Ensifer sp. NJF-2 of the genus Ensifer and its application in degrading polyfluoroalkyl compounds

Abstract

The present invention discloses Ensifer sp. NJF-2 capable of biodefluorinating polyfluoroalkyl compounds. Ensifer sp. NJF-2 was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 12, 2024, and the deposit number is: CGMCC NO. 1.19397. The application of this strain in biodefluorination is also disclosed. In vivo and in vitro transformation experiments show that this bacterium can achieve biodefluorination of various PFAS such as monofluoro, trifluoro, and hexafluoro.

Description

Technical Field

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

[0002] Per- and polyfluoroalkyl substances (PFAS) are a class of organic compounds containing carbon-fluorine (C-F) bonds, which at least contain -CF2- and -CF3 functional groups. Their physicochemical properties are extremely stable and they are called "forever chemicals". Since the 1950s, PFAS have been widely used in industrial production such as firefighting, textile, and electroplating, as well as daily products. Research shows that PFAS have potential toxicity and carcinogenicity to animals, plants, and human health, and have become a new type of organic pollutant attracting global attention. Due to the high dissociation energy of the C-F bond, the biodegradation of PFAS in the environment is extremely difficult, and they have been detected in various environmental media such as water, soil, and air. Military bases, fluorochemical industrial areas, and airports using aqueous film-forming foam fire extinguishing agents are the main pollution sources of PFAS. After these compounds are discharged into the soil, they can seep into groundwater through leakage, ultimately affecting drinking water sources and seriously threatening human health and ecological safety. The microbial defluorination of PFAS has become a hot topic in international research. In-depth 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 time in nature is short and environmental microorganisms cannot effectively degrade the C-F bond. Currently, the research on biological defluorination mainly focuses on the mechanism by which microorganisms convert precursor substances into PFAS, but the understanding of its cleavage of the C-F bond is still insufficient. Although the previous research on polyfluoro- and perfluoro-PFAS of pure bacteria has not made substantial progress, recently, some researchers have started from monofluoro substances and discovered biological defluorinating bacteria for low-fluorinated alkanes such as Rhodococcus sp. NJF-7 and Pseudomonas sp. 273. This study reports a new monofluoro defluorinating bacterium NJF-2, which can not only defluorinate monofluoroalkanes but also effectively defluorinate polyfluoro-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] To solve the above technical problems, the present invention adopts the following technical solutions to achieve:

[0005] The present invention provides Ensifer sp. NJF-2. Ensifer sp. NJF-2 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (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 postal code 100101. The strain is classified and named as Ensifer, and the deposit number is: CGMCC NO. 1.19397.

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

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

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

[0009] Ensifer sp. NJF-2 proposed in the present invention was screened using 1-fluorodecane (FD), a monofluoroalkane, as a substrate, and it was confirmed through in vivo biological defluorination experiments that this strain can effectively break the C-F bond.

[0010] Furthermore, Ensifer sp. NJF-2 has significant biological defluorination ability for 4,5,5-trifluoro-4-pentenoic acid (TFE) and 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC) in PFAS.

[0011] Even further, the research used resting cells for in vitro conversion experiments to confirm the biological defluorination effect of this strain on FD, TFE, and SFC.

[0012] Ensifer sp. NJF-2 proposed in the present invention was inoculated into an inorganic salt medium of fluorinated alkyl compounds and cultured at 28 °C and 160 r / min to achieve biological defluorination.

[0013] Furthermore, the inorganic salt medium is MM medium and MM-F2 medium.

[0014] Even further, the formulation of MM medium is as follows: 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, dissolved in 1000 ml of deionized water, and the pH is 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 are dissolved in 1000 ml of deionized water. After sterilizing the culture medium, it is added to a 20 mmol / L HEPES buffer solution with a pH of 7.2.

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

[0017] 1. The present invention screens a PFAS biodefluorination bacterium NJF-2, which is identified as the genus Ensifer, and this is the first report on the biodefluorination of this strain.

[0018] 2. The strain NJF-2 screened by the present invention has a biodefluorination effect on various fluorinated alkane substances such as monofluoro, trifluoro, and hexafluoro. Under a specific culture period, the defluorination rate of FD is 71.4%, the defluorination rate of TFE is 18.0%, and the defluorination rate of SFC is 13.2%. The defluorination rate of resting cells for FD is 26.4%, the defluorination rate of TFE is 8.86%, and the defluorination rate of SFC is 5.53%, indicating that the constitutive expression enzyme system of this strain has the function of biotransformation and defluorination. Description of the Drawings

[0019] Figure 1 It is the morphology and phylogenetic tree of Ensifer sp. NJF-2;

[0020] Figure 2 It is the defluorination effect of Ensifer sp. NJF-2 on monofluoroalkane FD;

