Ensifer sp.NJF-2 and application of Ensifer sp.NJF-2 in degradation of polyfluoroalkyl compounds

Abstract

The invention discloses an ensifer sp.NJF-2 capable of performing biological defluorination on a polyfluoroalkyl compound, the ensifer sp.NJF-2 is preserved in China General Microbiological Culture Collection Center on September 12, 2024, and the preservation number is CGMCC NO.1. 19397. The invention also discloses an application of the strain in the aspect of biological defluorination, in-vivo and in-vitro transformation experiments show that the ensifer sp.NJF-2 can perform biological defluorination on the polyfluoroalkyl compound, and the ensifer sp.NJF-2 can perform biological defluorination on the polyfluoroalkyl compound. The strain can achieve the biological defluorination effect on various PFAS such as monofluorine, trifluorine, hexafluorine and the like.

Description

Technical Field

[0001] The invention relates to the field of environmental microorganisms, in particular to Ensifer sp. NJF-2 and application thereof in degrading polyfluoroalkyl compounds. Background Art

[0002] Perfluorinated and polyfluoroalkyl substances (PFAS) are a class of organic compounds containing carbon-fluorine (CF) bonds, at least -CF2- and -CF3 functional groups. Their physical and chemical properties are extremely stable and they are called "permanent compounds". Since the 1950s, PFAS has been widely used in industrial production such as firefighting, textiles and electroplating, as well as daily life products. Studies have shown that PFAS is potentially toxic and carcinogenic to animals, plants and human health, and has become a new type of organic pollutant of global concern. Due to the high dissociation energy of the CF bond, PFAS is extremely difficult to biodegrade in the environment and has been detected in a variety of environmental media such as water, soil and air. Military bases, fluorine chemical industrial parks and airports using aqueous film-forming foam firefighting agents are the main sources of PFAS pollution. After these compounds are discharged into the soil, they can enter the groundwater through leakage, eventually affecting drinking water sources and seriously threatening human health and ecological safety. Microbial defluorination of PFAS has become an international research hotspot, and a deep understanding of its biological defluorination mechanism is of great significance for evaluating whether PFAS will cause "permanent pollution". However, the biological defluorination of PFAS faces major challenges. The scientific community generally believes that its existence in nature is short and environmental microorganisms cannot effectively degrade CF bonds. At present, research on biological defluorination mainly focuses on the mechanism by which microorganisms convert precursor substances into PFAS, but there is still insufficient understanding of their breaking CF bonds. Although previous studies on pure bacteria for polyfluorinated and perfluorinated PFAS have not made substantial progress, researchers have recently started with monofluorinated substances and discovered biological defluorination bacteria for low-fluorinated alkanes such as Rhodococcus sp.NJF-7 and Pseudomonas sp.273. This study reports a new monofluorinated defluorinating bacterium NJF-2, which can not only defluorinate monofluorinated alkanes, but also effectively defluorinate polyfluorinated PFAS. Summary of the invention

[0003] The purpose of the present invention is to provide Ensifer sp. NJF-2 and its application in degrading polyfluoroalkyl compounds to solve the above technical problems.

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

[0005] The present invention proposes Ensifer sp. NJF-2, which was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Committee 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 is named Ensifer (Ensifer), and the preservation number is: CGMCC NO.1.19397.

[0006] The basic biological characteristics of Ensifer sp. NJF-2 screened by the present invention are as follows:

[0007] (1) Colony morphology: milky white, smooth and moist surface, and easy to diffuse at the edge;

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

[0009] The Ensifer sp. NJF-2 proposed in the present invention is obtained by screening using monofluoroalkane 1-fluorodecane (FD) as a substrate, and an in vivo biological defluorination experiment confirms that the strain can effectively break the CF bond.

[0010] It is further shown that Ensifer sp. NJF-2 has significant biodefluorination ability for 4,5,5-trifluoro-4-pentenoic acid (TFE) and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid (SFC) in PFAS.

