to provide a strain of Stenotrophomonas sp. BRS7-2 that degrades petroleum hydrocarbons in a saline-alkali environment. The Stenotrophomonas sp. BRS7-2 was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M 2025545.

[0013] The second object of the present invention is to provide a bacterial agent containing the Stenotrophomonas BRS7-2.

[0014] The third object of the present invention is to provide the use of the Stenotrophomonas BRS7-2 or the bacterial agent in the degradation of petroleum hydrocarbons.

[0015] The fourth object of the present invention is to provide the use of the Stenotrophomonas BRS7-2 or the bacterial agent in degrading petroleum hydrocarbons in a saline-alkali environment.

[0016] The fifth object of the present invention is to provide the use of the Stenotrophomonas BRS7-2 or the bacterial agent in repairing a petroleum-contaminated environment.

[0017] Preferably, the petroleum-contaminated environment is petroleum-contaminated water and / or soil.

[0018] The sixth object of the present invention is to provide the use of the Stenotrophomonas BRS7-2 or the bacterial agent in the production of glycolipid surfactants.

[0019] The seventh object of the present invention is to provide a method for degrading petroleum hydrocarbons using the Stenotrophomonas BRS7-2 or the bacterial agent.

[0020] The eighth object of the present invention is to provide a petroleum hydrocarbon degradation system, wherein the system comprises the Stenotrophomonas BRS7-2 or the bacterial agent.

[0021] The beneficial effects of the present invention are as follows: (1) The present invention provides a strain of Stenotrophomonas sp. BRS7-2 that degrades petroleum hydrocarbons in saline-alkali environments. The Stenotrophomonas sp. BRS7-2 was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M 2025545;

[0022] (2) The colonies of the described Stenotrophomonas BRS7-2 are off-white with smooth edges, produce glycolipid surfactants, and degrade crude oil, toluene, isooctane, n-hexadecane, naphthalene, phenanthrene, pyrene, and benzopyrene, with all test results being positive;

[0023] (3) The described Stenotrophomonas BRS7-2 can degrade 71.01% of petroleum hydrocarbons after 14 days of reaction under the conditions of pH 7.4 and crude oil concentration of 3% (v:v); it can degrade 54.16% of petroleum hydrocarbons after 14 days of reaction under the high salinity and alkaline conditions of pH 8.0, NaCl concentration of 7.5%, and crude oil concentration of 3% (v:v). The described Stenotrophomonas BRS7-2 has the characteristics of salt and alkali resistance and can tolerate the pH range of pH 5.0-10.0 and the salinity range of 0.5%-7.5%;

[0024] (4) It provides bacterial strain resources for the remediation of oil pollution in saline-alkali land. Its strong tolerance and high degradation efficiency are expected to be applied on a large scale in the fields of complex environmental governance and ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Strain colony morphology

[0026] Figure 2 Identification results and phylogenetic tree constructed based on the neighbor-joining method

[0027] Figure 3 Growth curve of Stenotrophomonas BRS7-2

[0028] Figure 4 Glycolipid surfactants produced by Stenotrophomonas sp. BRS7-2

[0029] Figure 5 Degradation characteristics of Stenotrophomonas sp. BRS7-2

[0030] Figure 6 Degradation rate of petroleum hydrocarbons by Stenotrophomonas BRS7-2 under different conditions

[0031] Note: Normal conditions: pH 7.4, 0% NaCl; saline conditions: pH 8.0, 7.5% NaCl

[0032] Figure 7 Growth and degradation characteristics of Stenotrophomonas sp. BRS7-2 in saline-alkali environment

[0033] Note: a. Growth curve; b. Petroleum hydrocarbon degradation curve DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0035] It should be noted that, in the following examples, unless otherwise specified, the methods described are conventional methods, and the reagents described can be purchased from the market.

[0036] The culture medium formula involved in this embodiment is as follows:

[0037] BH inorganic salt culture medium: KH2PO4 1.0g, K2HPO4 1.0g, NH4NO3 1.0g, MgSO4.7H2O 0.2g, FeCl3 0.1g, CaCl2 0.03g, 1000mL distilled water, pH = 7.4.

[0038] MSM+trace element enriched culture medium: NH4SO4 1.0g, NaNO3 2.0g, KH2PO4 10g, Na2HPO4 4.0g, MgSO4.7H2O 0.3g, NaCl 5.0g, trace elements 0.5ml, distilled water 1000ml, trace element solution: FeSO4 0.054g, Mn(SO4)2 0.3g, CuSO4 0.032g, 1000mL distilled water.

[0039] Blue gel agar medium: 1 g beef extract, 20 g glucose, 5 g peptone, 0.2 g yeast extract powder, 18 g agar, 0.2 g hexadecyltrimethylammonium bromide, 0.005 g methylene blue, 1000 mL distilled water.

