A soil LiTFSI-degrading bacterium and its application in environmental remediation.

Abstract

This invention relates to the field of microbial technology, and particularly to a soil LiTFSI-degrading bacterium and its application in environmental remediation. The LiTFSI-degrading bacterium is *Pseudomonas nitroreductoides* (Nitroreducing Pseudomonas). Pseudomonas nitroreducens LiT2 or LiT3 were deposited on December 1, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, with accession numbers GDMCC No: 67389 and GDMCC No: 67390, respectively. This soil LiTFSI-degrading bacterium can use LiTFSI as its sole carbon source and exhibits excellent degradation efficiency for LiTFSI. This invention also provides an environmental remediation agent that can precisely and rapidly intervene in and enhance the degradation process of LiTFSI in soil, thereby significantly reducing the pollution levels of LiTFSI and its intermediate metabolites TFNH2 and TFOH.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a soil LiTFSI-degrading bacterium and its application in environmental remediation. Background Technology

[0002] The global transition to renewable energy has made lithium-ion batteries (LIPBs) central to electric vehicles, renewable energy storage, and consumer electronics. However, the large-scale dismantling, crushing, sorting, and purification processes involved in LIPB recycling release vast amounts of organic and inorganic pollutants. These substances exhibit environmental persistence, mobility, and potential biotoxicity, and our understanding of their environmental behavior, degradation patterns, and health risks remains insufficient.

[0003] Perfluorinated and polyfluoroalkyl substances (PFAS) are widely used in electrolyte systems due to their excellent conductivity and chemical stability. Among them, ultrashort-chain PFAS (such as trifluoromethanesulfonamide, TFNH2; trifluoromethanesulfonic acid, TFOH) pose more prominent ecological and health risks due to their higher water solubility and mobility. In environmental monitoring conducted on LIPB recycling sites, TFNH2 was confirmed as one of the highly toxic PFAS; despite a systematic investigation of battery raw materials and LIPBCP, its source remains unclear. Structural comparison indicates that lithium bis(trifluoromethanesulfonyl)imide (LiTFSI, molecular skeleton LiN(SO2CF3)2) can be hydrolyzed to generate TFNH2 (CF3SO2NH2) and TFOH (CF3SO3H), thus potentially being an important precursor. As a new generation of electrolyte lithium salts, the environmental behavior, biotransformation pathways, and association with ultrashort-chain PFAS in soil media of LiTFSI have not yet been systematically elucidated.

[0004] Soil microbial communities are the most diverse biological systems on the Earth's surface, driving organic matter decomposition and geochemical cycles. While PFAS pollution can suppress community diversity and abundance, some groups, including Pseudomonas and Acidobacteria, have shown degradation potential. Literature reports that Pseudomonas can degrade sulfonamide organofluorine compounds under aerobic conditions and has revealed key genes for desulfurization and defluorination; Acidobacteria have also been shown to promote defluorination through oxidation pathways in PFAS-stressed soils. However, the microbial degradation conditions, mechanisms of action, and transformation pathways of LiTFSI in soil remain largely unknown. Furthermore, how to avoid or reduce the accumulation of secondary pollutants (such as TFNH2 and TFOH) in engineering remediation remains a key challenge in the field of environmental bioremediation.

[0005] Therefore, it is urgent to establish an integrated technical system that integrates environmental monitoring, toxicological assessment, functional bacteria screening, pathway analysis and synergistic intervention, and to discover and optimize functional bacteria and compound bacterial agents that can efficiently degrade LiTFSI and its related ultra-short chain PFAS, so as to achieve precise, rapid and environmentally friendly in-situ remediation of contaminated soil (and water bodies and sediments). Summary of the Invention

[0006] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a LiTFSI degrading bacterium.

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

[0008] Another object of the present invention is to provide an environmental remediation microbial agent.

[0009] The fourth object of the present invention is to provide the application of the above-mentioned environmental remediation microbial agent.

[0010] The fifth objective of this invention is to provide a method for remediating soil using the aforementioned environmental remediation microbial agent.

