Chryseobacterium TF-12 and its application in biological defluorination

Abstract

The invention discloses a Chryseobacterium Chryseobacterium sp. TF-12 and its application in biological defluorination, the Chryseobacterium Chryseobacterium sp. TF-12 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on October 23, 2024, with the deposit number: CGMCC NO. 1.62207. The strain of the present invention can effectively utilize polyfluoroalkyl compounds (PFASs) and achieve biological defluorination, such as trifluoro-substituted 4,5,5-trifluoro-4-pentenoic acid and hexafluoro-substituted 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid. At the same time, the substitution of fluorine atoms inhibits the growth and utilization of structurally similar substrates by the microorganism.

Description

Technical Field

[0001] The invention belongs to the field of environmental engineering, and specifically relates to Chryseobacterium TF-12 and application thereof in biological defluorination. Background Art

[0002] Perfluorinated and polyfluoroalkyl compounds (PFASs) are a new class of organic pollutants that contain at least -CF2- and -CF3 functional groups. They are called "permanent compounds" because of their extremely stable carbon-fluorine (CF) bonds. The small atomic radius of fluorine enables it to significantly change the physical and chemical properties of PFASs. The low surface tension and polarity of PFASs make them widely used in chemical fields such as surfactants and lubricants. Over the past half century, these compounds have been often used to produce waterproof, oil-proof, and anti-fouling fabrics and carpets, and also as coatings for non-stick cookware. In addition, PFASs are also widely used in firefighting foams, cosmetics, and food packaging materials, becoming one of the key ingredients.

[0003] The CF bond cleavage energy is as high as 485kJ / mol, making PFASs difficult to be biodegraded in the environment. These substances have accumulated in a variety of environmental media such as soil, water, and lake sediments, attracting great attention from environmental workers around the world. Biodefluorination has become a hot topic in international research, especially in predicting whether PFASs will become "permanent pollutants" and developing biodegradable green fluorine chemical products. However, the amount of fluorinated organic matter in the natural environment is extremely small, and artificial PFASs (persistent fluorides) have been in nature for a short time. The scientific community generally believes that microorganisms in the environment have not yet evolved a biological enzyme system that can effectively break CF bonds. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Chryseobacterium with fluoride degradation function. Chryseobacteriumsp. TF-12.

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

[0006] The technical problem to be solved by the present invention is to provide a kind of Chryseobacterium Chryseobacteriumsp. TF-12. Application of the microbial preparation in biological defluorination.

[0007] Technical solution: To solve the above technical problems, the present invention provides a strain of Chryseobacterium Chryseobacteriumsp. TF-12, Chryseobacterium Chryseobacteriumsp.TF-12 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on October 23, 2024, with the deposit number: CGMCC NO.1.62207. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

[0008] The present invention also includes a microbial preparation containing the Chryseobacterium Chryseobacteriumsp. TF-12, its fermentation broth or its supernatant.

[0009] Wherein, the preparation includes a single preparation or a compound preparation, and the preparation type includes a liquid preparation or a solid preparation.

[0010] The present invention also includes the Chryseobacterium Chryseobacteriumsp. TF-12. Application of the microbial preparation in biological defluorination.

[0011] Wherein, the Chryseobacteriumsp. The concentration of TF-12 is 2.8×10 8 ~9.5×10 9 cfu / ml.

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

[0013] The monofluoro compound includes the alkane 1-fluorodecane (FD), and the polyfluoroalkyl compound includes 4,5,5-trifluoropent-4-enoic acid (TFE) and 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC).

[0014] Wherein, the application includes the use of Chryseobacterium Chryseobacteriumsp. TF-12 biodefluorinates mono- or polyfluoroalkyl compounds in an inorganic salt medium.

[0015] Among them, the inorganic salt culture medium includes inorganic salt culture medium MM-F2, and its culture medium composition includes MgSO4·2H2O, CaCl2·2H2O, MnSO4, NaNO3, KNO3, (NH4)2SO4, and HEPES buffer is added to the culture medium in water for sterilization.

