A polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing, and its screening method and application

Abstract

The present invention discloses a polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing, a screening method, and an application thereof. The polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing is Acinetobacter sp. YY-1, with a deposit number of GDMCC No: 63126 and a deposit date of January 9, 2023. The polycyclic aromatic hydrocarbon-degrading strain provided by the present invention has an enhanced ability to degrade polycyclic aromatic hydrocarbons under quorum sensing control and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of environmental protection technology, and in particular to a polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing, a screening method and an application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of aromatic compounds composed of two or more benzene rings, characterized by high melting and boiling points, low solubility, and strong hydrophobicity. Once PAHs enter the environment, they readily adsorb onto solid surfaces, making them difficult for microorganisms to access. Furthermore, due to their stable structure and low natural degradation rate, PAHs persist in environmental media for long periods of time. PAHs can enter the human body through the respiratory tract, skin, and digestive tract, and exhibit three toxic effects: teratogenicity, carcinogenicity, and mutagenicity. In recent years, the incomplete combustion of fossil fuels such as coal and oil, as well as leaks and discharges during crude oil extraction, transportation, and production, have led to a surge in PAH emissions to the environment. Environmental PAHs are impacting the safety of humans and the entire ecosystem.

[0003] To control PAHs' environmental pollution, remediation of PAH-contaminated media is necessary. Early attempts at physical and chemical remediation, such as thermal desorption, solvent extraction, and electrochemical remediation, yielded effective remediation results but were costly and prone to secondary contamination. In recent years, PAHs bioremediation has gained increasing attention due to its environmental friendliness, cost-effectiveness, and renewable nature. The effectiveness of PAHs bioremediation is primarily influenced by the performance of the degradation strains. Therefore, identifying efficient PAH-degrading strains is crucial for bioremediation of PAHs contamination.

[0004] Quorum sensing (QS) is a mechanism by which microorganisms control collective behavior by synthesizing and recognizing extracellular chemical molecules, sensing the density of surrounding populations, and regulating gene expression. Most Gram-negative bacteria utilize molecules called acyl-homoserine lactones (AHLs) for intraspecific communication. AHLs can freely enter and exit the cell membrane. When the concentration of AHLs in the bacterial environment exceeds a certain threshold, AHLs bind to receptor proteins to form a complex, which then interacts with promoters to initiate transcription of genes supporting various physiological activities. Many bacterial functions, such as the synthesis of extracellular polymers and degradative enzymes, are regulated by QS.

[0005] Bacteria that are regulated by the AHL-QS system and can degrade PAHs exist in different environmental media, such as Pseudomonas aeruginosa, Croceicoccus naphthovorans, Novosphingobium pentaromativorans, Pseudomonas putida, etc. However, the resources of such bacteria are very limited, and how to enhance the degradation of PAHs through QS regulation has not been explored. Summary of the Invention

[0006] The main purpose of the present invention is to propose a polycyclic aromatic hydrocarbons-degrading strain regulated by quorum sensing and its screening method and application, aiming to solve the problem of environmental pollution and further study how to enhance the degradation of polycyclic aromatic hydrocarbons through quorum sensing regulation.

[0007] To achieve the above objectives, in the first aspect of the present invention, a polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing is proposed. The polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing is Acinetobacter sp. YY-1, with a preservation number of GDMCC No: 63126 and a preservation time of January 9, 2023.

[0008] Optionally, the 16S rDNA sequence of the polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing is shown as SEQ ID NO: 1.

[0009] In a second aspect of the present invention, a method for screening polycyclic aromatic hydrocarbon-degrading strains regulated by quorum sensing as described above is provided, which comprises the following steps:

[0010] Collecting sludge and stabilizing it with artificial wastewater to obtain stabilized sludge;

[0011] The stabilized sludge was diluted with sterile water and then spread on LB medium agar plates to form the first single colony;

[0012] Pick the first single colony and expand it in LB medium to prepare a test bacterial solution;

[0013] Agrobacterium tumefaciens A136 was expanded and cultured in LB medium to prepare A136 bacterial liquid;