[0040] R2A culture medium: 0.1 g tryptone, 0.1 g yeast extract powder, 0.1 g acid hydrolyzed casein, 0.1 g soluble starch, 0.06 g sodium pyruvate, 0.06 g K2HPO4, 0.01 g MgSO4.7H2O, add 1000 ml distilled water, pH = 7.4, add 20 g agar to the solid culture medium.

[0041] LB medium: 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, plus 1000 ml of distilled water, pH = 7.4, and 20 g of agar was added to the solid medium.

[0042] Slant preservation medium: same as LB medium.

[0043] Example 1: Isolation, screening and identification of petroleum hydrocarbon-degrading strains

[0044] 1. Strain Isolation and Culture

[0045] 10 g of petroleum hydrocarbon-containing soil from an oilfield platform was added to 250 ml of BH inorganic salt medium and cultured in an enrichment incubator at 28°C and 180 rpm for 21 days. The culture medium was replaced every 7 days, and the crude oil concentration was increased (0.5%, 1.0%, and 3.0%). After the culture was completed, the flask was allowed to stand for 1 hour. 1 mL of the suspension was diluted and plated onto R2A solid medium, resulting in a strain named BRS7-2.

[0046] Filter results such as Figure 1 As shown, the colonies of the strain are milky white with smooth edges.

[0047] 2. Strain Identification

[0048] Genomic DNA from strain BRS7-2 was extracted using a bacterial genomic DNA extraction kit (DP302) and polymerase chain reaction (PCR) amplification was performed using universal bacterial primers (27F and 1492R). The amplified product was sequenced by Sangon Biotech (Shanghai) Co., Ltd. Sequencing results were compared at NCBI, and a phylogenetic tree was constructed using the neighbor-joining method based on the blast results.

[0049] The strain BRS7-2 was identified as Stenotrophomonas lactitubi (NR 179509.1) with a similarity of 98.96%. It is a Stenotrophomonas sp., so it was named Stenotrophomonas sp. BRS7-2. The phylogenetic tree is as follows: Figure 2 shown.

[0050] The Stenotrophomonas sp. BRS7-2 was deposited in the China Center for Type Culture Collection on March 20, 2025, with the deposit number CCTCC NO: M 2025545. The address is Wuhan University, Wuhan City, Hubei Province, Tel: 027-68754052, Fax: 027-68754833.

[0051] It should be noted that in the following examples, the Stenotrophomonas sp. BRS7-2 is abbreviated as Stenotrophomonas sp. BRS7-2, and both have the same meaning.

[0052] Example 2: Characteristics of Stenotrophomonas BRS7-2

[0053] 1. Strain Growth Curve

[0054] Strain growth curve determination: The activated Stenotrophomonas BRS7-2 was inoculated into LB liquid medium at a 2% inoculum volume and cultured at 28°C for 48 hours. Samples were taken every 2 hours and 200 μL of the bacterial solution was placed in a 96-well plate. The blank medium was used as a control and the absorbance OD was measured using a microplate reader. 600 , according to OD 600 Draw a growth curve.

[0055] The results are as follows Figure 3 As shown in Figure 2, the growth curve of Stenotrophomonas BRS7-2 cultured in LB liquid medium at 28°C for 48 hours shows typical microbial growth characteristics. The initial stage (0-8 hours) is the lag phase, and the OD 600 The value increases slowly; then enters the logarithmic growth period (8-24h), OD 600 It increased significantly, indicating that the bacteria proliferated rapidly; after 24 hours, it entered the stable period, and OD 600 The growth and death rates of the bacteria tended to be stable, and a balance was reached; no obvious decline period was observed during the 48h culture period.

[0056] 2. Salt tolerance

[0057] Stenotrophomonas BRS7-2 was inoculated into enriched culture medium with final NaCl concentrations of 0.5%, 5.0%, 7.5% and 10.0%, respectively. Three parallel groups were set up for each concentration. The culture was carried out in a 2 mL sterile centrifuge tube at 28°C and 180 r / min for 48 h. The growth of the strain was observed, and the uninoculated sterilized culture medium was used as a blank control.

[0058] The results showed that Stenotrophomonas BRS7-2 grew well at NaCl concentrations ranging from 0.5% to 7.5%, but its growth was significantly inhibited or stopped at 10% NaCl.

[0059] 3. Acid and alkali resistance

[0060] To investigate the effect of pH on the tolerance of Stenotrophomonas BRS7-2, the pH of the enrichment medium was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 using sterile NaOH or HCl, with triplicate samples set for each gradient. The strain was inoculated into a 2 mL sterile centrifuge tube at a specific concentration and incubated at 28°C and 180 rpm for 48 hours. The growth of the strain was observed, and uninoculated sterile medium served as a blank control.