[0011] The objective of this invention is achieved through the following technical solution:

[0012] A LiTFSI-degrading bacterium, named Nitroreducing Pseudomonas ( Pseudomonas nitroreducens LiT2 or LiT3 were deposited on December 1, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, with accession numbers GDMCC No: 67389 and GDMCC No: 67390 respectively.

[0013] The method for culturing LiTFSI-degrading bacteria includes the following steps:

[0014] The above-mentioned LiTFSI-degrading bacteria were inoculated into a basic salt solution containing LiTFSI and cultured.

[0015] The preferred culture conditions are an inoculum size of 4-6%, a pH of 6-8, and a temperature of 20-40℃.

[0016] The preferred culture conditions are an inoculum size of 6%, a pH of 7, and a temperature of 30°C.

[0017] The basic salt solution comprises the following components: 1.5 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, 0.2 g / L magnesium sulfate heptahydrate, 0.1 g / L sodium chloride, 0.01 g / L calcium chloride, and 1 mL / L trace element stock solution.

[0018] The trace element mother liquor contains 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.

[0019] The base salt solution preferably also contains an additional 0.01% by mass of ferrous sulfate (FeSO4).

[0020] The application of the LiTFSI degrading bacteria in the field of environmental remediation.

[0021] The environmental remediation mentioned includes, but is not limited to, soil, water bodies, and sediments.

[0022] The application of the LiTFSI degrading bacteria in the degradation of perfluorinated and polyfluoroalkyl compounds (PFAS) and their precursor compounds.

[0023] The perfluorinated and polyfluoroalkyl compounds mentioned include short-chain PFAS.

[0024] The short-chain PFAS include, but are not limited to, trifluoromethanesulfonamide (TFNH2) and trifluoromethanesulfonic acid (TFOH).

[0025] The precursor compounds include, but are not limited to, LiTFSI.

[0026] An environmental remediation microbial agent comprising the aforementioned LiTFSI-degrading bacteria.

[0027] The environmental remediation microbial agent preferably also contains other strains that can express key degrading enzymes.

[0028] The key degrading enzyme may be an N / S bond cleavage enzyme. 、 At least one of denitrase, desulfonase, and dehalogenase.

[0029] The other strains that can express the key degrading enzymes are preferably Pseudomonas.

[0030] The other strains that can express the key degrading enzymes are further preferably *Pseudomonas putida* KT2440, which can efficiently express denitrases.

[0031] The application of the aforementioned environmental remediation microbial agent in the field of environmental remediation.

[0032] A method for remediating soil using the above-mentioned soil remediation microbial agent includes the following steps:

[0033] (1) LiTFSI degrading bacteria and Pseudomonas putida KT2440 were cultured separately to obtain corresponding seed solutions;

[0034] (2) After centrifuging the seed culture and washing it with PBS solution, add it to the basic salt solution containing LiTFSI and culture for 12-24 h to activate the enzyme expression function of strains LiT3 and Pseudomonas putida KT2440.

[0035] (3) After centrifuging the bacterial culture in step (2) and washing it with PBS solution, resuspend it with basic salt solution;

[0036] (4) Mix the LiTFSI degrading bacteria solution obtained in step (3) with the Pseudomonas malodorans KT2440 bacteria solution to obtain a soil remediation agent;

[0037] (5) Mix the soil to be treated with the soil remediation agent prepared in step (4) and carry out remediation.

[0038] The culture medium used in step (1) is preferably LB liquid medium.

[0039] The concentration of LiTFSI in the LiTFSI-containing base salt solution mentioned in step (2) is preferably 1 mg / L.

[0040] In step (4), the density of LiTFSI degrading bacteria and Pseudomonas malodorosa KT2440 bacteria in the soil remediation microbial agent is preferably the same.

[0041] The preferred mass-to-volume ratio of soil and soil remediation microbial agent in step (5) is 10:(0.5-1) (g:mL).

[0042] Preferably, a solution containing glucose and KH2PO4 is also added to the mixed system described in step (5).

[0043] The preferred volume ratio of the solution containing glucose and KH2PO4 to the soil remediation microbial agent is 0.5:3.

