Strain TJFP1, screening and separating method thereof and application of strain TJFP1 in degradation of polycyclic aromatic hydrocarbon

Abstract

The invention relates to the technical field of environmental microorganisms, and discloses a strain TJFP1, a screening and separating method thereof and application of the strain TJFP1 in degradation of polycyclic aromatic hydrocarbon. According to the invention, a strain TJFP1 capable of degrading phenanthrene is separated from leather wastewater, and the strain is identified as a mycobacterium according to sequencing analysis of a strain 16S rDNA (ribosomal Deoxyribose Nucleic Acid) gene. The TJFP1 has a good degradation effect on polycyclic aromatic hydrocarbon phenanthrene, has good adaptability to the environment and is excellent in stress resistance.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to a strain TJFP1 and a screening and separation method thereof, and an application thereof in degrading polycyclic aromatic hydrocarbons. Background Art

[0002] PAHs are persistent organic pollutants (POPs) formed by the fusion of two or more benzene rings. They have bioaccumulation and "three hazard" (i.e., mutagenicity, teratogenicity and carcinogenicity) effects, and are potentially harmful to ecology and human health. They are widely present in media such as the atmosphere, soil, water and food. Safely and effectively repairing water and soil environments contaminated by PAHs and reducing ecological risks are currently the focus of common concern in related fields and industries.

[0003] The removal of PAHs mainly adopts three methods: physical, chemical and biological. Among them, physical and chemical technologies such as photocatalytic oxidation, chemical oxidation, electric and thermal technology and soil leaching have disadvantages such as complex technology, high cost and serious secondary pollution. Compared with physical and chemical technologies, biodegradation technology is cost-effective and environmentally friendly. Therefore, microbial degradation and remediation of PHAs is considered to be the most important way to remove PHAs in the environment.

[0004] Microorganisms that degrade PAHs mainly include bacteria, fungi, algae, etc. Among them, bacteria are the most widely studied category. They reproduce quickly and simply, have strong metabolic capacity and rich metabolic pathways. Currently, Pseudomonas, Rhodococcus, Bacillus, etc. have been reported. However, existing research is mostly aimed at laboratory environments, and there are few application cases in field remediation. In addition, due to the hydrophobicity and difficulty in degradation of PAHs themselves, as well as the fact that microorganisms are restricted by factors such as temperature, humidity, pH, oxygen content of the soil environment, and competition between microorganisms, the remediation effect cannot fully meet people's requirements. Therefore, obtaining PAH-degrading bacteria with excellent stress resistance, high degradation efficiency and stability is the key to biodegradation remediation. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of the lack of existing resources of polycyclic aromatic hydrocarbons degrading bacteria with excellent stress resistance, high degradation efficiency and stability.

[0006] In order to achieve the above object, the first aspect of the present invention provides a method for screening and isolating strain FPTJ1, the method comprising the following steps:

[0007] (1) preparing LB solid medium, LB liquid medium I, LB liquid medium II, LB liquid medium III, inorganic salt medium, and screening medium with phenanthrene as the sole carbon source respectively;

[0008] (2) Under aseptic conditions, inoculate leather wastewater into LB liquid medium I, culture it at a temperature of 30°C and a rotation speed of 200 rpm for 2 days. Then, take 2 mL of the culture solution from this LB liquid medium I, add it to LB liquid medium II, and culture it under the same conditions for 2 days to obtain an enriched culture solution, thus completing the enrichment process;

[0009] Under aseptic conditions, use a pipette to aspirate 100 μL of the above enriched culture solution, dilute it stepwise with sterile water, and spread it on LB solid medium. Culture it in a constant temperature incubator at 30°C. After colonies grow on the plate, isolate and select the colonies;

[0010] Perform streak plate isolation on the colonies obtained above until a single strain is obtained;

[0011] Inoculate the above single strain into LB liquid medium III. After activation, store it with glycerol at a ratio of 1:1;

[0012] Perform streak plate isolation to obtain a purified single strain, and inoculate the purified single strain into the screening medium with phenanthrene as the sole carbon source. Culture it at 30°C and 200 rpm for 10 days. Measure the remaining content of phenanthrene in the culture solution of the screening medium with phenanthrene as the sole carbon source, calculate the degradation rate, and screen for degrading bacteria based on the degradation rate;

[0013] (3) Perform continuous streak plate isolation on the degrading bacteria obtained in the above steps to obtain a pure culture. Identify it by 16S rRNA gene sequencing analysis. Perform BLAST analysis on the obtained homologous sequences on the NCBI website to obtain the length of the 16S rDNA gene sequence of this strain. Through sequencing and alignment of the 16S rDNA sequence of this strain, preliminarily determine that this strain belongs to the genus Mycobacterium, denoted as strain TJFP1.

