Facultative anaerobic iron-reducing bacteria and application thereof in treatment of emerging pollutants

Abstract

The present application belongs to the field of biological remediation of perfluorooctanoic acid and its salt and related compounds, and particularly relates to a facultative anaerobic iron-reducing bacteria and its application in treating new pollutants (perfluorooctanoic acid and its salt and related compound pollution) in contaminated soil and water. The facultative anaerobic iron-reducing bacteria (Clostridium diolis) FLP22 has been preserved in China Center for Type Culture Collection on November 21, 2023, and the preservation registration number is CCTCC NO: M 20232292. The strain is suitable for metabolic degradation of PFOA in soil and water under aerobic conditions and anaerobic conditions. Therefore, the strain has the ability to completely mineralize and degrade PFOA in soil and water and reduce the toxicity of the environmental medium.

Description

Technical Field

[0001] This invention belongs to the field of bioremediation of perfluorooctanoic acid (PFOA) and its salinization and related compounds, and specifically relates to a facultative anaerobic iron-reducing bacterium and its application in treating new pollutants (PFOA and its salinization and related compounds) in contaminated soil and water. Background Technology

[0002] Emerging pollutants (ECs) refer to newly discovered or identified pollutants that pose risks to the ecological environment or human health, and which are not yet included in management or whose risks are not effectively controlled by existing management measures. In May 2022, the "Action Plan for the Governance of Emerging Pollutants" outlined the overall requirements, action measures, and safeguards for the governance of emerging pollutants. In 2023, multiple national departments jointly proposed the "List of Key Emerging Pollutants under Control," which specifically includes dichloromethane, antibiotics, pentachlorophenol and its salts and esters, perfluorooctanesulfonic acid and its salts and perfluorooctanesulfonyl fluoride (PFOS), perfluorooctanoic acid and its salts and related compounds (PFOA), etc. Studies have shown that ECs possess significant characteristics such as biotoxicity, environmental persistence, and bioaccumulation. Although their concentrations in the environment are very low, their chemical stability, long-term environmental persistence, and easy accumulation in organisms pose a significant risk to the ecological environment and human health. Therefore, research on practical remediation technologies for emerging pollutants is urgently needed.

[0003] Soil is a significant reservoir of new pollutants. While ECs from other media often eventually migrate to soil and aquatic sediments, new pollutants in soil can also be released back into other media over time due to factors such as groundwater flow. Photocatalytic degradation, zero-valent metal reduction, and electrochemical oxidation-reduction techniques have shown promising results in the remediation of new pollutants in aquatic bodies. Due to the immobility and heterogeneity of soil, microbial degradation technology demonstrates its technological and economic viability. However, currently, few microbial strains are known to efficiently degrade PFOA. Existing strains, while capable of degradation, exhibit slow degradation rates and low efficiency. Therefore, further research is needed to develop highly efficient degrading strains and enhance their degradation effects. Summary of the Invention

[0004] The purpose of this invention is to provide a facultative anaerobic iron-reducing bacterium and its application in treating new pollutants (perfluorooctanoic acid and its salinization and related compound pollution) in contaminated soil and water.

[0005] To achieve the above objectives, the present invention employs the following technology:

[0006] A facultative anaerobic iron-reducing bacterium, Clostridium diolis FLP22, was deposited at the China Center for Type Culture Collection on November 21, 2023, with accession number CCTCC NO.: M 20232292.

[0007] An application of the aforementioned facultative anaerobic iron-reducing bacteria is characterized by the long-term and stable application of the strain in the degradation of perfluorooctyl sulfonic acid and its salts in the environment.

[0008] The strain has a long-lasting and stable application in the degradation of perfluorooctyl sulfonic acid and its salts in soil or water.

[0009] The strain is used for the long-term and stable degradation of perfluorooctyl sulfonic acid and its salts in soil or water under aerobic or anaerobic conditions.

[0010] A microbial agent for repairing PFOA, the agent containing the strain described above.

