Bacterial composition for promoting petroleum degradation based on quorum sensing and application of bacterial composition in low-temperature petroleum degradation

Abstract

The invention discloses a bacterial composition for promoting petroleum degradation based on quorum sensing and application of the bacterial composition in low-temperature petroleum degradation, and belongs to the field of environmental bioremediation. The bacterial composition provided by the invention is composed of pseudomonas ANFP-BB-8 and sphingomonas ANFP-OD-3, and the two strains of bacteria can generate AHLs signal molecules and have a quorum sensing system. Petroleum degradation experiment results show that the petroleum degradation rate of the pseudomonas ANFP-BB-8 at low temperature can be remarkably improved by culturing and stimulating the efficient petroleum degradation bacterium pseudomonas ANFP-BB-8 by taking the sphingomonas ANFP-OD-3 as a fermentation supernatant of AHLs donor bacteria, or co-culturing the pseudomonas ANFP-BB-8 and the sphingomonas ANFP-OD-3, and the petroleum degradation rate of the pseudomonas ANFP-BB-8 at the low temperature can be remarkably improved by taking the sphingomonas ANFP-OD-3 as the fermentation supernatant of the AHLs donor bacteria to culture and stimulate the efficient petroleum degradation bacterium pseudomonas ANFP-BB-8 and the sphingomonas ANFP-OD-3 as the fermentation supernatant of the AHLs donor bacteria. The invention provides an efficient and synergistic bioremediation strategy for low-temperature petroleum pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental bioremediation, in particular to a bacterial composition for promoting oil degradation based on quorum sensing and its application in low-temperature oil degradation. BACKGROUND

[0002] Oil, as one of the most important energy sources, its leakage in the process of exploitation, transportation, processing and use has caused serious and lasting pollution to soil, water and marine ecosystems. Traditional physical remediation methods (such as fencing, adsorption) and chemical remediation methods (such as dispersants, incineration) not only have high costs, but also easily cause secondary pollution or damage to the ecological system, so it is urgent to develop green and sustainable treatment technology.

[0003] Microbial remediation (Bioremediation) technology uses the metabolic activity of microorganisms to degrade pollutants such as petroleum hydrocarbons into harmless carbon dioxide and water. Because of its environmental friendliness and low cost, it has become a research hotspot for oil pollution remediation. However, in practical application, microbial remediation technology faces many challenges. In particular, in winter or high-latitude areas, the environmental temperature is often low throughout the year, which will significantly inhibit the physiological activity of microbial cells, leading to slow degradation rate, prolonged remediation period, and seriously restricting its application effect in the whole year. Therefore, how to effectively activate the metabolic potential of oil-degrading bacteria in low-temperature environments is a key scientific problem that needs to be solved in the field of bioremediation.

[0004] In order to cope with the challenge of low-temperature environment, researchers have screened and isolated many low-temperature resistant strains (Psychrophilic / psychrotrophic bacteria) with oil degradation ability from low-temperature environments such as the North and South Poles, deep sea, and high mountain permafrost. For example, the invention patent (CN105907675A) discloses a strain of Rhodococcus sp. QY-2 that can efficiently degrade oil at 0℃. Other studies have also isolated various low-temperature oil-degrading strains such as Pseudomonas sp., Agrobacterium sp., and Sphingomonas sp. These indigenous or exogenous low-temperature strains provide valuable strain resources for bioremediation of low-temperature oil pollution. However, the colonization, survival and degradation efficiency of single strains in actual complex environments are often limited, and the "bioaugmentation" strategy that simply relies on adding strains has limited effect.

[0005] Quorum sensing (QS) is a communication mechanism between microorganisms, which assesses its population density by synthesizing, releasing and sensing chemical signal molecules called autoinducers, and when the density reaches a threshold (i.e. "quorum"), it coordinates the initiation of a series of population density-related gene expressions, thus exhibiting collective behaviors such as biofilm formation, virulence factor secretion, antibiotic production and bioluminescence, etc. In Gram-negative bacteria, the most classic QS signal molecule is N-acyl homoserine lactone (AHL). Biofilm is a microbial community formed after microorganisms adhere to solid surfaces, which is wrapped by extracellular polymeric substances (EPS) and can significantly enhance the resistance of microorganisms to antibiotics, toxic substances and harsh environmental pressures. The QS system is the key to regulating biofilm formation, maturation and dispersion. In petroleum-contaminated environments, degrading bacteria exist in the form of biofilms, which can effectively overcome the hydrophobicity and toxicity of petroleum hydrocarbons. Studies have found that after adding C4-HSL and C12-HSL signal molecules to the electrically active biofilm (EAB) of petroleum hydrocarbon degrading bacteria, the thickness and density of the biofilm increase significantly, and the survival rate and activity of the cells are improved. One of the mechanisms by which EME promotes diesel oil degradation is by enhancing the QS system, up-regulating the expression of genes related to biofilm formation and extracellular polymeric substance (EPS) synthesis. The EPS matrix of the biofilm not only emulsifies or disperses petroleum hydrocarbons, increasing their contact area with microbial cells, but also acts as a "protective cover", reducing direct damage to the cell membrane by petroleum hydrocarbons, thus creating a favorable microenvironment for collaborative degradation within the community.