[0014] Preferably, in step (1), the LB liquid medium I, the LB liquid medium II, and the LB liquid medium III are the same LB liquid medium, and all include: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1000 mL of distilled water.

[0015] More preferably, in step (1), the LB solid medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, and 2% agar powder.

[0016] Preferably, in step (1), the inorganic salt medium includes: 1.0 g of Na2SO4, 6.8 g of K2HPO4·3H2O, 3.7 g of KH2PO4, 2.0 g of NH4Cl, 0.1 g of MgSO4, 1 mL of trace metal ion buffer solution, and 1000 mL of distilled water;

[0017] Among them, the trace metal ion buffer solution: 300.0 mg of FeCl2·4H2O, 38.0 mg of CoCl2·6H2O, 20.0 mg of MnCl2·4H2O, 14.0 mg of ZnCl2, 12.4 mg of H3BO3, 40.0 mg of Na2MoO4·2H2O, 3.4 mg of CuCl2·H2O, and 1000 mL of distilled water.

[0018] Preferably, in step (1), the method for preparing the screening medium with phenanthrene as the sole carbon source is as follows: In a laminar flow hood, use a pipette to transfer the stock solution of phenanthrene and place it in a pre-sterilized test tube. Seal the test tube with a heat-resistant and breathable sealing film and place it in a fume hood. Wait until the ethyl acetate in the test tube has completely evaporated, and then add the sterilized inorganic salt medium to obtain the screening medium with phenanthrene as the sole carbon source.

[0019] Stock solution of phenanthrene: Weigh 50.0 mg of solid phenanthrene and dissolve it in 50 mL of ethyl acetate. After filtering with a 0.22 μm organic phase filter membrane, store it in a brown reagent bottle for later use.

[0020] According to a particularly preferred specific embodiment, in step (2), measuring the remaining content of phenanthrene in the culture solution in the screening medium with phenanthrene as the sole carbon source includes the following steps:

[0021] S1: Establish a method for determining phenanthrene by ultra-high performance liquid chromatography:

[0022] First, determine the detection conditions: Select a Waters Premier UPLC ultra-high performance liquid chromatography instrument, with a TUV detector. The chromatographic column is a Waters Symeetry C18. The mobile phase is acetonitrile: ultrapure water = 85:15. The flow rate is set at 0.4 mL / min, the wavelength is set at 254 nm, the injection volume is 5 μL, the elution time is 10 min, and the retention time of the phenanthrene chromatographic peak is 7.2 min.

[0023] Secondly, draw a standard curve: Take the 1 g / L stock solution of phenanthrene in proportion and prepare phenanthrene standard solutions with concentrations of 1, 5, 10, 25, 50, and 100 mg / L respectively. After filtering and sterilizing with a 0.22 μm organic filter membrane, perform detection according to the above UPLC detection method. Draw a standard curve with the concentration of phenanthrene as the abscissa and the chromatographic peak area as the ordinate.

[0024] S2: Measure the remaining phenanthrene concentration in the culture solution:

[0025] The content of phenanthrene was detected by using the method of whole-bottle extraction. After the detection was completed, the test tube was taken out, 10 mL of ethyl acetate was added, vortexed and mixed evenly, ultrasonically treated in a water bath for 1 h, left standing for 30 min, the upper organic phase and the lower aqueous phase were separated, the upper organic phase was filtered through a 0.22-μm organic phase filter membrane, placed in a 2-mL brown sample vial, and the phenanthrene content was detected by UPLC.

[0026] The second aspect of the present invention provides the strain TJFP1 prepared by the method described in the first aspect of the present invention. The length of the 16S rDNA gene sequence of this strain is 1394 bp.

[0027] The third aspect of the present invention provides the application of the strain TJFP1 described in the second aspect of the present invention in degrading polycyclic aromatic hydrocarbons.

[0028] According to a particularly preferred specific embodiment, the application includes the following steps:

[0029] A1: Prepare tryptic soy broth liquid medium according to the following formula: 30 g of tryptic soy broth liquid medium dry powder and 1000 mL of distilled water;

[0030] A2: Inoculate the strain TJFP1 into a conical flask containing 100 mL of the tryptic soy broth liquid medium, culture at 30 °C and 200 rpm for 3 d, take the bacterial liquid, centrifuge at 4 °C and 8000 rpm for 5 min, remove the supernatant medium, wash three times with sterile inorganic salt medium, and resuspend with the liquid medium of the inorganic salt medium to prepare a bacterial suspension;

[0031] Add 0.25 - 1 mL of a stock solution containing phenanthrene at a concentration of 1 - 10 g / L into a sterilized test tube, place it in a fume hood, wait for the organic solvent to completely evaporate, add 9 mL of an inorganic salt medium with a pH of 4 - 10, and then inoculate 1 mL of the above bacterial suspension to form a culture system with a substrate concentration of 50 mg / L and an inoculation amount of 10%;

[0032] A3: Incubate the above culture system at 20 - 37 °C, set the speed of the shaker to 200 rpm, sample after 5 - 10 d, measure OD 600 , and use UPLC to detect the remaining phenanthrene concentration and calculate the degradation rate.