[0061] The results showed that Stenotrophomonas BRS7-2 could grow normally in the pH range of 5.0 to 10.0, and the bacterial solution was obviously turbid; under pH 11.0 conditions, the turbidity of the bacterial solution was significantly reduced, the growth of the strain was inhibited, and there was almost no obvious proliferation phenomenon.

[0062] 4. Ability to produce glycolipid surfactants

[0063] The morphological characteristics, surfactant production and degradation characteristics of Stenotrophomonas BRS7-2 were analyzed. The activated bacterial solution was adjusted to OD600 of 1.0 (colony count 2*10 8 CFU / mL), then aspirate 10 μL of bacterial solution and inoculate it onto blue gel agar medium. If a blue circle appears around the colony, it means that the bacteria produces glycolipid surfactants.

[0064] like Figure 4 As shown, the Stenotrophomonas sp. BRS7-2 produces glycolipid surfactants.

[0065] 5. Substrate degradation ability

[0066] The hydrocarbon degradation ability of Stenotrophomonas sp. BRS7-2 was evaluated using the DCPIP (dichlorophenol indophenol) assay. Crude oil, toluene, n-hexadecane, isooctane, phenanthrene, naphthalene, pyrene, and benzo[a]pyrene were added to BH medium containing DCPIP as the sole carbon source. The reaction system consisted of 3 μL of bacterial suspension, 2 μL of hydrocarbon substrate, 150 μL of culture medium, and 45 μL of a 1.0 g / L DCPIP solution. The strain's ability to utilize different hydrocarbons and its metabolic activity were assessed by observing the color change of DCPIP from blue (oxidized state) to colorless (reduced state). After sealing the 96-well plate, the plate was incubated at 25°C for 24, 48, 72, and 96 hours, and the DCPIP color change was qualitatively recorded. Two control groups were set up: Control 1 (CK1) consisted of DCPIP and culture medium plus bacterial suspension to evaluate the effect of the strain itself on DCPIP; Control 2 (CK2) consisted of DCPIP and culture medium plus various hydrocarbon substrates to eliminate interference between the substrate and the indicator. All experiments were performed in 96-well plates.

[0067] The results are shown in Table 1 and Figure 5 As shown, the Stenotrophomonas sp. BRS7-2 can degrade crude oil and hydrocarbons such as toluene, isooctane, n-hexadecane, naphthalene, phenanthrene, pyrene, and benzopyrene.

[0068] Table 1 Identification of surfactant production and degradation characteristics of strains

[0069]

[0070] Note: “+” indicates positive, and “-” indicates negative.

[0071] Example 3: Crude Oil Degradation Characteristics of Stenotrophomonas BRS7-2

[0072] 1. Determination of strain degradation rate

[0073] After culturing the activated Stenotrophomonas BRS7-2 in 100 mL LB medium for 48 h, the activated bacterial solution was adjusted to OD600 of 1.0 (colony count 2*10 8 CFU / mL), and inoculated with an inoculum size of 5% into an inorganic salt culture medium with 3% crude oil as the sole carbon source. The experiment set up two groups of conditions: normal conditions (NaCl concentration of 0%, pH 7.4) and high saline and alkaline conditions (NaCl concentration of 7.5%, pH 8.0). Three parallel samples were set up in each group and cultured for 14 days at 28°C and 180r / min. Then 25ml of n-hexane was used for extraction, and each sample was extracted three times and the extracts were combined. The crude oil extracted from the crude oil culture medium without inoculation of bacterial agents was used as the control group. After the extract was cultured, the degradation rate of petroleum hydrocarbons was determined: the culture medium was shaken with n-hexane to extract the residual petroleum hydrocarbons and filtered. After the n-hexane was completely evaporated, it was weighed and the degradation rate of petroleum hydrocarbons of the strain was calculated. The calculation of the degradation rate of petroleum hydrocarbons by the strain is as shown in formula (1).

[0074]

[0075] Where m1 is the petroleum hydrocarbon content in the control group after treatment; m2 is the petroleum hydrocarbon content in the microbial treatment. Three parallel experiments were performed for each sample, and the average value was taken to calculate the petroleum degradation rate.

[0076] The results are as follows Figure 6 As shown in the figure, it was calculated that under normal conditions and high saline-alkali conditions, Stenotrophomonas BRS7-2 degraded 71.01% and 54.16% of petroleum hydrocarbons within 14 days, respectively.