[0006] Although the exogenous addition of AHLs shows great potential for strengthening bioremediation, its application in the field of low-temperature petroleum degradation has not been studied. Currently, low temperature is still the main bottleneck limiting the activity of petroleum-degrading bacteria. Existing technologies mainly focus on the screening of low-temperature degrading bacteria, but do not provide specific methods to effectively activate their degradation potential. At the same time, it is not clear how exogenous AHLs overcome the low-temperature limitation and thus improve the efficiency of low-temperature petroleum degradation. SUMMARY

[0007] The purpose of the present application is to provide a bacterial composition based on quorum sensing for promoting petroleum degradation and its application in low-temperature petroleum degradation, to solve the problems existing in the prior art. The bacterial composition provided by the present application consists of Pseudomonas ANFP-BB-8 with a preservation number of CGMCC No. 36385 and Sphingomonas ANFP-OD-3 with a preservation number of CGMCC No. 36384. The two petroleum-degrading bacteria exhibit a synergistic effect in low-temperature petroleum degradation. The present application provides an efficient and synergistic bioremediation strategy for low-temperature petroleum pollution.

[0008] To achieve the above object, the present application provides the following scheme:

[0009] The present application provides a Pseudomonas sp. ANFP-BB-8, which has been preserved in the China General Microbiological Culture Collection Center on October 28, 2025, with a preservation number of CGMCC No. 36385 and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0010] The present application provides a Sphingomonas sp. ANFP-OD-3, which has been preserved in the China General Microbiological Culture Collection Center on October 28, 2025, with a preservation number of CGMCC No. 36384 and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0011] The present application also provides a bacterial composition for promoting oil degradation, which comprises the above-mentioned Pseudomonas sp. ANFP-BB-8 and the above-mentioned Sphingomonas sp. ANFP-OD-3.

[0012] The present application also provides a fermentation broth for promoting oil degradation, which is obtained by fermentation of the above-mentioned Pseudomonas sp. ANFP-BB-8, the above-mentioned Sphingomonas sp. ANFP-OD-3, or the above-mentioned bacterial composition.

[0013] The present application also provides an application of the above-mentioned Pseudomonas sp. ANFP-BB-8, the above-mentioned Sphingomonas sp. ANFP-OD-3, the above-mentioned bacterial composition, the above-mentioned fermentation broth, or the above-mentioned product in the preparation of a product for promoting oil degradation.

[0014] The present application also provides a product for promoting oil degradation, which comprises the above-mentioned Pseudomonas sp. ANFP-BB-8, the above-mentioned Sphingomonas sp. ANFP-OD-3, the above-mentioned bacterial composition, or the above-mentioned fermentation broth.

[0015] The present application also provides an application of the above-mentioned Pseudomonas sp. ANFP-BB-8, the above-mentioned Sphingomonas sp. ANFP-OD-3, the above-mentioned bacterial composition, the above-mentioned fermentation broth, or the above-mentioned product in promoting oil degradation.

[0016] Further, the promotion of oil degradation is the promotion of low-temperature oil degradation; the low temperature is 5℃.

[0017] The present application also provides a method for promoting oil degradation, which comprises the step of applying the above-mentioned fermentation broth or the above-mentioned product to oil.

[0018] Further, the promoting oil degradation is promoting low-temperature oil degradation; the low-temperature is 5℃.

[0019] The present application discloses the following technical effects:

[0020] The bacterial composition provided by the present application is composed of Pseudomonas ANFP-BB-8 with the preservation number of CGMCC No. 36385 and Sphingomonas ANFP-OD-3 with the preservation number of CGMCC No. 36384. The oil degradation experiment results show that culturing and stimulating Pseudomonas ANFP-BB-8 with the fermentation supernatant of Sphingomonas ANFP-OD-3 or co-culturing Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 can significantly improve the oil degradation rate of Pseudomonas ANFP-BB-8 at low temperature and increase the oil components degradable by Pseudomonas ANFP-BB-8 at low temperature. In summary, compared with single low-temperature oil degrading bacteria, the combination of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 provided by the present application produces obvious synergistic effect induced by quorum sensing signals, shows significantly enhanced degradation effect on various typical oil components at low temperature, and significantly widens the range of oil components that can be efficiently degraded at low temperature. The present application provides an efficient and synergistic bioremediation strategy for low-temperature oil pollution. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0022] Figure 1 The phylogenetic tree of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3;

