Microbacterium TF-9 and Its Application in Biological Defluorination

Abstract

The present invention discloses a strain of Microbacterium ( Microbacterium Microbacterium sp.) TF-9 and its application in biological defluorination. The Microbacterium ( Microbacterium Microbacterium sp.) TF-9 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on April 3, 2025, with the deposit number: CGMCC NO. 1.64745. The strain of the present invention was obtained by screening using trifluorinated 4,5,5-trifluoro-4-pentenoic acid as a substrate, and can utilize and achieve defluorination of various organic fluorides such as 1-fluorodecane, 4,5,5-trifluoro-4-pentenoic acid, and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid. The substitution of fluorine atoms significantly inhibits the utilization of trifluorinated substrates by TF-9.

Description

Technical Field

[0001] The present invention belongs to the field of environmental engineering, and relates to Microbacterium TF-9 and its application in biological defluorination. Specifically, it relates to a strain of Microbacterium TF-9 capable of biological defluorination of trifluorinated PFAS and its application in biological defluorination of other PFAS. Background Art

[0002] Per- and polyfluoroalkyl substances (PFAS) are a class of emerging organic pollutants that contain at least -CF2- and -CF3 functional groups and are known as "forever chemicals" due to their extremely stable carbon-fluorine (C-F) bonds. The small atomic radius of fluorine enables it to significantly alter the physical and chemical properties of PFAS. The low surface tension and polarity of PFAS have led to their widespread use in chemical industries such as surfactants and lubricants. Over the past half century, these compounds have often been used in the production of waterproof, oil-proof, and stain-resistant fabrics and carpets, as well as coatings for non-stick cookware. In addition, PFAS are also widely used in fire-fighting foams, cosmetics, and food packaging materials and have become one of the key components.

[0003] The high bond dissociation energy of the C-F bond, up to 485 kJ / mol, makes PFAS difficult to be biodegraded in the environment. These substances have accumulated in various environmental media such as soil, water bodies, and lake bottom sediments, attracting great attention from environmental scientists worldwide. Biological defluorination has become a hot topic in international research, especially in predicting whether PFAS will become "permanent pollutants" and developing biodegradable green fluorochemical products, which has important scientific research significance. However, the number of fluorine-containing organic compounds in the natural environment is extremely small, and the time for artificial PFAS to enter the natural world is relatively short. The scientific community generally believes that microorganisms in the environment have not yet evolved a biological enzyme system capable of effectively breaking the C-F bond. Therefore, screening for new strains with fluorine degradation function has extremely important practical significance. Summary of the Invention

[0004] Object of the Invention: The technical problem to be solved by the present invention is to provide a strain of Microbacterium ( Microbacterium sp. ) TF-9 with fluorine degradation function.

[0005] Another technical problem to be solved by the present invention is to provide a microbial preparation.

[0006] The last technical problem to be solved by the present invention is to provide the application of the above-mentioned Microbacterium ( Microbacterium sp. ) TF-9 or the above-mentioned microbial preparation in biological defluorination.

[0007] Technical Solution: To solve the above technical problems, the present invention provides a strain of Microbacterium ( Microbacterium sp. ) TF-9, and the Microbacterium ( Microbacterium sp.)(TF-9) was deposited with the China General Microbiological Culture Collection Center on April 3, 2025, with the deposit number: CGMCC NO. 1.64745, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also includes a microbial preparation, which contains the Microbacterium ( Microbacterium sp. ), TF-9, its fermentation broth or its supernatant.

[0009] Among them, the preparation includes a single-agent or compound preparation, and the preparation type includes a liquid preparation or a solid preparation.

[0010] The present invention also includes the application of the Microbacterium ( Microbacterium sp. ), TF-9 and the microbial preparation in biological defluorination.

[0011] Among them, the concentration of the Microbacterium ( Microbacterium sp. ), TF-9 is 1.2×10 8 ~ 3.2×10 8 cfu / mL.

[0012] Among them, the biological defluorination includes defluorination of monofluoro compounds or polyfluoroalkyl compounds.

[0013] Among them, the monofluoro compound includes alkane 1-fluorodecane (FD), and the polyfluoroalkyl compound includes 4,5,5-trifluoro-4-pentenoic acid (TFE) or 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid (SFC).

[0014] Among them, the application includes subjecting the Microbacterium ( Microbacterium sp. ), TF-9 to biological defluorination of monofluoro compounds or polyfluoroalkyl compounds in an inorganic salt medium.

[0015] Among them, the application includes biological defluorination of FD in the form of the sole carbon source or defluorination of TFE or SFC in the form of co-metabolism.