[0033] Preferably, in step (A2), the stock solution is polycyclic aromatic hydrocarbon fluorene, polycyclic aromatic hydrocarbon anthracene, polycyclic aromatic hydrocarbon fluoranthene, polycyclic aromatic hydrocarbon pyrene, polycyclic aromatic hydrocarbon benzo[α]anthracene or polycyclic aromatic hydrocarbon benzo[α]pyrene.

[0034] More preferably, in step (A2), the pH value of the inorganic salt medium is 9.

[0035] Preferably, in step (A3), the temperature of the cultivation is 30 °C.

[0036] The strain TJFP1 capable of degrading polycyclic aromatic hydrocarbons provided by the present invention has at least the following beneficial effects:

[0037] The present invention isolates a strain TJFP1 capable of degrading phenanthrene from leather wastewater. According to the 16S rDNA gene sequencing analysis of the strain, the strain is identified as the genus Mycobacterium. TJFP1 has a good degradation effect on the polycyclic aromatic hydrocarbon phenanthrene, has good adaptability to the environment, and excellent stress resistance. It is manifested as:

[0038] (1) It can have a certain degradation effect on the polycyclic aromatic hydrocarbon phenanthrene at 20-37 °C, and has the best degradation effect at 30 °C. 50 mg·L -1 of phenanthrene can be completely degraded in 10 days.

[0039] (2) When the pH is 5-9, the average degradation rate of 50 mg·L -1 of phenanthrene is above 85% at 10 days.

[0040] (3) When the pH is 4-10, the average degradation rate of 50 mg·L -1 of phenanthrene is above 20% at 5 days, and under certain acid-base stress, the degradation rate will increase to a certain extent. Under the condition of pH = 9, the degradation rate is 99.22%.

[0041] (4) When the concentration of phenanthrene is 25, 50, 100, 200, 500 mg·L -1 , TJFP1 all has a good degradation effect. When the concentration of phenanthrene is 100 mg·L -1 , the 5-day degradation rate of TJFP1 to phenanthrene is 99.26%. When the concentration is 500 mg·L -1 , the maximum average degradation rate is 55.68 mg / L·d -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the standard curve graph of Example 1 of the present invention with the concentration of phenanthrene as the abscissa and the chromatographic peak area as the ordinate;

[0043] Figure 2 is the graph of the degradation rate and growth difference of the strain TJFP1 obtained in Example 1 of the present invention to phenanthrene at different temperatures;

[0044] Figure 3 is the graph of the degradation rate and growth difference of the strain TJFP1 obtained in Example 1 of the present invention to phenanthrene when the pH is 5-9 and the degradation period is 10 days;

[0045] Figure 4It is the degradation rate and growth difference diagram of phenanthrene by the strain TJFP1 obtained in Example 1 of the present invention under the conditions of pH 4-10 and a degradation period of 5 days;

[0046] Figure 5 It is the degradation rate and growth difference diagram of phenanthrene by the strain TJFP1 obtained in Example 1 of the present invention under different phenanthrene concentrations;

[0047] Figure 6 It is the degradation effect diagram of different polycyclic aromatic hydrocarbons by the strain TJFP1;

[0048] Figure 7 It is the colony morphology diagram of the strain TJFP1 on TSB solid medium;

[0049] Figure 8 It is the phylogenetic tree diagram of the strain TJFP1. Detailed implementation manners

[0050] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0051] In the present invention, a highly efficient degrading bacterium strain TJFP1 for degrading polycyclic aromatic hydrocarbon pollutants belongs to Mycobacterium sp.F104, and its preservation number is CCTCC M 20242378, and it was deposited with the China Center for Type Culture Collection (CCTCC) on October 30, 2024.

[0052] In the present invention, the 16S rDNA gene sequence of the strain TJFP1 is shown as SEQ ID NO:1.

[0053] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, the raw materials are commercially available products.

[0054] In the following examples, unless otherwise specified, the tryptic soy broth liquid medium powder includes: tryptone 17.0 g / L, sodium chloride 5.0 g / L, soy peptone hydrolyzate 3.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L.