[0011] The bacterial agent may also be a mixture of the strain and the bacterial source mixture; or a mixture of mixed bacterial groups; wherein, the bacterial source mixture may be activated sludge from a sewage treatment plant, soil from an organically contaminated site, river sediment, etc.; the mixed bacterial group is a mixture of at least two of the following genera: Pseudomonas, Acidimicrobiales, Sporomusa, Acinetobacter, Megamonas, Rhizobium, Citrobacter, Desulfovibrio, Dehalococcoides, and Chrysosporium.

[0012] When the bacterial agent is a mixture of bacterial strains and bacterial sources, the bacterial strains account for approximately 10% to 30% of the total bacterial abundance in the bacterial agent.

[0013] If the bacterial mixture contains activated sludge from a wastewater treatment plant, this sludge is the residual activated sludge after sequential anaerobic and aerobic treatment. The bacteria in this sludge can degrade both aerobic and anaerobic pollutants. The sludge settling ratio (SV%) is 20%–30%, the suspended solids concentration (MLSS) is 2000–6000 mg / L, and the organic carbon content is less than 60%. If the sludge is soil or river sediment, it needs to be acclimatized to either anaerobic or aerobic conditions for one week before use. After acclimatization, the culturable bacterial count per gram of solids should be greater than 3 × 10⁶ CFU.

[0014] The bacterial strain in the bacterial agent is a culture, suspension, fermentation broth, or immobilized preparation of the strain.

[0015] A method for remediating PFOA-contaminated soil or water using a microbial agent involves inoculating the microbial agent into the contaminated soil or water to be remediated, achieving pollutant degradation under aerobic conditions; or, under anaerobic conditions, achieving pollutant degradation through anaerobic stirring.

[0016] The initial bacterial concentration after inoculation with the added microbial agent in the environment is 1×10⁶ to 5×10⁷ CFU per gram of soil or per milliliter of water.

[0017] Before adding the bacterial agent, iron minerals or salts containing ferric iron need to be added to the water or soil to promote the degradation and metabolism of PFOA by the bacterial agent. After addition, the Fe element mass ratio should be 1% to 3%.

[0018] Advantages of this invention:

[0019] The strain of this invention is a facultative anaerobic iron-reducing Clostridium, a Gram-positive rod-shaped bacterium without a capsule, and its spores are often larger than the bacterial cell, giving the cell a spindle shape. It can utilize acetate, citrate, sugars, and soil humus as carbon sources for growth, and can also ferment to produce hydrogen under anaerobic conditions. The humic acid-like substances and hydrogen produced through fermentation can serve as electron transport media, making it suitable for the metabolic degradation of PFOA in soil and water under both aerobic and anaerobic conditions. It can also enhance the PFOA degradation function of organic matter-degrading bacteria in other media. Therefore, this strain has the ability to completely mineralize and degrade PFOA in soil and water and reduce its environmental toxicity. Attached Figure Description

[0020] Figure 1 Phylogenetic analysis of the 16S rDNA gene sequence of Clostridium diolis FLP22, a facultative anaerobic iron-reducing bacterium provided in the embodiments of the present invention.

[0021] Figure 2 A photograph of the colony morphology of Clostridium diolis FLP22, a facultative anaerobic iron-reducing bacterium, provided in an embodiment of the present invention.

[0022] Figure 3 A scanning electron microscope image of Clostridium diolis FLP22, a facultative anaerobic iron-reducing bacterium, provided for an embodiment of the present invention.

[0023] Figure 4 The graph shows the change of PFOA content over time in each treatment group under anaerobic conditions when Clostridium diolis FLP22, a facultative anaerobic iron-reducing bacterium provided in this embodiment of the invention, is applied to the soil.

[0024] Figure 5 The graph shows the change in defluorination rate after anaerobic treatment of PFOA-contaminated soil with facultative anaerobic iron-reducing Clostridium diolis FLP22 provided in this embodiment of the invention.

[0025] Figure 6 The graph shows the change in bacterial abundance over time in each treatment group under anaerobic conditions when Clostridium diolis FLP22, a facultative anaerobic iron-reducing bacterium provided in this embodiment of the invention, is applied to the soil.