[0016] Among them, the defluorination of TFE or SFC in the form of co-metabolism includes adding glucose as a co-metabolic substrate.

[0017] Among them, the inorganic salt medium includes inorganic salt medium MM-F2 or inorganic salt medium MM-G. Among them, the composition of inorganic salt medium MM-F2 includes MgSO4·2H2O, CaCl2·2H2O, MnSO4, NaNO3, KNO3, (NH4)2SO4. Add them to water, and after the medium is sterilized, add HEPES buffer; among them, the composition of inorganic salt medium MM-G includes MgSO4·2H2O, CaCl2·2H2O, FeSO4·7H2O, MnSO4·H2O, (NH4)2SO4, NaNO3, KNO3, NaCl. Add them to water, and after the medium is sterilized, add HEPES buffer.

[0018] The content of the present invention also includes screening for defluorinating bacterium TF-9 using TFE as a substrate, including the following steps: (1) Enrichment of TFE-degrading bacterial flora; (2) Screening and identification of TFE defluorinating bacteria.

[0019] The content of the present invention also includes studying the defluorination effect of strains on TFE or SFC in the form of co-metabolism, including the following steps: (1) Culturing single colonies of Microbacterium TF-9 in LB medium to obtain an inoculum. (2) In an experimental flask containing MM-F2 medium, perform PFAS biodegradation test using glucose as a co-metabolic substrate.

[0020] The content of the present invention also includes a method for biological defluorination of FD in the form of a sole carbon source, including the following steps: (1) Culturing single colonies of Microbacterium TF-9 in LB medium to obtain an inoculum; (2) In an experimental flask containing MM-F2 medium, perform PFAS biodegradation test using FD as the sole carbon source.

[0021] The Microbacterium TF-9 of the present invention is screened and obtained using 4,5,5-trifluoropent-4-enoic acid (TFE) as a substrate. Through in vivo biological defluorination experiments, it is clear that this strain has the function of breaking the C-F bond; verifying the defluorination effect of this strain on 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC); verifying the biological defluorination effect of this strain on 1-fluorodecane (FD).

[0022] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention screens a PFAS biological defluorinating bacterium Microbacterium for the first time ( Microbacterium sp.)TF-9, identified as Microbacterium, is the first report of this type of strain for biological defluorination so far.

[0024] (2)The strain TF-9 of the present invention has a biological defluorination effect on various organic fluorides such as monofluoro, trifluoro, and hexafluoro. The defluorination rate of the strain for FD is 10.2% on the 7th day, the defluorination rate for TFE is 18.0% within 14 days, and the defluorination rate for SFC is 9.4% on the 45th day. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a morphological and phylogenetic tree diagram of Microbacterium TF-9;

[0026] Figure 2 It is a result diagram of the biological defluorination effect of Microbacterium TF-9 on trifluorocarboxylic acid TFE;

[0027] Figure 3 It is a result diagram of the biological defluorination effect of Microbacterium TF-9 on hexafluorocarboxylic acid SFC;

[0028] Figure 4 It is a result diagram of the biological defluorination effect of Microbacterium TF-9 on monofluoroalkane FD;

[0029] Figure 5 Result diagram of the influence of different organic fluorides on the growth of Microbacterium TF-9. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present invention will be further described in detail below with reference to the drawings, embodiments and experiments, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Those of ordinary skill in the art can implement it with reference to various commonly used reference books, scientific and technological literature or relevant specifications and manuals before the filing date of the present invention application.

[0031] Example 1 Isolation and Identification of Microbacterium TF-9

[0032] (1)The soil sample is from the polluted soil of a chemical plant in Suzhou City (soil depth > 6 meters).

[0033] (2)Enrichment of defluorinating bacteria: In a 250 mL Erlenmeyer flask, 20 g of soil was mixed with 50 mL of MM medium to prepare a soil slurry. 200 μM of TFE was added, and enrichment culture was carried out at 28 °C and 160 rpm for 1 month. The components of MM (1 L) were 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 the pH was adjusted to 7.0 - 7.2.

[0034] (3)Primary screening of defluorinating bacteria: The soil suspension in step (2) was taken and serially diluted. The diluted solution was spread on a double-layer plate with TFE as the sole carbon source. The lower layer of the double-layer plate was MM solid medium containing 1.5% agar, and the upper layer plate was MM solid medium containing 1% agar with 3.24 mM TFE as the sole carbon source. The plate was inverted and cultured at 28 °C until single colonies formed. Single colonies were picked for streak isolation.

