Rhodococcus and screening and application thereof
By screening and identifying Rhodococcus NJPE1, using magnetic nanoparticle enrichment culture technology, efficient degradation of pollutants such as PAEs in the environment is achieved, and the problem of difficult degradation of PAEs in the existing technology is solved, and an effective microbial repair is provided. method.
Patent Information
- Application Number
- CN202411099285.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-12
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art is difficult to effectively degrade the difficult-to-degrade organic pollutants phthalate (PAEs), which are widely present in the environment, which pose a threat to human and ecological health.
A strain of Rhodococcus NJPE1 was screened and identified. This strain had a good degradation effect on PAEs of different structures. The mud was prepared by mixing the soil of the contaminated site with a specific culture medium, adding magnetic nanoparticles for enrichment and culture, and using DEHP as the only carbon source. The active degrading bacteria were isolated and purified.
Rheumatoidea NJPE1 has a significant degradation effect on PAEs, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and polybrominated diphenyl ethers of different structures, with a degradation rate of 94.2% to 99.6%, which solves the problem of difficult degradation of PAEs in the environment and provides an efficient Microbial repair technology.
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Figure CN119120260A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microorganisms, and more specifically, relates to a rhodococcus and its screening and application. Background Art
[0002] Phthalate esters (PAEs) are widely used as plasticizers in polyvinyl chloride plastics and are a class of organic pollutants that are difficult to degrade. They have low solubility in water and high octanol / water partition coefficients. They easily accumulate in the environment and have been detected in sediments, water bodies, soils, and aquatic organisms. This type of pollutant is considered to be an endocrine disrupting chemical. Some PAEs are mutagenic and carcinogenic, which endangers human and ecological health. Microbial metabolism of PAEs is the main way for these pollutants to be removed from the environment. Studies have shown that in soil, water bodies, and wastewater, a variety of PAEs can be aerobically degraded by microorganisms. Screening strains that can effectively degrade a variety of PAEs is crucial for the development of efficient microbial remediation technologies. Summary of the invention
[0003] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.
[0004] One object of the present invention is to provide a strain of Rhodococcus, which has a good degradation effect on PAEs of different structures.
[0005] In order to achieve these purposes and other advantages of the present invention, a Rhodococcus sp. NJPE1 is provided, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on July 20, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 1.64773.
[0006] The screening method of Rhodococcus that degrades phthalates comprises the following steps: 1) Mixing the soil from the contaminated site with MM culture medium to prepare slurry, adding DEHP as a substrate to enrich and culture phthalate-degrading bacteria to obtain enriched culture; 2) The enriched culture was centrifuged to obtain an initial enriched bacterial solution, magnetic nanoparticles were added to the initial enriched bacterial solution to prepare a magnetized bacterial population, and active degrading bacteria were enriched from the magnetized bacterial population using MM medium with DEHP as the sole carbon source; 3) Phthalate-degrading bacteria were isolated and purified in MM solid medium with DEHP as the sole carbon source, and 16S rRNA gene sequencing and DEHP degradation experiments were used to obtain Rhodococcus that can degrade phthalates.
[0007] Preferably, each liter of MM culture medium contains the following raw material components: MgSO4·2H2O 0.2 g, CaCl2·2H2O20 mg, FeSO4·7H2O 10 mg, KH2PO40.4 g, Na2HPO40.6 g, MnSO420 mg, NaNO30.8 g, KNO30.3g, (NH4)2SO41 g, and the pH is adjusted to 7.0~7.2.
[0008] Preferably, in step 1), the temperature of the enrichment culture is 25-28° C., the rotation speed of the shaking culture is 150-200 rpm, and the culture time is 15-30 days.
[0009] Preferably, in step 2), the enriched culture is centrifuged at a speed of 1000-2000 rpm for 3-5 min.
[0010] Preferably, the specific method for preparing the magnetized bacterial colony is to add magnetic nanoparticles to the initial enrichment culture and then culture at 25-28°C and 150-200 rpm for 1 hour.