[0026] Figure 7 The graph shows the change of PFOA concentration over time in each treatment group under anaerobic conditions when Clostridium diolis FLP22, a facultative anaerobic iron-reducing bacterium, is applied to water as provided in the embodiments of the present invention.

[0027] Figure 8 The graph shows the change in defluoridation rate after anaerobic treatment of PFOA-contaminated water with facultative anaerobic iron-reducing Clostridium diolis FLP22, as provided in this embodiment of the invention.

[0028] Figure 9 The graph shows the change of PFOA concentration over time in each treatment group under aerobic conditions when Clostridium diolis FLP22, a facultative anaerobic iron-reducing bacterium, is applied to water as provided in the embodiments of the present invention. Detailed Implementation

[0029] To make the objectives and technical solutions of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] This invention provides a facultative anaerobic iron-reducing Clostridium diolis FLP22 strain, obtained from river sediment receiving wastewater treatment plant effluent. This strain can efficiently degrade PFOA in water and soil, and its degradation rate can be improved through optimization of its application methods. Therefore, the strain provided by this invention, suitable for PFOA degradation in soil and water, has significant market potential and will contribute to the development of the remediation industry for persistent pollutants in soil and water.

[0031] Example 1: Domestication and induction of PFOA degradation function of (Clostridium diolis) FLP22.

[0032] Strain FLP22 is a facultative anaerobic iron-reducing Clostridium diolis, and its 16S rDNA gene sequence is shown below. It was deposited at the China Center for Type Culture Collection (CCTCC) on November 21, 2023, with accession number CCTCC NO: M20232292. Upon identification, Clostridium diolis FLP22 belongs to the genus Bacillus. Figure 1 Morphological and physiological-biochemical characteristics of the facultative anaerobic iron-reducing bacterium *Clostridium diolis* FLP22 were observed. These characteristics indicate that it is a Gram-positive bacterium that forms round or irregular colonies with smooth surfaces, produces spores, and can utilize glucose, sodium acetate, and sodium pyruvate as carbon sources for anaerobic fermentation or aerobic metabolism. Figure 2 ), the cell morphology is rod-shaped ( Figure 3 ).

[0033] FLP 22

[0034]

[0035] The selected facultative anaerobic iron-reducing Clostridium diolis (FLP22) was cultured anaerobicly and aerobically in LB medium. At the logarithmic growth phase, the bacterial cells were collected by centrifugation and washed with an inorganic salt solution. The washed cells were then vortexed in the inorganic salt solution and inoculated into PFOA-contaminated inorganic salt culture medium (PFOA concentration 30 mg / L) under anaerobic and aerobic conditions, respectively, and cultured for one month. The inorganic salt culture medium consisted of: 0.15 g / L magnesium sulfate (MgSO4·7H2O), 0.06 g / L calcium chloride (CaCl2), 0.25 g / L ammonium chloride (NH4Cl), 0.6 g / L magnesium sulfate (MgSO4·7H2O), 0.24 g / L dipotassium hydrogen phosphate (K2HPO4·3H2O), 0.08 g / L potassium dihydrogen phosphate (KH2PO4), and 0.5% glucose. For the anaerobic culture medium, add 0.5 g / L L-cysteine ​​and deoxygenate with nitrogen. Then sterilize the medium at high temperature. After sterilization, add PFOA to a final concentration of 35 mg / L, and simultaneously inoculate with 5% bacterial suspension. Incubate at 30°C for 30 days, then centrifuge to collect a small amount of cells for scale-up culture in LB medium for later use.

[0036] Example 2: Characteristics of facultative anaerobic iron-reducing Clostridium falciparum FLP22 in anaerobic remediation of PFOA-contaminated soil