[0014] The test bacterial solution and A136 bacterial solution were spread on LB medium agar plates containing 5-bromo-4-chloro-3-indole-β-D-galactoside, and then cultured. The bacteria that made the base of the plate of A136 colony turn blue were the quorum sensing bacteria;

[0015] The quorum sensing bacteria were cultured in LB medium, and then the bacterial solution was washed with basal salt medium;

[0016] The cleaned bacterial liquid was inoculated into a basal salt medium containing phenanthrene, cultured, and then transferred to culture under the same conditions;

[0017] After the culture is completed, the OD600nm of the bacterial solution is analyzed, and the strains with good growth are selected and the bacterial solution is gradiently diluted with sterile water to obtain dilutions;

[0018] The diluted solution was spread on a basic salt medium agar plate containing polycyclic aromatic hydrocarbons, and cultured to form a second single colony;

[0019] The second single colony was picked up with a sterile needle and cultured again in the basal salt medium containing the above-mentioned polycyclic aromatic hydrocarbons. The OD600nm of the bacterial solution was analyzed, and the strain with the best growth was selected to obtain the polycyclic aromatic hydrocarbons-degrading strain regulated by quorum sensing.

[0020] Optionally, the steps of inoculating the cleaned bacterial liquid into a basal salt medium containing phenanthrene, culturing, and then transferring the culture under the same conditions specifically include:

[0021] The bacterial suspension was inoculated into a basal salt medium containing 100 mg / L phenanthrene at a ratio of 10% (V / V), cultured at 30°C for 7 days, and then transferred to culture under the same conditions for three times.

[0022] Optionally, after the culture is completed, the OD600nm of the bacterial solution is analyzed, strains with good growth are selected, and the bacterial solution is gradiently diluted with sterile water. The steps specifically include:

[0023] Step A: After the last culture is completed, analyze the bacterial solution OD600nm and select 5 strains with good growth;

[0024] Step B: dilute the bacterial solution in a gradient manner with sterile water.

[0025] Optionally, in the step of spreading the dilution on a basal salt culture medium agar plate containing polycyclic aromatic hydrocarbons, culturing, and forming a second single colony, the polycyclic aromatic hydrocarbons include phenanthrene, pyrene, and benzo[a]pyrene, and the concentrations of phenanthrene, pyrene, and benzo[a]pyrene are 100 mg / L, 100 mg / L, and 50 mg / L, respectively.

[0026] Optionally, the step of picking a second single colony with a sterile needle and culturing it again in a basal salt medium containing the polycyclic aromatic hydrocarbons, analyzing the bacterial solution OD600nm, selecting the best growing strain, and obtaining a polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing specifically includes:

[0027] Use a sterile needle to pick up the second single colony and culture it again on the basal salt medium containing the above-mentioned polycyclic aromatic hydrocarbons. After culturing again at 30°C for 7 days, analyze the OD600nm of the bacterial solution and select the strain with the best growth.

[0028] In a third aspect, the present invention provides a use of the polycyclic aromatic hydrocarbons-degrading strain regulated by quorum sensing as described above, wherein the polycyclic aromatic hydrocarbons-degrading strain regulated by quorum sensing is used to degrade polycyclic aromatic hydrocarbons.

[0029] Optionally, the polycyclic aromatic hydrocarbons-degrading strain regulated by quorum sensing enhances the degradation of polycyclic aromatic hydrocarbons under quorum sensing regulation.

[0030] Beneficial effects: The technical solution provided by the present invention provides a new polycyclic aromatic hydrocarbons-degrading strain regulated by quorum sensing. Compared with the reported strains, this strain has an enhanced ability to degrade polycyclic aromatic hydrocarbons under quorum sensing regulation and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 This is the Gram staining image of the bacteria to be tested obtained in Example 3 of the present invention;

[0033] Figure 2 This is the LB agar plate image of the bacteria to be tested obtained in Example 3 of the present invention;

[0034] Figure 3 This is a scanning electron microscope image of the test bacteria obtained in Example 3 of the present invention;

[0035] Figure 4 This is the phylogenetic tree diagram of YY-1 obtained in Example 3 of the present invention;

[0036] Figure 5 Growth diagram of YY-1 obtained in Example 4 of the present invention under different pH (left), salinity (middle) and temperature (right) conditions;

[0037] Figure 6 This is a graph of signal molecules produced by using reporter bacteria to detect YY-1 obtained in Example 5 of the present invention;

[0038] Figure 7 This is a diagram of biofilm formation of YY-1 under QS regulation obtained in Example 5 of the present invention;

[0039] Figure 8 Figure 6 shows the biofilms formed by YY-1 using different concentrations of phenanthrene (left) and different PAHs (center), and the degradation of different PAHs by YY-1 (right).