[0023] Figure 2 The result statistical diagram of degradation rate and residual concentration of n-alkanes (C9-C36) and polycyclic aromatic hydrocarbons after Pseudomonas ANFP-BB-8 is cultured in 1% concentration of oil medium for 14 days; wherein, A is the total n-alkane degradation rate; B is the total polycyclic aromatic hydrocarbon degradation rate; C is the n-alkane residual concentration; D is the polycyclic aromatic hydrocarbon residual concentration;

[0024] Figure 3The degradation rate and residual concentration of n-alkanes (C9-C36) and polycyclic aromatic hydrocarbons after Sphingomonas ANFP-OD-3 was cultured in 1% concentration of oil medium for 14 days; wherein, A is the total n-alkanes degradation rate; B is the total polycyclic aromatic hydrocarbons degradation rate; C is the n-alkanes residual concentration; D is the polycyclic aromatic hydrocarbons residual concentration;

[0025] Figure 4 The results of signal molecule quantification and species analysis of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3; wherein, A is the chromatographic detection results of Pseudomonas ANFP-BB-8; B is the chromatographic detection results of Sphingomonas ANFP-OD-3; C is the chromatographic detection results of standard; D is the detection results of signal molecule species produced by Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3;

[0026] Figure 5 The degradation rate and residual concentration of n-alkanes (C9-C36) and polycyclic aromatic hydrocarbons after Pseudomonas ANFP-BB-8 was cultured in the medium containing the fermentation supernatant of Sphingomonas ANFP-OD-3 with AHL signal molecules for 14 days; wherein, A is the total n-alkanes degradation rate; B is the total polycyclic aromatic hydrocarbons degradation rate; C is the n-alkanes residual concentration; D is the polycyclic aromatic hydrocarbons residual concentration;

[0027] Figure 6 The degradation rate and residual concentration of n-alkanes (C9-C36) and polycyclic aromatic hydrocarbons after Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 were mixed and cultured in equal proportions to degrade oil for 14 days; wherein, A is the total n-alkanes degradation rate; B is the total polycyclic aromatic hydrocarbons degradation rate; C is the n-alkanes residual concentration; D is the polycyclic aromatic hydrocarbons residual concentration. DETAILED DESCRIPTION

[0028] The various illustrative embodiments of the present application will now be described in detail in connection with the accompanying drawings. This description is made for the purpose of illustrating the certain aspects, features and embodiments of the present application and is not intended to limit the scope of the application, which is defined solely by the appended claims.

[0029] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intermediate value in the stated range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless clearly indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict between the content of the specification and that of any incorporated literature, the content of the specification controls.

[0031] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0032] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0033] The culture medium used in the present application is as follows:

[0034] Inorganic salt medium: On the basis of the purchased inorganic salt medium (CHINOOK, China), the carbon source was added, and the following is the type and content of nutrients added in 1 L of inorganic salt medium: sodium acetate 2 g, proteose peptone 0.5 g, yeast extract 0.5 g, potato infusion powder 0.5 g, glucose 0.2 g, sucrose 0.2 g, sodium malate 0.05 g, sodium citrate 0.05 g, sodium tartrate 0.05 g, pH adjusted to 7.2-7.4, then 121℃, sterilized for 15 min.

[0035] Petroleum medium: On the basis of the above inorganic salt medium, 1% volume of petroleum was added; the petroleum used was a mixture of 1:1 of BELANAK crude oil from Malaysia and diesel oil.

[0036] M8 liquid medium: as shown in Table 1. In Table 1, the method of Dyksterhouse et al. refers to the method described in the literature “Dyksterhouse S E, Gray J P, Herwig R P, et al. Cycloclasticus pugetii gen. nov., sp. nov., an aromatic hydrocarbon-degrading bacterium from marine sediments[J]. International Journal of Systematic Bacteriology, 1995, 45(1): 116-123.”:

