Soil LiTFSI degrading bacterium and application thereof in field of environmental restoration

Abstract

The invention relates to the technical field of microorganisms, in particular to a soil LiTFSI degrading bacterium and application thereof in the field of environmental remediation. The LiTFSI degrading bacteria are Pseudomonas nitroreducens LiT2 or LiT3, and the LiTFSI degrading bacteria are Pseudomonas nitroreducens LiT2 or LiT3, and the LiTFSI degrading bacteria are Pseudomonas nitroreducens LiT2 or LiT3. The strain is preserved in the Guangdong Microbial Culture Collection Center on December 01, 2025, the preservation address is the 5th floor of the building 59, 100 courtyard, Xianlie Middle Road, Guangzhou City, China, and the preservation numbers are respectively GDMCC No: 67389 and GDMCC No: 67390. The soil LiTFSI degrading bacterium can take LiTFSI as a unique carbon source, and has excellent degradation efficiency on LiTFSI. The invention also provides an environmental remediation microbial agent which can accurately and quickly intervene and strengthen the degradation process of LiTFSI in soil, so that the pollution level of LiTFSI and intermediate metabolites TFNH2 and TFOH thereof is obviously reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a soil LiTFSI degrading bacterium and its application in the field of environmental remediation. BACKGROUND

[0002] The global transition to renewable energy makes lithium-ion power batteries (LIPB) play a core role in electric vehicles, renewable energy storage and consumer electronics. During the recycling process of LIPB, a large number of organic and inorganic pollutants will be released in the process of large-scale disassembly, crushing, sorting and purification. These substances have environmental persistence, migration and potential biological toxicity, and the environmental behavior, degradation rule and health risk cognition are still insufficient.

[0003] Perfluoro and polyfluoroalkyl substances (PFAS) are widely used in electrolyte systems due to their excellent electrical conductivity and chemical stability. Among them, ultra-short chain PFAS (such as trifluoromethanesulfonamide, TFNH2; trifluoromethanesulfonic acid, TFOH) have more prominent ecological and health risks due to their higher water solubility and migration. In the environmental detection of LIPB recycling area samples, TFNH2 was confirmed to be one of the highly toxic PFAS; although the battery raw materials and crushed powder (LIPBCP) were systematically investigated, the source was still unknown. Structure comparison shows that lithium bis(trifluoromethanesulfonyl) imide (LiTFSI, molecular skeleton LiN(SO2CF3)2) can be hydrolyzed to generate TFNH2 (CF3SO2NH2) and TFOH (CF3SO3H), and thus may be an important precursor. LiTFSI as a new generation of electrolyte lithium salt, the environmental behavior, biological transformation pathway and correlation with ultra-short chain PFAS in soil media have not been systematically elucidated so far.

[0004] Soil microbial community is the most diverse biological system on the earth, driving the decomposition of organic matter and geochemical cycle. PFAS pollution can inhibit community diversity and abundance, but some groups including Pseudomonas and Acidobacteria show degradation potential. Literature reported Pseudomonas can degrade sulfonamide organic fluorides under aerobic conditions, and revealed the key genes of desulfonation and defluorination; Acidobacteria was also pointed out to promote defluorination through oxidation pathway in PFAS stressed soil. However, the microbial degradation conditions, mechanism and transformation pathway of LiTFSI in soil are still blank; at the same time, how to avoid or reduce the accumulation of secondary pollutants (such as TFNH2, TFOH) in engineering remediation is a key difficulty in the field of environmental bioremediation.

[0005] Therefore, it is urgent to establish an integrated technology system from environmental detection, toxicological evaluation, functional bacteria screening to pathway analysis and collaborative intervention, to explore and optimize functional bacteria and composite bacterial agents capable of efficiently degrading LiTFSI and related ultra-short-chain PFAS, and to realize accurate, rapid and environmentally friendly in-situ remediation of contaminated soil (and water body, sediment). SUMMARY

[0006] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present application is to provide a LiTFSI degrading bacteria.

[0007] Another purpose of the present application is to provide the application of the above-mentioned LiTFSI degrading bacteria in the field of environmental remediation.

[0008] Still another purpose of the present application is to provide an environmental remediation bacterial agent.

[0009] The fourth purpose of the present application is to provide the application of the above-mentioned environmental remediation bacterial agent.

[0010] The fifth purpose of the present application is to provide a method for repairing soil by using the above-mentioned environmental remediation bacterial agent.

[0011] The purpose of the present application is achieved by the following technical solutions: A LiTFSI degrading bacteria, named Pseudomonas nitroreducens (Pseudomonas nitroreducens) Pseudomonas nitroreducens LiT2 or LiT3, deposited in the Guangdong Microbial Culture Collection Center on December 1, 2025, with the address of 59 Building, 5th Floor, 100 Institute, Xianlie Middle Road, Guangzhou, China, and the preservation numbers of GDMCC No: 67389 and GDMCC No: 67390, respectively.