[0044] The glucose and KH2PO4 solution contains glucose and KH2PO4 at mass percentages of 0.5% and 0.1%, respectively.

[0045] The conditions for the repair described in step (5) are pH 7, room temperature, and darkness.

[0046] Technical principle of the invention:

[0047] This invention constructs a complete "detection-toxicology-screening-optimization-analysis-intervention" microbial remediation technology system, which includes, in sequence: (1) detection of environmental distribution of LiTFSI, TFNH2, and TFOH; (2) toxicological effect assessment; (3) enrichment, isolation, and purification of Nitro-reducing Pseudomonas LiT2 and LiT3; (4) system optimization of degradation kinetic conditions; (5) analysis of LiTFSI microbial degradation pathways and screening of key functional enzymes; and (6) construction and degradation intervention of compound microbial agents. The details are as follows:

[0048] 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.

[0049] 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.

[0050] The present invention has the following advantages and effects compared with the prior art:

[0051] (1) This invention provides a soil LiTFSI degrading bacterium: Nitro-reducing Pseudomonas (N-N-N-N) Pseudomonas nitroreducensLiT2 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.

[0052] (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 M max This provides technical support for the application of soil LiTFSI degrading bacteria in the field of environmental remediation.

[0053] (3) This invention establishes a degradation pathway of soil LiTFSI degrading bacteria, which proceeds through four consecutive steps, including hydrolysis, deamination, stepwise deamination and sulfonation, ultimately leading to mineralization and integration into the central metabolic cycle (glyoxylic acid and tricarboxylic acid cycle). This pathway is supported by genome annotation and highlights strain-specific enzymatic activity.

[0054] (4) Based on the toxicological effects of LiTFSI, TFNH2, and TFOH and the key enzymes for LiTFSI degradation, this invention screens strains that synergistically enhance soil LiTFSI degradation bacteria. This screening method has better scientific rigor, systematicity, and targeting. The soil remediation compound microbial agent obtained by this invention based on this screening strategy can accurately and quickly intervene in and enhance the degradation process of LiTFSI in the soil, thereby significantly reducing its pollution level.

[0055] (5) This invention provides a novel soil LiTFSI degrading bacteria and a scientific bioremediation strategy for solving LiTFSI soil pollution, which has important practical significance and application prospects for ensuring environmental safety and human health. Attached Figure Description

[0056] Figure 1 These are colony morphology diagrams of nitroreducing Pseudomonas LiT2 and LiT3.

[0057] Figure 2 These are transmission electron microscope images of nitroreducing Pseudomonas LiT2 and LiT3.

[0058] Figure 3 This is a sequencing image of the 16S-rDNA genes of Nitroreducing Pseudomonas LiT2 and LiT3.

[0059] Figure 4This is a graph showing the average nucleotide identity between Nitroreducing Pseudomonas LiT2 and LiT3 and related Pseudomonas strains.

[0060] Figure 5 This is the complete genome map of Nitroreducing Pseudomonas LiT2 and LiT3.

[0061] Figure 6 This is a degradation kinetic diagram of nitroreducing Pseudomonas LiT2 and LiT3.

[0062] Figure 7 The figure shows the optimization results of degradation conditions for Nitroreducing Pseudomonas LiT2 and LiT3, where A: pH, B: inoculum size, and C: trace metals.

[0063] Figure 8 This is a mass spectrum and biodegradation pathway diagram of LiTFSI degradation products, where A: mass spectrometry, B: biodegradation pathway.

[0064] Figure 9 This is a graph showing the changes in the concentrations of LiTFSI, TFNH2, and TFOH in the soil under the intervention of compound microbial agents. In the graph, A represents LiTFSI, B represents TFOH, and C represents TFNH2. Detailed Implementation

[0065] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0066] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0067] The base salt solution in the examples contains the following components: 1.5 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, 0.2 g / L magnesium sulfate heptahydrate, 0.1 g / L sodium chloride, 0.01 g / L calcium chloride, and 1 mL / L trace element stock solution (2.78 g / L ferrous sulfate heptahydrate, 1.98 g / L manganese chloride tetrahydrate, 2.87 g / L zinc sulfate heptahydrate, and 0.17 g / L copper chloride dihydrate, all of which are final concentrations in the stock solution).