[0037] Table 1 Medium formula

[0038] Culture medium Configuration method solution <![CDATA[Each 250 ml contains the following ingredients: 0.325 g of sodium fluoride (NaF), 3.375 g of boric acid (H3BO3), 10.375 g of sodium bromide (NaBr), 11.125 g of sodium dihydrogen phosphate heptahydrate (Na2HPO4·7H2O), 33.75 g of ammonium chloride (NH4Cl), 3.875 g of sodium bicarbonate (NaHCO3). Then, sterilization treatment is carried out.]]> solution Each 250 ml contains the following ingredients: 139.75 g of magnesium chloride hexahydrate (MgCl2-6H2O), 18.54 g of calcium chloride (CaCl2), 0.3 g of strontium chloride hexahydrate (SrCl2-6H2O). Subsequently, sterilization is performed. solution Each 100 ml contains 0.28 g of ferrous sulfate heptahydrate (FeS04-7H20). Subsequently, sterilization is performed. M8 culture medium Each liter contained the following ingredients: 0.5 g peptone, 0.2 g glucose, 3.98 g sodium sulfate (Na2SO4), 0.05 g sodium malate, 0.2 g sucrose, 22.79 g sodium chloride (NaCl), 2 g sodium acetate, 0.72 g potassium chloride (KCl), 0.5 g yeast extract, 0.05 g potassium sodium tartrate, 0.05 g trisodium citrate, 0.5 g potato infusion powder, 1.30 g TAPSO. The pH was between 7.6 and 7.8. Before use, 2 ml of previously prepared solution I was added and autoclaved (121 degrees Celsius, 15 minutes). Subsequently, sterile solutions II and III were added in certain proportions and concentrations according to Dyksterhouse et al.

[0039] The main instruments used in the present application are as follows:

[0040] Constant temperature culture shaker: THZ-100 type, Shanghai Yiheng Scientific Instrument Co., Ltd.; electric heating constant temperature drying oven: Shanghai Yiheng Scientific Instrument Co., Ltd.; electronic balance: BSA224S-CW, Sartorius Company; gas chromatograph mass spectrometer: Agilent6890A GC / 5973 MSD, American Agilent Company; liquid chromatograph mass spectrometer: 1100 LC-MS, American Agilent Company.

[0041] Example 1

[0042] 1. Enrichment and separation of petroleum degrading bacteria

[0043] The petroleum degrading bacteria of the present application are screened from the Antarctic low temperature environment samples obtained in the 36th Antarctic Scientific Expedition of China. The specific method is as follows:

[0044] Take 5 g of mud samples from different stations and add them to 100 mL of petroleum medium that has been completed high pressure sterilization, and set three parallel samples for each station. Place the petroleum medium on a 150 r·min -1 shaker for enrichment culture, and the environmental temperature is 25℃.

[0045] After 14 days, take 2% volume of the culture medium from the first enrichment culture and add it to the new sterilized petroleum medium for the second enrichment culture, and the enrichment culture time is 14 days each time, and a total of 3 times. The blank control is the petroleum medium that has been completed sterilization without adding sediments.

[0046] After the third enrichment culture was completed, the medium was gradiently diluted, and the dilutions of different concentrations were plated on solid M8 medium plates and cultured in a constant temperature incubator at 25°C. After the colonies grew, different morphological colonies were selected using a loop to streak on solid M8 medium plates and cultured in a constant temperature incubator for 7-14 days. The purpose of this process was to purify different petroleum-degrading bacteria into single colonies.

[0047] Two petroleum-degrading bacteria were purified and named ANFP-BB-8 and ANFP-OD-3.

[0048] 2. Strain screening and identification

[0049] The 16S rRNA gene sequences of the two petroleum-degrading bacteria ANFP-BB-8 and ANFP-OD-3 were sequenced. The 16S rRNA gene sequencing results showed that ANFP-BB-8 belonged to Pseudomonas, and ANFP-OD-3 belonged to Sphingomonas.

[0050] The 16S rRNA gene sequences of 15 strains similar to the above two strains were selected for ClustalW multiple alignment and construction of a neighbor-joining phylogenetic tree. The phylogenetic tree construction results are shown in Figure 1 The strain ANFP-BB-8 had the closest genetic relationship with Pseudomonas migulae CIP105470 (AF074383), with a sequence similarity of 99.59%, and was determined to be a Pseudomonas sp. strain, named Pseudomonas sp. ANFP-BB-8. The strain ANFP-OD-3 had the closest genetic relationship with the model strain Sphingomonas faeni MAolki (AJ429239), with a sequence similarity of 99.71%, and was named Sphingomonas sp. ANFP-OD-3.

[0051] 3. Strain preservation

[0052] Pseudomonas sp. ANFP-BB-8 was preserved at the China General Microbiological Culture Collection Center on October 28, 2025, with the preservation number CGMCC No. 36385 and the preservation address No. 3, Beichen West Road, Chaoyang District, Beijing.