[0055] Example 1

[0056] (1) Prepare LB solid medium, LB liquid medium (LB liquid medium I, LB liquid medium II, LB liquid medium III), inorganic salt medium, and screening medium with phenanthrene as the sole carbon source according to the following recipes respectively:

[0057] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water;

[0058] LB solid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, 2% (m:v) agar powder;

[0059] The inorganic salt medium: 1.0 g of Na2SO4, 6.8 g of K2HPO4·3H2O, 3.7 g of KH2PO4, 2.0 g of NH4Cl, 0.1 g of MgSO4, 1 mL of trace metal ion buffer solution, 1000 mL of distilled water;

[0060] Among them, the trace metal ion buffer solution: 300.0 mg of FeCl2·4H2O, 38.0 mg of CoCl2·6H2O, 20.0 mg of MnCl2·4H2O, 14.0 mg of ZnCl2, 12.4 mg of H3BO3, 40.0 mg of Na2MoO4·2H2O, 3.4 mg of CuCl2·H2O, 1000 mL of distilled water;

[0061] The screening medium with phenanthrene as the sole carbon source: Use a pipette to transfer the corresponding volume of the phenanthrene stock solution into a pre-sterilized 30 mL test tube in a laminar flow hood, seal the test tube with a heat-resistant breathable sealing film, and place it in a fume hood; wait until the ethyl acetate in the test tube has completely evaporated, and then add the sterilized inorganic salt medium to obtain the screening medium with the required concentration;

[0062] Phenanthrene stock solution: Weigh 50.0 mg of phenanthrene solid and dissolve it in 50 mL of ethyl acetate. After filtering with a 0.22 μm organic phase filter membrane, store it in a brown reagent bottle for later use.

[0063] (2) Under sterile conditions, inoculate leather wastewater into LB liquid medium I, culture it at a temperature of 30 °C and a rotation speed of 200 rpm for 2 d. Then, take 2 mL of the culture solution from this LB liquid medium I and add it to LB liquid medium II, and culture it under the same conditions for 2 d to obtain an enriched culture solution, thus completing the enrichment process;

[0064] Under sterile conditions, use a pipette to aspirate 100 μL of the above enriched culture solution, dilute it stepwise with sterile water, and spread it on LB solid medium. Culture it in a constant temperature incubator at 30 °C for an appropriate time. After colonies grow on the plate, isolate and select colonies with good growth status and different morphologies;

[0065] The obtained colonies above are separated by streak plate method for multiple times until single strains are obtained;

[0066] The above single strains are inoculated into LB liquid medium III for activation and then preserved with glycerol at a ratio of 1:1;

[0067] By streak plate separation, purified single strains are obtained, and the purified single strains are inoculated into the screening medium with phenanthrene as the sole carbon source (meanwhile, strains FPTJ1 and other strains preserved in the laboratory are compared). They are cultured at 30 °C and 200 rpm for 10 d. The remaining content of phenanthrene in the culture solution of the screening medium with phenanthrene as the sole carbon source is measured, the degradation rate is calculated, and degrading bacteria are screened according to the degradation rate.

[0068] Among them, measuring the remaining content of phenanthrene in the culture solution of the screening medium with phenanthrene as the sole carbon source includes the following steps:

[0069] S1: Establish a method for measuring phenanthrene by ultra-high performance liquid chromatography:

[0070] First, determine the detection conditions: The instrument selected is Waters Premier UPLC ultra-high performance liquid chromatography, the detector is TUV detector, the chromatographic column is Waters Symeetry C18 (5 μm, 4.6×100 mm), the mobile phase is acetonitrile: ultrapure water = 85:15, the flow rate is set at 0.4 mL / min, the wavelength is set at 254 nm, the injection volume is 5 μL, the elution time is 10 min, and the retention time of the chromatographic peak of phenanthrene is 7.2 min;

[0071] Second, draw a standard curve: Take the stock solution of 1 g / L phenanthrene and prepare phenanthrene standard solutions with concentrations of 1, 5, 10, 25, 50, and 100 mg / L respectively according to the ratio. After filtering and sterilizing with a 0.22 μm organic filter membrane, detect them according to the above UPLC detection method. Draw a standard curve with the concentration of phenanthrene as the abscissa and the chromatographic peak area as the ordinate (as Figure 1 shown).

[0072] S2: Determine the remaining concentration of phenanthrene in the culture solution:

[0073] Due to the property that phenanthrene is insoluble in water, the whole-bottle extraction method is used to detect the content of phenanthrene. After the detection is completed, take out the test tube, add 10 mL of ethyl acetate, vortex and mix evenly, perform water bath ultrasonic treatment for 1 h, then let it stand for 30 min, separate the upper organic phase and the lower aqueous phase, filter the upper organic phase through a 0.22 μm organic phase filter membrane into a 2 mL brown injection vial, and then detect the phenanthrene content by UPLC.

[0074] (3) The strain TJFP1 obtained from the above steps was streaked on a plate continuously for multiple times to obtain a pure culture, which was sent to Shanghai Ruixing Biotechnology Co., Ltd. for identification by 16S rRNA gene sequencing analysis. The obtained homologous sequences were analyzed by BLAST on the NCBI website. The sequencing results showed that the length of the 16S rDNA gene sequence of this strain was 1394 bp. By sequencing and aligning its 16S rDNA sequence (the sequencing result had the highest similarity with the sequence with the accession number OQ996658.1 on GenBank, up to 99.86%), it could be preliminarily determined that this strain belonged to the genus Mycolicibacterium sp., denoted as strain TJFP1;

[0075] Molecular identification result: The 16S rDNA gene sequence is as shown in SEQ ID NO:1.

