Nocardioides sp. NJF-4 and its application in biodefluorination of fluorinated alkyl compounds

Abstract

The invention discloses Nocardioides sp.NJF‑4 and its application in biological defluorination of fluorinated alkyl compounds. Nocardioides sp.NJF‑4 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on September 12, 2024, with a deposit number of CGMCC NO.4.7760. The obtained strain has the function of breaking C‑F bonds, has biological defluorination effect on 1‑fluorodecane, 4,5,5‑trifluoro‑4‑pentenoic acid, and 4,4,4‑trifluoro‑3‑(trifluoromethyl)crotonic acid, and has a co-metabolism-promoting defluorination effect on hexafluoro PFASs.

Description

Technical Field

[0001] The invention relates to the field of environmental microorganisms, in particular to Nocardioides sp. NJF-4 and its application in biological defluorination of fluorinated alkyl compounds. Background Art

[0002] Perfluorinated and polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals containing carbon-fluorine (CF) bonds. They contain an artificial structure with an alkyl chain as the skeleton and hydrogen atoms completely or partially replaced by fluorine atoms. The chemical structure of PFAS is extremely stable and is internationally known as a "permanent compound." Since 1950, PFAS and its derived polymers and surfactants have been widely used in multiple industrial and commercial fields. These applications include fluoropolymer processing aids, coatings, and aqueous film-forming foams for fire extinguishing. In addition, PFAS is also used in polymer products such as textiles and oil-resistant food contact paper. PFAS causes serious damage to the immune system and reproductive function of mammals. Its widespread use and environmental persistence have made its environmental impact increasingly concerned.

[0003] Fluorine is the smallest halogen element and the atom with the highest electronegativity known so far. The CF bond has a high bond energy (485 kJ / mol), which makes PFAS difficult to be biodegraded after entering the environment. Therefore, this type of substance tends to remain persistently in the environment. The accumulation of this type of substance has been found in soil, water, lake sediments, and even Antarctic ice cores. Compared with other halogen elements, there are very few fluorine-containing organic substances in the natural environment, and artificial PFAS has only recently entered the natural world. At present, the scientific community widely believes that microorganisms in the environment have not yet evolved a biological enzyme system that can effectively deal with CF bonds. Therefore, biological defluorination is currently an international research hotspot. Research on microbial defluorination is of great significance for solving PFAS environmental pollution from a biological perspective. At present, there are no reports on biological defluorination by Nocardia. Summary of the invention

[0004] The object of the present invention is to provide Nocardia sp. NJF-4 and its application in biological defluorination of fluorinated alkyl compounds to solve the above technical problems.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The present invention proposes Nocardioides sp. NJF-4, which was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on September 12, 2024, with the address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the strain classification was named Nocardioides, and the preservation number was: CGMCC NO.4.7760.

[0007] The basic biological characteristics of Nocardioides sp.NJF-4 of the present invention are:

[0008] (1) Colony morphology: milky white, smooth and moist surface;

[0009] (2) Biological and biochemical characteristics: Gram staining is negative and the bacterial morphology is rod-shaped.

[0010] The Nocardioides sp. NJF-4 proposed in the present invention is obtained by screening using the monofluoroalkane 1-fluorodecane as a substrate, and the biological defluorination experiment confirms that the strain has the function of breaking the CF bond.

[0011] It was further demonstrated that Nocardioides sp. NJF-4 had a biodefluorination effect on 1-fluorodecane, 4,5,5-trifluoro-4-pentenoic acid, and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid.

[0012] It is further demonstrated that the cometabolism of hexafluoro PFAS by Nocardioides sp. NJF-4 promotes the defluorination.

[0013] The Nocardioides sp. NJF-4 proposed in 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.

[0014] To further illustrate, the inorganic culture medium is inorganic salt medium MM and inorganic salt medium MM-G.

[0015] To further illustrate, the ratio of inorganic salt medium MM is: MgSO 4 ·2H 2 O 0.2 g, CaCl 2 ·2H 2 O 20mg, FeSO 4 7H 2 O 10 mg, KH 2 PO 4 0.4 g, Na 2HPO 4 0.6 g, MnSO 4 20 mg, NaNO 3 0.8 g, KNO 3 0.3 g, (NH 4 ) 2 SO 4 1 g, dissolved in 1000 ml of deionized water, adjust pH to 7.0-7.2.

[0016] To further illustrate, the ratio of the inorganic salt medium MM-G is: MgSO 4 ·2H 2 O 0.2 g, CaCl 2 ·2H 2 O 20mg, FeSO 4 7H 2 O 10 mg, 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 was dissolved in 1000 ml of deionized water. After the culture medium was sterilized, it was added into 20 mmol / L HEPES buffer at pH 7.2.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention screened a PFAS biological defluorination bacteria strain NJF-4 and identified it as Nocardia, which is the first study on biological defluorination of this strain.