[0053] Sphingomonas sp. ANFP-OD-3 was preserved in China General Microbiological Culture Collection Center on October 28, 2025, with a preservation number of CGMCC No. 36384 and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0054] Example 2

[0055] 1. Experimental method

[0056] 1.1 Petroleum degradation characteristics of Pseudomonas sp. ANFP-BB-8 and Sphingomonas sp. ANFP-OD-3

[0057] 1.1.1 Single-bacterium degradation experiment

[0058] After Pseudomonas sp. ANFP-BB-8 and Sphingomonas sp. ANFP-OD-3 were respectively expanded in M8 liquid medium to the logarithmic growth phase (OD 600 =0.8), the bacterial solution at this time was added to 100 mL of petroleum medium that had been sterilized by high pressure, and the petroleum medium was placed at 5°C, 10°C, 15°C, and 20°C, respectively, on a 150 r·min -1 shaker for degradation for 14 d. The blank control was the petroleum medium that had not been added with the bacterial solution and had been sterilized. Three parallel groups were set for each group. For the single-bacterium degradation experiment, the added bacterial solution was 1%, that is, 1 mL of the bacterial solution was added to 100 mL of the petroleum medium for degradation.

[0059] 1.1.2 Co-culture experiment of fermentation supernatant of Pseudomonas sp. ANFP-BB-8 and Sphingomonas sp. ANFP-OD-3

[0060] Pseudomonas sp. ANFP-BB-8 and Sphingomonas sp. ANFP-OD-3 were respectively inoculated in M8 liquid medium and expanded to the logarithmic growth phase (OD 600 =0.8). The Sphingomonas sp. ANFP-OD-3 culture solution was centrifuged at 4°C, and the supernatant was filtered through a 0.22 μm microporous filter to remove bacteria, thereby preparing the fermentation supernatant. Subsequently, 1 mL of the logarithmic-phase Pseudomonas sp. ANFP-BB-8 bacterial solution and 1 g of petroleum were added to 100 mL of the above fermentation supernatant at an inoculation amount of 1% (v / v). The inoculated system was placed in a 150 r·min -1 shaker in the dark at 5°C, 10°C, 15°C, and 20°C, respectively, for degradation for 14 d. The experiment also set the petroleum medium that had not been inoculated and had been sterilized as a blank control (CK), and three parallel repeats were set for each group.

[0061] 1.1.3 Co-culture experiment of Pseudomonas sp. ANFP-BB-8 and Sphingomonas sp. ANFP-OD-3

[0062] Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 were respectively expanded in M8 liquid medium to the logarithmic growth phase of OD 600 =0.8, and then added to 100 mL of sterilized petroleum medium in a volume ratio of 1:1. The petroleum medium was placed at 5℃, 10℃, 15℃, and 20℃, respectively, on a 150 r·min -1 -1 shaker for degradation for 14 days. The blank control was the sterilized petroleum medium without the addition of bacteria. Each group had three replicates. The total amount of bacteria added was 1%, i.e., 1 mL of mixed bacteria was added to 100 mL of petroleum medium for degradation.

[0063] 1.1.4 Pretreatment method for residual petroleum

[0064] First, pour the petroleum medium into a clean and dried separatory funnel. Use 100 mL of dichloromethane to extract the residual petroleum, and extract it twice, each time using 50 mL of dichloromethane. Pour 50 mL of dichloromethane into the conical flask containing the petroleum medium, and elute the petroleum adsorbed on the inner wall of the conical flask in a short time. Then pour the dichloromethane in the conical flask into the separatory funnel, shake and oscillate the separatory funnel to make the residual petroleum completely dissolve in the dichloromethane phase, and then collect the dichloromethane phase at the bottom of the separatory funnel. Repeat this process twice to collect a total of 100 mL of dichloromethane extract. Generally, the petroleum medium with high bacterial content and rich in surfactants will show emulsification effect after extraction with dichloromethane. In this case, the dichloromethane extract needs to be transferred to a centrifuge bottle and centrifuged to separate the impurities.

[0065] 1.2 GC-MS analysis

[0066] The instrument conditions for detecting n-alkanes by gas chromatography-mass spectrometry are as follows: the carrier gas is helium (99.999%). The injection volume is 1 μL, without splitting, and the flow rate is 1 mL·min -1 -1. The injection port temperature is 290℃. The temperature program starts at 50℃, maintains for 2 min, rises at a rate of 6℃·min -1 -1 to 300℃, and maintains for 16 min. The type of chromatographic column is Agilent HP-5MS (30 m×0.25 mm×0.25 μm).

[0067] The instrument conditions for detecting polycyclic aromatic hydrocarbons (PAHs) by gas chromatography-mass spectrometry are as follows: the carrier gas is helium (99.999%). The injection volume is 1 μL, in constant flow mode, and the flow rate is 1 mL·min -1, the injection port temperature was 290℃. The temperature program started at 50℃, held for 2 min, increased at a rate of 6℃·min -1 to 300℃, and held for 27 min. The model of the chromatographic column was Agilent HP-5MS (30 m x 0.25 mm x 0.25 μm).