[0012] The culture method of the LiTFSI degrading bacteria comprises the following steps: The above-mentioned LiTFSI degrading bacteria is inoculated into a basic salt solution containing LiTFSI for culture.

[0013] The culture conditions are preferably inoculation amount of 4-6%, pH value of 6-8, and temperature of 20-40℃.

[0014] The culture conditions are preferably inoculation amount of 6%, pH value of 7, and temperature of 30℃.

[0015] The basic salt solution comprises the following components: dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium sulfate 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.1 g / L, calcium chloride 0.01 g / L, and 1 mL / L trace element mother liquor.

[0016] The trace element mother liquor comprises the following components: ferrous sulfate heptahydrate 2.78 g / L, manganese chloride tetrahydrate 1.98 g / L, zinc sulfate heptahydrate 2.87 g / L, and copper chloride dihydrate 0.17 g / L.

[0017] The base salt solution preferably additionally comprises 0.01% of ferrous sulfate (FeSO4) by mass percentage.

[0018] The application of the LiTFSI degrading bacteria in the field of environmental remediation.

[0019] The environmental remediation includes but is not limited to soil, water body, sediment, etc.

[0020] The application of the LiTFSI degrading bacteria in degrading perfluoro and polyfluoro alkyl compounds (PFAS) and precursor compounds thereof.

[0021] The perfluoro and polyfluoro alkyl compounds include short-chain PFAS.

[0022] The short-chain PFAS includes but is not limited to trifluoromethanesulfonamide (TFNH2) and trifluoromethanesulfonic acid (TFOH).

[0023] The precursor compounds include but are not limited to LiTFSI.

[0024] An environmental remediation bacterial agent comprising the above LiTFSI degrading bacteria.

[0025] The environmental remediation bacterial agent preferably further comprises other bacterial strains capable of expressing key degrading enzymes.

[0026] The key degrading enzyme can be at least one of N-S bond breaking enzyme, denitrifying enzyme, desulfonating enzyme, and dehalogenase. 、

[0027] The other bacterial strains capable of expressing key degrading enzymes are preferably Pseudomonas.

[0028] The other bacterial strains capable of expressing key degrading enzymes are further preferably Pseudomonas putida KT2440, which can efficiently express denitrifying enzyme.

[0029] The application of the environmental remediation bacterial agent in the field of environmental remediation.

[0030] A method for repairing soil by using the above soil remediation bacterial agent, comprising the following steps: (1) respectively culturing the LiTFSI degrading bacteria and Pseudomonas putida KT2440 to obtain corresponding seed liquids; ​(2) centrifuging the seed liquid and washing with PBS solution, then adding into the basic salt solution containing LiTFSI to culture for 12-24 h to activate the enzyme expression function of the strains LiT3 and Pseudomonas putida KT2440; (3) centrifuging the bacterial liquid in step (2) and washing with PBS solution, then resuspending with the basic salt solution; (4) mixing the LiTFSI degrading bacteria liquid and Pseudomonas putida KT2440 liquid obtained in step (3) to obtain a soil remediation bacteria agent; (5) mixing the soil to be treated and the soil remediation bacteria agent prepared in step (4) and performing remediation.

[0031] The culture medium in step (1) is preferably LB liquid culture medium.

[0032] The concentration of LiTFSI in the basic salt solution containing LiTFSI in step (2) is preferably 1 mg / L.

[0033] The densities of LiTFSI degrading bacteria and Pseudomonas putida KT2440 in the soil remediation bacteria agent in step (4) are preferably the same.

[0034] The mass-volume ratio of the soil and the soil remediation bacteria agent in step (5) is preferably 10: (0.5-1) (g:mL).

[0035] The system in step (5) is preferably further added with a solution containing glucose and KH2PO4.

[0036] The volume ratio of the solution containing glucose and KH2PO4 and the soil remediation bacteria agent is preferably 0.5:3.

[0037] The mass percentages of glucose and KH2PO4 in the solution containing glucose and KH2PO4 are 0.5% and 0.1%, respectively.

[0038] The remediation conditions in step (5) are pH 7, room temperature and dark conditions.

[0039] Technical principle of the application: The application constructs a complete microbial remediation technology system of “detection-toxicology-screening-optimization-analysis-intervention”, which comprises the following steps in sequence: (1) detection of environmental distribution of LiTFSI, TFNH2 and TFOH; (2) evaluation of toxicological effects; (3) enrichment and separation and purification of Pseudomonas nitroreducens LiT2 and LiT3; (4) system optimization of degradation kinetics conditions; (5) analysis of LiTFSI microbial degradation pathway and screening of key functional enzymes; (6) construction and degradation intervention of composite bacteria agent. This invention uses soil samples collected from a lithium-ion battery recycling site as the research object to detect the content of ultrashort chain PFAS (TFNH2, TFOH) and their precursor LiTFSI. LiTFSI showed the highest detection rate and concentration among all samples, reaching 100% detection. This invention further employs toxicological effect models constructed from multiple model organisms representing cardiotoxicity, metabolic toxicity, developmental toxicity, and neurotoxicity indicators to evaluate the toxicological effects of LiTFSI, TFNH2, and TFOH. Based on LiTFSI gradient concentration screening, isolation, and purification, this invention obtains a soil LiTFSI-degrading bacterium, namely *Pseudomonas nitroreductoides* (…). Pseudomonas nitroreducens LiT2 and LiT3 were used to successfully establish the degradation pathway of LiTFSI degrading bacteria in this soil and to preliminarily identify the key degrading enzymes involved in the LiTFSI degradation process.