[0037] The PFOA-contaminated soil was remediated using facultative anaerobic Clostridium FLP22. First, sterilized clean soil containing 20 mg / kg PFOA was prepared. In one treatment, 3% magnetite was added as an iron source. Then, the contaminated soil was inoculated with an inorganic salt culture medium containing the expanded culture of facultative anaerobic Clostridium FLP22. After mixing, the concentration of the target bacteria was adjusted to 2.4 × 10⁷ CFU / g soil, with a soil moisture content of 50%, forming a slurry state. The reaction system was placed in a 200 ml serum bottle. The inorganic salt culture medium was formulated as follows: 0.15 g / L magnesium sulfate (MgSO4·7H2O), 0.06 g / L calcium chloride (CaCl2), 0.25 g / L ammonium chloride (NH4Cl), 0.6 g / L magnesium sulfate (MgSO4·7H2O), 0.24 g / L dipotassium hydrogen phosphate (K2HPO4·3H2O), 0.08 g / L potassium dihydrogen phosphate (KH2PO4), and 0.5% glucose. Treatment T1 consisted of only functional bacteria; treatment T2 consisted of magnetite mixed at 0.5% (w:w) of the soil mass with functional bacteria; and treatment T0 consisted of unsterilized contaminated soil without either magnetite or functional bacteria. Remediation began after the functional bacteria were introduced. Samples were taken at 0, 10, 20, 40, 60, and 80 days after incorporation to determine the content of perfluorooctanoic acid (PFOA) and fluoride ions, as well as bacterial abundance in the soil.

[0038] The results showed that, with the extension of treatment time, the PFOA content in the control soil treatment (T0) remained essentially unchanged; however, in the T1 and T2 treatment groups mixed with functional bacteria, the PFOA content gradually decreased, especially in the treatment group mixed with magnetite, where the PFOA degradation rate was significantly improved. Figure 4 The degradation rate reached its highest level of 40.15% after 70 days. Analysis of the degradation products of PFOA revealed a significant increase in fluoride ion content.

[0039] Furthermore, the microbial content in the treatment group mixed with functional bacteria initially increased rapidly during the treatment process, then decreased slightly, but remained higher than the initial inoculum. This reflects that the facultative anaerobic Clostridium FLP22 provided by this invention can stably exist in the soil environment and has excellent anaerobic degradation ability for PFOA pollutants, demonstrating good application potential.

[0040] Example 3: Characteristics of facultative anaerobic Clostridium FLP22 in anaerobic and aerobic remediation of PFOA-contaminated water.

[0041] The facultative anaerobic bacterium FLP22 was used to remediate PFOA-contaminated water bodies.

[0042] First, a culture medium was prepared containing 1.25 g / L ferric citrate, 0.15 g / L magnesium sulfate (MgSO4·7H2O), 0.06 g / L calcium chloride (CaCl2), 0.25 g / L ammonium chloride (NH4Cl), 0.6 g / L magnesium sulfate (MgSO4·7H2O), 0.24 g / L dipotassium hydrogen phosphate (K2HPO4·3H2O), 0.08 g / L potassium dihydrogen phosphate (KH2PO4), and 0.5% glucose. After sterilizing the above culture medium, a 10 mg / L PFOA-contaminated water solution was prepared under aseptic conditions. Part of the solution was dispensed into anaerobic bottles for deoxygenation and later use, while the other part was dispensed into aerobic Erlenmeyer flasks for use. The untreated polluted water was designated as S0 (anaerobic) and X0 (aerobic). 1 ml of the FLP22 bacterial culture obtained from the above-mentioned expanded culture was mixed into the above polluted water, with an abundance of 3.8 × 10⁶ CFU / ml after inoculation, and this was designated as treatment S1 (anaerobic) and X1 (aerobic). The above-mentioned self-prepared high-concentration PFOA polluted water was mixed with excess sludge from a wastewater treatment plant. This wastewater treatment plant treats domestic sewage and some pre-treated industrial wastewater year-round. The sludge's settling ratio (SV%) was 20%–30%, and its suspended solids concentration (MLSS) was 2000–6000. The sludge concentration was mg / L, and the organic carbon content in the sludge was less than 60%. After repeated washing and settling, 2 ml of the washed sludge suspension was added to the PFOA-contaminated water body as treatment S2 (anaerobic) and X2 (aerobic). The initial bacterial abundance of the system after inoculation was 2.6 × 10⁷ CFU / ml. 2 ml of the above-mentioned washed wastewater treatment plant sludge suspension was mixed with the contaminated water body, and 1 ml of FLP22 bacterial culture obtained from the expansion culture was added simultaneously as treatment S3 (anaerobic) and X3 (aerobic). The initial abundance of FLP22 strain accounted for 15% of the initial total bacterial abundance. The above culture system was incubated at 30°C with shaking. Perfluorooctanoic acid (PFOA) content in the water body was measured at 0, 10, 20, 30, and 50 days after mixing, and the fluoride ion content in the solution after the reaction was also measured.