[0068] 1.3 UHPLC-TOF / MS analysis

[0069] The present application adopts the ultra-high performance liquid chromatography-time of flight mass spectrometry (UHPLC-TOF / MS, Agilent) to qualitatively and quantitatively detect the C6-HSL and C8-HSL signal molecules produced by Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3. The chromatographic analysis is performed at 40℃, and 10 μL of the AHLs sample is taken, gradient elution is performed through the Eclipse XDB-C 18 The pump 1 flow rate is 0.5 mL / min, the pump 2 flow rate is 0.3 mL / min, and the total elution time is 17 min. The mass spectrometry analysis is performed by using the electrospray ionization source (ESI) in the positive ion scanning mode. The detection conditions are specifically set as follows: the drying gas temperature is set to 350℃, the drying gas flow rate is 10 L / min, the capillary voltage is 4000 V, the nebulizer pressure is 10 bar, and the mass range is 50-500 m / z.

[0070] 2. Experimental results and analysis

[0071] 2.1 Degradation characteristics of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3

[0072] The degradation effect of a single strain and the degradation characteristics of n-alkanes and polycyclic aromatic hydrocarbons are determined by using a petroleum medium. The petroleum used in the petroleum medium is a 1:1 mixture of Malaysian BELANAK crude oil and diesel oil. The single strain is cultured in the M8 medium and concentrated to an OD 600 of 0.8, 1 mL of the bacterial solution is added to the petroleum medium, and the degradation is cultured under the set conditions for 14 d. After the degradation is completed, the degradation effect is determined by extraction and GC-MS.

[0073] The degradation characteristics of the petroleum degrading bacteria are evaluated by the GC-MS method, and Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 are analyzed by GC-MS.

[0074] The results are as follows Figure 2As shown, *Pseudomonas* ANFP-BB-8 exhibited efficient and stable degradation capacity for n-alkanes over a wide temperature range, with no significant difference in degradation rate. At 5℃, the average degradation rate of total n-alkanes reached 66.31%, demonstrating the strain's good adaptability to low-temperature environments. For polycyclic aromatic hydrocarbons (PAHs), the strain showed high degradation rates at 5℃, 10℃, 15℃, and 20℃, approximately 48.80%, 55.85%, 52.38%, and 64.26%, respectively. Overall, the degradation effects varied relatively little across temperatures, indicating that *Pseudomonas* ANFP-BB-8 maintains stable and efficient degradation capacity for PAHs over a wide temperature range of 5-20℃, demonstrating its good adaptability to low-temperature environments and its wide-temperature range advantage in PAH degradation.

[0075] The results are as follows Figure 3 As shown, the degradation performance of n-alkanes by *Sphingomonas ANFP-OD-3* exhibits a significant temperature dependence. At a low temperature of 5°C, its degradation ability is the weakest, at 37.44%. However, when the temperature rises to 20°C, its degradation rate significantly increases, reaching a total degradation rate of 51.68%. This indicates that *Sphingomonas ANFP-OD-3* can effectively degrade n-alkanes at temperatures of 10°C and above, but is extremely sensitive to the low-temperature environment of 5°C.

[0076] Compared to Pseudomonas ANFP-BB-8, Sphingomonas ANFP-OD-3 exhibits significantly weaker degradation ability for PAHs, and this degradation is strictly temperature-dependent. Figure 3 As shown, at 5℃, the degradation rate of *Sphingomonas ANFP-OD-3* was 37.45%, indicating virtually no degradation activity. With increasing temperature, its degradation ability gradually improved, reaching 38.25%, 35.67%, and 54.39% at 10℃, 15℃, and 20℃, respectively. This trend indicates that the degradation activity of *Sphingomonas ANFP-OD-3* against PAHs is highly temperature-dependent, exhibiting considerable degradation ability only above 10℃, and reaching its optimal level at 20℃.

[0077] Further analysis of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 degradation rate of different carbon chain length alkane and isomeric alkane. Pseudomonas ANFP-BB-8 degradation rate of short chain alkane (C9-C13) was 82.79%, 79.23%, 76.29%, 70.94%, respectively, and degradation rate of long chain alkane (C14-C36) was 63.61%, 56.21%, 47.20%, 34.61%, respectively. In addition, its degradation rate of pristane (Pr) was 75.41%, 70.37%, 67.37%, 61.56%, respectively, and degradation rate of phytane (Ph) was 72.49%, 66.75%, 34.79%, 51.47%, respectively.