[0040] Based on the toxicological effects of LiTFSI, TFNH2, and TFOH, and combined with the key degradation enzymes of soil LiTFSI-degrading bacteria, this invention identifies strains that can be synergistically enhanced with soil LiTFSI-degrading bacteria, meeting the following conditions: (1) belonging to the genus *Pseudomonas*, and (2) capable of efficiently expressing denitrases. Among these, the synergistic strains belonging to the same genus as the soil LiTFSI-degrading bacteria provide compatibility for the compound microbial agent. TFNH2 has the lowest biological toxicity, meaning it is the most toxic. By using synergistic strains that can express denitrases, their metabolic activity is complementary to that of the soil LiTFSI-degrading bacteria, thereby converting LiTFSI into products with lower toxicity than those downstream of TFNH2 and TFOH. The compound microbial agent provided based on the above strategy can precisely and rapidly intervene in and enhance the degradation process of LiTFSI in soil, thereby significantly reducing its pollution level.

[0041] The present invention has the following advantages and effects compared with the prior art: (1) This invention provides a soil LiTFSI degrading bacterium: Nitro-reducing Pseudomonas (N-N-N-N) Pseudomonas nitroreducens LiT2 and LiT3. This soil LiTFSI degrading bacteria can use LiTFSI as the sole carbon source and degrade LiTFSI through four enzymes: NS bond cleavage enzyme, denitrase, desulfonase, and dehalogenase. It has excellent degradation efficiency for pollutants such as LiTFSI, TFNH2, and TFOH, especially LiTFSI.

[0042] (2) The present invention optimized the degradation kinetics of soil LiTFSI degrading bacteria and determined the optimal inoculum size of nitrifying Pseudomonas nitroreductoids LiT2 and LiT3. I max Optimal pH value P max Optimal metal salt type Mmax , and provides technical support for the application of soil LiTFSI degrading bacteria in the field of environmental remediation.

[0043] (3) The present application establishes the degradation pathway of soil LiTFSI degrading bacteria, which is carried out through four consecutive steps, including hydrolysis, deamination, step-by-step deamination and sulfonation, finally leading to mineralization and integration into central metabolic cycles (glyoxylate and tricarboxylic acid cycles), which is supported by genome annotation, highlighting the strain-specific enzymatic action.

[0044] (4) Based on the toxicological effects of LiTFSI, TFNH2 and TFOH and the key enzymes for LiTFSI degradation, the present application screens strains that can synergistically enhance the degradation of soil LiTFSI degrading bacteria, and the screening method has better scientificity, systematicness and targetability. The soil remediation composite microbial agent obtained based on the screening strategy of the present application can precisely and quickly intervene and enhance the degradation process of LiTFSI in soil, thereby significantly reducing the pollution level.

[0045] (5) The present application provides a new soil LiTFSI degrading bacteria and a scientific bioremediation strategy for solving LiTFSI soil pollution, which has important practical significance and application prospect for protecting environmental safety and human health. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is the colony morphology diagram of nitroreducing pseudomonas LiT2 and LiT3.

[0047] Figure 2 is the perspective electron microscope diagram of nitroreducing pseudomonas LiT2 and LiT3.

[0048] Figure 3 is the 16S-rDNA gene sequencing diagram of nitroreducing pseudomonas LiT2 and LiT3.

[0049] Figure 4 is the average nucleotide identity diagram between nitroreducing pseudomonas LiT2 and LiT3 and related pseudomonas type strains.

[0050] Figure 5 is the whole genome diagram of nitroreducing pseudomonas LiT2 and LiT3.

[0051] Figure 6 is the degradation kinetics diagram of nitroreducing pseudomonas LiT2 and LiT3.

[0052] Figure 7 is the degradation condition optimization result diagram of nitroreducing pseudomonas LiT2 and LiT3, wherein A: pH, B: inoculum size, C: trace metal.

[0053] Figure 8 is LiTFSI degradation product mass spectrum and biodegradation pathway diagram, wherein A: mass spectrum, B: biodegradation pathway.

[0054] Figure 9 is LiTFSI, TFNH2, TFOH concentration change diagram in soil under the intervention of composite microbial agent, wherein A: LiTFSI, B: TFOH, C: TFNH2. DETAILED DESCRIPTION

[0055] The application will be further described in conjunction with the examples and drawings, but the embodiments of the application are not limited thereto.