[0011] Preferably, in step 2), the specific method for enriching active degrading bacteria is to use the magnetized bacterial group as an inoculum, inoculate it into 5 mL-10 mL of MM culture medium with DEHP as the sole carbon source at a 5-10% inoculation ratio, culture it at 25-28 ° C and 150-200 rpm for 5-10 days, and centrifuge it at 1000-2000 rpm for 3-5 min. The supernatant obtained is the active degrading bacteria.
[0012] Preferably, isolating and purifying phthalate-degrading bacteria includes primary screening and secondary screening; The specific method of the initial screening is as follows: the active degrading bacteria obtained in step 2) are taken for gradient dilution, and the diluted bacterial solution is spread on a double-layer plate with DEHP as the sole carbon source, and inverted culture is carried out at 25-28°C until a single colony is formed on the plate, and the single colony is separated and purified on the LB plate by the continuous streaking method, and the single colony is selected according to the colony morphology on the LB plate; The preparation method of the double-layer plate is as follows: the lower plate is a MM solid culture medium containing 1.5% agar, and the upper plate contains a MM solid culture medium containing 1% agar and 800 mg / L DEHP.
[0013] The specific method of rescreening is: select the single colonies of different morphologies obtained in the initial screening, and culture them in LB liquid medium until OD 600To 0.6-0.8, collect the bacterial sludge by centrifugation, wash it three times with MM medium, and then resuspend the bacteria in an equal volume of MM medium as inoculum; inoculate it into MM medium with DEHP as the sole carbon source at a 10% inoculation ratio for cultivation; after the cultivation, add acetonitrile to the bottle to dissolve the residual pollutants in the bottle, aspirate the mixed solution to pass through a 0.22 μm organic phase filter membrane, determine the DEHP concentration by high performance liquid chromatography, pick the pure strain with the highest DEHP degradation rate, and obtain the phthalate-degrading bacteria.
[0014] The use of Rhodococcus sp. NJPE1 is to degrade phthalates, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and polybrominated diphenyl ethers.
[0015] The present invention has at least the following beneficial effects: First, the Rhodococcus NJPE1 screened by the present invention has good degradation effects on PAEs of different structures, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, polybrominated diphenyl ethers, etc. Among them, the degradation rates of DBP and DOP with straight-chain alkyl groups are 94.2-99.6%, the degradation rate of BBP with benzene ring groups is 99.5%, the degradation rate of DEHP with branched alkyl groups is 42.3%, the degradation rate of DCHP with cycloalkyl groups is 24.7%, the degradation effect on Phe and Pyr is close to 100%, the degradation effect on PCB28 is 91.0%, and the degradation effects on BDE15 and BDE28 are 93.3% and 81%, respectively.
[0016] Second, the present invention is the first to use magnetic nanoparticle-mediated separation technology to screen and separate Rhodococcus that can degrade PAEs.
[0017] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The morphology and phylogenetic tree diagram of the bacterial colony of the present invention; wherein A is a plate bacterial colony morphology diagram; B is an electron microscope limited morphology diagram; C is a phylogenetic tree diagram; Figure 2 This is the result of the DEHP degradation test by Rhodococcus NJPE1; Figure 3 The figure shows the degradation test results of PAEs with straight-chain alkyl groups by Rhodococcus sp. NJPE1; Sole refers to DBP or DOP only; Mixture refers to the degradation of DBP or DOP in a mixed pollutant of DBP, DOP, DEHP, DCHP and BBP; Figure 4The graph shows the degradation test results of Rhodococcus sp. NJPE1 on DEHP with branched alkyl groups, BBP with benzene ring groups, and DCHP with cycloalkyl groups; where Sole refers to BBP, DEHP, or DCHP only; Mixture refers to the degradation of BBP, DEHP, or DCHP in a mixed pollutant of DBP, DOP, DEHP, DCHP, and BBP; Figure 5 This is the degradation test result of Rhodococcus sp. NJPE1 on PAHs, PCBs and PBDEs. DETAILED DESCRIPTION
[0019] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.
[0020] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.
[0021] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.
[0022] The present invention provides a Rhodococcus sp. NJPE1, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 20, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 1.64773.