[0043] The anaerobic treatment experiment results showed that, with the extension of treatment time, the PFOA content in the control group S0 did not change significantly; in the treatment group S2, which incorporated activated sludge, the PFOA content decreased slightly, with a degradation rate of approximately 12%; in the treatment groups S1 and S3, which incorporated functional bacteria, the PFOA content decreased significantly, especially in the treatment group S3, which simultaneously incorporated activated sludge and degrading functional bacteria, where the highest PFOA degradation rate reached 68.31%; the degradation rate in S2 was approximately 49.97%. This may be because the functional bacteria FLP22 forms a relatively stable degradation community with the degrading bacteria in the activated sludge, exhibiting a better PFOA degradation effect compared to single-strain bacteria. In addition, due to the excellent iron reduction function of FLP22, low-valent reduced iron is provided for the dehalogenation of organic matter, promoting the degradation effect of dehalogenating bacteria in the sludge. Further analysis of the PFOA degradation products by ion chromatography showed an increase in fluoride ion content, with a defluorination rate of approximately 24%, proving that the PFOA underwent a defluorination reaction.

[0044] The results of the aerobic treatment experiment showed that this strain also has the function of degrading PFOA, with a maximum degradation rate of about 30%, which is slightly lower than that of the anaerobic treatment group.

[0045] The experimental results show that the facultative anaerobic Clostridium FLP22 provided by this invention can degrade PFOA under both aerobic and anaerobic conditions, and has good application potential.

Claims

1. Use of a facultative anaerobic iron-reducing bacteria for degrading PFOA in soil, characterized in that: The Latin name of the strain is Clostridium diolis FLP22, which was preserved in the China Center for Type Culture Collection on November 21, 2023, and the preservation registration number is CCTCC NO.: M 20232292.

2. Use of a facultative anaerobic iron-reducing bacteria according to claim 1, characterized in that: The application of the strain to degrade PFOA in soil under aerobic or anaerobic conditions.

3. A method for remediating PFOA contaminated soil or water using a microbial agent, characterized by: The bacterial agent is inoculated into contaminated soil or water to be remediated, and the degradation of pollutants is realized under aerobic conditions or through anaerobic stirring under anaerobic conditions. The bacterial agent contains the facultative anaerobic iron-reducing bacteria in claim 1. Before inoculating the bacterial agent, iron minerals or salts containing trivalent iron are added to the water body or soil to promote the degradation metabolism of the bacterial agent to PFOA, and the mass ratio of Fe element after addition is 1% to 3%.

4. The method for repairing PFOA contaminated soil or water body by using microbial agent according to claim 3, characterized in that: The bacterial agent is a mixture of the facultative anaerobic iron-reducing Clostridium and a bacterial source mixture according to claim 1; or mixed with a mixed bacterial population; wherein the bacterial source mixture is activated sludge from a sewage treatment plant, soil from an organic pollution site, river sediment; the mixed bacterial population is at least two kinds of mixtures of Pseudomonas ( Pseudomonas ), Acidiphilium ( Acidimicrobiales ), Muscodor ( poromusa ), Acinetobacter ( Acinetobacter ), Megamonospora ( Megamonas ), Rhizobium ( Rhizobium ), Citrobacter ( Citrobacter ), Desulfovibrio ( Desulfovibrio ), Dehalococcoides ( Dehalococcoides ), Chrysosporium ( Chrysosporium ).

5. The method for repairing PFOA contaminated soil or water body by using microbial agent according to claim 4, characterized in that: The bacterial concentration after inoculation with the added microbial agent in the environment was 1×10⁻⁶. 6 ~5×10 7 CFU per gram of soil or per milliliter of water.

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