Achromobacter sp. TF-6 and application thereof in degrading fluorinated alkyl compound

Abstract

The invention discloses Achromobacter sp. TF-6 and application of the Achromobacter sp. TF-6 in degradation of fluorinated alkyl compounds, the Achromobacter sp. TF-6 is preserved in China General Microbiological Culture Collection Center on September 12, 2024, Institute of Microbiology, Chinese Academy of Sciences, the postal code is 100101, and the preservation number of the Achromobacter sp. TF-6 is CGMCC No. 1.62204. The Achromobacter sp. TF-6 can be used for degrading fluorinated alkyl compounds. The strain disclosed by the invention can be used for carrying out biological defluorination on 4, 5, 5-trifluoro-4-pentenoic acid or 4, 4, 4-trifluoro-3-(trifluoromethyl) crotonic acid, and the biological defluorination efficiency of the strain on the 4, 4, 4-trifluoro-3-(trifluoromethyl) crotonic acid is remarkably improved under a co-metabolism condition.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental engineering, and in particular to a non-chromogenic bacillus Achromobacter sp. TF-6 and its application in degrading fluorinated alkyl compounds. BACKGROUND

[0002] Per- and polyfluoroalkyl substances (PFASs) are chemically stable and non-degradable due to the presence of -CF2- and -CF3 functional groups, and have been widely used in consumer and industrial products such as non-stick cookware, raincoats, camera film, and fire-fighting foam for nearly a century. Their persistence in the environment has led to their being referred to as "permanent compounds". As research into the environmental behavior and biological toxicity of PFASs has deepened, governments around the world have become aware of their health risks. These substances enter the human body through the water and food chain, and are detected in almost all organisms, despite the increasing regulatory efforts, there are still thousands of related compounds on the market.

[0003] The high electronegativity of fluorine atoms in PFASs significantly increases the dissociation energy of C-F bonds, and as the number of F substitutions increases, the dissociation energy of C-C and C-F bonds is further enhanced, exacerbating their chemical inertness. Current global research focuses on microbial defluorination, aiming to alleviate environmental pollution by analyzing the biological degradation mechanism of C-F bonds, which is crucial for predicting the "permanent pollutant" properties of PFASs, developing degradable fluorinated products, and developing bioremediation technologies. However, there are very few reports of existing biological defluorination microorganisms, and in particular, there is no published literature or patent disclosing the defluorination function of non-chromogenic bacillus. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a non-chromogenic bacillus with fluorine degradation function Achromobacter sp. TF-6 and related applications of the bacteria.

[0005] To solve the above technical problems, the following technical solutions are adopted: The present application provides a non-chromogenic bacillus Achromobacter sp. TF-6, non-chromogenic bacillus Achromobacter sp. TF-6 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on September 12, 2024, at address No. 3, Beichen West Road, Haidian District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101, and the strain preservation number is CGMCC No. 1.62204.

[0006] The present application also includes a microbial preparation containing the non-chromogenic bacillus Achromobacter sp. TF-6 or its fermentation broth, specifically, the preparation includes single or compound preparations, and the preparation types include liquid preparations or solid preparations.

[0007] The present application also includes the non-chromogenic bacillusAchromobacter sp. TF-6 and the microbial preparation are applied to biological defluorination of polyfluoroalkyl compounds, in particular, 4,5,5-trifluoro-4-pentenoic acid or 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid.

[0008] The present application also particularly studies the Achromobacter Achromobacter sp. TF-6 supplements ammonium acetate as a co-metabolic substrate to perform co-metabolic defluorination on 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid.

[0009] The present application provides an Achromobacter Achromobacter sp. TF-6 performs biological defluorination on polyfluoroalkyl compounds in an inorganic salt culture medium.

[0010] The inorganic salt culture medium is inorganic salt culture medium MM-G, and components of the inorganic salt culture medium MM-G include MgSO4·2H2O 0.2 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, and are dissolved in 1000 ml of deionized water, and after sterilization of the culture medium, 20 mmol / L of HEPES buffer with a pH of 7.2 is added.

[0011] The Achromobacter TF-6 of the present application is screened by taking 3-fluoro PFAS (4,5,5-trifluoropent-4-enoic acid, TFE) as a substrate, the strain can degrade 3-fluoro TFE and 6-fluoro 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC) under the condition of a unique carbon source, and can effectively improve the biological defluorination efficiency of SFC under the condition that ammonium acetate is a co-metabolic substrate.