[0023] A method for screening a strain of Rhodococcus comprises the following steps: 1) Mixing the soil from the contaminated site with MM culture medium to prepare slurry, adding DEHP as a substrate to enrich and culture phthalate-degrading bacteria to obtain enriched culture; 2) The enriched culture was centrifuged to obtain an initial enriched bacterial solution, magnetic nanoparticles were added to the initial enriched bacterial solution to prepare a magnetized bacterial population, and active degrading bacteria were enriched from the magnetized bacterial population using MM medium with DEHP as the sole carbon source; 3) The phthalate-degrading bacteria were isolated and purified in MM solid culture medium with DEHP as the sole carbon source, and the Rhodococcus was obtained by 16S rRNA gene sequencing and DEHP degradation experiments.
[0024] In another technical scheme, each liter of MM culture medium contains the following raw material components: MgSO4·2H2O 0.2 g, CaCl2·2H2O 20 mg, FeSO4·7H2O 10 mg, KH2PO4 0.4 g, Na2HPO4 0.6 g, MnSO4 20 mg, NaNO3 0.8 g, KNO3 0.3 g, (NH4)2SO4 1 g, and the pH is adjusted to 7.0~7.2.
[0025] In another technical solution, in step 1), the temperature of the enrichment culture is 25-28°C, the rotation speed of the shaking culture is 150-200 rpm, and the culture time is 15-30 days.
[0026] In another technical solution, in step 2), the centrifugation speed of the enriched culture is 1000-2000 rpm, and the centrifugation time is 3-5 min.
[0027] In another technical solution, the specific method for preparing the magnetized bacterial colony is to add magnetic nanoparticles to the initial enrichment culture and then culture it at 25-28°C and 150-200 rpm for 1 hour.
[0028] In another technical scheme, in step 2), the specific method for enriching active degrading bacteria is to use the magnetized bacterial group as an inoculum, inoculate it into 5 mL-10 mL of MM culture medium with DEHP as the sole carbon source at a 5-10% inoculation ratio, culture it at 25-28 ° C and 150-200 rpm for 5-10 days, and centrifuge it at 1000-2000 rpm for 3-5 min. The supernatant obtained is the active degrading bacteria.
[0029] In another technical solution, isolating and purifying phthalate-degrading bacteria includes primary screening and secondary screening; The specific method of the initial screening is as follows: the active degrading bacteria obtained in step 2) are taken for gradient dilution, and the diluted bacterial solution is spread on a double-layer plate with DEHP as the sole carbon source, and inverted culture is carried out at 25-28°C until a single colony is formed on the plate, and the single colony is separated and purified on the LB plate by the continuous streaking method, and the single colony is selected according to the colony morphology on the LB plate; The preparation method of the double-layer plate is as follows: the lower plate is a MM solid medium containing 1.5% agar, and the upper plate contains a MM solid medium containing 1% agar and 800 mg / L DEHP; The specific method of rescreening is: select the single colonies of different morphologies obtained in the initial screening, and culture them in LB liquid medium until OD 600To 0.6-0.8, collect the bacterial sludge by centrifugation, wash it three times with MM medium, and then resuspend the bacteria in an equal volume of MM medium as inoculum; inoculate it into MM medium with DEHP as the sole carbon source at a 10% inoculation ratio for cultivation; after the cultivation, add acetonitrile to the bottle to dissolve the residual pollutants in the bottle, aspirate the mixed solution to pass through a 0.22 μm organic phase filter membrane, determine the DEHP concentration by high performance liquid chromatography, pick the pure strain with the highest DEHP degradation rate, and obtain the phthalate-degrading bacteria.
[0030] The use of Rhodococcus sp. NJPE1 is to degrade phthalates, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and polybrominated diphenyl ethers. Phthalates include butyl phthalate (DBP) and dioctyl phthalate (DOP) with straight-chain alkyl groups, di(2-ethylhexyl) phthalate (DEHP) with branched alkyl groups, dicyclohexyl phthalate (DCHP) with cycloalkyl groups, and butyl benzyl phthalate (BBP) with benzene ring groups. Polycyclic aromatic hydrocarbons include typical phenanthrene (Phe), anthracene (Ant), and pyrene (Pyr); polychlorinated biphenyls include PCB28, PCB77, and PCB110; polybrominated diphenyl ethers include BDE15, BDE28, and BDE47.