[0040] Figure 9 These are the degradation of phenanthrene by YY-1 under the enhanced QS regulation of adding mixed AHLs obtained in Example 6 of the present invention (left) and the degradation of phenanthrene by YY-1 under the enhanced QS regulation of adding different AHLs (right).

[0041] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] To control PAHs' environmental pollution, remediation of PAH-contaminated media is necessary. Early attempts at physical and chemical remediation, such as thermal desorption, solvent extraction, and electrochemical remediation, yielded effective remediation results but were costly and prone to secondary contamination. In recent years, PAHs bioremediation has gained increasing attention due to its environmental friendliness, cost-effectiveness, and renewable nature. The effectiveness of PAHs bioremediation is primarily influenced by the performance of the degradation strains. Therefore, identifying efficient PAH-degrading strains is crucial for bioremediation of PAHs contamination.

[0044] In view of this, the present invention proposes a PAHs-degrading strain regulated by QS to solve the problem of environmental pollution and further enhance the degradation of PAHs through QS regulation.

[0045] The present invention proposes a PAHs-degrading strain regulated by QS, wherein the strain is Acinetobacter sp. YY-1, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 9, 2023, with a deposit number of GDMCC No: 63126.

[0046] The technical solution provided by the present invention provides a new PAHs-degrading strain regulated by QS. Compared with reported strains, this strain can enhance the degradation efficiency of PAHs under QS regulation and has good application prospects.

[0047] In some embodiments, the 16S rDNA sequence of the strain is shown as SEQ ID NO: 1.

[0048] The Acinetobacter sp. YY-1 proposed in the present invention is screened from sewage treatment plants and has a wide range of sources. The present invention proposes a screening method for the above-mentioned strain, which includes the following steps:

[0049] S1. Collect sludge and stabilize it with artificial wastewater;

[0050] S2. Dilute the stabilized sludge with sterile water and then spread it on an LB (tryptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L, pH 7.0) agar plate to form the first single colony;

[0051] S3, picking the first single colony and expanding it in LB medium to prepare the test bacterial solution;

[0052] S4, expanding the culture of Agrobacterium tumefaciens A136 in LB medium to prepare A136 bacterial liquid;

[0053] S5. Spread the test bacterial solution and A136 bacterial solution on LB medium agar plates containing 5-bromo-4-chloro-3-indole-β-D-galactoside (X-Gal), and then incubate. The bacteria that make the base of the plate of A136 colony appear blue are QS bacteria;

[0054] S6. Cultivate the QS bacteria in LB medium, and then wash the bacterial solution with basal salt medium (MSM, pH 7.0);

[0055] S7. Inoculate the cleaned bacterial solution into MSM containing phenanthrene, culture, and then transfer culture under the same conditions;

[0056] S8. After the culture is completed, analyze the OD600nm of the bacterial solution, select the strains with good growth, and dilute the bacterial solution in a gradient manner with sterile water;

[0057] S9, spreading the diluted solution on an MSM agar plate containing polycyclic aromatic hydrocarbons, culturing to form a second single colony;

[0058] S10. Use a sterile needle to pick up the second single colony and culture it again in MSM containing the above-mentioned polycyclic aromatic hydrocarbons. Analyze the OD600nm of the bacterial solution, select the strain with the best growth, and obtain the PAHs-degrading strain regulated by QS.

[0059] The present invention utilizes the color reaction of AHLs reporter bacteria, Agrobacterium tumefaciens A136, when encountering AHLs to screen out QS bacteria from sludge. The PAHs-degrading strain YY-1 regulated by QS is further obtained through enrichment and purification. The biofilm formation of this strain is affected by QS regulation, and it can utilize the degradation of multiple PAHs for its own growth.

