Feammox flora for anaerobic reduction defluorination of perfluorooctanoic acid and application of Feammox flora

Abstract

The invention belongs to the field of environmental biotechnology and wastewater biological treatment, and discloses a Feammox flora for anaerobic reduction defluorination of perfluorooctanoic acid and application of the Feammox flora. The Fammox flora is obtained by taking activated sludge as an inoculum, taking ammonium salt as an electron donor under an anaerobic condition and taking a ferric iron compound as an electron acceptor through enrichment and domestication; and the Feammox flora is used for reducing, defluorinating and degrading the perfluorooctanoic acid under an anaerobic condition. The method comprises the following steps: inoculating the Fammox flora into perfluorooctanoic acid-containing water to be treated, and reacting under an anaerobic condition in the presence of ammonium salt and ferric iron compounds. According to the invention, the Fammox composite flora which is domesticated from common activated sludge and does not depend on Acidimicrobium sp. A6 is used for the first time, efficient anaerobic biodegradation of PFOA is realized, microbial resources for PFAS bioremediation are broadened, and the Fammox composite flora has the characteristics of high efficiency, tolerance, wide application prospect and the like.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology and wastewater biological treatment, and relates to a Feammox (iron ammonia oxidation) bacterial community for anaerobic reduction defluorination of perfluorooctanoic acid (PFOA), and its application in the remediation of water bodies contaminated with perfluorinated and polyfluoroalkyl substances (PFAS). Background Technology

[0002] Perfluorinated and polyfluoroalkyl substances (PFAS), especially perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), are known as "permanent chemicals" due to their high CF bond energy and extremely stable chemical properties, making them difficult to degrade in the environment. They can travel long distances across media in the environment, leading to widespread pollution of drinking water, livestock, and crops, posing a serious threat to ecosystems and human health. Traditional physicochemical treatment methods (such as adsorption and advanced oxidation) are costly and prone to secondary pollution. Although studies have reported that Acidimicrobium sp. Strain A6 can mediate the reductive defluorination of PFOA / PFOS during the Feammox process, its application suffers from limitations such as single strain, limited function, and poor adaptability to complex environments.

[0003] Whether and how the enriched and domesticated Feammox microbial community, possessing greater complexity and adaptability, from engineering systems (such as activated sludge in wastewater treatment plants) can efficiently degrade PFAS (such as PFOA) was previously unclear, and corresponding microbial agents and application methods were lacking. Therefore, developing a stable and effective biological treatment technology for the reduction, defluorination, and degradation of PFOA is particularly important. Summary of the Invention

[0004] The purpose of this invention is to overcome the limitations of single strains and provide a Feammox composite microbial community enriched and domesticated from activated sludge, which has a highly efficient PFOA anaerobic reduction defluorination function, and the application of this community in the degradation of PFAS pollutants.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A Feammox microbial community for anaerobic reduction and defluorination of perfluorooctanoic acid (PFOA) was obtained by enriching and domesticating activated sludge under anaerobic conditions using ammonium salts as electron donors and ferric compounds as electron acceptors. The microbial community is capable of reducing, defluorinating, and degrading PFOA under anaerobic conditions.

[0007] The Feammox microbial community for anaerobic reduction defluorination of perfluorooctanoic acid includes the following microbial groups: UBA12294, OLB14, Desulfobacillus, CAADGL01, Brocadia, Rudaea, Rhodanobacter, and Zeimonas.

[0008] When the bacterial community degrades PFOA, it can produce the following intermediate products: perfluoroheptanoic acid (PFHpA), perfluorohexanoic acid (PFHxA), perfluorovalerate (PFPeA), and perfluorobutyric acid (HFBA).

[0009] A microbial agent comprising the above-mentioned Feammox bacteria that undergo anaerobic reduction defluorination of perfluorooctanoic acid.

[0010] The application of the Feammox bacterial community in the degradation of perfluorooctanoic acid.

[0011] A method for degrading perfluorooctanoic acid (PFOA) in water includes: inoculating the above-mentioned Feammox bacteria into water to be treated containing PFOA, and carrying out the reaction under anaerobic conditions in the presence of ammonium salts and ferric compounds.

[0012] The trivalent iron compound is ferrous ore.

[0013] The pH value of the reaction system is 5.0-5.5, and the reaction temperature is 20-30℃.