[0056] Unless specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. Unless specifically indicated, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field.

[0057] The base salt solution in the examples comprises the following components: dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium sulfate 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.1 g / L, calcium chloride 0.01 g / L, 1 mL / L trace element stock solution (ferrous sulfate heptahydrate 2.78 g / L, manganese chloride tetrahydrate 1.98 g / L, zinc sulfate heptahydrate 2.87 g / L and copper chloride dihydrate 0.17 g / L, all are final concentrations in the stock solution).

[0058] The LB liquid medium in the examples comprises the following components: tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, and the LB solid medium is obtained by adding 15-20 g / L agar to the LB liquid medium.

[0059] LiTFSI, TFNH2, TFOH are purchased from Spectrum.

[0060] Example 1 Toxicological effect evaluation 1. LC50 toxicological effect concentration H9C2 myocardial cells were selected to represent cardiac toxicity h , HEK-293T kidney cells to represent metabolic toxicity m , zebrafish (wild type AB strain) embryos to represent developmental toxicity d , and Caenorhabditis elegans (wild type N2 strain) to represent neurotoxicity v . According to conventional methods, four biological indicators representing cardiac toxicity h , metabolic toxicity m , developmental toxicity d , and neurotoxicity v were used to establish PFAS substance li i =1,2,3, l 1 = LiTFSI, l 2 = TFNH2, l 3 = LC of TFOH 50 Toxic effect concentration Q ij wherein: (1) For H9C2 myocardial cells and HEK-293T kidney cells: cells were seeded in DMEM medium containing different concentrations of pollutants for 24 h according to the conventional method, cell viability was determined by MTT method, and LC50value was calculated. 50

[0061] (2) For zebrafish embryos: zebrafish embryos (4-6 hpf) were seeded in E3 medium containing different concentrations of pollutants for 96 h, and the solution was updated every day, and the LC50value was calculated. 50

[0062] (3) For Caenorhabditis elegans: synchronized L1 stage nematodes were seeded in Caenorhabditis elegans growth medium containing different concentrations of pollutants for 24 h, and the LC50value was calculated. 50

[0063] 2. Toxic effect evaluation Based on LC50 toxic effect concentration Comprehensive evaluation of the organism toxic effect of LiTFSI, TFNH2, and TFOH The organism toxic effect evaluation formula of LiTFSI, TFNH2, and TFOH is as follows:

[0064] wherein: is the weight coefficient of the biological index of PFAS substance , and .

[0065] is the LC50 toxic effect concentration of the i-th biological index of PFAS substance , .

[0066] is the LC50 toxic effect concentration of the i-th biological index of PFAS substance , ​​​​​​LC50 toxicological effect concentration of the biological indicator, .

[0067] is the Hill coefficient.

[0068] is the non-linear mapping index.

[0069] is the interaction coefficient.

[0070] is the comprehensive toxicological effect coefficient of PFAS substances , , .

[0071] is the average value of the biological indicators of PFAS substances .

[0072] is the toxicological effect of PFAS substances on organisms, , .

[0073] wherein, .

[0074] The organism toxicological effect parameters of LiTFSI, TFNH2, and TFOH are shown in Table 1.

[0075] Table 1. Organism toxicological effect table of LiTFSI, TFNH2, and TFOH Example 2

[0076] 1. Environmental distribution detection of LiTFSI, TFNH2, and TFOH (1) Soil samples (a total of 6 samples) collected from the area of a lithium-ion battery recycling site in Huizhou, Guangdong Province were freeze-dried and weighed. Soil without PFAS direct pollution collected outside the battery recycling site area was used as a control.

[0077] (2) The soil samples were subjected to power ultrasonic extraction using methanol containing 0.1% (volume percentage) ammonium hydroxide (0.1% NH4OH-MeOH), wherein the power of the ultrasonic extraction was 100 kHz, the time was 30 min, and the extract was collected.

[0078] ​(3) The extraction solution collected in step (2) is loaded onto an ENVI-Carb solid-phase extraction column, and LiTFSI, TFNH2, and TFOH in the extraction solution are separated and purified using 0.1% NH4OH-MeOH as the eluent, and the purified solution is collected.

[0079] (4) The purified solution collected in step (3) is blown to dryness with nitrogen, and 0.1% NH4OH-MeOH is used to reconstitute the sample to be tested C e (e = 1, 2, … n), and n is the total number.

[0080] (5) The LiTFSI, TFNH2, and TFOH concentrations in the sample to be tested are detected using an ultra-high performance liquid chromatography-quadrupole-electric field orbitrap high-resolution mass spectrometer C e The detection method is as follows: a C18 chromatographic column (150 mm × 2.1 mm, 2.6 μm) is used, mobile phase A is 5 mM ammonium acetate, and mobile phase B is methanol. The chromatographic separation gradient program is as follows: 0-1 min, 5% (volume percent, same below) B; 1-6 min, 5-70% B; 6-14 min, 70-95% B; 14-21 min, 95% B; 21-25 min, 95-5% B; and the environmental distribution detection concentration table of LiTFSI, TFNH2, and TFOH is obtained.