[0068] The LB liquid culture medium in the examples contains the following components: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride. The LB solid culture medium is the LB liquid culture medium with 15-20 g / L agar added.

[0069] LiTFSI, TFNH2, and TFOH were purchased from Ampro.

[0070] Example 1: Toxicological Effects Assessment

[0071] 1. LC50 toxicological effect concentration

[0072] H9C2 cardiomyocytes were selected to represent cardiotoxicity. h HEK-293T kidney cells represent metabolic toxicity. m Zebrafish (wild-type AB strain) embryonic developmental toxicity d Caenorhabditis elegans (wild-type N2 strain) represents neurotoxicity. v Following standard methods, using methods representing cardiotoxicity... h Metabolic toxicity m Developmental toxicity d Neurotoxicity v Four biomarkers were used to establish PFAS substances l i ( i =1,2,3 l 1 = LiTFSI, l 2 = TFNH2, l 3 = LC of TFOH 50 Toxicological effect concentration Q ij ,in:

[0073] (1) For H9C2 cardiomyocytes and HEK-293T kidney cells: following standard procedures, cells were seeded in DMEM medium containing different concentrations of contaminants and treated for 24 h. Cell viability was determined using the MTT assay, and LC50 was calculated. 50 value.

[0074] (2) For zebrafish embryos: Zebrafish embryos (4-6 hpf) were inoculated in E3 medium containing different concentrations of contaminants and treated for 96 h. The solution was changed daily and the LC was calculated. 50 value.

[0075] (3) For Caenorhabditis elegans: L1 stage nematodes that have undergone synchronous treatment were inoculated into Caenorhabditis elegans growth medium containing different concentrations of pollutants and treated for 24 h. The LC ratio was calculated. 50 value.

[0076] 2. Toxicological Effects Assessment

[0077] Based on LC50 toxicological effect concentration A comprehensive assessment of the biotoxicological effects of LiTFSI, TFNH2, and TFOH. The formulas for assessing the biological toxicological effects of LiTFSI, TFNH2, and TFOH are as follows:

[0078]

[0079] In the formula:

[0080] PFAS substance biomarkers The weighting coefficients, and .

[0081] PFAS substance The LC50 toxicological effect concentrations of several biological indicators .

[0082] PFAS substance The LC50 toxicological effect concentrations of several biological indicators .

[0083] This is the Hill coefficient.

[0084] It is a non-linear mapping exponent.

[0085] This is the interaction coefficient.

[0086] PFAS substance The overall toxicological effect coefficient, , .

[0087] PFAS substance biomarkers The average value.

[0088] PFAS substance The biological toxicological effects, , .

[0089] in, .

[0090] The biological toxicological parameters of LiTFSI, TFNH2, and TFOH are shown in Table 1.

[0091] Table 1. Toxicological effects of LiTFSI, TFNH2, and TFOH on organisms.

[0092] Example 2

[0093] 1. Environmental distribution detection of LiTFSI, TFNH2, and TFOH

[0094] (1) Soil samples (a total of 6 samples) collected from a lithium-ion battery recycling site in Huizhou City, Guangdong Province were freeze-dried and weighed. Soil samples collected outside the battery recycling site that were not directly contaminated by PFAS were used as a control.

[0095] (2) Soil samples were subjected to power ultrasonic extraction using methanol (0.1% NH4OH-MeOH) containing 0.1% (volume percentage) ammonium hydroxide. The ultrasonic extraction power was 100 kHz and the time was 30 min. The extract was then collected.

[0096] (3) Load the extract collected in step (2) onto an ENVI-Carb solid phase extraction column and use 0.1% NH4OH-MeOH as the eluent to separate and purify LiTFSI, TFNH2 and TFOH in the extract, and collect the purified solution.

