Brucella sp. TF-10 and its application in degrading polyfluorinated PFAS

Abstract

The present invention discloses Brucella sp. TF-10 and its application in degrading per- and polyfluoroalkyl substances (PFAS). Brucella sp. TF-10 was deposited at the General Microbiological Center of the China General Microbiological Culture Collection Center on September 12, 2024, with the deposit number: CGMCC NO. 1.62206. The screened strain can achieve biological defluorination using PFAS such as 4,5,5-trifluoro-4-pentenoic acid and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid, and the biological defluorination efficiency of hexafluorinated PFAS is significantly improved under co-metabolic conditions.

Description

Technical Field

[0001] The present invention relates to the field of environmental microorganisms, and particularly to Brucella sp. TF-10 and its application in degrading perfluoroalkyl substances (PFAS). Background Art

[0002] Perfluoroalkyl and polyfluoroalkyl substances (PFAS) have attracted much attention due to their stable carbon-fluorine (C-F) bonds, specifically referring to compounds containing at least perfluoromethyl (-CF3) or methylene (-CF2-). The introduction of fluorine endows PFAS with excellent surface activity, hydrophobicity and stability, enabling their wide application in multiple industrial fields such as textiles, leather, electroplating, petroleum, mining, aerospace and semiconductors. In addition, PFAS are also commonly found in commercial products such as aqueous film-forming foams, pesticides, personal care products and non-stick cookware. Toxicological studies have shown that PFAS may cause multiple hazards such as hepatotoxicity, neurotoxicity, endocrine disruption and carcinogenicity, seriously threatening the health of humans and the ecosystem, and becoming the focus of global environmental risks. The C-F bond has a high dissociation energy, and PFAS exhibit extremely strong chemical stability, making them difficult to degrade in the environment. Due to their persistence, long-range migration and bioaccumulation, PFAS are widely present in soil, atmosphere, water bodies and sediments, and have been detected in plants, animals and humans. Researchers around the world are committed to the study of microbial defluorination mechanisms, aiming to alleviate the environmental hazards of PFAS by biodegradation of the C-F bond, explore the potential of microbial defluorination, which is particularly important for the development of biodegradable fluorinated chemical products. At present, the research on biological defluorination is still in its infancy, and most of the defluorination evidence comes from data on microbial communities in environmental samples, with few reports on pure strains capable of defluorination. In this study, a Brucella TF-10 strain with biological defluorination function was screened. According to literature research, this is the first report on biological defluorination of Brucella. Summary of the Invention

[0003] The object of the present invention is to provide Brucella sp. TF-10 and its application in degrading perfluoroalkyl substances (PFAS) to solve the above technical problems.

[0004] To solve the above technical problems, the present invention adopts the following technical solutions to achieve:[[]]END]]

[0005] The present invention provides Brucella sp. TF-10. Brucella sp. TF-10 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 12, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the postcode 100101. The strain is classified and named as Brucella, and the deposit number is: CGMCC NO. 1.62206.

[0006] The basic biological characteristics of Brucella sp. TF-10 of the present invention are as follows:

[0007] (1) Colony morphology: light yellow, flat and moist on the surface;

[0008] (2) Biochemical characteristics: Gram-negative staining, and the cell morphology is spherical.

[0009] Brucella sp. TF-10 screened in the present invention was obtained by screening using trifluorinated PFAS as a substrate. This strain can degrade 4,5,5-trifluoro-4-pentenoic acid (TFE) with three fluorines and 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC) with six fluorines under the condition of the sole carbon source, and simultaneously break the C-F bond.

[0010] Brucella sp. TF-10 screened in the present invention has a promoting defluorination effect on the co-metabolism of hexafluorinated PFAS.

[0011] Nocardioides sp. NJF-4 screened in the present invention was inoculated into an inorganic salt medium for fluorinated alkyl compounds and cultured at 28 °C and 160 r / min to achieve biological defluorination.

[0012] In the present invention, the inorganic medium is the inorganic salt medium MM-G. The ratio of the inorganic salt medium MM-G is as follows: 0.2 g of MgSO4·2H2O, 20 mg of CaCl2·2H2O, 10 mg of FeSO4·7H2O, 20 mg of MnSO4, 0.7 g of NaNO3, 0.3 g of KNO3, 0.3 g of NaCl, and 1 g of (NH4)2SO4. They are dissolved in 1000 ml of deionized water, and after the medium is sterilized, it is added to a 20 mmol / L HEPES buffer solution with pH = 7.2.

[0013] The beneficial effects of the present invention are as follows:

[0014] 1. The present invention screens a PFAS biological defluorination bacterium TF-10, which is identified as Brucella. This is the first study on biological defluorination of this strain.