[0081] The environmental distribution detection concentration results of LiTFSI, TFNH2, and TFOH are shown in Table 2. As can be seen from the table, in all samples, the detection rate and concentration of LiTFSI are the highest, reaching a detection rate of 100%.

[0082] Table 2 LiTFSI, TFNH2, and TFOH environmental detection concentration table

[0083] Note: n.d. = below the detection limit.

[0084] 2. Enrichment and separation and purification of Pseudomonas nitroreducens LiT2 and LiT3 (1) 5 g of soil containing LiTFSI, TFNH2, and TFOH and having the highest concentration of TFNH2 and TFOH in step 1 C1 is taken in a conical flask, 100 mL of a basic salt solution is added, and the culture is incubated at 30°C, 160 rpm for 24 h to reach the logarithmic phase.

[0085] (2) Take 1 mL of the culture obtained in step (1) and inoculate into 100 mL of the base salt solution containing 20 mg / L LiTFSI, and shake culture at 30°C and 160 rpm for 24 h to make the culture reach the logarithmic phase.

[0086] (3) Repeat step (2) in turn, and increase the LiTFSI concentration in the culture medium by 20 mg / L each time until the LiTFSI concentration in the culture medium is increased to 200 mg / L.

[0087] (4) Take 1 mL of the culture obtained in step (3), dilute it by a certain multiple, and evenly spread it on LB solid medium for culture, and according to the differences in color, morphology, etc. of different colonies, different strains are purified by repeated plate streaking.

[0088] 3. Morphological feature identification The strains separated in step 1 are cultured on LB solid medium, and the colony color, morphological characteristics are observed and recorded, and the cell morphology is observed under a microscope.

[0089] 4. Physiological and biochemical identification Referring to the Common Bacteria System Identification Manual, the strains separated in step 1 are used as objects, and methyl red test, etc. are performed.

[0090] 5. Sequence homology analysis, phylogenetic tree construction, and whole genome sequencing, assembly and annotation (1) The strains separated in step 1 are used as objects, genomic DNA is extracted using a DNA extraction kit (such as Omega Bio-tek DNA kit), and PCR amplification is performed using 515F (5'-GTGCCAGCAGCCGCGGTAA-3') and 806R (5'-GGACTACCAGGGTATCTAA-3'). The obtained PCR product is purified and sequenced, and according to the sequencing results, blast is performed and a phylogenetic tree is constructed (Meiji Biological Company) to confirm the genus of strains LiT2 and LiT3.

[0091] (2) Genomic DNA was further extracted and purified using a high molecular weight DNA extraction kit (e.g., Nanobind Plant Nuclei DNA kit). Whole-genome sequencing and functional annotation were performed using the PacBio analysis and detection platform, which has established a third-generation sequencing library. The sequencing results were imported into the Majorbio website (https: / / www.majorbio.com / ), where strains LiT2 and LiT3 were compared with other typical strains of the same genus (e.g., Pseudomonas trichloromethane ZM23, Pseudomonas nitroreductoides NBRC12694, and Pseudomonas deltae CCM7361) using the Majorbio CloudPlatform. The species and subspecies of strains LiT2 and LiT3 were confirmed by comparing the average nucleotide identity between the two strains and with other similar Pseudomonas species to see if it was greater than or equal to 95% and greater than 99%. Finally, the whole genome sequence was compared on the NCBI-BLAST website to definitively identify the subspecies of strains LiT2 and LiT3.

[0092] After screening with LiTFSI gradient concentrations, two strains were isolated and purified, named LiT2 and LiT3, respectively. Figure 1 As shown, the colonies of strains LiT2 and LiT3 on LB solid medium were both round, slightly convex in the center, milky yellow in color, with smooth and regular edges, and were methyl red negative. Further microscopic observation of the isolated and purified strains using transmission electron microscopy was performed, and the results are shown below. Figure 2 .

[0093] Based on the 16S rDNA sequences (SEQ ID No:1 and SEQ ID No:2) of strains LiT2 and LiT3, BLAST alignment analysis was performed, and a phylogenetic tree was constructed. The results are shown in Table 3. Figure 3 .from Figure 3 It can be seen that strains LiT2 and LiT3 are most closely related to the genus *Pseudomonas*. Table 3 confirms that strains LiT2 and LiT3 belong to the genus *Pseudomonas*, combined with... Figure 3 As a result, strains LiT2 and LiT3 were preliminarily identified as *Nitroreducing Pseudomonas* (…). Pseudomonas nitroreducens ).