[0011] Further, the study used resting cells for in vitro transformation experiments, confirming the biodefluorination effect of the strain on FD, TFE and SFC.

[0012] The Ensifer sp. NJF-2 proposed 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.

[0013] To further illustrate, the inorganic salt culture medium is MM culture medium and MM-F2 culture medium.

[0014] To further illustrate, the ratio of MM culture medium is: MgSO4·2H2O 0.2g, CaCl2·2H2O 20mg, FeSO4·7H2O 10mg, KH2PO40.4g, Na2HPO40.6g, MnSO420mg, NaNO30.8g, KNO30.3g, (NH4)2SO41g, dissolved in 1000ml of deionized water, and the pH was adjusted to 7.0-7.2.

[0015] To further illustrate, 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, and 1 g of (NH4)2SO4 were dissolved in 1000 ml of deionized water, and after the culture medium was sterilized, it was added to 20 mmol / L HEPES buffer with a pH of 7.2.

[0016] The beneficial effects of the present invention are:

[0017] 1. The present invention screened a PFAS biological defluorination bacteria strain NJF-2 and identified it as the genus Xylocystis. This is the first report on biological defluorination of this strain.

[0018] 2. The strain NJF-2 screened by the present invention has a biological defluorination effect on a variety of fluorinated alkanes such as monofluorinated, trifluorinated, and hexafluorinated. Under a specific culture cycle, the FD defluorination rate is 71.4%, the TFE defluorination rate is 18.0%, and the SFC defluorination rate is 13.2%. The resting cells have a FD defluorination rate of 26.4%, a TFE defluorination rate of 8.86%, and a SFC defluorination rate of 5.53%, indicating that the strain constitutively expresses an enzyme system that has a biological transformation defluorination function. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The morphology and phylogenetic tree of NJF-2 of the genus Xiphium;

[0020] Figure 2 The defluorination of monofluoroalkane FD by NJF-2 of the genus Xiphium;

[0021] Figure 3 The defluorination of trifluoroalkane TFE by NJF-2 of the genus Xiphium;

[0022] Figure 4 Defluorination of hexafluoroalkane SFC by NJF-2 of Xylocystis sp.

[0023] Figure 5 Defluorination of FD, TFE and SFC by NJF-2 resting cells. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] The specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0027] Example 1

[0028] NJF-2 of the genus Xylocystis was isolated from the contaminated soil (depth > 6 meters) of a chemical plant in Suzhou City. The specific steps are as follows:

[0029] (1) FD defluorination bacteria enrichment: 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 the mixture was enriched and cultured at 28°C and 160 rpm for 1 month. After the culture was completed, the supernatant was filtered through a 0.22 μm water filter membrane, and ion chromatography (IC) was used to detect the release of fluoride ions to determine the biological defluorination effect. The components of 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 7.0-7.2.

[0030] (2) Preliminary screening of FD defluorinating bacteria: The above enrichment solution was taken, and after gradient dilution, it was coated on a double-layer plate with monofluoroalkane 1-fluorodecane (FD) as the sole carbon source. The double-layer plate preparation scheme was as follows: the lower plate was pre-spread with MM solid medium with 1.5% agar, and the upper plate contained MM solid medium with 1% agar. 0.2% FD was introduced into the upper plate through acetone as a carbon source, and 0.05% yeast extract was added as a co-metabolism substrate. Inverted culture was carried out at 28°C until a single colony was formed, and single colonies were separated by continuous streaking on the LB plate, wherein the LB medium components (1L) were 10g of tryptone, 5g of yeast extract, and 5g of NaCl.

[0031] (3) Rescreening of FD defluoridation bacteria: Single colonies with different morphologies in the above steps were selected and cultured in LB liquid medium until OD 600 =0.6-0.8, collect the bacterial sludge by centrifugation at 5000rpm for 4min, wash three times with MM medium and then suspend the bacteria in equal volumes, inoculate them into MM medium with 5mmol / L FD as the sole carbon source at a 10% inoculation ratio, and culture them at 28℃ and 160rpm for 7 days. After the culture is completed, IC is used to detect the release of fluoride ions in the culture system.