[0021] Figure 3 It is the defluorination effect of Ensifer sp. NJF-2 on trifluoroalkane TFE;

[0022] Figure 4 It is the defluorination effect of Ensifer sp. NJF-2 on hexafluoroalkane SFC

[0023] Figure 5 It is the defluorination effect of NJF-2 resting cells on FD, TFE, and SFC. Detailed Embodiments

[0024] To facilitate the understanding of 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 shown 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.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

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

[0027] Example 1

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

[0029] (1) Enrichment of FD defluorination flora: 20 g of soil was mixed with 50 mL of MM medium in a 250 mL Erlenmeyer flask to prepare soil slurry, and 0.1% (v / v) of FD was added. 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-based filter membrane, and ion chromatography (IC) was used to detect the fluoride ion release to judge the biological defluorination effect. Among them, the components of the MM medium (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.

[0030] (2) Primary screening of FD defluorination bacteria: The above enrichment solution was aspirated, gradient diluted and then spread on a double-layer plate with monofluoroalkane 1-fluorodecane (FD) as the sole carbon source. The preparation scheme of the double-layer plate was as follows: The lower layer plate was pre-laid with MM solid medium containing 1.5% agar, and the upper layer plate was MM solid medium containing 1% agar. 0.2% of FD was introduced as a carbon source through acetone in the upper layer plate, and 0.05% yeast extract was added as a co-metabolic substrate. It was incubated at 28 °C in an inverted position until single colonies were formed, and the single colonies were continuously streaked and isolated on an LB plate. Among them, the components of the LB medium (1 L) were 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl.

[0031] (3) Re-screening of FD defluorination bacteria: Select single colonies with different morphologies in the above steps, and culture them in LB liquid medium until OD 600 = 0.6 - 0.8. The bacterial sludge was collected by centrifugation at 5000 rpm for 4 min, washed three times with MM medium and then suspended in the same volume of bacterial cells. According to the inoculation ratio of 10%, it was inoculated into MM medium with 5 mmol / L FD as the sole carbon source, and cultured at 28 °C and 160 rpm for 7 days. After the culture was completed, IC was used to detect the fluoride ion release in the culture system.

[0032] (4) Identification of FD-fluoride-removing bacteria: The 16S rRNA gene was amplified by PCR using primers 27F and 1492R. The amplification product was sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for sequencing. After the sequence was aligned by BLAST, a phylogenetic tree was constructed using MEGA software. The phylogenetic tree is as shown in Figure 1 .

[0033] (5) Fluoride removal effect of fluoride-removing bacteria NJF-2 on FD: A single colony of NJF-2 was picked, and the biological fluoride removal effect of this strain on FD under the condition of the sole carbon source was studied with reference to step (3).

[0034] Through the above steps, it was found that 1.98 mmol / L of fluoride was released in the enrichment system, approximately equivalent to a fluoride removal rate of 31.7%. The fluoride-removing bacterium NJF-2 was screened from the enrichment solution. Sequencing showed that it belongs to the genus Ensifer, as shown in Figure 1 . The colony morphology of this strain is milky white, with a smooth and moist surface and an edge that is easy to disperse. Please refer to Figure 2 . NJF-2 can effectively remove fluoride from the monofluoroalkane 1-fluorodecane (FD). The fluoride removal efficiency is 71.4% within a one-week culture cycle, and the fluoride removal effect is the strongest around 3 days.

[0035] Example 2

[0036] TFE was selected for the study of biological defluorination of trifluorinated PFAS. Based on previous literature reports, this substance can be co-metabolized and biologically defluorinated by the activated sludge microbial community. However, there is currently no research on the biological defluorination of pure cultures of such substances. The study found that during the co-metabolism of polyfluorinated PFAS, because some metabolites would interfere with the detection of fluoride ions by IC, a specific colorimetric method based on fluorine-lanthanide chelate was used for the study of biological defluorination. The specific steps are as follows:

[0037] (1) Preparation of inoculum: A single colony of NJF-2 was picked and cultured in LB liquid medium until OD 600 = 0.6 - 0.8. The bacterial sludge was collected by centrifugation at 5000 rpm for 4 min, washed with MM-F2 medium, and the cells were resuspended with an equal volume of MM-F2. The components of MM-F2 (1 L) 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, and 1 g of (NH4)2SO4. After the medium was sterilized, 20 mmol / L HEPES (pH = 7.2) was added.

[0038] (2) TFE degradation system: Inoculate the bacterial liquid from step (1) at an inoculation ratio of 10%. The degradation system is carried out in MM-F2 medium, and 100 μmol / L TFE and 5 mmol / L glucose are added respectively. Meanwhile, 100 μmol / L of the fluorine-free structural analog 4-pentenoic acid is added as a blank control. Incubate at 28 °C and 160 rpm for 30 days. During the incubation period, samples are taken at intervals. After centrifugation of the samples, 100 μL of the sample is taken and the fluoride ion concentration in the culture solution is detected by the colorimetric method.