[0060] In some embodiments, in the step of spreading the dilution on an MSM agar plate containing polycyclic aromatic hydrocarbons, culturing, and forming a second single colony, the polycyclic aromatic hydrocarbons include phenanthrene, pyrene, and benzo[a]pyrene, and the concentrations of phenanthrene, pyrene, and benzo[a]pyrene are 100 mg / L, 100 mg / L, and 50 mg / L, respectively.

[0061] In some embodiments, the step of picking a second single colony with a sterile needle and culturing it again in MSM containing the PAHs, analyzing the bacterial solution OD600nm, and selecting the best growing strain to obtain the PAHs-degrading strain regulated by QS specifically includes: picking a second single colony with a sterile needle and culturing it again in MSM containing the PAHs, culturing at 30°C for 7 days, analyzing the bacterial solution OD600nm, and selecting the best growing strain.

[0062] The present invention also provides a use of the above-mentioned strain, which is used to degrade PAHs.

[0063] The polycyclic aromatic hydrocarbons-degrading strain YY-1 provided by the present invention can utilize polycyclic aromatic hydrocarbons as a carbon source for its own growth. Experiments have shown that the strain can produce more biofilms as the concentration of polycyclic aromatic hydrocarbons in the environment increases, indicating that the strain consumes polycyclic aromatic hydrocarbons during its growth process, thereby removing polycyclic aromatic hydrocarbons in the soil.

[0064] Furthermore, the polycyclic aromatic hydrocarbon-degrading strain is used to enhance the degradation of polycyclic aromatic hydrocarbons under QS regulation. QS regulation affects the biofilm formation of YY-1. When QS is inhibited, the biofilm produced per unit bacterial amount decreases accordingly. When the AHLs concentration increases, the biofilm produced per unit bacterial amount increases. Strengthening the QS effect can improve the degradation efficiency of YY-1 for polycyclic aromatic hydrocarbons.

[0065] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0066] Example 1 Screening of QS-regulated bacterial flora

[0067] (1) Sludge was collected from a biochemical pool at a sewage treatment plant and stabilized with artificial wastewater for one week. The diluted sludge was spread on LB agar plates (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, pH 7.0). After colonies formed, 30 single colonies were randomly selected and expanded in LB medium to obtain the test bacterial solution.

[0068] (2) Simultaneously, the reporter bacterium Agrobacterium tumefaciens A136, which detects AHLs, was cultured in LB medium. The test bacteria and A136 cultures were plated in parallel on LB agar plates coated with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). After 24 hours of incubation, bacteria that caused the base of the A136 colony to appear blue were considered QS bacteria.

[0069] When AHLs are present in the environment, A136 can synthesize β-galactosidase, which degrades X-Gal to produce a blue product. Therefore, the presence of AHLs in the environment can be determined by the color reaction of A136.

[0070] Composition of artificial wastewater: glucose 500 mg / L, yeast extract 100 mg / L, (NH4)2SO4 236 mg / L, MgSO4·7H2O 22.5 mg / L, NH4Cl 1.7 mg / L, CaCl2·2H2O 27.5 mg / L, FeCl3·6H2O 0.25 mg / L, NaHCO3840 mg / L, K2HPO4·12H2O 21.75 mg / L, KH2PO48.5 mg / L, Na2HPO4·12H2O 44.6 mg / L.

[0071] Example 2 Enrichment and purification of PAHs-degrading strains

[0072] The QS bacteria from Example 1 were cultured overnight in LB medium and then washed three times with minimal salt medium (MSM) at pH 7.0. The culture was inoculated at a 10% (v / v) ratio into MSM containing 100 mg / L phenanthrene and cultured at 30°C for 7 days. The culture was then transferred to MSM and cultured three times under the same conditions. After the final culture, the OD600nm of the culture was analyzed, and five strains with good growth (determined by the OD600nm value) were selected. The culture was diluted and evenly spread onto MSM agar plates containing phenanthrene, pyrene, and benzo[a]pyrene (100 mg / L phenanthrene, 100 mg / L pyrene, and 50 mg / L benzo[a]pyrene) and cultured in a 30°C incubator. When single colonies formed, they were picked with a sterile needle and transferred to MSM containing the aforementioned PAHs. After another 7 days of culture at 30°C, the OD600nm of the culture was analyzed, and the strain with the best growth was selected. The purified bacterial liquid of QS bacteria using PAHs as carbon source was diluted and spread on LB agar plate culture medium. After forming single colonies, it was stored below 4°C for subsequent research.