[0016] The Chryseobacterium TF-12 of the present invention is obtained by screening with 3-fluoro PFASs (4,5,5-trifluoro-4-pentenoic acid; 4,5,5-trifluoropent-4-enoic acid, TFE) as a substrate. The strain can degrade TFE and 6-fluoro 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid, SFC) under the condition of glucose as co-metabolism; it can degrade the monofluoroalkane 1-fluorodecane (1-fluorodecane, FD) under the condition of the only carbon source. While degrading the substrate, the strain realizes the biological cleavage of the CF bond, but the trifluoro substitution has an inhibitory effect on the growth of the strain.

[0017] The strain screening process of the present invention is as follows: using TFE as a substrate to screen strains, including the following steps: (1) enriching TFE-degrading bacteria: mixing soil from a contaminated area with MM culture medium to make slurry, and adding TFE to promote the enrichment of defluoridation bacteria. (2) screening and identifying TFE defluoridation bacteria: isolating and purifying defluoridation strains on a culture medium with TFE as the sole carbon source, and screening strains with high-efficiency defluoridation capabilities through 16S rRNA gene sequencing and TFE biological defluoridation experiments.

[0018] The application of the strain specifically includes: studying the defluorination effect of the strain on TFE and SFC in the form of co-metabolism, including the following steps: (1) culturing a single colony on Luria-Bertani (LB) medium, collecting the bacteria in the logarithmic growth phase, and washing with MM-F2 medium to obtain an inoculum. (2) Conducting a co-metabolism degradation experiment: adding MM-F2 medium, glucose and the PFASs to be tested to the experimental bottle, inoculating the bacterial solution at a ratio of 10% for constant temperature culture. The culture time for the trifluoro substance TFE is 1 month, the culture time for the hexafluoro substance SFC is 2 months, and the culture time for the monofluoro substance FD is 7 days. Samples are taken regularly during the culture process to test the release of fluoride ions.

[0019] The application of the strain specifically includes: studying the defluorination effect of the strain on FD as the sole carbon source, including the following steps: (1) culturing a single colony on LB medium to the logarithmic growth phase, and washing the bacteria with MM-F2 medium. (2) conducting a sole carbon source degradation experiment: adding MM-F2 medium and FD to the experimental bottle, inoculating the bacterial solution at a ratio of 10% for constant temperature culture. The culture time is 7 days, and regular sampling is performed to test the release of fluoride ions.

[0020] The application of the strain specifically includes: studying the effects of FD, TFE, and SFC on bacterial growth as the sole carbon source, including the following steps: (1) culturing a single colony on LB medium, collecting the bacteria in the logarithmic growth phase, and washing with MM-G medium to obtain an inoculum. (2) Conducting a sole carbon source degradation experiment: adding MM-G medium to an experimental bottle, inoculating FD, TFE, SFC, and their corresponding fluorine-free structural analog substrates (decane, trans-2-pentenoic acid, 3,3-dimethylacrylic acid), inoculating the bacterial solution at a ratio of 10% for constant temperature culture. The culture time of the trifluoro substance TFE and the corresponding 3-fluorine substrate is 1 month, the culture time of the hexafluoro substance SFC and the corresponding 6-fluorine substrate is 2 months, and the culture time of the monofluoro substance FD and the corresponding monofluoro substrate is 7 days. After the culture is completed, the bacterial solution is collected to test the bacterial density.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] (1) The present invention screened a PFASs bio-defluorinating bacterium TF-12 and identified it as Chryseobacterium truncatum. This is the first report on bio-defluorinating activity of this strain.