[0097] (4) Blow the purified liquid nitrogen collected in step (3) to dryness, and then re-adjust the volume with 0.1% NH4OH-MeOH to prepare the sample to be tested. C e (e=1, 2, ..., n), where n is the total number.

[0098] (5) Targeted detection of the test sample was performed using an ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometer. C e The concentrations of LiTFSI, TFNH2, and TFOH in the atmosphere were determined using the following method: a C18 column (150 mm × 2.1 mm, 2.6 μm) was used, with mobile phase A being 5 mM ammonium acetate and mobile phase B being methanol. The chromatographic separation gradient program was as follows: 0–1 min, 5% (volume percentage, the 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. The environmental distribution concentration table of LiTFSI, TFNH2, and TFOH was obtained.

[0099] The environmental distribution and concentration results of LiTFSI, TFNH2, and TFOH are shown in Table 2. As can be seen from the table, LiTFSI had the highest detection rate and concentration among all samples, reaching a detection rate of 100%.

[0100] Table 2 Environmental Concentration Table of LiTFSI, TFNH2, and TFOH

[0101]

[0102] Note: nd = below the detection limit.

[0103] 2. Enrichment, isolation, and purification of Nitroreducing Pseudomonas LiT2 and LiT3

[0104] (1) Take 5g of the sample from step 1 containing the three substances LiTFSI, TFNH2, and TFOH, with the highest sum of TFNH2 and TFOH concentrations. C1 Soil was placed in an Erlenmeyer flask, 100 mL of basic salt solution was added, and the culture was shaken at 30 °C and 160 rpm for 24 h to allow the culture to reach the logarithmic phase.

[0105] (2) Take 1 mL of the culture obtained in step (1) and inoculate it into 100 mL of a basic salt solution containing 20 mg / L LiTFSI. Shake and culture at 30 °C and 160 rpm for 24 h to allow the culture to reach the logarithmic phase.

[0106] (3) Repeat step (2) in sequence, increasing the LiTFSI concentration in the culture medium by 20 mg / L each time, until the LiTFSI concentration in the culture medium increases to 200 mg / L.

[0107] (4) Take 1 mL of the culture obtained in step (3), dilute it by a certain factor, spread it evenly on LB solid medium, and culture it. Based on the differences in colony color, morphology, etc., different strains are isolated and purified by repeated streak plating.

[0108] 3. Morphological characteristics identification

[0109] The strains isolated in step 1 were cultured on LB solid medium, and the colony color and morphological characteristics were observed and recorded. Cell morphology was also observed under a microscope.

[0110] 4. Physiological and biochemical identification

[0111] Referring to the "Manual for Systematic Identification of Common Bacteria", the strains isolated in step 1 were used as the subjects for methyl red tests, etc.

[0112] 5. Sequence homology analysis, phylogenetic tree construction, and whole-genome sequencing, assembly, and annotation.

[0113] (1) Using the strains isolated in step 1 as the target, genomic DNA was extracted using a DNA extraction kit (e.g., Omega Bio-tek DNA kit), and PCR amplification was performed using 515F (5'-GTGCCAGCAGCCGCGGTAA-3') and 806R (5'-GGACTACCAGGGTATCTAA-3'). The obtained PCR products were purified and sequenced. Based on the sequencing results, blast was performed and a phylogenetic tree was constructed (Meiji Biotechnology Co., Ltd.) to confirm the genus of strains LiT2 and LiT3.

[0114] (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.

[0115] 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 .

[0116] 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 ).

[0117] 16S rDNA sequence of strain LiT2 (SEQ ID No:1):

[0118]

[0119] 16S rDNA sequence of strain LiT3 (SEQ ID No:2):

[0120]

[0121] Table 3. Comparison of 16S rDNA results between strains LiT2 and LiT3

[0122]

[0123] Further analysis of the average nucleotide identity between strains LiT2 and LiT3 and related Pseudomonas strains is shown in the figure. Figure 4 As can be seen from the figure, the average nucleotide identity values ​​of Pseudomonas LiT2 and LiT3 with other similar Pseudomonas strains (LiT2: 86.9%-91.85%, LiT3: 86.95%-92.13%) are all lower than the species classification threshold (95%), indicating that Pseudomonas LiT2 and LiT3 are newly discovered Pseudomonas species. The average nucleotide identity value between Pseudomonas LiT2 and LiT3 is less than the species classification threshold (95%) < 95.42% < 99%, further indicating that strains LiT2 and LiT3 are the same species but different subspecies.