[0073] MSM ingredients: NaHPO4 2800mg / L, KH2PO4 1000mg / L, (NH4)2PO4 500mg / L, MgCl2 53mg / L, Ca(NO3)2 50mg / L, EDTA-2Na 0.5mg / L, FeSO4·7H2O 0.2mg / L, ZnSO4 0.01mg / L, MnCl20.003mg / L, H3BO3 0.03mg / L, CoCl2 0.02mg / L, CuCl2·2H2O 0.001mg / L, NiCl2·6H2O0.001mg / L, NaMoO4·2H2O 0.003mg / L.

[0074] Example 3 Bacterial species identification

[0075] (1) Morphological characteristics identification

[0076] Gram staining: Culture the PAHs-degrading strain obtained in Example 2 overnight in LB medium. Take a glass slide and drop 20 μL of the cleaning solution, smear the solution with a sterile needle; let it dry naturally, dry and fix it with an alcohol lamp; stain with 1% crystal violet solution for 2 minutes, wash with pure water, and add iodine solution to stain for 2 minutes; wash again, decolorize with 95% alcohol for 10 seconds; finally, counterstain with safranin dye (2.5% (W / V) safranin ethanol solution: pure water = 1:4 (V / V)) for 1 minute; after washing, remove the water and observe with an optical microscope. Figure 1 The bacteria are stained red and are Gram-negative bacteria.

[0077] Plate colonies: dilute the bacterial solution, spread it on LB agar plate by streaking, and culture it in a 30℃ incubator. Figure 2 The colonies have clear edges and appear as opaque, raised milky white dots.

[0078] Scanning electron microscopy: Add 2.5% glutaraldehyde solution to the cleaned bacterial solution and let it stand at 4℃ for 24 hours to complete bacterial fixation; centrifuge to remove the residual glutaraldehyde solution and then wash the bacterial solution, transfer a small amount of bacterial solution to a glass slide; let it dry naturally, dehydrate it with a gradient of 50% to 95% alcohol solution, and finally dehydrate it again with anhydrous ethanol, dehydrating it for 20 minutes at each concentration; after dehydration, freeze-dry the sample for 48 hours; after gold spraying, observe it with a scanning electron microscope. Figure 3 The bacterial body size is about (0.5~1.0)×(1.0~1.5)μm, and it is a short rod with rounded ends.

[0079] (2) Molecular biological identification

[0080] DNA of the test strain was extracted using an Ezup column-based bacterial genomic DNA extraction reagent, and 16S rDNA was amplified using PCR primers 7F (5'-CAGAGTTTGATCCTGGCT-3') and 1540R (5'-AGGAGGTGATCCAGCCGCA-3'). The 1.5 kbp electrophoresis band was purified and recovered, and 16S rDNA sequencing was commissioned to Sangon Biotechnology Co., Ltd. The 16S rRNA gene sequence of the test strain is shown in SEQ ID NO. 1.

[0081] The sequencing results are as follows:

[0082] >Test bacteria (5'→3')

[0083]

[0084] The 16S rDNA sequence of the obtained strain was entered into the National Center for Biotechnology Information (NCBI) GenBank database (GenBank). The obtained sequence was compared with known 16S rDNA sequences using the local comparison search tool BLAST on the website. Multiple known sequences with high sequence similarity were downloaded and a phylogenetic tree was drawn using MEGA 11. After sequence alignment, the neighbor-joining method was selected to construct the phylogenetic tree. The bootstrap method was selected to test the established phylogenetic tree model. The phylogenetic tree was generated after setting the number of evaluations to 1000. Figure 4 .