[0035] (4)Rescreening and identification of defluorinating bacteria: Different-shaped single colonies were selected from step (3) and cultured in LB liquid medium until OD 600 OD = 0.6 - 0.8. The bacterial sludge was collected by centrifugation at 5000 rpm for 4 minutes, washed three times with MM medium, and then suspended in an equal volume of MM medium as the inoculum. The inoculum was inoculated into MM medium containing 100 μM TFE at an inoculation ratio of 10%, and 5 mM of glucose was added as a co-metabolic substrate. The culture was carried out at 28 °C and 160 rpm for one month. After the culture was completed, the release of fluoride ions in the culture system was preliminarily evaluated by ion chromatography. Pure strains with defluorination effects were selected and sent to Shanghai Majorbio Co., Ltd. for 16S rRNA gene sequencing, and a phylogenetic tree was constructed using MEGA software.

[0036] Through the above steps, a highly efficient defluorinating bacterium TF-9 was screened. The 16S rRNA sequencing results are shown in SEQ ID NO.1, and comparison on NCBI indicated that this strain belongs to the genus Microbacterium. The colony morphology was light yellow, the surface was moist, and the edge was regular; Gram staining was positive, and the cell morphology was rod-shaped ( Figure 1 ). This strain was deposited in the China General Microbiological Culture Collection Center on April 3, 2025. Its taxonomic name is Microbacterium sp. , and the deposit number is: CGMCC No. 1.64745. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101.

[0037] Example 2 Biological defluorination of trifluorinated PFAS by Microbacterium TF-9

[0038] TFE was selected for the study of biological defluorination of trifluorinated PFAS, and the fluoride ion release was tested by a fluorine-lanthanide chelate specific colorimetric method.

[0039] (1)Preparation of inoculum: Pick a single colony of TF-9 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-F2 medium, and then resuspend the cells with an equal volume of MM-F2 medium as the inoculum. The components of MM-F2 medium (1L) are 0.2 g of MgSO4·2H2O, 20 mg of CaCl2·2H2O, 20 mg of MnSO4, 0.7 g of NaNO3, 0.3 g of KNO3, 1 g of (NH4)2SO4. After sterilization of the medium, 20 mM HEPES (pH = 7.2) is added.

[0040] (2)Cometabolic degradation system: Inoculate the bacterial solution from step (1) into 5 mL of MM-F2 medium containing 4,5,5-trifluoropent-4-enoic acid (TFE) (100 μM) at an inoculation ratio of 10% (v / v), and add 5 mM glucose as the cometabolic substrate. The fluorine-free TFE structural analog trans-2-pentenoic acid at the same concentration is used as the fluorine-free blank control. Incubate at 28°C and 160 rpm for 30 days, and take 1 mL of culture medium samples on days 0, 7, 14, 21, and 28 of the culture. After centrifugation of the samples, take 100 μL of the supernatant to detect the fluoride ion release.

[0041] (3)Detection of fluoride ions by microplate colorimetry: Mix 10 μL of acetate buffer (1.68 M, pH 5.2), 20 μL of alizarin (50 μM), and 20 μL of lanthanum nitrate (50 μM) in sequence. In the microplate, add 100 μL of the supernatant treated in step 2, 50 μL of the colorimetric solution, and 50 μL of acetone in sequence. The color reaction lasts for more than half an hour, and record A 620 and A 530 by a microplate reader, and calculate A 620 / A 530Ratio. Detection of fluoride ion concentration based on the fluorine-lanthanide chelate microplate colorimetric method (refer to the method for detecting fluoride ions in the Chinese patent application with the publication number CN118909876A).

[0042] The results are as Figure 2 shown. Microbacterium TF-9 can biodefluorinate trifluoroalkyl carboxylic acid TFE, and no fluoride ions were detected in the experiment without fluorine analogs. The maximum defluorination effect can be achieved on the 14th day of cultivation. The detected fluoride ion concentration in the system is 54 μM, and the defluorination rate is 18.0%. The defluorination effect is the strongest on the 7th day.

[0043] Example 3 Biodefluorination of hexafluorinated PFAS by Microbacterium TF-9

[0044] 4,4,4-Trifluoro-3-(trifluoromethyl)crotonic acid (SFC) was selected for the study of biodefluorination of hexafluorinated PFAS, and the colorimetric method was used to detect the biodefluorination of polyfluorinated PFAS. The preparation of the Microbacterium TF-9 inoculum and the co-metabolic degradation system was referred to Example 2. The test substrate was 200 μM SFC, and the fluorine-free SFC structural analog 3-methylcrotonic acid (3,3-dimethylacrylic acid) was used as the fluorine-free blank control. The culture solution was cultured at 28 °C and 160 rpm for 60 days, and samples were taken at intervals. After centrifugation of the samples, the supernatant was used for the colorimetric reaction to detect the release of fluoride ions in the system, and the test method was the same as that in Example 2.