[0094] 16S rDNA sequence of strain LiT2 (SEQ ID No:1): 16S rDNA sequence of strain LiT3 (SEQ ID No: 2): Table 3 Comparison results of 16S rDNA of strains LiT2 and LiT3

[0095] Further analysis of the average nucleotide identity between strains LiT2 and LiT3 and related Pseudomonas type strains is shown in Table 4. Figure 4 As can be seen from the figure, the average nucleotide identity of Pseudomonas LiT2 and LiT3 with other similar Pseudomonas strains (LiT2: 86.9%-91.85%, LiT3: 86.95%-92.13%) is lower than the species division threshold (95%), indicating that Pseudomonas LiT2 and LiT3 are new species of Pseudomonas discovered, and the average nucleotide identity between Pseudomonas LiT2 and LiT3 is 95.42%<99%, further indicating that strains LiT2 and LiT3 are the same species but different subspecies.

[0096] The whole genome map of strains LiT2 and LiT3 is shown in Figure 2. Figure 5 As shown in the figure, the whole gene chain of Pseudomonas LiT2 and LiT3 is shown. By comparing the whole gene chain sequence with the gene chain sequence in the NCBI-BLAST website, it is further confirmed that strains LiT2 and LiT3 are new subspecies of Pseudomonas nitroreducens. Pseudomonas nitroreducens

[0097] In combination with the morphological characteristic identification results, physiological and biochemical identification results and biomolecular identification results of strains LiT2 and LiT3, strains LiT2 and LiT3 are attributed to Pseudomonas nitroreducens, Pseudomonas nitroreducens respectively named Pseudomonas nitroreducens Pseudomonas nitroreducens LiT2 and Pseudomonas nitroreducens Pseudomonas nitroreducens LiT3, deposited in Guangdong Microbial Culture Collection Center on December 01, 2025, with the address of 59# Building, 5th Floor, 100 Institute, Xianlie Middle Road, Guangzhou, China, and the preservation numbers are GDMCC No: 67389 and GDMCC No: 67390, respectively.

[0098] Example 3 1. Degradation kinetics condition optimization (1) The Pseudomonas nitroreducens LiT2 and LiT3 obtained by separation and purification in Example 2 were respectively cultured in LB liquid medium at 30°C, 160 rpm for 12h to prepare seed liquid of different strains.

[0099] ​(2) The seed liquid prepared in step (1) is inoculated into a basic salt solution containing 100 mg / L LiTFSI at a volume ratio of 4% inoculation amount, and cultured at pH=7, 30°C, 160 rpm for 96 h, with a basic salt solution containing 100 mg / L LiTFSI without any bacteria as a control.

[0100] (3) 20 mL of the solution is taken at time points of 0, 3, 6, 12, 24, 48, 72 and 96 h for detection of LiTFSI concentration.

[0101] (4) The inoculation amount (4, 5, 6%) of step (2), the pH value (6, 7, 8) and the additional addition of 0.01% (mass percentage) of FeSO4, CuSO4, MnSO4, ZnSO4 in the basic salt solution (dibasic potassium phosphate 1.5 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium sulfate 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.1 g / L, calcium chloride 0.01 g / L) without adding trace metal mother liquor are changed respectively, and the LiTFSI concentration of the culture medium at time points of 0, 3, 6, 12, 24, 48, 72 and 96 h under each variable is detected, so as to explore the best degradation inoculation amount of Pseudomonas nitroreducens LiT2 and LiT3. I max , the best pH value P max , the best metal salt type M max .

[0102] The degradation kinetics of Pseudomonas nitroreducens LiT2 and LiT3 are shown in Figure 6 , wherein the LiTFSI degradation rate of Pseudomonas nitroreducens LiT2 is 51.52% and the LiTFSI degradation rate of Pseudomonas nitroreducens LiT3 is 56.19% at 96 h of degradation, and Pseudomonas nitroreducens LiT3 has a better degradation effect than Pseudomonas nitroreducens LiT2. The degradation condition optimization of Pseudomonas nitroreducens LiT2 and LiT3 is shown in Figure 7 . The best degradation inoculation amount I max of Pseudomonas nitroreducens LiT2 and LiT3 is 6%, the best pH value P max is 7, and the best metal salt type M max is FeSO4.

[0103] 2. Degradation pathway establishment and suspicious enzyme screening (1) Based on the optimization results of step 1, taking Nitroreducing Pseudomonas LiT3, which has a better degradation effect, as an example, in a basic salt solution containing 100 mg / L LiTFSI with an additional 0.01% (mass percentage) FeSO4, an inoculation amount of 6% (volume percentage) was added, and OD was inoculated. 600 The seed culture of *Pseudomonas nitroreductoids* LiT3 with a concentration of 1 was cultured at pH 7, 30°C, and 160 rpm for 96 h.

[0104] (2) The LiTFSI degradation products trifluoromethanesulfonic acid, trifluoromethanesulfonamide, difluoromethanesulfonic acid, monofluoromethanesulfonic acid, hydroxymethanesulfonic acid, and formaldehyde in the culture medium were detected by non-targeting using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry. The detection conditions were as follows: a C18 column (150 mm × 2.1 mm, 2.6 μm) was used, mobile phase A was pure water, mobile phase B was methanol, and the chromatographic separation gradient program was as follows: 0-3 min, 5% (volume percentage, the same below) B; 3-18 min, 5-95% B; 18-20 min, 95% B; 20-20.5 min, 95-5% B; 20.5-25 min, 5% B.