[0085] Identification results: Based on the comprehensive morphological and molecular biological identification results, the obtained strain had a sequence similarity of more than 99% with the Acinetobacter genus, and the strain was named Acinetobacter sp.YY-1.

[0086] Example 4 Optimal Growth Conditions for YY-1

[0087] A single colony of the YY-1 strain was picked and cultured in LB medium at 30°C overnight. The culture was centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the culture was washed with fresh LB medium and resuspended.

[0088] (1) Optimal growth pH: LB culture medium with pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 was prepared using 0.1 mol / L citric acid-sodium citrate buffer system, 0.1 mol / L KH2PO4 and NaOH, and 0.1 mol / L HCl and NaHCO3, respectively. The above-mentioned LB culture medium with a pH value of 4.0 to 11.0 and the washed bacterial solution were added to a 96-well plate. Three replicates were set for each pH condition. The OD600nm value of YY-1 bacteria was measured continuously for 24 hours using a microplate reader to obtain the growth curve of YY-1 bacteria under different initial pH (30°C) culture conditions. Figure 5 (Left).

[0089] (2) Optimum salinity: NaCl was added to LB medium (containing 1% NaCl) to prepare LB medium with salinity of 2%, 3%, 4%, 5%, 6%, 7% and 8% NaCl. The above LB medium with different salinity and the washed bacterial solution were added to a 96-well plate. Three replicates were set for each salinity condition. The OD600nm value of YY-1 bacteria was measured continuously for 24 hours using a microplate reader. The growth curve of YY-1 strain under different salinity culture conditions (30°C, pH 7.0) was drawn to obtain the optimal salinity. Figure 5 (middle).

[0090] (3) Optimum growth temperature: Add LB medium (pH 7.0) and washed bacterial solution to a 50 mL sterilized centrifuge tube and place it in a shaker at 15°C, 20°C, 25°C, 30°C, 35°C, and 40°C. Set three replicates for each temperature condition, culture at 200 rpm for 24 h, and then analyze the OD600nm to determine the optimal growth temperature of the strain. Figure 5 (right).

[0091] Depend on Figure 5 It can be seen that YY-1 can maintain good growth in the range of pH 5.0-9.0, salinity <4% and temperature 20-40℃, and its optimal pH, salinity and temperature conditions are 7.0, 1% and 30℃ respectively.

[0092] Example 5 QS bacteria performance of YY-1

[0093] (1) AHLs reporter bacteria detection

[0094] The reporter bacteria A136 and Chromobacterium violaceum CV026 were used to detect AHLs produced by YY-1. A136 and CV026 detect medium-chain (C8-HSL and above) and short-chain (C4-HSL, C6-HSL) signaling molecules, respectively. Neither A136 nor CV026 produces AHLs themselves. However, in the presence of AHLs, A136 degrades X-gal to produce a blue substrate, while CV026 produces a purple pigment.

[0095] Single colonies of YY-1, A136, and CV026 were picked and cultured in LB medium overnight; antibiotics were added to the molten LB agar medium (20 μL of 4.5 g / L tetracycline and 100 μL of 10 g / L spectinomycin were added to the A136 chromogenic plate; 10 mg / L kanamycin was added to the CV026 chromogenic plate), mixed well, and poured into a culture dish. After the agar plate solidified, an inoculation stick was used to dip the reporter bacteria and YY-1 bacterial solution (the A136 plate also needed to be coated with 10 μL of 50 g / L X-Gal), and parallel short lines were drawn on the LB medium agar plate. The A136 color plate includes A136+C8-HSL (positive control), A136+A136 (negative control) and A136+YY-1 (experimental group); the CV026 color plate includes CV026+C6-HSL (positive control), CV026+CV026 (negative control) and CV026+YY-1 (experimental group). Seal the culture dish with sealing film and place it in a 30℃ incubator for 24 hours to obtain Figure 6 .

[0096] Depend on Figure 6It can be seen that the YY-1 strain only produces medium- and long-chain signal molecules, but does not produce short-chain signal molecules.

[0097] (2) Biofilm formation of YY-1 under QS regulation

[0098] In this experiment, AHLs and QS inhibitors were added to enhance and inhibit the QS effect, respectively, to test whether QS regulates the formation of YY-1 biofilm.