[0078] Sphingomonas ANFP-OD-3 degradation rate of short chain alkane (C9-C13) was 39.17%, 67.21%, 61.18%, 60.32%, respectively, and degradation rate of long chain alkane (C14-C36) was 41.08%, 54.42%, 52.70%, 51.68%, respectively. In addition, its degradation rate of pristane (Pr) was 34.86%, 29.25%, 25.44%, 22.46%, respectively, and degradation rate of phytane (Ph) was 43.34%, 27.50%, 39.91%, 27.64%, respectively.

[0079] For polycyclic aromatic hydrocarbons, Pseudomonas ANFP-BB-8 degradation rate of naphthalene was 43.47%, 49.83%, 56.63%, 68.78%, respectively, at 5℃-20℃; degradation rate of fluorene was 56.20%, 56.97%, 52.95%, 49.23%, respectively; degradation rate of phenanthrene was 17.03%, 49.99%, 40.17%, 65.26%, respectively; degradation rate of dibenzothiophene was 42.73%, 51.49%, 58.89%, 65.26%, respectively; degradation rate of pyrene was 44.29%, 58.8%, 58.26%, 65.23%, respectively.

[0080] Sphingomonas ANFP-OD-3 degradation rate of naphthalene was 53.63%, 61.45%, 63.83%, 72.07%, respectively, at 5℃-20℃; degradation rate of fluorene was 29.10%, 62.09%, 60.167%, 69.97%, respectively; degradation rate of phenanthrene was 42.04%, 50.70%, 49.09%, 51.55%, respectively; degradation rate of dibenzothiophene was 42.16%, 51.17%, 58.89%, 63.61%, respectively; degradation rate of pyrene was 44.29%, 58.80, 58.26%, 62.03%, respectively.

[0081] 2.2 HPLC-MS analysis of AHL signal molecules produced by Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3

[0082] As shown in Figure 4 , the two signal molecules produced by Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 were successfully identified as C8-HSL and C6-HSL by comparing the chromatograms of the samples with those of the standards, especially according to the time of elution. The yield of C8-HSL produced by Sphingomonas ANFP-OD-3 was measured to be 512.4 ng / L, while that produced by Pseudomonas ANFP-BB-8 was 4.95 ng / L.

[0083] 2.3 Effect of Sphingomonas ANFP-OD-3 fermentation supernatant on the degradation efficiency of Pseudomonas ANFP-BB-8 for petroleum

[0084] As shown in Figure 5 , it was found that the degradation rate of Pseudomonas ANFP-BB-8 for n-alkanes was the highest, reaching 90.26%, in the environment containing the fermentation supernatant of Sphingomonas ANFP-OD-3 at 5°C, which was 23.95% higher than that of Pseudomonas ANFP-BB-8 alone. The degradation rates of Pseudomonas ANFP-BB-8 for short-chain n-alkanes at 5, 10, 15, and 20°C were 81.07%, 77.90%, 76.26%, and 70.94%, respectively, and for medium- and long-chain n-alkanes were 76.32%, 56.54%, 46.45%, and 56.81%, respectively. In addition, the degradation rates of Pseudomonas ANFP-BB-8 for Pr were 77.62%, 69.68%, 67.37%, and 61.56%, respectively, and for Ph were 86.07%, 80.51%, 78.11%, and 73.62%, respectively.

[0085] For polycyclic aromatic hydrocarbons, in the environment containing the fermentation supernatant of Sphingomonas ANFP-OD-3, the degradation rates of Pseudomonas ANFP-BB-8 for naphthalene at 5-20°C were 52.52%, 63.78%, 69.39%, and 81.18%, respectively, for fluorene were 21.63%, 38.61%, 21.78%, 59.41%, respectively, for phenanthrene were 36.45%, 59.07%, 59.47%, and 69.11%, respectively, for dibenzothiophene were 80.26%, 84.03%, 81.34%, and 90.47%, respectively, and for pyrene were 77.19%, 85.45%, 73.07%, and 87.63%, respectively.

[0086] 2.4 Petroleum degradation characteristics of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3

[0087] As shown in the results Figure 6 It can be found that the co-culture (forming the composition) of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 has the highest degradation rate of n-alkanes at 5℃, reaching 83.96%, which is increased by 26.63% compared with the degradation rate of single bacteria. The degradation rates of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 co-culture at 5, 10, 15, and 20℃ are 81.94%, 78.13%, 77.56%, and 71.83% for short-chain, and 74.05%, 54.75%, 57.58%, and 60.39% for medium and long-chain, respectively. In addition, the degradation rates of pristane (Pr) are 76.86%, 62.22%, 68.75%, and 65.19%, respectively, and the degradation rates of phytane (Ph) are 85.82%, 78.26%, 78.40%, and 75.08%, respectively.