[0012] The strain screening process is: TFE is used as a substrate for screening, including the following steps: (1) enriching TFE degrading bacteria: mixing contaminated site soil and MM culture medium to prepare mud slurry, and adding TFE to enrich defluorination bacteria; (2) screening and identifying TFE defluorination bacteria: separating and purifying defluorination bacteria in a MM solid plate with TFE as the only carbon source, and using 16S rRNA gene sequencing and TFE biological defluorination experiment to determine the strain with high defluorination effect.

[0013] The application of the strain specifically includes: studying the defluorination of the strain on TFE and SFC, including the following steps: (1) culturing a single colony in Luria-Bertani (LB) medium, collecting the logarithmic growth phase bacteria, and obtaining inoculum after washing with MM-G medium; (2) degradation experiment: adding MM-G medium and test PFASs into the experimental bottle, adding a co-metabolic substrate according to the situation, inoculating bacteria liquid at a proportion of 10%, and incubating at a constant temperature for 1 month, sampling during the incubation process, and testing the release of fluoride ions by using a colorimetric method.

[0014] The beneficial effects of the present application are: 1. The present application screens a PFASs biological defluorination strain TF-6, which is identified as achromobacter, and is the first case of the strain about biological defluorination.

[0015] 2. The achromobacter TF-6 screened by the present application has biological defluorination effect on PFAs such as trifluoro and hexafluoro, and the defluorination rate of the strain on TFE is 11.7% and the defluorination rate on SFC is 24.0% under the condition of a unique carbon source. Ammonium acetate as a co-metabolic substrate significantly improves the SFC defluorination rate to 47.6%. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is the morphology and phylogenetic tree of achromobacter TF-6; Figure 2 It is the growth defluorination effect of achromobacter TF-6 on trifluoroalkane TFE; Figure 3 It is the growth defluorination effect of achromobacter TF-6 on hexafluoroalkane SFC; Figure 4 It is the co-metabolic defluorination effect of achromobacter TF-6 on SFC. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the related drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0019] The specific embodiments of the present application will be described below with reference to the accompanying drawings.

[0020] Example 1: Isolation and identification of TFE defluorination bacteria Achromobacter sp. TF-6 was screened from the contaminated soil of a chemical plant in Suzhou, and the specific steps are as follows: (1) Enrichment of TFE-defluorinating bacterial community: 20 g of soil was mixed with 50 mL of MM culture medium to make soil slurry in a 250 mL flask, and 0.1% (v / v) of FD was added, and the enrichment culture was carried out at 28°C and 160 rpm for 1 month. After the culture was completed, the soil suspension was centrifuged at 1400 rpm for 3 min, and the supernatant was the initial enrichment culture. The composition of the MM medium (1L) was 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 the pH was adjusted to 7.0-7.2.

[0021] (2) Preliminary screening of defluorinating bacteria: The soil suspension in step (1) was gradient diluted, and the diluted liquid 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 MM solid medium containing 1% agar and 3.24 mmol / L TFE. The plate was inverted and cultured at 28°C until single colonies were formed, and the single colonies were isolated on LB plates. The composition of the LB medium (1L) was tryptone 10 g, yeast extract 5 g, and NaCl 5 g. -1 (2) Preliminary screening of defluorinating bacteria: The soil suspension in step (1) was gradient diluted, and the diluted liquid 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 MM solid medium containing 1% agar and 3.24 mmol / L TFE. The plate was inverted and cultured at 28°C until single colonies were formed, and the single colonies were isolated on LB plates. The composition of the LB medium (1L) was tryptone 10 g, yeast extract 5 g, and NaCl 5 g.