[0099] A mixture containing five AHLs, C4-, C6-, C8-, C10-, and C12-HSL, was prepared (using acetonitrile as the solvent). YY-1 cells were cultured overnight in LB medium, washed three times with fresh LB, and adjusted to an OD600nm of 0.01 for later use. The AHL mixture (a gradient of five AHL concentrations, 0.1 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L) was added to a 96-well plate. After evaporation of the solvent, 200 μL of the diluted bacterial solution was added. As controls, tannic acid (a QS inhibitor, 25 mg / L) or acyltransferase (a QS inhibitor, which disrupts AHLs, 1 mg / L) solution was added to 200 μL of the bacterial solution to inhibit the QS effect. A blank control group containing only 200 μL of the bacterial solution was set up. The plates were sealed with parafilm and incubated in a 30°C incubator.

[0100] After 24 hours of incubation, the OD600nm was measured. The plate was washed three times with phosphate buffer (pH 7.0), the biofilm was fixed in a 60°C oven for 1 hour, and 1% crystal violet solution was added for staining in the dark for 30 minutes; the crystal violet staining solution was aspirated and washed with distilled water; the plate was dried at room temperature, 200 μL of 95% ethanol was added, and the solution was redissolved for 30 minutes, and the OD595nm value was measured. The OD595nm / OD600nm was calculated to investigate the effect of different concentrations of AHLs on the biofilm formation of strain YY-1. Figure 7 .

[0101] Depend on Figure 7 As shown, as AHLs concentration increased, the biofilm production per unit bacterial load increased; however, when QS was inhibited, the biofilm production per unit bacterial load decreased. The experimental results show that YY-1 biofilm formation is affected by QS regulation, indicating that YY-1 activity is regulated by QS.

[0102] Example 6 Application of YY-1 in Degrading PAHs

[0103] A single colony of strain YY-1 was cultured overnight in LB medium (pH 7.0) at 30°C. The supernatant was then centrifuged at 4000 rpm for 15 minutes and the supernatant discarded. The culture was washed three times with MSM and the OD600nm value was adjusted to 1.0. A diluted culture was prepared by mixing MSM and the washed culture at a volume ratio of 4:1 for subsequent PAH degradation experiments.

[0104] (1) YY-1 uses PAHs to form biofilm

[0105] The phenanthrene concentrate was added to a 96-well plate. After the solvent was evaporated, 200 μL of diluted bacterial solution was added. The phenanthrene concentration was set to 0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L and 400 mg / L. A blank control group (LB alone) was set to test the contamination. After culturing at 30°C for 3 days, the suspended bacterial solution was removed and the plate was washed three times with phosphate buffer (pH 7.0). The biofilm amount was analyzed according to the method described in Example 5, and the obtained results were as follows: Figure 8 (Left).

[0106] At the same time, the concentrated solutions of carbazole, anthracene and pyrene were added to a 24-well plate. After the solvent evaporated, 1 mL of diluted bacterial solution was added. A blank control group without PAHs was set up. The concentrations of carbazole, anthracene and pyrene were all 100 mg / L. After culturing at 30°C for 3 days, the biofilm amount was analyzed according to the method described in Example 5, and the results were as follows: Figure 8 (middle).

[0107] Depend on Figure 8 As can be seen (left), as the concentration of phenanthrene increases, YY-1 produces more biofilms, indicating that YY-1 uses phenanthrene as a carbon source for its own growth; Figure 8 (Middle) In the experimental group with carbazole, anthracene, and pyrene added, YY-1 produced more biofilms, indicating that YY-1 can use carbazole, anthracene, and pyrene as carbon sources for its growth. These results demonstrate that YY-1 can utilize a variety of PAHs for its growth.

[0108] (2) Degradation of different PAHs by YY-1

[0109] Carbazole, anthracene, and pyrene were added separately to 50 mL centrifuge tubes (all at a concentration of 20 mg / L). After evaporation of the solvent, 5 mL of the previously diluted bacterial solution was added to the tubes, mixed thoroughly, and incubated at 30°C and 200 rpm for 3 days. Three replicates were set up for each group, and a blank control group without bacterial solution was also established. After the experiment, 10 mL of dichloromethane was added to extract the remaining PAHs. 20 mg / L of benz[a]anthracene was also added as an internal standard. After 2 hours, 1 mL of the extract was transferred to a chromatographic injection vial. After evaporation with nitrogen, the extract was reconstituted with 1 mL of acetonitrile, filtered through a 0.22 μm organic phase filter, and quantitatively analyzed by high-performance liquid chromatography (HPLC).