[0015] 2. The screened Brucella TF-10 of the present invention has a biological defluorination effect on PFAS such as trifluoro and hexafluoro. In the case of the sole carbon source, the defluorination rate of this bacterium for TFE is 14.4%, and the defluorination rate for SFC is 23.3%. Ammonium acetate as a co-metabolic substrate significantly increases the dechlorination rate of SFC to 57.2%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the morphology and phylogenetic tree of Brucella sp. TF-10;

[0017] Figure 2 It is the growth and defluorination effect of strain TF-10 on trifluoroalkane TFE;

[0018] Figure 3 It is the growth and defluorination effect of strain TF-10 on hexafluoroalkane SFC;

[0019] Figure 4 It is the co-metabolic defluorination effect of strain TF-10 on SFC. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] The specific embodiments of the present invention will be described below with reference to the drawings.

[0023] Example 1

[0024] Brucella TF-10 screened the polluted soil of a chemical plant in Suzhou. The specific steps are as follows:

[0025] (1) Enrichment of defluorinating bacteria: Mix 20 g of soil with 50 mL of MM medium in a 250 mL Erlenmeyer flask to make soil slurry, add 0.1% (v / v) of FD, and perform enrichment culture at 28 °C and 160 rpm for 1 month. After the culture is completed, take 1400 rpm centrifugation of the soil suspension for 3 min, and take the supernatant as the initial enrichment culture. Among them, the MM component (1 L) is 0.2 g of MgSO4·2H2O, 20 mg of CaCl2·2H2O, 10 mg of FeSO4·7H2O, 0.4 g of KH2PO4, 0.6 g of Na2HPO4, 20 mg of MnSO4, 0.8 g of NaNO3, 0.3 g of KNO3, 1 g of (NH4)2SO4, and adjust the pH to 7.0 - 7.2.

[0026] (2) Primary screening of defluorinating bacteria: Pipette the soil suspension in step (1) for gradient dilution, and spread the diluted solution on a double-layer plate with TFE as the sole carbon source. The lower layer of the double-layer plate is MM solid medium containing 1.5% agar, and the upper layer plate is MM solid medium containing 1% agar with 3.24 mmol / L -1 TFE. Invert the plate and culture it at 28 °C until single colonies appear. Continuously streak and isolate single colonies on an LB plate. Among them, the LB medium component (1 L) is 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl.

[0027] (3) Re-screening of defluorinating bacteria: Select single colonies with different morphologies in step (2) and culture them in LB liquid medium until the OD 600 value reaches 0.6 - 0.8. Subsequently, centrifuge the culture solution at 5000 rpm for 4 minutes, collect the bacterial sludge, and wash it three times with MM medium. Finally, suspend the washed cells in an equal volume of liquid. Then, according to an inoculation ratio of 10%, inoculate the suspension into MM medium containing 100 μmol / L -1 TFE, and simultaneously add 100 μmol / L -1 glucose as a co-metabolic substrate, and culture it at 28 °C and 160 rpm for one month. After the culture is completed, use ion chromatography to preliminarily evaluate the release of fluoride ions in the culture system.

[0028] (4) Identification of defluorinating bacteria: Use 27F and 1492R primers to perform PCR amplification of the 16S rRNA gene. The amplified product is then sent to Shanghai Majorbio Co., Ltd. for sequencing. Finally, compare the obtained sequence through BLAST and use MEGA software to construct a phylogenetic tree. The phylogenetic tree is as Figure 1 shown.

[0029] Through the above steps, a highly efficient defluorinating bacterium TF-10 was screened. The sequencing results showed that this strain belongs to Brucella. Please refer to Figure 1, the colony morphology is light yellow, the surface is flat and moist, the Gram stain is negative, and the cell morphology is spherical.

[0030] Example 2

[0031] Select 4,5,5-trifluoro-4-pentenoic acid (TFE) for the study of biological defluorination of trifluoro PFAS. Based on literature reports, this substance can be co-metabolically defluorinated by the activated sludge microbial community. At present, there is no research on the biological defluorination of this substance by pure microbial cultures. It should be noted that previous studies have found that small molecules that strongly affect the detection of fluoride ions by IC are generally produced during the co-metabolic degradation of polyfluoro PFAS by this substance. Therefore, a fluorine-lanthanide chelate-specific colorimetric method was used to study the biological defluorination of polyfluoro PFAS. The specific steps are as follows:

[0032] (1) Preparation of inoculum: Pick a single colony of TF-10 on an 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 the inorganic salt medium MM-G, and then resuspend the cells with an equal volume of the inorganic salt medium MM-G as the inoculum. Among them, the components of the inorganic salt medium MM-G (1 L) are 0.2 g of MgSO4·2H2O, 20 mg of CaCl2·2H2O, 10 mg of FeSO4·7H2O, 20 mg of MnSO4, 0.7 g of NaNO3, 0.3 g of KNO3, 0.3 g of NaCl, and 1 g of (NH4)2SO4. After the medium is sterilized, 20 mmol / L HEPES (pH = 7.2) is added. The components of the LB medium (1 L) are 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl.