[0124] The complete genome diagrams of strains LiT2 and LiT3 are shown below. Figure 5 As shown in the figure. The figure displays the complete genome chains of Pseudomonas LiT2 and LiT3. By comparing the complete genome sequences with the genome sequences on the NCBI-BLAST website, it was further confirmed that strains LiT2 and LiT3 are nitro-reducing Pseudomonas (Nitro-reducing Pseudomonas). Pseudomonas nitroreducens A new subspecies of ).

[0125] Based on the morphological, physiological, biochemical, and biomolecular identification results of strains LiT2 and LiT3, strains LiT2 and LiT3 are classified as *Pseudomonas nitroreductans* (Nitroreductobacteria). Pseudomonas nitroreducens ), respectively named Nitroreducing Pseudomonas ( Pseudomonas nitroreducens LiT2 and Nitroreducing Pseudomonas ( Pseudomonas nitroreducens LiT3 was deposited on December 1, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, with accession numbers GDMCC No: 67389 and GDMCC No: 67390.

[0126] Example 3

[0127] 1. Optimization of degradation kinetic conditions

[0128] (1) Nitro-reducing Pseudomonas LiT2 and LiT3 obtained by isolation and purification in Example 2 were cultured in LB liquid medium at 30°C and 160 rpm for 12 h to prepare seed liquids of different strains.

[0129] (2) The seed culture obtained in step (1) was inoculated into a basic salt solution containing 100 mg / L LiTFSI at a volume ratio of 4%, and cultured for 96 h at pH=7, 30℃ and 160 rpm. A basic salt solution containing 100 mg / L LiTFSI without any bacteria was used as a control.

[0130] (3) Take 20 mL of solution at time points of 0, 3, 6, 12, 24, 48, 72 and 96 h to detect the LiTFSI concentration.

[0131] (4) The inoculum size (4%, 5%, 6%), pH value (6%, 7%, 8) and the addition of 0.01% (mass percentage) of FeSO4, CuSO4, MnSO4 and ZnSO4 to the base salt solution (1.5 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium sulfate, 0.2 g / L magnesium sulfate heptahydrate, 0.1 g / L sodium chloride, 0.01 g / L calcium chloride) without the addition of trace metal mother liquor were measured. The LiTFSI concentration in the culture medium at 0, 3, 6, 12, 24, 48, 72 and 96 h was measured under each variable to explore the optimal inoculum size for degradation of Pseudomonas nitroreductoids LiT2 and LiT3. I max Optimal pH value P max Optimal metal salt type M max .

[0132] Nitroreducing Pseudomonas degradation kinetics of LiT2 and LiT3 are as follows: Figure 6 As shown, after 96 hours of degradation, the LiTFSI degradation rate of *Pseudomonas nitroreductoids* LiT2 was 51.52%, and that of *Pseudomonas nitroreductoids* LiT3 was 56.19%. *Pseudomonas nitroreductoids* LiT3 showed superior degradation performance compared to *Pseudomonas nitroreductoids* LiT2. The optimization of degradation conditions for *Pseudomonas nitroreductoids* LiT2 and LiT3 is as follows: Figure 7 As shown. Optimal inoculum sizes for degradation by *Pseudomonas nitroreductoids* LiT2 and LiT3. I max All are 6%, optimal pH value P max All are 7, the best type of metal salt. M max All are FeSO4.

[0133] 2. Establishment of degradation pathways and screening of suspected enzymes

[0134] (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.

[0135] (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.

[0136] (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.

[0137] Mass spectrometry and biodegradation pathways of LiTFSI degradation products of Nitroreducing Pseudomonas LiT3, such as 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.