[0023] (2) The strain TF-12 of the present invention exhibited the ability to biodefluorinate polyfluorinated PFASs such as trifluoromethane and hexafluoromethane. The defluorination rate of TFE was 25.8% (after one month), and the defluorination rate of SFC was 8.95% (2 months). In addition, the strain also had a biodefluorination effect on monofluorinated long-chain alkanes, and the defluorination rate of FD reached 11.3% within seven days (7 days). Cell growth experiments showed that during the defluorination process of TFE, the strain had a certain inhibitory effect on cell growth, while during the biodefluorination process of SFC and FD, cell growth was not significantly affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The morphology and phylogenetic tree of Chryseobacterium TF-12;

[0025] Figure 2 The growth and defluorination of trifluoroalkane TFE by Chryseobacterium TF-12;

[0026] Figure 3 The growth and defluorination of hexafluoroalkane SFC by Chryseobacterium TF-12;

[0027] Figure 4 The growth and defluorination of monofluoroalkane FD by Chryseobacterium TF-12;

[0028] Figure 5 The effects of FD, TFE and SFC biodefluorination on the growth of TF-12 strain. DETAILED DESCRIPTION

[0029] Below in conjunction with accompanying drawings and Examples and test, the present invention is further described in detail, but the present invention is not limited to the following technical scheme. Unless otherwise specified, the reagent, method and equipment adopted in the present invention are conventional reagents, methods and equipment in the art. Those of ordinary skill in the art can implement with reference to various commonly used reference books, scientific and technological literature or relevant specification sheets, manuals etc. before the present invention's application date.

[0030] Example 1 Isolation and identification of TFE defluorinating bacteria

[0031] Chryseobacterium TF-12 was obtained by screening from the contaminated soil of a chemical plant in Suzhou using TFE as a substrate. The specific steps are as follows:

[0032] (1) Enrichment of TFE defluorinating bacteria: 20 g of soil was mixed with 50 mL of MM liquid medium to prepare soil slurry, and a final concentration of 200 μmol L -1 TFE was added and enriched at 28 °C and 160 rpm for 1 month. The composition of MM medium (1L) is: 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, added to 1L water, and adjusted to pH 7.0-7.2.

[0033] (2) Initial screening of TFE defluorinating bacteria: The soil suspension in step (1) was taken for gradient dilution, and 100 μL of the dilution 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 was 3.24 mmol L -1 MM solid medium containing 1% agar in TFE. The plate was inverted and cultured at 28°C until a single colony was formed, and a single colony was separated by continuous streaking on the LB plate. The components of the LB liquid medium (1L) were 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl.

[0034] (3) Rescreening of TFE defluorinating bacteria: Select single colonies of different morphologies in step (2) 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 suspended in equal volumes. Inoculated with TFE (final concentration of 100 μmol L -1 ) in MM medium, glucose (final concentration 100 μmol L-1 ) as a co-metabolism substrate. The culture was carried out at 28 °C and 160 rpm for one month. After the culture was completed, ion chromatography was used to preliminarily determine the release of fluoride ions in the culture system.

[0035] (4) Identification of TFE defluorinating bacteria: Pure strains with defluorinating effects were selected, and 16SrRNA gene was amplified by PCR using primers 27F and 1492R. The amplified products were then sent to the company for sequencing. The sequences were compared using BLAST, and a phylogenetic tree was constructed using MEGA software.

[0036] Through the above steps, a highly efficient defluoridation bacterium TF-12 was screened and obtained. The sequencing results showed that the strain belonged to Chryseobacterium. The colony morphology was golden yellow, with a smooth and sticky surface; Gram staining was negative, and the bacterial morphology was short rod-shaped ( Figure 1 ). Phylogenetic tree analysis of the 16S rRNA gene sequence determined that the strain belonged to Chryseobacterium. Chryseobacteriumsp. TF-12 was deposited on October 23, 2024 at the General Microbiology Center of the China Microbiological Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No. 1.62207.

[0037] Example 2: Co-metabolism of trifluoro PFASs by Chryseobacterium TF-12

[0038] The present invention found that in the process of TFE co-metabolism, the ion chromatography method cannot accurately detect fluoride ions (small molecules interfere with the fluoride ion peak), so a fluorine-lanthanide chelate-specific colorimetric method was subsequently used to conduct biological defluorination research.

