Establishment and application of microbial inorganic salt medium MM-F2 for detecting fluoride ion concentration based on fluorine-lanthanide chelate microplate colorimetric method

Abstract

The present invention discloses the establishment and application of a microbial inorganic salt culture medium MM-F2 for detecting fluoride ion concentration based on a fluorine-lanthanide chelate microplate colorimetric method. The present invention solves the problem that the microplate colorimetric method cannot detect defluorination that depends on the bacterial growth process. The present invention makes the detection process more efficient and convenient, and can be used to screen more PFASs defluorination bacteria in the future, accelerating the research on the biological defluorination mechanism.

Description

Technical Field

[0001] The invention relates to the field of environmental microbial culture medium, and in particular to the establishment and application of a microbial inorganic salt culture medium MM-F2 for detecting fluoride ion concentration based on a fluorine-lanthanide chelate microplate colorimetric method.

[0002] This application claims priority. The application number of the prior application is: 2024110804436, the name is: Sphingopyxis sp. NJF-3 and its application in degrading fluorinated alkyl compounds, and the priority date is: 2024.08.08. Background Art

[0003] Perfluorinated and polyfluoroalkyl compounds (PFASs) are a series of non-natural synthetic organic compounds with hydrophilic functional groups and hydrophobic alkyl side chains. They are fire-resistant, highly stable and durable. Due to their stable chemical properties, PFASs are widely used in industrial and commercial products. So far, the presence of PFASs has been widely detected in various environmental media. Currently, common PFASs remediation technologies include granular activated carbon (GAC), biochar, ion exchange (IX) technology, membrane filtration, etc., but the above methods only transfer the compounds from the environment and cannot achieve a good degradation effect on pollutants. Bioremediation is an effective and non-destructive technology, especially microbial remediation technology, which can degrade pollutants and reduce the toxic effects of pollutants in the environment. At the same time, bioremediation can also achieve in situ remediation and save costs.

[0004] The commonly used monitoring methods for PFASs are liquid chromatography or gas chromatography and mass spectrometry to detect the degradation of fluoride, but due to extraction losses, volatility of the substance itself and other reasons, the detection results will cause large deviations. Fluoride ions produced during the degradation process of PFASs can also be used as an indicator for the determination of fluoride degradation to test the biodegradation effect of CF compounds. Commonly used fluoride ion detection methods are ion chromatography (IC) and fluoride ion-specific selective electrode determination. The microplate colorimetric method reported in 2022 provides a detection method for fluoride ion concentration based on the fluorine-lanthanide chelate colorimetric reaction microplate colorimetric method ("Microwell Fluoride Screen for Chemical, Enzymatic, and Cellular Reactions Reveals Latent Microbial Defluorination Capacity for-CF3 published by Bygd in Applied and Environmental Microbiology in 2022). Groups.), this method has the advantages of small size, fast detection, high sensitivity, and large detection volume, and can speed up the screening of defluorinating bacteria. However, phosphate ions in common inorganic salt culture media will have a great impact on the detection results of this method, so it is necessary to use resting cells for microbial defluorination. Due to the high chemical stability of PFASs, microorganisms cannot degrade some PFASs under a single carbon source, and require cometabolic defluorination, which cannot be achieved by resting cell culture. Therefore, we improved an inorganic salt culture medium that does not affect the detection of microplate colorimetry, and verified that defluorinating bacteria can grow and defluorinate in this culture medium, making the detection process more efficient and convenient. In the future, it can be used to screen more PFASs defluorinating bacteria and accelerate the study of biological defluorination mechanisms. Summary of the invention

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

[0006] The present invention establishes a novel microbial inorganic salt culture medium MM-F2, which can directly detect the fluoride ion concentration generated by defluorination during the bacterial growth process by using a fluorine-lanthanide chelate microplate colorimetric method.

[0007] The preparation method of the inorganic salt culture medium MM-F2 used in the present invention comprises the following components (1 L): MgSO4·2H2O0.2 g, CaCl2·2H2O 20 mg, MnSO420 mg, NaNO30.7 g, KNO30.3 g, (NH4)2SO41 g, sterilized at 121° C. for 20 min, and HEPES (pH=7.2) 50 mmol / L was added after sterilization.

[0008] The defluorinating bacteria used in the present invention are Sphingopyxis sp. NJF-3, which was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on May 22, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the strain classification is named Sphingopyxis, and the deposit number is CGMCC No. 1.19398.

[0009] The preparation of the inoculum of Sphingopyxis sp. NJF-3 comprises the following steps:

[0010] 1) Plate culture: Sphingopyxis sp. NJF-3 was inoculated on LB solid plates and cultured at 28°C for 24-48 hours until a single colony was obtained;

[0011] 2) Expanding the culture: Use an inoculating loop to pick up a single colony in step 1) and inoculate it into LB liquid culture medium. Incubate the culture in a constant temperature shaking incubator at 28°C and 160 r / min for 24 to 36 h to obtain a bacterial solution with an OD600 of 0.6 to 0.8. Collect the bacteria by centrifugation, wash them three times with sterile MM-F2 culture medium, and resuspend the bacteria in an equal volume to obtain an inoculum.

[0012] Luria-Bertani (LB) medium (1 L) components: NaCl 5 g, tryptone 10 g, yeast extract 5 g; sterilized at 121° C. for 20 min; natural pH, solid medium was prepared by adding 1.5% agar powder.