[0077] 2. Growth curve and degradation ability of strains in saline-alkali environment

[0078] After culturing the activated BRS7-2 in 100 mL LB medium for 48 h, the activated bacterial solution was adjusted to OD600 of 1.0 (colony count 2*10 8 CFU / mL) were inoculated at a 5% inoculum into an inorganic salt medium containing 3% crude oil as the sole carbon source. The solution was adjusted to a pH of 8.0 and a NaCl concentration of 7.5% in a highly saline and alkaline environment. Three replicates were set up for each group and cultured at 28°C and 180 rpm for 12 days. Every other day, the absorbance at 600 nm was measured using a UV-visible spectrophotometer to plot the strain growth curve. The oil degradation rate was then determined the following day using the same method to plot the strain degradation curve.

[0079] like Figure 7As shown, in a saline-alkali environment (pH 8.0, 7.5% NaCl), Stenotrophomonas sp. BRS7-2 exhibited significant petroleum degradation ability when fed 3% crude oil as the sole carbon source. The petroleum hydrocarbon concentration gradually decreased with culture time. By day 12, the petroleum hydrocarbon concentration in the BRS7-2 group had dropped from an initial 0.3 g / kg to 0.13 g / kg, demonstrating significantly higher degradation efficiency than the control group (CK). The strain growth curve revealed that BRS7-2 entered a logarithmic growth phase during the initial culture period (days 0-2), with a rapid increase in OD600 values. This activity subsequently entered a stable phase and remained high, consistent with the increasing degradation rate.

[0080] In summary, the present invention provides a strain of Stenotrophomonas sp. BRS7-2 that degrades petroleum hydrocarbons in a saline-alkali environment. The Stenotrophomonas sp. BRS7-2 was deposited in the China Center for Type Culture Collection on March 20, 2025, with a deposit number of CCTCC NO: M 2025545. The colony of the Stenotrophomonas sp. BRS7-2 is off-white with smooth edges, produces glycolipid surfactants, degrades crude oil, degrades toluene, isooctane, n-hexadecane, naphthalene, phenanthrene, pyrene, and benzopyrene, and the test results are all positive. The Stenotrophomonas sp. BRS7-2 can degrade 71.01% of petroleum hydrocarbons after 14 days of reaction under the conditions of pH 7.4 and crude oil concentration of 3% (v:v); it can degrade 71.01% of petroleum hydrocarbons after 14 days of reaction at pH 7.4 and crude oil concentration of 3%. Under the high salinity and alkaline conditions of 8.0, NaCl concentration of 7.5%, and crude oil concentration of 3% (v:v), 54.16% of petroleum hydrocarbons were degraded after 14 days of reaction. The described stenotrophomonas BRS7-2 has the characteristics of salt and alkali resistance and can tolerate the pH value of 5.0-10.0 and the salinity range of 0.5%-7.5%. It provides a strain resource for the remediation of petroleum pollution in saline-alkali land. Its strong tolerance and high degradation efficiency are expected to achieve large-scale application in the fields of complex environmental governance and ecological restoration.

[0081] The above description is merely an exemplary embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any equivalent modification, simple variation or replacement made without departing from the core principle of the present invention shall be deemed to fall within the scope of protection of the claims of the present invention.

Claims

1. A strain of Stenotrophomonas sp. BRS7-2 that degrades petroleum hydrocarbons in saline-alkali environments, characterized by: The described Stenotrophomonas BRS7-2 was deposited in the China Center for Type Culture Collection on March 20, 2025, with the deposit number being CCTCC NO: M 2025545.

2. A bacterial agent, characterized in that The bacterial agent contains the Stenotrophomonas BRS7-2 according to claim 1.

3. Use of the Stenotrophomonas BRS7-2 according to claim 1 or the bacterial agent according to claim 2 in the degradation of petroleum hydrocarbons.

4. Use of the Stenotrophomonas BRS7-2 according to claim 1 or the bacterial agent according to claim 2 in degrading petroleum hydrocarbons in a saline-alkali environment.

5. Use of the Stenotrophomonas BRS7-2 according to claim 1 or the bacterial agent according to claim 2 in repairing a petroleum-contaminated environment.

6. The use according to claim 5, characterized in that The oil-polluted environment is oil-polluted water and / or soil.

7. Use of the Stenotrophomonas BRS7-2 according to claim 1 or the bacterial agent according to claim 2 in producing glycolipid surfactants.

8. A method for degrading petroleum hydrocarbons, characterized in that: Degradation is carried out using the Stenotrophomonas BRS7-2 described in claim 1 or the bacterial agent described in claim 2.

9. A petroleum hydrocarbon degradation system, characterized in that: The system comprises the Stenotrophomonas BRS7-2 according to claim 1 or the bacterial agent according to claim 2.

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