[0039] The specific steps are as follows:

[0040] (1) Preparation of inoculum: Pick a single TF-12 colony and culture it in LB medium until OD 600 = 0.6~0.8, centrifuge at 5000rpm for 4 min to collect the bacterial sludge, wash it three times and resuspend the bacteria in an equal volume of MM-F2 liquid culture medium. The strain concentration is 2.8*108cfu / mL.

[0041] The components of MM-F2 liquid culture medium (1L) are MgSO4·2H2O 0.2 g, CaCl2·2H2O 20 mg, MnSO420 mg, NaNO30.7 g, KNO30.3 g, (NH4)2SO41 g, added to 1L of water. After the culture medium is sterilized, HEPES (pH=7.2) is added with a final concentration of 20mmol / L.

[0042] (2) Cometabolic degradation system: The bacterial liquid was inoculated into MM-F2 medium with a final concentration of 100 μmol / L TFE at a 10% inoculation ratio, and glucose with a final concentration of 5 mmol / L was added as a cometabolic substrate. At the same time, a fluorine-free TFE-similar substrate (trans-2-pentenoic acid) with a final concentration of 100 μmol / L was added as a blank control. The culture medium was cultured at 28 °C and 160 rpm for 30 days and samples were taken at intervals. After the sample was centrifuged, 100 μL of the sample was taken and the fluoride ion concentration in the culture medium was detected by colorimetric method.

[0043] (3) Detection of fluoride ions by microplate colorimetric method: Preparation of colorimetric detection solution: Mix 10 μL of acetate buffer (pH 5.2, 1.68 mol / L), 20 μL of alizarin (50 μmol / L), and 20 μL of lanthanum nitrate (50 μmol / L). In a 96-well microplate, add 100 μL of sample, 50 μL of colorimetric detection solution, and 50 μL of acetone in sequence. After half an hour of reaction, obtain A620 / A530 using an ELISA reader. A standard curve was established for standard fluoride ion samples in the range of 0-80 μmol / L, and the fluoride ion concentration in the sample was calculated by regression method.

[0044] The study showed that Chryseobacterium TF-12 was able to biodefluorinate TFE, while no fluoride release was detected in trans-2-pentenoic acid. During the one-month incubation period, the maximum fluoride release in the culture medium occurred on the 21st day, releasing 77.4 μmol / L fluoride ions, and the TFE defluorination rate reached 25.8% ( Figure 2 ), among which the defluorination rate was the fastest on the 7th day.

[0045] Example 3: Biodefluorination of hexafluoro PFASs by co-metabolism of Chryseobacterium TF-12

[0046] The preparation of the inoculum of Chryseobacterium TF-12 and the cometabolism degradation system was carried out according to Example 2. The test substrate was 200 μmol / L SFC, and the cometabolism substrate was 5 mmol / L glucose. During the same period, 200 μmol / L of the fluorine-free SFC structural analog 3,3-dimethylacrylic acid was added as a fluorine-free blank control. The culture medium was cultured at 28°C and 160 rpm for 60 days, with samples taken at intervals during the period. After the sample was centrifuged, the supernatant was used to detect the release of fluoride ions in the system by a colorimetric reaction, and the test method was the same as in Example 2. Figure 3The results showed that Chryseobacterium TF-12 was able to perform cometabolic biofluorination on SFC, while no fluoride release was detected in 3,3-dimethylacrylic acid. During the 2-month incubation period, the maximum release occurred on the 45th day, releasing 107.4 μmol / L fluoride ions, and the SFC defluorination rate was 8.95%, with the fastest defluorination rate on the 30th day.