[0076] Application Example 1

[0077] This example was used to provide the detection of the degradation effect of the strain TJFP1 obtained in Example 1 of the present invention on phenanthrene at different temperatures, speculate on the adaptability of the strain TJFP1 to temperature conditions, and explore the optimal temperature for culturing the strain TJFP1. The results are as Figure 2 shown;

[0078] Specifically, it included:

[0079] A1: Prepare tryptic soy broth liquid medium according to the following formula: 30 g of tryptic soy broth liquid medium dry powder and 1000 mL of distilled water;

[0080] A2: Inoculate the strain TJFP1 obtained in the above Example 1 into a conical flask containing 100 mL of tryptic soy broth liquid medium, culture it at 30 °C and 200 rpm for 3 d, take the bacterial liquid, centrifuge it at 4 °C and 8000 rpm for 5 min, remove the supernatant medium, wash it three times with sterile inorganic salt medium, and resuspend it with the liquid medium of inorganic salt medium to prepare a bacterial suspension;

[0081] Add 0.5 mL of the mother liquor with a phenanthrene concentration of 1 g / L into a sterilized test tube, place it in a fume hood, wait for the organic solvent to completely dry, add 9 mL of inorganic salt medium, and then inoculate 1 mL of the above bacterial suspension to form a culture system 1 with a substrate concentration of 50 mg / L and an inoculation amount of 10%;

[0082] A3: Prepare culture system 2, culture system 3, and culture system 4 according to the above steps, and then culture them at temperature gradients of 20 °C, 25 °C, 30 °C, and 37 °C respectively. The rate of the shaker is set to 200 rpm. After 10 d, take samples and use an ultraviolet spectrophotometer to measure the OD at the end of the culture cycle 600, and the remaining substrate phenanthrene concentration was detected by UPLC, and the degradation rate was calculated to speculate the adaptability of the strain to temperature conditions, and at the same time, the optimal culture temperature was obtained.

[0083] It can be seen from Figure 2 that at 20 °C, 25 °C, 20 °C, and 37 °C, the strain TJFP1 has a certain degradation effect on phenanthrene. It can be seen that the strain TJFP1 has good adaptability to different temperatures, and its degradation ability is the strongest at 30 °C, and it can completely degrade 50 mg / L of phenanthrene. Moreover, OD 600 is the highest. Although there is no significant difference, it can still be judged that the growth state of the strain is the best at 30 °C.

[0084] Application Example 2

[0085] This example is used to provide the detection of the degradation effect of the strain TJFP1 obtained in Example 1 of the present invention on phenanthrene at different pH values, speculate the adaptability of the strain TJFP1 to acid-base conditions, and explore the optimal pH for culturing the strain TJFP1. The results are as Figure 3 and Figure 4 shown;

[0086] Specifically, it includes:

[0087] A1: Prepare tryptic soy broth liquid medium according to the following formula: 30 g of tryptic soy broth liquid medium dry powder and 1000 mL of distilled water;

[0088] A2: Inoculate the strain TJFP1 obtained in Example 1 above into a conical flask containing 100 mL of tryptic soy broth liquid medium, culture it at 30 °C and 200 rpm for 3 days, take the bacterial liquid, and centrifuge it at 4 °C and 8000 rpm for 5 minutes to remove the supernatant medium. After washing three times with sterile inorganic salt medium, resuspend it with the liquid medium of the inorganic salt medium to make a bacterial suspension;

[0089] Add 0.5 mL of the mother liquor with a phenanthrene concentration of 1 g / L to a sterilized test tube, place it in a fume hood, wait for the organic solvent to completely dry, add 9 mL of inorganic salt medium 1, and then inoculate 1 mL of the above bacterial suspension to obtain a culture system A with a pH of 5, a substrate concentration of 50 mg / L, and an inoculation amount of 10%;

[0090] Among them, the pH value of the inorganic salt medium is adjusted with 1 mol / L HCl or 1 mol / L NaOH; the pH value of the inorganic salt medium 1 is 5;

[0091] The pH value of the inorganic salt medium 2 is 6;

[0092] The pH value of the inorganic salt medium 3 is 7;

[0093] The pH value of the inorganic salt medium 4 is 8;

[0094] The pH value of the inorganic salt medium 5 is 9;

[0095] A3: Prepare culture systems B to E according to the above steps, place them in an incubator at 30 °C and 200 rpm for cultivation. After 10 days, take samples and use an ultraviolet spectrophotometer to measure the OD at the end of the cultivation period 600 , and use UPLC to detect the concentration of the remaining substrate phenanthrene, calculate the degradation rate, so as to infer the adaptability of the strain to acid-base conditions, and at the same time obtain the optimal cultivation pH.