[0019] 2. The strain NJF-4 screened by the present invention has a biological defluorination effect on various fluorinated alkanes such as monofluorinated, trifluorinated, and hexafluorinated substances. Under the condition of the only carbon source, the biological defluorination rate of the bacteria for FD is 70.5%, the defluorination rate for TFE is 11.7%, and the defluorination rate for SFC is 17.8%. Ammonium acetate, as a co-metabolism substrate, can significantly improve the biological defluorination of SFC, and the dechlorination rate is increased to 61.2%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The morphology and phylogenetic tree of strain NJF-4;

[0021] Figure 2 The defluorination of monofluoroalkane FD by strain NJF-4;

[0022] Figure 3 It is the growth defluorination of trifluoroalkane TFE by strain NJF-4;

[0023] Figure 4 It is the growth defluorination of hexafluoroalkane SFC by strain NJF-4;

[0024] Figure 5 This is the cometabolic defluorination effect of strain NJF-4 on SFC. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] The specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0028] Example 1

[0029] Nocardia NJF-4 was screened from contaminated soil of a chemical plant in Suzhou City. The specific steps are as follows:

[0030] (1) Enrichment of FD defluoridation bacteria: 20 g of soil and 50 mL of inorganic salt culture medium MM were placed in a 250 mL Erlenmeyer flask and mixed to make soil slurry. 0.1% (v / v) FD was added and enriched and cultured at 28°C and 160 r / min for 1 month. After the culture, the soil suspension was centrifuged at 1400 rpm for 3 min, and the supernatant was taken for initial enrichment culture. After the culture, 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 defluoridation effect. It should be noted that the inorganic salt culture medium MM component (1 L) is MgSO 4 ·2H 2 O 0.2 g, CaCl 2 ·2H 2 O 20 mg, FeSO 4 7H 2 O 10 mg, KH 2 PO 4 0.4 g, Na 2 HPO 4 0.6 g, MnSO 4 20 mg, NaNO 3 0.8 g, KNO 30.3 g, (NH 4 ) 2 SO 4 1 g, adjust pH 7.0-7.2.

[0031] (2) Initial screening of FD defluorinating bacteria: The supernatant in step (1) was taken for gradient dilution, and 100 μL of the dilution was spread on a double-layer plate with FD as the sole carbon source. The double-layer plate preparation scheme was as follows: the lower plate was MM solid medium containing 1.5% agar, and the upper plate was MM solid medium containing 1% agar (0.2% FD was introduced as a carbon source through acetone, and 0.05% yeast extract was added to promote strain growth). The plate was inverted and cultured at 28°C until a single colony was formed on the plate. The single colony was separated by continuous streaking on the LB plate. The LB medium components (1 L) were 10 g peptone, 5 g yeast extract, 5 g NaCl, pH 7.0.

[0032] (3) Rescreening of FD defluoridation bacteria: Select single colonies of different morphologies in step (2) and culture them in LB liquid medium until OD 600 =0.6-0.8, centrifuge at 5000rpm for 4min to collect the bacterial sludge, wash it three times with inorganic salt culture medium MM, and then suspend the bacteria in equal volumes. Inoculate them into inorganic salt culture medium MM (5mL) with FD (0.1%) 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, refer to step (1) to detect the release of fluoride ions in the culture system.

[0033] (4) Identification of FD defluoridation bacteria: A pure strain with defluoridation effect was selected, and the 16SrRNA gene was amplified by PCR using 27F and 1492R primers. 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 1 shown.

[0034] (5) Defluorination of FD by Nocardia sp. NJF-4: A single colony of Nocardia sp. NJF-4 was selected and the biodefluorination of FD by this strain was studied in the presence of the sole carbon source according to step (3).

[0035] Through the above steps, a highly efficient defluoridation bacterium NJF-4 was screened and obtained. The sequencing results showed that the strain belonged to the genus Nocardia, the colony morphology was milky white, the surface was smooth and moist, the Gram staining was negative, and the bacterial morphology was Figure 1 The strain is shown in rod-like form and is able to efficiently defluorinate the monofluoroalkane FD, see Figure 2 The defluorination efficiency was 70.5% within a one-week culture period, and the defluorination effect was strongest around 3 days.

[0036] Example 2

[0037] 4,5,5-trifluoro-4-pentenoic acid (TFE) was selected for the biodefluorination study of trifluoro PFAS. Based on previous studies, small molecules that strongly affect IC detection of fluoride ions will be produced during the degradation of polyfluorinated PFAS. Therefore, a fluorine-lanthanide chelate-specific colorimetric method was used to study the release of fluoride ions. The specific steps are as follows:

[0038] (1) Preparation of inoculum: Pick a single colony of NJF-4 from the LB plate and culture it in LB liquid medium until the 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 inoculum; it should be noted that the composition of inorganic salt medium MM-G (1 L) is MgSO 4 ·2H 2 O 0.2 g, CaCl 2 ·2H 2 O 20 mg, FeSO 4 7H 2 O 10 mg, 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 culture medium after sterilization, and the components of the LB culture medium were consistent with those of the LB culture medium in Example 1.

[0039] (2) 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 the fluorine-free TFE structural analog trans-2-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.