[0105] (3) The detected LiTFSI degradation products were tandemly linked to form a LiTFSI biodegradation pathway. Combined with the whole genome information of the strain detected in Example 2, the NS bond cleavage enzymes of Nitroreducing Pseudomonas LiT3 involved in the LiTFSI degradation process were screened. 、 Denitrase, desulfonase 、 Dehalogenase.

[0106] LiTFSI degradation products and biodegradation pathways of *Pseudomonas nitroreductoids* LiT3: Figure 8 As shown, Figure 8 Mass spectrometry information of LiTFSI degradation products trifluoromethanesulfonic acid (TFOH), trifluoromethanesulfonamide (TFNH2), difluoromethanesulfonic acid, monofluoromethanesulfonic acid, and hydroxymethanesulfonic acid was obtained, and the degradation pathway of "LiTFSI→trifluoromethanesulfonic acid→trifluoromethanesulfonamide→difluoromethanesulfonic acid→monofluoromethanesulfonic acid→hydroxymethanesulfonic acid→formaldehyde" was cascaded. The results also show that the degradation process of soil LiTFSI degrading bacteria includes four consecutive steps: hydrolysis, deamination, stepwise deammoniation, and sulfonation, which ultimately leads to mineralization and integration into the central metabolic cycle (glyoxylic acid and tricarboxylic acid cycle). This pathway is supported by genome annotation and highlights the strain-specific enzymatic activity.

[0107] The key degradation enzymes mainly involved in the LiTFSI degradation process can be determined by screening enzymes involved in the degradation of LiTFSI degradation products through changes in the groups of the degradation products in the degradation pathway, wherein the N-S bond breaking enzymes include amidases, alkyl sulfonic acid monooxygenases, etc., the denitrification enzymes include biuret amide hydrolase, nitro monooxygenase, etc., the dehalogenases include halogenated acetic acid dehalogenase, etc., and the desulfonases include alkyl sulfonic acid monooxygenase, taurine dioxygenase, etc., and the specific results are shown in Table 4.

[0108] Table 4 Statistics of key degradation enzymes involved in the LiTFSI degradation process Example 4

[0109] In order to improve the remediation efficiency of PFAS and minimize the accumulation of secondary pollutants, this embodiment further studies the synergistic degradation effect of Pseudomonas nitroreducens LiT3 or LiT2 and other degrading strains (named strain B in this embodiment) to form a composite microbial agent in combination with toxicological effect evaluation. The specific method is as follows: 1. Strain screening Pseudomonas nitroreducens LiT3 or LiT2 and strain B are combined to form a composite microbial agent, wherein the key degradation enzymes expressed by strain B need to meet the following conditions: (1) If the biological toxicological effect of LiTFSI is the lowest value among LiTFSI, TFNH2 and TFOH, the key degradation enzyme Z that strain B needs to express dominantly is N-S bond breaking enzyme; (2) If the biological toxicological effect of TFNH2 is the lowest value among LiTFSI, TFNH2 and TFOH, the key degradation enzyme Z that strain B needs to express dominantly is denitrification enzyme; (3) If the biological toxicological effect of TFOH is the lowest value among LiTFSI, TFNH2 and TFOH, the key degradation enzyme Z that strain B needs to express dominantly is desulfonase.

[0110] According to the above three conditions combined with the toxicological effect evaluation results in Example 1, it is confirmed that the key degradation enzyme expressed by strain B is denitrification enzyme.

[0111] 2. Intervention of composite microbial agent in the biotransformation process of LiTFSI in soil In order to improve the compatibility of the composite microbial agent, the strain of Pseudomonas is preferentially selected as strain B. Based on the above conditions, Pseudomonas nitroreducens LiT3 and Pseudomonas putida KT2440 capable of expressing denitrification enzyme are selected as strain B to form a composite microbial agent, and the intervention degradation is carried out, and the specific method is as follows: (1) The strains LiT3 and Pseudomonas putida KT2440 (purchased from Bio-sci Company) were respectively cultured in LB liquid medium at 30°C, 160 rpm to the optical density OD 600 =1 to prepare the seed liquid of each strain.

[0112] (2) 6mL of the seed liquid was respectively centrifuged at 8000 rpm for 3 min to precipitate the bacteria, washed with PBS solution for three times, and then added to the basic salt solution containing 1 mg / L LiTFSI for 24h to activate the enzyme expression function of the strains LiT3 and Pseudomonas putida KT2440.

[0113] (3) After centrifugation at 8000 rpm for 3 min to precipitate the bacteria and washing with PBS solution for three times, the bacteria were resuspended with the basic salt solution to prepare the bacterial liquid with OD 600 =1.1.