[0096] From Figure 3 it can be seen that when the pH is between 5 and 9, the degradation rate is above 85% and there is no significant difference. It can be seen that it is speculated that the strain TJFP1 has good tolerance to acid-base conditions, which is a prominent advantage of the strain TJFP1 compared with other degradation strains. Its stress resistance to acid-base conditions implies the practical feasibility of its application in actual complex polluted environments; at the same time, according to OD 600 it can be roughly estimated that the optimal growth and degradation pH is 9.

[0097] In order to further explore the tolerance and degradation effect of the strain TJFP1 to pH, the pH gradient was broadened to 4-10, and the degradation period was shortened to 5 days. Again, according to the above steps, measure the OD at the end of its cultivation period 600 and the concentration of the remaining substrate phenanthrene and calculate the degradation rate.

[0098] From Figure 4 it can be seen that for the 5-day degradation of phenanthrene by the strain TJFP1 at different pH values, when pH = 9, the growth state of TJFP1 is the best and the degradation rate is the highest, which is 99.23%.

[0099] Application Example 3

[0100] This example is used to provide the detection of the degradation effect of the strain TJFP1 obtained in Example 1 of the present invention on phenanthrene at different phenanthrene concentrations, and the results are as Figure 5 shown;

[0101] Specifically include:

[0102] A1: Prepare tryptic soy broth liquid medium according to the following formula: 30 g of tryptic soy broth liquid medium powder and 1000 mL of distilled water;

[0103] A2: Inoculate the strain TJFP1 obtained in Example 1 above into a conical flask containing 100 mL of tryptic soy broth liquid medium, cultivate at 30 °C and 200 rpm for 3 days, take the bacterial liquid, and centrifuge at 4 °C and 8000 rpm for 5 min to remove the supernatant medium. After washing three times with sterile inorganic salt medium, resuspend with the liquid medium of the inorganic salt medium to prepare a bacterial suspension;

[0104] 0.25 mL of the mother liquor 1 with a phenanthrene concentration of 1 g / L, 0.5 mL of the mother liquor 1 with a phenanthrene concentration of 1 g / L, 1 mL of the mother liquor 1 with a phenanthrene concentration of 1 g / L, 0.2 mL of the mother liquor 2 with a phenanthrene concentration of 10 g / L, and 0.5 mL of the mother liquor 2 with a phenanthrene concentration of 10 g / L were separately added into sterilized test tubes and placed in a fume hood. After the organic solvents were completely dried, 9 mL of the inorganic salt medium with a pH of 9 was added separately, and then 1 mL of the above-mentioned bacterial suspension was inoculated respectively to form culture system I with a substrate concentration of 25 mg / L and an inoculation amount of 10%, culture system II with a concentration of 50 mg / L and an inoculation amount of 10%, culture system III with a concentration of 100 mg / L and an inoculation amount of 10%, culture system IV with a concentration of 200 mg / L and an inoculation amount of 10%, and culture system V with a concentration of 500 mg / L and an inoculation amount of 10%.

[0105] A3: The above-mentioned culture systems were placed at 30 °C and 200 rpm. After culturing for 5 d, the OD at the end of the culture period was measured using an ultraviolet spectrophotometer. 600 The remaining substrate phenanthrene concentration was detected by UPLC, and the degradation rate was calculated.

[0106] It can be seen from Figure 5 that when the concentration of phenanthrene is 100 mg / L or less, the strain TJFP1 can almost completely degrade phenanthrene within a 5-d culture period. Moreover, in a high-concentration environment, the degradation effect is also remarkable. Although the degradation rate decreases, the amount of substrate degraded per unit time is still increasing, which is also a major feature of this strain superior to other degrading bacteria.

[0107] Application Example 4

[0108] This example is used to provide the detection of the degradation effect of the strain TJFP1 obtained in Example 1 of the present invention on different polycyclic aromatic hydrocarbons, and the results are as Figure 6 shown.

[0109] Specifically, it includes:[[]]

[0110] A1: Prepare tryptic soy broth liquid medium according to the following formula: 30 g of tryptic soy broth liquid medium dry powder and 1000 mL of distilled water.

[0111] A2: The strain TJFP1 obtained in Example 1 above was inoculated into a conical flask containing 100 mL of tryptic soy broth liquid medium. After culturing at 30 °C and 200 rpm for 3 d, the bacterial liquid was taken, centrifuged at 4 °C and 8000 rpm for 5 min, the supernatant medium was removed, washed three times with sterile inorganic salt medium, and then resuspended with the liquid medium of the inorganic salt medium to prepare a bacterial suspension.