[0040] (3) Detection of fluoride ions by microplate colorimetric method: To prepare the colorimetric detection solution, 10 μL of acetate buffer (1.68 mol / L, sodium acetate and glacial acetic acid in a ratio of 9:1), 20 μL of alizarin (500 μmol / L, alizarin-3-methyliminodiacetic acid; prepared freshly from 50 mmol / L stock solution), and 20 μL of lanthanum nitrate (500 μmol / L, lanthanum(III) nitrate hexahydrate; prepared freshly from 50 mmol / L stock solution) were sequentially mixed. 100 μL of sample, 50 μL of colorimetric solution, and 50 μL of acetone were sequentially added to the microplate. The colorimetric reaction lasted for more than half an hour. A620 / 530 was calculated using an ELISA reader. A standard curve was established using standard fluoride ion samples in the range of 0-80 μmol / L. The fluoride ion concentration in the sample was calculated using the regression method.

[0041] Studies have shown that Nocardia sp. NJF-4 is able to biodefluorinate trifluoroalkyl compounds TFE, and no fluoride ion release was detected in the fluorine-free structural analogs. Figure 3 The maximum defluorination effect can be achieved in the second week. After the culture, the fluoride ion concentration in the system was detected to be 35.1μmol / L, and the defluorination rate was 11.7%.

[0042] Example 3

[0043] 4,4,4-Trifluoro-3-(trifluoromethyl)crotonic acid (SFC) was selected for the study of biodefluorination of hexafluoro PFAS. This substance is also reported in the literature as a substance that can be biodefluorinated, but there is currently no research on pure culture of microorganisms.

[0044] The study adopted a colorimetric method to detect the biological defluorination of polyfluorinated PFAS. The preparation of Nocardia NJF-4 inoculum and the degradation system was carried out according to Example 2. The test substrate was 200 μmol / L SFC. During the same period, the same concentration of fluorine-free SFC structural analog 3-methylcrotonic acid was added as a fluorine-free blank control. The culture medium was cultured at 28 °C and 160 rpm for 1 month. Samples were taken at intervals during the period. 300 μL of the sample was centrifuged and the supernatant was used to detect the fluoride ion concentration by a microplate colorimetric method based on the colorimetric reaction of fluorine-lanthanide chelates. The test method was the same as in Example 2.

[0045] Studies have shown that Nocardia sp. NJF-4 is able to biodefluorinate hexafluoroalkyl compounds (SFCs), and no fluoride ion release was detected in the fluorine-free structural analogs. Figure 4 The maximum defluorination effect was basically achieved in the third week. After the culture was completed, the fluoride ion concentration in the system was detected to be 213.2 μmol / L, and the defluorination rate was 17.8%.

[0046] Example 4

[0047] Ammonium acetate was selected as the co-metabolism biological defluorination study of SFC. The study used a colorimetric method to detect the biological defluorination effect of polyfluorinated PFAS. The inoculum was carried out with reference to Example 2, and the degradation system was also carried out with reference to Example 2, except that ammonium acetate was additionally supplemented as the co-metabolism substrate. The test substrate was 200 μmol / L SFC, and the co-metabolite was 5 mmol / L ammonium acetate. The culture medium was cultured at 28°C and 160 rpm for 1 month. Samples were taken at intervals during the period, and 300 μL of the sample was centrifuged. The supernatant was used to detect the fluoride ion concentration by a microplate colorimetric method based on the colorimetric reaction of fluorine-lanthanide chelates. The test method was the same as in Example 2.

[0048] Studies have shown that ammonium acetate can significantly promote the biodefluorination of hexafluoroalkyl compounds (SFC) by Nocardia sp. NJF-4. Figure 5 The maximum defluorination effect can be achieved in the third week, and the fluoride ion concentration detected in the system after the culture is 734.9μmol / L.

[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. Nocardioides sp. NJF-4, characterized in that Nocardioides sp. NJF-4 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 12, 2024, with the deposit number: CGMCC NO.4.7760.

2. Use of Nocardioides sp. NJF-4 as described in claim 1 for biological defluorination of fluorinated alkyl compounds, wherein the fluorinated alkyl compounds are 1-fluorodecane, 4,5,5-trifluoro-4-pentenoic acid, and 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid.

3. The use according to claim 2, characterized in that: Nocardioides sp. NJF-4 was inoculated into an inorganic salt medium containing fluorinated alkyl compounds for culture and biological defluorination was achieved.

4. The use according to claim 3, characterized in that: The inorganic culture medium is inorganic salts medium MM and inorganic salts medium MM-G.

5. The use according to claim 4, characterized in that: The ratio of inorganic salt culture medium MM is: MgSO4·2H2O0.2 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, dissolved in 1000 ml of deionized water, and adjusted to pH 7.0-7.

2.

6. The use according to claim 4, characterized in that: The ratio of the inorganic salt culture medium MM-G is: MgSO4·2H2O0.2 g, CaCl2·2H2O 20 mg, FeSO4·7H2O 10 mg, MnSO4 20 mg, NaNO3 0.7 g, KNO3 0.3 g, NaCl 0.3 g, (NH4)2SO4 1 g, dissolved in 1000 ml of deionized water, and after the culture medium is sterilized, it is added to 20 mmol / L HEPES buffer with pH = 7.2.

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