[0045] The results are as Figure 3 shown. Microbacterium TF-9 can biodefluorinate the hexafluoroalkyl compound SFC, and no fluoride ions were detected in the fluorine-free analogs. The best defluorination effect was achieved on the 45th day. The detected fluoride ion concentration in the system was 112.8 μM, and the defluorination rate was 9.4%. The defluorination effect was the strongest on the 14th day ( Figure 3 ).

[0046] Example 4 Biodefluorination of monofluoro long-chain alkane FD by Microbacterium TF-9

[0047] The Microbacterium TF-9 inoculum was referred to Example 2, and 1 mM monofluoroalkane FD was added as the sole carbon source. The culture solution was cultured at 28 °C and 160 rpm for 7 days. After centrifugation of the samples, the supernatant was taken and the colorimetric method was used to detect the release of fluoride ions, and the test method was the same as that in Example 2. The results are as Figure 4 shown. Microbacterium TF-9 can biodefluorinate the monofluoro long-chain alkane FD, and no fluoride ion release was detected in the fluorine-free analogs. During the cultivation period, the best defluorination effect was achieved on the 7th day. The detected fluoride ion concentration in the system was 102 μM, and the defluorination rate was 10.2%. The defluorination effect was the strongest on the 3rd day.

[0048] Example 5 Effect of Organofluorides on the Growth of Microbacterium TF-9 Cells

[0049] The preparation of the Microbacterium TF-9 inoculum and the sole carbon source degradation system was carried out with reference to Example 4. The medium used was MM-G medium, and the final concentrations of the test substrates were 1 mM FD (monofluoro), 1 mM decane, 100 μM TFE (trifluoro), 100 μM trans-2-pentenoic acid, 200 μM SFC (hexafluoro), and 200 μM 3,3-dimethylacrylic acid, and no co-metabolic substrate was provided in the culture system. The culture solution was cultured at 28 °C and 160 rpm for 7 days (monofluoro), 30 days (trifluoro), and 60 days (hexafluoro) respectively. After the culture was completed, 1 mL of the bacterial solution was collected to measure the cell density ( OD 600 ). Among them, the inorganic salt medium MM-G, its components (1 L) were 0.2 g MgSO4·2H2O, 20 mg CaCl2·2H2O, 10 mg FeSO4·7H2O, 20 mg MnSO4·H2O, 1 g (NH4)2SO4, 0.7 g NaNO3, 0.7 g KNO3, 0.3 g NaCl, added to 1 L of water. After the medium was sterilized, HEPES with a final concentration of 20 mM (pH = 7.2) was added.

[0050] The results were as Figure 5 shown. In the MM-G medium, TFE and SFC could support the growth of Microbacterium TF-9 cells. When the inoculation concentration ( OD 600 ) was 0.06, the cell concentration could reach about 2 times or more after the culture was completed. Among them, SFC had no effect on cell growth, while the trifluoro substance TFE had a significant inhibitory effect on cell growth ( Figure 5 ).

Claims

1. A Microbacterium TF-9, characterized in that, The Microbacterium TF-9 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on April 3, 2025, with the deposit number: CGMCC NO. 1.64745.

2. A microbial preparation, characterized in that, It contains the Microbacterium TF-9 or its fermentation broth described in claim 1.

3. The microbial agent according to claim 2, wherein The preparation includes a single-agent or compound preparation, and the preparation types include liquid preparations or solid preparations.

4. Use of the Microbacterium TF-9 described in claim 1 and the microbial preparation described in claim 2 or 3 in biological defluorination, wherein the biological defluorination includes defluorination of monofluoro compounds or polyfluoroalkyl compounds, the monofluoro compounds include 1-fluorodecane, and the polyfluoroalkyl compounds include 4,5,5-trifluoro-4-pentenoic acid or 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid.

5. The application according to claim 4, characterized in that, The concentration of the Microbacterium TF-9 is 1.2×10 8 ~3.2×10 8 cfu / mL.

6. The application according to claim 4, characterized in that, The application includes biodefluorination of monofluoro compounds or polyfluoroalkyl compounds by Microbacterium ( Microbacterium sp. ) TF-9 in an inorganic salt medium.

7. The application according to claim 6, characterized in that, The use includes defluorination of 1-fluorodecane in the form of the sole carbon source or defluorination of 4,5,5-trifluoro-4-pentenoic acid or 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid in the form of co-metabolism.

8. The application according to claim 7, characterized in that, The defluorination of 4,5,5-trifluoro-4-pentenoic acid or 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid in the form of co-metabolism includes adding glucose as a co-metabolic substrate.

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