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

[0014] The present invention establishes a novel microbial inorganic salt culture medium MM-F2, defluorination bacteria can be cultured using the culture medium, the supernatant can be directly centrifuged after the defluorination bacteria are cultured, and the fluorine ion concentration generated by defluorination during the bacterial growth process is detected using a fluorine-lanthanide chelate microplate colorimetric method. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 To identify the interference factors of traditional microplate colorimetry in inorganic salts;

[0016] Figure 2 The pH, OD600 and defluorination of MM-F2 after culture in different buffers (pH 7.2);

[0017] Figure 3 Defluorination effect of HEPES buffer with different concentrations. DETAILED DESCRIPTION

[0018] It should be noted that the following specific descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all scientific and technical terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. If it is not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0019] The specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0020] Example 1

[0021] This example screened the interference factors of traditional inorganic salt culture medium components on microplate colorimetry.

[0022] The transmission inorganic salt medium is MM inorganic salt medium, and the components of the MM inorganic salt medium are as follows:

[0023] MgSO4·2H2O 0.2g, CaCl2·2H2O 20mg, FeSO4·7H2O 10mg, KH2PO40.4g, Na2HPO40.6g, MnSO420mg, NaNO30.8g, (NH4)2SO41g.

[0024] The steps for screening the interference factors of traditional inorganic salt culture medium components on microplate colorimetry are as follows:

[0025] 1) Prepare the above MM inorganic salt medium by dissolving each component in 10 mL ultrapure water to obtain a 100-fold concentration of the mother solution, and take 100 μL of each mother solution and dilute it to 5 mL as the screening sample for the interference factor of the microplate colorimetric method;

[0026] 2) Add 50 μL of diluent and 50 μL of ultrapure water to the sample without NaF;

[0027] 3) Add 50 μL of diluent and 50 μL of NaF solution (100 μmol / L) to the sample containing NaF;

[0028] 4) The fluoride ion concentrations of the above samples were determined by microplate colorimetry.

[0029] See also Figure 1 The results showed that MM inorganic salt medium would have a significant impact on the microplate colorimetry, the specific impacts are as follows:

[0030] See also Figure 1In Figure a, when NaF is not present, the interfering factors that have a significant impact on the microplate colorimetry are FeSO4·7H2O, KH2PO4, and Na2HPO4.

[0031] See also Figure 1 In Figure b, in the presence of NaF (50 μmol / L), the significant influencing factors are KH2PO4 and Na2HPO4.

[0032] Example 2

[0033] This example investigates bacterial growth, culture system pH, and defluorination effects after culture in different buffer solutions.

[0034] In this example, based on the results in Example 1, the three components that affect color development are removed. Based on the role of the two inorganic phosphate salts in the culture medium as buffers, glycine, Tris-HCl, and HEPES buffer are added to the culture medium as new buffer systems to form a new culture medium MM-F2: MgSO4·2H2O 0.2g, CaCl2·2H2O 20mg, MnSO420mg, NaNO30.7g, KNO30.3g, (NH4)2SO41g; the culture medium is sterilized at 121°C for 20min, and after sterilization, 5mmol / L of sterile buffer (pH=7.2) is added. The experimental process is as follows:

[0035] 1) In the experiment, a single colony of NJF-3 was picked and placed in LB liquid medium, cultured at 28°C and 160rpm to the logarithmic phase, centrifuged at 5000rpm for 5min, the bacteria were collected and inoculated with a 10% (v / v) inoculation ratio in the degradation experiment;

[0036] 2) The experiment was carried out in a 20 mL sterile vial containing 5 mL of MM-F2 medium, with 1-FD (1 mmol / L) added as the sole carbon source, and incubated at 28°C and 160 rpm for 7 days. Three replicates were set for each treatment group.

[0037] 3) Take 1 mL of each sample and pass it through a 0.22 μm water filter membrane for ion chromatograph (IC) to detect the fluoride ion concentration. Take 200 μL and centrifuge at 5000 rpm for 5 min. Take 100 μL of the supernatant to detect the fluoride ion concentration using a microplate colorimetric method based on a fluorine-lanthanide chelate colorimetric reaction. Take 2 mL to determine the OD600 value of bacterial growth. Use the remaining sample to determine the pH value.

[0038] See also Figure 2 Figure a shows that the pH of the three buffers decreased and became acidic after culture, and glycine as a buffer was significantly less effective than Tris-HCl and HEPES for bacterial growth.

[0039] See also Figure 2 In Figure b, the fluoride ion concentrations measured by the microplate colorimetric method and the IC detection method are basically consistent. The defluorination effects of the three buffers are HEPES>Tris-HCl>glycine, and HEPES has the best defluorination effect.

[0040] Example 3

[0041] This example investigates the defluorination effects of HEPES buffer solutions with different concentrations.

[0042] The experimental system establishment and sampling detection in this example are consistent with those in Example 2. The concentrations of HEPES (pH 7.2) are selected as 20 mmol / L and 50 mmol / L. Five sampling time points are set in the experiment, with 3 replicates at each point.

[0043] See also Figure 3 ( Figure 3 The concentration corresponding to Figure a is 20mmol / L. Figure 3 The concentration corresponding to Figure b is 50mmol / L) The results show that the fluoride ion concentrations measured by the microplate colorimetric method and the IC detection method are basically the same, and the defluorination effect in Example 2 is significantly improved. Among them, the defluorination effect at 50mmol / L is slightly better than that at 20mmol / L, and the detection results of the two methods are more compatible at 50mmol / L.

[0044] The above-described embodiments are only preferred implementation schemes given to better explain the present invention. Therefore, the above-described embodiments are not used to limit the present invention. Modifications, improvements and substitutions made within the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Sphingobacteria Sphingopyxis sp. )NJF-3, characterized in that Sphingobacterium Sphingopyxis sp. )NJF-3, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 22, 2024, with the deposit number: CGMCC No.1.19398.

2. The sphingosine box bacteria according to claim 1 ( Sphingopyxis sp .)Application of NJF-3 in the degradation of 1-fluorodecane.

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