[0110] The HPLC operating parameters (Agilent Ultimate 3000, ZORBAX Eclipse PAH column, 4.6 × 150 mm × 3.5 μm) were as follows: mobile phase: acetonitrile and ultrapure water, 62% acetonitrile (0-0.75 min), 62% → 100% acetonitrile (0.75-5.25 min), 100% acetonitrile (5.25-7.5 min), 100% → 62% acetonitrile (7.5-9.75 min), and 62% acetonitrile (9.75-11 min); flow rate: 2 mL / min, retention time: 12 min, column temperature: 25°C, injection volume: 5 μL. Spiked recovery for all samples was between 90% and 110%, and the lower limit of quantification was 0.01 mg / L.

[0111] The analysis results are as follows Figure 8 As shown in (right), within 3 days, YY-1 degraded anthracene, pyrene and carbazole to a certain extent, among which the degradation rate of carbazole reached about 20%. The bacterium showed the ability to degrade multiple PAHs.

[0112] (3) QS regulates PAH degradation by YY-1

[0113] A 50 mL centrifuge tube was added with the AHLs mixture (prepared in Example 5) and phenanthrene concentrate. After evaporation of the solvent, 5 mL of diluted bacterial solution (final concentration of each of the five AHLs was 8 μmol / L, and the final concentration of phenanthrene was 100 mg / L) was added. After incubation at 30°C and 200 rpm for 3 and 7 days, three tubes were randomly selected and analyzed for residual phenanthrene concentration. A blank control group was established without bacterial solution. Each experiment was repeated three times.

[0114] To a 50 mL centrifuge tube, add C4-HSL, C8-OXO-HSL, or C12-HSL, along with phenanthrene concentrate. After evaporation of the solvent, add 5 mL of diluted bacterial solution (final concentrations of 10 μmol / L for each of the three AHLs and 100 mg / L for phenanthrene). After incubation at 30°C and 200 rpm for 3 days, analyze the residual phenanthrene concentration. A blank control group was established without bacterial solution. Each experiment was repeated three times.

[0115] Add 10 mL of dichloromethane to the centrifuge tube to terminate the experiment, and add internal standards (carbazole and benz[a]anthracene) at the same time. After extraction for 2 hours, draw 1 mL into a chromatographic injection bottle. After blowing the solvent dry with nitrogen, reconstitute it with 1 mL of chromatographic grade acetonitrile and filter it with a 0.22 μm nylon filter membrane. Then transfer the filtrate to a new chromatographic injection bottle. Quantitative analysis of phenanthrene by HPLC yields Figure 9 .

[0116] Depend on Figure 9 (Left) It can be seen that the degradation rate of phenanthrene by YY-1 was higher in the experimental group with AHLs added than in the control group; Figure 9(Right) Compared to the control group, the experimental groups treated with different signaling molecules showed higher phenanthrene degradation rates by YY-1. This result suggests that enhancing the QS effect can improve the degradation efficiency of YY-1 for phenanthrene.

[0117] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing, characterized in that: The polycyclic aromatic hydrocarbons degradation strain regulated by quorum sensing is Acinetobacter ( Acinetobacter sp.) YY-1, the deposit number is GDMCC No: 63126, and the deposit date is January 9, 2023.

2. A use of the polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing as claimed in claim 1, characterized in that: The polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing is used to degrade at least one polycyclic aromatic hydrocarbon among anthracene, pyrene and carbazole.

3. A use of the polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing as claimed in claim 2, characterized in that: The polycyclic aromatic hydrocarbon-degrading strain regulated by quorum sensing enhances the degradation of at least one polycyclic aromatic hydrocarbon among anthracene, pyrene and carbazole under the regulation of quorum sensing.

Images