[0031] Experiment 1: Isolation, purification and identification of DEHP-degrading bacteria The strain NJPE1 was obtained from the soil of a chemical plant in Tianjin. The specific steps are as follows: (1) Enrichment of DEHP-degrading bacteria: 20 g of soil and 100 mL of MM culture medium were mixed in a 250 mL Erlenmeyer flask to prepare soil slurry, 500 mg / L of DEHP was added as substrate, and the DEHP-degrading bacteria were enriched at 28 °C and 160 rpm for 1 month. After the culture, the soil suspension was centrifuged at 1400 rpm for 3 min, and the supernatant was the initial enrichment culture. The components of the MM culture medium (1 L) were MgSO4·2H2O 0.2 g, CaCl2·2H2O 20 mg, FeSO4·7H2O 10 mg, KH2PO4 0.4 g, Na2HPO4 0.6 g, MnSO4 20 mg, NaNO3 0.8 g, KNO3 0.3 g, (NH4)2SO4 1 g, and the pH was adjusted to 7.0.
[0032] (2) Obtaining active DEHP-degrading bacterial flora: Add 300 μL of magnetic nanoparticles (8 gL -1Fe3O4), and cultured at 28 ℃ and 160 rpm for 1 h to magnetize the microbial flora. After the magnetization treatment, centrifuge at 1400 rpm for 3 min to collect the magnetized precipitate, wash the magnetized flora 2-3 times with MM medium to remove the unadsorbed microorganisms, and finally resuspend the magnetized flora with an equal volume of MM medium. The magnetized flora was used as an inoculum and inoculated into 5 mL of MM medium with DEHP (100 mg / L) as the sole carbon source at a 10% inoculation ratio. It was cultured at 28 ℃ and 160 rpm for 7 days, centrifuged at 1400 rpm for 3 min, and the supernatant contained active degrading bacteria.
[0033] (3) Initial screening of DEHP-degrading bacteria: The supernatant in step (2) was taken for gradient dilution, and 100 μL of the diluted bacterial solution was spread on a double-layer plate with DEHP as the sole carbon source to screen single bacteria. The double-layer plate was prepared according to the following scheme: the lower plate was MM solid medium containing 1.5% agar, and the upper plate was MM solid medium containing 1% agar (800 mg / L DEHP was introduced as a screening substrate through acetone). The plate was inverted and cultured at 28°C until a single colony was formed on the plate. The single colony was isolated and purified on the LB plate by continuous streaking. The single colony was selected according to the colony morphology on the LB plate and stored separately. The LB culture components (1L) were 10 g tryptone, 5 g yeast extract, 5 g NaCl, pH 7.0.
[0034] (4) Rescreening of DEHP-degrading bacteria: Select single colonies of different morphologies and culture them in LB liquid medium until OD 600 = 0.6~0.8, centrifuged at 5000 rpm for 4 min to collect the bacterial sludge, washed three times with MM medium, and then resuspended with an equal volume of MM medium as an inoculum. Inoculated into 5 mL of MM medium (20 mL culture bottle) with DEHP (5 mg / L) as the sole carbon source at a 10% inoculation ratio, and cultured at 28 ℃ and 160 rpm for 7 days. After the culture was completed, 2 volumes of acetonitrile were added to the bottle to dissolve the residual pollutants in the bottle, 1 mL of the mixed solution was aspirated to pass through a 0.22 μm organic phase filter, and the DEHP concentration was determined by high performance liquid chromatography (HPLC).