[0033] (2) Sole carbon source degradation system: According to the inoculation ratio of 10%, inoculate the bacterial solution in step (1) into the inorganic salt medium MM-G (5 mL) containing TFE (100 μmol / L). At the same time, add a fluorine-free TFE structural analog, 4-pentenoic acid, with the same concentration as the fluorine-free blank control. The culture solution is cultured at 28 °C and 160 rpm for 30 days. Samples are taken at intervals during the culture period. Take 300 μL of the sample and centrifuge it. The supernatant is used to detect the fluoride ion concentration by the microplate colorimetric method based on the color reaction of fluorine-lanthanide chelate.

[0034] (3) Detection of fluoride ions by microplate colorimetric method: Preparation of colorimetric detection solution: 10 μL of acetate buffer (1.68 mol / L), 20 μL of alizarin (500 μmol / L), and 20 μL of lanthanum nitrate (500 μmol / L) were mixed in sequence. 100 μL of sample, 50 μL of colorimetric solution, and 50 μL of acetone were added to the microplate in sequence. The color development reaction lasted for more than half an hour. A620 / 530 was calculated by an ELISA instrument. A standard curve was established in the range of 0-80 μmol / L using standard fluoride ion samples. The fluoride ion concentration in the sample was calculated by regression method.

[0035] See also Figure 2 The study showed that Brucella TF-10 was able to biologically defluorinate the trifluoroalkyl compound TFE, and no fluoride ion release was detected in the fluorine-free structural analogues. The maximum defluorination effect was achieved in the second week. After the culture was completed, the fluoride ion concentration detected in the system was 43.2 μmol / L, and the defluorination rate was 14.4%.

[0036] Example 3

[0037] 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC) was selected for the study of biodefluorination of hexafluoro PFAS, and the colorimetric method was used to detect the biodefluorination of polyfluorinated PFAS. The preparation of Brucella TF-10 inoculum and degradation system was carried out according to Example 2. The test substrate was 200 μmol / L SFC, and the same concentration of fluorine-free SFC structural analog 3-methyl crotonic acid was added at the same time as a fluorine-free blank control. The culture medium was cultured at 28 ° C and 160 rpm for 1 month, and samples were taken at intervals during the period. Take 300 μL of sample and centrifuge it, and the supernatant uses a microplate colorimetric method based on the color reaction of fluorine-lanthanide chelates to detect the fluoride ion concentration. The test method is the same as Example 2.

[0038] See also Figure 3 The study showed that Brucella TF-10 was able to biologically defluorinate the trifluoroalkyl compound TFE, and no fluoride ion release was detected in the fluorine-free structural analogues. The maximum defluorination effect was basically achieved in the third week. After the culture was completed, the fluoride ion concentration detected in the system was 279.3 μmol / L, and the defluorination rate was 23.3%.

[0039] Example 4

[0040] Ammonium acetate was selected for the study of co-metabolic biological defluorination of SFC. The colorimetric method was used to detect the biological defluorination of polyfluorinated PFAS. The inoculum was prepared according to Example 2; the degradation system was also prepared according to Example 2, except that ammonium acetate was additionally supplemented as a co-metabolic substrate. The test substrate was 200 μmol / L SFC, and the co-metabolic substance was 5 mmol / L ammonium acetate. The culture solution was cultured at 28 °C and 160 rpm for 1 month. Samples were taken at intervals. 300 μL of the sample was centrifuged, and the supernatant was detected for fluoride ion concentration by the microplate colorimetric method based on the color reaction of fluoride-lanthanide chelate. The test method was the same as that in Example 2.

[0041] Please refer to Figure 4 , the research shows that ammonium acetate can significantly promote the biological defluorination of hexafluoroalkyl compound SFC by Brucella TF-10. The maximum defluorination effect can be basically achieved in the 3rd week. After the culture, the detected fluoride ion concentration in the system is 686.0 μmol / L, and the defluorination rate is 57.2%.

[0042] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0043] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it cannot be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Brucella Brucella sp.)TF-10, characterized in that Brucella ( Brucella sp.)TF-10, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 12, 2024, with the deposit number: CGMCC NO.1.62206.

2. Brucella according to claim 1 ( Brucella sp.)TF-10 for the biodefluorination of 4,5,5-trifluoro-4-pentenoic acid and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid.

3. The use according to claim 2, characterized in that: Brucella ( Brucella sp.)TF-10 for biodefluorination of 4,5,5-trifluoro-4-pentenoic acid and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid in an inorganic salt medium.

4. The use according to claim 3, characterized in that: The inorganic salt culture medium is the inorganic salt culture medium MM-G. The ratio of the inorganic salt culture medium MM-G is: MgSO4·2H2O 0.2 g, CaCl2·2H2O 20 mg, FeSO4·7H2O 10 mg, MnSO4 20 mg, NaNO3 0.7 g, KNO3 0.3 g, NaCl 0.3 g, (NH4)2SO4 1 g, dissolved in 1000 ml of deionized water, and after the culture medium is sterilized, it is added to 20 mmol / L HEPES buffer with pH=7.2.

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