[0114] (4) The bacterial liquid was mixed according to the volume ratio of LiT3 : KT2440 = 1:1 to prepare the composite microbial agent.

[0115] (5) 40g of the mixed C1, C2 and C3 soil collected in Example 2 was taken into a centrifugal tube, and the mixed bacterial liquid was added according to the ratio of soil: composite microbial agent = 10g: 0.75mL, and the same volume of sterile basic salt solution was added to the control group.

[0116] (6) 0.5mL of pure water containing 0.5% (mass percentage, the same below) glucose and 0.1% KH2PO4 was added to promote the survival of bacteria and enzyme expression, and the soil was shaken uniformly.

[0117] (7) The centrifugal tube was sealed with a sealing film and punctured for ventilation, and cultured at the optimal pH value of 7, room temperature and in the dark.

[0118] (8) The soil was sampled every 24h, and the concentrations of LiTFSI, TFNH2 and TFOH in the soil were detected according to the method of step 1.

[0119] (9) If the reduction amounts of LiTFSI, TFNH2 and TFOH concentrations are all >50%, it is considered that the intervention effect is good; if the reduction amounts of LiTFSI, TFNH2 and TFOH concentrations are ≤50%, the intervention time is prolonged until the intervention effect is good. The calculation method is: concentration reduction amount = (initial concentration - concentration detected every 24h) x 100% ÷ initial concentration.

[0120] The concentration graphs of LiTFSI, TFNH2 and TFOH are as follows: Figure 9As shown in the figure, under the intervention of Pseudomonas nitroreducens LiT3 alone, the concentration of LiTFSI in the system decreased significantly, and the concentrations of TFNH2 and TFOH increased, indicating that the addition of Pseudomonas nitroreducens LiT3 would promote the degradation of LiTFSI into TFNH2 and TFOH. Under the intervention of Pseudomonas putida KT2440 alone, the concentrations of LiTFSI and TFNH2 decreased, but there was no significant difference in TFOH compared with the control. Under the intervention of the complex microbial agent composed of LiT3 and KT2440, the concentrations of LiTFSI, TFNH2 and TFOH all decreased significantly, among which the reduction amounts of LiTFSI, TFNH2 and TFOH were 58.3%, 62.4% and 65.3% respectively, all > 50%, indicating that the intervention effect of the complex microbial agent was good.

[0121] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A LiTFSI-degrading bacterium, characterized in that Pseudomonas nitroreducens (ATCC 25259) Pseudomonas nitroreducens ) LiT2 or LiT3; deposited on December 01, 2025 at Guangdong Microbial Culture Collection Center, located at 59# Building, 5th Floor, 100 Institute, Guangzhou, Guangdong, China, with accession numbers GDMCC No: 67389 and GDMCC No: 67390, respectively.

2. The culture method of the LiTFSI-degrading bacteria according to claim 1, characterized by comprising the following steps: The LiTFSI-degrading bacteria of claim 1 are inoculated into a LiTFSI-containing basic salt solution for culture.

3. The culture method of the LiTFSI-degrading bacteria according to claim 2, characterized in that: The culture conditions are as follows: inoculation amount of 4-6%, pH value of 6-8, and temperature of 20-40°C.

4. Use of the LiTFSI-degrading bacteria of claim 1 in the field of environmental remediation.

5. Use of the LiTFSI-degrading bacteria of claim 1 in degrading perfluoro- and polyfluoroalkyl compounds and precursor compounds thereof.

6. An environmental remediation bacteria agent, characterized by The LiTFSI-degrading bacteria of claim 1.

7. The environmental remediation bacteria of claim 6, wherein Other strains that can express key degrading enzymes are also included; The key degrading enzyme is an N-S bond breaking enzyme 、 at least one of a deaminase, a desulfonase, and a dehalogenase.

8. The environmental remediation bacteria agent according to claim 7, characterized in that: The other strains that can express key degrading enzymes are Pseudomonas putida KT2440.

9. Use of the environmental remediation bacteria agent of any one of claims 6-8 in the field of environmental remediation.

10. A method for remediation of soil using a soil remediation bacterial agent, characterized by comprising the following steps: (1) The LiTFSI-degrading bacteria of claim 1 and Pseudomonas putida KT2440 are respectively cultured to obtain corresponding seed liquids; (2) The seed liquids are centrifuged and washed with PBS solution, and then added into a LiTFSI-containing basic salt solution for culture for 12-24 h to activate the enzyme expression function of the strains LiT3 and Pseudomonas putida KT2440; (3) The bacterial liquids in step (2) are respectively centrifuged and washed with PBS solution, and then resuspended with a basic salt solution; (4) The LiTFSI-degrading bacteria liquid and Pseudomonas putida KT2440 liquid obtained in step (3) are mixed to obtain a soil remediation bacteria agent; (5) The soil to be treated is mixed with the soil remediation bacteria agent prepared in step (4) and subjected to remediation.

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