[0138] By screening enzymes involved in LiTFSI degradation through group changes in LiTFSI degradation products in the degradation pathway, key degradation enzymes mainly involved in the LiTFSI degradation process can be identified. Among them, NS bond cleavage enzymes include amidases and alkyl sulfonate monooxygenases, denitrases include biuretamide hydrolases and nitro monooxygenases, dehalogenases include haloacetic acid dehalogenases, and desulfonases include alkyl sulfonate monooxygenases and taurine dioxygenases. The specific results are shown in Table 4.

[0139] Table 4. Statistical table of key degrading enzymes involved in the LiTFSI degradation process

[0140] Example 4

[0141] To improve the remediation efficiency of PFAS and minimize the accumulation of secondary pollutants, this embodiment, combined with toxicological effect assessment, further investigates the synergistic degradation effect of a composite agent composed of Nitroreducing Pseudomonas LiT3 or LiT2 and other degrading strains (named strain B in this embodiment). The specific method is as follows:

[0142] 1. Strain screening

[0143] A compound bacterial agent is prepared by combining Nitrorereducing Pseudomonas LiT3 or LiT2 with strain B, wherein the key degrading enzyme expressed by strain B must meet the following conditions:

[0144] (1) If the biological toxicological effects of LiTFSI If the value of LiTFSI, TFNH2, and TFOH is the lowest, then the key degradative enzyme Z that strain B needs to express dominantly is an N-S bond cleavage enzyme.

[0145] (2) If the biological toxicological effects of TFNH2 If the values ​​of LiTFSI, TFNH2, and TFOH are the lowest, then the key degradative enzyme Z that strain B needs to express dominantly is denitrase;

[0146] (3) If the biological toxicological effects of TFOH If the values ​​of LiTFSI, TFNH2, and TFOH are the lowest, then strain B needs to express the key degradative enzyme Z, which is a desulfonase.

[0147] Based on the above three conditions and the toxicological effect assessment results in Example 1, it was confirmed that the key degrading enzyme expressed by strain B is a denitrase.

[0148] 2. Intervention of compound microbial agents in the biotransformation process of LiTFSI in soil

[0149] To improve the compatibility of the compound microbial agent, strains of the genus *Pseudomonas* were preferentially selected as strain B. Based on the above conditions, using *Pseudomonas nitroreductoides* LiT3 and *Pseudomonas putida* KT2440, which expresses denitrase, as strain B to form the compound microbial agent, intervention degradation was carried out. The specific method is as follows:

[0150] (1) Strains LiT3 and Pseudomonas putida KT2440 (purchased from Bio-sci) were cultured in LB liquid medium at 30°C and 160 rpm until the bacterial culture optical density OD reached the specified values. 600 =1 to prepare seed solutions for each strain.

[0151] (2) Centrifuge 6 mL of seed culture at 8000 rpm for 3 min to precipitate bacteria. After washing three times with PBS solution, add them to a basic salt solution containing 1 mg / L LiTFSI and culture for 24 h to activate the enzyme expression function of strains LiT3 and Pseudomonas putida KT2440.

[0152] (3) After centrifuging at 8000 rpm for 3 min to precipitate bacteria and washing three times with PBS solution, the bacteria were resuspended in basal salt solution to prepare OD. 600 =1.1 bacterial solution.

[0153] (4) Mix the bacterial culture according to the ratio of LiT3:KT2440 = Mix them in a 1:1 volume ratio to prepare a compound microbial agent.

[0154] (5) Weigh 40g of the soil samples collected in Example 2 from C1, C2 and C3 and put them into a centrifuge tube. Add the mixed bacterial solution according to the ratio of soil: compound bacterial agent = 10g: 0.75mL. Add the same volume of sterile basic salt solution to the control group.

[0155] (6) Add 0.5 mL of pure water containing 0.5% (mass percentage, the same below) glucose and 0.1% KH2PO4 to promote bacterial survival and enzyme expression, and shake the soil evenly.

[0156] (7) Seal the centrifuge tube opening with sealing film and poke holes to allow air to pass through. Incubate at the optimal pH of 7, room temperature and in the dark.