[0035] (5) Identification of DEHP-degrading bacteria: Combined with step (4), a pure strain with the highest DEHP degradation rate was selected and named NJPE1. The 16S rRNA gene was amplified by PCR using 27 F and 1492 R primers. The amplified product was sent to Shanghai Meiji Company for sequencing to obtain the sequence SEQ ID No. 1. After BLAST alignment, the sequence was used to construct a phylogenetic tree using MEGA software (such as Figure 1-C). Sequencing results showed that the strain belongs to Rhodococcus. The colony morphology is orange-yellow ( Figure 1 -A), with a smooth, opaque surface, positive Gram staining, and a spherical shape ( Figure 1 -B); the indole test and catalase test were positive, while the methyl red test, VP (Voges-Proskaurer) test, citrate test, starch hydrolysis test, and cellulose hydrolysis test were all negative.
[0036] The strain was able to grow with DEHP as the sole carbon source. After one week of cultivation, the pollutant concentration decreased from the initial 5 mg / L to 2.82 mg / L. Figure 2 shown.
[0037] The sequence of 16S rRNA gene SEQ ID No.1 is as follows:
[0038] Experiment 2: Degradation of linear alkyl PAEs by Rhodococcus sp. NJPE1 as the sole carbon source Linear alkyl DBP and DOP with different chain lengths were selected for microbial degradation test, where DBP had a C4 linear alkyl on one side and DOP had a C8 linear alkyl on one side. The specific test steps are as follows: (1) Preparation of inoculum of NPJE1: Pick a single colony of NPJE on the LB plate and culture it in LB liquid medium until OD 600 = 0.6~0.8, centrifuge at 5000 rpm for 4 min to collect the bacterial sludge, wash it three times with MM medium, and then resuspend the bacteria in an equal volume of MM medium as inoculum.
[0039] (2) Preparation of the sole carbon source degradation system: Use acetone to dissolve DBP and DOP respectively, add the acetone solution into a sterilized Erlenmeyer flask (100 mL), and let it stand in a sterile clean bench until the acetone is completely evaporated. Add 10 mL of MM culture medium (the final concentration of the pollutants is 20 mg / L), inoculate the NJPE1 bacterial solution in step (1) at a ratio of 10%, and culture at 28 °C and 160 rpm for 7 days. After the culture is completed, add 2 times the volume of acetonitrile to the bottle to dissolve the residual pollutants in the bottle, aspirate 1 mL of the mixed solution through a 0.22 μm organic phase filter, and use HPLC to determine the concentrations of DBP and DOP.
[0040] The results are as follows Figure 3 As shown in the figure, Rhodococcus sp. NJPE1 has a good degradation effect on DBP and DOP, that is, it has a good degradation effect on PAEs with straight-chain alkyl groups. Among them, the degradation efficiency of BOP is 99.6%. The chain length of the alkyl group has a certain effect on the biodegradation efficiency. Under the same culture conditions, the degradation efficiency of DOP is 94.2%.
[0041] Experiment 3: Degradation of other PAEs by Rhodococcus sp. NJPE1 as the sole carbon source Other PAEs with different chemical structures were selected for research, including DEHP with branched alkyl groups, BBP with benzene ring groups, and DCHP with cycloalkyl groups.
[0042] (1) Preparation of inoculum of NPJE1: Pick a single colony of NPJE on the LB plate and culture it in LB liquid medium until OD 600 = 0.6~0.8, centrifuge at 5000 rpm for 4 min to collect the bacterial sludge, wash it three times with MM medium, and then resuspend the bacteria in an equal volume of MM medium as inoculum.
[0043] (2) Preparation of the sole carbon source degradation system: DEHP, DCHP and BBP were dissolved in acetone, respectively. The pollutants were introduced into a sterile Erlenmeyer flask (100 mL) through acetone. After the acetone was completely evaporated, 10 mL of MM culture medium (the final concentration of the pollutants was 20 mg / L) and NJPE1 bacterial solution (10% inoculation ratio) were added to the flask. The PAEs concentration in the flask was analyzed after culturing at 28 °C and 160 rpm for 7 days.