[0022] (3) Re-screening of defluorinating bacteria: Different morphological single colonies in step (2) were selected and cultured in LB liquid medium until the OD600 value reached 0.6-0.8. Then, the bacterial slurry was collected by centrifugation at 5000 rpm for 4 min, and washed with MM medium three times, and finally the washed bacterial suspension was suspended in an equal volume of liquid. Then, according to the inoculation ratio of 10%, the suspension was inoculated into MM medium containing 100 μmol / L TFE, and 100 μmol / L glucose was added as a co-metabolic substrate, and cultured at 28°C and 160 rpm for one month. After the culture was completed, the release of fluoride ions in the culture system was preliminarily evaluated by ion chromatography. -1 (3) Re-screening of defluorinating bacteria: Different morphological single colonies in step (2) were selected and cultured in LB liquid medium until the OD600 value reached 0.6-0.8. Then, the bacterial slurry was collected by centrifugation at 5000 rpm for 4 min, and washed with MM medium three times, and finally the washed bacterial suspension was suspended in an equal volume of liquid. Then, according to the inoculation ratio of 10%, the suspension was inoculated into MM medium containing 100 μmol / L TFE, and 100 μmol / L glucose was added as a co-metabolic substrate, and cultured at 28°C and 160 rpm for one month. After the culture was completed, the release of fluoride ions in the culture system was preliminarily evaluated by ion chromatography. -1 (3) Re-screening of defluorinating bacteria: Different morphological single colonies in step (2) were selected and cultured in LB liquid medium until the OD600 value reached 0.6-0.8. Then, the bacterial slurry was collected by centrifugation at 5000 rpm for 4 min, and washed with MM medium three times, and finally the washed bacterial suspension was suspended in an equal volume of liquid. Then, according to the inoculation ratio of 10%, the suspension was inoculated into MM medium containing 100 μmol / L TFE, and 100 μmol / L glucose was added as a co-metabolic substrate, and cultured at 28°C and 160 rpm for one month. After the culture was completed, the release of fluoride ions in the culture system was preliminarily evaluated by ion chromatography.

[0023] (4) Identification of defluorinating bacteria: The pure strain with good defluorination effect was selected, and 27F and 1492R primers were used for PCR amplification of 16S rRNA gene. The amplification product was then sent to Shanghai Meiji Company for sequencing. Finally, the obtained sequence was compared by BLAST, and the phylogenetic tree was constructed using MEGA software.

[0024] Reference Example 1: Isolation of a strain of Achromobacter sp. capable of defluorination Figure 1 By the above steps, a strain of defluorination bacteria TF-6 was screened, which belongs to Achromobacter sp. The colony morphology is light yellow, the surface is flat, smooth and moist; Gram staining is negative, and the bacterial morphology is short rod. Through phylogenetic tree analysis of 16S rRNA gene sequence, it is determined that the strain is Achromobacter sp. The Achromobacter sp. was preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 12, 2024, located at No. 1, Beichen West Road, Haidian District, Beijing, China, and the Institute of Microbiology of the Chinese Academy of Sciences, Beijing 100101, China. The strain preservation number is CGMCC No. 1.62204.

[0025] Example 2: Biological defluorination of Achromobacter sp. TF-6 on trifluoro PFASs TFE was selected for biological defluorination research of trifluoro PFASs. Based on previous research, small molecule substances will be produced during the degradation of polyfluoro PFASs, which will strongly affect the detection of fluoride ions by IC. Therefore, a fluorine-lanthanide chelate specific colorimetric method was used to study the release of fluoride ions.

[0026] The specific steps are as follows: (1) Preparation of inoculum: Achromobacter sp. TF-6 single colony was picked from LB plate, cultured in LB liquid medium to OD600 = 0.6~0.8, centrifuged at 5000 rpm for 4 min to collect the bacterial slurry, washed with MM-G medium for three times, and then resuspended the bacterial cells with equal volume of MM-G medium as inoculum.

[0027] The composition of MM-G medium (1L) is MgSO4·2H2O 0.2 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, add 1L water, after sterilization of the medium, add 20 mmol / L HEPES (pH=7.2); the composition of LB medium is as described in Example 1.

[0028] (2) Degradation system: according to 10% inoculation ratio, the bacterial solution of step (1) was inoculated into MM-G medium (5 mL) containing TFE (100 μmol / L). At the same time, the same concentration of TFE structural analog without fluorine, trans-2-pentenoic acid, was added as a blank control without fluorine. The culture solution was cultured at 28 ℃, 160 rpm for 30 days, and samples were taken at intervals during the culture. 300 μL of sample was centrifuged, and the supernatant was detected by microwell plate colorimetric method based on fluorine-lanthanide chelate color reaction to detect the concentration of fluoride ions.

[0029] (3) Fluoride ion detection by microplate colorimetry: The colorimetric detection solution was prepared by mixing 10 μL acetate buffer (1.68 mol / L), 20 μL alizarin (500 μmol / L), and 20 μL lanthanum nitrate (500 μmol / L) in sequence. In the microplate, 100 μL sample, 50 μL colorimetric solution, and 50 μL acetone were added in sequence, and the colorimetric reaction lasted for more than half an hour. A620 / 530 was calculated by an enzyme-labeled instrument. A standard curve was established in the range of 0-80 μmol / L of the standard fluoride ion sample, and the fluoride ion concentration in the sample was calculated by the regression method.

