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

Abstract

The invention discloses Brucella sp. TF-10 and application thereof to degradation of polyfluorinated PFAS (perfluoro-perfluoro-aspartic acid), the Brucella sp. TF-10 is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 12, 2024, and the preservation number is CGMCC NO.1.62206. The screened strain can utilize PFAS such as 4, 5, 5-trifluoro-4-pentenoic acid and 4, 4, 4-trifluoro-3-(trifluoromethyl) crotonic acid to realize biological defluorination, and the Brucella sp. TF-10 can be applied to degradation of polyfluorinated PFAS. And the biological defluorination efficiency of the hexafluoro PFAS under the co-metabolism condition is obviously improved.

Description

Technical Field

[0001] The present invention relates to the field of environmental microorganisms, and in particular to Brucella sp. TF-10 and application thereof in degrading polyfluorinated PFAS. Background Art

[0002] Per- and polyfluoroalkyl compounds (PFAS) have attracted much attention due to their stable carbon-fluorine (CF) bonds, specifically compounds containing at least perfluoromethyl (-CF3) or methylene (-CF2-). The introduction of fluorine gives PFAS excellent surface activity, hydrophobicity and stability, making it widely used in many industrial fields such as textiles, leather, electroplating, petroleum, mining, aerospace and semiconductors. In addition, PFAS is 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 ecosystems, and becoming the focus of global environmental risks. The CF bond has a high dissociation energy, and PFAS exhibits extremely strong chemical stability, which makes it difficult to degrade in the environment. Due to its persistence, long-distance migration and bioaccumulation, PFAS is widely present in soil, atmosphere, water and sediments, and has been detected in plants, animals and humans. Researchers around the world are working on the mechanism of microbial defluorination, hoping to mitigate the environmental hazards of PFAS by biodegrading CF bonds and exploring the potential of microbial defluorination, which is of great practical significance for the development of biodegradable fluorinated chemical products. At present, research on biological defluorination is still in its infancy, and most of the evidence for defluorination comes from data on microbial communities in environmental samples. There are few reports on pure strains capable of defluorination. This study screened a strain of Brucella TF-10 with biological defluorination function. According to literature retrieval and research, this is the first report on biological defluorination of Brucella. Summary of the invention

[0003] The purpose of the present invention is to provide Brucella sp. TF-10 and its application in degrading polyfluorinated PFAS to solve the above-mentioned technical problems.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] The present invention proposes Brucella sp. TF-10, which was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on September 12, 2024, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the strain classification was named Brucella (Brucella), and the preservation number was: CGMCCNO.1.62206.

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

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

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

[0009] The Brucella sp. TF-10 screened by the present invention is obtained by screening with trifluoro PFAS as a substrate. The strain can degrade trifluoro 4,5,5-trifluoro-4-pentenoic acid (TFE) and hexafluoro 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC) while achieving the breaking of CF bonds in the case of a sole carbon source.

[0010] The Brucella sp. TF-10 screened by the present invention has a defluorination promoting effect on the cometabolism of hexafluoro PFAS.

[0011] The Nocardioides sp. NJF-4 screened by the present invention is inoculated into an inorganic salt culture medium of a fluorinated alkyl compound, cultured at 28° C. and 160 r / min to achieve biological defluorination.

[0012] The inorganic medium in the present invention is an inorganic salt medium MM-G, and the ratio of the inorganic salt medium MM-G is: MgSO 4 ·2H 2 O 0.2g, CaCl 2 ·2H 2 O 20mg, FeSO 4 7H 2 O 10mg, MnSO 4 20 mg, NaNO 3 0.7 g, KNO 3 0.3 g, NaCl 0.3 g, (NH 4 ) 2 SO 41 g was dissolved in 1000 ml of deionized water, and after sterilization of the culture medium, it was added into 20 mmol / L HEPES buffer at pH 7.2.

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

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

[0015] 2. The Brucella TF-10 screened by the present invention has a biological defluorination effect on PFAS such as trifluoromethane and hexafluoromethane. Under the condition of the only carbon source, the defluorination rate of TFE by the bacteria is 14.4%, and the defluorination rate of SFC is 23.3%. Ammonium acetate is used as a co-metabolism substrate to significantly increase the SFC dechlorination rate to 57.2%. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0018] Figure 3 for the growth and defluorination of hexafluoroalkane SFC by strain TF-10;

[0019] Figure 4 This is the cometabolic defluorination effect of strain TF-10 on SFC. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more fully 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, the purpose of providing these embodiments is 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 those commonly understood by those skilled in the art of the present invention. 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 are described below with reference to the accompanying drawings.

[0023] Example 1

[0024] Brucella TF-10 was used to screen the contaminated soil of a chemical plant in Suzhou. The specific steps are as follows:

[0025] (1) Enrichment of defluorinated bacteria: 20 g of soil and 50 mL of MM culture medium were mixed in a 250 mL Erlenmeyer flask to prepare soil slurry, 0.1% (v / v) FD was added, and enrichment culture was carried out 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 taken as the initial enrichment culture, wherein the MM component (1 L) was MgSO 4 ·2H 2 O 0.2g, CaCl 2 ·2H 2 O20mg, FeSO 4 7H 2 O 10mg, KH 2 PO 4 0.4 g, Na 2 HPO 4 0.6 g, MnSO 4 20 mg, NaNO 3 0.8 g, KNO 3 0.3 g, (NH 4 ) 2 SO 4 1g, adjust pH 7.0-7.2.