[0044] The results are as follows Figure 4 As shown in the figure, Rhodococcus NJPE1 also has a good degradation effect on BBP with a benzene ring. Within a one-week culture period, almost all BBP was degraded (99.5%). The degradation rate of branched alkyl DEHP was relatively lower (42.3%), but the degradation efficiency was also close to 50%. In comparison, NJPE1 had a lower degradation efficiency for cycloalkyl DCHP, with a degradation efficiency of 24.7% under the sole carbon source condition. Experiment 4: Degradation of other PAEs by Rhodococcus sp. NJPE1 in the form of complex carbon sources.
[0045] All the above PAEs with different structures were selected for experiments to test the degradation effect of NJPE1 on pollutants when they were used as composite carbon sources. PAEs with different chemical structures include DBP and DOP with straight-chain alkyl groups, DEHP with branched alkyl groups, DCHP with cycloalkyl groups, and BBP with benzene ring groups.
[0046] (1) Preparation of inoculum of NPJE1: Pick a single colony of NPJE on the LB plate and culture it in LB liquid medium until OD 600 = 0.6~0.8, centrifuge at 5000 rpm for 4 min to collect the bacterial sludge, wash it three times with MM medium, and then resuspend the bacteria in an equal volume of MM medium as inoculum.
[0047] (2) Preparation of composite carbon source degradation system: Use acetone to dissolve DBP, DOP, DEHP, DCHP and BBP respectively, and mix the pollutants. Introduce the mixed pollutants into a sterile conical flask (100 mL). After the acetone is completely evaporated, add 10 mL of MM culture medium (the final concentration of the pollutants is 20 mg / L) and NJPE1 bacterial solution (10% inoculation ratio) into the flask. After culturing at 28 °C and 160 rpm for 7 days, analyze the PAEs concentration in the flask.
[0048] The results are as follows Figure 3 , Figure 4As shown, when pollutants were used as composite carbon sources, Rhodococcus NJPE1 had no effect on the degradation of DBP, BBP and DCHP, the degradation rate of DOP decreased (down to 70%), while it promoted the degradation of DEHP (increased to 52.6%).
[0049] Experiment 5: Degradation of other organic pollutants by Rhodococcus sp. NJPE1 in the form of a composite carbon source Other organic pollutants tested included PAHs, PCBs and PBDEs, which were classified and combined to test the degradation effect of NJPE1 on them under the condition of composite carbon source. The PAH degradation group included a mixture of Phe, Ant and Pyr; the PCB degradation group included a mixture of PCB28, PCB77 and PCB110; the PBDE degradation group included a mixture of BDE15, BDE28 and BDE47.
[0050] (1) Preparation of inoculum of NPJE1: Pick a single colony of NPJE on the LB plate and culture it in LB liquid medium until OD 600 = 0.6~0.8, centrifuge at 5000 rpm for 4 min to collect the bacterial sludge, wash it three times with MM medium, and then resuspend the bacteria in an equal volume of MM medium as inoculum.
[0051] (2) Preparation of composite carbon source degradation system: Phe, Ant and Pyr were dissolved in acetone respectively, and the pollutants were mixed as the PAH degradation group; PCB28, PCB77 and PCB110 were dissolved separately and mixed as the PCB degradation group; BDE15, BDE28 and BDE47 were dissolved separately and mixed as the PBDE degradation group. The mixed pollutants were introduced into sterile triangular flasks (100 mL) (i.e. three independent experiments). After the acetone was completely evaporated, 10 mL of MM culture medium (the final concentration of pollutants was 20 mg / L) and NJPE1 bacterial solution (10% inoculation ratio) were added to the flasks. After culturing at 28 °C and 160 rpm for 7 days, the concentration of organic pollutants in the flasks was analyzed.
[0052] The results are as follows Figure 5 As shown in the figure, when pollutants are used as composite carbon sources, Rhodococcus NJPE1 has a good degradation effect on PAHs, PCBs and PBDEs, among which the degradation effect on Phe and Pyr is close to 100%; the degradation effect on PCB28 is 91.0%; the degradation effect on BDE15 and BDE28 is 93.3% and 81% respectively ( Figure 5 ) Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.
Claims
1. Rhodococcus sp. NJPE1, characterized in that The bacteria was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 20, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.1.64773.