[0030] See Figure 2 , the study showed that A. nonchromogenes TF-6 could biodefluorinate trifluoroalkyl compound TFE, and no fluoride ion release was detected in the structure analog without fluorine. The maximum defluorination effect was achieved in the second week, and the fluoride ion concentration detected in the system after the culture ended was 35.0 μmol / L, and the defluorination rate was 11.7%.

[0031] Example 3: Biodefluorination of hexafluoro PFASs by A. nonchromogenes TF-6 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid (SFC) was selected for the biodefluorination of hexafluoro PFASs. This substance is also reported in the literature to be biodefluorinated, but there is currently no research on pure microbial culture. The colorimetric method was used to detect the biodefluorination of polyfluoro PFASs. The preparation of A. nonchromogenes TF-6 inoculum and the degradation system was referred to Example 2. The test substrate was 200 μmol / L SFC, and the same concentration of SFC structure analog 3-methyl crotonic acid without fluorine was added at the same time as the fluorine-free blank control. The culture solution was cultured at 28 ℃, 160 rpm for 1 month, and samples were taken at intervals during the period. 300 μL of sample was centrifuged, and the supernatant was detected for fluoride ion concentration by microplate colorimetric method based on fluorine-lanthanide chelate color reaction. The test method was the same as Example 2.

[0032] See Figure 3 , the study showed that A. nonchromogenes TF-6 could biodefluorinate trifluoroalkyl compound TFE, and no fluoride ion release was detected in the structure analog without fluorine. The maximum defluorination effect was achieved in the second week, and the fluoride ion concentration detected in the system after the culture ended was 35.0 μmol / L, and the defluorination rate was 11.7%.

[0033] Example 4: Co-metabolic biodefluorination of hexafluoro PFASs by A. nonchromogenes TF-6 Ammonium acetate was selected as the co-metabolic substrate for the study of biological defluorination of SFC by SFC. The colorimetric method was used to detect the biological defluorination of PFASs. The inoculum was prepared according to Example 2; the degradation system was also prepared according to Example 2, except that ammonium acetate was added as a co-metabolic substrate. The test substrate was 200 μmol / L SFC, and the co-metabolic substrate was 5 mmol / L ammonium acetate. The culture solution was incubated at 28 ℃ and 160 rpm for 1 month, and samples were taken at intervals. 300 μL of sample was centrifuged, and the supernatant was detected for fluoride ion concentration by the microplate colorimetric method based on the color reaction of fluorine-lanthanide chelate. The test method was the same as Example 2.

[0034] Please refer to Figure 4 The results showed that ammonium acetate could significantly promote the biological defluorination of SFC by Achromobacter TF-6. The maximum defluorination effect was basically achieved in the third week, and the fluoride ion concentration in the system was 571.1 μmol / L after incubation, with a defluorination rate of 47.6%.

[0035] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered as within the scope of the present disclosure.

[0036] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. Achromobacter Achromobacter sp. TF-6 characterized in that, Achromobacter Achromobacter sp. TF-6, deposited on September 12, 2024 at China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 1.62204.

2. A microbial preparation, characterized in that, which contains the achromobacter of claim 1 Achromobacter sp. TF-6 or a fermentation broth thereof.

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

4. The Achromobacter of claim 1 Achromobacter sp. TF-6, the use of the microbial preparation of claim 2 or claim 3 for the biological defluorination of polyfluoroalkyl compounds.

5. Use according to claim 4, characterized in that, The polyfluoroalkyl compound is 4,5,5-trifluoro-4-pentenoic acid or 4,4,4-trifluoro-3-(trifluoromethyl) crotonic acid.

6. Use according to claim 5, characterized in that: Acinetobacter Achromobacter sp. Co-metabolic defluorination of 4,4,4-trifluoro-3-(trifluoromethyl)crotonic acid by TF-6 with ammonium acetate.

7. Use according to claim 5 or 6, characterized in that: ochrobactrum Achromobacter sp. TF-6 biodefluorinates polyfluoroalkyl compounds in inorganic salt media.

8. Use according to claim 7, characterized in that: The inorganic salt medium is inorganic salt medium MM-G, and components of the inorganic salt medium MM-G include MgSO4·2H2O 0.2 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 into 1000 ml of deionized water, and after sterilization of the medium, 20 mmol / L of HEPES buffer solution with pH=7.2 is added.

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