[0088] For polycyclic aromatic hydrocarbons, the degradation rates of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 co-culture at 5-20℃ are 56.85%, 70.13%, 69.11%, and 95.50% for naphthalene, 28.50%, 40.67%, 36.59%, and 57.86% for fluorene, 34.75%, 56.56%, 54.49%, and 69.19% for phenanthrene, 69.74%, 87.71%, 85.65%, and 96.40% for dibenzothiophene, and 68.00%, 70.24%, 64.05%, and 73.44% for pyrene, respectively.

[0089] 3. Summary

[0090] Compared with single low-temperature petroleum-degrading bacteria, the composition of Pseudomonas ANFP-BB-8 and Sphingomonas ANFP-OD-3 provided by the present application shows significantly enhanced degradation effect on various typical petroleum components under low-temperature conditions.

[0091] The total degradation rate data results are shown in Table 2, and the experimental results show that under the condition of 5 DEG C, the fermentation supernatant of sphingomonas ANFP-OD-3 has the best promotion effect on the total n-alkane degradation rate of pseudomonas ANFP-BB-8. The n-alkane degradation rate of pseudomonas ANFP-BB-8 is increased to 90.26% by the fermentation supernatant of sphingomonas ANFP-OD-3, which is increased by 23.95% compared with the single pseudomonas system, and the degradation ability of the components such as medium-long chain n-alkanes (C14-C36) and phytane which are stable in structure and have strong hydrophobicity is significantly improved, which is increased by 12.71% and 13.58%, respectively.

[0092] The n-alkane degradation rate of the combination of pseudomonas ANFP-BB-8 and sphingomonas ANFP-OD-3 is increased to 83.96%, which is increased by 17.65% compared with the single pseudomonas system, and the degradation ability of the components such as medium-long chain n-alkanes (C14-C36) and phytane which are stable in structure and have strong hydrophobicity is significantly improved, which is increased by 10.44% and 13.03%, respectively. The limitation that the degradation rate decreases obviously with the increase of carbon chain in the single bacterium system is effectively weakened.

[0093] Meanwhile, the combination of pseudomonas ANFP-BB-8 and sphingomonas ANFP-OD-3 significantly enhances the degradation ability of polycyclic aromatic hydrocarbons, especially the degradation rate of triphenyl compounds such as phenanthrene which is difficult to be effectively degraded by a single strain at low temperature is increased by more than one time compared with the single bacterium system, and the removal efficiency of naphthalene, dibenzothiophene and other typical pollutants is higher and more stable. The above results show that the strain combination of the application produces obvious synergistic effect by quorum sensing signal induction, and significantly widens the range of petroleum components that can be efficiently degraded at low temperature.

[0094] Table 2 Total degradation rate statistical results of pseudomonas ANFP-BB-8, sphingomonas ANFP-OD-3 and combination

[0095]

[0096] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A strain of Pseudomonas sp. ANFP-BB-8, characterized in that, The aforementioned Pseudomonas ANFP-BB-8 was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36385, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A strain of Sphingomonas sp. ANFP-OD-3, characterized in that, The sphingomonas ANFP-OD-3 was deposited on October 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36384, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

3. A bacterial composition for promoting petroleum degradation, characterized in that, It includes the Pseudomonas ANFP-BB-8 as described in claim 1 and the Sphingomonas ANFP-OD-3 as described in claim 2.

4. A fermentation broth for promoting petroleum degradation, characterized in that, The fermentation broth is obtained by fermentation of the Pseudomonas ANFP-BB-8 of claim 1, the Sphingomonas ANFP-OD-3 of claim 2, or the bacterial composition of claim 3.

5. The use of the Pseudomonas ANFP-BB-8 of claim 1, the Sphingomonas ANFP-OD-3 of claim 2, the bacterial composition of claim 3, or the fermentation broth of claim 4 in the preparation of products that promote petroleum degradation.

6. A product that promotes the degradation of petroleum, characterized in that, It contains the Pseudomonas ANFP-BB-8 of claim 1, the Sphingomonas ANFP-OD-3 of claim 2, the bacterial composition of claim 3, or the fermentation broth of claim 4.

7. The use of the Pseudomonas ANFP-BB-8 of claim 1, the Sphingomonas ANFP-OD-3 of claim 2, the bacterial composition of claim 3, the fermentation broth of claim 4, or the product of claim 6 in promoting petroleum degradation.

8. The application as described in claim 7, characterized in that, The term "promoting petroleum degradation" refers to promoting low-temperature petroleum degradation; the low temperature is 5°C.

9. A method for promoting petroleum degradation, characterized in that, The step includes applying the fermentation broth of claim 4 or the product of claim 6 to petroleum.

10. The method as described in claim 9, characterized in that, The term "promoting petroleum degradation" refers to promoting low-temperature petroleum degradation; the low temperature is 5°C.

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