[0014] The beneficial effects of this invention are:

[0015] (1) Originality: For the first time, the Feammox complex microbial community, which is domesticated from ordinary activated sludge and does not depend on Acidimicrobium sp. A6, was disclosed. It can achieve efficient anaerobic biodegradation of PFOA and broaden the microbial resources that can be used for PFAS bioremediation.

[0016] (2) High efficiency and tolerance: This bacterial group has high tolerance to PFOA (500 μg / L) and can achieve a removal rate of more than 77% at this concentration, showing its potential for practical application.

[0017] (3) The mechanism is clear: Through intermediate product tracing and metagenomic analysis, the degradation pathway of stepwise reduction and defluorination (decarboxylation-hydroxylation-fluorine elimination-hydrolysis) was clarified, and the mechanism of the transformation of recalcitrant pollutants was revealed through functional division of labor (iron ammonia oxidation, iron reduction, dehalogenation, fluorine tolerance) within the microbial community was revealed.

[0018] (4) Broad application prospects: It provides a new environmentally friendly bioremediation strategy for PFAS contaminated media based on the Feammox principle, which is especially suitable for anaerobic groundwater or sediment environments rich in iron and ammonium. Attached Figure Description

[0019] Figure 1 This is a diagram showing the morphology and activity of Feammox bacterial community in this invention.

[0020] Figure 2 This is a diagram of the microbial composition of the Feammox bacterial community based on metagenomic analysis, as presented in this invention.

[0021] Figure 3 The figure shows the results of the bio-defluorination and degradation of PFOA by Feammox according to the present invention;

[0022] Figure 4 This is a graph showing the detection results of intermediate products of Feammox defluorination and degradation of PFOA according to the present invention.

[0023] Figure 5 This is a graph showing the fluoride ion detection results of Feammox defluorination and degradation of PFOA according to the present invention;

[0024] Figure 6 This is a metagenomic functional gene-species annotation diagram of the Feammox bacterial community according to the present invention. Detailed Implementation

[0025] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0026] Part 1: Enrichment and domestication methods for Feammox microbiota.

[0027] Inoculation source: Activated sludge from the secondary sedimentation tank of Lingshuihe Wastewater Treatment Plant in Dalian, Liaoning Province.

[0028] Reactor: Upflow anaerobic reactor, 40 cm high, 9 cm in diameter, with an effective volume of 2.5 L.

[0029] Culture medium: A synthetic culture medium containing ammonium salts (NH4Cl 177 mg / L and (NH4)2SO4 78 mg / L) as electron donors, ferrous sulfate (6.5 g) as electron acceptors, bicarbonates (KHCO3 140 mg / L and NaHCO3 40 mg / L) as inorganic carbon sources, and KH2PO4 9 mg / L, MgSO4·7H2O 100 mg / L, CaCl2·2H2O 60 mg / L, AQDS 9 mg / L, ZnSO4·7H2O 430 μg / L, CoCl2·6H2O 240 μg / L, MnCl2·4H2O 990 μg / L, CuSO4·5H2O 250 μg / L, NaMoO4·2H2O 220 μg / L, and NaSeO4·10H2O. 210μg / L, NaWO4·2H2O 50μg / L, EDTA·2Na10mg / L.

[0030] Operating conditions: Anaerobic (N2 / CO2 (90 / 10, v / v) mixture introduced), pH maintained at 5.0~5.5, temperature at approximately 25℃, hydraulic retention time (HRT) of 16 hours.

[0031] Domestication markers: The system stably exhibits a coupling phenomenon between ammonia nitrogen removal and Fe(II) formation, i.e., Feammox activity; through 15 Nitrogen isotope labeling experiments confirmed that the product was... 30 N2.

[0032] Part Two: Composition and Functional Characteristics of the Microbial Community.