[0047] Example 4: Biodefluorination of monofluorinated long-chain alkanes FD by Chryseobacterium TF-12 as the sole carbon source

[0048] The inoculum of Chryseobacterium TF-12 was carried out according to Example 2, and 1 mmol / L monofluoroalkane 1-fluorodecane (FD) was added as the sole carbon source. The culture medium was cultured at 28 °C and 160 rpm for 7 days. Samples were collected on the 5th and 7th days, and the supernatant was taken after centrifugation to detect the release of fluoride ions by colorimetric method. The test method was the same as that in Example 2. The study showed that Chryseobacterium TF-12 was able to biologically defluorinate FD under the sole carbon source situation. On the 7th day, the fluoride ion release was 113 μmol / L, and the FD defluorination rate was 11.3% ( Figure 4 ).

[0049] Example 5: Effects of FD, TFE, and SFC biodefluorination on the growth of Chryseobacterium TF-12 cells

[0050] The preparation of the inoculum of Chryseobacterium TF-12 and the sole carbon source degradation system was carried out according to Example 4. The culture medium used MM-G medium, and the final concentrations of the test substrates were 1 mmol / L FD (monofluoride), 1 mmol / L decane, 100 μmol / LTFE (3 fluoride), 100 μmol / L trans-2-pentenoic acid, 200 μmol / L SFC (6 fluoride) and 200 μmol / L 3,3-dimethylacrylic acid, respectively. No co-metabolism substrate was provided in the culture system. The culture solution was cultured at 28 °C and 160 rpm for 7 days (monofluoride), 30 days (3 fluoride), and 60 days (6 fluoride), respectively. After the culture was completed, 1 mL of the bacterial solution was collected to test the bacterial density ( OD 600 ). Among them, the inorganic salt medium MM-G, whose components (1L) are 0.2 g MgSO42H2O, 20 mg CaCl22H2O, 10 mg FeSO47H2O, 20 mg MnSO4H2O, 1 g (NH4)2SO4, 0.7 g NaNO3, 0.7 g KNO3, 0.3 g NaCl, is added to 1L water, and HEPES (pH=7.2) with a final concentration of 20 mmol / L is added after the medium is sterilized.

[0051] The study showed that these substances can support the growth of Chryseobacterium TF-12 cells in MM-G medium at inoculation concentration ( OD 600 ) was 0.06, the cell concentration could reach about 2 times higher after the culture. Among them, the biological defluorination of FD and SFC did not affect cell growth, while the trifluoro substance TFE had a certain inhibitory effect on cell growth ( Figure 5 ).

Claims

1. A strain of Chryseobacterium Chryseobacteriumsp. TF-12, characterized in that Chryseobacterium Chryseobacteriumsp. TF-12 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 23, 2024, with the deposit number: CGMCC NO. 1.62207.

2. A microbial preparation, characterized in that: It contains the Chryseobacterium described in claim 1 Chryseobacteriumsp. TF-12 or its fermentation broth.

3. The microbial preparation according to claim 2, characterized in that The preparation includes a single preparation or a compound preparation, and the preparation type includes a liquid preparation or a solid preparation.

4. Chryseobacterium according to claim 1 Chryseobacteriumsp. TF-12. Use of the microbial preparation according to claim 2 or 3 in biological defluorination, wherein the biological defluorination is the defluorination of monofluoro compounds or polyfluoroalkyl compounds, the monofluoro compound is the alkane 1-fluorodecane, and the polyfluoroalkyl compound is 4,5,5-trifluoro-4-pentenoic acid or 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid.

5. The use according to claim 4, characterized in that: Chryseobacterium Chryseobacteriumsp. The concentration of TF-12 is 2.8×10 8 ~9.5×10 9 cfu / mL.

6. The use according to claim 4, characterized in that: The application comprises the following steps: Chryseobacteriumsp. TF-12 biodefluorinates mono- or polyfluoroalkyl compounds in an inorganic salt medium.

7. The use according to claim 6, characterized in that: The inorganic salt culture medium includes the inorganic salt culture medium MM-F2, and its culture medium composition includes MgSO4·2H2O, CaCl2·2H2O, MnSO4, NaNO3, KNO3, (NH4)2SO4, which are added into water, and HEPES buffer is added after the culture medium is sterilized.

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