[0032] (4) Identification of FD defluorinating bacteria: 16S rRNA gene was amplified by PCR using primers 27F and 1492R. The amplified product was sent to Shanghai Meiji Company for sequencing. After BLAST comparison, the phylogenetic tree was constructed using MEGA software. The phylogenetic tree is shown in Figure 2. Figure 1 shown.

[0033] (5) Defluorination effect of defluorinating bacteria NJF-2 on FD: Pick a single colony of NJF-2 and refer to step (3) to study the biological defluorination effect of this strain on FD under the condition of sole carbon source.

[0034] Through the above steps, it was found that 1.98mmol / L of fluorine was released in the enrichment system, which was equivalent to a defluorination rate of approximately 31.7%. Defluorinating bacteria NJF-2 was screened from the enrichment solution, and sequencing showed that it belonged to the genus Swordbacterium. Figure 1 As shown, the colony morphology of this strain is milky white, with a smooth and moist surface and easy diffusion at the edge. Figure 2 NJF-2 can effectively defluorinate the monofluoroalkane 1-fluorodecane (FD), with a defluorination efficiency of 71.4% within a one-week culture period, and the defluorination effect is the strongest around 3 days.

[0035] Example 2

[0036] TFE was selected for the biodefluorination study of trifluoro PFAS. Based on previous literature reports, this substance can be biodefluorinated by cometabolism of activated sludge microbial communities, but there is currently no research on the biodefluorination of pure cultures of such substances. The study found that in the process of cometabolism of polyfluorinated PFAS, some metabolites will interfere with the detection of fluoride ions by IC. Therefore, a specific color development method based on fluorine-lanthanide chelates was used for biodefluorination research. The specific steps are as follows:

[0037] (1) Preparation of inoculum: Pick a single colony of NJF-2 and culture it in LB liquid medium until OD 600 =0.6-0.8, collect the bacterial sludge by centrifugation at 5000rpm for 4min, wash with MM-F2 medium, and resuspend the bacteria in an equal volume of MM-F2. The components of MM-F2 (1L) are MgSO4·2H2O 0.2g, CaCl2·2H2O 20mg, MnSO420mg, NaNO30.7g, KNO30.3g, (NH4)2SO41g. After the culture medium is sterilized, 20mmol / L HEPES (pH=7.2) is added.

[0038] (2) TFE degradation system: The bacterial solution of step (1) was inoculated at a 10% inoculation ratio. The degradation system was carried out in MM-F2 medium, and 100 μmol / L TFE and 5 mmol / L glucose were added respectively. At the same time, 100 μmol / L of the fluorine-free structural analog 4-pentenoic acid was added as a blank control. The culture was cultured at 28°C and 160 rpm for 30 days. Samples were taken at intervals during the culture period. After the samples were centrifuged, 100 μL of the samples were taken and the fluoride ion concentration in the culture medium was detected by colorimetric method.

[0039] (3) Colorimetric detection of fluoride ions: 10 μL of acetate buffer (1.68 mol / L), 20 μL of alizarin (50 μmol / L), and 20 μL of lanthanum nitrate (50 μmol / L) were mixed in sequence to prepare a colorimetric detection solution. 100 μL of sample, 50 μL of colorimetric solution, and 50 μL of acetone were added to a 96-well microplate. After half an hour of reaction, A620 / A530 was obtained by an ELISA reader. A standard curve was established for standard fluoride samples in the range of 0-80 μmol / L, and the fluoride ion concentration in the sample was calculated by regression method.

[0040] See also Figure 3 The results showed that NJF-2 of the genus Xanthoceras was able to biodefluorinate the trifluoroalkyl compound TFE, while no fluoride ion release was detected in 4-pentenoic acid. During the 1-month incubation period, the fluoride ion release in the culture medium was 54 μmol / L, and the TFE defluorination rate reached 18.0%, with the fastest defluorination rate on the 7th day.