[0033] The Feammox microbial community is a complex microbial community. Metagenomic analysis shows that it includes the following functional groups: (1) genera with Feammox functions, such as Brocadia (anaerobic ammonia oxidizer) and Rudaea, which are responsible for coupling ammonia oxidation and iron reduction; (2) genera with symbiotic bacteria that provide nutrients for Brocadia, such as Desulfobacillus and CAADGL01; (3) USA12294 and OLB14, which have nitrite removal and aggregate formation functions; (4) genera with dissimilar iron reduction functions, such as Rhodanobacter, whose genome contains genes related to extracellular electron transport (such as DmkB, FmnB), which can reduce Fe(III); (5) genera with potential dehalogenation / degradation functions, such as Zeimonas, whose genome contains genes related to dehalogenation under PFOA stress (such as dhaA, The relative abundance of dehH increases; (6) Bacterial genera with fluoride efflux function: such as Rhodanobacter, under PFOA stress, the expression of genes encoding fluoride efflux proteins (such as crcB, FEX) is upregulated, helping the bacterial community to tolerate the F produced by defluorination. - .

[0034] Functional validation: The bacterial community maintained its activity under initial PFOA stress of 500 μg / L and achieved a PFOA removal rate of 77.3% within 100 days; the defluorination process was confirmed by the detection of fluoride ion release and four distinct defluorination intermediates (PFHpA, PFHxA, PFPeA, HFBA).

[0035] Part 3: Application methods of microbial communities in the degradation of PFOA.

[0036] Application targets: Water bodies, groundwater or sediments contaminated with PFOA or other structurally similar perfluorinated or polyfluoroalkyl substances.

[0037] Application conditions: (1) Anaerobic environment: The reaction system needs to be kept anaerobic; (2) Electron donor: Provide ammonium salts (such as NH4Cl, (NH4)2SO4) as electron donors for microbial metabolism; (3) Electron acceptor: Provide trivalent iron minerals (preferably amorphous ferrohydrate) as terminal electron acceptors; (4) pH and temperature: The optimal pH range is 5.0-5.5, and the optimal temperature range is 20-30℃.

[0038] Inoculation and reaction: The enriched and domesticated Feammox microbial community was inoculated into the treatment system for reaction, and ammonium salts and iron sources were replenished regularly.

[0039] Examples and Experimental Data

[0040] Example 1: Enrichment and activity verification of Feammox bacterial community.

[0041] (1) The activated sludge inoculum was taken from the secondary sedimentation tank of Lingshuihe Wastewater Treatment Plant in Dalian, Liaoning Province.

[0042] (2) Enrichment of Feammox microorganisms: 1 L of activated sludge was added to an upflow anaerobic reactor with an effective volume of 2.5 L. Artificial synthetic culture medium was added in a continuous flow manner. The medium contained ammonium salts (NH4Cl 177 mg / L and (NH4)2SO4 78 mg / L) as electron donors, ferrohydrate (6.5 g) as electron acceptors, bicarbonate (KHCO3 140 mg / L and NaHCO3 40 mg / L) as inorganic carbon sources, and KH2PO4 9 mg / L, MgSO4·7H2O 100 mg / L, CaCl2·2H2O 60 mg / L, AQDS 9 mg / L, ZnSO4·7H2O 430 μg / L, CoCl2·6H2O 240 μg / L, MnCl2·4H2O 990 μg / L, CuSO4·5H2O The concentrations of the following ingredients were used: 250 μg / L NaMoO4·2H2O, 220 μg / L NaSeO4·10H2O, 210 μg / L NaWO4·2H2O, and 10 mg / L EDTA·2Na. The culture medium was anaerobic (purged with a 90 / 10, v / v N2 / CO2 mixture), with pH maintained at 5.0–5.5 and temperature at approximately 25°C. The hydraulic retention time (HRT) was 16 hours, and the enrichment culture lasted for 255 days.

[0043] (3) Validation of Feammox bacterial activity: The enrichment and acclimatization process was monitored by measuring ammonia nitrogen removal, Fe(II) formation, and 15 N-labeled experiments produced 30 N2 confirmed the existence of the Feammox pathway.

[0044] (4) 15 N-labeling experiment: 2g of wet weight Feammox bacterial culture was added to a 100mL anaerobic flask, mixed with 80mL of synthetic culture medium, and then... 15 NH4Cl 177 mg / L and ( 15 NH4)2SO4 78 mg / L was used as the electron donor, 0.21 g of ferrohydrate was used as the electron acceptor, and bicarbonate (KHCO3 140 mg / L and NaHCO3 40 mg / L) was used as the inorganic carbon source. High-purity helium (99.999%) was introduced to maintain the anaerobic environment, the pH was maintained at 5.0~5.5, the temperature was about 25℃, and the culture was carried out for 1 week.