2. A method for screening Rhodococcus, characterized in that: Screening the Rhodococcus described in claim 1 comprises the following steps: 1) Mixing the soil from the contaminated site with MM culture medium to prepare slurry, adding DEHP as a substrate to perform enrichment culture on phthalate-degrading bacteria to obtain enrichment culture; 2) centrifuging the enriched culture to obtain an initial enriched bacterial solution, adding magnetic nanoparticles to the initial enriched bacterial solution to prepare a magnetized bacterial population, and using MM medium with DEHP as the only carbon source to enrich active degrading bacteria from the magnetized bacterial population; 3) Phthalate-degrading bacteria were isolated and purified in MM solid medium with DEHP as the sole carbon source, and 16SrRNA gene sequencing and DEHP degradation experiments were performed to obtain the phthalate-degrading Rhodococcus.
3. The method for screening Rhodococcus according to claim 2, characterized in that: Each liter of MM culture medium contains the following raw material components: MgSO4·2H2O 0.2g, CaCl2·2H2O 20mg, FeSO4·7H2O 10mg, KH2PO4 0.4g, Na2HPO40.6g, MnSO4 20mg, NaNO3 0.8g, KNO3 0.3g, (NH4)2SO4 1g, and the pH is adjusted to 7.0-7.
2.
4. The method for screening Rhodococcus according to claim 2, characterized in that: In step 1), the temperature of the enrichment culture is 25-28° C., the rotation speed of the shaking culture is 150-200 rpm, and the culture time is 15-30 days.
5. The method for screening Rhodococcus according to claim 2, characterized in that: In step 2), the centrifugal speed of the enriched culture is 1000-2000 rpm, and the centrifugal time is 3-5 min.
6. The method for screening Rhodococcus according to claim 2, characterized in that: The specific method for preparing the magnetized bacterial community is to add magnetic nanoparticles to the initial enriched bacterial solution and then culture it at 25-28° C. and 150-200 rpm for 1 hour.
7. The method for screening Rhodococcus according to claim 2, characterized in that: In step 2), the specific method for enriching active degrading bacteria is to use the magnetized bacterial group as an inoculum, inoculate it into 5mL-10mL of MM culture medium with DEHP as the sole carbon source at a 5-10% inoculation ratio, culture it at 25-28°C and 150-200rpm for 5-10 days, and centrifuge it at 1000-2000rpm for 3-5min after the culture is completed. The supernatant obtained is the active degrading bacteria.
8. The method for screening Rhodococcus according to claim 2, characterized in that: Isolation and purification of phthalate-degrading bacteria includes primary screening and secondary screening; The specific method of the initial screening is: taking the active degrading bacteria obtained in step 2) and performing gradient dilution, respectively taking the diluted bacterial solution and coating it on a double-layer plate with DEHP as the sole carbon source, inverting and culturing at 25-28°C until a single colony is formed on the plate, using the continuous streaking method to separate and purify the single colony on the LB plate, and selecting the single colony according to the colony morphology on the LB plate; The preparation method of the double-layer plate is as follows: the lower plate is a MM solid culture medium containing 1.5% agar, and the upper plate contains a MM solid culture medium containing 1% agar and 800 mg / L DEHP; The specific method of rescreening is: select the single colonies of different morphologies obtained in the initial screening, and culture them in LB liquid medium until OD 600 to 0.6-0.8, collect bacterial sludge by centrifugation, wash three times with MM culture medium, and then resuspend the bacteria in an equal volume of MM culture medium as inoculum; inoculate into MM culture medium with DEHP as the sole carbon source at a 10% inoculation ratio for cultivation; after the cultivation, add acetonitrile into the bottle to dissolve the residual pollutants in the bottle, draw the mixed solution through a 0.22 μm organic phase filter membrane, determine the DEHP concentration by high performance liquid chromatography, pick out the pure strain with the highest DEHP degradation rate, and obtain phthalate degrading bacteria.
9. Use of Rhodococcus sp. NJPE1, characterized in that: The bacteria is used to degrade phthalates, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and polybrominated diphenyl ethers.