[0039] (3) Detection of fluoride ions by colorimetric method: Sequentially mix 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) to prepare the colorimetric detection solution. In a 96-well microplate, add 100 μL of the sample, 50 μL of the colorimetric solution, and 50 μL of acetone. After reacting for half an hour, obtain A620 / A530 through an enzyme-linked immunosorbent assay reader. A standard curve is established for the standard fluoride ion sample in the range of 0 - 80 μmol / L, and the fluoride ion concentration in the sample is calculated by the regression method.

[0040] Please refer to Figure 3 , Pseudomonas sp. NJF-2 can biodefluorinate the trifluoroalkyl compound TFE, and no fluoride ion release is detected in 4-pentenoic acid. During the 1-month incubation period, the fluoride ion release in the medium is 54 μmol / L, and the TFE defluorination rate reaches 18.0%. The defluorination rate is the fastest on the 7th day.

[0041] Example 3

[0042] SFC was selected for the study of the biodefluorination of hexafluorinated PFAS, and the colorimetric method was used to detect the biodefluorination of polyfluorinated PFAS. The preparation of the defluorinating bacteria NJF-2 inoculum and the degradation system refers to Example 2. The test substrate is 200 μmol / L SFC, the co-metabolite is 5 mmol / L glucose, and 3,3-dimethylacrylic acid is added as a fluoride-free blank control. Incubate at 28 °C and 160 rpm for 60 days. Samples are taken at intervals during this period, and the fluoride ions in the system are detected with reference to Example 2. The results show that, please refer to Figure 4 , Pseudomonas sp. NJF-2 can biodefluorinate SFC, and no fluoride ion release is detected in 3,3-dimethylacrylic acid. During the 2-month incubation period, the SFC defluorination rate is 13.2%. The defluorination rate is the fastest around the 14th day.

[0043] Example 4

[0044] Study the biodefluorination of FD, TFE, and SFC by NJF-2 resting cells. The steps are as follows:

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

[0046] (2) Resting cell experiment: Pre-add 50 μL of the test PFAS into a 96-deep well plate. For FD, it is introduced with acetone, and for TFE and SFC, they are added through sterile water. Take 950 μL of the bacterial solution from step (1) and add it to the deep well plate. After covering the plate with the lid, place it horizontally on a shaker and incubate at a constant temperature for 96 hours. The usage concentrations of FD, TFE, and SFC are 1 mmol / L, 100 μmol / L, and 200 μmol / L respectively.

[0047] (3) After the culture is completed, detect fluoride ions in the detection system with reference to Example 2.

[0048] Please refer to Figure 5 , research shows that the defluorination enzyme of Pseudomonas NJF-2 belongs to constitutive expression, and in vitro experiments can defluorinate various PFAS. The defluorination efficiency is generally consistent with the in vivo transformation experiment. Among them, the defluorination rate in the FD system is 26.4%, the defluorination rate in the TFE system is 8.9%, and the defluorination rate in the SFC system is 5.5%.

[0049] 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 embodiments are described. However, as long as these technical feature combinations do not conflict, they should be considered as the scope described in this specification.

[0050] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting 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 invention patent should be subject to the appended claims.

Claims

1. Ensifer sp. NJF-2, characterized in that, Ensifer sp. NJF-2 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 12, 2024, with the deposit number: CGMCC NO. 1.19397.

2. The application of Ensifer sp. NJF-2 described in claim 1 in the biological defluorination of 1-fluorodecane, 4,5,5-trifluoro-4-pentenoic acid, and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid.

3. The application according to claim 2, characterized in that, Ensifer sp. NJF-2 performs biological defluorination in an inorganic salt medium.

4. The application according to claim 3, characterized in that The inorganic salt medium is MM medium and MM-F2 medium.

5. The application according to claim 4, wherein Using MM medium, the defluorination effect of Ensifer sp. NJF-2 on 1-fluorodecane was studied.

6. The application according to claim 4, characterized in that, Using MM-F2 medium, the defluorination effect of Ensifer sp. NJF-2 on 4,5,5-trifluoro-4-pentenoic acid and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid was studied.

7. The application according to claim 5, wherein The formulation of MM medium is as follows: 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, dissolved in 1000 ml of deionized water, and the pH was adjusted to 7.0 - 7.

2.

8. The application according to claim 6, wherein The formulation of MM-F2 medium is as follows: 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, dissolved in 1000 ml of deionized water, and after sterilizing the medium, it was added to 20 mmol / L HEPES buffer solution with pH = 7.2.

Images