[0112] Add 0.5 mL of mother liquor a with a concentration of 1 g / L to the sterilized test tube, place it in the fume hood, wait for the organic solvent to completely dry, add 9 mL of inorganic salt medium, and then inoculate 1 mL of the above-mentioned bacterial suspension to form culture system i with a substrate concentration of 50 mg / L and an inoculation amount of 10%.

[0113] Among them, mother liquor a is polycyclic aromatic hydrocarbon fluorene;

[0114] Mother liquor b is polycyclic aromatic hydrocarbon anthracene;

[0115] Mother liquor c is polycyclic aromatic hydrocarbon fluoranthene

[0116] Mother liquor d is polycyclic aromatic hydrocarbon pyrene;

[0117] Mother liquor e is polycyclic aromatic hydrocarbon benzo[α]anthracene;

[0118] Mother liquor f is polycyclic aromatic hydrocarbon benzo[α]pyrene;

[0119] A3: Prepare culture systems ii, iii, iv, v, vi, and vii according to the above steps, and then place the above culture systems at 30 °C and 200 rpm respectively. After culturing for 5 days, use an ultraviolet spectrophotometer to measure the OD at the end of the culture cycle 600 , and use UPLC to detect the remaining substrate phenanthrene concentration and calculate the degradation rate.

[0120] It can be seen from Figure 6 that the strain TJFP1 has a certain degradation effect on tricyclic polycyclic aromatic hydrocarbons (anthracene, fluorene) and some tetracyclic polycyclic aromatic hydrocarbons (fluoranthene, pyrene). This characteristic is superior to some reported degrading bacteria, but it has basically no degradation effect on high-molecular-weight polycyclic aromatic hydrocarbons (benzo[α]anthracene, benzo[α]pyrene).

[0121] In order to more clearly express the strain TJFP1 of the present invention, the present invention shows the colony morphology of the strain TJFP1 on the TSB solid medium through Figure 7 , and shows the phylogenetic tree of the strain TJFP1 through Figure 8 .

[0122] It can be seen from Figure 7 that the colony morphology of the strain TJFP1 on the TSB plate is round, light yellow, with regular colony edges, smooth texture, and wet surface.

[0123] It can be seen from Figure 8 that according to the phylogenetic tree, it shows that the strain has the highest similarity with the genus Mycolicibacteriumsp. and has a close genetic relationship, and can be preliminarily identified as the genus Mycolicibacterium.

[0124] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for screening and isolating strain FPTJ1, characterized in that, The method comprises the following steps: (1) Prepare LB solid medium, LB liquid medium I, LB liquid medium II, LB liquid medium III, inorganic salt medium, and screening medium with phenanthrene as the sole carbon source respectively; (2) Under sterile conditions, inoculate leather wastewater into LB liquid medium I, culture at 30 °C and 200 rpm for 2 d, then take 2 mL of the culture solution from the LB liquid medium I, add it to LB liquid medium II, and culture under the same conditions for 2 d to obtain an enriched culture solution, thus completing the enrichment process; Under sterile conditions, pipette 100 μL of the above enriched culture solution, dilute it stepwise with sterile water and spread it on the LB solid medium, culture it in a constant temperature incubator at 30 °C, and after colonies grow on the plate, isolate and select the colonies; Perform streak plate isolation on the obtained colonies above until a single strain is obtained; Inoculate the above single strain into LB liquid medium III, after activation, store it with glycerol at a ratio of 1:1; Perform streak plate isolation to obtain a purified single strain, and inoculate the purified single strain into the screening medium with phenanthrene as the sole carbon source, culture at 30 °C and 200 rpm for 10 d, measure the remaining content of phenanthrene in the culture solution of the screening medium with phenanthrene as the sole carbon source, calculate the degradation rate, and screen the degrading bacteria according to the degradation rate; (3) Perform continuous streak plate isolation on the degrading bacteria obtained in the above steps to obtain a pure culture, identify it by 16S rRNA gene sequencing analysis, perform BLAST analysis on the obtained homologous sequences on the NCBI website to obtain the length of the 16S rDNA gene sequence of the strain, and through sequencing and alignment of the 16S rDNA sequence of the strain, preliminarily determine that the strain belongs to the genus Mycobacterium, denoted as strain TJFP1.

2. The method according to claim 1, wherein In step (1), the LB liquid medium I, the LB liquid medium II, and the LB liquid medium III are the same LB liquid medium, and all include: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1000 mL of distilled water; And / or, in step (1), the LB solid medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 1000 mL of distilled water, and 2% agar powder.

3. The method according to claim 1, characterized in that, In step (1), the inorganic salt medium includes: 1.0 g of Na2SO4, 6.8 g of K2HPO4·3H2O, 3.7 g of KH2PO4, 2.0 g of NH4Cl, 0.1 g of MgSO4, 1 mL of trace metal ion buffer solution, and 1000 mL of distilled water; Among them, the trace metal ion buffer solution: 300.0 mg of FeCl2·4H2O, 38.0 mg of CoCl2·6H2O, 20.0 mg of MnCl2·4H2O, 14.0 mg of ZnCl2, 12.4 mg of H3BO3, 40.0 mg of Na2MoO4·2H2O, 3.4 mg of CuCl2·H2O, and 1000 mL of distilled water.