[0157] (8) Take samples every 24 hours and test the concentrations of LiTFSI, TFNH2 and TFOH in the soil according to the method in step 1.

[0158] (9) If the reduction in the concentrations of LiTFSI, TFNH2, and TFOH is greater than 50%, the intervention effect is considered good; if the reduction in the concentrations of LiTFSI, TFNH2, and TFOH is less than or equal to 50%, the intervention time should be extended until the intervention effect is good. The calculation method is: concentration reduction = (initial concentration - concentration measured by sampling every 24 hours) × 100% ÷ initial concentration.

[0159] The concentrations of LiTFSI, TFNH2, and TFOH are shown in the graph below. Figure 9 As shown in the figure, the concentration of LiTFSI in the system was significantly reduced and the concentrations of TFNH2 and TFOH increased under the intervention of *Pseudomonas nitroreductoids* LiT3 alone, indicating that the addition of *Pseudomonas nitroreductoids* LiT3 promoted the degradation of LiTFSI into TFNH2 and TFOH. Under the intervention of *Pseudomonas putida* KT2440 alone, the concentrations of LiTFSI and TFNH2 decreased, but the concentrations of TFOH were not significantly different from the control. Under the intervention of the combined bacterial agent composed of LiT3 and KT2440, the concentrations of LiTFSI, TFNH2, and TFOH were all significantly reduced, with reductions of 58.3%, 62.4%, and 65.3% respectively, all greater than 50%, indicating that the combined bacterial agent had a good intervention effect.

[0160] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A LiTFSI-degrading bacterium, characterized in that The names are *Pseudomonas nitroreducens* LiT2 or LiT3; they were deposited on December 1, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, 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 It includes the following steps: The LiTFSI-degrading bacteria of claim 1 were inoculated into a basic salt solution containing LiTFSI and cultured.

3. The method for culturing LiTFSI-degrading bacteria according to claim 2, characterized in that: The culture conditions are: inoculum size of 4-6%, pH value of 6-8, and temperature of 20-40℃.

4. The application of the LiTFSI degrading bacteria according to claim 1 in the field of environmental remediation, characterized in that: The remediation involves degrading or eliminating the contamination caused by LiTFSI.

5. The application of the LiTFSI-degrading bacteria according to claim 1 in the degradation of LiTFSI.

6. An environmental remediation microbial agent, characterized in that... It contains the LiTFSI degrading bacteria as described in claim 1.

7. The environmental remediation microbial agent according to claim 6, characterized in that... It also includes other strains that can express key degrading enzymes; The key degrading enzyme is at least one of N / S bond cleavage enzyme, denitrase, desulfonase, and dehalogenase.

8. The environmental remediation microbial agent according to claim 7, characterized in that: The other strain that can express the key degrading enzyme is *Pseudomonas putida* KT2440.

9. The application of the environmental remediation microbial agent according to claim 8 in the field of environmental remediation, characterized in that: The remediation involves degrading or eliminating contamination caused by at least one of TFNH2, TFOH, and LiTFSI.

10. A method for remediating soil using soil remediation microbial agents, characterized in that... It includes the following steps: (1) The LiTFSI degrading bacteria and Pseudomonas putida KT2440 described in claim 1 were cultured respectively to obtain the corresponding seed liquid; (2) After centrifuging the seed culture and washing it with PBS solution, add it to the basic salt solution containing LiTFSI and culture for 12-24 h to activate the enzyme expression function of strains LiT3 and Pseudomonas putida KT2440. (3) After centrifuging the bacterial culture in step (2) and washing it with PBS solution, resuspend it with basic salt solution; (4) Mix the LiTFSI degrading bacteria solution obtained in step (3) with the Pseudomonas malodorans KT2440 bacteria solution to obtain a soil remediation agent; (5) Mix the soil to be treated with the soil remediation agent prepared in step (4) and carry out remediation; The remediation involves degrading or eliminating contamination caused by at least one of TFNH2, TFOH, and LiTFSI.

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