[0026] (2) Initial screening of defluoridation bacteria: The soil suspension in step (1) was taken for gradient dilution, and the dilutions were 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 The MM solid medium containing 1% agar in TFE was inverted and cultured at 28°C until a single colony was formed. The single colony was separated by continuous streaking on the LB plate. The components of the LB medium (1 L) were 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl.

[0027] (3) Rescreening of defluorinated bacteria: Select single colonies of different morphologies in step (2) and culture them in LB liquid medium until OD 600 The value reached 0.6-0.8, then the culture solution was centrifuged at 5000 rpm for 4 minutes, the bacterial sludge was collected, and washed three times with MM medium. Finally, the washed bacteria were suspended in an equal volume of liquid. Then, the suspension was inoculated with 100 μmol L -1 In the MM medium containing TFE, 100 μmol L -1 Glucose was used as the co-metabolism substrate and cultured at 28°C and 160rpm for one month. After the culture was completed, ion chromatography was used to preliminarily evaluate the release of fluoride ions in the culture system.

[0028] (4) Identification of Defluorinated Bacteria: 16S rRNA gene was amplified by PCR using primers 27F and 1492R. The amplified product was then sent to Shanghai Meiji Company for sequencing. Finally, the obtained sequences were aligned by BLAST and a phylogenetic tree was constructed using MEGA software. The phylogenetic tree is shown in Figure 1 shown.

[0029] Through the above steps, a highly efficient defluoridation bacterium TF-10 was screened and obtained. The sequencing results showed that the strain belonged to Brucella. Figure 1 The colony morphology is light yellow, the surface is flat and moist, Gram staining is negative, and the bacterial body morphology is spherical.

[0030] Example 2

[0031] 4,5,5-trifluoro-4-pentenoic acid (TFE) was selected for the study of biodefluorination of trifluoro PFAS. Based on literature reports, this substance can be biodefluorinated by cometabolism of activated sludge microbial communities. Currently, there is no study on the biodefluorination of this substance by pure microbial culture. It should be noted that previous studies have found that the process of cometabolism of polyfluorinated PFAS by this substance generally produces small molecules that strongly affect the IC detection of fluoride ions. Therefore, a specific color development method based on fluorine-lanthanide chelates was used to study the biodefluorination of polyfluorinated PFAS. The specific steps are as follows:

[0032] (1) Preparation of inoculum: Pick a single TF-10 colony from the LB plate and culture it 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 inorganic salt medium MM-G, and then resuspended with an equal volume of inorganic salt medium MM-G as an inoculum, wherein the inorganic salt medium MM-G component (1 L) is MgSO 4 ·2H 2 O0.2g, CaCl 2 ·2H 2 O 20mg, FeSO 4 7H 2 O10mg, MnSO 4 20 mg, NaNO 3 0.7 g, KNO 3 0.3 g, NaCl 0.3 g, (NH 4 ) 2 SO 4 1 g, 20 mmol / L HEPES (pH = 7.2) was added to the sterilized culture medium, and the LB culture medium components (1 L) were tryptone 10 g, yeast extract 5 g, and NaCl 5 g.

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

[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 as the cometabolism biodefluorination study of SFC, and the study used a colorimetric method to detect the biodefluorination of polyfluorinated PFAS. The inoculum was carried out with reference to Example 2; the degradation system was also carried out with reference to Example 2, except that ammonium acetate was additionally supplemented as the cometabolism substrate, the test substrate was 200 μmol / L SFC, the cometabolism substance was 5 mmol / L ammonium acetate, the culture medium was cultured at 28°C and 160 rpm for 1 month, and samples were taken at intervals during the period. 300 μL of the sample was centrifuged, and the supernatant was detected for fluoride ion concentration by a microplate colorimetric method based on the colorimetric reaction of fluorine-lanthanide chelates, and the test method was the same as Example 2.

[0041] See also Figure 4 The study showed that ammonium acetate can significantly promote the biological defluorination of hexafluoroalkyl compounds SFC by Brucella TF-10, and the maximum defluorination effect can be basically achieved in the third week. After the culture, the fluoride ion concentration detected in the system was 686.0 μmol / L, and the defluorination rate was 57.2%.

[0042] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, 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, they should be considered to be within the scope of this specification.

[0043] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Brucella sp. TF-10, characterized in that Brucella (Brucellas.) TF-10 was 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. Application of Brucella sp. TF-10 described in claim 1 to biological defluorination of polyfluorinated PFAS.

3. The use according to claim 2, characterized in that: The fluorinated alkyl compounds are 4,5,5-trifluoro-4-pentenoic acid and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid.

4. The use according to claim 3, characterized in that: Brucella sp. TF-10 biodefluorinates polyfluorinated PFAS in the inorganic salt medium MM-G.

5. The use according to claim 4, characterized in that: The ratio of the inorganic salt culture medium MM-G is: MgSO4·2H2O0.2g, CaCl2·2H2O 20mg, FeSO4·7H2O 10mg, MnSO420mg, NaNO30.7g, KNO30.3g, NaCl0.3g, (NH4)2SO41g, dissolved in 1000ml of deionized water, and after the culture medium is sterilized, it is added to 20mmol / L HEPES buffer with pH=7.2.

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