4. The method according to claim 1, wherein In step (1), the preparation method of the screening medium with phenanthrene as the sole carbon source is as follows: In a laminar flow hood, use a pipette to transfer the mother liquor of phenanthrene and place it in a pre-sterilized test tube. Seal the test tube with a heat-resistant breathable sealing film and place it in a fume hood. Wait until the ethyl acetate in the test tube has completely volatilized, then add the sterilized inorganic salt medium to obtain the screening medium with phenanthrene as the sole carbon source. Mother liquor of phenanthrene: Weigh 50.0 mg of solid phenanthrene and dissolve it in 50 mL of ethyl acetate. After filtering with a 0.22 μm organic phase filter membrane, store it in a brown reagent bottle for later use.

5. The method according to claim 1, wherein In step (2), measuring the remaining content of phenanthrene in the culture solution of the screening medium with phenanthrene as the sole carbon source includes the following steps: S1: Establish a method for determining phenanthrene by ultra-high performance liquid chromatography: First, determine the detection conditions: Select a Waters Premier UPLC ultra-high performance liquid chromatography instrument, with a TUV detector. The chromatographic column is a Waters Symeetry C18. The mobile phase is acetonitrile: ultrapure water = 85:15, the flow rate is set at 0.4 mL / min, the wavelength is set at 254 nm, the injection volume is 5 μL, the elution time is 10 min, and the retention time of the phenanthrene chromatographic peak is 7.2 min. Secondly, draw a standard curve: Take the mother liquor of 1 g / L phenanthrene in proportion and prepare phenanthrene standard solutions with concentrations of 1, 5, 10, 25, 50, and 100 mg / L respectively. After filtering and sterilizing with a 0.22 μm organic filter membrane, perform detection according to the above UPLC detection method. Draw a standard curve with the concentration of phenanthrene as the abscissa and the chromatographic peak area as the ordinate. S2: Measure the remaining phenanthrene concentration in the culture solution: Adopt the method of whole-bottle extraction to detect the content of phenanthrene. After the detection is completed, take out the test tube, add 10 mL of ethyl acetate, vortex and mix well, perform ultrasonic bath for 1 h, let it stand for 30 min, separate the upper organic phase and the lower aqueous phase, filter the upper organic phase with a 0.22 μm organic phase filter membrane, place it in a 2 mL brown injection vial, and detect the phenanthrene content by UPLC.

6. The strain TJFP1 prepared by the method according to any one of claims 1-5, characterized in that, The length of the 16S rDNA gene sequence of this strain is 1394 bp.

7. The application of the strain TJFP1 described in claim 6 in degrading polycyclic aromatic hydrocarbons.

8. The application according to claim 7, wherein Including the following steps: A1: Prepare tryptic soy broth liquid medium according to the following formula: 30 g of tryptic soy broth liquid medium powder and 1000 mL of distilled water. A2: Inoculate the strain TJFP1 into a conical flask containing 100 mL of the above tryptic soy broth liquid medium, culture it at 30 °C and 200 rpm for 3 d, then take the bacterial liquid, centrifuge it at 4 °C and 8000 rpm for 5 min, remove the supernatant medium, wash it three times with sterile inorganic salt medium, and resuspend it with the liquid medium of the inorganic salt medium to prepare a bacterial suspension. Add 0.25 - 1 mL of the mother liquor with a phenanthrene concentration of 1 - 10 g / L to a sterilized test tube, place it in a fume hood, wait until the organic solvent has completely dried, add 9 mL of the inorganic salt medium with a pH of 4 - 10, and then inoculate 1 mL of the above bacterial suspension to form a culture system with a substrate concentration of 50 mg / L and an inoculation amount of 10%. A3: Incubate the above culture system at 20 - 37°C with the shaker set at a rate of 200 rpm. Take samples after 5 - 10 days and measure the OD using an ultraviolet spectrophotometer. 600 Also, detect the remaining phenanthrene concentration using UPLC and calculate the degradation rate.

9. The application according to claim 8, characterized in that, In step (A2), the mother liquor is polycyclic aromatic hydrocarbon fluorene, polycyclic aromatic hydrocarbon anthracene, polycyclic aromatic hydrocarbon fluoranthene, polycyclic aromatic hydrocarbon pyrene, polycyclic aromatic hydrocarbon benzo[α]anthracene or polycyclic aromatic hydrocarbon benzo[α]pyrene; And / or, in step (A2), the pH value of the inorganic salt medium is 9.

10. The application according to claim 8, characterized in that, In step (A3), the temperature of the culture is 30°C.

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