[0041] Example 3

[0042] SFC was selected for the study of biodefluorination of hexafluoro PFAS, and the colorimetric method was used to detect the biodefluorination of polyfluoro PFAS. The preparation of the defluorination bacteria NJF-2 inoculum and the degradation system was carried out according to Example 2. The test substrate was 200 μmol / L SFC, the co-metabolite was 5 mmol / L glucose, 3,3-dimethylacrylic acid was added as a fluorine-free blank control, and the culture was carried out at 28°C and 160 rpm for 60 days. Samples were taken at intervals during the period, and the fluoride ions in the system were detected according to Example 2. The results showed that Figure 4 The Xanthophyll sp. NJF-2 was able to biodefluorinate SFC, and no fluoride ion release was detected in 3,3-dimethylacrylic acid. During the 2-month incubation period, the SFC defluorination rate was 13.2%, with the fastest defluorination rate around the 14th day.

[0043] Example 4

[0044] The biodefluorination of FD, TFE and SFC by NJF-2 resting cells was studied as follows:

[0045] (1) Preparation of NJF-2 inoculum: Pick a single NJF-2 colony 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 with MM-F2 medium, and resuspend the bacteria in an equal volume of MM-F2.

[0046] (2) Resting cell experiment: 50 μL of the PFAS to be tested was pre-added to a 96-well deep-well plate, of which FD was introduced using acetone, and TFE and SFC were added using sterile water. 950 μL of the bacterial solution in step (1) was added to the deep-well plate. After the plate was covered, it was placed horizontally on a shaker and cultured at a constant temperature for 96 hours. The concentrations of FD, TFE and SFC were 1 mmol / L, 100 μmol / L and 200 μmol / L, respectively.

[0047] (3) After the incubation, the fluoride ions in the system were detected according to Example 2.

[0048] See also Figure 5 The study showed that the defluorination enzyme NJF-2 of the genus Xiphium was constitutively expressed and could defluorinate a variety of PFAS in in vitro experiments. The defluorination efficiency was generally consistent with the in vivo conversion experiment. The defluorination rate was 26.4% in the FD system, 8.9% in the TFE system, and 5.5% in the SFC system.

[0049] 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.

[0050] 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. Ensifer sp. NJF-2, characterized in that Ensifer sp. NJF-2 was deposited in the General Microbiology Center of China Culture Collection Administration on September 12, 2024, with the deposit number: CGMCCNO.1.19397.

2. Application of Ensifer sp. NJF-2 as claimed in claim 1 for biodefluorination of various fluorinated alkyl compounds.

3. The use according to claim 2, characterized in that: The fluorinated alkyl compounds are 1-fluorodecane, 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: Ensifer sp. NJF-2 was used for biological defluorination in inorganic salt medium.

5. The use according to claim 4, characterized in that: The inorganic salt culture medium is MM medium and MM-F2 medium.

6. The use according to claim 5, characterized in that: The defluorination of 1-fluorodecane by Ensifer sp. NJF-2 was studied using MM medium.

7. The use according to claim 5, characterized in that: The defluorination of 4,5,5-trifluoro-4-pentenoic acid and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid by Ensifer sp. NJF-2 was studied using MM-F2 medium.

8. The use according to claim 6, characterized in that: The ratio of MM culture medium is: MgSO4·2H2O0.2g, CaCl2·2H2O 20mg, FeSO4·7H2O 10mg, KH2PO40.4 g, Na2HPO40.6 g, MnSO420 mg, NaNO30.8g, KNO30.3 g, (NH4)2SO41 g, dissolved in 1000ml of deionized water, and adjusted to pH 7.0-7.

2.

9. The use according to claim 7, characterized in that: The ratio of MM-F2 culture medium is: MgSO4·2H2O0.2g, CaCl2·2H2O 20mg, MnSO420mg, NaNO30.7g, KNO30.3g, (NH4)2SO41g, dissolved in 1000ml of deionized water. After the culture medium is sterilized, it is added to 20mmol / L HEPES buffer with pH=7.2.

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