[0045] Example 2: Tolerance and degradation effect of Feammox bacteria on PFOA.

[0046] (1) Implementation method: The domesticated Feammox bacterial culture (wet weight 1 g / L) was placed in a 100 mL anaerobic bottle containing 500 μg / L PFOA for defluorination and degradation experiments. The anaerobic bottle contained 80 mL of artificially synthesized culture medium. 15 NH4Cl 177 mg / L and ( 15 NH4)2SO4 78 mg / L was used as the electron donor, 0.21 g of ferrohydrate was used as the electron acceptor, and bicarbonate (KHCO3 140 mg / L and NaHCO3 40 mg / L) was used as the inorganic carbon source. An anaerobic environment was maintained by introducing a N2 / CO2 (90 / 10, v / v) mixture, the pH was maintained at 5.0~5.5, the temperature was about 25℃, and the culture was carried out for 100 days.

[0047] (2) Data results: After 100 days of cultivation, the removal rate of 500 μg / L PFOA reached 77.3%.

[0048] (3) Evidence of defluorination: 25.8 μg / L of F was detected in the defluorination degradation experiment of 500 μg / L PFOA using ion chromatography. - The stepwise reduction defluorination products of PFOA were clearly detected by UPLC-MS / MS: PFHpA → PFHxA → PFPeA → HFBA. The cumulative product amount was positively correlated with the initial PFOA concentration. No PFOA was detected in the sterilized control group and the control group without added ferrous sulfate. - The aforementioned intermediates demonstrate that the degradation is bio-driven and dependent on the Feammox process.

[0049] Example 3: Analysis of bacterial community structure and functional genes.

[0050] The Feammox bacterial community was sent to Shanghai Lingen Biotechnology Co., Ltd. for metagenomic sequencing. The raw sequencing data has been stored in the NCBI Biotechnology Project Database, with the number PRJNA1345523. The raw sequence data fragments of the metagenomic genome can be obtained in SRA with the number SRR35926746.

[0051] The Feammox microbial community comprises the following microbial groups: UBA12294, OLB14, Desulfobacillus, CAADGL01, Brocadia, Rudaea, Rhodanobacter, and Zeimonas.

[0052] The functional genes of the Feammox microbial community include the following groups: carbon fixation genes (cbbL, cbbG), iron reduction genes (DmkB, FmnB), nitrogen transformation genes (hao, hzsABC, narGHIJ, nirBDKS, nosZ, norB), dehalogenation genes (dhaA, dehH) and fluoride efflux genes (crcB, FEX).

[0053] The above are preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made to the structure, principle and method of this application should be covered within the scope of protection of this application.

Claims

1. A Feammox bacterial community for anaerobic reduction defluorination of perfluorooctanoic acid, characterized in that, The Feammox microbial community was obtained by enriching and domesticating activated sludge as an inoculum under anaerobic conditions using ammonium salts as electron donors and ferric compounds as electron acceptors; the Feammox microbial community reduced, defluorinated and degraded perfluorooctanoic acid under anaerobic conditions.

2. The Feammox bacterial community for anaerobic reduction defluorination of perfluorooctanoic acid according to claim 1, characterized in that, The Feammox microbial community comprises the following microbial groups: UBA12294, OLB14, Desulfobacillus, CAADGL01, Brocadia, Rudaea, Rhodanobacter, and Zeimonas.

3. A microbial inoculant, characterized in that, The Feammox microbial community comprising the perfluorooctanoic acid anaerobic reduction defluorination as described in claim 1 or 2.

4. The application of the Feammox microbial community for anaerobic reduction defluorination of perfluorooctanoic acid as described in claim 1 or 2 in the degradation of perfluorooctanoic acid.

5. A method for degrading perfluorooctanoic acid (PFOA) in water, characterized in that, include: The Feammox bacteria as described in claim 1 or 2 are inoculated into water to be treated containing perfluorooctanoic acid, and the reaction is carried out under anaerobic conditions in the presence of ammonium salts and ferric compounds.

6. The method according to claim 5, characterized in that, The trivalent iron compound is ferrous ore.

7. The method according to claim 5, characterized in that, The pH value of the reaction system is 5.0-5.5, and the reaction temperature is 20-30℃.

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